Prolactin-Induced Protein Is Required for Cell Cycle Progression in Breast Cancer

Abstract

Prolactin-induced protein (PIP) is expressed in the majority of breast cancers and is used for the diagnostic evaluation of this disease as a characteristic biomarker; however, the molecular mechanisms of PIP function in breast cancer have remained largely unknown. In this study, we carried out a comprehensive investigation of PIP function using PIP silencing in a broad group of breast cancer cell lines, analysis of expression microarray data, proteomic analysis using mass spectrometry, and biomarker studies on breast tumors. We demonstrated that PIP is required for the progression through G1 phase, mitosis, and cytokinesis in luminal A, luminal B, and molecular apocrine breast cancer cells. In addition, PIP expression is associated with a transcriptional signature enriched with cell cycle genes and regulates key genes in this process including cyclin D1, cyclin B1, BUB1, and forkhead box M1 (FOXM1). It is notable that defects in mitotic transition and cytokinesis following PIP silencing are accompanied by an increase in aneuploidy of breast cancer cells. Importantly, we have identified novel PIP-binding partners in breast cancer and shown that PIP binds to β-tubulin and is necessary for microtubule polymerization. Furthermore, PIP interacts with actin-binding proteins including Arp2/3 and is needed for inside-out activation of integrin-β1 mediated through talin. This study suggests that PIP is required for cell cycle progression in breast cancer and provides a rationale for exploring PIP inhibition as a therapeutic approach in breast cancer that can potentially target microtubule polymerization.

Full text

Prolactin-Induced Protein Is
Required for Cell Cycle
Progression in Breast Cancer
1,2
Ali Naderi and Marion Vanneste
Holden Comprehensive Cancer Center, Medical Education
and Research Facility, University of Iowa, Iowa City, IA, USA
Abstract
Prolactin-induced protein (PIP) is expressed in the majority of breast cancers and is used for the diagnostic evaluation
of this disease as a characteristic biomarker; however, the molecular mechanisms of PIP function in breast cancer
have remained largely unknown. In this study, we carried out a comprehensive investigation of PIP function using
PIP silencing in a broad group of breast cancer cell lines, analysis of expression microarray data, proteomic analysis
using mass spectrometry, and biomarker studies on breast tumors. We demonstrated that PIP is required for the
progression through G
1
phase, mitosis, and cytokinesis in luminal A, luminal B, and molecular apocrine breast cancer
cells. In addition, PIP expression is associated with a transcriptional signature enriched with cell cycle genes and
regulates key genes in this process including cyclin D1, cyclin B1, BUB1, and forkhead box M1 (FOXM1). It is notable
that defects in mitotic transition and cytokinesis following PIP silencing are accompanied by an increase in
aneuploidy of breast cancer cells. Importantly, we have identified novel PIP-binding partners in breast cancer and
shown that PIP binds to β-tubulin and is necessary for microtubule polymerization. Furthermore, PIP interacts with
actin-binding proteins including Arp2/3 and is needed for inside-out activation of integrin-β1mediatedthroughtalin.
This study suggests that PIP is required for cell cycle progression in breast cancer and provides a rationale for exploring
PIP inhibition as a therapeutic approach in breast cancer that can potentially target microtubule polymerization.
Neoplasia (2014) 16, 329–342.e14
Introduction
Prolactin-induced protein (PIP) is widely expressed in breast cancer and
has been used as a characteristic biomarker for the diagnostic evaluation of
this disease [1]. Genomic studies have revealed that PIP is highly
expressed in luminal A and molecular apocrine subtypes of breast cancer
[2–4]. Molecular apocrine is a subtype of estrogen receptor (ER)–negative
breast cancer that is characterized by the overexpression of steroid
response genes such as androgen receptor (AR) and forkhead box A1
(FOXA1) [3,5,6]. Notably, a recent study has shown that PIP is one of the
best biomarkers for the immunohistochemica l identifi cation of molecular
apocrine tumors [7]. It is known that PIP expression is regulated by
prolactin and androgen hormones [8]. In particular, AR engages in a
transcriptional cooperation with prolactin-activated Stat5 and Runx2 to
regulate PIP expression [8,9]. In addition, we have demonstrated that PIP
is a cAMP responsive element binding protein 1 (CREB1)targetgenethat
is induced by a positive feedback loop between AR and extracellular
signal-regulated kinase (ERK) [10].
There is limited knowledge regarding the molecular function and
binding partners of PIP in breast cancer. The available data indicate
that PIP is a secreted protein with aspartyl protease activity that can
degrade fibronectin and has the ability to bind and modulate CD4
receptor in T lymphocytes [11,12]. In addition, early studies using
actin-sepharose columns have shown possible binding of PIP to actin in
seminal fluid [13]; however, this finding has not been validated using
more modern proteomic methods. Furthermore, the importance of PIP
in cell proliferation has been demonstrated by the fact that purified PIP
promotes growth of breast cancer cells and PIP expression is necessary
for the proliferation of T-47D and MDA-MB-453 cell lines [9,10,14].
www.neoplasia.com
Volume 16 Number 4 April 2014 pp. 329–342.e14 329
Address all correspondence to: Ali Naderi, MD, Holden Comprehensive Cancer
Center, 3202 Medical Education and Research Facility, University of Iowa, 375
Newton Road, Iowa City, IA 52242. E-mail: ali-naderi@uiowa.edu
1
Research reported in this publication was supported by the Holden Comprehensive
Cancer Center at the University of Iowa and the National Cancer Institute of the
National Institutes of Health under Award No. P30CA086862. Authors have no
conflict of interests to disclose.
2
This article refers to supplementary materials, which are designated by Tables W1 to
W9 and Figures W1 and W2 and are available online at www.neoplasia.com.
Received 16 January 2014; Revised 6 March 2014; Accepted 24 March 2014
Copyright © 2014 Neoplasia Press, Inc. All rights reserved 1476-5586/14
http://dx.doi.org/10.1016/j.neo.2014.04.001

Moreover, we have recently demonstrated that PIP mediates invasion
of breast cancer cells in a process that partially depends on the
degradation of fibronectin by this protein [10].
It is notable that the extracellular effects of PIP on fibronectin
degradation are necessary for the outside-in activation of integrin-β1,
which, in addition to the regulation of invasion, has a role in promoting
cell proliferation [10]. Furthermore, it has been shown that coculture of
PIP-silenced and naive T-47D cells does not reverse the growth
inhibition induced by PIP silencing, which suggests a potential
intracellular function for this protein [4]. Despite these findings, the
underlying molecular mechanisms of PIP function in cell proliferation
have remained largely unknown and require further studies.
In this study, we investigated PIP function in breast cancer using
small interfering RNA (siRNA) silencing in a broad group of breast
cancer cell lines, analysis of expression microarray data, proteomic
analysis by mass spectrometry (MS), and biomarker studies on primary
breast tumors. We demonstrated that PIP is required for the progression
through different phases of cell cycle and identified key molecular
mechanisms and binding partners for this protein in breast cancer.
Materials and Methods
Cell Culture
Breast cancer cell lines MCF-7, T-47D, BT-474, HCC-202,
HCC-1954, MDA-MB-453, SK-BR-3, MFM-223, and MDA-MB-
231 were obtained from American Type Culture Collection
(Manassas, VA) and cultured as recommended by the provider.
RNA Interference
PIP knockdown (KD) by siRNA silencing was performed as
described before [15]. The following two siRNA-duplex oligos
(Sigma-Aldrich, St Louis, MO) were applied: duplex 1—sense, 5′
CUCUACAAGGUGCAUUUAA and antisense, 5′UUAAAUG -
CACCUUGUAGAG; and duplex 2—sense, 5′CCUCUACAAG-
GUGCAUUUA and antisense, 5′ UAAAUGCACCUUGUAGAGG.
Transfections with siRNA Universal Negative Control No. 1 (Sigma-
Aldrich) were used as controls. The effect of KD was assessed
72 hours after transfections. The average changes obtained for two
duplexes are pres ented in manuscript.
Quantitative Real-Time Reverse
Transcription–Polymerase Chain Reaction
Quantitative real-time reverse transcription–polymerase chain reaction
(qRT-PCR) to assess the expression levels of PIP (assay ID:
Hs00160082_m1 ), cyclin D1 (Hs00765553 _m1), cyclin E1
(Hs0102653 6_m1) , cyclin B1 (Hs01565448_g1), forkhead box M1
(FOXM1) (Hs01073586_m1), TTK (Hs01009870_m1), BUB1
(Hs01557695_m1), and cell division cycle 20 (CDC20)
(Hs00426680_ mH) was carried out using TaqMan Gene Expression
Assays (Applied Biosystems, Grand Island, NY). Housekeeping gene
ribosomal protein, large, P0 (RPLP0) was used as a control. Relative gene
expression = gene expression in the KD group / average gene expression in
the control group.
Western Blot Analysis
Rabbit monoclonal PIP antibody (Novus Biologicals, Littleton,
CO); rabbit antibodies for ERK1/2, phospho-ERK1/2 (Thr
202
/
Tyr
204
), c-Jun, phospho–c-Jun (Se r
63
), Stat3, phospho-Stat3 (Try
705
),
Cdc2, phospho-Cdc2 (Tyr
15
), focal adhesion kinase (FAK), and Talin-1
(Cell Signaling Technology, Danvers, MA); rabbit polyclonal integrin- β1
and mouse monoclonal Arp2/3 antibo dies (Millipore, Billerica, MA);
rabbit monoclonal phospho-FAK (Tyr
397
) antibody (Life Technologies,
Grand Island, NY); and mouse monoclonal β-tubulin antibody (Sigma-
Aldrich) were applied at 1:1000 dilutions using 20 μgofeachcelllysate.
Rabbit α-tubulin antibody (Abcam, Cambridge, United Kingdom) was
applied to assess loading. To extract protein from media, cell lines were
cultured for 48 hours in serum-free media, followed by concentration
using Amicon Ultra-15 (3 K) centrifugal filters (Millipore). A total of
100 μg from each concentrated sample was precipitated and used for
immunoblot analysis.
Cell Proliferation Assay
Cell proliferation assays were performed using Vybrant MTT
Proliferation Assay Kit (Life Technologies) in eight replicates as
previously published [10].
Cell Cycle Analysis
Cell cycle analysis with propidium iodide was performed as
described before [16]. Data analysis was carried using ModFit LT
software (Verity Software House, Topsham, ME).
Immunohistochemistry
Three sets of breast cancer tissue microarray (TMA) slides that are
constituted of duplicate cores for a total of 210 malignant breast
tumors (BRC1501-3) were obtained from Pantomics (Richmond,
CA). Immunohistochemist ry (IHC) staining was performed as
described before [17]. Staining was carried out with rabbit PIP
antibody at 1:100 dilution and mouse monoclonal antibodies (Dako,
Carpinteria, CA) for AR (1:75 dilution), Ki-67 (1:100 dilution), and
cytokeratin 5/6 (1:100 dilution). PIP score was defined as the
percentage of PIP-positive cells (0-100) multiplied by the intensity of
PIP cytoplasmic staining (1-3).
Immunofluorescence
Immunofluorescence (IF) staining was performed as previously
described [15,17], with mouse monoclonal β-actin and α-tubulin
antibodies (Abcam) at 1:200 dilution, mitotic protein monoclonal 2
(MPM-2; Abcam) at 1:500 dilution, and rabbit polyclonal
pericentrin antibody (Abcam) at 1:1000. Alexa 488 anti-mouse and
Alexa 594 anti-rabbit (Life Technologies) were used as secondary
antibodies. Quantification of pericentrin/nuclear ratio was perform ed
on 100 nuclei, and experiments were carried out in duplicates.
Percentage of multinucleated cells and percentage of cells stained with
MPM-2 were assessed in PIP-KD and control experiments on 200
cells, and each experiment was carried out in four replicates.
Immunoprecipitation
Immunoprecipitation (IP) of integrin-β1 using CHAPS buffer was
performed as previously published [10]. To perform PIP-IP, T-47D cells
were grown in 10-cm dishes to 60% confluency in full media and further
cultured in serum-free media containing 100 nM dihydrotestosterone,
(Thermo Fisher Scientific, Waltham, MA) for 48 hours. Conditioned
mediawerethenconcentratedwithAmiconUltra-15filters,andvolume
was adjusted to 500 μl with a nondenaturing lysis buffer containing
20 mM Tris-HCl (pH 8), 137 mM NaCl, 10% glycerol, 2 mM
EDTA, and 1% NP-40. Cell lysate from each dish was extracted using
500 μl of lysis buffer. Subsequently, the extracted medium and lysate for
each sample were mixed and subjected to IP as described [10].PIP-IP
was carried out using 2 μg of rabbit monoclonal PIP antibody, and
control experiments followed the same process without a PIP antibody.
330 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

Proteomics
Following PIP-IP, protein bands were separated using sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and
Coomassie R-250 staining (Bio-Rad Laboratories, Hercules, CA).
Control experiments included pulldown with Protein A-Sepharose
beads (Life Technologies) without a PIP antibody. Processing of gels for
MS and database search were carried out by the Proteomics Facility at
the University of Iowa (Iowa City, IA). In summary, each PIP-IP and
Figure 1. PIP silencing in breast cancer cell lines and the effect of PIP expression on cell proliferation. (A and B) qRT-PCR demonstrates
PIP-KD efficiencies with siRNA-duplex 1 (D1) and siRNA-duplex 2 (D2). PIP expression is relative to nontargeting siRNA (CTL).
(C) Immunoblot analysis shows PIP protein following PIP-KD using cell extracts or (D) conditioned media. Fold changes (RR) in band
density were measured relative to the control and represent the average change for two siRNA duplexes. (E and F) MTT assay measures
cell proliferation following PIP-KD. The asterisk (*) is P value for each PIP-KD versus control (CTL).
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 331

control IP lane was sliced to 16 separate bands and analyzed by MS. In-
gel digestion and sample processing were carried out as described before
[18]. Database searching was performed by Mascot search engine
(Matrix Science, Boston, MA), Spectrum Mill MS Prote omics
Workbench (Agilent Technologies, Santa Clara, CA), and X! Tandem
[The Global Proteome Machine Organization (The GPM), thegpm.
org; version CYCLONE (2010.12.01.1)]. Scaffold (Proteome Software
Inc, Portland, OR) was used to validate MS/MS-based peptide and
protein identifications. Protein identifications were accepted at greater
than 99.0% probability with at least four identified peptides [19].Two
replicates of PIP-IP and control experiments were analyzed by MS, and
only hits that were present in both PIP-IP replicates and absent in the
controls were accepted for further analysis.
Tubulin Polymerization Assay
Quantitation of polymerized and soluble tubulin was carried out as
described before [20]. Immunoblot analysis was performed using
mouse monoclonal β-tubulin antibody and the band intensities of
polymerized and soluble β-tubulin in each PIP-KD experiment were
normalized to that of control siRNA.
Bioinformatics and Statistical Analysis
Analysis of Gene Expression Data. Gene expression for 52 breast
cancer cell lines was extracted from published microarray data by Neve et
al. [21]. PIP transcriptional signature included genes that showed Pearson
correlation coefficients (CCs) ≥ 0.5 with PIP expression (P b .001).
Pearson CC analysis, proxim ity matrix, and clustering algorithms were
performed using IBM SPSS Statistics 20 (Armonk, NY). Hierarchical
clustering of the PIP signature was carried out using centroid linkage
method, and intervals were measured by CC values. Functional
annotation of the PIP signature based on Gene Ontology was performed
using The Database for Annotation, Visualization and Integrated
Discovery (DAVID) Bioinformatics Resources (National Institute of
Allergy and Infectious Diseases, Bethesda, MD) [22,23 ].
Analysis of Proteomics Data. Functional classification of PIP-
binding partners was carried out using DAVID Bioinformatics
Resources. The following parameters were used for the analysis: κ
similarity overlap = 4, similarity threshold = 0.35, and multiple
linkage threshold = 0.50. Enrichment score was obtained for each
functional cluster. Canonical pathways associated with PIP-binding
partners were derived using Ingenuity Pathway Analysis (Ing enuity
Systems, Redwood City, CA).
Statistical Analysis. Biostatistics was carried out using IBM SPSS
Statistics 20. Student's t test and paired sample t test were applied for
calculating the statistical significance. All error bars depict ± 2 SEM.
Results
PIP Expression Is Necessary for Cell Proliferation
We first characterized PIP expression in nine breast cancer cell
lines from different molecular subtypes (Table W1). These included
luminal A lines MCF-7 and T-47D, luminal B line BT-474, ER-
negative luminal lines MDA-MB-453, HCC-202, MFM-223, and
SK-BR-3, and ER-negative basal lines HCC -1954 and MDA-MB-
231. PIP expression using qRT-PCR was high in HCC-202, T-47D,
MDA-MB-453, and HCC-1954 cells, intermediate in BT-474,
Figure 2. PIP expression in breast tumors. (A and B) PIP expression using IHC in breast tumors. Magnifications are at 10X. (C) PIP
expression scores using IHC in molecular subtypes of breast cancer is shown. *P b .01 is for ER −/AR− versus other groups. (D) PIP-IHC
scores in ER-negative tumors stratified on the basis of AR and CK5/6 status are shown. *P b .01 is for AR − tumors versus other groups.
332 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

MFM-223, and SK-BR-3 cell lines, and very low to undetectable in
MCF-7 and MDA-MB-231 cells (Table W1 and Figure W1A).
Notably, PIP-KD resulted in ≥ 80% reduction in PIP transcripts
across seven cell lines with measurable PIP (Figure 1, A and B). In
addition, PIP-KD was validated at the protein level using cell extracts
in cell lines with high PIP expression (Figure 1C). In cell lines with an
intermediate PIP expression, due to low levels of cellular PIP (Figure
W1B), conditioned media were used to measure secreted PIP levels
(Figure 1D). Impo rtantly, all seven cell lines with measurable PIP
levels showed a marked reduction of this protein following PIP-KD
by ≥ 80% (Figure 1, C and D).
We next examined the effect of PIP silencing on the proliferation of
breast cancer cells using MTT (3-[4,5-dimet hylthiazo l-2-yl]- 2,5 diphenyl
tetrazolium bromide) assay and observed a significant reduction in
cell proliferation following PIP-KD in T-47D, BT-474, MFM-223,
HCC-1954, HCC-202, and SK-BR-3 cell lines ( P b .01-.0 3; Figure 1,
E and F). These results are similar to the effect of PIP silencing on
MDA-MB-453 cells [10] and suggest that PIP expression is necessary
for cell proliferation across different molecular subtypes of breast cancer.
PIP Expression in Molecular Subtypes of Breast Cancer
We next investigated PIP expression in primary breast tumors to
identify the pattern of PIP expression in various molecular subtypes of
breast cancer and to study the association of biomarkers with PIP
expression. In this process, we carried out IHC in a TMA cohort of 210
primary breast tumors (Table W2). To classify the cohort into
established molecular subtypes, ER-positive tumors were subdivided
into luminal A and luminal B groups using ErbB2 and Ki-67 expression
patterns [24]. On the basis of this classification, luminal B was defined as
ER-positive tumors with either ErbB2 overexpression or a Ki-67 index
≥ 14% (Table W3). In addition, ER-negative tumors were subdivided on
the basis of their AR status [25], and cytokeratin 5/6 (CK5/6) staining
was employed as a marker of basal-type tumors. Furthermore, we
obtained IHC scores for PIP on the basis of the percentage and intensity
of staining for this protein in each tumor (Figure 2, A and B).
Next, we assessed the association of P IP expression with
biomarkers and molecular subtypes. We observed that AR-positive
tumors have a significantly higher PIP expression compared to AR
negatives ( P b .01; Table W3). In addition, PIP was highly expressed
in lumina l A, luminal B, and ER −/AR + molecular subtypes
(Figure 2C and Table W3). Furthermore, among ER-negative
tumors, PIP expression was associated with AR-positive status and
was unrelated to CK5/6 staining (Figure 2D). It is notable that PIP
expression was not associated with either tumor size or grade in this
cohort (P N .1). These findings suggest that PIP is widely expressed in
luminal A, luminal B, and ER −/AR + (molecular apocrine) subtypes
of breast cancer. Moreover, PIP expression is associated with AR and
is present in both luminal and basal ER-negative tumors.
PIP Transcriptional Signature Is Enriched with Cell
Cycle Genes
To investigate the functional role of PIP in breast cancer, we
carried out a nonbiased genomic approach to study the transcriptional
signature of this gene using a microarray data set of 52 breast cancer
cell lines [21]. To identify PIP coregulated genes, we first calculated
the Pearson CC for each gene expression in the data set with that of
PIP and then obtained the list of genes that had Pearson CC values
≥ 0.5 (P b .001) with PIP expression. This PIP transcriptional
signature is composed of 136 genes that had a highly coregulated
expression pattern with PIP (Tables W4 and W5 ). We next
performed hierarchical clustering analysis of PIP transcriptional
signature and observed two main gene clusters in this signature on the
basis of the direction of CC values with PIP expression (Figure 3A
and Table W6).
Figure 3. PIP transcriptional signature. (A) Hierarchical clustering analysis of PIP transcriptional signature was performed using centroid
linkage method, and intervals were measured by Pearson CCs. Functional annotations for gene clusters are demonstrated. FE, fold
enrichment. (B) Functional annotation of PIP transcriptional signature based on Gene Ontology is presented. FEs and P values are shown.
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 333

We subsequently conducted functional annotation of the PIP
signature. Notably, this signature was highly enriched with cell
cycle genes and in particular those related to mitotic transition and
spindle checkpoint [Figure 3B, fold enrichment (FE) values = 11-56;
P b .001]. These findings suggest a strong transcriptional coregulation
between PIP and cell cycle–related genes that is most pronounced in
relation to mitotic transition.
PIP Expression Is Required for G
1
-S Progression
In view of the fact that PIP transcriptional signature is highly
saturated with cell cycle genes and PIP expression is necessary for cell
proliferation in brea st cancer, we investigated the role of PIP in cell
cycle progression using flow cytometry analysis on PIP-silenced cells.
We observed that T-47D and MDA-MB-453 cells underwent a
profound G
1
arrest following PIP-KD manifested by a 10% to 20%
increase in G
0
-G
1
cell population compared to the controls with a
corresponding decrease in the percentage of cells in S phase (P b .01;
Figure 4, A and B). Furthermore, this G
1
arrest was associated with a
marked decrease in cycli n D1 and cyclin E1 expression following PIP-
KD (P b .01; Figure 4, C and D).
Moreover, we examined the effect of PIP-KD on the level of ERK
phosphorylation that is a required step for the transcriptional
activation of cyclin D1 [26]. In addition, we have previously
demonstrated a reduction in ERK phosphorylation in MDA-MB-
453 cells following PIP silencing [10]. Notably, phospho-/total ERK
was markedly reduced by 0.14-fold following PIP-KD in T-47D cells
(Figure 4E), which represents a similar pattern to that observed in
MDA-MB-453 [10]. We also examined the effect of PIP silencing on
AB
C
PI Channel-T47D (control)
Cell Number
0
0
40 80 120 160
200
800
1600
G0-G1: 62.4%
S: 21.4%
G2-M: 16.3%
PI Channel-T47D (PIP-KD)
0
40
80
120
160
200
0 1200 2400
Cell Number
G0-G1: 82.1%
S: 9.8%
G2-M: 8%
63
83
23
14
8
9
CTL KD
T-47D
0
20
40
60
80
100
G0-1
S
G2M
61
28
11
73
14
13
CTL KD
MDA-MB-453
N= 4
ΔG0-1 p< 0.01
Relative Cyclin D1 Expression
using qRT-PCR
0
0.2
0.4
0.6
0.8
1
1.2
T-47D
CTL
PIP-KD
N= 3
p< 0.01
MDA-MB-453
E
Relative Cyclin E1 Expression
using qRT-PCR
0
0.2
0.4
0.6
0.8
1
1.2
T-47D
MDA-MB-453
CTL
PIP-KD
N= 3
p< 0.01
D
α−Tubulin
Ph/T-RR:
42 KD
Ph-ERK
42 KD
T-ERK
0.14
T-47D
CTL PIP-KD
50 KD
Ph-c-Jun
50 KD
c-Jun
80 KD
Stat3
Ph-Stat3
Figure 4. The effect of PIP expression on G
1
phase. (A) Cell cycle histograms following PIP-KD in T-47D are shown. (B) The percentage of
cells in different phases of cell cycle following PIP-KD is shown. P b .01 is for ΔG
0-1
between PIP-KD and CTL groups. (C) Cyclin D1 and (D)
cyclin E1 expression using qRT-PCR for PIP-KD relative to control is shown. (E) Immunoblot analysis measures the ratio of phospho (Ph)-
ERK to total (T) ERK, Ph–c-Jun to T–c-Jun, and Ph-Stat3 to T-Stat3 following PIP-KD. Fold changes were assessed relative to control. The
average changes obtained for two duplexes are presented.
334 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

phosphorylation of c-Jun and STAT3 that are also involved in G
1
/S
transition; however, we did not find any significant changes in the
level of these proteins (Figure 4E). These data suggest that PIP
silencing results in a profound G
1
arrest in T-47D and MDA-MB-
453 cells associated with a reduction in the levels of cyclin D1 , cyclin
E1, and ERK phosphorylation.
PIP Silencing Leads to Mitotic Arrest and Aneuploidy
Cell cycle studies in MFM-223, SK-BR-3, HCC-1954, HCC-202,
and BT-474 cell lines following PIP silencing revealed that these lines
undergo a G
2
/M arrest manifested by a significant increase in G
2
/M
phase and a ma rked increase in the percentage of aneuploidy by
approximately 15% to 30% (P b .01; Figure 5, A–C). In addition,
A
B
0
0
1000
03060
90 120
3000 5000
03060
90 120
1000
2000
3000
4000
PI Channel-BT-474 (control)
PI Channel-BT-474 (PIP-KD)
Cell NumberCell Number
0
20
40
60
80
100
G0-1
S
G2M
CT
CT
CT
KD
KD
KD
MFM223 SKBR3 BT474
C
N= 4
CT KD
HCC1954
53
30
17
62
17
21
49
53
40
30
11 17
CT KD
HCC202
50
33
29
21
37
30
52 58
30
14
18
28
58 63
40
32
2
5
CTL
PIP-KD
N= 3, p< 0.01
D
Relative Cyclin B1 Expression
using qRT-PCR
0
0.2
0.4
0.6
0.8
1
1.2
B
T474
MFM223
SKBR3
HCC1954
HCC202
Relative Cyclin D1 Expression
using qRT-PCR
0
0.2
0.4
0.6
0.8
1.2
1
BT474
MFM223
SKBR3
HCC1954
HCC202
N= 3,
CTL
PIP-KD
E
ΔG2/M p< 0.01
ΔG0-1 p< 0.01
(KD-CT) Aneuploid= 32%
Δ
Percentage increase in aneuploidy
with PIP-KD
0
10
20
30
40
T-47D
(KD-CT) p< 0.01
MDA453
MFM223
SKBR3
B
T474
HCC1954
HCC202
Aneuploid-G2/M
Δ
N= 4
F
α−Tubulin
T-Cdc2
Ph-Cdc2
T-RR:
34 KD
34 KD
Ph-RR:
BT474 MFM223 HCC1954
HCC202
CT CT CT CTKD KD KD KD
1.2
1.2
1
0.9
0.33
0.27
0.42
0.48
*
*
*
*
*
p< 0.01
Figure 5. The effect of PIP expression on G
2
/M and aneuploidy. (A) The percentage of cells in different phases of cell cycle following PIP-
KD is shown. P values are for ΔG
0-1
and ΔG
2
/M between PIP -KD and control groups. (B) The change in percentage of aneuploidy between
PIP-KD and control (CT) experiments is presented. (C) Cell cycle histograms following PIP-KD in BT-474 cell line are shown. (D) Cyclin D1
expression using qRT-PCR for PIP-KD relative to control (CTL) is shown. *P b .01 is for PIP-KD versus CTL groups. (E) T- and Ph-Cdc2
protein levels by immunoblot analysis for PIP-KD relative (RR) to control are shown. (F) Cyclin B1 expression for PIP-KD as explained in D is
shown. The average changes obtained for two duplexes are presented.
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 335

there was a moderate degree of G
1
arrest following PIP-KD in four of
these cell lines (P b .01; Figure 5A). In contrast, T-47D and MDA-
MB-453 cells did not have an increase in aneuploidy following PIP
silencing (Figure 5, A and B). These data suggest that PIP expression
is required for progression through both G
1
and G
2
/M phases of the
cell cycle and the occurrence of G
2
/M arrest following PIP silencing is
accompanied by an increase in aneuploidy.
To identify a molecular basis for the observed cell cycle findings,
we assessed the effect of PIP silencing on the expression of key genes
involved in G
1
,G
2
/M checkpoint, and mitotic transition. We
observed that cyclin D1 expression, a key regulator of G
1
, was
significantly reduced in MFM-223, HCC1954, HCC202, and BT-
474 cells following PIP-KD (P b .01; Figure 5D). We next examined
the effect of PIP silencing on Cdc2 (Cdk1) and cyclin B1 as main
regulators of G
2
/M checkpoint [27].Notably,therewasa
proportionate reduction in total and phospho-Cdc2 protein levels
following PIP-KD in HCC-1954 and HCC-202 cell lines
that suggests a decrease in the expression of this protein following
PIP silencing (Figure 5E). Furthermore, cyc lin B1 expression
was markedly reduced by approxim ately 50% to 80% in all five
lines (P b .01; Figure 5F).
In view of the fact that functional annotation showed a strong
correlation between PIP and mitotic transition (Figure 3), we also
studied the effect of PIP silencing on some of the key mitotic genes in
PIP signature. In this resp ect, we examined FOXM1 , TTK, BUB1,
and CDC20, which have a critical role in mitotic transition [28–30].
Expression levels of these genes were assessed following PIP-KD in
BT-474, HCC-1954, MMF-223, SK-BR-3, HCC-202, and MDA-
MB-453 cell lines. Importantly, there was a significant reduction in
FOXM1, TTK, BUB1, and CDC20 expression following PIP-KD in
these cells that supports a functional role for PIP in the regulation of
mitotic transition (P b .01; Figure 6, A–D).
We next investigated whether the observed G
2
/M accumulation
following PIP silencing is a result of an arrest in G
2
or M phase of the
cell cycle. This was studied using IF staining with the mitotic marker
MPM-2 that stains mitotic cells after G
2
phase [31]. IF for MPM-2 was
carried out in MFM-223, BT-474, and HCC-1954 cell lines, and the
percentage of nuclei stained with MPM-2 was assessed for PIP-KD and
control experiments (Figure 6, E and F). We observed a significant
increase in MPM-2 staining by two- to three-fold in PIP-KD cells
compared to the control, suggesting an arrest in mitotic transition
following PIP silencing (P b .01; Figure 6, E and F). Overall, these
findings indicate that PIP expression is required for mitotic transition
in breast cancer and the effect of PIP silencing on cell cycle corresponds
to the transcriptional changes in key cell cycle genes.
PIP Silencing Results in a Cytokinesis Defect
We subsequently studied the effect of PIP expression on cytokinesis.
It is established that microtubules and actin organization are essential
for this process [32]. Therefore, we first examined the effect of PIP
silencing on actin microfilaments using IF in BT-474, HCC-1954, and
MFM-223 cell lines. We observed that actin organization was
markedly disrupted after PIP-KD, resulting in abnormally shaped
and large filopodia protrusions and irregular lamellipodia that were
formed in multinucleated cells (Figures 7A and W2, A and B). In
addition, as opposed to the control cells that demonstrated polarity in
actin organization as evidenced by the orientation of filopodia and
retraction fibers, there was a loss of polarity in actin filaments following
PIP silencing (Figure 7A). Furthermore, formation of cleavage furrow,
an essential step in cytokinesis, was disrupted in some dividing PIP-KD
cells (Figure W2A).
We next assessed the formation of microtubules and peric entrin to
nuclear ratio following PIP silencing in these cells, because
supernumerary percentrins during cell division are associated with
cytokinesis defect and multinucleation [33]. Notably, pericentrin/
nuclear ratios significantly increa sed following PIP-KD in all three
cell lines by 1.4- to 2.2-fold compared to controls, suggesting that
there are supernumerary percentrins (P b .03; Figures 7, B and C,
and W2C). In addition, α-tubulin staining revealed that PIP
silencing leads to multipolar spindle formation during cell division
and an absence of distinct microtubules in multinuclear cells
(Figures 7B and W2 C).
Moreover, we observed that there is a 20% to 60% increase in the
number of multinucleated cells following PIP silencing in MFM-223,
SK-BR-3, BT-474, HCC-1954, and HCC-202 cell lines (P b .01;
Figure 7D). However, T-47D and MDA-MB-453 cells did not
demonstrate a significant increase in the number of multinucleated
cells after PIP -KD. Impo rtantly, these findings are in agreement with
the occurrence of aneuploidy among these cell lines. Overall, our data
suggest that PIP silencing disrupts the organization of actin
microfilaments and microtubules and leads to an increase in the
number of pericentrins and multinucleated cells that are all indicators
of a cytokinesis defect.
PIP Is Required for Inside-Out Activation of
Integrin-β1 Signaling
It is known that dysregulation of integrin-β1 signaling results in
cell cycle defects in G
1
progression and cytokinesis [34]. Therefore,
we investigated the effect of PIP silencing on FAK phosphorylation
(Tyr
397
), which is a key downstream mediator of integrin-β1
signaling and integrin-β1 binding to talin-1 that is a required step for
inside-out activation of integrins [35,36].
We observed that the level of phospho-FAK was generally low in
breast cancer cell lines and was only detectable in HCC-1954 and
HCC-202 cells. In addition, PIP-KD resulted in the reduction of
phospho-/total FAK ratios by approximately 50-70% in these two
lines, which was partly related to a relative increase in the total FAK
levels following PIP-KD (Figure 7E). Furthermore, we found a
baseline interaction between integrin-β1 and talin-1 in T-47D and
MFM-223 cell lines using IP with integrin-β1 and immunoblot
analysis with talin-1 (Figure 7F). Importantly, integrin-β1 binding to
talin-1 was abrogated in T-47D cells and reduced by 0.5-fold in
MFM-233 following PIP silencing (Figure 7E). These data suggest
that PIP expression is necessary for inside-out activa tion and signaling
effects of integrin-β1.
Identification of PIP-Binding Partners
To identify protein-binding partners for endogenous PIP, we
carried out MS. These experiments were performed in T-47D cell
line, which has a high level of endogenous PIP expre ssion that was
further induced using dihydrotestosterone. Because PIP is a secreted
protein, IP was performed using a combination of cell extracts and
conditioned me dia. The result of PIP pulldown was first validated
using immunoblot analysis with PIP antibody (Figure 8A). PIP-IP
bands were then separated using SDS-PAGE and Coomassie staining,
followed by MS analysis (Figure 8B).
We identified a total of 156 protein-binding partners for PIP that
were reproducible between two replicate experiments (Table W7).
336 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

Figure 6. The effect of PIP expression on mitosis. (A–D) The effects of PIP silencing on mitotic transition genes are presented. Expression of
FOXM1, TTK, BUB1,andCDC20 are measured using qRT-PCR for PIP-KD relative to control. P values are for PIP-KD versus CTL groups in each cell
line. The asterisk (*) denotes P b .01 in BUB1 experiments. The average changes obtained for two duplexes are presented. (E and F) IF with the
mitotic marker MPM-2. IF staining for MPM-2/Alexa 488 was carried for PIP-KD and control siRNA experiments in MFM-223, BT-474, and HCC-1954
cell lines. Percentage of MPM-2 staining was calculated in 200 nuclei for each experiment, and the average changes obtained for two duplexes are
presented. 4',6-diamidino-2-phenylindole (DAPI) DAPI staining was used to assess the nuclei. *P value is for PIP-KD versus control groups.
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 337

Figure 7. The effect of PIP on cytokinesis and integrin signaling. (A) IF staining for β-actin/Alexa488 following PIP-KD in BT-474 cells is shown.
Control and PIP-KD cells are shown during cell division (top panels), and a multinucleated cell following PIP-KD is shown in bottom panel.
White arrow, filopodia (fil); yellow arrow, lamellipodia (lam); orange arrow, retraction fibers (rf); and magenta arrow, direction of actin polarity.
(B) IF staining for α-tubulin (Tub) and pericentrin following PIP-KD in MFM-223 cells is shown. (C) Pericentrin to nuclear ratios following PIP-
KD are presented. DAPI staining was used to assess the nuclei. *P value is for PIP-KD versus control groups. (D) Change in percentage of
multinucleated cells between PIP-KD and control cell lines is shown. IF following β-actin and DAPI staining was used to assess
multinucleated cells. *P value is for PIP-KD versus control groups. (E) Immunoblot analysis measures the ratio of Ph-FAK (Tyr
397
)toT-FAK
following PIP-KD in cell lines. Fold changes were assessed r elative to control. E xperiments were carried out in four replicates using two
PIP-siRNA duplexes or control siRNA, and mean changes (± SEM) were s hown. (F) IP a ssesses integrin- β1(ITG-β1)bindingtotalin-1
following PIP- KD. IP and immunoblot an alysis were carried out with ITG-β1 and talin-1 antibodies, respectively. Membrane was stripped,
and immunoblot analysis for ITG-β1 was used to assess loading. ITG-β1 immunoblot for input con trol is shown. Fold cha nges were
assessed relative to control. Experiments were carried out in four replicates using two PIP-siRNA duplexes or control siRNA, and mean
change (± SEM) is shown for each cell line.
338 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

Functional classification of these binding partners using bioinformatics
revealed six highly significant clusters (Figure 8C and Table W8). These
functional clusters included translational elonga tion, ribonuclear
protein, nucleosome and chromatin assembly, regulation of actin and
cytoskeleton, clathrin-coated pit, and small guanosine diphosphate
(GDP)-binding protein. In addition, we studied the functional
association of PIP-binding partners with the canonical pathways
using Ingenuity Pathway Analysis. The top identified pathways
included eukaryotic Initiation Factor 2 (eIF2) and eIF4 signaling,
followed by remodeling of epithelial adherens junctions, clathrin-
mediated endocytosis, regulation of actin-based motility by Rho, and
integrin signaling (Table W9).
We further validated two of the identified PIP-binding partners
that have key functions in cell cycle (Table W7). Notably, we
identified β-tubulin as one of the top PIP-binding partners , which has
a well-established role in mitosis [37]. In addition, Arp2/3 protein is
another PIP-binding partner with a significant role in cytokinesis and
promoting talin binding to integrins [38,39]. To validate these
interactions, we carried out PIP-IP and performed immunoblot
analysis with β-tubulin an d Arp2/3 antibodie s on pulldowns.
Immunoblot analysis with PIP antibody was used to confirm a
successful PIP pulldown (Figure 8D). Notably, we observed a strong
interaction between PIP and β-tubulin and detected a specific
interaction between PIP and Arp2/3 protein in PIP pulldown
Figure 8. Identification of PIP-binding partners. (A) IP and immunoblot analysis (IB) with PIP antibody. The low molecular weight band may
represent a PIP fragment product. (B) Coomassie staining of SDS-PAGE for PIP-IP and control pulldowns is shown. (C) Functional
classification of PIP-binding partners is shown. Enrichment score and P value are shown. (D) IP with PIP antibody and IB with β-tubulin,
PIP, and Arp2/3 antibodies in T-47D cells are shown. A nonspecific rabbit IgG was used for control IP. Membrane was stripped, and IB for
PIP was used to assess loading. PIP immunoblot for input control is shown. (E) Microtubule polymerization assay measures polymerized
and soluble tubulin fractions following PIP-KD. The amount of each fraction following PIP-KD was normalized to that of control, and the
relative ratio of Pol/Sol fractions was obtained for each cell line. The average changes obtained for two duplexes are presented.
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 339

(Figure 8D). Taken together, our proteomic studies identified novel
PIP-binding protein s.
PIP Is Necessary for Microtubule Polymerization
To assess whether PIP binding to β-tubulin has a regulatory effect on
microtubule polymerization, we measured the effect of PIP silencing on
the polymerized and soluble fractions of tubulin in T-47D and HCC-
1954 cell lines. Immunoblot analysis was carried out using a β-tubulin
antibody to measure polymerized and soluble tubulin fractions. The
amount of each fraction following PIP silencing was normalized to that of
siRNA control, and the relative ratio of polymerized to soluble (Pol/Sol)
fractions was obtained for each cell line. We observed that Pol/Sol tubulin
ratio was markedly reduced following PIP-KD to 0.31- and 0.06-fold of
controls in T-47D and HCC-1954 cell lines, respectively (Figure 8E).
These findings suggest that PIP is required for tubulin polymerization in
breast cancer cells.
Discussion
PIP is widely expressed in breast cancer and is used as a characteristic
biomarker in this disease; however, the molecular functions of PIP
have remained largely unknown . Therefore, we carried out a
comprehensive study to identify the underlying mechanisms for
PIP function in breast cancer. In this process, we employed seven
breast cancer cell lines that encompass luminal A, luminal B, and
molecular apocrine subtypes. It is notable that these molecular
subtypes also correspond to the pattern of PIP expression among
primary breast tumors. Previous studies have shown a positive
regulatory role for PIP in cell prol iferation [9,10]. In this study, we
demonstrated that PIP expression is necessary for the proliferation of
all brea st cancer cell lines that have a detectable level of PIP. Although
PIP expression varied among different cell lines, the impact of PIP on
cell proliferation was similar across these cells. Moreover, the only two
cell lines with undetectable levels of PIP were MCF-7 and MDA-MB-
231, which do not have AR expression. This association between AR
and PIP expression is also present among primary breast tumors and
can be explained by the fact that AR is a transcriptional regulator of
PIP [8–10]. Overall, the pattern of PIP association with molecular
subtypes and biomarkers are similar between breast cancer cell lines
and breast tumors.
The effect of PIP on proliferation is explained by the fact that PIP
expression is required for cell cycle progression in breast cancer cells
and PIP silencing leads to defects in G
1
, mitosis, and cytokinesis. In
addition, we show that PIP interacts with β-tubulin and is necessary
for tubulin polymerization. This interaction is particularly significant
because microtubules are kno wn to have a critical role in mitotic
transition, spindle assembly, and cytokinesis [37,40] . Moreover,
proteomic data provide further evidence for the importance of PIP
interaction with β-tubulin in the functional characterization of PIP-
binding partners. In fact, a major portion of PIP interactions that
contrib ute to the observed functional classification are known
“Tubulin-Binding Proteins” (Figure 8C and Table W7). These
include RNA-binding proteins, proteins involved in translation such
as eIF4, heat shock proteins, clathrin, and 14-3-3 protein family [41–
43]. It is notable that 14-3-3 protein is a key regulator of cell cycle and
clathrin is required for mitotic spindle function and endocytosis
[42,43]. In addition, mRNA localization to microtubules contributes
to the translation of genes involved in cell division such as cyclin B1
[44]. Therefore, PIP regulation of microtubular polymerization and
its interaction with other tubulin-binding proteins would have a
profound effect on cell cycle (Figure 9).
Another major group of PIP interactions involves actin-binding
proteins. Among these, Arp2/3 has a critical role in actin
polymerization, and along with vinculin and talin, it provides a
physical link between actin cytoskeleton and the integrin scaffold,
which is needed to transform talin binding to integrin-β1 from a low-
to high-affinity state [38]. Therefore, PIP inter action with Arp2/3 can
Fn
Fn-f
Talin
FAK
vinculin
Arp2/3
ITG
PIP
PIP
Histones
PIP
FOXM1
β-tubulin
Figure 9. A schematic model for PIP regulation of cell cycle. The proposed mechanisms by which extracellular and intracellular PIP can
regulate cell cycle are depicted. Fn, fibronectin; Fn-f, fibronectin fragments; ITG, integrin-β1. Red arrows indicate positive regulation. Cell
membrane has been depicted by a circular line. Arp2/3, β-tubulin, and histones interact with PIP based on our study.
340 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

explain the marked reduction in talin binding to integrin-β 1, which is
a required step for inside-out activation of integrins, and a decrease in
FAK phosphorylation following PIP silencing. It is notable that
outside-in signaling of integrin-β1 binding to integrin-linked kinase 1
(ILK1) is also regulated by PIP in a process that partially depends on
fibronectin fragmentation (Figure 9), [10]. In addition to Arp2/3,
some of the other PIP-binding partners including gelsolin, cofilin 1,
F-actin–capping protein, α-actinin, and small GTP-binding proteins
have key roles in actin organization, formation of foca l adhesions, and
cytokinesis [39,45]. In fact, our study provides functional evidence
for the importance of these interactions as shown by a defect in
cytokinesis following PIP silencing that is associated with abnormal
actin organization (Figures 7 and W2). Im portantly, some of the PIP-
binding partners such as Rho-GTPase are known to coordinate
cytokinesis and cell polarity [46]. Overall, our findings indicate that a
key feature of PIP function is the regulation of cytoskeleton
(Figure 9).
Our data suggest that PIP expression is necessary for both G
1
/S and
mitotic progression in breast cance r cells. However, the impact of PIP
on each phase of cell cycle varies among breast cancer lines. In this
respect, we observed two main patterns for the effect of PIP silencing
on cell cycle. In one group constituted of T-47D and MDA-MB-453
cell lines, there was a severe degree of G
1
arrest that was not associated
with mitotic arrest or aneuploidy. Importantly, this pattern was
associated with a profound reduction in cyclin D1 exp ression
following PIP silencing. In comparison, the remaining cell lines
developed a moderate degree of G
1
arrest accompanied by mitotic
arrest and a marked increase in aneuploidy (Figure 5, A and B).
Furthermore, our genomics data on PIP transcriptional signature and
a marked reduction in key mitotic transition genes such as cyclin B1,
BUB1, and CDC20 following PIP down-regulation suggest the
importance of PIP expression in mitosis. It is notable that the
emergence of aneuploidy can be a consequence of defects in both
mitotic checkpoint and cytokinesis [47] . In particular, dysregulation
of mitotic and spindle checkpoint genes such as BUB1 and FOXM1
have been associated with aneuploidy [48,49]. Importantly, this
increase in aneuploidy can further contribute to a reduction in cell
proliferation [50].
We observed that PIP expression is necessary for the transcription
of multiple genes involved in cell cycle progression. Some of these
effects can be explained by PIP regulation of the upstream signaling
pathways. For example, cyclin D1 is a target of integrin-β1 mediated
through the activation of ERK [34]. Therefore, the effect of PIP on
cyclin D1 can be explained by the fact that G
1
arrest in T-47D cells
is accompanied by a marked reduction in ERK phosphorylation
associated with an abrogation of talin binding to integrin-β1. In
addition, PIP transcriptional signature shows a robust pattern of
coregulation between PIP and mitotic transition genes. This finding
along with a profound decrease in the expression of mitotic genes
observed following PIP silencing suggest that PIP is required to
maintain a balance in the expression of key genes involved in mitotic
transition. This is especially important because both overexpression
and down-regulation of some of the mitotic genes such as BUB1 can
lead to abnormal mitosis and aneuploidy [48,51]. Notably, FOXM1
is known to be a transcription factor for multiple genes i nvolved
in cell cycle progression including cyclin B1, BUB1, CDC20, and
polo-like kinase (PLK) [28]. Therefore, PIP regulation of FOXM1
expression can explain many of the transcriptional changes observed
following PIP silencing. In addition, soluble tubulin has been shown
to interact with histones and regulate transcription [52]. In view of
the fact that PIP interacts with histone s (Table W7) and also regulates
soluble tubulin levels, the possibility of a transcriptional role for PIP
deserves further studies (Figure 9).
In summary, we propose that PIP has a versatile function in breast
cancer resulting from a diverse range of both intracellular and
extracellular binding partners (Figure 9). As a consequence of these
functional interactions, PIP can regulate key cellular processes
including outside-in and inside-out activation of integrin-β1,
transcription of key cell cycle genes such as FOXM1, and cytoskeletal
organization including microtubule polymerization. The net effect of
these molecular functions is the fact that PIP can profoundly
influence cell cycle progression in breast cancer cells (Figure 9).
Importantly, our findings provide a rationale for the tantalizing
possibility of explorin g PIP as a therapeutic target in breast cancer.
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Figure W1. PIP expression in breast cancer cell lines. (A) Relative PIP expression in breast cancer cell lines to that of HCC202 cells
presented in a logarithmic scale (base 2). (B) PIP protein expression in BT-474 and MFM-223 cell lines following PIP-knockdown (KD) using
cell lysate samples. CTL: cells transfected with the control-siRNA. RR: relative ratio to control.
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 342.e1

Figure W2. PIP effect on cytokinesis. (A-B) Immunofluorescence (IF) staining for β-Actin in HCC-1954 and MFM-223 cell lines. White
arrow: filopodia (fil), blue arrow: cleavage furrow (cf). (A) Cleavage furrow formation is present in dividing control cells (CT) and is absent in
dividing cells with PIP-knockdown (KD). Multinucleated cells are shown following PIP-KD in HCC-1954 (A) and MFM-223 cells (B). IF was
carried out with β-Actin antibody and Alexa488 was used as secondary antibody. (C) IF staining for α-Tub/Pericentrin. IF was carried out
with, α-Tubulin and Pericentrin antibodies in BT-474 cell line. Alexa488 and Alexa 594 antibodies were used as secondaries. There is an
absence of distinct microtubules and presence of supernumerary pericentrins in PIP-KD cells. Magnifications are shown for each panel.
342.e2 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

Table W1. Characteristics of Breast Cancer Cell Lines Used in the Functional Study of PIP.
Cell Line ER ErbB2 AR Subtype PIP − ΔCT
MCF-7 POS NEG NEG Luminal A − 17.5 (±0.08)
T-47D POS NEG POS Luminal A − 4.9 (±0.04)
BT-474 POS POS POS Luminal B − 12.2 (±0.08)
HCC-202 NEG POS POS Luminal − 1.1 (±0.13)
HCC-1954 NEG POS POS Basal A − 7.3 (±0.02)
MDA-MB-453 NEG POS POS Luminal − 6.2 (±0.06)
SK-BR-3 NEG POS NEG Luminal − 11.1 (± 0.08)
MFM-223 NEG NEG POS Luminal − 11.8 (± 0.03)
MDA-MB-231 NEG NEG NEG Basal B − 20 (±0) *
Cell subtype and AR status were obtained from Lehmann BD et al. (2011) [53], Magklara A et al.(2002) [54], Heiser LM et al. (2012) [55], Nade ri A et al. (2010) [56], Naderi A et al. (2008) [57], Doane
AS et al. (2006) [58], and Neve RM et al. (2006) [59]. ErbB2 status was obtained from Subik K et al. (2010) [60], Ginestier C et al. (2007) [61], Lombardi DP et al. (2004) [62], and Agarwal R et al.
(2009) [63]. POS, positive; NEG, negative. PIP − ΔCT is − Δ cycle threshold value (±SEM) for PIP expression using qRT-PCR.
* PIP expression was not detectable after 40 cycles of qRT-PCR.
Table W2. Characteristics of the TMA Cohort.
Feature Status Percentage
Histology IDC * 85%
Others ** 15%
ER Negative 51%
Positive 49%
ErbB2 0-1 52%
2-3 48%
p53 0-1 68%
N 2 32%
Percentage is calculated in a total of 210 primary breast tumors.
* IDC, invasive ductal carcinoma.
** Others: Ductal carcinoma in situ, Paget disease, invasive lobular carcinoma, invasive micropapillary
carcinoma, invasive tubulo-lobular carcinoma, invasive papillary carcinoma, lobular carcinoma in situ,
invasive carcinoma with apocrine features, invasive tubular carcinoma, intraductal carcinoma, and tubular
mixed carcinoma.
Table W3. Association of PIP Expres sion with Molecular Features in Breast Cancer Cohort.
Biomarkers
Marker ErbB2 ER AR p53
Status b 2 2-3 Neg Pos Neg Pos Neg Pos
PIP (SEM) 112 (8) 126 (9) 111 (9) 126 (9) 80 (11) 132 (7) 119 (7) 118 (11)
P value N .1 N .1 b .01 * N .1
Molecular Subtypes
Subtype Luminal A Luminal B ER−/AR+ ER−/AR−
Frequency 28% 21% 31% 20% (Basal: 10%)
PIP (SEM) 116 (12) 136 (12) 141 (11) 62 (12)
P value P b .01 ** P b .01 ** P b .01 **
Mean scores for PIP expression are demonstrated in 210 primary breast tumors. Luminal B, ER+
with Ki-67 index ≥ 14% or ErbB2 staining score of 3; AR +, ≥ 10% nuclear staining; Basal, CK5/6+;
SEM, standard error of mean.
* P value for AR-negative (Neg) versus AR-positive (Pos) tumors.
** P values versus ER−/AR− group.
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 342.e3

Table W4. Proximity Matrix for PIP Coregulated Genes. Each Gene has Pearson CC ≥ 0.5 with PIP Expression at a Significant Level of P b .001.
342.e4 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

Table W4. (continued)
(continued on next page)
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Table W4. (continued)
342.e6 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

Table W4. (continued)
(continued on next page)
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Table W4. (continued)
342.e8 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

Table W5. Transcriptional Signature of PIP Coregulated Genes.
Gene CC P b Function *
ACOX1 0.518 .001 Fatty acid pathway
ACY1 0.504 .001 Hydrolysis
AGA 0.525 .001 Lysosomal
AGR2 0.592 .001 Cell migration
ALDH6A1 0.515 .001 Mitochondrial
AR 0.59 .001 Steroid receptor
ARHGEF16 0.5 .001 Cell migration
ARHH 0.572 .001 Survival and migration
ARRB1 0.547 .001 G-protein receptor
ATP2A3 0.554 .001 Ca2+ function
BIK 0.545 .001 Apoptosis
ANKRA2 0.527 .001 Cytoskeletal
CACFD1 0.522 .001
CPD 0.606 .001 Peptidase
CRAT 0.51 .001 Mitochondrial
SGSM3 0.702 .001 neurofibromatosis 2 (NF-2) signaling
LRP10 0.603 .001 Lipid metabolism
DUSP4 0.51 .001 mitogen-activated protein kinase
(MAPK) signaling
EGFL3 0.506 .001
ERRB3 0.501 .001 epidermal growth factor receptor
(EGFR) signaling
FKSG28 0.583 .001 Proliferation
SRD5A3 0.572 .001 Androgen metabolism
TMEM132A 0.563 .001 Cell death
TCTN1 0.523 .001 Hedgehog signaling
RHBDF1 0.523 .001 EGFR signaling
FMO4 0.502 .001 Metabolism
FRAT1 0.503 .001 Wnt signaling
FZD4 0.569 .001 Wnt signaling
GABRA3 0.513 .001 Neurotransmitter
HMIC 0.504 .001 Metabolism
HNF3A 0.518 .001 Steroid response
HOXC10 0.501 .001 Transcription factor
HPIP 0.564 .001 estrogen receptor-alpha (ESR) signaling
ICA1 0.517 .001 Secretory function
ITPR1 0.545 .001 Ca2+ signaling
KHNYN 0.562 .001
TRIL 0.587 .001 Cytokine secretion
KIF13B 0.632 .001 Cytoskeletal
KMO 0.52 .001 Metabolism
LDB1 0.532 .001 Transcription
LDB3 0.598 .001 Cytoskeletal
LFG 0.501 .001 Apoptosis
NFATC4 0.513 .001 Transcription
P24B 0.561 .001 Protein trafficking
P2RX4 0.547 .001 Ion channel
P2RY6 0.504 .001 G-protein receptor
PAPSS2
0.521 .001 Metabolism
PCK2 0.501 .001 Metabolism
PDEF 0.521 .001 Transcription
PISD 0.565 .001 Mitochondrial
PRKAG1 0.514 .001 Metabol ism
PRKCH 0.641 .001 Protein kinase
PRSS8 0.535 .001 Sserine protease
PXMP4 0.518 .001
RAB5B 0.552 .001 Protein transport
SEMA3F 0.511 .001 Cell motility
SERF2 0.513 .001
SERHL 0.518 .001 Serine hydrolase
SH3GLB2 0.512 .001
NHE2 0.517 .001 Channel protein
SPRY1 0.521 .001 Fibroblast growth factor signaling
SPTLC2 0.645 .001 Metabolism
SSBP2 0.502 .001 Genome stability
SUOX 0.595 .001 Mitochondrial
TJP3 0.52 .001 Cytoskeletal mitosis
TM7SF1 0.505 .001
TM7SF2 0.646 .001 Sterol met abolism
TM9SF1 0.503 .001 Autophagy
TMEM8 0.506 .001 Adhesion
TSPAN1 0.552 .001 Growth and moti lity
TST 0.506 .001 Mitochondrial
ULK1 0.527 .001 Autophagy
(continued on next page)
Table W5.(continued)
Gene CC P b Function *
VIPR1 0.5 .001 G-protein receptor
WSB1 0.551 .001 Proteasome
XBP1 0.505 .001 Transcription
ZDHHC3 0.538 .001 Cell surface stability
PATZ1 0.516 .001 Transcription
BUB1 − 0.607 .001 Mitosis
CCNA2 − 0.584 .001 Cell cycle
CCNB2 − 0.574 .001 Mitosis
CDC20 − 0.588 .001 Mitosis
CDC5L − 0.531 .001 Cell cycle
CENPE − 0.565 .001 Mitosis
CTPS − 0.531 .001 Cell growth
DDX18 − 0.54 .001 Cell growth and division
HJURP − 0.662 .001 Centrosome function
DNMT1 − 0.563 .001 Epigenetics
E2_EPF − 0.52 .001
E2F3 − 0.504 .001 Cell cycle
FBXO5 − 0.566 .001 Mitosis
FAM64A − 0.521 .001 Mitosis
PRR11 − 0.599 .001
QTRTD1 − 0.503 .001 RNA synthesis
NCAPG2 − 0.589 .001 Mitosis
SPDL1 − 0.536 .001 Mitosis
IARS − 0.504 .001 RNA synthesis
FASTKD1 − 0.512 .001
FOXM1 − 0.54 .001 Cell cycle
GLS_C − 0.52 .001 Metabolism
HCAP_G − 0.557 .001 Mitosis
HNRPD_E − 0.562 .001 RNA function
HNRPH1 − 0.613 .001 RNA function
DLGAP5 − 0.548 .001 Mitosis
NUP205 − 0.54 .001 Nuclear transport
EHBP1 −
0.542 .001 Cytoskeletal actin
KIF4A − 0.515 .001 Cytokinesis
MAD2L1 − 0.561 .001 Mitosis
METAP1 − 0.515 .001 Cell cycle
MSH2 − 0.507 .001 Genome stability
NCL − 0.559 .001 Transcription
NUP54 − 0.578 .001 Nuclear transport
OSBPL11 − 0.559 .001 Lipid metabolism
PDCD5 −0.64 .001 Apoptosis
PLK − 0.535 .001 Cell cycle
PLS3 − 0.616 .001 Actin binding
PMS1 − 0.514 .001 Genome stability
PMSCL1 − 0.578 .001 RNA function
PSMD1 − 0.522 .001 Proteasome
RAB6KIFL − 0.522 .001 Cytokinesis
RHEB2 −0.518 .001 Ras-GTPase
RUVBL2 − 0.521 .001 DNA repair
SFRS10 − 0.533 .001 Splicing factor
SFRS7 − 0.539 .001 Splicing factor
SIL − 0.59 .001 Mitosis
ORNT1 − 0.53 .001 Mitochondrial
SMC4L1 − 0.578 .001 Mitosis
SNRPD1 − 0.584 .001 RNA function
STK12 (Aurora B) − 0.5 .001 Mitosis and cytokinesis
TSN − 0.512 .001 Chromosomal function
TTK − 0.639 .001 Mitosis
UBA2 − 0.581 .001 Protein modification
UBE2C − 0.541 .001 Mitosis
UPF3B − 0.539 .001 RNA function
USP1 − 0.555 .001 DNA repair
XPO1 − 0.563 .001 Protein export
ZRF1 − 0.562 .001 Transcription
List of genes that have Pearson CCs ≥ 0.5 with PIP
expression at a significance level of P b .001.
Raw data for gene expression values were extracted from Affymetrix microarray data set published by Neve
RM et al. 2006.
* Proposed gene Function is derived from GeneCards (www.genecards.org).
Table W5. (continued )
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 342.e9

Table W7. Identified PIP-Binding Proteins (Protein Threshold at 99% and Peptide Threshold at
0.1% false discovery rate [FDR]).
No Accession No.
1 Cluster of tubulin β chain;
Organism Species (OS) = Homo sapiens;
Gene Name (GN) = TUBB; PE = 1;
Splice Variant (SV) = 2 (TBB5_HUMAN)
TBB5_HUMAN [2]
1.1 Tubulin β chain; OS = H sapiens ;
GN = TUBB; PE = 1; SV = 2
TBB5_HUMAN
1.2 Tubulin β-4B chain; OS = H sapiens;
GN = TUBB4B; Protein Existence (PE) = 1; SV = 1
TBB4B_HUMAN
2 Heat shock 70-kDa protein 1A/1B;
OS = H sapiens ; GN = HSPA1A; PE = 1; SV = 5
HSP71_HUMAN
3 Unconventional myosin- Id; OS = H sapiens;
GN = MYO1D; PE = 1; SV = 2
MYO1D_HUMAN
4 Cluster of heat shock protein HSP 90-β;
OS = H sapiens ; GN = HSP90AB1; PE = 1;
SV = 4 (HS90B_HUMAN)
HS90B_HUMAN
4.1 Heat shock protein HSP 90-β;OS=H sapiens;
GN = HSP90AB1; PE = 1; SV = 4
HS90B_HUMAN
5 Heterogeneous nuclear ribonucleoproteins A2/B1;
OS = H sapiens ; GN = HNRNPA2B1; PE = 1; SV = 2
ROA2_HUMAN
6 Neuroblast differentiation-associated protein AHNAK;
OS = H sapiens ; GN = AHNAK; PE = 1; SV = 2
AHNK_HUMAN
7 Cluster of prelamin-A/C; OS = H sapiens;
GN = LMNA; PE = 1; SV = 1 (LMNA_HUMAN)
LMNA_HUMAN
7.1 Prelamin-A/C; OS = H sapiens; GN = LMNA;
PE = 1; SV = 1
LMNA_HUMAN
8 Cluster of 40S ribosomal protein S3a; OS = H sapiens;
GN = RPS3A; PE = 1; SV = 2 (RS3A_HUMAN)
RS3A_HUMAN
8.1 40S ribosomal protein S3a; OS = H sapiens;
GN = RPS3A; PE = 1; SV = 2
RS3A_HUMAN
940S ribosomal protein S3; OS = H sapiens;
GN = RPS3; PE = 1; SV = 2
RS3_HUMAN
10 Cluster of α-enolase; OS = H sapiens ; GN = ENO1;
PE = 1; SV = 2 (ENOA_HUMAN)
ENOA_HUMAN
10.1 α
-enolase; OS = H sapiens; GN = ENO1;
PE = 1; SV = 2
ENOA_HUMAN
11 60S ribosomal protein L10a; OS = H sapiens;
GN = RPL10A; PE = 1; SV = 2
RL10A_HUMAN
12 Cluster of serine/arginine-rich splicing factor 6;
OS = H sapiens ; GN = SRSF6; PE = 1;
SV = 2 (SRSF6_HUMAN)
SRSF6_HUMAN [4]
12.1 Serine/arginine-rich splicing factor 6;
OS = H sapiens ; GN = SRSF6; PE = 1; SV = 2
SRSF6_HUMAN
12.2 Serine/arginine-rich splicing factor 4;
OS = H sapiens ; GN = SRSF4; PE = 1; SV = 2
SRSF4_HUMAN (+ 2)
13 60S ribosomal protein L8; OS = H sapiens;
GN = RPL8; PE = 1; SV = 2
RL8_HUMAN
14 40S ribosomal protein S6; OS = H sapiens;
GN = RPS6; PE = 1; SV = 1
RS6_HUMAN
15 Pyruvate kinase PKM; OS = H sapiens;
GN = PKM; PE = 1; SV = 4
KPYM_HUMAN
16 40S ribosomal protein S18; OS = H sapiens;
GN = RPS18; PE = 1; SV = 3
RS18_HUMAN
17 Ras GTPase-activating protein-binding protein 1;
OS = H sapiens ; GN = G3BP1; PE = 1; SV = 1
G3BP1_HUMAN
18 UPF0568 protein C14orf166; OS = H sapiens;
GN = C14orf166; PE = 1; SV = 1
CN166_HUMAN
19 Heterogeneous nuclear ribonucleoprotein A1;
OS = H sapiens ; GN = HNRNPA1; PE = 1; SV = 5
ROA1_HUMAN
20 Heat shock protein β-1; OS = H sapiens;
GN = HSPB1; PE = 1; SV = 2
HSPB1_HUMAN
21 60S ribosomal protein L10; OS = H sapiens;
GN = RPL10; PE = 1; SV = 4
RL10_HUMAN
22 Cluster of histone H1.3; OS = H sapiens;
GN = HIST1H1D; PE = 1; SV = 2 (H13_HUMAN)
H13_HUMAN [2]
22.1 Histone H1.3; OS = H sapiens; GN = HIST1H1D;
PE = 1; SV = 2
H13_HUMAN
22.2 Histone H1.2; OS = H sapiens; GN = HIST1H1C;
PE = 1; SV = 2
H12_HUMAN
23 60S acidic ribosomal protein P0; OS =
H sapiens;
GN = RPLP0; PE = 1; SV = 1
RLA0_HUMAN (+1)
24 tRNA-splicing ligase RtcB homolog;
OS = H sapiens ; GN = C22orf28; PE = 1; SV = 1
RTCB_HUMAN
25 Serine/arginine-rich splicing factor 9;
OS = H sapiens ; GN = SRSF9; PE = 1; SV = 1
SRSF9_HUMAN
Table W6. Dendrogram Using Centroid Linkage.
342.e10 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

Table W7.(continued)
No Accession No.
26 40S ribosomal protein S11; OS = H sapiens;
GN = RPS11; PE = 1; SV = 3
RS11_HUMAN (+1)
27 Clathrin heavy chain 1; OS = H sapiens; GN = CLTC;
PE = 1; SV = 5
CLH1_HUMAN
28 40S ribosomal protein S16; OS = H sapiens;
GN = RPS16; PE = 1; SV = 2
RS16_HUMAN (+1)
29 Cluster of annexin A2; OS = H sapiens; GN = ANXA2;
PE = 1; SV = 2 (ANXA2_HUMAN)
ANXA2_HUMAN [4]
29.1 Annexin A2; OS = H sapiens; GN = ANXA2;
PE = 1; SV = 2
ANXA2_HUMAN (+3)
30 Cluster of Q5VU59_HUMAN Q5VU59_HUMAN [2]
30.1 Q5VU59_HUMAN Q5VU59_HUMAN
30.2 Tropomyosin α-3 chain; OS = H sapiens;
GN = TPM3; PE = 1; SV = 2
TPM3_HUMAN
31 ADP/ATP translocase 2; OS = H sapiens;
GN = SLC25A5; PE = 1; SV = 7
ADT2_HUMAN
32 Cluster of eukaryotic translation initiation
factor 4 γ 1; OS = H sapiens; GN = EIF4G1;
PE = 1; SV = 4 (IF4G1_HUMAN)
IF4G1_HUMAN
32.1 Eukaryotic translation initiation factor 4 γ 1;
OS = H sapiens; GN = EIF4G1; PE = 1; SV = 4
IF4G1_HUMAN
33 60S ribosomal protein L19; OS = H sapiens;
GN = RPL19; PE = 1; SV = 1
RL19_HUMAN
34 Cluster of dystonin; OS = H sapiens; GN = DST;
PE = 1; SV = 4 (DYST_HUMAN)
DYST_HUMAN
34 Cluster of dystonin; OS = H sapiens; GN = DST;
PE = 1; SV = 4 (DYST_HUMAN)
DYST_HUMAN
35 EH domain-containing protein 1; OS = H sapiens;
GN = EHD1; PE = 1; SV = 2
EHD1_HUMAN
36 Peptidyl-prolyl cis-trans isomerase A; OS = H sapiens;
GN = PPIA; PE = 1; SV = 2
PPIA_HUMAN (+4)
37 GTP-binding nuclear protein Ran; OS = H sapiens;
GN = RAN; PE = 1; SV = 3
RAN_HUMAN
38 60S ribosomal protein L23a; OS =
H sapiens;
GN = RPL23A; PE = 1; SV = 1
RL23A_HUMAN
39 Cluster of AP-2 complex subunit α-1; OS = H sapiens;
GN = AP2A1; PE = 1; SV = 3 (AP2A1_HUMAN)
AP2A1_HUMAN
39.1 AP-2 complex subunit α-1; OS = H sapiens;
GN = AP2A1; PE = 1; SV = 3
AP2A1_HUMAN
40 Elongation factor 2; OS = H sapiens; GN = EEF2;
PE = 1; SV = 4
EF2_HUMAN
41 60S ribosomal protein L13a; OS = H sapiens;
GN = RPL13A; PE = 1; SV = 2
RL13A_HUMAN
42 40S ribosomal protein S25; OS = H sapiens;
GN = RPS25; PE = 1; SV = 1
RS25_HUMAN
43 EF-hand domain-containing protein D1;
OS = H sapiens; GN = EFHD1; PE = 1; SV = 1
EFHD1_HUMAN
44 60S ribosomal protein L26; OS = H sapiens;
GN = RPL26; PE = 1; SV = 1
RL26_HUMAN
45 AP-2 complex subunit μ;OS=H sapiens;
GN = AP2M1; PE = 1; SV = 2
AP2M1_HUMAN (+2)
46 Ataxin-2-like protein; OS = H sapiens; GN = ATXN2L;
PE = 1; SV = 2
ATX2L_HUMAN
47 60S ribosomal protein L15; OS = H sapiens;
GN = RPL15; PE = 1; SV = 2
RL15_HUMAN (+2)
48 60S ribosomal protein L24; OS = H sapiens;
GN = RPL24; PE = 1; SV = 1
RL24_HUMAN (+2)
49 Cluster of thyroid hormone receptor–associated
protein 3; OS = H sapiens; GN = THRAP3;
PE = 1; SV = 2 (TR150_HUMAN)
TR150_HUMAN [5]
49.1 Thyroid hormone receptor–associated protein 3;
OS = H sapiens; GN = THRAP3; PE = 1; SV = 2
TR150_HUMAN
49.2 THRAP3 protein (fragment); OS = H sapiens;
GN = THRAP3; PE = 2; SV = 1
Q05D20_HUMAN (+3)
50 Gelsolin; OS = H sapiens; GN = GSN; PE = 1; SV = 1 GELS_HUMAN
51 Nucleophosmin; OS = H sapiens; GN = NPM1;
PE = 1; SV = 2
NPM_HUMAN
52 Histone H4; OS = H sapiens; GN = HIST1H4A;
PE = 1; SV = 2
H4_HUMAN
53 Cofilin 1; OS = H sapiens; GN = CFL1;
PE = 1; SV = 3
COF1_HUMAN
54 Glutathione S-transferase μ 3; OS = H sapiens;
GN = GSTM3; PE = 1; SV = 3
GSTM3_HUMAN
55 Prolactin-inducible protein; OS = H sapiens;
GN = PIP; PE = 1; SV = 1
PIP_HUMAN
(continued on next page)
Table W7.(continued)
No Accession No.
56 Cluster of F-actin–capping protein subunit β ;
OS = H sapiens ; GN = CAPZB;
PE = 1; SV = 4 (CAPZB_HUMAN)
CAPZB_HUMAN
56.1 F-actin–capping protein subunit β;
OS = H sapiens ; GN = CAPZB; PE = 1; SV = 4
CAPZB_HUMAN
57 Cluster of peroxiredoxin-1; OS = H sapiens;
GN = PRDX1; PE = 1; SV = 1 (PRDX1_HUMAN)
PRDX1_HUMAN [2]
57.1 Peroxiredoxin-1; OS = H sapiens; GN = PRDX1;
PE = 1; SV = 1
PRDX1_HUMAN
57.2 Peroxiredoxin-2; OS = H sapiens; GN = PRDX2;
PE = 1; SV = 5
PRDX2_HUMAN
58 Fructose-bisphosphate aldolase A; OS = H sapiens;
GN = ALDOA; PE = 1; SV = 2
ALDOA_HUMAN
59 40S ribosomal protein S15a; OS = H sapiens;
GN = RPS15A; PE = 1; SV = 2
RS15A_HUMAN
60 KH domain-containing, RNA-binding,
signal transduction-associated protein 1;
OS = H sapiens ; GN = KHDRBS1;
PE = 1; SV = 1
KHDR1_HUMAN
61 Leucine-rich repeat-containing protein 59;
OS = H sapiens ; GN = LRRC59; PE = 1; SV = 1
LRC59_HUMAN
62 60S ribosomal protein L12; OS = H sapiens;
GN = RPL12; PE = 1; SV = 1
RL12_HUMAN
63 Tropomodulin-3; OS = H sapiens; GN = TMOD3;
PE = 1; SV = 1
TMOD3_HUMAN
64 Heterogeneous nuclear ribonucleoprotein H;
OS = H sapiens ; GN = HNRNPH1; PE = 1; SV = 4
HNRH1_HUMAN (+ 2)
65 60S ribosomal protein L21; OS = H sapiens;
GN = RPL21; PE = 1; SV = 2
RL21_HUMAN (+1)
66 Cluster of ADP-ribosylation factor 1;
OS = H sapiens ; GN = ARF1; PE = 1;
SV = 2 (ARF1_HUMAN)
ARF1_HUMAN [4]
66.1 ADP-ribosylation factor 1; OS = H sapiens;
GN = ARF1; PE = 1; SV = 2
ARF1_HUMAN (+1)
66.2 ADP-ribosylation factor 4; OS = H sapiens;
GN = ARF4; PE = 1; SV = 3
ARF4_HUMAN (+1)
67 Cluster of eukaryotic initiation factor 4A-I;
OS = H sapiens ; GN = EIF4A1;
PE = 1; SV = 1 (IF4A1_HUMAN)
IF4A1_HUMAN
67.1 Eukaryotic initiation factor 4A-I; OS = H sapiens;
GN = EIF4A1; PE = 1; SV = 1
IF4A1_HUMAN
68 60S ribosomal protein L14; OS = H sapiens;
GN = RPL14; PE = 1; SV = 4
RL14_HUMAN
69 ATP synthase subunit α, mitochondrial;
OS = H sapiens ; GN = ATP5A1; PE = 1; SV = 1
ATPA_HUMAN
70 40S ribosomal protein S7; OS = H sapiens;
GN = RPS7; PE = 1; SV = 1
RS7_HUMAN
71 Histone H2A type 1-B/E; OS = H sapiens;
GN = HIST1H2AB; PE = 1; SV = 2
H2A1B_HUMAN (+14)
72 AP-2 complex subunit β;OS=H sapiens ;
GN = AP2B1; PE = 1; SV = 1
AP2B1_HUMAN
73 ELAV-like protein 1; OS = H sapiens;
GN = ELAVL1; PE = 1; SV = 2
ELAV1_HUMAN
74 Peroxiredoxin-6; OS = H sapiens; GN = PRDX6;
PE = 1; SV = 3
PRDX6_HUMAN
75 60S ribosomal protein L11; OS = H sapiens;
GN = RPL11; PE = 1; SV = 2
RL11_HUMAN (+2)
76 78-kDa glucose-regulated protein; OS = H sapiens;
GN = HSPA5; PE = 1; SV = 2
GRP78_HUMAN
77 40S ribosomal protein S17-like; OS = H sapiens;
GN = RPS17L; PE = 1; SV = 1
RS17L_HUMAN (+1)
78 Cluster of F-actin–capping protein subunit α-1;
OS = H sapiens ; GN = CAPZA1;
PE = 1; SV = 3 (CAZA1_HUMAN)
CAZA1_HUMAN
78.1 F-actin–capping protein subunit α-1;
OS = H sapiens ; GN = CAPZA1; PE = 1; SV = 3
CAZA1_HUMAN
79 Phosphoglycerate mutase 1; OS = H sapiens;
GN = PGAM1; PE = 1; SV = 2
PGAM1_HUMAN
80 Phosphoglycerate kinase 1; OS = H sapiens
;
GN = PGK1; PE = 1; SV = 3
PGK1_HUMAN
81 Cluster of stress-70 protein, mitochondrial;
OS = H sapiens ; GN = HSPA9; PE = 1;
SV = 2 (GRP75_HUMAN)
GRP75_HUMAN
81.1 Stress-70 protein, mitochondrial; OS = H sapiens;
GN = HSPA9; PE = 1; SV = 2
GRP75_HUMAN
(continued on next page)
Table W7. (continued ) Table W7. (continued )
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 342.e11

Table W7.(continued)
No Accession No.
82 Cluster of transformer-2 protein homolog β;
OS = H sapiens ; GN = TRA2B; PE = 1;
SV = 1 (TRA2B_HUMAN)
TRA2B_HUMAN [2]
82.1 Transformer-2 protein homolog β;OS=H sapiens;
GN = TRA2B; PE = 1; SV = 1
TRA2B_HUMAN
82.2 Transformer-2 protein homolog α;OS=H sapiens;
GN = TRA2A; PE = 1; SV = 1
TRA2A_HUMAN
83 Heterogeneous nuclear ribonucleoprotein M;
OS = H sapiens ; GN = HNRNPM; PE = 1; SV = 3
HNRPM_HUMAN
84 Heterogeneous nuclear ribonucleoprotein A3;
OS = H sapiens ; GN = HNRNPA3; PE = 1; SV = 2
ROA3_HUMAN
85 40S ribosomal protein S10; OS = H sapiens ;
GN = RPS10; PE = 1; SV = 1
RS10_HUMAN
86 60S ribosomal protein L18a; OS = H sapiens;
GN = RPL18A; PE = 1; SV = 2
RL18A_HUMAN
87 Malate dehydrogenase, mitochondrial; OS = H sapiens;
GN = MDH2; PE = 1; SV = 3
MDHM_HUMAN (+5)
88 Cluster of 14–3–3 protein β/α;OS=H sapiens;
GN = YWHAB; PE = 1; SV = 3 (1433B_HUMAN)
1433B_HUMAN [3]
88.1 14-3-3 protein β/α;OS=H sapiens; GN = YWHAB;
PE = 1; SV = 3
1433B_HUMAN (+1)
88.2 14-3-3 protein ζ/δ;OS=H sapiens; GN = YWHAZ;
PE = 1; SV = 1
1433Z_HUMAN
89 Ras GTPase-activating protein-binding protein 2;
OS = H sapiens ; GN = G3BP2; PE = 1; SV = 2
G3BP2_HUMAN (+1)
90 Ras-related protein Rab-1B; OS = H sapiens;
GN = RAB1B; PE = 1; SV = 1
RAB1B_HUMAN (+1)
91 Catenin α-1; OS = H sapiens
; GN = CTNNA1;
PE = 1; SV = 1
CTNA1_HUMAN (+1)
92 RNA-binding protein EWS; OS = H sapiens;
GN = EWSR1; PE = 1; SV = 1
EWS_HUMAN (+3)
93 60S ribosomal protein L3; OS = H sapiens;
GN = RPL3; PE = 1; SV = 2
RL3_HUMAN (+6)
94 Cathepsin D; OS = H sapiens; GN = CTSD;
PE = 1; SV = 1
CATD_HUMAN
95 Chloride intracellular channel protein 1;
OS = H sapiens ; GN = CLIC1; PE = 1; SV = 4
CLIC1_HUMAN
96 Fatty acid synthase; OS = H sapiens; GN = FASN;
PE = 1; SV = 3
FAS_HUMAN
97 Guanine nucleotide-binding protein subunit β-2–like 1;
OS = H sapiens ; GN =; GNB2L1; PE = 1; SV = 3
GBLP_HUMAN
98 Α-actinin-4; OS = H sapiens; GN = ACTN4;
PE = 1; SV = 2
ACTN4_HUMAN (+2)
99 Splicing factor, proline- and glutamine-rich;
OS = H sapiens ; GN = SFPQ; PE = 1; SV = 2
SFPQ_HUMAN
100 ARF6 protein; OS = H sapiens; GN = ARF6;
PE = 2; SV = 1
Q6FH17_HUMAN
101 40S ribosomal protein S5; OS = H sapiens;
GN = RPS5; PE = 1; SV = 4
RS5_HUMAN (+4)
102 Barrier-to-autointegration factor; OS = H sapiens ;
GN = BANF1; PE = 1; SV = 1
BAF_HUMAN
103 60S ribosomal protein L28; OS = H sapiens;
GN = RPL28; PE = 1; SV = 3
RL28_HUMAN (+1)
104 Ribosomal protein S19 (Fragment); OS = H sapiens;
PE = 2; SV = 1
Q8WVX7_HUMAN
105 Histone H1.5; OS = H sapiens; GN = HIST1H1B;
PE = 1; SV = 3
H15_HUMAN
106 Single-stranded DNA-binding protein, mitochondrial;
OS = H sapiens ; GN = SSBP1; PE = 1; SV = 1
SSBP_HUMAN
107 Transferrin receptor protein 1; OS = H sapiens;
GN = TFRC; PE = 1; SV = 2
TFR1_HUMAN (+1)
108 40S ribosomal protein S14; OS = H sapiens;
GN = RPS14; PE = 1; SV = 3
RS14_HUMAN
109 60S ribosomal protein L22; OS = H sapiens;
GN = RPL22; PE = 1; SV = 2
RL22_HUMAN (+3)
110 60S ribosomal protein L31; OS = H sapiens;
GN = RPL31; PE = 1; SV = 1
RL31_HUMAN
111 60S ribosomal protein L35; OS = H sapiens;
GN = RPL35; PE = 1; SV = 2
RL35_HUMAN
112 THO complex subunit 4; OS = H sapiens;
GN = ALYREF; PE = 1; SV = 3
THOC4_HUMAN (+1)
113 Serine/arginine repetitive matrix protein 2;
OS = H sapiens ; GN = SRRM2; PE = 1; SV = 2
SRRM2_HUMAN
114 Peptidyl-prolyl cis-trans isomerase FKBP4;
OS = H sapiens ; GN = FKBP4; PE = 1; SV = 3
FKBP4_HUMAN
Table W7.(continued)
No Accession No.
115 Actin-related protein 2/3 complex subunit 3;
OS = H sapiens ; GN = ARPC3; PE = 1; SV = 3
ARPC3_HUMAN (+2)
116 40S ribosomal protein S26; OS = H sapiens;
GN = RPS26; PE = 1; SV = 3
RS26_HUMAN
117 Transcription factor A, mitochondrial;
OS = H sapiens ; GN = TFAM; PE = 1; SV = 1
TFAM_HUMAN
118 Polypyrimidine tract-binding protein 1;
OS = H sapiens ; GN = PTBP1; PE = 1; SV = 1
PTBP1_HUMAN
119 Adenine phosphoribosyltransferase; OS = H sapiens ;
GN = APRT; PE = 1; SV = 2
APT_HUMAN
120 Protein FAM98A; OS = H sapiens; GN = FAM98A;
PE = 1; SV = 1
FA98A_HUMAN (+4)
121 Galectin-7; OS = H sapiens; GN = LGALS7;
PE = 1; SV = 2
LEG7_HUMAN
122 Triosephosphate isomerase; OS = H sapiens;
GN = TPI1; PE = 1; SV = 3
TPIS_HUMAN
123 Histone H2B type 1-A; OS = H sapiens;
GN = HIST1H2BA; PE = 1; SV = 3
H2B1A_HUMAN (+19)
124 DNA repair protein XRCC1; OS = H sapiens;
GN = XRCC1; PE = 1; SV = 2
XRCC1_HUMAN (+ 3)
125
L-lactate dehydrogenase A chain; OS = H sapiens;
GN = LDHA; PE = 1; SV = 2
LDHA_HUMAN (+ 1)
126 Cluster of serine/threonine-protein phosphatase
PP1-α catalytic subunit; OS = H sapiens;
GN = PPP1CA; PE = 1; SV = 1 (PP1A_HU
PP1A_HUMAN
126 Cluster of serine/threonine-protein phosphatase
PP1-α catalytic subunit; OS = H sapiens;
GN = PPP1CA; PE = 1; SV = 1 (PP1A_HU
PP1A_HUMAN
127 Cluster of chloride intracellular channel protein 3;
OS = H sapiens ; GN = CLIC3; PE = 1;
SV = 2 (CLIC3_HUMAN)
CLIC3_HUMAN
127.1 Chloride intracellular channel protein 3;
OS = H sapiens ; GN = CLIC3; PE = 1; SV = 2
CLIC3_HUMAN
128 14-3-3 protein ε;OS=H sapiens; GN = YWHAE;
PE = 1; SV = 1
1433E_HUMAN
129 MUC1 isoform J14; OS = H sapiens; GN = MUC1;
PE = 2; SV = 1
B6ECA3_HUMAN
130 cDNA FLJ59433, highly similar to elongation
factor 1-γ;OS=H sapiens; PE = 2; SV = 1
B4DUP0_HUMAN
131 Non-POU domain-containing octamer-binding
protein; OS = H sapiens; GN = NONO;
PE = 1; SV = 4
NONO_HUMAN (+ 1)
132 Actin-related protein 2/3 complex subunit 4;
OS = H sapiens ; GN = ARPC4; PE = 1; SV = 3
ARPC4_HUMAN (+1)
133 60-kDa heat shock protein , mitochondrial;
OS = H sapiens ; GN = HSPD1 ; PE = 1; SV = 2
CH60_HUMAN
134 DNA ligase 3; OS = H sapiens; GN = LIG3;
PE = 1; SV = 2
DNLI3_HUMAN
135 40S ribosomal protein S15; OS = H sapiens;
GN = RPS15; PE = 1; SV = 2
RS15_HUMAN (+4)
136 Matrin-3; OS = H sapiens ; GN = MATR3;
PE = 1; SV = 2
MATR3_HUMAN (+3)
137 Serine/arginine-rich splicing factor 10;
OS = H sapiens ; GN = SRSF10; PE = 1; SV = 1
SRS10_HUMAN
138 Cluster of Ras-related protein Rab-10;
OS = H sapiens ; GN = RAB10; PE = 1;
SV = 1 (RAB10_HUMAN)
RAB10_HUMAN [3]
138.1 Ras-related protein Rab-10; OS = H sapiens;
GN = RAB10; PE = 1; SV = 1
RAB10_HUMAN
138.2 Ras-related protein Rab-13; OS =
H sapiens;
GN = RAB13; PE = 1; SV = 1
RAB13_HUMAN (+ 1)
139 Actin-related protein 3; OS = H sapiens;
GN = ACTR3; PE = 1; SV = 3
ARP3_HUMAN (+2)
140 U1 small nuclear ribonucleoprotein A;
OS = H sapiens ; GN = SNRPA;
PE = 1; SV = 3
SNRPA_HUMAN (+6)
141 Fragile X mental retardation syndrome–related
protein 2; OS = H sapiens; GN = FXR2;
PE = 1; SV = 2
FXR2_HUMAN
142 Anterior gradient protein 2 homolog;
OS = H sapiens ; GN = AGR2; PE = 1; SV = 1
AGR2_HUMAN
143 Clathrin interactor 1; OS = H sapiens;
GN = CLINT1; PE = 1; SV = 1
EPN4_HUMAN
144 Cell division control protein 42 homolog;
OS = H sapiens ; GN = CDC42; PE = 1; SV = 2
CDC42_HUMAN
342.e12 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014

Table W8. Functional Classification of PIP Proteomics Data.
Cluster 1 Enrichment Score = 33.10409316169969 P Value
UNIPROT_ID Protein Name 9.3E-76
823648 Ribosomal protein L23a pseudogene 63
779090 Ribosomal protein L15 pseudogene 22
801125 Ribosomal protein L8; ribosomal protein L8 pseudogene 2
781935 Ribosomal protein S25 pseudogene 8; ribosomal protein S25
783263 Ribosomal protein L12 pseudogene 2; ribosomal protein L12
793235 Ribosomal protein L21 pseudogene 134; ribosomal protein L21
772335 Ribosomal protein S26 pseudogene 38; ribosomal protein S26
782492 Ribosomal protein L22 pseudogene 11; ribosomal protein L22
778688 Ribosomal protein L18a pseudogene 6; ribosomal protein L18a
797877 Ribosomal protein S5
808990 Ribosomal protein S10; ribosomal protein S10 pseudogene 4
796446 Ribosomal protein L31 pseudogene 49; ribosomal protein L31
810885 Ribosomal protein L11
808508 Ribosomal protein S6 pseudogene 25; ribosomal protein S6
803377 Ribosomal protein L35; ribosomal protein L35 pseudogene 1
802424 Ribosomal protein L3; similar to 60 S ribosomal protein L3 (L4)
796817 Ribosomal protein L10a pseudogene 6; ribosomal protein L10a
791016 Ribosomal protein L13a pseudogene 7; ribosomal protein L13a
816191 Ribosomal protein S3 pseudogene 3; ribosomal protein S3
789968 Ribosomal protein S3A pseudogene 5; ribosomal protein S3a
799749 Ribosomal protein S19 pseudogene 3; ribosomal protein S19
800698 Ribosomal protein L24; ribosomal protein L24 pseudogene 6
784832 Ribosomal protein S15 pseudogene 5; ribosomal protein S15
821689 Fragile X mental retardation, autosomal homolog 2
796227 Ribosomal protein, large, P0 pseudogene 2; ribosomal protein
817032 Ribosomal protein S14
785872 Ribosomal protein L26 pseudogene 33; ribosomal protein L26
777984 Ribosomal protein L14
773042 Ribosomal protein S7; ribosomal protein S7 pseudogene 11
786531 Ribosomal protein L19; ribosomal protein L19 pseudogene 12
788428 Ribosomal protein S15a pseudogene 17; ribosomal protein S15a
790706 Ribosomal protein S16 pseudogene 1
786137 Ribosomal protein S11 pseudogene 5; ribosomal protein S11
799716 Ribosomal protein L10; ribosomal protein L10 pseudogene 15
814248 Ribosomal protein S18 pseudogene 12
820040 Ribosomal protein L28
Cluster 2 Enrichment Score: 13.296661105241336 P Value
UNIPROT_ID Protein Name 5.14E-32
814227 Heterogeneous nuclear ribonucleoprotein A2/B1
824519 Heterogeneous nuclear ribonucleoprotein A1-like 3
816714 FUS-interacting protein (serine/arginine-rich) 1
806558 THO complex 4
786723 Matrin 3
826437 Heterogeneous nuclear ribonucleoprotein H1 (H)
810069 Heterogeneous nuclear ribonucleoprotein A3
797048 Transformer 2 β homolog (Drosophila)
825011 Splicing factor, arginine/serine-rich 4
826656 Splicing factor proline/glutamine-rich
825901 Polypyrimidine tract binding protein 1
800520 Serine/arginine repetitiv e matrix 2
776456 Non-POU domain containing, octamer-binding
798354 Small nuclear ribonucleoprotein polypeptide A
776781 Transformer 2 α homolog (Drosophila)
806156 Splicing factor, arginine/serine-rich 6
791398 Heterogeneous nuclear ribonucleoprotein M
802485 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1
816065 Splicing factor, arginine/serine-rich 9
Cluster 3 Enrichment Score: 6.102262729332349 P Value
UNIPROT_ID Protein Name 2.47E-12
775719 Histone cluster 1, H2ae; histone cluster 1, H2ab
784692 Histone cluster 1, H4l; histone cluster 1
781820 Histone cluster 1, H1d
784865 Histone cluster 1, H1c
799393 Histone cluster 1, H1b
783255 Histone cluster 1, H2ba
(continued on next page)
Neoplasia Vol. 16, No. 4, 2014 PIP Is Required for Cell Cycle Progression Naderi and Vanneste 342.e13

Table W9. Canonical Pathways Associated with PIP-Binding Partners.
Canonical Pathways − Log (P Value) Ratio to Total Molecules
EIF2 Signaling 4.37E+01 1.94E-01 RPL11, RPL24, RPL22, RPS18, RPL14, RPL26, EIF4G1
RPS17/RPS17L, RPS11, RPS7, RPL35, RPS3A, RPL18A, RPL19
RPL12, RPL8, PPP1CA, RPS5, RPS3, RPS10, RPL31, RPL3, RPS19
RPL21, RPL23A, RPLP0 , RPL10A, RPS6, RPL15, RPS16, RPS26
RPL28, RPL10, EIF4A1, RPS15, RPS15A, RPS25, RPL13A, RPS14
Regulation of eIF4 and p70S6K signaling 1.55E+01 1.03E-01 RPS18, RPS19, RPS17/RPS17L, EIF4G1, RPS11, RPS7, RPS6
RPS6, RPS3A, RPS16, RPS26, EIF4A1, RPS15, RPS15A, RPS25
RPS5, RPS3, RPS10, RPS14
Remodeling of epithelial adherens junctions 7.03E+00 1.14E-01 ARF6, ACTR3, TUBB4B, CTNNA1, ARPC3, ACTN4, TUBB, ARPC4
Clathrin-mediated endocytosis signaling 5.46E+00 5.05E-02 AP2B1, AP2M1, AP2A1, ARF6, ACTR3, CLTC, TFRC, ARPC3, CDC42 ARPC4
Regulation of actin-based motility by Rho 4.20E+00 6.59E-02 ACTR3, CFL1, ARPC3, CDC42, GSN, ARPC4
Integrin signaling 3.60E+00 3.85E-02 ARF1, ARF6, ACTR3, ARF4, ARPC3, ACTN4, CDC42, ARPC4
Table W8.(continued)
Cluster 4 Enrichment Score: 5.218508620408835 P Value
UNIPROT_ID Protein Name 6.01E-08
775114 Gelsolin (amyloidosis, Finnish type)
819298 Capping protein (actin filament) muscle Z-line, β
799685 Capping protein (actin filament) muscle Z-line, α 1
773758 Similar to actin related protein 2/3 complex subunit 3
Cluster 5 Enrichment Score: 3.1707699720411564 P Value
UNIPROT_ID Protein Name 4.18E-09
777280 Adaptor-related protein complex 2, β 1 subunit
797086 Adaptor-related protein complex 2, α 1 subunit
789199 Adaptor-related protein complex 2, mu 1 subunit
783500 Clathrin, heavy chain (Hc)
Cluster 6 Enrichment Score: 2.0551591175667023 P Value
UNIPROT_ID Protein Name 9.54E-11
794683 ADP-ribosylation factor 4
824702 RAB13, member RAS oncogene family; similar to hCG24991
823808 RAB10, member RAS oncogene family
819977 RAB1B, member RAS oncogene family
825257 ADP-ribosylation factor 6
803034 ADP-ribosylation factor 1
Table W8. (continued )
342.e14 PIP Is Required for Cell Cycle Progression Naderi and Vanneste Neoplasia Vol. 16, No. 4, 2014