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Functions of the CXC ligand Family in the
Pancreatic Tumour Microenvironment
Nien-Hung Lee
MDHS
ORCID ID: orcid.org/0000-0001-5423-8880
Doctor of Philosophy
September 2021
Department of Surgery, Austin Health
Faculty of Medicine, Dentistry and Health Sciences
The University of Melbourne
Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy
September 2021
I
Abstract
Chemoresistance is the major contributor to the low survival of pancreatic cancer
(PC). PC progression is a complex process reliant on interactions between tumour and
tumour microenvironment (TME). A family of structurally similar inflammatory
chemokines, namely CXC ligands (CXCLs), were recently discovered to play
important roles in various cancer types, including PC. This thesis aimed to investigate
the role of CXCL5 in chemoresistance of PC.
In both human and mice PC cell lines tested, CXCL5 expression was dramatically
upregulated. The expressions of CXCL5, CXCL10 and selected CSC genes were
various in gemcitabine resistant cell lines, and gemcitabine treated cells. However, in
mouse xenografted tumour samples, which was generated from a patient-derived cell
line, gemcitabine alone or in combination with other chemotherapeutic reagents led to
increased CXCL5 protein level while CXCL10 level remained unchanged. These
results suggested that expression of CXCL5 may be stimulated upon administration of
gemcitabine or other chemotherapeutic reagents. Therefore, CXCL5 has a role in
chemoresistance and clinical importance in PC; however, the mechanisms involved
deserves a careful investigation.
To determine whether CXCL5 mediates chemoresistance in PC, CXCL5 expression
in MiaPaCa-2 cells was knocked down by shRNA. To determine whether CXCL5
mediated chemoresistance in vitro, two chemotherapeutic drugs, were used to treat a
negative control (NC) and CXCL5 knockdown (KD) clones. In the cell proliferation
assays, CXCL5 was found to mediate the resistance to gemcitabine and 5-fluouracil (5-
FU). Mice carrying xenografted tumours inoculated by either NC or CXCL5 KD cells
II
were treated with gemcitabine. CXCL5 KD suppressed tumour growth and enhanced
the inhibitory effect of gemcitabine by decreasing proliferation and promoting
apoptosis These results indicated that knockdown of CXCL5 sensitized PC cell
response to gemcitabine and 5-FU, suggesting that CXCL5 mediates chemoresistance
in PC.
Finally, the global proteomic analysis showed CXCL5 knockdown resulted in
significant changes in expression of several proteins. Each of these proteins had a
distinct biological function in cancer as determined with KEGG pathway analysis and
NCBI. From the phosphor-proteomic analysis, CXCL5 knockdown induced significant
changes of certain phosphorylated proteins. Cross-referencing with the database of
NCBI clearly identified the biological functions of these proteins. Although
experimental and clinical validation are necessary, CXCL5 serves as a pivotal
molecular target in overcoming chemoresistance and eliminating PC tumours in clinical
practices.
In summary, these studies have revealed that CXCL5 plays an important role in
chemoresistance and activates several intracellular pathways that contribute to
resistance to therapeutic treatments and PC progression. Therefore, CXCL5 could serve
as a potential molecular target in reversing chemoresistance in pancreatic cancer.
III
Declaration
This is to certify that:
• The thesis comprises only my original work towards the PhD except where indicated
in the Preface.
• Due acknowledgement has been made in the text to all other material used.
• The thesis is fewer than 100,000 words in length, exclusive of tables, maps,
bibliographies and appendices.
Nien-Hung Lee
July 2021
IV
Preface
Chapter 1 of this particular thesis represents literature review that has been
published (Pancreatology. 2018 Oct; 18(7): 705-716. doi: 10.1016/j.pan.2018.07.011).
This work was jointly conducted by Dr. Hong He and Assoc. Prof. Mehrdad Nikfarjam
who aided in manuscript drafting and were both involved in manuscript revision and
final approval before publication. I independently reviewed all the literature, drew and
placed all the figures, wrote the initial manuscript, answered reviewers’ questions,
revised and corrected the final manuscript according to editor’s suggestions. Prof
Graham Baldwin provided valuable comments in manuscripts revision. The Kinghorn
Center Cell Line 15 (TKCC15) which was generated by and obtained from The
Kinghorn Cancer Center, Garvan Institute of Medical Research, was a generous gift
from Dr. Marina Pajic’s laboratory.
For Chapter 4, Kai Wang, Nhi Huynh and Dr. Hong He assisted me in part of the in
vivo experiments, data collection and analysis. I independently carried out all the rest
in vitro experiments, analyzed the data, assembled and drew the figures and tables and
wrote the initial manuscript. Dr. Oneel Patel assisted the creation of competent E. coli
bacterial cells for the amplification of CXCL5 shRNA plasmids. Dr. Hong He and
Chelsea Dumesny assisted me with generating CXCL5 knockdown and negative
control clones of MiaPaCa-2 cells. I independently conducted the
immunohistochemical staining experiments and analyzed the subsequent data as well
as plotting the figures and tables. Therefore, it is estimated that my contribution to the
work done in this thesis exceeds 85%. And I acknowledge all the indispensable
assistance and important contribution from those people mentioned above in
conducting this study for my PhD project.
V
Acknowledgements
This particular doctor degree could not possibly be completed without the non-
conditional support from all the ones who offered guidance, mentor, inspiration and
friendship along the way. First of all, I would like to thank my supervisors, Assoc Prof
Mehrdad Nikfarjam and Dr Hong He, for giving me the opportunity to conduct research
into chemoresistance in pancreatic cancer in this fascinating project. Your kind
guidance in the research field of both clinical and basic sciences in pancreatic cancer,
the ability to always make time for me, patience, and ongoing support were
indispensable and have provided me with a solid foundation to continue my future in
the field of biomedical research. Lastly, I would like to show my gratitude to you both
for your mentorship and friendship over the last few years.
I would also like to personally thank the Department of Surgery for allowing me to
conduct research in the laboratory. Special thanks to Chelsea Dumesny, Kai Wang and
Nhi Huynh who was always willing to share their time to go through my work with me
or provide me a hand in the laboratory. In addition, the authors would like to show
gratitude to Mr. Kai Wang and Ms. Nhi Huynh for sharing their pearls of wisdom with
them during the establishment of the mouse model of PC in this research. Also to Prof
Graham Baldwin for his invaluable advice, scientific support and guidance. Thank you
to the rest of the department, past and present, animal facility, and tissue biobank for all
your help and the friendships I have gained.
I would also like to thank Dr Chin-Seng An and the Bio21 Molecular Science and
Biotechnology Institute. Dr An has offered a help for the proteomic analyses conducted
in the laboratory and have contributed to the vital context of my research into this cancer.
VI
Finally, I would like to thank my parents for their unconditional love and support
throughout my life, and everything they have done for me. And I also would like to
thank all my friends for understanding and providing good help in fitting in a different
country.
VII
Table of Contents
Abstract………………………………………………………………………..……....I
Declaration…………………………………………………………………………..III
Preface…………………………………………………………………………...…..IV
Acknowledgements…………………………….………………………………...…..V
List of Figures…………………………..……….………………………….……….XI
List of Tables…..……………...…………………………………………..………..XIII
Abbreviations……...…………………………………………………...….……....XIV
Chapter 1: Functions of the CXC ligand (CXCL) family in the pancreatic tumour
microenvironment……………………………………………………………………1
1.1 Introduction………………………………………………………………..…..2
1.2 Roles of CXCLs in pancreatic cancer…………………………………………..3
1.2.1 CXCL1………………………………………………………………….6
1.2.2 CXCL2…………………………………………..…………..…….…....8
1.2.3 CXCL5…………………………………………………………....…….9
1.2.4 CXCL6………………….……………………………………………..10
1.2.5 CXCL7……………………………………………………………...…11
1.2.6 CXCL8…………………………………………………………...……11
1.2.7 CXCL9……………..………………………………………………….12
1.2.8 CXCL10………………………...………………………………..……13
1.2.9 CXCL11……………………………………………………………….14
1.2.10 CXCL12……………………………………………………….……..15
1.2.11 CXCL13……………………...…………………………………..…..16
1.3 Current findings of the relationship between CXCLs and cancer stem cell
characteristics…………………………………………………………………….17
VIII
1.4 Roles of CXCL family members in chemoresistance………………………....18
1.5 Roles of CXCRs in CSC phenotypes and chemoresistance…..……………….18
1.5.1 CXCR1…………………………………………………………….…….19
1.5.2 CXCR2…………………………………………………………………..19
1.5.3 CXCR3…………………………………………………………………..20
1.5.4 CXCR4………………………………………………………….……….21
1.5.5 CXCR7…………………………………………………………………..22
1.6 Targeting CXCLs in PC………………………………………………….……23
1.7 Conclusion…………………………………………………..………………..24
Chapter 2: Materials and methods…………………………………………………26
2.1. Materials………..……………………………………………………………27
2.1.1. Reagents………….…………………………………………...………27
2.1.2. Chemotherapeutic reagents……..………………………………...…..28
2.1.3. Reverse transcription polymerase chain reaction (RT-PCR)………….28
2.1.4. Immunohistochemistry…………………………………………….…31
2.1.5. CXCL5 knockdown with shRNA…………………………………….33
2.1.6. MTT (thiazolyl blue tetrazolium bromide) assay……………………..34
2.1.7. Tumour tissue embedding....………………………………………….35
2.1.8. Proteomic analysis….………………………………………..……….35
2.1.9. Instruments………………………………………………….………..37
2.2. Methods………………………………………………………………..…….38
2.2.1. Cell culture.………….………………………………………………..38
2.2.2 Reverse transcription polymerase chain reaction (RT-PCR)..……...….39
2.2.3. Immunohistochemistry…………..………………………….………..43
2.2.4. CXCL5 knockdown with shRNA.……………………………………45
2.2.5. MTT assay.……………….………………………………….………..49
IX
2.2.6. Mice experiment.…..….……………………………….……………..50
2.2.7. Tumour tissue embedding.………………..………………..…..……..51
2.2.8. Patient Samples...…..…………………………………………………51
2.2.9. Proteomic Analysis.……………………………..…………………....52
2.2.10. Statistical Analysis…….…………………………………………….54
Chapter 3: Involvement of CXC ligands (CXCLs) and cancer stem cell (CSC)
markers in chemoresistance of pancreatic cancer….…...……………………...….55
3.1 Abstract……………………………………………………………………….56
3.2 Introduction…………………………………………………………………..56
3.3 Scientific method……………………………………………………………..59
3.4 Results…………….………………………………………………………….59
3.4.1 Expression of CXCLs and cancer stem cell (CSC) genes in human and
mouse pancreatic cancer (PC) cells.……………...…………………………….59
3.4.2 Correlations between PAK1, CXCLs and selected CSC genes..…..……...64
3.4.3 Effect of PAK1 knockdown on expression of selected CXCL members.....66
3.4.4 Effects of gemcitabine resistance on CXCL5, CXCL10 and the CSC
genes……………….…………………………………………………..………69
3.4.5. Expression of CXCL5 in tumour samples from PC patients.…………….73
3.5 Discussion.……………………………………………………………………76
Chapter 4: The role of CXC ligand 5 in chemoresistance of pancreatic cancer......80
4.1 Abstract………….……………………………………………………………81
4.2 Introduction…………………………………………………………………..81
4.3 Scientific method………………………………………………………….….84
4.4 Results………………………………………………………………………..84
4.4.1 Establishment of CXCL5 knockdown (KD) MiaPaCa-2 cell lines…….……84
4.4.2 Effects of gemcitabine and 5-FU on growth of CXCL5 knockdown cell
X
lines…………….………………………………………….……………………..87
4.4.3 CXCL5 protects PC tumours against gemcitabine in vivo…………………..89
4.4.4 Effects of CXCL5 on PC cell proliferation and apoptosis in the presence of
gemcitabine in vivo…….…...…………….…………………………………..…..93
4.5 Discussion….…………………………………………………..……………..96
Chapter 5: Mechanism(s) involved in the effect of CXCL5 on chemoresistance in
pancreatic cancer……………….………………………………..………………….99
5.1 Abstract………………………………………………………………….…..100
5.2 Introduction…………………………………………………………...…….100
5.3 Scientific method….………………………………………………………...103
5.4 Results…………………………………………………………………..…..104
5.4.1 Difference in global proteins between negative control (NC) and CXCL5
knockdown (KD) clones of MiaPaCa-2 cells………………………..……….104
5.4.2 Difference in phosphorylation of proteins between NC and CXCL5 KD
clones of MiaPaCa-2 cells………………………………………………...….108
5.3 Discussion………..………………………………………………………….113
Chapter 6: General discussions and future directions……………...…………...118
6.1 CXCL5 has a potential role in chemoresistance in PC……………..…….…119
6.2 CXCL5 is a gene that mediates chemoresistance in PC………………..…...121
6.3 Proteomic analysis of pathways involved in CXCL5 functions in PC……..122
6.4 Limitations and future directions…………………………………...………122
6.5 Conclusion…………………………………………………………………..124
References…………………………………………………………………...……..124
Appendix: Paper publication…………………………………….………………….164
XI
List of Figures
Figure 1.1.1 Interactions between tumour cells and stromal cells contributes to
chemoresistance and other behaviors of cancer…………………………………..……3
Figure 1.2.1 CXCL molecular structures………………………………………………4
Figure 1.2.2. Effects of binding of CXCLs to their corresponding CXCRs in cancer…..5
Figure 1.4.1 Binding of CXCLs to CXCRs leads to interactions with TME cellular
components…………………………………………………………………………...25
Figure 3.4.1. Expression patterns of PAK1, CXCLs and selected CSC genes in human
PC cell lines…………………………………………………………………………..61
Figure 3.4.2. Expression patterns of PAK1, CXCLs and selected CSC genes in mouse
PC cell lines…………………………………………………………………….…….63
Figure 3.4.3. Effect of PAK1 knockdown on expression of selected CXCL members..68
Figure 3.4.4. Effect of gemcitabine resistance on expression of CXCL5, CXCL10,
ALDH1, CD24, CD44 and CD133 in MiaPaCa-2, Pan02 and TB117 cells…………...70
Figure 3.4.5. Effect of gemcitabine on CXCL5 and CXCL10 in BxPC-3, MiaPaCa-2,
Panc-1 and TB117 PC cell lines……………………………………………………....71
Figure 3.4.6. Gemcitabine along or in combination with abraxane or PF378309
increased CXCL5 expression while CXCL10 remained unchanged in TKCC15
xenografts………………………………………………………………………….....73
Figure 3.4.7. CXCL5 expression may contribute to death and T stage in a cohort of 33
patients……………………………………………………………………………….75
Figure 3.4.8. CXCL5 expression may contribute to death and T stage in a cohort of 33
patients……………………………………………………………………………….76
Figure 4.4.1 CXCL5 knockdown by CXCL5 human shRNA plasmids……………….86
Figure 4.4.2 Effects of gemcitabine and 5-FU on proliferation of NC1, NC2, CXCL5-
XII
KD12 and CXCL5-KD22 MiaPaCa-2 clones………………………………………...88
Figure 4.4.3 CXCL5 knockdown inhibited tumour growth in the presence of
gemcitabine in
vivo…………………………………………………………………………………...91
Figure 4.4.4 Expression of CXCL5 in NC and CXCL5KD tumours in the presence or
absence of gemcitabine treatment…………………………………………………….92
Figure 4.4.5 Expression of Ki-67 in NC and CXCL5KD tumours in the presence or
absence of gemcitabine…………………………………………………..………...…94
Figure 4.4.6 Expression of Caspase 3 in NC and CXCL5KD tumours in the presence
or absence of gemcitabine in
vivo……..………………….…………………………………………………….…...95
Figure 5.4.1 Differences in the global intracellular protein expression between NC and
CXCL5KD MiaPaCa-2 clones……………………………………………………....105
Figure 5.4.2. Differences in the phosphorylated intracellular protein expression
between NC and CXCL5KD MiaPaCa-2 clones…………………………………….110
XIII
List of Tables
Table 1.2.1 The roles of various CXCLs in PC……………………..…………………..6
Table 2.1.1 Primers……………..………………………………………………….....30
Table 2.2.2 Annealing temperatures for distinct pairs of primers..……………………42
Table 2.2.3 Antibodies used for immunohistochemical staining……..…………….....44
Table 3.4.1.………………………………………………………………………...…64
Table 4.4.1 IC50 concentrations of gemcitabine and 5-FU in cell proliferation..……....89
Table 5.4.1 Proteins with most significant differences between NC and CXCL5 KD
cells....……………………………………………………………………………….106
Table 5.4.2. Cellular functions of proteins with most significant differences between
NC and CXCL5 KD cells…………………………………………………………....107
Table 5.4.3 Biological functions of proteins with most significant differences between
NC and CXCL5 KD cells……………………………………………………………108
Table 5.4.4. Phosphorylated Proteins with most significant differences between NC and
CXCL5 KD cells………………………………………………………………….....111
Table 5.4.5. Cellular functions of phosphorylated proteins with most significant
differences between NC and CXCL5 KD cells……………………………………...112
Table 5.3.6. Biological functions of phosphorylated proteins with most significant
differences between NC and CXCL5 KD cells……………………………………...113
XIV
Abbreviations
ACN acetonitrile
ADD2 β-adducin
Akt protein kinase B
ALDH1 aldehyde dehydrogenase 1
ANKRD26 ankyrin repeat domain-containing protein 26
AP-1 activator protein 1
ARID3A AT-rich interactive domain-containing protein 3A
BIN1 Myc box-dependent-interacting protein 1/ bridging integrator 1
CAF cancer-associated fibroblast
CD11b cluster of differentiation molecule 11B
CD24 heat stable antigen CD24
CD44 P-glycoprotein 1
CD133 prominin-1
CDA cytidine deaminase
COX-2 cyclooxygenase-2
CSC cancer stem cell
CT control
CXCL CXC ligand
CXCL5KD CXCL5 knockdown
CXCR CXC receptor
DAB 3,3′-diaminobenzidine
DC dendritic cell
DDW distilled deionized water
DKC1 H/ACA ribonucleoprotein complex subunit 4
XV
DMEM Dulbecco’s modified Eagle’s medium
DPX dibutylphthalate polystyrene xylene
EC endothelial cell
E. coli Escherichia coli
ECM extracellular matrix
EDTA ethylenediaminetetraacetic acid
EGFR epidermal growth factor receptor
EGTA ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
ELR Glu-Leu-Arg
EMT epithelial-mesenchymal transition
ERK1/2 extracellular signal-regulated kinase 1/2
FAK focal adhesion kinase
FAP fibroblast activation protein-α
FB fibroblast
FBS fetal bovine serum
FOXO3A forkhead box O3-A
5-FU 5-fluorouracil
Gαi Gi protein α subunit
GATA1 GATA Binding Protein 1
GCLM glutamate-cysteine ligase regulatory subunit
GEM gemcitabine
GEMR gemcitabine resistance
GPCR G protein-coupled 7-transmembrane receptor
hCNT concentrative nucleotide transporter
hENT human equilibrate nucleotide transporter
HLA-B HLA Class I histocompatility B-14 α chain
XVI
HPDE human pancreatic duct epithelial
HIF-1α hypoxia-inducible factor 1-α
IFN interferon
IKKα IκB kinase α
IL interleukin
KD knockdown
KEGG Kyoto Encyclopedia of Genes and Genomes
Ki-67 marker of proliferation Ki-67
KPC LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre
Kras Kirsten Rat Sarcoma
LB medium Luria-Bertani medium
LIN28 Lin-28 homolog A
Ly6G lymphocyte antigen 6 complex locus G6D
Mφ macrophage
MAPK mitogen-activated protein kinase
MCT1 monocarboxylate transporter 1
MDSC myeloid-derived suppressive cell
MEK mitogen-activated protein kinase kinase
miRNA microRNA
MMP2 matrix metallopeptidase 2
MMP9 matrix metallopeptidase 9
MO monocyte
MSC mesenchymal stem cell
mRNA messenger RNA
MRP5 multi-drug resistance-associated protein5
MRP8 multi-drug resistance-associated protein8
XVII
mTOR mammalian target of rapamycin
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MyD88 myeloid differentiation primary response 88
NAB2 NGFI-A-binding protein 2
NC negative control
NCBI National Center for Biotechnology Information
NDPK nucleotide diphosphate kinase
NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells
NMPK pyrimidine nucleotide monophosphate kinase
OXCT1 succinyl-CoA:3-ketoacid coenzyme A transferase 1, mitochondrial
PAK1 p21-activated kinase 1
PanIN pancreatic intraepithelial neoplasia
PC pancreatic cancer
PCR polymerase chain reaction
PDAC pancreatic ductal adenocarcinoma
PHLDB2 pleckstrin homology-like domain family B member 2
PI3K phosphatidylinositol 3-kinase
PRDX6 peroxiredoxin 6
RANBP2 E3 SUMO-protein ligase RanBP2
RR ribonucleoside reductase
PSC pancreatic stellate cell
RSK1/2 ribosomal s6 kinase 1/2
RT room temperature
RT-PCR reverse transcription polymerase chain reaction
SCID severe combined immunodeficient
SF1 splicing factor 1
XVIII
shRNA short hairpin RNA
SLC16A1 monocarboxylate transporter 1
Src proto-oncogene tyrosine-protein kinase Src
SRM spermine synthase
STAT1 signal transducer and activator of transcription 1
STAT3 signal transducer and activator of transcription 3
TAE Tris acetate EDTA
TBS-T Tris-buffered saline-Tween
TEAB triethylammonium bicarbonate buffer
TFA trifluoroacetic acid
TGF-β transforming growth factor β
TKCC15 The Kinghorn Center Cell Line 15
TME tumour microenvironment
TNF-α tumour necrosis factor α
Tris HCl Tris hydrochloride
TTC28 tetratricopeptide repeat protein 28
VEGF vascular endothelial growth factor
WT wild type
1
Chapter 1:
Functions of CXC Ligand Family in Pancreatic Tumour
Microenvironment
2
1.1 Introduction
Pancreatic cancer (PC) is one of the most prevalent cancers, with an increasing
incidence worldwide 1,2. Pancreatic ductal adenocarcinoma (PDAC) constitutes more
than 90% of PC, and has an 8% 5-year survival at diagnosis 1. Most patients have
metastatic disease at diagnosis or develop it following surgical resection.
Chemotherapy remains the mainstay of treatment, but response rates are generally poor.
Therapeutic resistance represents a major problem that limits the outcomes of
cancer therapy. Tumour cells either become intrinsically resistant to a cancer therapy 3,
or acquire resistance via different methods, such as DNA repair 4, enzymatic
inactivation, altered membrane transport or activation of an upstream molecular marker
to ensure cell survival 5. However, because of the side-effects of chemotherapy 6,
alternative methods, such as targeted therapy in PC or naturally-derived compounds,
may be applied to cure cancer 7,8.
The tumour stroma or tumour microenvironment (TME) is the microscopic location
where a tumour resides, and is composed of cancer cells, neighboring non-
cancerous/stromal cells (such as endothelial cells (ECs), fibroblasts (FBs) and
macrophages (Ms)), and the extracellular matrix (ECM) 9. Tumours are associated
with stromal cells which further enhance angiogenesis, chemoresistance, growth,
invasion, metastasis and survival of cancer cells (Fig. 1.1.1) 10. PC cells interact with
ECM, ECs, FBs, Ms, neutrophils and stellate cells 11,12. Cancer and stromal cells
communicate with each other by secreting signal molecules to either support or
suppress tumour progression 13. Moreover, the TME also enhances chemoresistance by
alternating cell adhesion and the structure of the ECM, by secreting growth factors and
3
by activation of chemoresistance pathways in tumour cells 9. Consequently, a successful
treatment, in addition to eliminating all cancer cells, could be the reprograming the
TME.
Figure 1.1.1 Interactions between tumour cells and stromal cells contributes to
chemoresistance and other behaviours of cancer. Tumour and stromal cells interact
with each other by secreting chemokines (e.g. CXCLs), growth factors or other peptidyl
signals. Such communications lead to chemoresistance, angiogenesis, metastasis,
proliferation and cell survival. Abbreviations: ECs, endothelial cells; FBs, fibroblasts;
Ms, macrophages; PSCs, pancreatic stellate cells.
1.2 Roles of CXCLs in pancreatic cancer
A family of structurally similar inflammatory chemokines, namely CXC ligands
(CXCLs), in which C stands for cysteine and X stands for any amino acid (Fig. 1.2.1),
were recently discovered to play important roles in various cancer types, including brain,
colon, liver, lung and pancreas 13-16. These CXCLs are separated into two structurally
distinct groups, ELR+ and ELR-, by the presence or absence of a Glu-Leu-Arg (ELR)
4
motif at the N-terminus. CXCLs bind to specific CXC receptors (CXCRs), which are
G protein-coupled receptors, and cause chemotaxis of stromal cells (Fig. 1.2.2). The
ELR+ members, including CXCL1 to CXCL3 and CXCL5 to CXCL8, bind to CXCR1
and/or CXCR2 and contribute to angiogenesis in tumours. On the other hand, the ELR-
members (CXCL4, CXCL4L1, and CXCL9 to CXCL14) bind to CXCR3, CXCR4,
CXCR5 or CXCR7, are angiostatic, and have anti-tumour functions, with one exception,
CXCL12, which binds to CXCR4 or CXCR7 to promote metastasis. CXCR3, which
exhibits anti-tumour abilities, induces chemotaxis of ECs, Ms, monocytes (MOs),
platelets and T lymphocytes 16. Finally, each CXCL-CXCR combination attracts a
distinct range of stromal cells. For example, CXCL2-CXCR2 causes chemotaxis of ECs,
eosinophils, Ms, MOs and neutrophils 13,16.
Figure 1.2.1 CXCL molecular structures. CXCLs are separated into two structurally
distinct groups, ELR+ and ELR-, by the presence or absence of a Glu-Leu-Arg amino
acid sequence (ELR in the one letter amino acid code) at the N-terminus.
5
Figure 1.2.2. Effects of binding of CXCLs to their corresponding CXCRs in cancer.
ELR positive CXCLs (e.g. CXCL1 and CXCL5) bind to CXCR1 and/or CXCR2,
resulting in pro-tumour effects (such as chemoresistance, angiogenesis and metastasis)
and chemotaxis of a unique set of stromal cell types, including neutrophils, endothelial
cells, etc. On the contrary, binding of ELR negative CXCLs to either CXCR3, 4, 5 or 7
leads to distinct anti-cancer effects, for instance, reduced angiogenesis and decreased
tumour growth as well as recruiting a different range of TME cellular components (e.g.
T lymphocytes, macrophages and platelets). Abbreviations: ECs, endothelial cells;
EOSs, eosinophils; Ms, macrophages; MOs, monocytes.
Most members of the chemokine family, including CXCL1, CXCL2, CXCL5,
CXCL9, CXCL10 and CXCL13, have been found in PC 17-21, where they are secreted
by cancer or stromal cells, such as cancer-associated fibroblasts (CAFs) and dendritic
cells (DCs) 17,19,22. Most of these ligands have been reported to promote
chemoresistance, immunosuppression, tumour proliferation and metastasis 17,20,23,24.
However, certain CXCL chemokines, such as CXCL9 and CXCL10, may lead to
elimination of tumour cells as they evoke chemotaxis of CD4+ and CD8+ T lymphocytes,
DCs, natural killer and Th1 cells 25-27. The exact mechanism of how these ligands
6
modify the TME remains poorly understood, although modulation of the tumour stroma
has been reported to contribute to better cancer treatment 28,29. Ideally, targeting various
constituents of the TME, including CAFs, ECM and CXCL members that promote
tumour progression, would more effectively treat cancer. The CXCLs of interest in PC
are listed in Table 1.2.1 and discussed below.
Table 1.2.1 The Roles of Various CXCLs in PC. This table summarizes currently
known functions and intracellular molecules involved in signaling of CXCLs in PC.
1.2.1 CXCL1
CXCL1 is also known as Growth-Related Oncogene α (GROα) and its secreted
form was originally discovered in melanoma cell extracts and found to be involved in
7
oncogenic transformation 30. The ligand stimulates chemotaxis of CAFs, neutrophils
and myeloid-derived suppressive cells (MDSCs) toward tumours by binding to its
receptor(s) CXCR1 and/or CXCR2 31-33. In diverse cancer types, this ligand promotes
or is correlated with angiogenesis, metastasis, proliferation, resistance to chemotherapy,
tumour cell survival and tumourigenesis 20,31,34-36. In colorectal cancer, CXCL1 is
important for the formation of the premetastatic niche in the liver by recruiting CXCR2+
MDSCs to the destination site 33. The source of CXCL1 in this case is tumour-
associated Ms, which are stimulated by VEGF-A secreted by the primary tumour. In
a study of bladder cancer by Miyake et al., CXCL1 expression in cancer cells leads to
recruitment of both tumour-associated Ms and CAFs, which are known to extracellular
matrix components (e.g. collagen I and IV, fibronectin, tenascin-C) as well as
substances that attract ECs and pericytes 34. CXCL1 secreted from gastric tumour cells
stimulates the CXCR2-bearing bone marrow-derived mesenchymal cells infiltration to
gastric TME, inducing cancer cell proliferation and lymph node metastasis 37.
CXCL1 is found to be highly expressed in a panel of PC cell lines, and its expression
leads to increased tumour proliferation and angiogenesis as well as poor prognosis in
patients 20,38, via binding to its receptor, CXCR2 39. In the study by Lesina et al., the
CXCL1/CXCR2 axis caused chemotaxis of tumour-associated Ms, which promoted
tumour cell survival and growth by suppressing anti-tumour immunity 40. Clinically,
high expression of CXCL1 in both cancer cell cytoplasm and stroma of PC specimens,
is associated with carcinogenesis, tumour classification and TNM stage 38,40. Here T
refers to the size and extent of the primary tumour, N refers to the number of nearby
lymph nodes that have cancer and M refers to whether the cancer has metastasized. In
addition, the ligand promotes pancreatic oncogenesis and is also secreted by CAFs to
promote PC cell mobility 41,42. Recently, the promotor sequence of this gene was
8
discovered to contain binding sites for NF-κB, a vital transcription factor which is
involved in inflammation, tumour initiation and chemoresistance 43. In a LSL-KrasG12D
Ptf1a-Creex1 mouse model, the CXCL1/CXCR2 axis, which is downstream to NF-κB,
was shown to induce pancreatic carcinogenesis during development of PDAC 40. In a
study by Seifert et al., CXCL1 serves as a mediator of gemcitabine resistance, immune
suppression and the subsequent tumourigenesis in a LSL-KrasG12D/+; LSL-
Trp53R172H/+; Pdx1-Cre (KPC) mouse model and cell lines derived from the KPC
mice 41. The expression of CXCL1 was upregulated after depleting a necrosis-related
pathway in Ms, resulting in an immunosuppressive, tumour-promoting TME. Finally,
in a study involving xenografting a panel human PC cell lines and mouse Pan02 cells,
expression of CXCL1 was induced by IL-35, signaling via STAT1 and STAT3, and
CXCL1 mediated resistance to gemcitabine and monocyte infiltration, and stimulated
angiogenesis and xenograft tumour growth 44.
1.2.2 CXCL2
CXCL2, also known as Macrophage Inflammatory Protein-2α, was initially found
in supernatants of melanoma cell lines 45. It binds to CXCR2 and promotes chemotaxis
of neutrophils and ECs, contributing to angiogenesis, chemoresistance, transformation
and growth of tumours 13,31,46. In bladder cancer, CXCR2+ MDSCs are attracted to the
TME by CXCL2 secreted from the cancer cells 47. The MDSCs then secrete several
immunosuppressive molecules which reduce T cell proliferation, via activation of
MAPK and NF-κB by the CXCL2/CXCR2 axis. CXCL2 is highly expressed in tumours
in a KPC mouse model 48, and in human PC samples, CAFs of PC, and a panel of human
PC cell lines 39,40,49-51. From study of biopsies, binding of CXCL2 to its receptor,
CXCR2, is positively correlated with inflammation, and significantly reduces survival
9
of patients with PDAC. In a study by Kumar et al., smoking-induced inflammation
resulted in up-regulated CXCL2 expression, activation of DCs, Ms and PSCs and
tumourigenesis 18. In this study, the authors found that smoking induced inflammation
and secretion of inflammatory markers, such as CXCL1 and CXCL2. In addition,
smoking is known to cause oxidative damage by producing reactive oxidative species
52, and inflammation 18, in which the latter leads to more oxygen-containing free
radicals from Ms and subsequent carcinogenesis. PSCs participate in chronic
pancreatitis and the desmoplastic reaction 53. Activated PSCs can enhance proliferation,
migration, production of ECM by secreting cytokines and chemokines in PC.
1.2.3 CXCL5
In several cancer types, Epithelial Neutrophil-Activating Peptide-78 or CXCL5, is
associated with angiogenesis, chemoresistance, proliferation, migration and invasion,
and poor patient survival 51,54-58. In an intrahepatic cholangiocarcinoma study by Zhou
et al., CXCL5 induces infiltration of Ly6G+ neutrophils into the tumour stroma via
PI3K/Akt, ERK1/2 and RSK1/2 pathways in vivo 59. Then, the neutrophils mediate
cancer cell proliferation and motility and a similar TME pattern appears in human
samples. In colon cancer, platelet-derived CXCL5 recruits CD11b+Ly6G+ granulocytes
to lungs to establish early metastatic niches and subsequently leads to metastasis of
cancer cells 60.
CXCL5 is highly expressed in PC cell lines, including MiaPaCa-2 and Capan-2, as
well as in patient tumours 51. In pancreatic tumours, elevated levels of CXCL5 mRNA
are correlated with advanced clinical stages (stages T3 and 4), tumour progression and
poor patient survival 51. Moreover, CXCL5 from the cell-conditioned media of the PC
10
cell lines AsPC-1, BxPC-3 and Capan-2 induced angiogenesis by activation of Akt and
ERK through binding to the receptor CXCR2. Recently, CXCL5 was found to be co-
expressed with 3 markers of cancer stem cells (CSCs), namely ALDH1, CD44 and
CD133, which contributed to gemcitabine resistance 58. Finally, the CXCL5/CXCR2
axis in PC was also found to reinforce proliferation of HPAC-1 and Colo-357 cell lines
61.
1.2.4 CXCL6
CXCL6, also known as Granulocyte Chemotactic Protein-2, is expressed in several
types of cancers, including human bone, liver and lung cancers, as well as mouse
melanoma 62-65. High expression has been noted in pediatric osteosarcoma and is
correlated with poor survival 62. In hepatocarcinoma, transcription of CXCL6 can be
triggered by hypoxia and contributes to increased cancer invasion and migration 63,64.
In a study by Gijsbers et al., CXCL6 expression by ECs within gastrointestinal tumours
contributes to tumour development through angiogenesis due to EC chemotaxis and to
cell motility by recruiting neutrophils containing proteases that degrades extracellular
matrix 66.
In PC, CXCL6 is secreted by CAFs along with CXCL1, and CXCL8, and its
expression is higher in low grade PanIN (pancreatic intraepithelial neoplasia, defined
as microscopic flat or papillary lesions arising in small intralobular pancreatic ducts,
which are usually less than 5 mm in diameter) than in human PC 40,50,67. Currently, this
ligand is known to cause angiogenesis, inflammation and metastasis in PC 68; however,
it may not be important for advanced pancreatic tumours or in the context of
chemoresistance.
11
1.2.5 CXCL7
CXCL7, is expressed in breast cancer, colorectal adenocarcinoma, leukemia, lung
cancer, neuroblastoma, papillomas and renal cancer as well as in PC 48,69-75. In some
cases, CXCL7 seems to promote proliferation and/or cell mobility 70,75,76; however, its
expression level in PC is very low in all stages and it was therefore assumed to be
involved only in early carcinogenesis 74. The observation that CXCL7 expression was
induced after chemotherapy in colon cancer suggesting its role in chemoresistance [82].
Kruidenier et al. discovered that overexpression of CXCL7 reduced decitabine-induced
melanoma cell apoptosis and rescued decitabine-treated cell colonies via MAPK
pathways 77. In leukemia, addition of CXCL7 promoted resistance to decitabine in
chronic myelomonocytic leukemia cells with the involvement of MAPK pathways 78.
Overexpression of CXCL7 resulted in chemotaxis of CD206+ M2 type tumour-
associated Ms in lung cancer 79, and positively correlated with increased proliferation
of tumours. Results from these studies suggest a role of CXCL7 in chemoresistance;
however, the exact mechanism needs thorough investigation.
1.2.6 CXCL8
Originally known as IL-8, CXCL8 is a well-studied member of the CXCL family
which has multiple roles in tumour promotion by binding to CXCR1 and/or CXCR2 80.
Consistent with the fact that the promoter sequence of the CXCL8 gene contains an
NF-κB binding site, CXCL8 has been noted to promote or up-regulate angiogenesis,
cell mobility and establishment of a pro-tumoural microenvironment in breast, ovarian
and prostate tumours after its transcription and translation 81-83. In other cases, CXCL8
activates various intracellular pathways, including ERK1/2, PI3K/Akt, VEGF and NF-
12
κB 84-86, which have tumour-promoting effects and can cause chemoresistance 87. In
lung carcinoma, NF-κB activation leads to increased CXCL8 expression, which then
causes increased blood vessel formation in the bulk of the tumour 81.
Finally, CXCL8 also induces expression of CSC genes in PC in addition to its
known pro-tumoural functions 88-91. In breast cancer, the CXCL8/CXCR1/CXCR2 axis
mediates formation of a tumour sphere which serves as an indicator of self-renewal, a
characteristic of CSCs, partially via EGFR 92. ECs in the brain interact with
glioblastoma CSCs, and promote angiogenesis, metastasis and proliferation through
CXCL8 signaling 93. In a study by Chen et al., CXCL8 increases numbers of
subpopulation in tumour spheres formed from CD24+CD44+ in PC Capan-1 cells
binding to CXCR1 91.
1.2.7 CXCL9
The reported roles of CXCL9 in cancer have been contradictory. In some types of
cancer, including breast tumours, hepatocellular carcinoma, lung cancer, lymphoma and
melanoma, CXCL9 was shown to promote tumourigenesis, enhance metastasis and
reduce patient survival 94-97. On the other hand, CXCL9 also resulted in reduction of
cancer progression by stimulating anti-tumour immunity 97-100. Since CXCL9 has
displayed both tumour promoting and suppressing effects, its role in PC is uncertain. In
a study by Bronger et al., interferon (IFN)-γ-induced CXCL9 exerts tumour-
suppressing function by recruiting tumour-infiltrating lymphocytes after exposing a
COX inhibitor in ovarian cancer 101. Hu et al. discovered that doxorubicin alone or plus
IFN-12 induced elevation of CXCL9 102. This led to chemotaxis of tumour infiltrating
T cells into melanoma TME and caused increased apoptosis in tumour cells. In another
13
study, IFN-γ-induced CXCL9 also caused an anti-cancer response in lung TME in
which neutralization of the chemokine led to a reduced frequency of CXCR3+ T cells
and DCs at the tumour site 27.
A study by Thakur et al. showed a surge of CXCL9 expression in cancer cells after
treatment with antibodies against CD3 and HER2 plus Th1 cytokines, and a consequent
significant reduction in differentiation of MDSCs and their immunosuppressive
function in PC cells 103. In another study, IFN-γ, which was secreted by DCs, exerted
an anti-angiogenetic effect through induction of CXCL9 22, after inducing chemotaxis
of CD4+ T lymphocytes. As it has both tumour-promoting and anti-cancer effects in
different cancer types, its role in development and treatment in PC needs further
investigation.
1.2.8 CXCL10
CXCL10, also known as IFN-γ-inducible Protein 10, exerts anti-tumour function
by promoting the homing of immune cells, such as lymphocytes, into sites where
tumours are located 101,104-107. CXCL10 also acts through its receptor, CXCR3, to induce
adhesion to laminin, angiogenesis, metastasis and growth of cancer cells 108-112. In the
study by Bronger et al., in addition to CXCL9, CXCL10 also recruits lymphocytes into
ovarian TME to exert its tumour-eliminating effects 101. In study in lung cancer,
CXCL10 results in chemotaxis of CXCR3+ T lymphocytes and CD11c+ DCs into the
TME to eliminate tumour cells after IFN-γ treatment 27. In breast cancer, it was found
that tumour infiltration of T cells caused the subsequent death of cancer cells, which
was mediated by CXCL10 113.
CXCL10 is secreted by human PSCs, contributes to pancreatic tumour development
14
and positively correlates with the presence of intratumoural CXCR3+ regulatory T cells
114. Despite having a tumour promoting role, in an experimental model involving ex
vivo samples from 48 patients with resectable PC, CXCL10 showed little or no
enhancement of PC cell proliferation and migration 19. However, expression of the
mRNAs for CXCL10 and its receptor, CXCR3, was found to be high in patients with
PC. In a study with resected PC specimens and supernatants from co-cultures of PC cell
lines and primary tumour-associated PSCs, elevated CXCL10 concentrations were
correlated with increased chemotaxis of CD4+ and CD8+ T cells, leading to
immunosuppression and subsequent reduced tumour cell viability, and higher patient
survival 23. Furthermore, binding of CXCL10 to CXCR3 resulted in resistance to
gemcitabine. Finally, the elevated level of CXCL10 also led to down-regulated
differentiation and activation of MDSCs, which further enhanced its effect on
immunosuppression and chemoresistance in a 3-demensional co-culture model of
peripheral blood mononuclear cells with PC cells 103.
1.2.9 CXCL11
CXCL11, also known as IFN-Inducible Protein 9, is another ELR- ligand with both
cancer-promoting and anti-tumour functions. In basal cell carcinoma, colorectal
carcinoma, ovarian, prostate and renal cancers 115-118, this ligand contributes to
angiogenesis, invasion, migration and tumour growth by binding to CXCR7. The
associated pro-tumoural behaviors are shared by another tumour-promoting, ELR-
member of the CXCL chemokine family, CXCL12 119. On the other hand, in breast and
colon cancers CXCL11 causes chemotaxis of CXCR3+ cytotoxic T lymphocytes into
the tumour stroma and reduced angiogenesis by binding to CXCR3 on cancer cells 120.
Recently, Huang et al., discovered that in melanoma, CXCL11 significantly induced
15
infiltration of CD3+ T-lymphocytes into TME under influences of TNF-α and NF-κB
121, which was reversed by VEGF and rescued by addition of a chemotherapeutic
reagent, sunitinib. In prostate cancer, its expression is lower in tumours than in normal
prostate epithelial cells 122. In PC, although both CXCL11 and CXCL12 bind to CXCR4
and CXCR7, only CXCL11 drives proliferation of PC cells 123.
1.2.10 CXCL12
CXCL12 is found in a wide variety of cancer types, including brain, breast, colon,
gastric, lung, pancreatic, prostatic and thyroid cancers 124-131. CXCL12 binds to CXCR4
and CXCR7 to promote cancer progression 125,126. CXCL12 activates a range of
intracellular signaling molecules, including Akt, EGFR, ERK1/2, Gαi, mTOR, NF-κB
and Src 125,132-134, to promote chemoresistance, chemotaxis of CAFs, invasion,
migration and proliferation as well as CSC phenotypes 130,134-138. By blocking CXCL12
with chemical inhibitors in a murine model, Zboralski et al. found that there was an
enhanced infiltration of T lymphocytes and natural killer cells and an increased
activation of the T lymphocytes in the TME 139. In ovarian cancer, targeting CXCR4
reduced the infiltration of numbers of ECs, myeloid cells and plasmacytoid DCs 140.
In PC, CXCL12 facilitates proliferation, invasion, migration, chemoresistance and
angiogenesis 89,126,133,141. In a KPC murine model, chemotaxis of Schwann cells toward
pancreatic TME is mediated by the CXCL12/CXCR4/CXCR7 axis 142. In another study
using KPC mice, Feig et al. discovered that CXCL12 was secreted by FAP+ CAFs and
led to significantly reduced cytotoxic T cell infiltration in the PC TME and reduced
anti-tumour immunity 143. This T cell exclusion was mediated by CXCL12/CXCR4. As
observed in clinical observations, FAP+ stromal cells appear in almost all PC tumours
16
in patients 144, this discovery has a clinical implication that CXCL12/CXCR4 axis and
the FAP+ cells can be potential for PC treatment and prevention.
1.2.11 CXCL13
CXCL13, originally termed B-Cell-Attracting Chemokine, is expressed by stromal
cells within B-cell follicles in secondary lymphoid organs 145. Currently, the
CXCL13/CXCR5 axis has been reported to contribute to cancer progression in breast,
gastric, lung, pancreatic and prostate cancers 146-150. However, like CXCL9 and
CXCL10, it also exerts tumour-suppressing effects in other cases 151,152. In breast cancer,
expression of CXCL13 in tumour cells attracted CXCR5+ T regulatory and Th2 cells
into the TME which contributed to upregulating EMT and lymph node metastasis 153.
The CXCL13/CXCR5 axis was activated by NF-κB and nuclear factor (erythroid-
derived 2)-like 2, which was associated with increased infiltrating T lymphocytes of
IFN-γ+/IL-10+, and reductions of proliferation and metastasis of ovarian CSCs. In
androgen-deprived prostate tumours, B lymphocytes are recruited into TME by
secretion of CXCL13 from tumour-associated myofibroblasts via activation of hypoxia-
inducible factor 1/TGF-β pathway 154.
In a study by Lee et al., expression of CXCL13 at both the mRNA and protein level
was elevated in the pancreas of KrasG12D mice and in patients with advanced PC 155,156.
This significant increase in CXCL13 resulted in infiltration of B cells, which was found
to contribute to tumourigenesis and cell mobility 157. CXCL13 is also secreted by the
fibro-inflammatory stroma in PanIN lesions in mouse and human 158. In addition, the
high expression level of CXCL13 in PC is mediated by a non-canonical NF-κB/IKKα
pathway 159.
17
1.3 Current findings of the relationship between CXCLs and cancer stem cell
characteristics
There is increasing evidence showing that the signals from the tumour stroma
regulate and promote CSC activity. Several studies in breast, colon and ovarian cancers
have demonstrated that silencing tumour-promoting CXCLs significantly reduced CSC
properties leading to markedly improved treatments 160-162. In a study of ovarian cancer,
CXCL8 was induced after treating mesenchymal stem cells with tumour-conditioned
media and implied to regulate expression of genes involved in cell mobility and CSC
phenotypes (ALDH1 and CD133) 160. In colorectal cancer, mesenchymal stem cells
(MSCs) were found to secrete CXCL1 and CXCL8, in which the former induced cell
invasion and tumour initiation, which are two important features of CSCs 161,163. In a
study by Liu et al., co-culture of breast cancer and MSCs stimulated activity of ALDH1
by CXCL1 and CXCLs 5-8, and CXCL7 seems to play an important role in interaction
between MSCs and breast CSCs after stimulating the co-cultured cells with IL-6 162.
As mentioned previously, CXCL5 contributes to expression of several CSC genes
and chemoresistance in PC 58. In the same cancer type, CXCL8 promotes expression of
CSC genes involved in formation of tumour spheres, CD24 and CD44, by binding to
CXCR1 91. Finally, by binding to CXCR4, CXCL12 induces CD133 expression, which
subsequently results in resistance to gemcitabine and increased metastatic capability 138.
However, even with current knowledge of the relationship between CXCL members
and CSC functions in cancer, the exact molecular mechanisms which link the TME and
pancreatic CSCs remain poorly understood.
18
1.4 Roles of CXCL family members in chemoresistance
According to recent research, particularly in breast cancer, several members of the
CXCL family, including CXCL1, CXCL2, CXCL8 and CXCL12, have been shown to
be involved in resistance to cancer chemotherapies 31,164,165. Based on their highly up-
regulated expression, the CXCLs were shown to promote angiogenesis, cell survival,
chemoresistance, metastasis and proliferation after treatment with chemotherapeutic
reagents 31,166,167. As part of their intracellular signaling, molecular markers such as
MEK/ERK, NF-κB and STAT1, were found to be involved in their gene transcription
and downstream effects 31,168,169. Similar events were observed in acute myeloid
leukemia, colorectal carcinoma, gastric cancer, melanoma, renal cell carcinoma and
small cell lung cancer 164,170-174.
Most work done on chemoresistance in PC has been based on the study of
gemcitabine, and the current understanding of other chemotherapeutic drugs is still
preliminary 175. Both PC stroma and secretions from PSCs contributed to the limitation
of treatment efficiency via several intracellular signaling pathways, such as HIF-1α,
NF-κB and miRNAs (Fig. 1.4.1) 175,176. In a study by Delitto et al., CXCL10 was shown
to induce resistance to gemcitabine with reduced patient survival and increased cancer
cell survival 23. In another study, CXCL12, secreted by stromal cells, resulted in
chemoresistance and desmoplasia as well as elevated cell survival, proliferation and
metastasis 177. How CXCLs contribute to resistance to chemotherapy in patients with
PC is unknown.
1.5 Roles of CXCRs in CSC phenotypes and chemoresistance
As the receptors for CXCLs, several members of CXCRs have been found to
19
mediate expression of CSC phenotypes in distinct cancer types. Chemoresistance is a
dominant limitation to complete elimination of tumours and TME represents a pivotal
contributor [182]. In recent years, investigation of CXCRs in the field of
chemoresistance shone some light in better cancer treatment.
1.5.1 CXCR1
CXCR1, originally known as IL-8 receptor A, binds CXCL6 and CXCR8 13,
induces and/or maintains CSC populations and phenotypes in breast and thyroid
cancers and genes such as ALDH1, CD24, CD44 and CD133 are downstream to the
receptor 178-180. CXCR1 acts through a range of intracellular signaling molecules,
including FAK/Akt/FOXO3A. In PC, CXCR1 also leads to expression of stem-cell-like
characteristics such as sphere formation and metastasis 91.
In colon cancer, bevacizumab resistance and VEGF-independent angiogenesis are
mediated by CXCR1 181. Inhibition of CXCR1 with an antibody against the receptor
protein, reversed temozolomide resistance in melanoma cells leading to a significant
reduction of sphere-population 182. CXCR1 knockdown led to improved sensitivity to
cisplatin and reduced Akt phosphorylation even in the presence of CXCL8 in
osteosarcoma cells 183.
1.5.2 CXCR2
CXCR2, also known as IL-8 receptor B, binds CXCLs 1 to 7 in addition to CXCL8
16. The receptor couples with Gi (a small G protein) and recruits Ly6G+ neutrophils,
which are the predominant CXCR2+ cells among blood leukocytes, and ECs in cancers
184. In a range of cancer types, CXCR2 is shown to induce several CSC markers 93,185
20
including ALDH1, Oct-4 and Sox-2, metastasis and proliferation. In cancer cells,
CXCR2 acts via distinct intracellular signaling cascades including STAT3, to exert its
functions 186.
Blocking the CXCL7/CXCR2 axis diminished resistance to 5-fluouracil, suggesting
that CXCR2 is a potential target of reversing therapy resistance 187. CXCR2 was shown
to mediate resistance to taxol in breast cancer by downregulating Akt1 and activating
COX-2 as well as promoting tumour growth and metastasis 188. In colorectal cancer,
although CXCL8 activates both CXCR1 and CXCR2, only the later mediated
chemoresistance in HCT116 cell line 189. CXCR2 has been shown to mediate resistance
to FOLFIRINOX in PC 190, indicating an important role of CXCR2 in chemoresistance
in PC treatment.
1.5.3 CXCR3
CXCR3, binding to CXCL4 and CXCLs 9 to 11 191, is found to have three different
isoforms, A, B and alternative 192. In hepatocellular carcinoma, CXCR3A promotes
metastatic ability of CD133+ cells via ERK1/2-MMP2/MMP9 signaling pathway after
binding to CXCL9 193. In breast cancer, CXCR3B functions to increase tumour sphere
formation and ALDH1 activity 194. However, it is also found that CXCR3B displays
anti-tumour function 195. In addition, the CXCR3 ligands, namely CXCL9 and CXCL10,
also play anti-cancer roles in a number of cancers 26,97, including PC 22,97,103.
CXCR3 is also involved in chemoresistance. In PC, the CXCL10/CXCR3 axis is
activated in gemcitabine resistant cells and patient samples 196. CXCL10 secreted by
gemcitabine-resistant cells activated CXCR3, stimulating tumour growth and resistance
to the chemotherapeutic drug 197.
21
1.5.4 CXCR4
CXCR4, binding to CXCL12 ligand, is an important molecular factor in TME for
tumour progression 198. In a variety of types of tumours, including brain, breast,
esophageal, gastric and ovarian, CXCR4 has been shown to be responsible for
aggressiveness of CSCs and expression of CSC phenotypes (e.g. ALDH1 activity,
colony formation and tumourigenicity) by signaling through distinct intracellular
pathways, such as ERK1/2 199-203. PC cells with active CXCL12/CXCR4 axis displayed
high metastatic, proliferative and tumourigenic potentials as well as activating sonic
hedgehog and NF-κB 134,138,204.
In addition to its effects on CSC gene expression, CXCR4 also influence
chemoresistance in cancer. In ovarian cancer, CXCR4 overexpression resulted in
resistance to cisplatin in 124 patients and expression of several CSC markers (ALDH1,
ALDH2, MyD88, and LIN28) 203,205. The interaction between CXCR4 and CXCL12 in
the lung TME caused a protective effect against etoposide-induced apoptosis and
adhesion of tumour cells to fibronectin, collagen, and stromal cells 171. Blockage of
CXCR4 significantly reversed resistance to dacarbazine in the CXCR4+/CD133+
subpopulation in melanoma 206. In a study by Xiao et al., tumourigenesis and resistance
to gemcitabine are caused by downregulation of an miRNA, Let -7a, by CXCR4 in
BxPC-3 and Panc-1 PC cell lines in vitro and in vivo 207. Moreover, it is found that the
CXCL12/CXCR4 axis activated several intracellular signaling cascades, including
FAK, ERK and Akt, enhanced transcriptional activities of β-catenin and NF-κB, and
expression of survival proteins in the presence of gemcitabine 135. In addition,
CXCL12/CXCR4 induces chemotaxis of CAFs or mesenchymal stem cells to further
support gemcitabine resistance by activating Akt, MEK and ERK 208.
22
1.5.5 CXCR7
Another CXCL12 receptor, CXCR7 also plays important roles in cancer. Silencing
CXCR7 has displayed downregulation of expression of a number of CSC genes, such
as ALDH1 and CD44, in breast cancer 209. In prostate cancer, CXCR7 altered CD44
expression as well as CXCL8 and VEGF in order to contribute to angiogenesis and
metastasis 210. By acting through ERK, CXCR7 induces differentiation of CSCs in
hepatocellular carcinoma 211. As for PC, binding of CXCL12 activates ERL1/2 via
CXCR7 123.
CXCR7+ population in the SW480 colorectal cancer cell line displayed a higher
level of resistance to 5-fluouracil than the CXCR4+ cells 212. In lung cancer, CXCR7 is
upregulated by TGF-β and is responsible for induction of tumour sphere formation,
stem-like properties, chemoresistance and tumourigenesis in vivo 213. The increased
CXCR7 expression also correlates with CD44 expression and metastasis. In the bone
marrow microenvironment, CXCL12/CXCR7 pathway mediates imatinib resistance
via ERK activation 214.
From the above discussion, the CXCRs members, namely, CXCR2, CXCR4 and
CXCR7, seem to display great importance in chemotherapeutic resistance in distinct
cancer types. As mentioned above, several CXCLs have shown to mediate
chemoresistance as well as expression of CSC phenotypes (please see section 1.2).
CSCs are also capable of interacting with the surrounding TME 52. A specific
combination of CXCL and CXCR appears to be an important link between CSCs, TME
and tumour progression. As the CXCL/CXCR axis may serve as a prognostic marker
for treatment responses.
23
1.6 Targeting CXCLs in PC
Current chemotherapeutic reagents have only modest effects in extending patient
survival and restricting disease progression in PC. Although various combinations of
chemotherapies, such as FOLFIRINOX (a mixture of leucovorin, folinic acid, 5-
fluouracil, irinotecan and oxaliplatin), or gemcitabine and nab-paclitaxel, improve
overall successive eliminations of PC, chemoresistance still limits effectiveness of these
drugs 215. As many CXCLs have been shown to mediate chemoresistance and
interaction between tumours and stroma, they potentially serve as therapeutic targets in
PC.
CXCR2, also known as IL-8 Receptor β, was originally found to bind IL-8 (CXCL8)
13. CXCR2 also binds CXCL1, CXCL2, CXCL3 and CXCL5 to CXCL8, and is found
in a wide range of cancers, including PC 13,216. The receptor couples with a small G
protein on the cytoplasmic side of the cell membrane, Gi, and functions to mediate
proliferation, angiogenesis, tumourigenesis and metastasis, as well as inducing
chemotaxis of neutrophils that carry a specific surface antigen, Ly6G 184,216. In PC,
CXCR2 is highly expressed in patient samples and cell lines and promotes angiogenesis
by binding ELR+ CXCLs 48,217.
Although CXCR2 appears to be involved in PC progression, the pancreatic stroma
and intracellular signaling pathways (e.g. NF-κB) must also be taken into account as
CXCR2 is expressed in both tumour and stromal cells 216. For example, among all
tumour promoting CXCLs expressed in PC, CXCL5 is not only co-expressed with
CXCR2 in human patients but also in KPC mice 39. In the mouse model, the
CXCL5/CXCR2 axis mediates chemotaxis of CD11b+Ly6G+ neutrophils to promote
24
proliferation of cancer cells and suppress T cell activation 39. In human PC,
CXCL5/CXCR2 induces chemoresistance and poor patient survival as well as other
pro-tumoural functions 39,58,61.
In cancer, expression of CXCLs depends on several intracellular pathways,
including AP-1, NF-κB and STAT1, via binding sites in the CXCL promotor regions
61,218-221. Together with its ligands, CXCR2 mediates pro-tumoural characteristics of
cancer, including chemoresistance 20,31,51,56,87,222 and expression of CSC phenotypes
92,93,223,224. As a G protein-coupled receptor, there are number of downstream proteins
that mediate the effects of a specific CXCL-CXCR2 combination, including Gαi, Akt,
ERK1/2 and NF-κB 51,84,184. In a mouse PC model, NF-κB activation led to increased
activity of CXCR2 in the presence of KrasG12D mutation, resulting in elevated tumour
cell proliferation and reduced survival 40. As NF-κB acts both up- and down-stream to
CXCR2, it may form a positive feedback loop with a CXCL-CXCR2 axis which
continuously supports and promotes tumour malignancy.
1.7 Conclusion
PC communicates with cellular components within TME by expressing chemokines
and their receptors. The CXCL ligand family is being extensively researched in cancer
and other diseases. CXCL8 and CXCL12 are now the two most well-understood CXCL
family members in terms of their function, signaling pathways, and interactions with
TME cellular components and CSCs. However, as each cancer has a unique genomic
makeup and mutations, the exact secretome varies and other chemokine may play
specific roles in controlling PC progression. CSCs also interact with the TME and
mediate chemoresistance 225. As only a specific range of CXCLs are secreted by a
25
distinct cancer type, targeting a unique CXCL and its receptor may provide an
opportunity to improve treatment outcomes 13,14,191. Therefore, further investigation of
the effects of CXCLs on CSCs and their roles in the chemoresistance of PC is necessary.
Figure 1.4.1 Binding of CXCLs to CXCRs leads to interactions with TME cellular
components.
Such interaction may lead to chemoresistance, metastasis and proliferation in PC as
well as expression of cancer stem cell (CSC) phenotypes; however, the exact
mechanisms remain to be investigated. Moreover, although different intracellular
pathways contribute to the pro-tumour behaviors, their association with CSCs also
deserves a thorough study.
This particular chapter is now published in Pancreatology (Pancreatology, 2018.
18(7): p. 705-716. doi: 10.1016/j.pan.2018.07.011.).
26
Chapter 2:
Materials and Methods
27
2.1. Materials
2.1.1. Reagents
Abraxane (nab-paclitaxel; NDC68817-134-50, Abraxis Bioscience Australia Pty Ltd,
Melbourne, Victoria, Australia)
Apo-transferrin, ≥ 98% (T4382, Sigma-Aldrich Inc., St. Louis, MO, USA)
Dulbecco’s modified Eagle’s medium (DMEM; 11965-092, GIBCO, Thermo-Fisher
Scientific Corp., Grand Island, NY, USA)
Dulbecco’s phosphate-buffered saline (PBS; 70011-044, GIBCO, Thermo-Fisher
Scientific Corp., Grand Island, NY, USA)
EGF recombinant human protein solution (PHG0311L, Thermo-Fisher Scientific
Corp., Grand Island, NY, USA)
F-12 (1×) (11150-59, GIBCO, Thermo-Fisher Scientific Corp., Grand Island, NY,
USA)
Fetal bovine serum (FBS; A15B017, Assay Matrix Pty Ltd., Ivanhoe North,
Melbourne, Victoria, Australia)
D-(+)-Glucose solution (10%) (G8644, Sigma-Aldrich Inc., St. Louis, MO, USA)
HEPES (ultra-pure) (11344-041, GIBCO, Thermo-Fisher Scientific Corp., Grand
Island, NY, USA)
Hydrocortisone (H0888, Sigma-Aldrich Inc., St. Louis, MO, USA)
Gentamicin reagent (15750-060, GIBCO, Thermo-Fisher Scientific Corp., Grand
Island, NY, USA)
L-Glutamine (200 mM) (25030-081, GIBCO, Thermo-Fisher Scientific Corp., Grand
Island, NY, USA)
Insulin human recombinant (91077C, SAFC Biosciences, Sigma-Aldrich Inc., St.
28
Louis, MO, USA)
Medium 199 (1×) (11150-59, GIBCO, Thermo-Fisher Scientific Corp., Grand Island,
NY, USA)
MEM vitamin solution (10×) (11120-052, GIBCO, Thermo-Fisher Scientific Corp.,
Grand Island, NY, USA)
O-Phosphorylethanolamine (P0503, Sigma-Aldrich Inc., St. Louis, MO, USA)
Penicillin-streptomycin (15140-122, GIBCO, Thermo-Fisher Scientific Corp., Grand
Island, NY, USA)
PF3758309 (A-1091; Active Biochemical Co., Maplewood, NJ, USA)
3, 3’, 5 - Triiodo-L-thyronine sodium salt (tri-iodothyronine; T5516, Sigma-Aldrich
Inc., St. Louis, MO, USA)
Trypsin (25300-062, GIBCO, Thermo-Fisher Scientific Corp., Grand Island, NY,
USA)
2.1.2. Chemotherapeutic Reagents
5-Fluorouracil, ≥ 99% (HPLC), powder (5-FU; F6627, Sigma-Aldrich Inc., St. Louis,
MO, USA)
Gemcitabine (AUST R 160204, Hospira Pty Ltd., Melbourne, Victoria, Australia)
2.1.3. Reverse Transcription-polymerase Chain Reaction (RT-PCR)
(A) Extraction of Total RNA
Chloroform (496189, Sigma-Aldrich Inc., St. Louis, MO, USA)
Diethyl pyrocarbonate (DEPC; D5758, Sigma-Aldrich Inc., St. Louis, MO, USA)
Ethanol (305935, Chem-Supply Pty Ltd., Gillman, South Australia, Australia)
29
Isopropanol (269722, Chem-Supply Pty Ltd., Gillman, South Australia, Australia)
TRIzol™ reagent (15596026, Life Technologies Corp., Carlsbad, CA, USA)
(B) Reverse Transcription
Deoxynucleotide (dNTP) Solution Mix (dNTPs; #N0447L, New England BioLab Inc.,
Ipswich, MA, USA)
Oligo-dT (15Mer) (29015, MDBio Inc., Qingdao, Shandong, China)
M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant (reverse transcriptase;
M368B, Promega Corp., MI, USA)
M-MLV Reverse Transcriptase 5× Reaction Buffer (5×RT buffer; M531A, Promega
Corp., Madison, WI, USA)
rRNasin RNase Inhibitor (RNase inhibitor; N251B, Promega Corp., Madison, WI,
USA)
(C) Polymerase Chain Reaction (PCR)
Deoxynucleotide (dNTP) Solution Mix (dNTPs; #N0447L, New England BioLab Inc.,
Ipswich, MA, USA)
10× Standard Taq Reaction Buffer (Taq buffer; #B90145S, New England BioLab Inc.,
Ipswich, MA, USA)
Taq DNA Polymerase (Taq polymerase; #M0273L, New England BioLab Inc.,
Ipswich, MA, USA)
30
(D) Table 2.1.1 Primers
31
(E) Gel Electrophoresis
Acetic acid (UN2789, Chem-Supply Pty Ltd., Gillman, South Australia, Australia)
Agarose (BIO-41025, BioLine Pty Ltd., Alexandra, New South Wales, Australia)
Blue/orange 6× loading dye (loading dye; 0000232261, Promega Corp., Madison, WI,
USA)
Ethylenediaminetetraacetic acid disodium salt (EDTA; EA023, Chem-Supply Pty Ltd.,
Gillman, South Australia, Australia)
GelRed nucleic acid gel stain, 10000× in water (41003, Biotium Inc., Fremont, CA,
USA)
DNA ladder (10787-018, Invitrogen, Thermo-Fisher Scientific Corp., Grand Island,
NY, USA)
Trizma®
base (Tris-base; T6066, Sigma-Aldrich Inc., St. Louis, MO, USA)
2.1.4. Immunohistochemistry
Anti-Cleaved Caspase 3 antibody (Asp175) (#9661, Cell Signaling Technology Inc.,
Danvers, MA, USA)
Anti-CXCL5 antibody (ab9802, Abcam PLC, Cambridge, United Kingdom)
Anti-CXCL10 antibody (ab9807, Abcam PLC, Cambridge, United Kingdom)
Anti-Ki67 antibody (RM9106S, Thermo-Fisher Scientific Corp., Grand Island, NY,
USA)
Bovine serum albumin (BSA; A3912, Sigma-Aldrich Inc., St. Louis, MO, USA)
Citric acid monohydrate (CA014, Chem-Supply Pty Ltd., Gillman, South Australia,
Australia)
Cover slips, 22×40 mm (CS2240100, Menzel-Glaser, Trajan Scientific Australia Pty
32
Ltd., Melbourne, Victoria, Australia)
DAB+ Chromogen (10100906, Dako, Agilent Technologies Inc., Santa Clara, CA,
USA)
DAB+ Substrate Buffer (10118936, Dako, Agilent Technologies Inc., Santa Clara,
CA, USA)
DPX mountant for microscopy (36022294H, VWR International Ltd., Poole, UK)
Ethanol (305935, Chem-Supply Pty Ltd., Gillman, South Australia, Australia)
Histolene (FNNJJ031, Fronine Pty Ltd., Riverstone, New South Wales, Australia)
Labelled Polymer-HRP Anti-Rabbit Secondary Antibody (10118936, Dako, Agilent
Technologies Inc., Santa Clara, CA, USA)
Hematoxylin (MHS32, Sigma-Aldrich Inc., St. Louis, MO, USA)
Microscope slides superfrost plus, 25x75mm (SF41296SP, Menzel-Glaser, Trajan
Scientific Australia Pty Ltd., Melbourne, Victoria, Australia)
Normal goat serum (RA227554, Thermo-Fisher Scientific Corp., Grand Island, NY,
USA)
Peroxidase Blocker (10106908, Dako Pty Ltd., Agilent Technologies Inc., Santa Clara,
CA, USA)
Rabbit (DA1E) mAb IgG XP® isotype control (anti-rabbit IgG; #3900, Cell Signaling
Technology Inc., Danvers, MA, USA)
Scott’s blue tap water (151051152, Amber Scientific Pty Ltd., Midvale, Western
Australia, Australia)
Sodium chloride (NaCl; K46997104547, Merck KGaA, Darmstadt, Germany)
Trisodium citrate (467, Ajax Finechem Pty Ltd., Taren Point, New South Wales,
Australia)
Trizma®
base (Tris-base; T6066, Sigma-Aldrich Inc., St. Louis, MO, USA)
33
Tween 20 (P1379, Sigma-Aldrich Inc., St. Louis, MO, USA)
2.1.5. CXCL5 Knockdown with shRNA
(A) Preparation of Electrocompetent Escherichia coli (E. coli) Cells
Glycerol (G7757, Sigma-Aldrich Inc., St. Louis, MO, USA)
Sodium chloride (NaCl; K46997104547, Merck KGaA, Darmstadt, Germany)
Sodium hydroxide (NaOH; UN1823, Merck KGaA, Darmstadt, Germany)
Tryptone (211705, BD Biosciences, Franklin Lakes, NJ, USA)
Yeast extract (212750, BD Biosciences, Franklin Lakes, NJ, USA)
(B) Electroporation and Bacterial Transformation with Plasmid DNA
Agarose (BIO-41025, BioLine Pty Ltd., Alexandra, New South Wales, Australia)
Chloramphenicol (C0378, Sigma-Aldrich Inc., St. Louis, MO, USA)
CXCL5 Human shRNA Plasmid Kit (TL313631, OriGene Technologies Inc.,
Rockville, MD, USA)
Gene Pulser electroporation cuvettes, 0.1cm (1652089, BIO-RAD Laboratories Inc.,
Hercules, CA, USA)
Scrambled shRNA cassette (TR30021, OriGene Technologies Inc., Rockville, MD,
USA)
Sodium chloride (NaCl; K46997104547, Merck KGaA, Darmstadt, Germany)
Sodium hydroxide (NaOH; UN1823, Merck KGaA, Darmstadt, Germany)
Tryptone (211705, BD Biosciences, Franklin Lakes, NJ, USA)
Yeast extract (212750, BD Biosciences, Franklin Lakes, NJ, USA)
34
(C) Plasmid Extraction
Ethanol (305935, Chem-Supply Pty Ltd., Gillman, South Australia, Australia)
Genopure Plasmid Midi Kit (03-143-414-001, Roche Diagnostics GmbH, Mannheim,
Germany)
Isopropanol (269722, Chem-Supply Pty Ltd., Gillman, South Australia, Australia)
(D) Establishment of CXCL5 knockdown MiaPaCa-2 Cell Line and Colony
Selection
Lipofectamine® 2000 Reagent (11668-019, Invitrogen, Thermo-Fisher Scientific
Corp., Grand Island, NY, USA)
Penicillin-streptomycin (14190-144, GIBCO, Thermo-Fisher Scientific Corp., Grand
Island, NY, USA)
Puromycin (A11138-03, GIBCO, Thermo-Fisher Scientific Corp., Grand Island, NY,
USA)
2.5% Trypsin (10X) (15090-046, GIBCO, Thermo-Fisher Scientific Corp., Grand
Island, NY, USA)
2.1.6. MTT (Thiazolyl Blue Tetrazolium Bromide) Assay
Hydrochloric acid, 36% (HCL; 1789, Ajax Finechem Pty Ltd., Taren Point, New
South Wales, Australia)
Isopropanol (269722, Chem-Supply Pty Ltd., Gillman, South Australia, Australia)
Thiazolyl blue tetrazolium bromide (MTT; M5655, Sigma-Aldrich Corp., St. Louis,
MO, USA)
35
2.1.7. Tumour Tissue Embedding
Biopsy pads (Trajan Scientific Australia Pty Ltd., Ringwood, Victoria, Australia)
Ethanol (305935, Chem-Supply Pty Ltd., Gillman, South Australia, Australia)
Formalin solution, neutral buffered, 10% (HT501128, Sigma-Aldrich Corp., St. Louis,
MO, USA)
Tissue processing cassettes (M509, Simport Scientific, Saint-Mathieu-de-Beloeil, QC,
Canada)
2.1.8. Proteomic Analysis
(A) Radioimmunoprecipitation assay (RIPA) Buffer
Complete Mini protease inhibitor (11836153001, Roche Diagnostics GmbH,
Mannheim, Germany)
Ethylene Glycol-bis (β-aminoethyl ether) - N, N, N’, N’- extraacetic acid (EGTA; E-
4378, Sigma-Aldrich Corp., St. Louis, MO, USA)
PhosSTOP EASYpack (phosphatase inhibitor; 04-906-845-001, Roche Diagnostics
GmbH, Mannheim, Germany)
Sodium chloride (NaCl; K46997104547, Merck KGaA, Darmstadt, Germany)
Sodium deoxycholate (D-6750, Merck KGaA, Darmstadt, Germany)
Sodium hydroxide (NaOH; UN1823, Merck KGaA, Darmstadt, Germany)
Sodium lauryl sulfate, 95%, extra pure (SDS; SO04501000, Chem-Supply Pty Ltd.,
Gillman, South Australia, Australia)
Triton X-100 (Iso-octylphenoxypolyethaoxyethanol) (6007902, Thermo-Fisher
Scientific Corp., Grand Island, NY, USA)
Trizma® hydrochloride, reagent grade, minimum 99% redox. titration (Tris-HCL;
36
T5253, Merck KGaA, Darmstadt, Germany)
(B) Preparation for Proteomic Analysis
Acetone (UN1090, Chem-Supply Pty Ltd., Gillman, South Australia, Australia)
Acetonitrile (ACN; 1041130-935, Merck KGaA, Darmstadt, Germany)
Ammonia, anhydrous, ≥99.98% (294993, Sigma-Aldrich Corp., St. Louis, MO, USA)
Bond-Breaker TCEP solution (TCEP; 77720, Thermo-Fisher Scientific Corp., Grand
Island, NY, USA)
DC™ Protein Assay Reagent A (500-113; BIO-RAD Laboratories Inc., Hercules, CA,
USA)
DC™ Protein Assay Reagent B (500-114; BIO-RAD Laboratories Inc., Hercules, CA,
USA)
Dulbecco’s phosphate-buffered saline (PBS; 70011-044, GIBCO, Thermo-Fisher
Scientific Corp., Grand Island, NY, USA)
Formic Acid, reagent grade, ≥ 95% (F0507, Merck KGaA, Darmstadt, Germany)
Iodoacetamide (2811, BDH Chemicals Limited, Poole, United Kingdom)
Oasis® HLB 3cc (60 mg) Extraction Cartridges (SPE cartridges; WAT094226, Waters
Corp., Milford, MA, USA)
Lactic acid, natural, ≥ 85% (W261114, Sigma-Aldrich Corp., St. Louis, MO, USA)
Parafilm M Laboratory Film (PM-996, Hach Pacific Pty Ltd., Dandenong South,
Victoria, Australia)
Pierce® Microplate BCA Protein Assay Kit - Reducing Agent Compatible (23252,
Thermo-Fisher Scientific Corp., Grand Island, NY, USA)
Titansphere® Phos-TiO Bulk, 10 μm (TiO beads; 5010-21315, GL Sciences Inc.,
Tokyo, Japan)
37
Triethylammonium bicarbonate buffer, 1.0 M, pH 8.5±0.1 (TEAB; T-7408, Sigma-
Aldrich Corp., St. Louis, MO, USA)
Trifluoroacetic acid, analytical standard (TFA; 74564, Merck KGaA, Darmstadt,
Germany)
Trypsin from porcine pancreas, bioreagent, proteomics grade, dimethylated (T6567,
Sigma-Aldrich Corp., St. Louis, MO, USA)
Urea, minimum 99.5% (U1250, Sigma-Aldrich Corp., St. Louis, MO, USA)
2.1.9. Instruments
Allegra® X-15R centrifuge (Beckman Coulter Inc., Indianapolis, IN, USA)
BioShake iQ high-speed thermoshaker (Quantifoil Instruments GmbH, Jena, Germany)
Cary 100 UV-Visible spectrophotometer (Agilent Technologies Inc., Santa Clara, CA,
USA)
Electrophoresis power supply EPS 600 (Pharmacia Biotech, Pfizer Inc., NY, USA)
Gene Pulser Xcell™ electroporation system (#165-2660, BIO-RAD Laboratories Inc.,
Hercules, CA, USA)
J2-MI Centrifuge (Beckman Coulter Life Sciences Inc., Indianapolis, IN, USA)
Medium orbital shaker/incubator (OM11, Ratek Instruments Pty Ltd., Boronia,
Victoria, Australia)
Mini-Sub® Cell GT Cell DNA electrophoresis system (BIO-RAD Laboratories Inc.,
Hercules, CA, USA)
ModulyoD Freeze Dryer (Thermo Electron Corp., Thermo-Fisher Scientific Corp.,
Grand Island, NY, USA)
NanoVue™ plus spectrophotometer (28923215PC, GE Healthcare Corp., Chicago, IL,
USA)
38
Olympus BX50 fluorescence microscope (Olympus Corp., Shinjuku, Tokyo, Japan)
PTC-100™ Programmable thermal controller (MJ Research Inc., BIO-RAD
Laboratories Inc., Hercules, CA, USA)
Sanyo MCO-20AIC CO2 incubator (Marshall Scientific Llc., Hampton, NH, USA)
Semi-automated Rotary Microtome (RM2245, Leica Microsystems Pty Ltd., Mount
Waverley, Victoria, Australia)
Sunrise™ absorbance microplate reader (16039400, Tecan Group Ltd., Männedorf,
Zürich, Switzerland)
Thermo Savant ISS110 SpeedVac System (Marshall Scientific Inc., Hampton, NH,
USA)
2.2. Methods
2.2.1. Cell Culture
The human PC cell lines utilized in this research were purchased from American
Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s modified Eagle’s
medium (DMEM) (Sigma-Aldrich, MO) with 5% fetal bovine serum (FBS) (HyClone,
UT) in humidified air with 5% CO2 at 37°C. The mouse cell lines, LM-P (donated from
Andrew Lowy, Moores Cancer Center, University of California, CA), Pan02 (Division
of Cancer Treatment and Diagnosis Tumour Repository, NCI, MD) and TB117 (isolated
from ascites of mouse primary pancreatic tumours) were also cultured in DMEM with
5% FBS in humidified air with 5% CO2 at 37°C as well as human pancreatic duct
epithelial (HDPE) cells (obtained from TS Tsao, Ontario Cancer Institute, Ontario,
Canada).
A patient-derived PC cell line, The Kinghorn Cancer Center Cell Line 15 (TKCC15),
39
was generated by Australian Pancreatic Cancer Genome Initiative (APGI, The
Kinghorn Cancer Center, Garvan Institute of Medical Research, Darlinghurst, New
South Wales, Australia) 226. The cell line was cultured in a culture medium consisting
of a mixture of medium 199 (GIBCO, Thermo-Fisher Scientific, NY) and F-12 medium
(GIBCO, Thermo-Fisher Scientific, NY) at 1:1 ratio (v/v), 15mM HEPES, 20nM L-
glutamine (GIBCO, Thermo-Fisher Scientific, NY), 20ng/mL human epidermal growth
factor (Thermo-Fisher Scientific, NY), 40ng/mL hydrocortisone (Sigma-Aldrich, MO),
25μg/mL apo-transferrin (Sigma-Aldrich, MO), 0.2IU/mL human insulin (Sigma-
Aldrich, MO), 0.06% D-(+)-glucose (Sigma-Aldrich, MO), 0.05μg/ml tri-
iodothyronine Sigma-Aldrich, MO), 1×MEM vitamin solution (GIBCO, Thermo-
Fisher Scientific, NY), 2μg/mL O-phosphorylethanolamine (Sigma-Aldrich, MO),
20ug/mL gentamicin reagent (GIBCO, Thermo-Fisher Scientific, NY) and 5% FBS.
Culture medium was renewed around 48 to 72 hours when cells grow to 80-90%
confluency.
2.2.2 Reverse Transcription-polymerase Chain Reaction (RT-PCR)
(A) Extraction of Total RNA
The PC cells were grown in 6cm culture dishes containing DMEM (5% FBS) to 80-
90% confluency. Then the culture medium was discarded and 300 μL TRIzol reagent
(Invitrogen, Thermo-Fisher Scientific, CA) per plate was added to lyse the cells and the
cell lysates were transferred to 1.5-mL microcentrifuge tubes. The lysates were
centrifuged for 5 minutes at 12000 ×g at 4°C. The supernatants were transferred to new
tubes and kept at room temperature for 5 minutes. 60 μL chloroform was added to each
sample and then the mixtures were incubated at RT for 3 minutes. The samples were
40
centrifuged for 15 minutes at 12000 ×g at 4°C. From each tube, the upper colorless
liquid phase (containing total RNA) was transferred to a new tube and mixed with 150
μL isopropanol. The resulting mixtures were centrifuged for 10 minutes at 12000 ×g at
4°C, generating white, gel-like pellets at bottoms of the tubes. Each pellet was
resuspended with 300 μL of 75% ethanol and centrifuged for 5 minutes at 8000 ×g at
4°C. Supernatants were carefully extracted and the samples were centrifuged for
another 2 minutes at 4°C to completely precipitate total RNA. The remaining ethanol
was carefully removed, and the pellets were dried for 10 minutes at RT. Finally, each
pellet was mixed with 50-100 μL of autoclaved DEPC-treated water. Then the total
RNA samples were heated at 57°C for 10 minutes, spun down and stored at -70°C.
Concentrations and purity of RNA samples were determined on the absorbance of 260
nm.
(B) Reverse Transcription
Firstly, 5 μL RNase inhibitor (Promega, WI), 200 μL 5×RT buffer (Promega, WI),
80 μL 10× dNTP Mixture (Takara Bio, Shiga, Japan) and 20 μL Oligo-dT (MDBio,
Shandong, China) were mixed in an autoclaved 1.5-mL microcentrifuge tube on ice,
creating reagent mixture. 51.5 μL of autoclaved milli-Q water was mixed with 4 μg of
each RNA sample in a PCR tube on ice. Resulting sample mixtures were heated for 5
minutes at 70°C to remove any secondary structure in the total RNA samples, added
with reverse transcriptase (Promega, WI) (2 μL/PCR tube), cooled on ice, and finally
spun down. 40.5 μL of reagent mixture was added to each PCR tube containing sample
mixture and the tubes were vortexed and spun down. The samples were placed in a hot
plate. Reverse transcription took 65 minutes at 42°C generating cDNA samples. Then
the sample mixtures were heated at 90°C for 5 minutes to inactivate reverse
41
transcriptase and then were cooled down at 4°C for 99 minutes.
(C) Polymerase Chain Reaction (PCR)
Prior to the experiment, for one PCR reaction, 5 μL 10× Standard Reaction Buffer
(Takara Bio, Shiga, Japan), 28 μL dNTP Mixture, 14 μL 10μM forward primer solution
and 14 μL 10μM reverse primer solution were mixed in 1.5-mL microcentrifuge tubes
for separate genes on ice and spun down. To each tube, 3.5 μL Taq polymerase (Takara
Bio, Shiga, Japan) was added and the PCR reagent mixtures were generated. 5 μL of
each cDNA sample was transferred to a new PCR tube, 13.5 μL of appropriate PCR
reagent mixture was added to PCR tube and resulting mixtures were vortexed, spun
down and transferred to PTC-100™ Programmable Thermal Controller (MJ Research,
CA). cDNA samples were amplified in 20-35 cycles of Taq polymerase activation for
5 minutes at 95°C, strand separation for 10 seconds at 98°C, primer annealing for 1
minute at melting temperatures of different primer pairs, extension of primers for 20
seconds at 72°C and final elongation for 6 minutes at 72°C. Resulting PCR products
were cooled down for 99 minutes at 4°C and then stored at -20°C.
42
Table 2.2.2 Annealing temperatures for distinct pairs of primers:
(D) Gel Electrophoresis
Concentrated TAE solution (2M Tris-base, 0.5M EDTA and 5.71% acetic acid) was
10× diluted to generate 0.5× TAE buffer. PCR products were run in gels containing 2%
agarose in 0.5× TAE buffer, the running process took 35 minutes at 100V, 400 amps,
powered by a power supply and then the resultant bands were visualized with GelRed
Nucleic Acid Gel Stain (Biotium, CA) and Quantity One 1-D Analysis Software
Version 4.6.9 (Bio-Rad Laboratories, CA).
43
2.2.3. Immunohistochemistry
For the patient-derived PC cell line, TKCC 15 was subcutaneously injected into the
flank of a SCID mouse was subcutaneously injected into the flank of a severe combined
immunodeficiency (SCID) mouse. Due to their immunodeficiency, SCID mice are able
for subsequent study of human cells in vivo without rejecting any xenograft 227-229. The
xenografted tumours were then treated with distinct chemotherapeutic reagents,
gemcitabine (Sigma-Aldrich, MO) along, gemcitabine plus abraxane (Abraxis
BioScience Pharmaceutical, CA) or gemcitabine plus PF-3758309 (Pfizer, NY). In the
process of de-waxing, slides with TKCC15 xenograft tissue sections were first heated
for 30 minutes at 60℃ and incubated in 100% histolene in two changes. In the
rehydration step, the slides were then immersed in 100% ethanol in two changes
followed by 70% ethanol solution. The slides were rinsed in distilled deionized water
(DDW) and shaken in 1×Tris-buffered saline-Tween (1×TBS-T) (20 mM Tris-base, 150
mM NaCl and 0.1% Tween 20, pH7.6) solution for 5 minutes. The tumour sections
were marked with a PAP pen and then treated with Peroxidase Blocker (Dako, Agilent
Technologies, CA) for 15 minutes avoiding light. Subsequently, the slides were
submerged in a 10mM citrate buffer (20mM citric acid and 11.4mM trisodium citrate,
pH 6.0) for 30 minutes at 99℃ in a hot water bath for antigen retrieval. The slides were
cooled at RT for 30 minutes, washed with DDW for 5 minutes once and then with TBS-
T for 5 minutes twice. The tissue sections were incubated with blocking buffer (5%
normal goat serum and 1% bovine serum albumin in 1×TBS-T) for 30 minutes. Then
the sections were incubated with primary antibodies in 1×TBS-T in humidified air at
4℃ overnight. After the incubation with appropriate primary antibodies, the slides were
rinsed with DDW and then washed with 1×TBS-T for 5 minutes for three times. The
tumour sections were incubated with Labelled Polymer-HRP Anti-Rabbit Secondary
44
Antibody (Dako, Agilent Technologies, CA) in 1×TBS-T in humidified air at RT for 1
hour. The slides were washed with 1×TBS-T for 5 minutes for three times. In a chemical
hood, one drop of DAB+ Chromogen (Dako, Agilent Technologies, CA) was mixed
with 1 mL DAB+ Substrate Buffer (Dako, Agilent Technologies, CA). The resulting
mixture was added to each tumour section separately, reacted for appropriate time for
each specific primary antibody and then washed off with running tap water for 1 to 2
minutes. In the counterstaining step, the slides were submerged in hematoxylin for 4
minutes and washed with running tap water until clear. Afterwards, the slides were
immersed in Scott’s blue tap water for 2 minutes and washed with running tap water
for 1 to 2 minutes. In the process of dehydration, the slides were submerged in 70%
ethanol solution once and then in 100% ethanol in two changes. Finally, the slides were
incubated in 100 % histolene for 10 to 15 minutes, mounted with excess DPX (VWR
International, Poole, UK) and covered with cover slips. The stained tumour sections
were observed under Olympus BX50 Fluorescence Microscope (Olympus, Shinjuku,
Tokyo, Japan).
Table 2.2.3 Antibodies used for immunohistochemical staining
45
2.2.4. CXCL5 Knockdown with shRNA
(A) Preparation of Electrocompetent E. coli Cells
Single colonies of E. coli bacteria were picked up and transferred to 300-ml conical
flasks each containing 50 ml LB medium followed by incubation overnight at 37℃
with shaking. On the next day, the overnight bacterial cultures were transferred to 2-L
conical flasks each containing 1L LB medium and incubated for 90 minutes at 37℃
with shaking. Then, OD values were measured at 550 nm. The cultures were transferred
to pre-chilled, autoclaved centrifuge bottles, chilled for 30 minutes on ice and
centrifuged for 15 minutes at 3500 rpm, 4℃. LB medium was completely discarded
and 500 ml cold, autoclaved milli-Q water was used to wash each pellet. The resultant
mixtures were centrifuged for 15 minutes at 3500 rpm, 4℃, supernatants were
discarded and each bacterial pellet was washed with 250 mL cold, autoclaved milli-Q
water. Supernatants were discarded completely, and each pellet was re-suspended with
10 mL cold, autoclaved 10% glycerol using pre-chilled 25-mL pipettes. Afterwards, the
resulting mixtures were transferred to chilled 50-ml centrifuge tubes and centrifuged
for 15 minutes at 3500 rpm, 4℃. Supernatants were discarded, each pellet was re-
suspended with 1 mL cold, autoclaved 10% glycerol and then the bacterial cells were
pooled together using pre-chilled 10-mL pipettes. The bacterial suspension was sub-
packaged to pre-chilled 1.5-mL microcentrifuge tubes (about 60 μL per tube) on ice,
snapped frozen with liquid nitrogen and stored at -80℃.
(B) Electroporation and Bacterial Transfection with Plasmid DNA
Electroporation cuvettes (BIO-RAD Laboratories, CA), electrocompetent cells,
1.5-mL microcentrifuge tubes and CXCL5 Human shRNA plasmid kit containing a
46
scrambled shRNA vector (OriGene Technologies, MD) and four unique 29-mer CXCL5
shRNA constructs (OriGene Technologies, MD) were placed on ice. In the Gene Pulser
Xcell™ Electroporation System (BIO-RAD Laboratories, CA), ‘Pre-set protocol’ and
‘0.1cm cuvettes’ were selected for the electroporation process. About 100 ng per 1-2
μL of each plasmid was mixed with 20 μL electrocompetent cells by pipetting in a 1.5-
mL microcentrifuge tube. The resultant mixtures were then transferred to appropriate
pre-chilled electroporation cuvettes. Each of the cuvettes was tapped a few times and
its exterior was wiped to eliminate moisture. Each cuvette was inserted to the shock
pod of the electroporation system and an electric current was applied. After
electroporation, each cuvette was removed from the shock pod and added with 1 mL
LB medium. The electroporated cells were mixed with LB medium by pipetting, the
resulting mixtures were transferred to pre-chilled 15-mL centrifuge tubes and shaken
for 1 hour at 150 rpm, 37℃. The transformed E. coli were streaked onto LB agar plates
containing 34μg/mL chloramphenicol and incubated overnight at 37 ℃. Single colonies
from the plates were picked and incubated in fresh 5 mL LB medium containing the
antibiotic overnight at 37℃ with shaking. Finally, 1 mL of each overnight culture was
transferred to fresh 50 mL LB medium containing 34 μg/mL chloramphenicol in a
conical flask to incubate overnight at 37℃ with shaking to obtain large quantities of
plasmids.
(C) Plasmid Extraction
The 50-mL overnight bacterial cultures were transferred to 50-mL centrifuge tubes,
centrifuged for 10 minutes at 5000 ×g at 4 °C and then mixed with 4 mL Suspension
Buffer containing RNase A (Genopure Plasmid Midi Kit; Roche Diagnostics,
Mannheim, Germany) per tube. Then, to each tube, 4 mL Lysis Buffer (Genopure
47
Plasmid Midi Kit; Roche Diagnostics, Mannheim, Germany) was added. The solutions
were mixed by inversion 6 to 8 times to generate homogeneous suspensions/lysates,
which were incubated for 5 minutes on ice. Filters were folded and inserted new 50-
mL centrifuge tubes and moistened with Equilibration Buffer (Genopure Plasmid Midi
Kit; Roche Diagnostics, Mannheim, Germany). The lysates were loaded to filters and
collected. Each column was mounted with a sealing ring, inserted a 50-mL collection
tube, equilibrated with 2.5 mL Equilibration Buffer and allowed to be emptied by
gravity flow. The flow through was discarded. Each filtered lysate was loaded to an
equilibrated column, which was emptied by gravity flow, and flow through was
discarded. Each column was washed with 5 mL Wash Buffer (Genopure Plasmid Midi
Kit; Roche Diagnostics, Mannheim, Germany) and allowed to empty by gravity, the
flow through was discarded. This step was repeated once and subsequently the flow
through, and the collection tubes were discarded. The columns were inserted to fresh
50-mL centrifuge bottles and plasmid DNA was eluted with prewarmed Elution Buffer
(Genopure Plasmid Midi Kit; Roche Diagnostics, Mannheim, Germany) (5 mL per
column). The eluted fluids were added with isopropanol (3.6 mL per bottle) and
immediately centrifuged for 40 minutes at 16000 ×g at 4°C, and supernatants were
discarded carefully. Plasmid DNA pellets were marked on the exterior of each bottle
and washed with 3 mL chilled 70% ethanol and centrifuged for 10 minutes at 16000 ×g
at 4°C. Ethanol from each tube was carefully removed with a pipette tip and plasmid
DNA was air-dried for 10 minutes. Finally, 100 μL sterile milli-Q water was added to
dissolve each DNA pellet and the samples were then stored at -20°C.
48
(D) Establishment of CXCL5 Knockdown in MiaPaCa-2 Cell Line and Colony
Selection
Prior to transfection, optimal dose of puromycin for MiaPaCa-2 PC cells was
determined. Untreated MiaPaCa-2 cells were seeded in a 6-well plate (1 × 105 cells/well)
containing DMEM. After 24 hours of incubation in humidified air with 5% CO2 at 37°C,
the cells were transfected with the plasmids described previously utilizing
Lipofectamine 2000 transfection reagent (Invitrogen, NY). Briefly, 4 μL of each
CXCL5 shRNA plasmid or a scrambled sequence (TR30021; OriGene Technologies,
MD) was mixed with 46 μL DMEM in a 1.5-mL microcentrifuge tube. For each
construct, 10 μL lipofectamine 2000 transfection reagent (Invitrogen, NY) was mixed
with 40 μL DMEM and the resultant mixture was incubated for 5 minutes at RT. Each
plasmid solution was mixed 100 μL lipofectamine-containing DMEM and the resultant
mixtures were incubated for 20 minutes at RT to generate plasmid DNA-lipofectamine
complex solutions. To cell well, 100 μL of each plasmid-lipofectamine mixture was
added with 2 mL DMEM containing 5% FBS. The cells were incubated for 6 hours in
humidified air with 5% CO2 at 37°C. Then, the media containing lipofectamine and the
plasmids were replaced with fresh DMEM containing 5% FBS to terminate the
transfection. After 24 hours of incubation, puromycin (GIBCO, Thermo-Fisher
Scientific, NY) was added to the transfected cells which were incubated for 24-48 hours
to select successfully transduced cells. Subsequently, puromycin-containing medium
was refreshed every 2-3 days to maintain the CXCL5 knockdown phenotype and
allowed the cells form colonies.
To pick up single colonies, 0.5 mL medium containing 0.5 μg/mL puromycin was
placed in each well of a 24-well plate while adding 150 μL trypsin to each well of a 96-
49
well plate first. Under the microscope, each colony was picked up with 200-μL tip and
then transferred to the 96-well plate containing trypsin to be broken down into single
cells. The resulting mixture was transferred to a well in the 24-well plate containing
medium with puromycin. After 2-3 days of incubation in humidified air with 5% CO2
at 37°C, the old medium was replaced by fresh DMEM containing puromycin.
When cells in each well of the 24-well plate reached at least 70% confluency, most
of them were transferred to a new 6-well plate for PCR analysis and the rest were kept
in a 5-mL culture flask, which were frozen down or discarded after determining whether
the corresponding knockdown clones had a significant reduction of CXCL5 expression
compared to wild type or scrambled PC cells.
2.2.5. MTT Assay
Prior to the experiments, wild type MiaPaCa-2 PC cells was first transfected with
scrambled or CXCL5 shRNA plasmid and then single knockdown clones were picked
after confirming with PCR, in which negative controls (NCs, ones transfected with
scramble shRNA plasmid) and the knockdown clones were compared with wild type
cells to compare CXCL5 mRNA expression (Section 2.2.4D). Negative control cell
lines, NC1 and NC2, and knockdown clones, D-12 and D-22, were selected in the
process. NC1, NC2, D-12 and D-22 MiaPaCa-2 PC cells were grown in 96-well plates
(3000 cells/well) containing DMEM (5% FBS). NC1 and NC2 indicate negative
control (NC clones of MiaPaCa-2), while D-12 and D-22 are the CXCL5 knockdown
clones. Twenty-four hours later, the cell lines were treated with different doses of
gemcitabine (Hospira, Melbourne, Victoria, Australia) (10, 50 and 100 nM) or 5-FU
(Sigma-Aldrich, MO) (100, 200 and 400 μM). Forty-eight or Seventy-two hours after
50
administration of gemcitabine or 5-FU, 100 μL of 0.5mg/mL MTT solution (5 mg
MTT in 1 mL 1×PBS) (Sigma-Aldrich, MO, USA) per well was added to the culture.
The cells were incubated for 2 hours. Medium from each well was discarded and the
plates were air-dried. Then 100 μL acidified isopropanol (0.04 M HCL in 100%
isopropanol) per well was added to the plates which were rocked for 20 minutes.
Absorbance of the samples was read at 570 nm.
2.2.6. Mice Experiment
Male SCID mice were purchased from Animal Resource Center (Murdoch, Western
Australia, Australia) and were in the xenografted mouse model (Chapter 2.2.7). The
animals were housed in standard cages at controlled constant temperatures and
humidity with alternating 12-hour long cycles of light and dark and were on a diet
containing an autoclaved chow with water as required. None of the mice had lesions of
any kind and were free of microbial infections prior to any experiment.
All mouse experiments were approved by Austin Health Animal Ethics Committee
with project number A2016/5317. Human pancreatic cancer cells were subcutaneously
injected into the flank of a SCID mouse. The dosage of gemcitabine (GEM) used was
referred to our previous publication 230, and was given twice weekly.
The flanks of the SCID mice were shaved, and NC and CXCL5 KD MiaPaCa-2
cells were injected into either flanks (5 × 106 cells/100 μL/injection) subcutaneously
with 27-gauge syringe. Each mouse had NC cells at one side of the flank and CXCL5
KD cells at the other side of the flank. When average tumour size between 50 to 100
mm3 (about two weeks after tumour cell injection), the mice were divided to control
(CT) and gemcitabine (GEM) treatment groups and treated for 6 weeks. GEM was
51
administered at 40 mg/Kg through peritoneal injection twice weekly. Tumour volumes
(V) were measured and determined with the following formula:
V = W2 × L/2, where W and L represent shortest and longest dimensions of a
tumour, respectively.
At the end point, the mice were culled, and tumours were isolated and weighted, and
fixed for subsequent immunohistochemical analyses.
2.2.7. Tumour Tissue Embedding
Tumours were fixed in 10% formalin at RT for 24 hours followed by incubation in
70% ethanol solution. The tumours were placed in biopsy pads soaked in 70% ethanol
in tissue processing cassettes. The cassettes were then processed by Melbourne
Histology Platform (School of Biomedical Sciences, The University of Melbourne,
Parkville, Melbourne, Victoria 3010, Australia). The tumour tissues were embedded in
paraffin wax blocks for preparation of tissue slides with a rotary microtome (Leica
Microsystems Pty Ltd., Mount Waverley, Victoria, Australia).
2.2.8. Patient Samples
The tumour microarrays were collected from tumour biopsies of 84 patients who
were diagnosed with the pancreatic cancer in Austin Hospital, Heidelberg, Melbourne,
Victoria, Australia. All patients received no neoadjuvant and/or adjuvant chemotherapy
prior to sample collection. Information regarding their survival time, death, recurrence
and tumour staging was followed during subsequent visits, and confirmed by the Austin
Hospital registrars. The tumours were collected from the patients after their admission
52
between 2007 and 2015.
The use of human tissue samples was approved by the Human Research Ethics
Committee at Austin Health (Ethics number: H2013-04953).
2.2.9. Proteomic Analysis
(A) Collection of Protein Samples
MiaPaCa-2 clones NC, D-12 and D-22 were initially seeded in 10-cm culture plates
and were grown to 80-90% confluency at 4 repeats per cell line. Protein samples were
collected by adding 1 mL RIPA cell lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1%
Triton X-100, 1% sodium deoxycholate, 0.5% SDS, 1 mM EGTA, protease inhibitor,
phosphatase inhibitor) to each plate on ice. The samples were spun down for 20 minutes
at 13000 rpm, 4°C. Subsequently, protein concentrations were accessed with a DC
protein assay kit (Bio-Rad Laboratories, USA).
(B) Processing Protein Samples for Proteomic Analysis
Acetone was added to the protein samples at volumes which were 5 times of that of
the samples. The mixtures were placed at -20°C overnight to precipitate proteins. On
the next day, the samples were centrifuged for 10 minutes at 13000 rpm, 4°C.
Supernatants were removed and 1.4 mL acetone was added to carefully wash each
sample. The samples were spun for 10 minutes at 13000 rpm, 4°C again, and
supernatants were discarded. Each protein pellet was re-suspended in 500 μL of 8M
urea in 50mM triethylammonium bicarbonate buffer (TEAB) plus DTT by vortexing
and incubated for 45 minutes at 37°C. The solutions were spun down and the
53
supernatants were transferred to new tubes. Then the protein concentration of each
sample was assessed with micro BCA assay (Pierce® Microplate BCA Protein Assay
Kit; Thermo-Fisher Scientific Corp., USA). 50 μL iodoacetamide was added to each
sample and the mixtures were incubated for 45 minutes at RT avoiding light. Dilute
each sample with 25mM TEAB to a solution containing 1M urea. Finally, the protein
samples were digested with trypsin with shaking overnight at 30°C.
On the following day, each SPE cartridge (Waters Corp., Milford, MA, USA) was
washed with 1 mL 80% acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA)
followed by 1.2 mL of 0.1% TFA twice and the flow-through from each wash was
discarded. The samples were acidified to 1% (v/v) by 100% formic acid and then loaded
to SPE cartridges. Flow-through liquids were discarded. Each sample was washed with
1.2 mL 0.1% TFA twice again and the flow-throughs were eliminated. Then each
sample was eluted with 800 mL of a solution consisting of 80% ACN and 0.1% TFA,
collected in a 2-mL Eppendorf tube. 20 μg of each sample was aliquoted to a separate
tube and the rest was transferred to a second one. All samples were subjected to the
Thermo Savant ISS110 SpeedVac System (Marshall Scientific, NH, USA) to eliminate
solvents for 20 minutes. The first tube of each sample was used for global mass
spectrum and the second ones for global phospho-proteomics.
On the fourth day, TiO beads (GL Sciences Inc., Tokyo, Japan) were washed with
500 μL TiO washing buffer (50% ACN and 5% TFA) once and then with 300 μL TiO
loading (2M lactic acid in 5% TFA) twice followed by pre-incubation with 20 μL TiO
loading buffer per sample for 20 minutes. To each of the second set of freeze-dried
tubes, 300 μL TiO loading buffer was added. 10 μL TiO beads was added to each sample
and the resulting mixtures were incubated for 1 hour with shaking at RT. To prepare c8
54
tips, 1-mL tips were used to pierce a parafilm membrane and then placed inside 200-μL
and a small of the membrane was passed from a 1-mL tip to a 200-μL one. Samples
were pipetted to the c8 tips in 150-μL batches and centrifuged for 10 minutes at 2000
rpm, 4°C. The flow-through was discarded. The TiO beads were washed by passing 200
μL washing buffer through newly formed TiO tips twice. The beads were washed with
washing buffer for another three times. In these washing steps, every flow-through
liquid was discarded.
The c8 tips containing samples were transferred to new collection tubes and
phosphopeptides were eluted with elution buffer (1% ammonia (v/v), pH11.3) three
times and then with 30% acetonitrile once. The eluted fractions of a sample were
collected a 1.5-mL eppendorf tube. Each sample was then acidified with 1 μL formic
acid per 10 μL eluent. The samples were placed in a freeze-dry machine and then stored
at -80°C. Finally, each sample was re-suspended in 15 μL of a solution consisting of
2% ACN and 0.05% TFA for mass spectrum analysis.
Finally, the proteomic data were prepared using Max Quant software and analyzed
with Perseus software version 1.6.14.0.
2.2.10. Statistical Analysis
The percentage of CXCL5 positivity of the tumour samples, less than 3.5% were
regarded as low expression and those higher than 3.5% were treated as high expression,
and the parameters in patient information, including survival group, death, recurrence,
T stage and N stage, were analyzed by the Kaplan-Meier method using the log-rank and
chi-square tests. P<0.05 was considered as statistically significant. All analyses were
conducted by utilizing SPSS 24.0 software (IBM, NY, USA).
55
Chapter 3:
Involvement of CXC Ligands (CXCLs) and Cancer
Stem Cell (CSC) Markers in Chemoresistance of
Pancreatic Cancer
56
3.1 Abstract
Therapeutic resistance is the major contributor to the low survival of pancreatic
cancer (PC). PC progression is a complex process reliant on interactions between
tumour and tumour microenvironment (TME). A unique chemokine family, CXC
ligands (CXCLs), seems to play important roles in regulating PC progression in
pancreatic TME. The expression of a panel of CXCLs and cancer stem cell (CSC) genes
in human and murine PC cell lines were determined by reverse transcription polymerase
chain reaction (RT-PCR). In both human and mice PC cell lines, CXCL5 expression
was dramatically up-regulated while the expression of CXCL10 was down-regulated.
The expression of CXCL5 and CXCL10 were varied in gemcitabine resistant cell lines,
and gemcitabine treated cells. However, in mouse xenografted tumour samples, which
was generated from a patient-derived cell line, gemcitabine alone or in combination
with other chemotherapeutic reagents led to increased CXCL5 protein level while
CXCL10 remained unchanged. These results suggested that expression of CXCL5 may
be stimulated upon administration of gemcitabine or other chemotherapeutic reagents,
and that CXCL5 is a candidate protein that mediates chemoresistance in PC.
3.2 Introduction
Pancreatic cancer (PC) is the most commonly diagnosed, with increasing
occurrences globally and in Australia 1,2. Pancreatic ductal adenocarcinoma (PDAC),
has more than 90% occurrence in PC and less than 10% of five-year survival rate 1.
Gemcitabine is used as a chemotherapeutic reagent in a range of tumour types
including PC 231-233. Gemcitabine is a synthetic pyrimidine nucleoside prodrug that
chemically binds to DNA molecules to abridge DNA synthesis 234. Gemcitabine is a
57
first line chemotherapeutic reagent in treating PC; however, it causes unintended
consequences, such as bone marrow suppression, liver and kidney problems, nausea
and neuropathy. In clinical practice, gemcitabine is co-administrated with different
chemotherapeutic drugs, such as cisplatin and paclitaxel.
Chemoresistance is a pivotal limiting factor of successful cancer treatment caused
by a number of factors, such as mutations 3,4, and activation of upstream cell survival
intracellular molecules 5.
Tumour microenvironment (TME) defines a microscopic location or niche in a
tissue or organ, composing of cancer cells, non-cancerous cells that surround the
tumour and the extracellular matrix 9. The two cellular components collaborate with
each other to promote tumour progression and chemoresistance by secreting signal
molecules 10. In PC, cancer cells interact with numbers of stromal cells, including
macrophages, neutrophils and stellate cells 11-13.
CXCLs, a group of chemokines that display diverse roles in cancer, are divided into
two structurally distinct groups, depending on the presence or absence of a Glu-Leu-
Arg motif 13-16. They bind to specific G protein-coupled receptors, namely CXC
receptors (CXCRs), summoning various types of non-cancerous cells, such as
fibroblasts, macrophages and neutrophils. Most CXCLs found in PC are secreted by
cancer or TME cells 17-22. Some of CXCLs have pro-tumoural functions, contributing
to chemoresistance, tumour proliferation and metastasis 17,20,23,24, while some have anti-
tumour functions by causing chemotaxis of T lymphocytes, dendritic and natural killer
cells 25-27. Targeting CXCLs contributes to better treatment outcome, but how they
regulate a TME deserves further investigation 28,29.
58
CXCL5 is involved in several pro-tumoural functions, such as angiogenesis,
chemoresistance and cell mobility 51,54-58. It binds to CXCR2, summons Ly6G+
neutrophils into the TME of cholangiocarcinoma and colon cancer to promote
proliferation and cell motility 59,235. In PC, CXCL5 is highly expressed in cell lines and
patient tumour samples, resulting in advanced clinical staging and progression 51.
CXCL5 causes elevated angiogenesis via CXCR2, Akt and ERK. CXCL5 is co-
expressed with 3 cancer stem cell (CSC) genes, ALDH1, CD44 and CD133, which
contribute to gemcitabine resistance 58. CXCL5 also binds to CXCR2 to enhance
proliferation and invasion in a mouse xenograft model 61.
In breast, colon and ovarian cancers, tumour-promoting CXCLs have been shown
to cause expression of CSC genes and subsequent chemoresistance as well as increased
cell mobility and tumour initiation 160-163. In PC, CXCL5, CXCL8 and CXCL12 induce
expression of several CSC genes and gemcitabine resistance as well as promoting
formation of tumour spheres and metastasis 58,91,138.
Finally, a family of p21-activated kinases (PAKs) were found to interact with small
Rho-like G proteins, whose dysregulation have been shown to initiate and promote
tumourigenesis 236. In PC, inhibition of PAK1, led to a significant reduction of tumour
cell proliferation and an increase of sensitivity to gemcitabine as well as chemotaxis of
pancreatic stellate cells to the proximity to pancreatic tumour 230,237. In addition,
mutations of Kras activate PAK1 via a Kras-dependent or independent pathway.
Jagadeeshan et al., showed that PAK1 conferred resistance to gemcitabine and
interacted with several intracellular signaling pathways, such as NF-κB and TGF-β 238.
In this chapter, possible involvement of CXCLs and CSC genes in PC was
59
investigated in the context of chemoresistance.
3.3 Scientific Method – quantification of IHC data
To quantify the IHC results in this chapter, 10 random fields were taken of each
tumour tissue at 200 x magnification. Positively stained areas were analyzed with the
Image Pro-Plus version 4.5.0.29 image analysis software (Media Cybernetics, MD,
USA)239 and the percentage of the positively stained area to the total area in each field
was calculated.
3.4 Results
3.4.1 Expression of CXCLs and Cancer Stem Cell (CSC) Genes in Human and
Mouse Pancreatic Cancer (PC) Cells
To determine mRNA expression levels in a panel of PC cell lines, reverse
transcription polymerase chain reaction (RT-PCR) was performed. As shown in Fig.
3.4.1A and 3.4.1B(ii), levels of CXCL1 were up-regulated in CFAC-1 and Panc-1
(53.97% and 39.98% increments compared to the non-cancerous pancreatic cell line,
human pancreatic duct epithelial (HDPE) cells, respectively), whereas its expression
was reduced at various degrees in AsPC-1, BxPC-3, Capan-2, HPAC-II, MiaPaCa-2,
PL45 and SW1990 compared to HDPE (8.29%, 20.39%, 11.47%, 88.01%, 74.3%,
88.15% and 72.37% decrements, respectively). In all 3 mouse PC cell lines tested, LM-
P (which was originated from liver metastasis in KPC mice) and TB117 displayed
higher CXCL1/18S ratios than Pan02 (Fig. 3.4.2A & B(ii)). There was a minor
elevation of CXCL2 in CFPAC-1 (8.07%) whereas its expression was lower in other
human PC cell lines (Fig. 3.4.1A & B(iii)). In addition, LM-P and Pan02 had higher
60
CXCL2 expression, whereas TB117 expressed little or no CXCL2 mRNA (Fig. 3.4.2A
& B(iii)).
CXCL5 expression was elevated in all PC cell lines tested by 957.06%, 298.49%,
356.45%, 381.6%, 867.49%, 611.38%, 625.03%, 577.79%, 430.96% and 847.13% in
AsPC-1, BxPC-3, Capan-2, CFPAC-1, HPAF-II, MiaPaCa-2, Panc-1, PL45 and
SW1990, respectively (Fig. 3.4.1A & B(iv)). LM-P cells had the highest CXCL5
expression among the murine PC cells tested (Fig. 3.4.2A & B(iv)). mRNA expression
of CXCL9 showed distinct levels of reductions in almost all the PC cell lines (3.34%,
39.28%, 20.15%, 31.86%, 50.69%, 37.96%, 21.28% and 29.62% reductions compared
to HDPE), except SW1990, which had an 18.68% increase (Fig. 3.4.1A & B(v)). TB117
showed highest CXCL9 expression level in all murine PC cell lines tested; however,
the murine cell lines generally expressed very low CXCL9 (Fig. 3.4.2A & B(v)).
In all human pancreatic cancer cell lines examined, there were reductions in
CXCL10 mRNA, especially in HPAF-II, MiaPaCa-2, Panc-1 and PL-45 cell lines that
decreased by 88.21%, 97.68%, 95.17% and 87.7% compared to HDPE, respectively
(Fig. 3.4.1A & B(vi)). Similarly, CXCL10 also had low expression levels in murine PC
cell lines tested (Fig. 3.4.2A & B(vi)). Finally, in BxPC-3, HPAF-II, PL45 and SW1990,
the expression of CXCL13 were significant increased by 636.87%, 1111.69%, 646.78%,
1170.71% and 1258.66%, respectively (Fig. 3.4.1A & B(vii)). However, in AsPC-1,
Capan-2, CFPAC-1 and MiaPaCa-2, the levels CXCL13 had insignificant changes. The
murine PC cells displayed weak or no CXCL13 expression (Fig. 3.4.2A & B(vii)).
61
A
B
62
Figure 3.4.1. Expression patterns of PAK1, CXCLs and selected CSC genes in
human PC cell lines. (A) RT-PCR analysis of mRNA expression of PAK1, CXCLs and
selected CSC genes in HDPE and a panel of human PC cell lines. (B) Quantifications
of mRNA expression of PAK1, CXCLs and selected CSC genes. The expression levels
of genes tested in HDPE were taken as 100%.
The p21-activated kinase 1 (PAK1), is up-regulated in PC cell lines and mediates
growth, survival and resistance to gemcitabine 230,238,240. PAK1 expression was detected
in both human and murine cell lines (Fig. 3.4.1A, B(i), C & D(i)). In AsPC-1, BxPC-3,
CFPAC-1 and Panc-1, PAK1 mRNA expression was significantly up-regulated
(30.35%, 77.55%, 53.43% and 39.82% increments compared to HDPE). In the murine
cell lines tested, LM-P and TB117 displayed high PAK1/18S ratios (Fig. 3.4.2A & B(i)).
Finally, expression levels of selected CSC genes, namely, ALDH1, CD24, CD44
and CD133, were determined in human and murine cell lines. Compared to HDPE,
ALDH1 mRNA was dramatically increased in AsPC-1, BxPC-3, Capan-2, CFPAC-1,
HPAF-II, MiaPaCa-2, Panc-1 and PL45 (555.12%, 176.57%, 322.8%, 311.56%,
588.7%, 907.79%, 236.06% and 471.15% increments, respectively) (Fig. 3.4.1A &
B(viii)). However, in SW1990, there was only 4.85% elevation of ALDH1. Two of the
murine cell lines tested, LM-P and TB117, had higher ALDH1/18S ratios (Fig. 3.4.2A
& B(viii)). Levels of CD24 in BxPC-3, CFPAC-1, HPAF-II, Panc-1 and PL45 were
apparently higher than that in HDPE (51.62%, 65.12%, 41.51% and 119.88%
increments, respectively) (Fig. 3.4.1A & B(ix)). On the contrary, in other human PC
cell lines examined, CD24 expression was lower than in HDPE. TB117 had a higher
CD24 expression than other murine PC cell lines tested (Fig. 3.4.2A & B(ix)).
CD44 mRNA expression levels were elevated in AsPC-1, BxPC-3, CFPAC-1,
MiaPaCa-2, Panc-1, PL45 and SW1990 (91.83%, 41.67%, 45.85%, 56.16%, 125.53%,
63
76.83% and 76.31% increments compared to HDPE, respectively) (Fig. 3.4.1A and
3.4.1B(x)). In LM-P, CD44/18S ratio seemed to be higher than that in Pano2 or TB117
(Fig. 3.4.2A & B(x)). CD133 expression in Capan-2, CFPAC-1, MiaPaCa-2, PL45 and
SW1990 were lower than in HDPE (23.05%, 16.99%, 39.17%, 50.38% and 63.57%
reductions, respectively) (Fig. 3.4.1A & B(xi)), while CD133 was raised in AsPC-1 and
Panc-1, by 147.29% and 142.5%, respectively. LM-P had higher CD133 mRNA
expression than the other two murine PC cell lines (Fig. 3.4.2A & B(xi)).
A
B
64
Figure 3.4.2. Expression patterns of PAK1, CXCLs and selected CSC genes in
mouse PC cell lines. (A) RT-PCR analysis of mRNA expression of PAK1, CXCLs and
selected CSC genes in LM-P, Pan02 and TB117 cells. (B) Quantifications of mRNA
expression of PAK1, CXCLs and selected CSC genes.
3.4.2 Correlations between PAK1, CXCLs and Selected CSC Genes
To determine whether there are correlations between PAK1 and the selected CXCLs
and CSC genes, regression analysis was carried out. Among human PC cell lines tested,
PAK1 and the selected CXCL genes had low correlations, determined by the Rsqr and
P values (Table 3.4.1A). There were low correlations between any two CXCL members
as well. Similarly, the correlations of PAK1 or selected CXCLs to the four CSC markers
were insignificant (Table 3.4.1B). Similar trends were obtained in the murine PC cell
lines tested (Table 3.4.1C&D). These results suggest that PAK1 and CXCLs may act
through separate pathways.
Table 3.4.1
A. Correlations between PAK1 and selected members from the CXCL chemokine
family in human PC cell lines tested.
PAK1 Rsqr P value CXCL1 Rsqr P value
CXCL1 0.5458 0.0147 CXCL2 0.4319 0.0389
CXCL2 0.4228 0.0418 CXCL5 0.0018 0.9084
CXCL5 0.0079 0.8066 CXCL9 0.0022 0.8982
CXCL9 0.0166 0.7227 CXCL10 0.065 0.4773
CXCL10 0.1059 0.359 CXCL13 0.5124 0.0199
CXCL13 0.1878 0.2109
CXCL2 Rsqr P value CXCL5 Rsqr P value
CXCL5 0.0095 0.7885 CXCL9 0.0129 0.7544
CXCL9 0.1911 0.2064 CXCL10 0.106 0.3586
CXCL10 0.4488 0.0341 CXCL13 0.0072 0.8162
CXCL13 0.258 0.1339
CXCL9 Rsqr P value CXCL10 Rsqr P value
CXCL10 0.2203 0.1711 CXCL13 0.0664 0.4722
CXCL13 0.0004 0.9547
65
B. Correlations between PAK1 or CXCLs and selected CSC genes in human PC
cell lines tested.
C. Correlations
between PAK1 and selected members from the CXCL chemokine family in mouse
PC cell lines tested.
PAK1 Rsqr
P value
CXCL1 Rsqr
P value
ALDH1 0.041 0.5747 ALDH1 0.037 0.5945
CD24 0.1943 0.2023 CD24 0.0711 0.4566
CD44 0.0293 0.6361 CD44 0.0476 0.5447
CD133 0.4515 0.0333 CD133 0.2877 0.1099
CXCL2 Rsqr
P value
CXCL5 Rsqr P value
ALDH1 0.2165 0.1753 ALDH1 0.1111 0.3467
CD24 0.0367 0.5962 CD24 0.001 0.9305
CD44 0.0024 0.8934 CD44 0.2323 0.1583
CD133 0.0226 0.6788 CD133 0.0016 0.9132
CXCL9 Rsqr P value CXCL10 Rsqr P value
ALDH1 0.3443 0.0746 ALDH1 0.5579 0.013
CD24 0.0248 0.6641 CD24 0.0157 0.7302
CD44 0.08 0.4283 CD44 0.1295 0.307
CD133 0.004 0.8618 CD133 0.0031 0.8782
CXCL13
Rsqr P value ALDH1 Rsqr P value
ALDH1 0.0333 0.6137 CD24 0.241 0.1497
CD24 0.1683 0.2389 CD44 0.0003 0.9624
CD44 0.0293 0.6361 CD133 3.21E-07 0.9988
CD133 0.1238 0.3187
CD24 Rsqr P value CD44 Rsqr P value
CD44 0.0415 0.5723 CD133 0.0493 0.5375
CD133 0.0524 0.5249
PAK1 Rsqr P value CXCL1 Rsqr P value
CXCL1 0.6028 0.4341 CXCL2 0.41 0.5576
CXCL2 0.0002 0.9917 CXCL5 0.9839 0.0811
CXCL5 0.7228 0.353 CXCL9 0.3948 0.5675
CXCL9 5.82E-06 0.9985
CXCL10
0.9709 0.1092
CXCL10 0.7614 0.3249
CXCL13
0.0933 0.8024
CXCL13 0.7011 0.3683
CXCL2 Rsqr P value CXCL5 Rsqr P value
CXCL5 0.289 0.6387 CXCL9 0.275 0.6486
CXCL9 0.9998 0.0099
CXCL10
0.9981 0.0281
CXCL10 0.2498 0.6668
CXCL13
0.1798 0.7213
CXCL13 0.287 0.64
CXCL9 Rsqr P value
CXCL10
Rsqr P value
CXCL10 0.2365 0.6767
CXCL13
0.2149 0.6931
CXCL13 0.3012 0.6302
66
D. Correlations between PAK1 or CXCLs and selected CSC genes in mouse PC
cell lines tested.
3.4.3 Effect of PAK1 knockdown on expression of selected CXCL members
CXCL ligands bind to specific G protein-coupled 7-transmembrane receptors
(GPCRs), namely CXCRs, in order to exert downstream effects 13. PAK1 is an effector
protein downstream to a GPCR and facilitates many pro-tumour effects, such as
metastasis and chemoresistance 238,240-243. To determine the effect of PAK1 on
expression of CXCLs, RT-PCR was performed following PAK1 knockdown.
PAK1 knockdown (KD) clones, KD3.09 and KD3.12 in MiaPaCa-2 had reduced
PAK1 mRNA by 28.38% and 90.02% compared to NC2 (control MiaPaCa-2 clone,
which received a scramble sequence inducing no changes in PAK1 mRNA),
respectively; and KD2.05 and KD2.10 in Panc-1, had reduced PAK1 mRNA by 89.25%
and 37.2% compared to NC (control Panc-1 which received a scramble sequence
PAK1 Rsqr P value CXCL1 Rsqr P value
ALDH1 0.8637 0.2408 ALDH1 0.239 0.6748
CD24 0.4024 0.5625 CD24 2.84E-05 0.9966
CD44 0.2119 0.6955 CD44 0.8406 0.2614
CD133 1.00E-01 0.7948 CD133 0.7119 0.3607
CXCL2 Rsqr P value CXCL5 Rsqr P value
ALDH1 0.1275 0.7676 ALDH1 0.3549 0.5937
CD24 0.5847 0.4458 CD24 0.0175 0.9155
CD44 0.7987 0.2962 CD44 0.7374 0.3425
CD133 0.9074 0.1969 CD133 0.5909 0.4418
CXCL9 Rsqr P value CXCL10 Rsqr P value
ALDH1 0.138 0.7577 ALDH1 0.3977 0.5656
CD24 0.6 0.4359 CD24 0.031 0.8874
CD44 0.7862 0.306 CD44 0.6977 0.3706
CD133 0.8982 0.2068 CD133 0.5472 0.4699
CXCL13
Rsqr P value
ALDH1 0.9604 0.1275
CD24 0.9098 0.1942
CD44 0.01 0.9362
CD133 0.0642 0.8369
67
inducing no changes in PAK1 mRNA), respectively (Fig. 3.4.3A & B(i)). Except for
KD3.09, which had reduced CXCL1 expression, CXCL1 expression was elevated in
KD3.12, KD2.05 and KD2.10 (26.89%, 58.28% and 107.48% increments, respectively)
(Fig. 3.4.3A & B(ii)). Levels of CXCL2 were raised in all four PAK1 KD clones
compared to the NCs (30.07%, 179.35%, 125.85% and 280.8% increments) (Fig.
3.4.3A & B(iii)).
CXCL5 expression was elevated in three out four PAK1 KD cell lines (52.78%,
41.38% and 28.29% increments in KD3.12, KD2.05 and KD2.10, respectively), except
for KD3.09, which had less CXCL5 than NC2 (55.24% reduction) (Fig. 3.4.3A & B(iv)).
In MiaPaCa-2, PAK1 knockdown resulted in a 24.08% decrement in CXCL9
expression by KD3.12, whereas it caused sharp increase in CXCL9 mRNA in Panc-1
(1071.36% and 617.96% increments in KD2.05 and KD2.10, respectively) (Fig. 3.4.3A
& B(v)). Except for KD3.12, CXCL10 expression was dramatically decreased due to
PAK1 knockdown (89.45%, 42.08% and 94.09% decrements in KD3.09, KD2.05 and
KD2.10, respectively) (Fig. 3.4.3A & B(vi)). As for CXCL13, knocking down PAK1
resulted in minor changes in expression of the gene (Fig. 3.4.3A & B(vii)). These results
indicate that PAK1 knockdown did not uniformly cause an increase or decrease of the
CXCL genes in MiaPaCa-2 and Panc-1 cell lines. Although changes in these genes may
indicate compensatory effects of the genetic alternation, PAK1 and the selected CXCLs
may represent independent intracellular signaling pathways.
68
A
B
Figure 3.4.3. Effect of PAK1 knockdown on expression of selected CXCL members.
(A) RT-PCR analysis of mRNA expression of PAK1 and CXCLs in NC2, KD3.09, KD
3.12 of MiaPaCa-2, and NC, KD2.05 and KD2.10 of Panc-1cell lines. KD: PAK1
knockdown. (B) Quantifications of mRNA expression of PAK1 and CXCLs. The
expresisons in NC cell lines were taken as 100%.
69
3.4.4 Effects of Gemcitabine Resistance on CXCL5, CXCL10 and the CSC Genes
The results that CXCL5 expression was increased in all human PC cell lines tested
while CXCL10 was decreased (results in section 3.4.1), and that ALDH1 expression
was increased in all human PC cell lines tested while CD24, CD44 and CD133
expression were various cross those PC cell lines, suggesting that CXCL5, CXCL10
and CSC markers may play roles in gemcitabine resistance (GEMR) in PC, three GEMR
cell lines were used, namely GEMR-MiaPaCa-2, GEMR-Pan02 and GEMR-TB117,
generated in the laboratory by another PhD student, Kai Wang 244. The effects of
gemcitabine resistance on the expression of CXCL5 and CXCL10, and CSC markers
were determined in these cell lines.
Surprisingly, CXCL5 expression was reduced in MiaPaCa-2 and Pan02 after the
acquirement of resistance to gemcitabine (Fig. 3.4.4A & B(i)). However, in TB117,
GEMR resulted in a 29.59% increase in an alternative form, which was between 200
and 300 base pairs. For CXCL10, GEMR-MiaPaCa-2 had a significantly less CXCL10
expression than the parental strain (Fig. 3.4.4A & B(ii)), whereas GEMR-Pan02 and
GEMR-TB117 had slight increases of CXCL10 mRNA. GEMR-MiaPaCa-2 and GEMR-
Pan02 also had reduced ALDH1 expression (Fig. 3.4.4A & B (iii)); however, GEMR-
TB117 had a 22.05% increase in ALDH1. The three GEMR cell lines all had
significantly less CD44 than their corresponding parental ones (Fig. 3.4.4A & B (v)).
On the contrary, expressions of CD24 and CD133 were significantly elevated in the
chemoresistance cell lines (Fig. 3.4.4A, B(iv) & B(vi)).
To further investigate how gemcitabine treatment affects the expression of CXCL5
& 10, and CSC markers, four PC cell lines, BxPC-3, MiaPaCa-2, Panc-1 and LM-P,
70
were treated with gemcitabine at their corresponding IC50 concentrations (calculated by
a previous PhD student, Dannel Yeo) 230. Gemcitabine treatment decreased CXCL5
A
B
Figure 3.4.4. Effect of gemcitabine resistance on expression of CXCL5, CXCL10,
ALDH1, CD24, CD44 and CD133 in MiaPaCa-2, Pan02 and TB117 cells. (A) RT-
PCR analysis of mRNA expression of CXCL5, CXCL10, ALDH1, CD24, CD44 and
CD133 in parental and gemcitabine-resistant (GEMR) MiaPaCa-2, Pan02 and TB117
cells. (B) Quantifications of mRNA expression of CXCL5, CXCL10, ALDH1, CD24,
CD44 and CD133. The values from the reletive parental lines were taken as 100%.
71
expression in BxPC-3 and MiaPaCa-2; however, increased its expression in Panc-1 and
TB117 (42.64% and 56.59% increments compared to control) (Fig. 3.4.5). Gemcitabine
treatment increased CXCL10 expression by 25.74 and 31.65% in MiaPaCa-2 and
TB117, respectively. In contrast, it reduced CXCL10 by 20.54% in Panc-1 cells.
A
B
Figure 3.4.5. Effect of gemcitabine on CXCL5 and CXCL10 in BxPC-3, MiaPaCa-
2, Panc-1 and TB117 PC cell lines. (A) 24 hours after seeding BxPC-3, MiaPaCa-2,
Panc-1 and TB117 cells in 6-cm culture plates, different doses of gemcitabine were
administrated. RT-PCR assay was conducted 24 hours after administration of
gemcitabine. (B) Quantifications of CXCL5 and CXCL10. The values from controls
(CT, non-gemcitabine-treated) lines were taken as 100%.
Next, the effects of gemcitabine alone or in combination with other chemo-reagents
on CXCL5 and CXCL10, and CSC markers were investigated in the xenografted
72
tumours generated by Nhi Huynh and Kai Wang using the TKCC 15 cell line, which
was injected in to 8-week-old SCID male mice 245. The Kinghorn Cancer Centre (TKCC)
15 cell line was originally isolated from a 66-year-old patient with a poorly
differentiated pancreatic adenocarcinoma and given by Dr. Marina Pajic of the Garvan
Institute of Medical Research (Sydney, NSW, Australia). The mice carrying
xenografted tumours were treated with gemcitabine, nab-paclitaxel (also called
abraxane, a microtubule inhibitor that interrupts mitosis in cancers) (Abraxis
Bioscience Australia, Melbourne, Victoria, Australia) and PF3758309 (Active
Biochemical, NJ, USA) alone or in combinations. Here, doses of gemcitabine, nab-
paclitaxel and PF3758309 were 40, 10 and 25 mg/kg, respectively.
As illustrated in Fig. 3.4.6 (top row), compared to control, expression of CXCL5 in
TKCC xenografted tumour was markedly increased by gemcitabine along (307.18%
increase) or in combination with nab-paclitaxel, CXCL5 level displayed a significant
elevation by 311.37%. When a PAK inhibitor, PF3758309, was co-administered with
gemcitabine, CXCL5 level in TKCC xenografts was increased by 287.95%. The
expression of CXCL10 seemed to be insignificantly altered by any of the drug
treatments in the TKCC15 xenografts (Fig. 3.4.6, bottom row). The results that
gemcitabine treatment increased CXCL5 in TKCC15 xenograft suggested that CXCL5
may mediate resistance to gemcitabine in PC.
73
A
B
Figure 3.4.6. Gemcitabine along or in combination with abraxane (nab-paclitaxel)
or PF378309 increased CXCL5 expression while CXCL10 remained unchanged in
TKCC15 xenografts. The mice carrying TKCC15 xenografted tumours were treated
with gemcitabine, gemcitabine plus abraxane or gemcitabine plus PF3758309 245. An
anti-rabbit IgG was applied as an isotype control in this experitment. (A) Gemcitabine
along, gemcitabine plus Abraxane or gemcitabine plus PF3758309 resulted in
significant elevation of CXCL5, whereas there was no noticeable change in CXCL10.
200x magnification. (B) Quantifications of protein expression of CXCL5 and CXCL10.
The values from controls (CT, non-drug-treated) tumour tissues were taken as 100%.
3.4.5. Expression of CXCL5 in tumour samples from PC patients
To demonstrate the importance of CXCL5 in PC patients, immunohistochemical
analysis of CXCL5 expression on tissue microarrays was performed. Kaplan-Meier
74
analysis was used to investigate the clinical relevance of expression of CXCL5 in PC.
A list of clinicopathological parameters, including patient death, recurrence, survival
group, T stage and N stage, were subjected to Kaplan-Meier analysis to determine any
correlation with CXCL5 expression levels in PC tumours of the patients.
T stage refers to tumour size, which ranges from 2 cm in diameter to over 4 cm
across 246. N stage is defined as various degrees of metastasis into lymph nodes.
Survival groups of patients means whether they survive over the course of the cancer.
Recurrence is the likelihood of resurgence of PC after tumour treatment or surgery.
In a cohort of 33 patients, CXCL5 expression levels, which were calculated as
percentage of stained area in a tissue section in the tissue microarray, were varied from
one of the patients to another (Fig. 3.4.7A and B). Samples with low CXCL5 expression
(i.e. CXCL5 positivity < 3.5%) were assigned with the grade of 0 in the Kaplan-Meier
analysis, while those with CXCL5 positivity > 3.5% were assigned with the grade of
1.0. As a result of the Kaplan-Meier analysis, there was no strong indication that
CXCL5 was correlated with most of the clinicopathological features of the patients,
such as recurrence.
75
A
B
Figure 3.4.7. Expression of CXCL5 protein in the samples derived from a cohort
of 33 patients with pancreatic cancer. (A) Samples with low CXCL5 expression (i.e.
CXCL5 positivity < 3.5%). Magnification: 100X. (B) Samples with high CXCL5
expression (i.e. CXCL5 positivity > 3.5%). Magnification: 100X.
76
Figure 3.4.8. CXCL5 expression may contribute to death and T stage in a cohort
of 33 patients. (A)-(E) Kaplan-Meier survival curves for patient death, recurrence,
survival group, T stage and N stage using SPSS version 24.0.
3.5 Discussion
CXCLs are found to display a range of cellular behaviors which lead to tumour
progression, including chemoresistance 41-43,58,61, or anti-cancer effects 22,103. CXCLs
bind to distinct CXCRs on the surface of a tumour or stromal cell, to exert their
functions, inducing chemotaxis of various types of cellular components to the tumour.
These chemokines are shown to induce CSC phenotypes as well as other pro-tumoural
characteristics 160-162,247. However, their functions in the TME of PC tumours require
careful investigations.
CXCL5 expression was significantly increased in almost all PC cells tested (Fig.
3.4.1 and 3.4.2). This result is consistent with previous reports that increased CXCL5
77
levels were observed in human PC cell lines and tumour specimens from PC patients 51
and other types of cancers, suggesting an important role of CXCL5 in cancer 54,56,248.
In contrast, CXCL10 displayed uniformly low mRNA levels in all the PC cell lines
utilized in this research and seemed to have an opposite trend to CXCL5. In current
findings, it seems to display tumour-promoting roles, such as immunosuppression
23,103,112. However, as CXCL10 has been shown to have both pro-tumoural and anti-
cancer functions in other cancer types, it may play a tumour suppressing role in PC as
it demonstrated such abilities in other types of cancer 27,101,105-107.
CSCs are capable of differentiation, metastasis, self-renewal and proliferation as
well as chemoresistance and interacting with TME 163. In PC, genes that display CSC
characteristics include ALDH1, CD24, CD44 and CD133 249, which were shown to be
important contributors to pro-tumoural behaviors of pancreatic tumours. In this study,
expressions of ALDH1, CD24 and CD44 in most of the human PC cell lines were
markedly increased (Fig. 3.4.1); however, CD133 levels were higher only in two cell
lines. In other findings, the selected CSC genes were found to be highly expressed in
PDAC and other distinct cancer types 250-255.
PAK1 is upregulated in PC and mediates growth, gemcitabine resistance and
survival 230,238,240. PAK1 expression was detected in both human and mouse cell lines
(Fig. 3.4.1). PAK1 mRNA expression was significantly up-regulated in several human
PC cell lines utilized in this research, as well as LM-P and TB117 (mouse PC cells).
Since the receptors of CXCLs are GPCRs and PAK1 is downstream to a particular
member of the receptor family 240, CXCLs and PAK1 may act via the same intracellular
signaling pathway. The results, however, showed no correlation any member of CXCLs
to PAK1 (Table 3.4.1). Furthermore, PAK1 knockdown also generated irregular
78
changes in expression of the CXCLs (Fig. 3.4.3). Similarly, there was no correlation
shown between the expressions of the CSC genes and PAK1 (Table 3.4.1). Although
these changes may indicate compensatory effects of the genetic alternation, PAK1 and
the selected genes may act via dependent intracellular signaling cascades. There are
evidences suggesting that CXCLs induce expression of CSC phenotypes 160-162,247.
Molecular mechanisms which link a CXCL and pancreatic CSC phenotypic behaviors
remain to be determined.
The changes in the expressions of CXCL5 and CXCL10 were not consistent across
the gemcitabine-resistant cell lines examined (Fig. 3.4.5). Similarly, the changes in the
expression of both CXCL5 and CXCL10 were varied in gemcitabine treated cells (Fig.
3.4.5). However, in the presence of the chemotherapeutic reagents, the CXCL5
expression was increased in tumour tissue. The differentiation of CXCL5 expression
between cell lines and tissue needs further investigation.
Different from the in vitro results, the protein expression of CXCL5 was increased
in all chemotherapeutic reagents treated xenograft tumours while the protein expression
of CXCL10 had not changed significantly (Fig. 3.4.6). These results suggest different
mechanisms involved in regulation of CXCL5 and CXCL10 in vitro and in vivo.
Moreover, expression of CXCL5 may be stimulated upon administration of
gemcitabine or other chemotherapeutic reagents and the ligand mediates resistance to
gemcitabine in PC. Similarly, in a study by Kashiwagi and Tanaka, CXCL5 was implied
in gemcitabine resistance in PC 58. In bladder, liver and ovarian cancers, CXCL5 was
identified or implied as a chemoresistance marker 256-258, as it was shown to activate
EMT and NF-κB pathway, expressed in chemoresistant cells with increased metastasis
and recruit tumour-associated neutrophiles as well as activating the HIF1α/NF-κB
79
pathway, respectively.
Finally, the expression of CXCL5 in human patients did not seem to show clear
connections with patient death and T stage (Fig. 3.4.7 and Fig. 3.4.8), which is probably
due to the small amount of patients’ samples. However, in other cancer types, such as
bile duct, colon, liver and prostate, CXCL5 was shown to have clinical importance
56,259,260. In colon cancer, CXCL5 is over-expressed in patient tumour tissues and
associated with advanced cancer stage as well as poor prognosis 56. In patients with
cholangiocarcinoma or hepatocellular carcinoma, high CXCL5 levels are inversely
correlated with overall patient survival 259. In prostate tumours, CXCL5 protein
expression is correlated with increased bone metastasis 260. Therefore, CXCL5 may
serve as a molecular marker in treating PC patients.
In conclusion, CXCL5 mRNA expression was increased dramatically in a panel of
human PC cell lines as well as the mouse cell lines, suggesting an important role of
CXCL5 in PC. As expression of both CXCL5 and the selected CSC genes was high in
most PC cell lines tested, CXCL5 may contribute to CSC characteristics such as
tumourigenicity, self-renewal and chemoresistance. In this study, expression of PAK1
and CXCL5 did not correlate, indicating that these two genes belong to separate
intracellular pathways. Furthermore, the levels of CXCL5 were increased in TKCC
xenograft tumours received gemcitabine and gemcitabine plus other chemo-reagents
treatments, suggesting a role of CXCL5 in chemoresistance. The clinical importance of
CXCL5; however, needs further investigation.
80
Chapter 4:
The Role of CXC Ligand 5 in Chemoresistance of
Pancreatic Cancer
81
4.1 Abstract
Chemoresistance in cancer treatment is a serious problem for the complete
eradication of the disease. To understand whether CXC ligand 5 (CXCL5) mediates
chemoresistance in PC, the expression of CXCL5 in MiaPaCa-2 cells was knocked
down by shRNA. Here, MiaPaCa-2 was chosen due to its moderately high CXCL5
expression (chapter 3) and IC50 concentrations of gemcitabine and 5-fluouracil (5-FU)
(determined in our laboratory previously). In all clones tested, KD-12 and KD-22 had
the lowest CXCL5 mRNA expression levels compared to negative controls transfected
with scrambled shRNA plasmids or the wild type cells, and therefore selected for later
experiments. To determine whether CXCL5 mediated chemoresistance in vitro, two
chemotherapeutic drugs, gemcitabine and 5-FU were used to treat a negative control
(NC) and the knockdown (KD) clones. CXCL5 was found to mediate the resistance to
these chemotherapeutic reagents. The xenografted tumours inoculated by either NC or
CXCL5 KD cells were treated with gemcitabine. CXCL5 KD reduced the tumour
growth significantly compared to NC. Gemcitabine further decreased the tumour
growth of CXCL5 KD. Gemcitabine increased CXCL5 and caspase 3 protein
expression and decreased Ki-67 expression. Gemcitabine treated CXCL5KD tumours
had the lowest Ki-67 and the highest caspase 3. These results indicated that knockdown
of CXCL5 sensitized PC cell response to gemcitabine and 5-FU, suggesting that
CXCL5 mediates chemoresistance in PC.
4.2 Introduction
In clinical practices, gemcitabine, 5-fluorouracil (5-FU), FOLFIRINOX (a
combination of folinic acid, 5-FU, irinotecan and oxaliplatin), erlotinib and nab-
82
paclitaxel, are administered to pancreatic cancer (PC) patients 1-5. These chemicals are
found to effectively eliminate most PC tumour cells; however, chemoresistance and
adverse effects limit effectiveness of chemotherapeutic treatments 6.
Chemoresistance leads to disease relapse, accelerated growth and metastasis, as
well as reduced patient survival 7. In advanced PC, the knowledge of chemoresistance
mostly came from studies using gemcitabine in clinical practices 6, 261-263. The
interaction of tumour cell with tumour microenvironment (TME) is a major
contributing factor in resistance to therapy 8. As cancer cells interact with TMEs, a list
of factors, including peptide signals, their receptors and tumour-associated, non-
cancerous cells, influence cancer cell growth, metastasis, survival and chemoresistance
9. Interactions between tumour and stromal cells play important roles in progression of
tumour in the context of chemotherapeutic treatments.
A family of chemokines, namely CXC ligands (CXCLs), has been shown to
contribute to chemoresistance after binding to their corresponding G-protein-coupled
receptors, namely CXC receptors (CXCRs) 11-16. Several members of this chemokine
family, including CXCL1, CXCL2, CXCL8 and CXCL12, have been found to mediate
resistance to a range of reagents, such as doxorubicin, cyclophosphamide, paclitaxel
and docetaxel, in breast cancer 17-19. In other cancer types, such as colon, ovarian and
leukemia, the pro-tumoural members of the CXCLs also have been demonstrated to
mediate or induce resistance to chemotherapy 20-27. Moreover, through binding to their
receptors CXCR1, CXCR2, CXCR4 and CXCR7, respectively, they also activate
pathways like PI3K/Akt, NF-κB and ERK1/2 12.
CXCL5 is a tumour promoting CXCL that is associated with chemoresistance as
83
well as angiogenesis, chemotaxis of CXCR2+ neutrophils (in which CXCR2 is the
CXCL5 receptor), growth and cancer cell mobility 28-33. In a study by King et al., high
CXCL5 expression is associated with aggressiveness and reduced survival in PC
patients 34. CXCL5 is currently known to mediate angiogenesis, metastasis, invasion,
proliferation and tumourigenesis in some cases 30-32, 35-38; and is found to affect tumour
metastasis, angiogenesis and proliferation by activating some signaling molecules or
pathways, including ERK1/2, PI3K/Akt, NF-κB, Snail and certain matrix
metalloproteinases (MMP)s after binding to its receptor, CXCR2. However, the
mechanism of how CXCL5 mediates chemoresistance in PC needs further study.
Gemcitabine is a synthetic pyrimidine nucleotide analog prodrug 39, 40, which is used
to treat a number of tumour types, including breast, ovarian, lung, pancreas and bladder
41-45. After entering a cancer cell, gemcitabine is phosphorylated three times at different
positions by the enzyme deoxycytidine kinase, becoming its active form, gemcitabine
triphosphate 46, 47. Then, gemcitabine is incorporated into a growing DNA chain, leading
to inhibition of further DNA synthesis by ribonucleotide reductase. Although
gemcitabine is a first-line treatment for PC, it does have side-effects, such as bone
marrow suppression, liver and kidney problems, nausea, fever, rash, shortness of breath,
and hair loss 48. In clinical practices, gemcitabine is co-administered with carboplatin
49, cisplatin 50 or nab-paclitaxel 51. However, resistance to gemcitabine represents as a
major obstacle 52.
5-FU is used to treat different types of cancer, including breast, cervical, colon,
esophageal, pancreas and stomach 53-58. 5-FU inhibits thymidylate synthase, blocking
synthesis of the pyrimidine thymidine, which is required for DNA replication. The
enzyme adds a methyl group to deoxyuridine monophosphate, producing thymidine
84
monophosphate 59. Like other chemotherapeutic drugs, 5-FU also causes different kinds
of side effects, such as cognitive impairment, diarrhea, nausea, mouth sores, loss of
appetite, sensitivity to light, taste changes, skin discoloration and low blood count 60, 61.
Clinically, 5-FU is often co-administered with other chemotherapeutic drugs, such as
doxorubicin, mitomycin, methotrexate, leucovorin, irinotecan and oxaliplatin 62, 63. As
a chemotherapeutic drug, resistance to 5-FU has been found in a variety of cancer types,
such as breast, colon, stomach and pancreas 56, 64-66.
From the results of Chapter 3, CXCL5 was selected for investigations of its
potential role in chemoresistance of PC. CXCL5 shRNA knockdown cells and two
chemotherapeutic reagents, gemcitabine and 5-FU, were utilized to determine effects
of CXCL5 knockdown on PC response to gemcitabine or 5-FU, and the mechanism
involved.
4.3 Scientific Method-quantification of IHC data
To quantify the results from IHC, 20 random fields were taken of each tumour tissue
sample at 200x magnification. Positively stained areas were analyzed with the Image
Pro-Plus version 4.5.0.29 image analysis software (Media Cybernetics, MD, USA) and
the percentage of the positively stained area to the total area in each field was calculated.
Positive stained cell (%) was calculated by the ratio of numbers of positive stained cells
to numbers of total cells per field.
4.4 Results
4.4.1 Establishment of CXCL5 Knockdown (KD) MiaPaCa-2 Cell Lines
In chapter 3, it was found that AsPC-1, CFPAC-1 and SW1990, had the highest
85
CXCL5 expression in the human PC cell lines tested, whereas MiaPaCa-2, HPAC-II
and Panc-1 had moderately high levels of CXCL mRNA (Fig. 3.4.1). Then, CXCL5
expression positively correlates with gemcitabine IC50 concentrations which were
previously determined in our laboratory as well as the selected CSC genes. In addition,
CXCL5 protein expression in TKCC15 xenografted tumours was increased due to the
treatments of distinct drug treatments (Fig. 3.4.6).
Here, MiaPaCa-2 was chosen for the experiments and the subsequent ones because it
grows well into xenografted tumours in SCID mice whiles other human PC cell lines,
such as AsPC-1, CFPAC-1 or SW1990, did not provide a stable growth. Therefore,
MiaPaCa-2 was selected for the mouse model.
MiaPaCa-2 cells were treated with different doses of puromycin, at 0.2, 0.5 and 1.0
μg/mL in a 6-well plate and incubated for 48-72 hours to find out the dose of puromycin
to kill >90% of cells. The optimal dose was 0.5 μg/mL, which was then used to select
the transfected cells and to maintain the CXCL5 KD phenotype. Single colonies were
picked from wells containing different amounts of CXCL5 shRNA constructs and
seeded in separate 24-well plates as various KD clones.
CXCL5 expression in the KD clones was tested with RT-PCR and it was found that
10 KD clones had significantly reduced CXCL5 mRNA expression (Figure 4.4.1A and
4.4.1B), namely C-9, C-14, C-18, C-20, C-22, D-12, D-15, D-20, D-21, D-22 and D-
23 (37.42%, 43.11%, 52.46%, 32.8%, 41.05%, 68.01%, 40.24%, 36.32%, 37.33%,
65.28% and 39.9% decrements compared to wild type (WT) MiaPaCa-2, respectively).
To further confirm this finding, the clones were compared with wild type and the
negative control (NC) clones transfected with scrambled shRNA plasmids. In Figure
86
4.4.1C and 4.4.1D, D-12, D-15 and D-22 showed markedly reduced CXCL5 expression
(66.28% 62.87% and 64.81% decrements compared to WT MiaPaCa-2, respectively),
while the 2 scrambled clones had roughly same CXCL5 level as the WT. As a result,
D-12, D-15 and D-22 were selected for later experiments and re-named as CXCL5-
KD12, CXCL5-KD15 and CXCL5-KD22, respectively.
A
B
C
D
87
Figure 4.4.1 CXCL5 knockdown by CXCL5 human shRNA plasmids. (A) PCR
analysis of CXCL5 expression after transfecting MiaPaCa-2 cells with CXCL5 human
shRNA plasmids and selection of knockdown clones with puromycin. “C” and “D”
represent separate batches of shRNA plasmid constructs. NC means “negative control”,
the clones transfected with scrambled shRNA plasmid. (B) Quantification of mRNA
expression of CXCL5. (C) Confirmation of CXCL5 knockdown effeciency in clones
D-12, D-15 and D-22 with PCR. (D) Quantifications of mRNA expression of CXCL5.
4.4.2 Effects of Gemcitabine and 5-FU on Growth of CXCL5 Knockdown Cell
Lines
Gemcitabine and 5-FU were used to treat four different clones of MiaPaCa-2 cells,
namely NC1, NC2, CXCL5-KD12 and CXCL5-KD22 for 48 or 72 hours, and MTT
assays were conducted upon completion. Here, the negative controls (NCs), namely
NC1 and NC2, served as parental controls because their CXCL5 levels are the same as
WT (Fig. 4.4.1) and they contain a scrambled sequence which does not target CXCL5
mRNA.
As shown in Table 4.4.1 IC50 values of gemcitabine in the negative control cell lines
were 122.18 nM for NC1 and 145.88 nM for NC2 by 72-hour treatment. However, the
IC50 values of gemcitabine in CXCL5 knockdown cell lines were 27.86 nM for CXCL5-
KD12 and 33.57 nM for CXCL5-KD22, which were approximately 5 to 6 times less
than those of the NCs. Therefore, CXCL5 knockdown enhanced the inhibitory effect of
gemcitabine on proliferation in the KD cell lines in vitro.
Similarly, when treated the cell lines with 5-FU for 48 hours, proliferation of all the
MiaPaCa-2 clones tested were decreased. 5-FU decreased the proliferation of CXCL5-
KD12 and CXCL5-KD22 cell lines more than in the NCs. As summarized in Table 4.4.1,
IC50 values of 5-FU were 263.03 mM and 159.96 mM for NC1 and NC2 respectively
88
whereas the IC50 values of 5-FU were 91.62 mM and 86.7 mM for CXCL5-KD12 and
CXCL5-KD22 respectively. The NCs had significantly higher IC50 values than those of
the KD clones. These results indicated that CXCL5 exerted a protective effect on PC
cell proliferation in the presence of gemcitabine or 5-FU in vitro.
A
B
Figure 4.4.2 Effects of gemcitabine and 5-FU on proliferation of NC1, NC2,
CXCL5-KD12 and CXCL5-KD22 MiaPaCa-2 clones. 3000 cells were seeded in
each well of a 96-well plate. (A) A range of doses of gemcitabine was administered 24
hours after cell seeding. The cells were incubated with the drug for 72 hours and then
MTT assay was conducted to access its effect on proliferation (n =3). (B) 5-FU was
administered at different concentrations 24 hours after cell seeding and MTT assay was
conducted after 48 hours of drug incubation (n=3).
89
Table 4.4.1 IC50 concentrations of gemcitabine and 5-FU in cell proliferation.
Gemcitabine
IC50
concentrations at
72 HRs (nM)
5-FU IC50
concentrations at
48 HRs (μM)
NC1
122.18
263.03
NC2
145.88
159.96
CXCL5-KD12
27.86
91.62
CXCL5-KD22
33.57
86.7
4.4.3 CXCL5 protects PC tumours against gemcitabine in vivo
The effect of CXCL5 on PC cells in response to gemcitabine in vivo was
investigated in a xenograft mouse model. Here, gemcitabine was applied in the mouse
model is commonly used adjuvant chemotherapy in treating PC patients in clinical
practices 264, whereas 5-FU is used in combination with several other chemotherapeutic
reagents. As mentioned previously, the understanding of chemoresistance in PC came
from the study of gemcitabine, whereas the knowledge of other chemotherapeutic drugs
remains scarce 175. NC2 and CXCL5-KD12 MiaPaCa-2 clone cells were
subcutaneously injected into the either flank of SCID mice. Gemcitabine (GEM)
treatments were started a week after tumour induction by peritoneal injection at 40
mg/kg. Saline was given to the control (CT) groups. The mice were divided into four
groups: NC-CT, NC-Gem, CXCL5-KD-CT and CXCL5-KD-Gem. Throughout the
animal experiment, no toxicity was found in any of the mice. Here, NC-CT refers to
mice injected with NC cells only, NC-Gem refers to mice injected with NC cells and
then with gemcitabine, CXCL5-KD-CT refers to mice injected with CXCL5-KD12
MiaPaCa-2 cells (CXCL5 knockdown) only and CXCL5-KD-Gem refers to mice
injected with the knockdown cell line and then with gemcitabine. NC-Gem, CXCL5-
KD-CT or CXCL5-KD-Gem was compared with NC-CT. In addition, CXCL5-KD-
90
Gem was compared with CXCL5-KD-CT.
Here, NC-CT was used as a control when comparing with other tissues. From day
45, volumes of NC xenografts were significantly larger than the CXCL5-KD ones (Fig.
4.4.3A). Gemcitabine significantly reduced the tumour volume of NC from day 51 and
further decreased the tumour growth of CXCL5-KD by reducing the tumour weight
(Fig. 4.4.3B). at the end point of the experiment.
The immunohistochemistry for CXCL5 expression of the tumours which were
isolated at the end of the experiment and fixed in Formalin, showed that CXCL5 protein
expression was significantly elevated in the NC tumour treated with gemcitabine (NC-
Gem) (30.73% increment compared to NC-CT) (Fig. 4.4.4). In contrast, its levels were
lower in the CXCL5-KD tumour with or without the gemcitabine treatment (62.18%
and 25.32% reduction compared to NC-CT, respectively). Although the knockdown did
result in reduced CXCL5 expression, gemcitabine was able to cause increased CXCL5
level in the CXCL5-KD-Gem group. These results indicate a potential role of CXCL5
in pancreatic tumour growth and gemcitabine-resistance.
91
A
B
Figure 4.4.3 CXCL5 knockdown inhibited tumour growth in the presence of
gemcitabine in vivo. MiaPaCa-2 clones, NC and KD-12, were subcutaneously injected
into flanks of SCID mice. The mice were treated with saline (CT) or gemcitabine (Gem).
(A) Gemcitabine significantly decreased volumes of both NC and CXCL5-KD
xenografts. **p < 0.01 vs NC-CT (decrease). (B) A similar pattern was observed in
tumour weight. ***p < 0.001 vs NC-CT (decrease). ##p < 0.01 vs CXCL5-KD-CT
(decrease). #p < 0.001 vs CXCL5-KD-CT (decrease).
92
A
B
Figure 4.4.4 Expression of CXCL5 in NC and CXCL5-KD tumours in the presence
or absence of gemcitabine treatment. Tumour tissue prepared from the experiment
described in the text, were stained for CXCL5 protein (A) Gemcitabine significantly
increased CXCL5 expression of tumour tissues from both NC-Gem and CXCL5-KD-
Gem xenografts when compared with NC-CT and CXCL5-KD-CT, respectively.
93
However, both CXCL5-KD-CT and CXCL5-KD-Gem had lower CXCL5 levels
compared to NC-CT. Magnification: 200X. (B) Quantifications of protein expression
of CXCL5 (n = 6 for NC-CT, n = 5 for NC-Gem, n = 5 for CXCL5-KD-CT, n = 4 for
CXCL5-Gem). # p < 0.05 compared to NC-CT. * p < 0.05, *** p < 0.001 compared to
NC-CT. ^^^ p < 0.001 compared to CXCL5-KD-CT.
4.4.4 Effects of CXCL5 on PC Cell Proliferation and Apoptosis in The Presence of
Gemcitabine in vivo.
To determine the effect of CXCL5 on PC cell response to Gem in vivo, cell
proliferation and apoptosis were measured by immunohistochemistry staining of Ki-67
and caspase 3 respectively.
As shown in Figure 4.4.5, CXCL5 knockdown significantly reduced Ki-67
expression (around 85.16% reduction when comparing CXCL5-KD to NC in the
absence of gemcitabine treatment). Gemcitabine decreased the expression of Ki-67 in
the tumour tissues from both NC and CXCL5-KD.
On the contrary, CXCL5 knockdown increased the cleaved caspase 3 level in the
PC tumours as well as gemcitabine-treated tumours (56.15%, 58.92% and 252.71%
increments compared to NC-CT, respectively) (Fig. 4.4.6). As seen in Figure 4.4.5,
although caspase 3 protein expression was increased in both gemcitabine-treated NC
and CXCL5-KD tumours (56.15% and 121.95% compared to the perspective control
of the MiaPaCa-2 clones), the latter seems to have a higher amplitude. These imply that
CXCL5 was required to protect PC tumours against the chemotherapy. These results
indicate that CXCL5 plays an important role in chemoresistance which is necessary to
promote tumour proliferation in vivo.
94
A
B
Figure 4.4.5 Expression of Ki-67 in NC and CXCL5-KD tumours in the presence
or absence of gemcitabine. Tumour tissues prepared from the experiment described in
section 4.4.3 were stained for Ki-67. (A) Representative images from staining of
negative control (NC) and CXCL5 knockdown (KD) tumours treated with gemcitabine
(Gem) and control (CT). Magnification: 200X. (B) Quantifications of protein
95
expression of Ki-67 (n = 6 for NC-CT, n = 5 for NC-Gem, n = 5 for CXCL5-KD-CT, n
= 4 for CXCL5-Gem). *** p < 0.001 compared to NC-CT. ### p < 0.001 compared to
CXCL5-KD-CT. ^^ p < 0.01 compared to NC-Gem. ^^^ p < 0.001 compared to NC-
Gem.
A
B
96
Figure 4.4.6 Expression of caspase 3 in NC and CXCL5-KD tumours in the
presence or absence of gemcitabine in vivo. Tumour tissues prepared from the
experiment described in section 4.4.3 were stained for caspase 3. (A) Representative
images from staining of negative control (NC) and CXCL5 knockdown (KD) tumours
treated with gemcitabine (Gem) and control (CT). Magnification: 200X. (B)
Quantifications of protein expression of Caspase 3 (n = 6 for NC-CT, n = 5 for NC-
Gem, n = 5 for CXCL5-KD-CT, n = 4 for CXCL5-Gem). ** p < 0.01 compared to NC-
CT. *** p < 0.001 compared to NC-CT.
4.5 Discussion
Chemoresistance is a major limiting factor in the complete eradication of PC in
clinical practices 175,265-269, and leads to disease relapse and other complications 270.
According to current understanding, several pathways, such as PI3K/Akt, NF-κB,
MAPK and autophagy, are found to mediate chemoresistance as well as markers of
cancer stem cells 270. Certain intracellular signaling pathways are found to mediate
resistance to gemcitabine, such as NF-κB, FAK, sonic hedgehog and HIF-1α, 271-274. In
interaction with TMEs, cancer cells engage associated, non-tumour cells to progress
and more importantly, to resist chemotherapy 275.
Members of CXCLs contribute to chemoresistance and other pro-tumoural
characteristics, including metastasis and proliferation 13-16,43,88,135,276-285. They also
activate intracellular pathways like PI3K/Akt, NF-κB and ERK1/2 277. CXCL5 is
currently found to display a number of pro-tumoural functions, such as metastasis,
invasion, proliferation and tumourigenesis 51,56,57,222,286-288. Moreover, CXCL5 is found
to activate some signaling molecules or pathways, CXCR2, including ERK1/2,
PI3K/Akt, NF-κB, Snail and certain matrix metalloproteinases (MMP)s. CXCL5
activates some signaling molecules after binding to CXCR2.
97
The effect of CXCL5 on chemoresistance were investigated in vitro and in vivo in
this chapter. First, from the results shown in Table 4.2.1, IC50 values of gemcitabine and
5-FU from CXCL5-KD clones are significantly lower than that in NC clones,
suggesting that CXCL5 protected PC cells from these chemo-reagents. From the in vivo
study, gemcitabine inhibited the tumour growth of both NC and CXCL5-KD, with more
reduction in CXCL5-KD tumour. However, the NC tumour still had better proliferative
ability than the CXCL5-KD one. In a study by Wang et al., CXCL5 was shown to
reduce sensitivity to a chemotherapeutic drug in patients with non-muscle invasive
bladder cancer 256. In another study by Roca et al., CXCL5 was required for prostatic
tumours to proliferate and metastasize to bone 260. These indicated that knockdown of
CXCL5 sensitized PC cells in response to gemcitabine, and that CXCL5 plays an
important role in PC resistance to chemotherapy.
The results from the immunohistochemical staining of Ki-67 and caspase 3 to
determine proliferation and apoptosis, in vivo respectively, had further indicated that
CXCL5 protected PC against chemo-treatment by promoting PC proliferation and
suppressing apoptosis. In a study by Wang et al., CXCL5 promoted resistance to
mitomycin C in non-muscle invasive bladder cancer 256. Similarly, CXCL5 was also
shown in a chemoresistant function in pancreatic cancer stem cells 289. In breast and
lung cancers, depletion or knockdown of CXCL5 caused an obvious reduction in cell
proliferation 290,291. In intrahepatic cholangiocarcinoma, overexpression of CXCL5 in
vivo led to enhanced proliferation as well as tumour metastasis and recurrence 292.
Therefore, CXCL5 plays an important role in survival and sustained or continued
proliferation of tumour cells under the pressure of chemotherapeutic treatments.
In conclusion, chemoresistance remains one of the obstacles in successful PC
98
treatment and is closely associated with TME. Chemokines are a group of extracellular
signaling molecules that connect a bulk of tumour cells and the TME where it is located.
A few of the CXCL family of chemokines were recently found to contribute to
chemoresistance as well as other pro-tumoural characteristics. The study in this chapter
has demonstrated that CXCL5 knockdown sensitized PC cell response to 5-fluouracil
or gemcitabine in vitro, and that CXCL5 protected pancreatic tumour from gemcitabine
treatment in vivo by stimulating proliferation and inhibiting apoptosis.
99
Chapter 5:
Mechanism(s) involved in the Effect of CXCL5 on
Chemoresistance in Pancreatic Cancer
100
5.1 Abstract
In previous chapters, CXCL5 was found to contribute to chemoresistance in PC.
However, its downstream signaling pathways remain to be investigated. In the
proteomic comparison between negative control (NC) and the CXCL5 knockdown
clone of MiaPaCa-2 cells, namely CXCL5KD22, CXCL5 knockdown resulted in up-
regulation of spermine synthase, peroxiredoxin 6, monocarboxylate transporter 1 and
glutamate-cysteine ligase regulatory subunit and down-regulation of HLA-B7 and
mitochondrial succinyl-CoA:3-ketoacid coenzyme A transferase 1. Each of these
proteins had a distinct biological function in cancer. From the phospho-proteomic data,
CXCL5 knockdown induced significant changes of certain phosphorylated proteins.
However, KEGG pathway analysis could not identify to all the phospho-proteins with
most significant differences between CXCL5 KD and NC cells. Cross-referencing with
the NCBI database clearly identified the biological functions of these proteins. CXCL5
activates several intracellular pathways in PC contributing to chemoresistance and other
biological processes. Although experimental and clinical validation are necessary,
CXCL5 serves as a pivotal molecular target in overcoming chemoresistance and
reversing the pancreatic tumour microenvironment, thus eliminating PC in clinical
practices.
5.2 Introduction
Chemoresistance has been a major obstacle in the treatment of all cancer types and
a leading cause of cancer-related deaths. In clinical observations, therapeutic drugs are
often rendered ineffective due to changes in cancer cell metabolism, including
increased cell survival and metastasis as well as sustained proliferation 293. In recent
101
years, there has been a number of signaling pathways which were found to mediate this
particular phenomena, such as NF-κB, PI3K/Akt and JAK/STAT3 270. Moreover, there
also has been an increasing number of discoveries that chemoresistance is linked to
local, microscopic locations where tumours reside, namely the tumour
microenvironment (TME) 294.
A structurally similar family of chemokines that is expressed in the TME, CXC
ligands (CXCLs), has been shown to play distinct roles in cancer, including
chemoresistance 295-297. In pancreatic cancer (PC), targeting CXCLs in combination
with conventional chemotherapeutic reagents such as gemcitabine is also an effective
treatment option 298,299. In the current understanding of CXCL pathways in cancer,
certain intracellular signaling molecules are known to mediate downstream effects of a
CXCL. For instance, the CXCL1/CXCR2 axis in PC induces carcinogenesis during
development of pancreatic ductal adenocarcinoma 40. In melanoma, CXCL7 has a
protective effect against decitabine-induced apoptosis via MAPK pathways 77. In
another example, CXCL8 exerts tumour-promoting effects and mediates
chemoresistance by activating several pathways, including ERK1/2, PI3K/Akt, VEGF
and NF-κB 300-303.
In the previous chapter, CXCL5 reduced the sensitivity to 5-FU and gemcitabine in
PC cells in vitro and protected pancreatic tumour from gemcitabine-induced apoptosis
while stimulating proliferation in vivo. In other cancer types, such as bladder 256 and
breast 304, CXCL5 was also found to mediate chemoresistance. Currently CXCL5 is
discovered to activate different intracellular signaling cascades in bladder, colon, liver,
lung, pancreatic, prostate and skin tumours, such as STAT3, NF-κB, ERK, PI3K/Akt,
Wnt/β-catenin and MAPK 51,56,260,292,305-308. The outcomes of these pathways which are
102
pro-tumoural in nature are diverse, including angiogenesis, cell motility, epithelial-
mesenchymal transition (EMT), immunosuppression, inflammation and proliferation.
In PC, most of these pathways can be activated in the resistance to gemcitabine 309-313,
which is a commonly used chemotherapeutic reagent in treating the cancer 314. In
addition, in minor cases, other signaling molecules, such as mTOR 313, GATA1 315,
Hedgehog 313 and Notch 313, were also found to mediate gemcitabine resistance in PC.
Since gemcitabine is used to treat PC, several proteins or genes which were found
to be involved in different stages of the metabolism of the drug may be involved in
resistance. For example, concentrative nucleotide transporters (hCNTs) 1, 2 and 3 in
human PC are responsible for intake of gemcitabine; in contrast, equilibrate nucleotide
transporters (hENTs) 1, 2 and 3 result in efflux of the chemical 263,316. Enzymes that are
parts of the machinery of DNA synthesis or inactivation of gemcitabine may also be
expressed as PC cells are under the stress from gemcitabine treatments, such as
pyrimidine nucleotide monophosphate kinase (NMPK), nucleotide diphosphate kinase
(NDPK), cytidine deaminase (CDA) and ribonucleoside reductase (RR) 263,317-319.
Moreover, other protein markers, involved in EMT 320 or gemcitabine sensitivity 312
were also found to be involved in chemoresistance in PC.
In clinical practices, 5-fluorouracil (5-FU) is used to treat PC as well as several
other types of tumours278,321-325. The mechanism of resistance to 5-FU in PC also
involves several molecular markers. In terms of 5-FU metabolism, multi-drug
resistance-associated protein (MRP)5 and MRP8 serve to exclude the chemical out of
a cancer cell 326-329. Reduced mRNA level of hENT1 resulted in elevated 5-FU
sensitivity and subsequent cancer cell death 330. In addition, thymidylate synthase
integrates 5-FU into a growing DNA chain during DNA replication, thus introducing a
103
genetic lesion into the genome 331-334. Currently, 5-FU resistance has been shown to be
mediated by distinct intracellular signaling molecules, including EMT proteins 335,336,
Src 337, Notch 338, sonic hedgehog 339,340 and NF-κB 341. These markers have diverse
functions, such as cell motility, resistance to gemcitabine, 5-FU and cisplatin, reduced
cell death, tumour sphere formation and proliferation.
In previous findings, CXCL5 was found to function in chemoresistance in PC.
However, its downstream signaling cascades and effects need a thorough investigation.
In this chapter, using CXCL5 knockdown (KD) cell lines, the intracellular molecules
that are involved in the effect of CXCL5 were identified through proteomic analysis.
5.3 Scientific Method
To quantify the results from the global and phospho-proteomic analyses, the raw
data was uploaded to Perseus version 1.6.14.0 (Max-Planck-Institute of Biochemistry,
Munich, Germany) first. In the process of global proteomic analysis, the raw data was
filtered to eliminate any contaminant and transformed first. Then, the samples were
grouped and named. The data was filtered according to distinct groups, imputed with
missing values, clustered, and subjected for t-test in order to compare NC and CXCL5-
KD-22 samples. Subsequently, a heatmap and summary matrix describing up- and
down-regulations of various proteins in distinct degrees were generated.
Similarly, the raw data from phospho-proteomics was conducted using the same
software. As a result, a heatmap and a summary matrix were generated, showing
changes of phosphorylation status of distinct proteins.
104
5.4 Results
5.4.1 Differences in global proteins between negative control (NC) and CXCL5
knockdown (KD) clones of MiaPaCa-2 cells
To determine differences in protein expression between CXCL5-KD and negative
control (NC) MiaPaCa-2 clones, protein samples from these clones were processed,
enriched and separated into two different fractions: one for general global mass spectra
and another for phosphoproteomic analysis. Proteomic analysis that determined
changes in protein expression of distinct intracellular cascades were assessed to provide
an insight to biological processes affected by CXCL5 knockdown.
Through statistical analysis using Perseus software (version 1.6.14.0), the
difference of protein expressions between NC and CXCL5 KD clones was summarized
as shown in a heatmap (Fig. 5.4.1). The proteins with most significant differences
between NC and CXCL5-KD were listed in Table 5.4.1. The cellular functions of these
proteins were predicted through KEGG as shown in Table 5.4.2. The KEGG analysis
revealed a crucial representation of biological process categories that are related to
several metabolic pathways, including amino acid, fatty acid, glucose, glutathione,
folate, iron and ketone bodies. Other processes were also revealed, including antigen
processing and presentation, cell adhesion, mRNA surveillance pathway, RNA
transport and natural killer cell mediated cytotoxicity as well as RNA and protein
processing. Finally, proteins with significant changes in expression were cross-
referenced with the database of the National Center for Biotechnology Information
(NCBI) for their biological functions. The biological functions of these proteins in
cancer were searched and summarized in Table 5.4.3, demonstrating some distinct
105
functions, including proliferation (spermidine synthase), redox regulation
(peroxiredoxin 6), chemoresistance (peroxiredoxin 6), invasion (myopalladin) and
anaerobic energy production (monocarboxylate transporter 1 or solute carrier family 16
member 1), tissue compatibility, chemotherapy response, immune escape, cell
migration and proliferation (HLA Class I B-14 α chain (HLA-B)) 342-344. The protein
samples subjected to protein mass spectrum analysis from another CXCL5-KD clone,
CXCL5-KD12, was an outlier as seen in the volcano plot (data not shown) and was
therefore removed from further analysis.
In summary, CXCL5 knockdown resulted in up-regulation of spermine synthase
(SRM), peroxiredoxin 6 (PRDX6), monocarboxylate transporter 1 (MCT1) and
glutamate-cysteine ligase regulatory subunit (GCLM) and down-regulation of HLA-B7
and OXCT1. Each of these proteins had a distinct biological function in cancer (Table
5.4.3) as indicated in the results here.
106
Figure 5.4.1 Differences in the global intracellular protein expression between NC
and CXCL5-KD MiaPaCa-2 clones. Protein samples from two MiaPaCa-2 clones,
NC and CXCL5-KD22 were extracted, processed, quantified and subjected to mass
spectrum analysis of total intracellular proteins. A heatmap showing a global proteomic
comparison between NC and CXCL5-KD22 is extracted from the statistical analysis
using Perseus software (version 1.6.14.0).
Table 5.4.1 Proteins with most significant differences between NC and CXCL5-
KD cells
Protein
Fold
Change
FDR
Spermine synthase (SRM)
+ 2.3784
0.02042
Peroxiredoxin 6 (PRDX6)
+ 2.4623
0.022763
Monocarboxylate Transporter 1 (MCT1)
+ 2.1435
0.04184
Glutamate-cysteine ligase regulatory subunit (GCLM)
+ 2.3784
0.082085
HLA Class I histocompatility B-14 𝛼 chain (HLA-B)
- 2.3784
0.030197
Succinyl-CoA:3-ketoacid coenzyme A transferase 1,
mitochondrial (OXCT1)
- 2.0000
0.18
+: increased expression in CXCL5 KD cells; -: decreased expression in CXCL5 KD
cells.
107
Table 5.4.2. Cellular functions of proteins with most significant differences
between NC and CXCL5-KD cells
Protein
Gene
Count
KEGG
Pathway
ID
Gene Ontology
ID(s)
Predicted Cellular
Function(s)
SRM
1490
hsa01100
0006749
Glutathione metabolism
pathways
56
hsa00270
0006534
0006555
Amino acid metabolism
180
hsa03013
0050658
RNA transport
91
hsa03015
0071028
mRNA surveillance
pathway
PRDX6
1490
hsa01100
0045454
0098869
0034599
0046475
0042744
0006979
Metabolic pathways
56
hsa00480
0006749
Glutathione metabolism
MCT1
18
hsa00061
0006629
Fatty acid biosynthesis
GCLM
1490
hsa00270
0006534
0006555
Cysteine and methionine
metabolism
56
hsa00480
0006749
Glutathione metabolism
40
hsa04216
0097707
Ferroptosis
HLA-B
148
hsa04514
0050839
Cell adhesion
78
hsa04612
0019885
0019886
0042591
Antigen processing and
presentation
131
hsa04650
0042267
Natural killer cell mediated
cytotoxicity
OXCT1
10
hsa00072
0008260
0008410
Synthesis and degradation
of ketone bodies
48
hsa00280
0006550
0006552
Valine, leucine and
isoleucine degradation
108
Table 5.4.3 Biological functions of proteins with most significant differences
between NC and CXCL5-KD cells
Protein Name
NCBI
Gene ID
Genomic
Location
Known Biological
Process(es) in Cancer
References
Spermine synthase
(SRM)
6723
1p36.22
Proliferation regulation
278,345,346
Peroxiredoxin 6
(PRDX6)
9588
1q25.1
Redox regulation;
chemoresistance
347,348
Monocarboxylate
Transporter 1
(MCT1)
6566
1p13.2
Anaerobic energy
production
349-351
Glutamate-cysteine
ligase regulatory
subunit (GCLM)
2730
1p22.1
Chemoresistance
352-354
HLA Class I
histocompatility B-
14 𝛼 chain (HLA-
B)
3106
6p21.33
Chemotherapy
response; immune
escape; cell migration;
proliferation
342-344
Succinyl-CoA:3-
ketoacid coenzyme
A transferase 1,
mitochondrial
(OXCT1)
5019
5p13.1
Proliferation; ketone
body metabolism;
tumourigenesis; cell
motility
355-358
5.4.2 Difference in phosphorylation of proteins between NC and CXCL5 KD
clones of MiaPaCa-2 cells
To determine differences in protein phosphorylation between CXCL5-KD and NC
MiaPaCa-2 clones, a phospho-proteomic analysis was performed.
As shown in Fig. 5.4.2, compared to NC, the phosphorylated ankyrin repeat
109
domain-containing protein 26 (ANKRD26), beta-adducin (ADD2), E3 SUMO-protein
ligase RanBP2 (RANBP2), H/ACA ribonucleoprotein complex subunit 4 (DKC1), Myc
box-dependent-interacting protein 1 (BIN1) and NGFI-A-binding protein 2 (NAB2),
were increased while the phosphorylated AT-rich interactive domain-containing protein
3A (ARID3A), monocarboxylate transporter 1 (SLC16A1), pleckstrin homology-like
domain family B member 2 (PHLDB2), splicing factor 1 (SF1) and tetratricopeptide
repeat protein 28 (TTC28), decreased in CXCL5-KD cells. The fold changes of
CXCL5-KD over NC in these phosphorylated proteins were summarized in Table 5.4.4.
The cellular functions of these proteins analyzed by KEGG were listed in Table 5.4.5.
The phosphorylated proteins with significant differences between NC and CXCL5-KD
were cross-referenced with the database of NCBI for their biological functions. The
biological functions of these proteins in cancer were searched and summarized in Table
5.4.6.
CXCL5-KD induced significant up- and down-regulation of certain phosphorylated
proteins as listed in Table 5.4.4. However, the pathways analyzed by KEGG could not
be identified to all the phospho-proteins with most significant differences between
CXCL5-KD and NC cells as shown in Table 5.4.5. The biological functions of these
proteins cross-referenced with the database of NCBI were clearly identified (Table
5.4.6).
110
Figure 5.4.2. Differences in the phosphorylated intracellular protein expression
between NC and CXCL5-KD MiaPaCa-2 clones. Protein samples from two
MiaPaCa-2 clones, NC and CXCL5-KD22 were extracted, processed, quantified and
subjected to mass spectrum analysis of total phosphorylated intracellular proteins. A
heatmap showing a phospho-protein proteomic comparison between NC and CXCL5-
KD22 is extracted from the statistical analysis using Perseus software (version 1.6.14.0).
111
Table 5.4.4. Phosphorylated Proteins with most significant differences between
NC and CXCL5-KD cells
Protein name
Fold
Change
FDR
Ankyrin repeat domain-containing protein 26
(ANKRD26)
+ 4.32854
0.00914
Beta-adducin (ADD2)
+ 5.07831
0.0136
E3 SUMO-protein ligase RanBP2 (RANBP2)
+ 4.76361
0.00908
H/ACA ribonucleoprotein complex subunit 4 (DKC1)
+ 4.35299
0.01067
Myc box-dependent-interacting protein 1 (BIN1)
+ 4.93132
0.00971
NGFI-A-binding protein 2 (NAB2)
+ 4.7498
0.00756
AT-rich interactive domain-containing protein 3A
(ARID3A)
- 6.03374
0
Monocarboxylate transporter 1 (SLC16A1)
- 4.72811
0.0068
Pleckstrin homology-like domain family B member 2
(PHLDB2)
- 5.27965
0.017
Splicing factor 1 (SF1)
- 5.43416
0.02267
Tetratricopeptide repeat protein 28 (TTC28)
- 6.00597
0
+: increased phosphorylation in CXCL5-KD cells; -: decreased phosphorylation in
CXCL5-KD cells.
112
Table 5.4.5. Cellular functions of phosphorylated proteins with most significant
differences between NC and CXCL5-KD cells
Protein
Genes
Count
KEGG
pathway
ID
G.O. ID(s)
Predicted Cellular Function(s)
by KEGG pathway analysis
ADD2
(no hit)
ANKRD26
(no hit)
ARID3A
(no hit)
BIN1
252
hsa04144
0006897
Endocytosis
97
hsa04666
0006909
Fc gamma R-mediated
phagocytosis
DKC1
110
hsa03008
0032212
1904851
0000495
1990481
Ribosome biogenesis in
eukaryotes
NAB2
(no hit)
PHLDB2
(no hit)
RANBP2
186
hsa03013
0050658
RNA transport
364
hsa05014
N/A
Amyotrophic lateral sclerosis
SLC16A1
18
hsa00061
0006629
Fatty acid biosynthesis
SF1
(no hit)
TTC28
(no hit)
Proteins that show no prediction by KEGG analysis are labeled as “no hit”, which
indicates no predicted role in human cells.
113
Table 5.4.6. Biological functions of phosphorylated proteins with most significant
differences between NC and CXCL5-KD cells
Name of Phospho-Protein
NCBI Gene
ID
Genomic
Location
Known Biological Role(s) in
Cancer
References
β-Adducin (ADD2)
119
2p13.3
Cell mobility
359,360
Ankyrin repeat domain-containing
protein 26 (ANKRD26)
22852
10p12.1
Tumourigenesis
361
AT-rich interactive domain-
containing protein 3A (ARID3A)
1820
19p13.3
Cell mobility;
chemosensitivity; proliferation;
stemness
362-364
E3 SUMO-protein ligase RanBP2
(RANBP2)
5903
2q13
Cell mobility; DNA replication;
proliferation
365,366
H/ACA ribonucleoprotein complex
subunit 4/Dyskerin pseudouridine
synthase 1 (DKC1)
1736
Xq28
Apoptosis; cell mobility;
chemoresistance; proliferation;
telomere maintenance;
tumourigenesis
367,368
Monocarboxylate transporter 1
(MCT1; SLC16A1)
6566
1p13.2
Cell mobility; chemoresistance;
oxidative metabolism;
proliferation; tumourigenesis
369-375
Myc box-dependent-interacting
protein 1/Bridging integrator 1
(BIN1)
274
2q14.3
Apoptosis; chemoresistance;
proliferation; tumourigenesis
376-383
NGFI-A-binding protein 2 (NAB2)
4665
12q13.3
Cell mobility; proliferation
384
Pleckstrin homology-like domain
family B member 2 (PHLDB2)
90102
3q13.2
Cell mobility
385,386
Splicing factor 1 (SF1)
7536
11q13.1
RNA splicing; tumourigenesis
387,388
Tetratricopeptide repeat protein 28
(TTC28)
23331
22q12.1
Tumourigenesis
389
5.5 Discussion
Knockdown of CXCL5 in PC cells caused up- and down-regulations of the
expressions of some non-phospho-proteins (Table 5.4.1) and phosphorylated proteins
(Table 5.4.3). KEGG pathway analysis identified the cellular functions to the non-
phospho-proteins with most significant changes between CXCL5-KD and NC cells
(Table 5.4.2), but to only certain phospho-proteins with most significant changes
between CXCL5-KD and NC cells (Table 5.4.4). However, the biological function
analyzed using the database of NCBI indicated distinctive functions in cancer for both
non-phospho- (Table 5.4.3) and phospho-proteins (Table 5.4.6) with most significant
114
changes between CXCL5 KD and NC cells. The discrepancy in the results of KEGG
pathway analysis and biological function analysis using the database of NCBI requires
further investigation.
Chemoresistance has been a major obstacle in cancer treatment and has been shown
to linked to tumour microenvironment (TME) 294. Numbers of proteins or genes
involved in different stages of the metabolism of chemo-reagents may be involved in
resistance. In PC, most of these pathways can be activated in the resistance to
gemcitabine 309-313. 5-FU resistance in PC involves a number of molecular markers,
such as MRP5 and MRP8 326-329 and hENT1 331-334, EMT proteins and NF-κB 341.
CXCLs has been shown to play distinct roles in cancer, including chemoresistance 295-
297. CXCL5 is known to activate different intracellular signaling cascades in distinct
cancer types 51,56,260,292,305-308, leading to distinct biological outcomes, such as
angiogenesis, cell mobility and proliferation, and chemoresistance.
The data here showed that CXCL5 KD increased the protein expressions of SRM,
PRDX6, MCT1 and GCLM while decreased the protein expressions of HLA-B and
OXCT1. The biological function analysis using the database of NCBI indicated that
PRDX6, HLA-B and OXCT1 were involved in chemoresistance, and that HLA-B and
OXCT1 were also involved in tumourigenesis. SRM maintains cancer cell survival 390,
promotes autophagy and proliferation 346, and contributes to chemoresistance 345. In a
study by Hu et al., PRDX6 was found to be responsible for proliferation, cell mobility,
and inhibition of apoptosis in cervical cancer 391. In other studies, PRDX6 was found
to mediate proliferation, mitochondrial homeostasis, chemoresistance and suppression
of apoptosis in other cancer types, including blood, liver and lung 392-395. HLA-B in
pancreatic cancer was found to influence integrin β-1 expression and migration 396. In
115
addition, in a study by Garcia-Lora et al., loss of HLA-B was also found to facilitate
escape from immune surveillance by tumours 397. MCT1 (or SLC16A1), which
transports lactate, pyruvate, and other monocarboxylates across the cell membrane 398,
promotes cell survival, glycolysis, metastasis, proliferation and oxidative
phosphorylation in cancer 399-402.
OXCT1, which plays a role in ketone body metabolism 355,356, function as a pro-
tumoural gene in mediating protection from autophagy, cell mobility, proliferation and
tumourigenesis in distinct cancer types, including, bladder, brain and liver 358,403,404. In
addition, chemical inhibition of OXCT1 led to retardation of ketone metabolism in
cancer stem cells 405. Finally, GCLM, a subunit of glutamate cysteine ligase, which was
firstly found in glutathione metabolism 406, promotes chemoresistance and
tumourigenesis 354,406, while suppressing oxidative stress 407, in cancer. Therefore, as
CXCL5 knockdown affected protein expression of the proteins listed above, CXCL5
could affect several behaviors of PC tumours via regulating expression levels of these
proteins.
Likewise, knockdown of CXCL5 reduced the phospho-proteins of ARID3A,
SLC16A1 (MCT1), PHLDB2, SF1 and TTC28 while increased the phospho-proteins
of ANKRD26, ADD2, RANBP2, DKC1, BIN1 and NAB2. The cross-referencing with
the database of NCBI revealed that ANKRD26 361, DKC1 368 and BIN1 377,381 as well
as SLC16A1 372, SF1 387,388 and TTC28 389 were involved in tumourigenesis, and that
ARID3A 363 and SLC16A1 369,375 were involved in chemosensitivity. ARID3A, which
was originally found in B cell activation, in distinct types of cancer, including colon,
neural and rectal, is found to be involved in cell mobility, chemoresistance, DNA
methylation, proliferation and tumourigenesis 363,408,409. In addition, in a study by An et
al., ARID3A also was shown to stimulate cancer stem cells 410. PHLDB2, a focal
116
adhesion protein which is inhibited by p53 386, mediates cell adhesion and epithelial-
mesenchymal transition in several cancer types, including colon and renal 385,411. SF1,
a component in spliceosome assembly 412, is an important contributor of testicular germ
cell tumourigenesis as well as colon cancer 387,388. These results suggest that CXCL5
acts its role in chemoresistance and tumourigenesis of PC through regulation the
phospho- or active status of these proteins identified from this proteomic analysis. In a
study by Pitkänen et al., TTC28 drives transposition of Long Interspersed Nuclear
Element 1 and contributes to tumourigenesis of colon cancer 389. ANKRD26 plays a
role in metastasis in papillary thyroid carcinoma 361.
ADD2, or β-Adducin, was shown to mediate cell mobility in endometrial cancer 360.
RANBP2, which suppresses topoisomerase-II by sumoylation in cancer 413, promotes
proliferation in bile duct cancer 366. In a study by Packham et al., RANBP2 causes
nuclear translocation of insulin-like growth factor-1 receptor, which has critical roles
in cancer cell proliferation, in distinct cancer cell lines 414. In addition, RANBP2
activity influences proliferation, cell mobility, apoptosis chemoresistance in blood,
cervical, and head and neck cancer 415-417. According to a study by Hou et al., DKC1
activates HIF-1α transcription in order to enhance angiogenesis in colon cancer 418.
DKC1 was found to induce cell mobility, cell cycle progression, proliferation and
tumourigenesis in various cancer types, including colon, lung, neural and pituitary
368,419-422, while inhibiting apoptosis 422. BIN1, which has a MYC-binding domain,
suppresses metastasis and proliferation, and induces apoptosis as well as T cell
activation in cervical, colon, liver, lymph and prostate cancer 376,423-425. However, its
phosphorylation by CDK5 neutralizes it anti-tumour effects in lung cancer 426. Finally,
expression of NAB2 promotes metastasis in head and neck cancer 384 as well as
proliferation of solitary fibrous tumours 427, as it fuses with STAT6. Therefore, as
117
CXCL5 knockdown changed the phosphorylation states of the above proteins, it is
implied that the CXCL5 pathway involves these proteins to exert its downstream effects
in PC through phosphorylation. However, the detailed mechanism(s) of how these
molecules work in a pathway require further study.
In conclusion, CXCL5 is a chemoresistance gene in PC. CXCL5 knockdown
resulted in changes in expression and phosphorylation states of distinct proteins. By
databases analysis, CXCL5 was shown to mediate a range of downstream intracellular
pathways. This chapter provides more clues of how CXCL5 acts in PC, which should
provide useful information for improvement of PC treatment.
118
Chapter 6:
General Discussions and Future Directions
119
This chapter of general discussions reviews the findings and potential implications
of this research and outlines its limitations and challenges, and future directions.
6.1 CXCL5 has a potential role in chemoresistance in PC
Chemoresistance is a main limiting factor in treating pancreatic cancer (PC) and is
contributed by interactions between tumour cells, non-cancerous cells and secreted
factors in a tumour microenvironment (TME) 9,10. CXC ligands (CXCLs) mediate
interactions between PC cells and other cellular components, having a number of
distinct functions, including chemoresistance 41-43,58,61 and cancer stem cell (CSC)
phenotypes as well as other pro-tumoural characteristics 160-162,247. However, exactly
how these ligands function in pancreatic CSCs and their relationships with the TME
require further investigations.
In the study of Chapter 3, CXCL5 expression was found to be significantly
upregulated in both human and mouse PC cell lines (Fig. 3.4.1); however, CXCL10
mRNA expression had the opposite trend. The expressions of CSC genes including
ALDH1, CD24, CD44 were markedly increased in most of human PC cell lines
compared to the non-cancerous HDPE cells (Fig. 3.4.1). PAK1 is upregulated in PC
and stimulates PC growth, contributes to gemcitabine resistance and reduced cancer
survival 230,238,240. PAK1 expression was significantly up regulated in several human PC
cell lines utilized in this research as well as two mouse PC cells (Fig. 3.4.1). However,
expressions of the CXCLs or CSC genes and PAK1 were shown to be non-correlated
(Table 3.4.1). Furthermore, PAK1 knockdown also generated irregular changes in
expression of the CXCLs (Fig. 3.4.2).
CXCL5 expression positively correlates with gemcitabine IC50 concentrations
120
which were previously determined in the laboratory (unpublished data calculated by a
previous PhD student, Dannel Yeo) as well as ALDH1, CD24 and CD44, with CXCL10
expression being negatively correlated. In the in vivo results, expression of CXCL5
protein were increased in all chemo-reagent treated TKCC15 xenograft tumours while
the protein expression of CXCL10 had not changed significantly (Fig. 3.4.6),
suggesting different mechanisms involved in regulation of these ligands. In addition,
expression of CXCL5 may be stimulated upon chemotherapy and mediates resistance
to gemcitabine in PC.
The expression of CXCL5 in human patients seemed to have connections with
patient death and T stage (Fig. 3.4.7). A large numbers of patients’ samples is needed
for further investigation before a solid conclusion can be drawn for PC although in other
cancer types, such as bile duct, colon, liver and prostate, CXCL5 was shown to have
clinical importance 56,259,260.
The roles of CXCLs in tumour and TMEs have been investigated in various cancer
types in recent years and play either pro- or anti-tumour roles by regulating the
interactions between cancer cells and TME via distinct intracellular signaling pathways.
However, their functions in PC remain poorly understood, especially in the context of
chemoresistance. In the study of chapter 3, CXCL5 mRNA expression were found to
increase dramatically in a panel of human PC cell lines as well as the mouse cell lines,
suggesting an important role of CXCL5 in PC. The positive correlation of CXCL5 to
gemcitabine IC50 in PC cells and the increased level of CXCL5 in TKCC xenograft
tumours received different chemotherapeutic treatments (including gemcitabine) have
further indicated the role of CXCL5 in chemoresistance of PC. Therefore, the
mechanism involved in CXCL5 functions in PC was investigated in the subsequent
121
chapters.
6.2 CXCL5 is a gene that mediates chemoresistance in PC
CXCL5 is known to activate distinct pathways such as STAT3, NF-κB, ERK,
PI3K/Akt, Wnt/β-catenin and MAPK 51,56,260,292,305-308 in bladder, colon, liver, lung,
pancreatic, prostate and skin tumours. The outcomes of these pathways which are pro-
tumoural in nature are diverse, including angiogenesis, cell motility, epithelial-
mesenchymal transition (EMT), immunosuppression, inflammation and proliferation.
In PC, most of these pathways can be activated in the resistance to gemcitabine 309-313,
which is a commonly used chemotherapeutic reagent in treating the cancer 314. In
addition, other signaling molecules, such as mTOR 313, GATA1 315, Hedgehog 313 and
Notch 313, were also found to mediate gemcitabine resistance in PC.
In the study of chapter 4, CXCL5 knockdown (KD) cell lines were generated in
MiaPaCa-2 to determine its role in chemotherapy of PC. Compared to a negative
control (NC) cells which was transfected with a scramble sequence, CXCL5 KD cells
had reduced IC50 values of both gemcitabine and 5-FU on their inhibition of PC cell
proliferation (Table 4.4.1). CXCL5 KD not only reduced the xenografted tumour
growth in SCID mice but also enhanced the inhibitory effect of gemcitabine on PC
tumour growth (Fig. 4.4.3), indicating a relationship between CXCL5 inhibition and
gemcitabine. The cellular mechanisms involved in the inhibition on PC growth by
CXCL5 KD, include decreased proliferation shown by reduced Ki-67 expression (Fig.
4.4.5) and increased apoptosis shown by increased caspase 3 expression (Fig. 4.4.6) in
PC cells. Similarly, CXCL5 KD enhanced the inhibitory effect of gemcitabine on PC
by reducing proliferation and promoting apoptosis.
122
One important finding in the study of chapter 4 demonstrated that the expression of
CXCL5 was increased by gemcitabine treatment in both NC and KD tumour tissue (Fig.
4.4.4), which is consistent with the finding in the study of chapter 3 showing an
increased CXCL5 level in gemcitabine-treated human PC xenografted tumour (Fig.
3.4.6). Together these findings indicate an important role of CXCL5 in PC growth and
chemoresistance.
6.3 Proteomic analysis of pathways involved in CXCL5 functions in PC
In the study of chapter 5, proteomic analysis was used to determine differences in
global and phosphorylated protein expression between CXCL5-KD and NC MiaPaCa-
2 clones, and changes in protein expression of distinct intracellular cascades were
assessed in order to provide an insight to biological processes affected by CXCL5
knockdown.
CXCL5 knockdown induced changes in protein expression and their
phosphorylation status, which are implicated in distinct pathways in cancer progression
and therefore may contribute to chemoresistance of PC. These results revealed the
possible roles of CXCL5 in PC progression, particularly in chemoresistance, suggesting
that CXCL5 is a potential target for PC treatment. However a more solid conclusion
about the clear roles of signaling molecules in CXCL5-mediated chemo-resistance of
PC, need to be validated and therefore to be confirmed by other experimental methods
such as Western blotting and immune-staining.
6.4 Limitations and future directions
In this thesis, CXCL5 is established as a gene that contributes to chemoresistance
123
of PC. It is known that CXCL5 in cancer plays roles in some pro-tumoural
characteristics 51,61,289 as well as activating some pathways 292. However, exactly what
intracellular mechanisms that are involved in either chemoresistance or other aspects
of PC remain to be investigated. In addition, the possible intracellular pathways
predicted by proteomic analyses in chapter 5 need to be further clarified by other
experimental methods such as Western blotting and immunohistochemical staining.
Since CXCL5 is a ligand found in the microenvironment in PC and other cancer
types, it requires more detailed investigation of how it influences the dynamics between
PC tumour and its surroundings (i.e. cellular and non-cellular components of tumour
microenvironment). It was previously found that CXCL5 induces chemotaxis of non-
cancerous cells bearing surface molecule Ly6G 60,292 to promote cancer progression. It
is also known that PI3K/Akt, ERK1/2 and RSK1/2 pathways was induced to cause
chemotaxis of Ly6G+ neutrophils into the tumour stroma in cholangiocarcinoma.
However, which similar events in PC with other non-cancerous cell types is mediated
by CXCL5 is unknown, such as that for pancreatic stellate cells, which was found to
mediate PC progression 433. In addition, in the context of chemoresistance, it is also
unknown about what roles surrounding cell types play and what signaling pathways can
be activated. This research has not touched on dynamic interactions between PC cells
and associated microenvironmental cell types that contribute chemoresistance and
possible changes in compositions of pancreatic microenvironment.
About the role of CXCL5 in chemoresistance of PC, the relationship between
CXCL5 and expression of the selected CSC genes needs to be established. Although
CXCL5 expression is increased upon treatments of chemotherapy in experimental
setups, its clinical applications remain unknown and therefore requires more
124
investigation. More importantly, the mechanism of how CXCL5 mediates
chemoresistance in PC in terms of how it regulates behaviors of tumour cells as well as
the surrounding non-cancerous cells that are associated with the tumour need further
study.
6.5 Conclusion
In this study, CXCL5 was found to mediate chemoresistance in PC both in vitro and
in vivo as CXCL5 knockdown significantly enhanced the inhibitory effects of
gemcitabine or 5-FU on proliferation of PC cells, and the inhibition by gemcitabine of
PC cell xenografted growth. In PC cells, CXCL5 is required for expression of distinct
proteins with diverse functions, implying multiple biological roles of CXCL5 in the
progression of PC as demonstrated by the results obtained from the proteomic analysis
of global- and phosphor-proteins. The results suggest CXCL5 activates several
intracellular pathways in PC contributing to chemoresistance and other biological
processes. Although experimental and clinical validation are necessary, CXCL5 serves
as a pivotal molecular target in overcoming chemoresistance and modulating pancreatic
tumour microenvironment, thus eliminating PC tumours in clinical practices.
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Appendix: Paper publication
Functions of the CXC ligand family in the pancreatic tumor
microenvironment
Nien-Hung Lee, Mehrdad Nikfarjam
1
, Hong He
*
,
1
Department of Surgery, University of Melbourne, Austin Health, Melbourne, Victoria, Australia
article info
Article history:
Received 9 May 2018
Received in revised form
6 July 2018
Accepted 30 July 2018
Available online 1 August 2018
Keywords:
Pancreatic cancer
Tumor microenvironment
Chemoresistance
CXC ligands
Cancer stem cells
abstract
Therapeutic resistance is the major contributor to the poor prognosis of and low survival from pancreatic
cancer (PC). Cancer progression is a complex process reliant on interactions between the tumor and the
tumor microenvironment (TME). Members of the CXCL family of chemokines are present in the
pancreatic TME and seem to play a vital role in regulating PC progression. As pancreatic tumors interact
with the TME and with PC stem cells (CSCs), determining the roles of specific members of the CXCL
family is vital to the development of improved therapies. This review highlights the roles of selected
CXCLs in the interactions between pancreatic tumor and its stroma, and in CSC phenotypes, which can be
used to identify potential treatment targets.
©2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.
Introduction
Pancreatic cancer (PC) is one of the most commonly diagnosed
cancers, with an increasing incidence worldwide [1,2]. Pancreatic
ductal adenocarcinoma (PDAC) constitutes more than 90% of PC,
and has an 8% 5-year survival at diagnosis [3]. Most patients have
metastatic disease at diagnosis or develop it following surgical
resection. Chemotherapy remains the mainstay of treatment, but
response rates are generally poor.
Therapeutic resistance represents a major problem that limits
the outcomes of cancer therapy. Tumor cells either become
intrinsically resistant to a cancer therapy [4], or acquire resistance
via different methods, such as DNA repair [5], enzymatic inactiva-
tion, altered membrane transport or activation of an upstream
molecular marker to ensure cell survival [6]. However, because of
the side-effects of chemotherapy [7], alternative methods, such as
targeted therapy in PC or naturally-derived compounds, may be
applied to cure cancer [8,9].
The tumor stroma or TME is the microscopic location where a
tumor resides, and is composed of cancer cells, neighboring non-
cancerous/stromal cells (such as endothelial cells (ECs),
fibroblasts (FBs) and macrophages (M
f
s)), and the extracellular
matrix (ECM) [10]. Tumors are associated with stromal cells which
further enhance angiogenesis, chemoresistance, growth, invasion,
metastasis and survival of cancer cells (Fig. 1)[11]. PC cells interact
with ECM, ECs, FBs, M
f
s, neutrophils and stellate cells [12,13].
Cancer and stromal cells communicate with each other by secreting
signal molecules to either support or suppress tumor progression
[14]. Moreover, the TME also enhances chemoresistance by alter-
nating cell adhesion and the structure of the ECM, by secreting
growth factors and by activation of chemoresistance pathways in
tumor cells [10]. Consequently, a successful treatment, in addition
to eliminating all cancer cells, should reprogram the TME.
Roles of CXCLs in pancreatic cancer
A family of structurally similar inflammatory chemokines,
namely CXC ligands (CXCLs), in which C stands for cysteine and X
stands for any amino acid (Fig. 2), were recently discovered to play
important roles in various cancer types, including brain, colon, liver,
lung and pancreas [14e17]. These CXCLs are separated into two
structurally distinct groups, ELR
þ
and ELR
, by the presence or
absence of a Glu-Leu-Arg (ELR) motif at the N-terminus. CXCLs bind
to specific CXC receptors (CXCRs), which are G protein-coupled
receptors, and cause chemotaxis of stromal cells (Fig. 3). The
ELR
þ
members, including CXCL1 to CXCL3 and CXCL5 to CXCL8,
bind to CXCR1 and/or CXCR2 and contribute to angiogenesis in
*Corresponding author. Dept. of Surgery, University of Melbourne, Austin Health,
Studley Rd., Heidelberg, Victoria 3084, Australia.
E-mail address: hong.he@unimelb.edu.au (H. He).
1
The last two authors share equal last authorship.
Contents lists available at ScienceDirect
Pancreatology
journal homepage: www.elsevier.com/locate/pan
https://doi.org/10.1016/j.pan.2018.07.011
1424-3903/©2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.
Pancreatology 18 (2018) 705e716
tumors. On the other hand, the ELR
members (CXCL4, CXCL4L1,
and CXCL9 to CXCL14) bind to CXCR3, CXCR4, CXCR5 or CXCR7, are
angiostatic, and have anti-tumor functions, with one exception,
CXCL12, which binds to CXCR4 or CXCR7 to promote metastasis.
CXCR3, which exhibits anti-tumor abilities, induces chemotaxis of
ECs, M
f
s, monocytes (MOs), platelets and T lymphocytes [17].
Finally, each CXCL-CXCR combination attracts a distinct range of
stromal cells. For example, CXCL2-CXCR2 causes chemotaxis of ECs,
eosinophils, M
f
s, MOs and neutrophils [14,17].
Most members of the chemokine family, including CXCL1,
CXCL2, CXCL5, CXCL9, CXCL10 and CXCL13, have been found in PC
[18e22], where they are secreted by cancer or stromal cells, such as
cancer-associated fibroblasts (CAFs) and dendritic cells (DCs)
[18,20,23]. Most of these ligands have been reported to promote
chemoresistance, immunosuppression, tumor proliferation and
metastasis [18,21,24,25]. However, certain CXCL chemokines, such
as CXCL9 and CXCL10, may lead to elimination of tumor cells as they
evoke chemotaxis of CD4
þ
and CD8
þ
T lymphocytes, DCs, natural
killer and Th1 cells [26e28]. The exact mechanism of how these
ligands modify TME remains poorly understood, although modu-
lation of the tumor stroma has been reported to contribute to better
cancer treatment [29,30]. Ideally, targeting various constituents of
the TME, including CAFs, ECM and CXCL members that promote
tumor progression, would more effectively treat cancer. The CXCLs
of interest in PC are listed in Table 1 and discussed below.
CXCL1
CXCL1 is also known as Growth-Related Oncogene
a
(GRO
a
) and
its secreted form was originally discovered in melanoma cell ex-
tracts and found to be involved in oncogenic transformation [31].
The ligand stimulates chemotaxis of CAFs, neutrophils and
myeloid-derived suppressive cells (MDSCs) toward tumors by
binding to its receptor(s) CXCR1 and/or CXCR2 [32e34]. In diverse
cancer types, this ligand promotes or is correlated with angiogen-
esis, metastasis, proliferation, resistance to chemotherapy, tumor
cell survival and tumorigenesis [21,32,35e37]. In colorectal cancer,
CXCL1 is important for the formation of premetastatic niche in the
Fig. 1. Interactions between tumor cells and stromal cells contributes to chemoresistance and other behaviors of cancer. Tumor and stromal cells interact with each other by
secreting chemokines (e.g. CXCLs), growth factors or other peptidyl signals. Such communications lead to chemoresistance, angiogenesis, metastasis, proliferation and cell survival.
Abbreviations: ECs, endothelial cells; FBs, fibroblasts; M
f
s, macrophages; PSCs, pancreatic stellate cells.
Fig. 2. CXCL molecular structures. CXCLs are separated into two structurally distinct groups, ELR
þ
and ELR
, by the presence or absence of a Glu-Leu-Arg amino acid sequence (ELR
in the one letter amino acid code) at the N-terminus.
N.-H. Lee et al. / Pancreatology 18 (2018) 705e716706
liver by recruiting CXCR2
þ
MDSCs to the destination site [38]. The
source of CXCL1 in this case is tumor-associated M
f
s, which are
stimulated by VEGF-A secreted by the primary tumor. In a study of
bladder cancer by Miyake et al., CXCL1 expression in cancer cells
leads to recruitment of both tumor-associated M
f
s and CAFs,
which are known to extracellular matrix components (e.g. collagen
I and IV, fibronectin, tenascin-C) as well as substances that attract
ECs and pericytes [35]. CXCL1 secreted from gastric tumor cells
stimulates the CXCR2-bearing bone marrow-derived mesenchymal
cells infiltration to gastric TME, inducing cancer cell proliferation
and lymph node metastasis [39].
CXCL1 is found to be highly expressed in a panel of PC cell lines,
and its expression leads to increased tumor proliferation and
angiogenesis as well as poor prognosis in patients [21,40], via
binding to its receptor, CXCR2 [41]. In the study by Lesina et al., the
CXCL1/CXCR2 axis caused chemotaxis of tumor-associated M
f
s,
which promoted tumor cell survival and growth by suppressing
anti-tumor immunity [42]. Clinically, high expression of CXCL1 in
both cancer cell cytoplasm and stroma of PC specimens, is associ-
ated with carcinogenesis, tumor classification and TNM stage
[40,42]. In addition, the ligand promotes pancreatic oncogenesis
and is also secreted by CAFs to promote PC cell mobility [43,44].
Recently, the promotor sequence of this gene was discovered to
contain binding sites for NF-
k
B, a vital transcription factor which is
involved in inflammation, tumor initiation and chemoresistance
[45]. In a LSL-KrasG12D Ptf1a-Creex1 mouse model, the CXCL1/
CXCR2 axis, which is downstream to NF-
k
B, was shown to induce
pancreatic carcinogenesis during development of PDAC [42]. In a
Fig. 3. Effects of binding of CXCLs to their corresponding CXCRs in cancer. ELR positive CXCLs (e.g. CXCL1 and CXCL5) bind to CXCR1 and/or CXCR2, resulting in pro-tumor effects
(such as chemoresistance, angiogenesis and metastasis) and chemotaxis of a unique set of stromal cell types, including neutrophils, endothelial cells, etc. On the contrary, binding of
ELR negative CXCLs to either CXCR3, 4, 5 or 7 leads to distinct anti-cancer effects, for instance, reduced angiogenesis and decreased tumor growth as well as recruiting a different
range of TME cellular components (e.g. T lymphocytes, macrophages and platelets). Abbreviations: ECs, endothelial cells; EOSs, eosinophils; MOs, monocytes; M
f
s, macrophages;
MOs, monocytes.
Table 1
The Roles of Various CXCLs in PC. This particular table summarizes currently known functions and intracellular molecules involved in signaling of CXCLs in PC.
CXCL member Known functions in PC Intracellular signaling molecule(s) found to be involved in PC References
CXCL1 (GRO
a
) Chemoresistance, proliferation, angiogenesis,
carcinogenesis
CXCR1, CXCR2, NF-
k
B, STAT1 [21,31e36]
CXCL2 (MIP-2
a
)Inflammation, proliferation, migration CXCR2, NF-
k
B[19,32,37]
CXCL3 (GRO
g
) Tumor progression CXCR2 [38]
CXCL4 (PF-4) Anti-angiogenesis, growth inhibition CXCR3 [14]
CXCL5 (ENA-78) Chemoresistance, CSC phenotypes, cancer staging,
tumorigenesis, invasion, angiogenesis, proliferation
CXCR2, Akt, ERK1/2, ALDH1, CD44, CD133 [39e41]
CXCL6 (GCP-2) Angiogenesis, inflammation, metastasis CXCR2 [33,38,42,43]
CXCL7 (CTAPIII) No known function in PC CXCR2 [44,45]
CXCL8 (IL-8) Chemoresistance, angiogenesis, proliferation, invasion,
metastasis
CXCR1, CXCR2, NF-
k
B, CD44, CD133 [46e50]
CXCL9 (MIG) Immunosensitivity, apoptosis, anti-angiogenesis, growth
inhibition, anti-carcinogenesis
CXCR3 [23,51]
CXCL10 (IF-10) Anti-cancer: immunosensitivity, apoptosis
Pro-tumor: immunosuppression, tumor progression,
chemoresistance
CXCR3 [20,24,51e53]
CXCL11 (IP-9) Angiogenesis, proliferation CXCR3, CXCR4, CXCR7, ERK1/2 [54]
CXCL12 (SDF-1) Proliferation, invasion, migration, chemoresistance,
angiogenesis
CXCR4, CXCR7, ERK1/2, mTOR, Akt, NF-
k
B, sonic hedgehog, Src [48,55e59]
CXCL13 (BCA-1) Tumorigenesis, proliferation CXCR5, NF-
k
B, IKK
a
[60e64]
N.-H. Lee et al. / Pancreatology 18 (2018) 705e716 707
study by Seifert et al., CXCL1 serves as a mediator of gemcitabine
resistance, immune suppression and the subsequent tumorigenesis
in a LSL-KrasG12D/þ; LSL-Trp53R172H/þ; Pdx1-Cre (KPC) mouse
model and cell lines derived from the KPC mice [46]. The expression
of CXCL1 was upregulated after depleting a necrosis-related
pathway in M
f
s, resulting in an immunosuppressive, tumor-
promoting TME. Finally, in a study involving xenografting a panel
human PC cell lines and mouse Pan02 cells, expression of CXCL1
was induced by IL-35, signaling via STAT1 and STAT3 [47], and
CXCL1 mediated resistance to gemcitabine and monocyte infiltra-
tion, and stimulated angiogenesis and xenograft tumor growth
[47].
CXCL2
CXCL2, also known as Macrophage Inflammatory Protein-2
a
,
was initially found in supernatants of melanoma cell lines [48]. It
binds to CXCR2 and promotes chemotaxis of neutrophils and ECs,
contributing to angiogenesis, chemoresistance, transformation and
growth of tumors [14,32,49]. In bladder cancer, CXCR2
þ
MDSCs are
attracted to the TME by CXCL2 secreted from the cancer cells [50].
The MDSCs then secrete several immunosuppressive molecules
which reduce T cell proliferation, via activation of MAPK and NF-
k
B
by the CXCL2/CXCR2 axis.
CXCL2 is highly expressed in tumors in the best mouse model of
PC (the KPC mice) [44], and in human PC samples, CAFs of PC, and a
panel of human PC cell lines [41,42,51e53]. From study of biopsies,
binding of CXCL2 to its receptor, CXCR2, is positively correlated
with inflammation, and significantly reduces survival of patients
with PDAC. In a study by Kumar et al., smoking-induced inflam-
mation resulted in up-regulated CXCL2 expression, activation of
DCs, M
f
s and PSCs and tumorigenesis [19]. In this study, the au-
thors found that smoking induced inflammation and secretion of
inflammatory markers, such as CXCL1 and CXCL2. In addition,
smoking is known to cause oxidative damage byproducing reactive
oxidative species [54], and inflammation [19], in which the latter
leads to more oxygen-containing free radicals from M
f
s and sub-
sequent carcinogenesis. PSCs participate in chronic pancreatitis and
the desmoplastic reaction [55]. Activated PSCs can enhance pro-
liferation, migration, production of ECM by secreting cytokines and
chemokines in PC.
CXCL5
In several cancer types, Epithelial Neutrophil-Activating Pep-
tide-78 or CXCL5, is associated with angiogenesis, chemoresistance,
proliferation, migration and invasion, and poor patient survival
[56e61]. In an intrahepatic cholangiocarcinoma research by Zhou
et al., CXCL5 induces infiltration of Ly6G
þ
neutrophils into the tu-
mor stroma via PI3K/Akt, ERK1/2 and RSK1/2 pathways in vivo [62].
Then, the neutrophils mediate cancer cell proliferation and motility
and a similar TME pattern appears in human samples. In colon
cancer, platelet-derived CXCL5 recruits CD11b
þ
Ly6G
þ
granulocytes
to lungs to establish early metastatic niches and subsequently leads
to metastasis of cancer cells [63].
CXCL5 is highly expressed in PC cell lines, including MiaPaCa-2
and Capan-2, as well as in patients' tumors [59]. In pancreatic tu-
mors, elevated levels of CXCL5 mRNA are correlated with advanced
clinical stages (stages T3 and 4), tumor progression and poor pa-
tient survival [59]. Moreover, CXCL5 from the cell-conditioned
media of the PC cell lines AsPC-1, BxPC-3 and Capan-2 induced
angiogenesis by activation of Akt and ERK through binding to the
receptor CXCR2. Recently, CXCL5 was found to be co-expressed
with 3 markers of cancer stem cells (CSCs), namely ALDH1, CD44
and CD133, which contributed to gemcitabine resistance [61].
Finally, the CXCL5/CXCR2 axis in PC was also found to reinforce
proliferation of HPAC-1 and Colo-357 cell lines [64].
CXCL6
CXCL6, also known as Granulocyte Chemotactic Protein-2, is
expressed in several types of cancers, including human bone, liver
and lung cancers, as well as mouse melanoma [65e68]. High
expression has been noted in pediatric osteosarcoma and is
correlated with poor survival [65]. In hepatocarcinoma, transcrip-
tion of CXCL6 can be triggered by hypoxia and contributes to
increased cancer invasion and migration [66,67]. In a study by
Gijsbers et al., CXCL6 expression by ECs within gastrointestinal
tumors contributes to tumor development through angiogenesis
due to EC chemotaxis and to cell motility by recruiting neutrophils
containing proteases that degrades extracellular matrix [69].
In PC, CXCL6 is secreted by CAFs along with CXCL1, and CXCL8,
and its expression is higher in low grade PanIN (pancreatic intra-
epithelial neoplasia, defined as microscopic flat or papillary lesions
arising in small intralobular pancreatic ducts, which are usually less
than 5 mm in diameter) than in human PC [42,53,70]. Currently,
this ligand is known to cause angiogenesis, inflammation and
metastasis in PC [71]; however, it may not be important for
advanced pancreatic tumors or in the context of chemoresistance.
CXCL7
CXCL7, is expressed in breast cancer, colorectal adenocarcinoma,
leukemia, lung cancer, neuroblastoma, papillomas and renal cancer
as well as in PC [72e79]. In some cases, CXCL7 seems to promote
proliferation and/or cell mobility [73,79,80]; however, its expres-
sion level in PC is very low in all stages and it was therefore
assumed to be involved only in early carcinogenesis [78]. The
observation that CXCL7 expression was induced after chemo-
therapy in colon cancer suggesting its role in chemoresistance [82].
Meldi et al. discovered that overexpression of CXCL7 reduced
decitabine-induced melanoma cell apoptosis and rescued
decitabine-treated cell colonies via MAPK pathways [81]. In leu-
kemia, addition of CXCL7 promoted resistance to decitabine in
chronic myelomonocytic leukemia cells with the involvement of
MAPK pathways [82]. Overexpression of CXCL7 resulted in
chemotaxis of CD206
þ
M2 type tumor-associated M
f
s in lung
cancer [83], and positively correlated with increased proliferation
of tumors. Results from these studies suggest a role of CXCL7 in
chemoresistance; however, the exact mechanism needs thorough
investigation.
CXCL8
Originally known as IL-8, CXCL8 is a well-studied member of the
CXCL family which has multiple roles in tumor promotion by
binding to CXCR1 and/or CXCR2 [84]. Consistent with the fact that
the promoter sequence of the CXCL8 gene contains an NF-
k
B
binding site, CXCL8 has been noted to promote or up-regulate
angiogenesis, cell mobility and establishment of a pro-tumoral
microenvironment in breast, ovarian and prostate tumors after its
transcription and translation [85e87]. In other cases, CXCL8 acti-
vates various intracellular pathways, including ERK1/2, PI3K/Akt,
VEGF and NF-
k
B[88e90], which have tumor-promoting effects and
can cause chemoresistance [91]. In nonesmall-cell lung carcinoma,
NF-
k
B activation leads to increased CXCL8 expression, which then
causes increased blood vessel formation in the bulk of tumor [85].
Finally, CXCL8 also induces expression of CSC genes in PC in
addition to its known pro-tumoral functions [92e95]. In breast
cancer, CXCL8/CXCR1/CXCR2 axis mediates formation of tumor
N.-H. Lee et al. / Pancreatology 18 (2018) 705e716708
sphere which serves as an indicator of self-renewal, a characteristic
of CSCs, partially via EGFR [96]. ECs in the brain interact with
glioblastoma CSCs, and promote angiogenesis, metastasis and
proliferation through CXCL8 signaling [97]. In a study by Chen et al.,
by binding to CXCR1, CXCL8 increases numbers of subpopulation in
tumor spheres formed from CD24
þ
CD44
þ
in PC Capan-1 cells [95].
CXCL9
The reported roles of CXCL9 in cancer have been contradictory.
In some types of cancer, including breast tumors, hepatocellular
carcinoma, lung cancer, lymphoma and melanoma, CXCL9 was
shown to promote tumorigenesis, enhance metastasis and reduce
patient survival [98e101]. On the other hand, CXCL9 also resulted
in reduction of cancer progression by stimulating anti-tumor im-
munity [101e104 ]. Since CXCL9 has displayed both tumor pro-
moting and suppressing effects, its role in PC is uncertain. In a
research by Bronger et al., interferon (IFN)-
g
-induced CXCL9 exerts
tumor-suppressing function by recruiting tumor-infiltrating lym-
phocytes after exposing a COX inhibitor in ovarian cancer [105]. Hu
et al. discovered that doxorubicin alone or plus IFN-12 induced
elevation of CXCL9 [106]. This led to chemotaxis of tumor infil-
trating T cells into melanoma TME and caused increased apoptosis
in tumor cells. In another study, IFN-
g
-induced CXCL9 also caused
an anti-cancer response in lung TME in which neutralization of the
chemokine led to a reduced frequency of CXCR3
þ
T cells and DCs at
the tumor site [28].
A study by Thakur et al. showed a surge of CXCL9 expression in
cancer cells after treatment with antibodies against CD3 and HER2
plus Th1 cytokines, and a consequent significant reduction in dif-
ferentiation of MDSCs and their immunosuppressive function in PC
cells [107]. In another study, IFN-
g
, which was secreted by DCs,
exerted an anti-angiogenetic effect through induction of CXCL9
[23], after inducing chemotaxis of CD4
þ
T lymphocytes. As it has
both tumor promoting and anti-cancer effects in different cancer
types, its role in development and treatment in PC needs further
investigation.
CXCL10
CXCL10, also known as IFN-
g
-inducible Protein 10, exerts anti-
tumor function by promoting the homing of immune cells, such
as lymphocytes, into sites where tumors are located [105,108e111 ].
On the other hand, CXCL10 also acts through its receptor, CXCR3, to
induce adhesion to laminin, angiogenesis, metastasis and growth of
cancer cells [112 e116 ]. In the study by Bronger et al., in addition to
CXCL9, CXCL10 also recruits lymphocytes into ovarian TME to exert
its tumor-eliminating effects [105]. In a research in lung cancer,
CXCL10 results in chemotaxis of CXCR3
þ
T lymphocytes and CD11c
þ
DCs into the TME to eliminate tumor cells after IFN-
g
treatment
[28]. In breast cancer, it was found that tumor infiltration of T cells
caused the subsequent death of cancer cells, which was mediated
by CXCL10 [117 ].
CXCL10 is secreted by human PSCs, contributes to pancreatic
tumor development and positively correlates with the presence of
intratumoral CXCR3
þ
regulatory T cells [118]. Despite having a tu-
mor promoting role, in an experimental model involving ex vivo
samples from 48 patients with resectable PC, CXCL10 showed little
or no enhancement of PC cell proliferation and migration [20].
However, expression of the mRNAs for CXCL10 and its receptor,
CXCR3, was found to be high in patients with PC. In a study with
resected PC specimens and supernatants from co-cultures of PC cell
lines and primary tumor-associated PSCs, elevated CXCL10 con-
centrations were correlated with increased chemotaxis of CD4
þ
and CD8
þ
T cells, leading to immunosuppression and subsequent
reduced tumor cell viability, and higher patient survival [24].
Furthermore, binding of CXCL10 to CXCR3 resulted in resistance to
gemcitabine. Finally, the elevated level of CXCL10 also led to down-
regulated differentiation and activation of MDSCs, which further
enhanced its effect on immunosuppression and chemoresistance in
a 3-demensional co-culture model of peripheral blood mono-
nuclear cells with PC cells [107].
CXCL11
CXCL11, also known as IFN-Inducible Protein 9, is another ELR
ligand with both cancer promoting and anti-tumor functions. In
basal cell carcinoma, colorectal carcinoma, ovarian, prostate and
renal cancers [119e122], this ligand contributes to angiogenesis,
invasion, migration and tumor growth by binding to CXCR7. The
associated pro-tumoral behaviors are shared by another tumor-
promoting, ELR
member of the CXCL chemokine family, CXCL12
[123]. On the other hand, in breast and colon cancers CXCL11 causes
chemotaxis of CXCR3
þ
cytotoxic T lymphocytes into the tumor
stroma and reduced angiogenesis by binding to CXCR3 on cancer
cells [124]. Recently, Huang et al., discovered that in melanoma,
CXCL11 significantly induced infiltration of CD3
þ
T-lymphocytes
into TME under influences of TNF-
a
and NF-
k
B[125], which was
reversed by VEGF and rescued by addition of a chemotherapeutic
reagent, sunitinib. In prostate cancer, its expression is lower in
tumors than in normal prostate epithelial cells [126]. In PC,
although both CXCL11 and CXCL12 bind to CXCR4 and CXCR7, only
CXCL11 drives proliferation of PC cells [127].
CXCL12
CXCL12 is found in a wide variety of cancer types, including
brain, breast, colon, gastric, lung, pancreatic, prostatic and thyroid
cancers [128e135]. CXCL12 binds to CXCR4 and CXCR7 to promote
cancer progression [129,130 ]. CXCL12 activates a number of intra-
cellular signaling molecules, including Akt, EGFR, ERK1/2, G
a
i,
mTOR, NF-
k
B and Src [129,136e138], to promote chemoresistance,
chemotaxis of CAFs, invasion, migration and proliferation as well as
CSC phenotypes [134,138e14 2]. By blocking CXCL12 with chemical
inhibitors in a murine model, Zboralski et al. found that there was
an enhanced infiltration of T lymphocytes and natural killer cells
and an increased activation of the T lymphocytes in the TME [143].
In ovarian cancer, targeting CXCR4 reduced the infiltration of
numbers of ECs, myeloid cells and plasmacytoid DCs [144].
In PC, CXCL12 facilitates proliferation, invasion, migration, che-
moresistance and angiogenesis [93,130,137,145]. In a KPC murine
model, chemotaxis of Schwann cells toward pancreatic TME is
mediated by the CXCL12/CXCR4/CXCR7 [14 6]. In another study
using KPC mice, Feig et al. discovered that CXCL12 was secreted by
FAP
þ
CAFs and led to significantly reduced cytotoxic T cell infil-
tration in the PC TME and reduced anti-tumor immunity [147]. This
T cell exclusion was mediated by CXCL12/CXCR4. As observed in
clinical observations, FAP
þ
stromal cells appear in almost all PC
tumors in patients [148], this discovery has a clinical implication
that CXCL12/CXCR4 axis and the FAP
þ
cells can be potential for PC
treatment and prevention.
CXCL13
CXCL13, originally termed B-Cell-Attracting Chemokine, is
expressed by stromal cells within B-cell follicles in secondary
lymphoid organs [149]. Currently, the CXCL13/CXCR5 axis has been
reported to contribute to cancer progression in breast, gastric, lung,
pancreatic and prostate cancers [150 e154]. However, like CXCL9
and CXCL10, it also exerts tumor-suppressing effects in other cases
N.-H. Lee et al. / Pancreatology 18 (2018) 705e716 709
[155 ,156]. In breast cancer, expression of CXCL13 in tumor cells
attracted CXCR5
þ
T regulatory and Th2 cells into the TME which
contributed to upregulating EMT and lymph node metastasis [157 ].
The CXCL13/CXCR5 axis was activated by NF-
k
B and nuclear factor
(erythroid-derived 2)-like 2, which was associated with increased
infiltrating T lymphocytes of IFN-
g
þ
/IL-10
þ
, and reductions of
proliferation and metastasis of ovarian CSCs. In androgen-deprived
prostate tumors, B lymphocytes are recruited into TME by secretion
of CXCL13 from tumor-associated myofibroblasts via activation of
hypoxia-inducible factor 1/TGF-
b
pathway [158].
In a study by Lee et al., expression of CXCL13 at both the mRNA
and protein level was elevated in the pancreas of KrasG12D mice
and in patients with advanced PC [15 9,160 ]. This significant in-
crease in CXCL13 resulted in infiltration of B cells, which was found
to contribute to tumorigenesis and cell mobility [161]. CXCL13 is
also secreted by the fibro-inflammatory stroma in PanIN lesions in
mouse and human [162 ]. In addition, the high expression level of
CXCL13 in PC is mediated by a non-canonical NF-
k
B/IKK
a
pathway
[163 ].
Current findings of the relationship between CXCLs and
cancer stem cell characteristics
There is increasing evidence showing that the signals from the
tumor stroma regulate and promote CSC activity. Several studies in
breast, colon and ovarian cancers have demonstrated that silencing
tumor-promoting CXCLs significantly reduced CSC properties
leading to markedly improved treatments [164e166]. In a study of
ovarian cancer, CXCL8 was induced after treating mesenchymal
stem cells with tumor-conditioned media and implied to regulate
expression of genes involved in cell mobility and CSC phenotypes
(ALDH1 and CD133) [164]. In colorectal cancer, mesenchymal stem
cells (MSCs) were found to secrete CXCL1 and CXCL8, in which the
former induced cell invasion and tumor initiation, which are two
important features of CSCs [165,167]. In a research by Liu et al., co-
culture of breast cancer and MSCs stimulated activity of ALDH1 by
CXCL1 and CXCLs 5e8, and CXCL7 seems to play an important role
in interaction between MSCs and breast CSCs after stimulating the
co-cultured cells with IL-6 [166 ].
As mentioned previously, CXCL5 contributes to expression of
several CSC genes and chemoresistance in PC [61]. In the same
cancer type, CXCL8 promotes expression of CSC genes involved in
formation of tumor spheres, CD24 and CD44, by binding to CXCR1
[95]. Finally, by binding to CXCR4, CXCL12 induces CD133 expres-
sion, which subsequently results in resistance to gemcitabine and
increased metastatic capability [142]. However, even with current
knowledge of the relationship between CXCL members and CSC
functions in cancer, the exact molecular mechanisms which link the
TME and pancreatic CSCs remain poorly understood.
Roles of CXCL family members in chemoresistance
According to recent research, particularly in breast cancer,
several members of the CXCL family, including CXCL1, CXCL2, CXCL8
and CXCL12, have been shown to be involved in resistance to cancer
chemotherapies [32,168 ,169]. Based on their highly up-regulated
expression, the CXCLs were shown to promote angiogenesis, cell
survival, chemoresistance, metastasis and proliferation after
treatment with chemotherapeutic reagents [32,17 0 ,171 ]. As part of
their intracellular signaling, molecular markers such as MEK/ERK,
NF-
k
B and STAT1, were found to be involved in their gene tran-
scription and downstream effects [32,172 ,173 ]. Similar events were
observed in acute myeloid leukemia, colorectal carcinoma, gastric
cancer, melanoma, renal cell carcinoma and small cell lung cancer
[168 ,174e178].
Most work done on chemoresistance in PC has been based on
the study of gemcitabine, and the current understanding of other
chemotherapeutic drugs is still preliminary [179 ]. Both PC stroma
and secretions from PSCs contributed to the limitation of treatment
efficiency via several intracellular signaling pathways, such as HIF-
1
a
, NF-
k
B and miRNAs (Fig. 4)[17 9 ,180]. In a study by Delitto et al.,
CXCL10 was shown to induce resistance to gemcitabine with
reduced patient survival and increased cancer cell survival [24]. In
another study, CXCL12, secreted by stromal cells, resulted in che-
moresistance and desmoplasia as well as elevated cell survival,
proliferation and metastasis [181 ]. How CXCLs contribute to resis-
tance to chemotherapy in patients with PC is unknown.
Roles of CXCRs in CSC phenotypes and chemoresistance
As the receptors for CXCLs, several members of CXCRs have been
found to mediate expression of CSC phenotypes in distinct cancer
types. Chemoresistance is a dominant limitation to complete
eliminations of tumors and TME represents a pivotal contributor
[182 ]. In recent years, investigation of CXCRs in the field of che-
moresistance shone some light in better cancer treatment.
CXCR1
CXCR1, originally known as IL-8 receptor A, binds CXCL6 and
CXCR8 [14], induces and/or maintains CSC populations and phe-
notypes in breast and thyroid cancers and genes such as ALDH1,
CD24, CD44 and CD133 are downstream to the receptor [182e184].
CXCR1 acts through a range of intracellular signaling molecules,
including FAK/Akt/FOXO3A. In PC, CXCR1 also leads to expression of
stem-cell-like characteristics such as sphere formation and
metastasis [95].
In colon cancer, bevacizumab resistance and VEGF-independent
angiogenesis are mediated by CXCR1 [185]. Inhibition of CXCR1
with an antibody against the receptor protein, reversed temozo-
lomide resistance in melanoma cells leading to a significant
reduction of sphere-population [186]. CXCR1 knockdown led to
improved sensitivity to cisplatin and reduced Akt phosphorylation
even in the presence of CXCL8 in osteosarcoma cells [187 ].
Fig. 4. Binding of CXCLs to CXCRs leads to interactions with TME cellular compo-
nents. Such interaction may lead to chemoresistance, metastasis and proliferation in
PC as well as expression of cancer stem cell (CSC) phenotypes; however, the exact
mechanisms remain to be investigated. Moreover, although different intracellular
pathways contribute to the pro-tumor behaviors, their association with CSCs also
deserves a thorough study.
N.-H. Lee et al. / Pancreatology 18 (2018) 705e716710
CXCR2
CXCR2, also known as IL-8 receptor B, binds CXCLs 1 to 7 in
addition to CXCL8 [17]. The receptor couples with Gi (a small G
protein) and recruits Ly6G
þ
neutrophils, which are the predomi-
nant CXCR2
þ
cells among blood leukocytes, and ECs in cancers
[188 ]. In a range of cancer types, CXCR2 is shown to induce several
CSC markers [97,189] including ALDH1, Oct-4 and Sox-2, metastasis
and proliferation. In cancer cells, CXCR2 acts via distinct intracel-
lular signaling cascades including STAT3, to exert its functions
[190 ].
Blocking the CXCL7/CXCR2 axis diminished resistance to 5-
fluouracil, suggesting that CXCR2 is a potential target of reversing
therapy resistance [191]. CXCR2 was shown to mediate resistance
to taxol in breast cancer by downregulating Akt1 and activating
COX-2 as well as promoting tumor growth and metastasis [192 ]. In
colorectal cancer, although CXCL8 activates both CXCR1 and CXCR2,
only the later mediated chemoresistance in HCT116 cell line [19 3].
CXCR2 has been shown to mediate resistance to FOLFIRINOX in PC
[194 ], indicating an important role of CXCR2 in chemoresistance in
PC treatment.
CXCR3
CXCR3, binding to CXCL4 and CXCLs 9 to 11 [195], is found to
have three different isoforms, A, B and alternative [196 ]. In hepa-
tocellular carcinoma, CXCR3A promotes metastatic ability of
CD133
þ
cells via ERK1/2-MMP2/MMP9 signaling pathway after
binding to CXCL9 [197]. In breast cancer, CXCR3B functions to in-
crease tumor sphere formation and ALDH1 activity [198]. However,
it is also found that CXCR3B displays anti-tumor function [199]. In
addition, the CXCR3 ligands, namely CXCL9 and CXCL10, also play
anti-cancer roles in a number of cancers [27,101], including PC
[23,101,10 7].
CXCR3 is also involved in chemoresistance. In PC, the CXCL10/
CXCR3 axis is activated in gemcitabine resistant cells and patient
samples [200]. CXCL10 secreted by gemcitabine-resistant cells
activated CXCR3, stimulating tumor growth and resistance to the
chemotherapeutic drug [201].
CXCR4
CXCR4, binding to CXCL12 ligand, is an important molecular
factor in TME for tumor progression [202]. In a variety of types of
tumors, including brain, breast, esophageal, gastric and ovarian,
CXCR4 has been shown to be responsible for aggressiveness of CSCs
and expression of CSC phenotypes (e.g. ALDH1 activity, colony
formation and tumorigenicity) by signaling through distinct
intracellular pathways, such as ERK1/2 [203e207]. PC cells with
active CXCL12/CXCR4 axis displayed high metastatic, proliferative
and tumorigenic potentials as well as activating sonic hedgehog
and NF-
k
B[138,142 ,208].
In addition to its effects on CSC gene expression, CXCR4 also
influence chemoresistance in cancer. In ovarian cancer, CXCR4
overexpression resulted in resistance to cisplatin in 124 patients
and expression of several CSC markers (ALDH1, ALDH2, MyD88, and
LIN28) [207,209]. The interaction between CXCR4 and CXCL12 in
the lung TME caused a protective effect against etoposide-induced
apoptosis and adhesion of tumor cells to fibronectin, collagen, and
stromal cells [175 ]. Blockage of CXCR4 significantly reversed resis-
tance to dacarbazine in the CXCR4
þ
/CD133
þ
subpopulation in
melanoma [210]. In a study by Xiao et al., tumorigenesis and
resistance to gemcitabine are caused by downregulation of an
miRNA, Let -7a, by CXCR4 in BxPC-3 and Panc-1 PC cell lines in vitro
and in vivo [211]. Moreover, it is found that the CXCL12/CXCR4 axis
activated several intracellular signaling cascades, including FAK,
ERK and Akt, enhanced transcriptional activities of
b
-catenin and
NF-
k
B, and expression of survival proteins in the presence of
gemcitabine [139]. In addition, CXCL12/CXCR4 induces chemotaxis
of CAFs or mesenchymal stem cells to further support gemcitabine
resistance by activating Akt, MEK and ERK [212].
CXCR7
Another CXCL12 receptor, CXCR7 also plays important roles in
cancer. Silencing CXCR7 has displayed downregulation of expres-
sion of a number of CSC genes, such as ALDH1 and CD44, in breast
cancer [213]. In prostate cancer, CXCR7 altered CD44 expression as
well as CXCL8 and VEGF in order to contribute to angiogenesis and
metastasis [214]. By acting through ERK, CXCR7 induces differen-
tiation of CSCs in hepatocellular carcinoma [215]. As for PC, binding
of CXCL12 activates ERL1/2 via CXCR7 [127].
CXCR7
þ
population in SW480 colorectal cancer cell line dis-
played a higher level of resistance to 5-fluouracil than the CXCR4
þ
cells [216]. In lung cancer, CXCR7 is upregulated by TGF-
b
and
responsible for induction of tumor sphere formation, stem-like
properties, chemoresistance and tumorigenesis in vivo [217]. The
increased CXCR7 expression also correlates with CD44 expression
and metastasis. In bone marrow microenvironment, CXCL12/CXCR7
pathway mediates imatinib resistance via ERK activation [218].
From the above discussion, the CXCRs members, namely, CXCR2,
CXCR4 and CXCR7, seem to display great importance in chemo-
therapeutic resistance in distinct cancer types. As mentioned
above, several CXCLs have shown to mediate chemoresistance as
well as expression of CSC phenotypes (please see the “Roles of
CXCLs in pancreatic cancer”section above). CSCs are also capable of
interacting with the surrounding TME [54]. A specific combination
of CXCL and CXCR appears to be an important link between CSCs,
TME and tumor progression. Such CXCL/CXCR axis may serve as a
prognostic marker for treatment responses.
Targeting CXCLs in PC
Current chemotherapeutic reagents have only modest effects in
extending patient survival and restricting disease progression in
PC. Although various combinations of chemotherapies, such as
FOLFIRINOX (a mixture of leucovorin, folinic acid, 5-fluouracil, iri-
notecan and oxaliplatin), or gemcitabine and nab-paclitaxel,
improve overall successive eliminations of PC, chemoresistance
still limits effectiveness of these drugs [219]. As many CXCLs have
been shown to mediate chemoresistance and interaction between
tumors and stroma, they potentially serve as therapeutic targets in
PC.
CXCR2, also known as IL-8 Receptor
b
, was originally found to
bind IL-8 (CXCL8) [14]. CXCR2 also binds CXCL1, CXCL2, CXCL3 and
CXCL5 to CXCL8, and is found in a wide range of cancers, including
PC [14,220]. The receptor couples with a small G protein on the
cytoplasmic side of the cell membrane, Gi, and functions to mediate
proliferation, angiogenesis, tumorigenesis and metastasis, as well
as inducing chemotaxis of neutrophils that carry a specific surface
antigen, Ly6G [188,220]. In PC, CXCR2 is highly expressed in patient
samples and cell lines and promotes angiogenesis by binding ELR
þ
CXCLs [74,221].
Although CXCR2 appears to be involved in PC progression, the
pancreatic stroma and intracellular signaling pathways (e.g. NF-
k
B)
must also be taken into account as CXCR2 is expressed in both
tumor and stromal cells [220]. For example, among all tumor pro-
moting CXCLs expressed in PC, CXCL5 is not only co-expressed with
CXCR2 in human patients but also in KPC mice [41]. In the mouse
model, the CXCL5/CXCR2 axis mediates chemotaxis of
N.-H. Lee et al. / Pancreatology 18 (2018) 705e716 711
CD11b
þ
Ly6G
þ
neutrophils to promote proliferation of cancer cells
and suppress T cell activation [41 ]. In human PC, CXCL5/CXCR2
induces chemoresistance and poor patient survival as well as other
pro-tumoral functions [41,61,64].
In cancer, expression of CXCLs depends on several intracellular
pathways, including AP-1, NF-
k
B and STAT1, via binding sites in the
CXCL promotor regions [64,222e225]. Together with its ligands,
CXCR2 mediates pro-tumoral characteristics of cancer, including
chemoresistance [21,32,58,59,91,226] and expression of CSC phe-
notypes [96,97,227,228]. As a G protein-coupled receptor, there are
number of downstream proteins that mediate the effects of a
specific CXCL-CXCR2 combination, including G
a
i, Akt, ERK1/2 and
NF-
k
B[59,88,18 8]. In a mouse PC model, NF-
k
B activation led to
increased activity of CXCR2 in the presence of KrasG12D mutation,
resulting in elevated tumor cell proliferation and reduced survival
[42]. As NF-
k
B acts both up- and down-stream to CXCR2, it may
form a positive feedback loop with a CXCL-CXCR2 axis which
continuously supports and promotes tumor malignancy.
Conclusion
PC communicates with cellular components within TME by
expressing chemokines and their receptors. The CXCL ligand family
is being extensively researched in cancer and other diseases. CXCL8
and CXCL12 are now the two most well-understood CXCL family
members in terms of their function, signaling pathways, and in-
teractions with TME cellular components and CSCs. However, as
each cancer has a unique genomic makeup and mutations, the
exact secretome varies and other chemokine may play specific roles
in controlling PC progression. CSCs also interact with the TME and
mediate chemoresistance [229]. As only a specific range of CXCLs
are secreted by a distinct cancer type, targeting a unique CXCL and
its receptor may provide an opportunity to improve treatment
outcomes [14,15,195]. Therefore, further investigation of the effects
of CXCLs on CSCs and their roles in the chemoresistance of PC is
necessary.
Conflict of interest
All the authors declare that there is no conflict of any kind
regarding the content of the manuscript.
Acknowledgements
The authors would like to acknowledge Pancare Foundation
(www.pancare.org.au) for supporting the pancreatic cancer
research program in the Department of Surgery.
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