EpCAM Signaling Promotes Tumor Progression and Protein Stability of PD-L1 through the EGFR Pathway

Abstract

Although epithelial cell adhesion molecule (EpCAM) has previously been shown to promote tumor progression, the underlying mechanisms remain largely unknown. Here, we report that the EGF-like domain I within the extracellular domain of EpCAM (EpEX) binds EGFR, activating both AKT and MAPK signaling to inhibit forkhead transcription factor O3a (FOXO3a) function and stabilize PD-L1 protein, respectively. Treatment with the EpCAM neutralizing antibody, EpAb2-6, inhibited AKT and FOXO3a phosphorylation, increased FOXO3a nuclear translocation, and upregulated high temperature requirement A2 (HtrA2) expression to promote apoptosis while decreasing PD-L1 protein levels to enhance the cytotoxic activity of CD8+ T cells. In vivo, EpAb2-6 markedly extended survival in mouse metastasis and orthotopic models of human colorectal cancer. The combination of EpAb2-6 with atezolizumab, an anti-PD-L1 antibody, almost completely eliminated tumors. Moreover, the number of CD8+ T cells in combination-treated tumors was increased compared with atezolizumab alone. Our findings suggest a new combination strategy for cancer immunotherapy in patients with EpCAM-expressing tumors.

Significance
This study shows that treatment with an EpCAM neutralizing antibody promotes apoptosis while decreasing PD-L1 protein to enhance cytotoxic activity of CD8+ T cells.

Full text

CANCER RESEARCH | TUMOR BIOLOGY AND IMMUNOLOGY
EpCAM Signaling Promotes Tumor Progression and
Protein Stability of PD-L1 through the EGFR Pathway
Hao-Nien Chen, Kang-Hao Liang, Jun-Kai Lai, Chun-Hsin Lan, Mei-Ying Liao, Shao-Hsi Hung,
Yi-Ting Chuang, Kai-Chi Chen, William Wei-Fu Tsuei, and Han-Chung Wu
ABSTRACT
◥
Although epithelial cell adhesion molecule (EpCAM) has pre-
viously been shown to promote tumor progression, the underlying
mechanisms remain largely unknown. Here, we report that the
EGF-like domain I within the extracellular domain of EpCAM
(EpEX) binds EGFR, activating both AKT and MAPK signaling to
inhibit forkhead transcription factor O3a (FOXO3a) function and
stabilize PD-L1 protein, respectively. Treatment with the EpCAM
neutralizing antibody, EpAb2-6, inhibited AKT and FOXO3a
phosphorylation, increased FOXO3a nuclear translocation, and
upregulated high temperature requirement A2 (HtrA2) expression
to promote apoptosis while decreasing PD-L1 protein levels to
enhance the cytotoxic activity of CD8
þ
T cells. In vivo, EpAb2-6
markedly extended survival in mouse metastasis and orthotopic
models of human colorectal cancer. The combination of EpAb2-6
with atezolizumab, an anti-PD-L1 antibody, almost completely
eliminated tumors. Moreover, the number of CD8
þ
T cells in
combination-treated tumors was increased compared with atezo-
lizumab alone. Our findings suggest a new combination strategy for
cancer immunotherapy in patients with EpCAM-expressing
tumors.
Significance: This study shows that treatment with an EpCAM
neutralizing antibody promotes apoptosis while decreasing PD-L1
protein to enhance cytotoxic activity of CD8
þ
T cells.
Introduction
Epithelial cell adhesion molecule (EpCAM) is the most frequently
expressed tumor-associated antigen; it is overexpressed in the majority
of human adenocarcinomas and squamous cell carcinomas and is
associated with poor prognosis in patients (1–4). EpCAM is sequen-
tially processed by a disintegrin and metalloproteinase 17 (ADAM17),
also called TNFaconverting enzyme (TACE), and g-secretase (5, 6).
Processing by these enzymes, respectively, releases the extracellular
domain (EpEX) and intracellular domain (EpICD). After release,
EpICD interacts with four and a half LIM domains protein 2 (FHL2)
and b-catenin to form a complex that translocates to the nucleus and
interacts with Lef-1, which binds to DNA (5, 6). The EpICD complex
promotes tumorigenesis of tumor-initiating cells through the upre-
gulation of reprogramming genes and epithelial–mesenchymal tran-
sition (EMT; ref. 7). In addition, the increased release of EpEX
enhances EpICD generation and upregulates the expression of repro-
gramming and EMT genes (5, 7–9). It was previously reported that
EpEX could directly bind to EGFR and stimulate EGFR phosphory-
lation and its downstream signaling pathway (5, 8, 10). Moreover,
EpEX-induced EGFR phosphorylation can activate ADAM17 and
g-secretase to further increase the shedding of EpEX and
EpICD (5, 8, 10). However, the domain of EpEX that binds and
activates EGFR has not been previously identified.
AKT is a primary mediator of EGFR signaling that promotes cell
survival partially by inactivating proapoptotic proteins. One such
proapoptotic protein that is inactivated by AKT phosphorylation is
forkhead transcription factor O3a (FOXO3a; ref. 11). FOXO3a is also
referred to as FKHRL-1 and is a member of the forkhead transcription
factor family (12). Because genes activated by FOXO proteins generally
function to limit cell growth and promote death, this family is thought
of as tumor suppressors. When activated, FOXO3a accumulates in the
nucleus, where it enhances the transcription of various genes involved
in apoptosis and the cell-cycle control, such as BIM, FasL, and p21 (13).
Furthermore, AKT inactivation is an essential step in anoikis, and
FOXO3a regulation by the PI3K/AKT pathway may be essential for
this inactivation (14, 15). Thus, AKT inactivation has been suggested
to be an essential step in apoptosis (11).
In addition to its mediation of survival signals, EGFR activation is
crucial for triggering immune escape (16). EGFR-activating mutations
were reported to be associated with increased PD-L1 expression in
mouse models of EGFR-driven lung cancer and bronchial epithelial
cells with the expression of mutant EGFR (16). Moreover, EGFR
inhibitors can reduce PD-L1 expression in non–small cell lung cancer
(NSCLC) cell lines with activated EGFR, suggesting that EGFR sig-
naling may trigger immune escape (16). Further investigations dem-
onstrated that EGFR ligands, such as EGF, induce PD-L1 expression
primarily at the level of posttranslational modifications. EGF was
shown to stabilize PD-L1 by inducing PD-L1 glycosylation, which
prevents GSK3-dependent proteasomal degradation of PD-L1 by
b-TrCP (17). Moreover, the MAPK signaling pathway, another impor-
tant axis of EGFR signaling, is associated with PD-L1 mRNA expres-
sion. It was reported that EGFR activation increases PD-L1 expression
through p-ERK1/2/p-c-Jun (18–20). In addition to promoting PD-L1
transcription, MAPK signaling also stabilizes PD-L1 mRNA by atten-
uating TPP activity (21). Because EpCAM is an activator of EGFR
signaling, EpCAM might promote escape from immune surveillance.
However, EpCAM-mediated immunosuppression and its mechan-
isms are almost completely undescribed.
Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei,
Taiwan.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
H.-N. Chen and K.-H. Liang contributed equally to this article.
Corresponding Author: Han-Chung Wu, Academia Sinica, 128 Academia Road,
Section 2, Nankang, Taipei 11529, Taiwan. Phone: 8862-2789-9528; Fax: 8862-
2788-0268; E-mail: hcw0928@gate.sinica.edu.tw
Cancer Res 2020;80:5035–50
doi: 10.1158/0008-5472.CAN-20-1264
2020 American Association for Cancer Research.
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In this study, we report that the EGF-like domain I within EpEX
binds to EGFR and activates both the AKT and ERK1/2 pathways.
EpEX-induced AKT activation inhibits FOXO3a activity, which
decreases high temperature requirement A2 (HtrA2) transcription.
Moreover, a therapeutic antibody directed against EpCAM (EpAb2-6)
promotes FOXO3a nuclear translocation, and increases HtrA2 expres-
sion. We also found that EpCAM inhibits antitumor immunity by
activating the PD-1/PD-L1 pathway to suppress T-cell function. EpEX
increases PD-L1 protein stability rather than mRNA level, while
blockade of EGFR or MAPK signaling pathways attenuates EpEX-
mediated stabilization of PD-L1 protein. Furthermore, treatment of
EpAb2-6 markedly prolongs the survival of mice in metastasis and in
orthotopic models of human colorectal cancer, and a combination of
atezolizumab and EpAb2-6 shows enhanced therapeutic efficacy and
high levels of tumor-localized CD8
þ
T cells in a peripheral blood
mononuclear cell (PBMC) cell line–derived xenograft (CDX) mouse
model. Our findings not only shed light on the molecular mechanisms
underlying EpCAM signaling in cancer malignancy, but also suggest
that therapeutic targeting of EpCAM may work well in combination
with current immunotherapies.
Materials and Methods
Chemicals and antibodies
Antibodies against human EpCAM and p84 were purchased from
Abcam, and antibody against b-catenin was from Santa Cruz Bio-
technology. Anti-a-tubulin antibody was from Sigma-Aldrich. Poly-
clonal antibodies detecting total ERK and Thr202/Tyr204-phosphor-
ylated ERK, total AKT and Ser473-phosphorylated AKT, total
FOXO3a, Ser253-phosphorylated FOXO3a, Ser318/321-phosphory-
lated FOXO3a, and The32-phosphorylated FOXO3a, and active (non-
phospho Ser33/Ser37/Thr41) b-catenin, HtrA2, cytochrome c, COX
IV, XIAP, and PD-L1 were from Cell Signaling Technology. U0126
(MEK inhibitor), wortmannin (PI3K inhibitor), and MG132 (protea-
some inhibitor) were obtained from Selleck Chemicals.
Cell culture
Human lung adenocarcinoma cells (H441), lung carcinoma cells
(H460), embryonic kidney cells (HEK293T), colorectal carcinoma
cells (HCT116), and colorectal adenocarcinoma cells (SW620) were
obtained from the ATCC. H441 and H460 cells were cultured in
RPMI1640 Medium (Gibco), and HEK293T, HCT116, and SW620
cells were cultured in DMEM (Gibco). All cells were maintained
in conditioned medium supplemented with 10% FBS (Gibco) and
100 mg/mL penicillin–streptomycin (Gibco) at 37C in a humidified
incubator with 5% CO
2
.
Western blotting
Cells were lysed with RIPA buffer (20 mmol/L Tris-HCl, pH 7.4,
150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Nonidet
P-40, 1% sodium deoxycholate, and 2.5 mmol/L sodium pyrophos-
phate) containing Protease Inhibitor (Roche) and Phosphatase Inhib-
itor (Roche) to extract whole-cell lysates. The lysates were then
quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher
Scientific). The lysates were mixed with 5sample buffer (50 mmol/L
Tris-HCl, pH 6.8, 2% SDS, b-mercaptoethanol, 0.1% bromophenol
blue, and 10% glycerol), separated by SDS-PAGE, and transferred to
polyvinylidene difluoride (PVDF) Membrane (Millipore). Nonspecific
antibody-binding sites on the PVDF membrane were blocked with 3%
BSA in TBST (TBS buffer containing 0.1% Tween 20) and membranes
were incubated with indicated antibody overnight at 4C, followed by
incubation with horseradish peroxidase–conjugated secondary anti-
bodies (Jackson ImmunoResearch Laboratories) at room temperature
for 1 hour. The protein bands were subsequently visualized with
Chemiluminescence Reagents (Millipore) and detected by UVP
BioSpectrum 600 Imagining System (UVP). The protein expression
was quantified by Gel-Pro Analyzer 3.1 (Media Cybernetics).
Production and purification of EpEX-6xHis recombinant protein
The DNA fragment encoding EpEX (amino acids 24–262 of
EpCAM) was amplified by PCR using PfuTurbo DNA polymerase
and cloned into pSecTag2 vector with C-terminal 6xHis tag to generate
pSecTag2-EpEX-6xHis. The EpEX-6xHis fusion protein was produced
with Expi293F Expression System (Thermo Fisher Scientific) and
purified by Ni-Affinity Column (GE Healthcare).
Extracellular interactions between EpEX-Fc and EGFR
HCT116 cells were harvested with 10 mmol/L EDTA in PBS, and
incubated with EpEX-Fc for 1 hour at 4C. After incubation, 2 mmol/L
DTSSP (Thermo Fisher Scientific) was used as a cross-linker to
stabilize the interaction between EpEX-Fc and EGFR. To stop the
cross-linking reaction, Tris, pH 7.5, was added to a final concentration
of 20 mmol/L. Membrane proteins were extracted using Mem-PER
Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Fisher
Scientific). Finally, the EpEX-Fc–EGFR complex was pulled down with
Dynabeads Protein G (Invitrogen), and probed by Western blotting.
Construction of the EpCAM EGF-like domain deletion
In its EpEX, EpCAM contains two EGF-like domains, at amino
acids 27–59 (first EGF-like domain) and 66–135 (second EGF-like
domain), and a cysteine-free motif (22). The EpCAM EGF-like
domain deletion was generated using the standard QuikChange
deletion mutation system with first forward mutagenic deletion primer
(50-GCAGCTCAGGAAGAATCAAAGCTGGCTGCC-30) and first
reverse mutagenic deletion primer (50-GGCAGCCAGCTTTGATTC-
TTCCTGAGCTGC-30); and second forward primer (50-AAGCTGG-
CTGCCAAATCTGAGCGAGTGAGA-30) and second reverse primer
(50-TCTCACTCGCTCAGATTTGGCAGCCAGCTT-30). The PCR
amplifications were performed using KAPA HiFi Hot Start DNA
Polymerase (Kapa Biosystems), and products were treated with restric-
tion enzyme, DpnI (Thermo Fisher Scientific), to digest methylated
parental DNAs.
Cycloheximide chase assay
Cycloheximide, a protein synthesis inhibitor, was used to evaluate
the stability of PD-L1. Cells were treated with cycloheximide for 0, 2, 4,
or 6 hours. Proteins were extracted and Western blot analysis was
performed to detect PD-L1 protein level.
Immunoprecipitation assay
Cells were lysed in lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 150
mmol/L NaCl, and 1% NP-40) with Protease Inhibitors (Roche). For
immunoprecipitation, cell lysates were incubated with antibodies for
6 hours at 4C. Then, 20 mL Dynabeads Protein G was added and the
mixture was incubated for 2 hours at 4C to pull-down the antibody-
bound protein. The immunoprecipitation samples were washed with
PBS three times, denatured in sample buffer, and analyzed by Western
blotting.
Generation of mAbs and purification of IgG
Generation of EpAb2-6 and control IgG was performed as described
previously (23). The protocol was approved by the Committee on the
Chen et al.
Cancer Res; 80(22) November 15, 2020 CANCER RESEARCH
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Ethics of Animal Experiments of Academia Sinica [Nankang, Taipei,
Taiwan, AS Institutional Animal Care and Use Committee (IACUC):
11-04-166].
Lentivirus-mediated short hairpin RNA knockdown
All lentiviral short hairpin RNA (shRNA) constructs were pur-
chased from the RNAi Core Facility at Academia Sinica (Nankang,
Taipei, Taiwan). Lentiviral production, infection, and selection were
performed according to protocols from the RNAi Consortium
(Academia Sinica, Nankang, Taipei, Taiwan). To produce the lenti-
virus, HEK293T cells were transiently cotransfected with shRNA
plasmid, packaging (pCMV-DR8.91) and envelope (pMD.G) expres-
sion plasmids, using PolyJet DNA Transfection Reagent (SignaGen
Laboratories). The next day, the transfection solution was replaced
with 1% BSA containing medium to improve virus yield. Two days
after transfection, medium containing lentivirus was collected. Cells
were cultured in lentivirus-containing medium, supplemented with
8mg/mL polybrene, for another 48 hours. The transduced cells were
selected with 2 mg/mL puromycin for 4 days and the knockdown
efficiency was measured by Western blotting. The target sequence for
human EpCAM–specific shRNA was shRNA 50-GCAAATGGACA-
CAAATTAC AA-30. Luciferase shRNA (shLuc) was used as a negative
control.
RNA extraction, cDNA synthesis, quantitative reverse-
transcription PCR
Total RNA extraction, first-strand cDNA synthesis, and SYBR
Green–based real-time PCR were performed as described in the
manufacturer’s instructions. To extract total RNA, cells were lysed
using TRIzol Reagent (Invitrogen), and proteins and phenol were
removed from TRIzol using chloroform. After centrifugation, the
top colorless layer was collected and mixed with isopropanol to
precipitate RNA pellet. Then, the RNA pellet was washed with
70% ethanol, air-dried at room temperature, and dissolved in
RNase-free water. For first-strand cDNA synthesis, 5 mgoftotal
RNA was used for reverse transcription with oligo(dT) primer
and SuperScriptIII Reverse Transcriptase (Invitrogen) at 50Cfor
60 minutes. Target gene levels were evaluated by quantitative PCR
(qPCR), using LightCycler 480 SYBR Green I Master Mix (Roche)
and a LightCycler480 System (Roche). GAPDH mRNA expression
was measured as endogenous housekeeping control to normalize
all qPCR reactions. The qPCR reaction was as follows: 95Cfor
5 minutes, followed by 40 cycles of denaturation at 95Cfor
10 seconds, annealing at 60C for 10 seconds, and extension at
72C for 30 seconds. Final results were calculated from three
independent experiments. Primer sequences used to detect the
mRNA expression of genes of interest are listed in Supplementary
Table S1.
Immunofluorescence assay
For immunofluorescence, 2 10
4
cells were seeded on 12-mm
cover slips in 24-well culture plate, fixed with 2% paraformalde-
hyde, and incubated with 0.1% Triton X-100 to increase the
permeability of cell membrane. Then, samples were blocked with
1% BSA for 1 hour at room temperature and stained with
primary antibodies at 4C overnight. Slides were washed and
stained with FITC or Alexa568-conjugated secondary antibodies
and DAPI for 40 minutes. Cover slips were then washed with
PBS and mounted in Mounting Solution (Vector Laboratories).
The slides were examined with a confocal microscope (Leica
TCS-SP5).
Apoptosis protein array
HCT116 cells were stimulated with 20 mg/mL EpAb2-6 for 6 hours,
and apoptosis arrays were performed according to the manufacturer’s
instructions (R&D System; ARY009). The membranes (arrays) were
detected by the UVP BioSpectrum 600 Imagining System (UVP). The
arrays were quantified by a Gel-Pro Analyzer 3.1 (Media Cybernetics,
Inc.).
Flow cytometry analysis
Cells were harvested, washed, and suspended in FACS buffer (PBS
with 1% FBS), and 10
5
cells were transferred to 96-well shrill-based
plates. Cells were stained with different antibodies for 1 hour at 4C,
and then subsequently stained with indicated PE-conjugated second-
ary antibodies for 1 hour at 4C. Then, cells were washed with FACS
buffer twice and suspended in 400 mL FACS buffer. Florescence signals
were analyzed using flow cytometry (BD FASCSC AntoII) and mea-
sured with FCS Express V3 software. The data were collected from
three independent experiments.
Chromatin immunoprecipitation
In brief, HCT116 cells (1 10
5
) were treated with 20 mg/mL EpAb2-
6 for 6 hours, followed by cross-linking fixation in 1% formaldehyde.
Fixation was quenched by the addition of glycine to a final concen-
tration of 200 mmol/L and fixed chromatin complexes were then
sonicated to an average length of 250 bp using an MISONIX Sonicator
3000. The sonicated protein–DNA complexes were subjected to
immunoprecipitation using 2 mg of antibodies against FOXO3a. The
immunoprecipitated DNA was recovered by a PCR Purification Kit
(Qiagen), and the amount of target DNA was detected by PCR using
the primers complementary to the sequence listed in Supplementary
Table S2.
Dual-luciferase reporter assays
The human HtrA2 proximal promoter fragments covering the
regions 1628 to þ86 and 700 to þ86 (with the transcriptional
start site denoted as þ1) were PCR amplified and then cloned into the
firefly luciferase reporter plasmid pGL4.18 Vector (Promega). To
assess the effect of EpA2-6 on HtrA2 promoter activity, HCT116 and
SW620 cells (1 10
4
/well), seeded onto 24-well dishes, were tran-
siently transfected with HtrA2 promoter reporter constructs in com-
bination with a plasmid expressing Renilla luciferase using the PolyJet
Transfection Reagent (SignaGen Laboratories) for 24 hours, followed
by 20 mg/mL EpAb2-6 for 6-hour stimulation. Cell lysates were
prepared and subjected to the luciferase activity assay using the
Dual-Luciferase Reporter Assay Kit (Promega). Firefly luciferase
activity was normalized to Renilla luciferase activity, and final data
were presented as the fold induction of luciferase activity compared
with EpAb2-6-IgG controls. Data are expressed as means SD from
three independent experiments. Primers used to amplify the human
HtrA2 proximal promoter fragment are listed in Supplementary
Table S3.
Apoptosis and mitochondrial membrane potential assay
Cells (2 10
5
) were seeded onto 24-well dishes and treated with
20 mg/mL control IgG or EpAb2-6 for 6 hours. The apoptotic cell and
mitochondrial membrane potentials were detected using FITC
Annexin V Apoptosis Detection Kit (BD Biosciences) and MitoStatus
Red (BD Biosciences), respectively. The apoptotic cell and mitochon-
drial membrane potentials were analyzed using a Flow Cytometer
(Thermo Fisher Scientific). Each measurement was carried out
at least three times to ensure reproducibility. The effect of gene
EpCAM Signaling Regulates Tumorigenesis
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knockdown on EpAb2-6–induced apoptosis is represented in Supple-
mentary Table S4.
IHC
Human lung cancer tissue microarray was purchased from Super
BioChip. After exhausting the endogenous hydroperoxidases with 3%
hydrogen peroxide in methanol for 30 minutes, the tissue microarray
was blocked with 1% BSA for 1 hour and exposed to anti-PD-L1 (Clone
28-2, Abcam) or EpAb3-5 (developed by our laboratory as described
previously; ref. 23) at 4C overnight. After washing with PBST, the
tissue microarray was processed using the standard procedure of Super
Sensitive IHC Detection System (Bio-Genex) and stained by DAB.
Nuclei were stained with Mayer’s hematoxylin solution (Wako).
PBMC and T-cell isolation and culture
For the experiments using PBMCs, blood samples were drawn from
healthy donors and collected into 10 mL Vacutainer tubes containing
the anticoagulant EDTA (BD Bioscience). After centrifugation at
1,500 rpm for 10 minutes, plasma was removed from the sample and
mixed with an equal volume of PBS. The mixture was layered on Ficoll-
Paque plus (Ficoll:blood ¼1:2) and centrifuged at 1,500 rpm for 30
minutes. The PBMC layer (buffy coat) was collected and washed with
PBS with 0.5% BSA and 2 mmol/L EDTA twice. To obtain purified
CD3
þ
T cells, PBMCs were positively selected by anti-CD3 magnetic
beads (MACS), according to the standard protocol. Isolated CD3
þ
T cells (1 10
6
) were cultured and activated by 25 mL anti-CD3/
anti-CD28–coated Dynabeads (Invitrogen) in RPMI1640 medium
supplemented with 10% FBS, 100 mg/mL penicillin–streptomycin,
12.5 ng/mL IL2 (Gibco), and 1 ng/mL IL15 (MACS) for 48 hours.
The protocol was approved by the Institutional Review Board (IRB)
of Academia Sinica (Nankang, Taipei, Taiwan, AS IRB: AS-IRB01-
19049).
Cell viability assays in vivo
NOD/SCID mice were intravenously injected with HCT116-GFP
cells; mice bearing circulating HCT116-GFP cells were intravenously
treated with EpAb2-6 or an equivalent dosage of control IgG at 1 hour
after cell injection (antibody was delivered at 20 mg/kg). Then, blood
samples were obtained from the facial vein of mice and the fluores-
cence intensity of whole blood was quantified at the indicated time-
points. The fluorescence was measured with a Microplate Reader
(Molecular Devices, SpectraMax M5) at an excitation wavelength of
355 nm and emission wavelength of 440 nm.
Subcutaneous human colorectal cancer studies
Subcutaneous studies were performed as reported previously (23).
The protocol was approved by the Committee on the Ethics of Animal
Experiments of Academia Sinica (Nankang, Taipei, Taiwan, AS
IACUC: 11-04-166).
Orthotopic implantation and therapeutic studies
Orthotopic studies were performed as reported previously (24).
Briefly, NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were used
for orthotopic implantation of HCT116 cells, which were previously
infected with Lenti-luc virus (lentivirus containing luciferase genes).
The mice were anesthetized by intraperitoneal injection of avertin,
2,2,2-Tribromo-Ethanol (Sigma-Aldrich) at a dose of 250 mg/kg.
Tumor development was monitored by bioluminescence imaging. For
the orthotopic therapeutic study, tumor-bearing mice were treated
with control IgG or EpAb2-6 (20 mg/kg). Tumor progression was
monitored by quantification of bioluminescence. Mouse body weight
and survival rate were measured. Animal care was carried out in
accordance with the guidelines of Academia Sinica (Nankang, Taipei,
Taiwan). The protocol was approved by the Committee on the Ethics
of Animal Experiments of Academia Sinica (Nankang, Taipei, Taiwan,
AS IACUC: 11-04-166).
Gene set enrichment analysis
The gene expression matrix was obtained from LUAD projects in
The Cancer Genome Atlas. The 25% of samples with highest EpCAM
expression were defined as the “EpCAM_High”group and the
25% samples with least EpCAM expression were defined as the
“EpCAM_Low”group. T-cell activation and proliferation gene sets
provided by gene set enrichment analysis (GSEA) website were then
used to analyze the data.
Statistical analyses
All data are represented as means SD from at least three
independent experiments. Significant differences from the respective
control for each experimental condition were calculated using
Student ttest, unless otherwise specified. ,P<0.05; ,P<0.01; or
,P<0.001 are indicated as significant. Survival analysis was
performed using a log-rank test. The correlation coefficient was
calculated using Spearman analysis.
Results
EpEX binds to EGFR through its EGF-like domain I
Previously, we reported that EpEX can bind to EGFR (10), and
functions as a growth factor and induces EGFR phosphorylation (5, 10).
To verify that EpEX interacts with EGFR, we used the cross-linker,
DTSSP, to stabilize the EpEX–EGFR complex. The binding of EpEX to
EGFR was confirmed by immunoprecipitation and Western blotting
(Fig. 1A). To evaluate the direct binding of recombinant EpEX to the
EpEX of EGFR (EGFR
ECD
), we performed ELISA to probe the direct
interaction between purified EpEX and EGFR
ECD
protein (Fig. 1B).
To test whether membrane-bound EpCAM could bind to EGFR, we
performed immunoprecipitation experiments using HEK293T cells
with EpCAM-V5 and EGFR-Flag overexpression. The interaction
between exogenous EpCAM and EGFR was detected by coimmuno-
precipitation (Fig. 1C). In EpCAM EpEX, it contains two EGF-like
domains, at amino acids 27–59 (first EGF-like domain) and 66–135
(second EGF-like domain), and a cysteine-free motif (22). To identify
the specific region of EpEX that binds to EGFR, we constructed
different EGF-like domain–deleted EpCAM mutants. Surprisingly,
the EGF-like domain I–deleted EpCAM mutant (EpCAM
DEGFI
)
showed decreased binding to EGFR, while the EGF-like domain II–
deleted EpCAM mutant (EpCAM
DEGFII
) exhibited an enhanced inter-
action with EGFR (Fig. 1D). To further evaluate the binding of soluble
EpEX to EGFR
ECD
, HEK293T cells were cotransfected with different
vectors expressing soluble EGFR
ECD
-Flag or EpEX-Fc, and the protein
complex was examined in culture media (Fig. 1E). Soluble EGF-like
domain II–deleted EpEX-Fc (EpEX
DEGFII
-Fc) showed significantly
increased affinity, while EGF-like domain I–deleted EpEX-Fc
(EpEX
DEGFI
-Fc) exhibited decreased affinity for EGFR
ECD
-Flag com-
pared with EpEX-Fc (Fig. 1F). Overall, we found that the membrane-
bound EpCAM and secreted EpEX both bind EGFR and deletion of the
EGF domain I diminished this binding (Fig. 1C–F).
Similar results were observed using purified wild-type or mutant
EpEX and EGFR
ECD
recombinant proteins. The recombinant
EpEX
DEGFII
protein had a stronger binding affinity for EGFR
ECD
than
wild-type controls, and the EpEX
DEGFI
protein lost its ability to bind
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Figure 1.
EpEX bindsto EGFR through its EGF-likedomain I. A, Immunoprecipitation (IP)of affinity cross-linkedEpEX-Fc bound to EGFR from HCT116cells. B, EpEX-Fc was added
to EGFR
ECD
-coated ELISA plates and detected by TMB (3,30,5,50-tetramethylbenzidine) colorimetric peroxidase assay. C, 293T cells were transfected with EGFR-Flag
and EpCAM-V5. Immunoprecipitation was performed with anti-V5 antibody (left) or anti-Flag antibody (right), followed by Western blotting. D, 293T cells were
transfected with EGFR-Flag and EGF domain–deleted mutant EpCAM-V5. The protein interaction was probed by immunoprecipitation with anti-V5 antibody and
Western blotting with anti-Flag antibody. E, In culture media from 293T cells with EGFR
ECD
-Flag and EpEX-Fc transfection, immunoprecipitation was performed with
DynabeadsProtein G and detected byWestern blotting withanti-Flag antibody.F, 293T cells were transientlytransfected with EGFR
ECD
-Flag and EGFdomain–deleted
mutant EpEX-Fc. The EGFR
ECD
-Flag and mutant EpEX-Fc interaction was examined by immunoprecipitation with Dynabeads Protein G and Western blotting with
anti-Flag antibody. G, Purified EGFR
ECD
-6xHis and EGF domain–deleted mutant EpEX-Fc were incubated and immunoprecipitation was performed with Dynabeads
Protein G and detected by Western blotting. H, HCT116 cells were starved and treated with wild-type or EGF domain–deleted mutant EpEX, and EGFR signaling was
analyzed by Western blotting.N¼3 independent experiments. Graphical data are shown as meanSEM. ,P<0.001, two-tailed Studentttest. IB, immunoblotting.
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EGFR
ECD
(Fig. 1G). Moreover, the EpEX
DEGFII
protein induced EGFR
signaling like the wild-type control, but EpEX
DEGFI
protein did not
(Fig. 1H). These results suggest that the EGF-like domain I in EpEX is
the major domain responsible for the EpEX–EGFR interaction and
activation of EGFR signaling.
Inhibiting EpEX increases FOXO3a nuclear accumulation and
induces apoptosis
Because we found that EpEX activates EGFR through EpEX domain
I, we further investigated whether EpEX-mediated EGFR activation is
important for cancer progression. AKT is a major downstream effector
of EGFR, which promotes cell survival partially by inactivating
FOXO3a (12). Inhibition of AKT/FOXO3a signaling has been sug-
gested an essential step in apoptosis (11) and could inhibit tumor-
sphere formation of the SKOV3 ovarian cancer cell line (25). To
evaluate whether EpEX affects FOXO3a function, we examined
FOXO3a phosphorylation and intracellular location. Immunoblotting
analysis demonstrated that EpEX induced the phosphorylation of AKT
and FOXO3a in a time-dependent manner (Fig. 2A), and EpEX-
induced phosphorylation of FOXO3a was inhibited by AG1478 (EGFR
inhibitor) or wortmannin (PI3K inhibitor; Fig. 2B). Moreover, after
EpEX treatment, FOXO3a translocated from the nucleus to the
cytoplasm (Fig. 2C). Because the EpEX acted through EGFR, we used
EGF, a well-known ligand for EGFR, as an inducer. Similar to EpEX
treatment, EGF induced nuclear exclusion of FOXO3a (Supplemen-
tary Fig. S1A–S1C). Therefore, we conclude that EpEX inhibits the
FOXO3a activity through the EGFR–AKT pathway.
Previously, we had developed a neutralizing antibody, EpAb2-6,
which targets EpEX and induces apoptosis (5, 23). EpAb2-6 was
employed to block the EpEX. By blocking the function of EpEX,
EpAb2-6 treatment is known to interrupt the EpEX/EGFR/ADAM17
axis, which represents a positive feedback loop promoting EpCAM
cleavage and subsequently increases EpEX and EpICD production (5).
Indeed, decreases in ADAM17 and g-secretase activity and soluble
EpEX release were observed after EpAb2-6 treatment (Supplementary
Fig. S2A–S2C). Moreover, nuclear-activated b-catenin and down-
stream genes, including reprogramming genes and EMT-related
genes, were also decreased in EpAb2-6–treated cells (Supplementary
Fig. S2D and S2E). Next, we examined whether EpCAM contributes to
anchorage-independent growth. For this purpose, a soft agar colony
formation assay was performed with colon cancer cell lines. EpAb2-6–
treated cells formed significantly smaller and fewer colonies compared
with the control cells (Supplementary Fig. S2F). We further cultivated
these attached colon cancer cells into tumorspheres and found that
EpAb2-6 treatment inhibited tumorsphere formation (Supplementary
Fig. S2G). These results suggest that targeting EpCAM with a specific
antibody, EpAb2-6, is a potentially viable strategy to suppress colon
cancer malignancy.
It has been previously reported that the nuclear accumulation of
FOXO3a promotes cancer cell apoptosis after treatment with various
chemotherapy drugs or EGFR inhibitors (26–28). Thus, we wanted to
clarify whether inhibiting the function of EpEX facilitates nuclear
accumulation of FOXO3a. Blocking the function of EpEX with EpAb2-
6 treatment also decreased the phosphorylation of AKT and FOXO3a
(Fig. 2D) and subsequently increased nuclear accumulation of
FOXO3a, while diminishing the nuclear translocation of b-catenin
(Fig. 2E). The downstream genes of FOXO3a (BIM, p21, and FASL)
were also upregulated in HCT116 cells with EpAb2-6 treatment
compared with IgG control (Fig. 2F). Using immunofluorescence
staining, FOXO3a and b-catenin were detected in both the cytoplasm
and nucleus of colon cancer cells. However, after EpAb2-6 treatment,
FOXO3a was increased in the nucleus, but nuclear b-catenin was
decreased both in vitro (Fig. 2G) and in vivo (Fig. 2H). Thus,
inhibiting EpEX enhances the nuclear translocation of FOXO3a and
consequently upregulates downstream genes, which are involved in
promoting apoptosis.
To evaluate whether EpAb2-6 induces apoptosis via EGFR/AKT/
FOXO3a, we used shRNA to knockdown EGFR, AKT, and FOXO3a
expression in colon cancer cells and examined the effect of EpAb2-6 on
cancer cell apoptosis. EGFR–, AKT–, and FOXO3a-knockdown cells
were all less sensitive to EpAb2-6–induced apoptosis than control cells,
and EpAb2-6–induced apoptosis was completely abrogated in
EpCAM-knockout cells (Supplementary Fig. S3A–S3D). Together,
our data show that EpEX induces phosphorylation of FOXO3a and
inactivates its biological function; meanwhile, blocking the function of
EpEX inhibits the phosphorylation of FOXO3a to promote nuclear
accumulation of FOXO3a and activates expression of its downstream
proapoptotic genes.
HtrA2 acts downstream of FOXO3a and contributes to
EpAb2-6–induced apoptosis
To gain further insight into the mechanisms of EpAb2-6–induced
apoptosis, we compared the expression of 35 apoptosis-related sig-
naling proteins in control IgG- and EpAb2-6–treated colon cancer
cells. We found that among the 35 apoptosis-related proteins, two
showed substantial increases in expression after EpAb2-6 treatment
compared with the IgG control. These proteins were HtrA2 and
X-linked inhibitor of apoptosis protein (XIAP; Fig. 3A).
Immunoblotting and qPCR experiments showed that EpAb2-6
treatment enhanced the protein and mRNA expression of HtrA2 in
HCT116 cells (Fig. 3B). According to previous studies, HtrA2 can be
released into the cytosol, where it contributes to apoptosis (29). As
expected, we found that EpAb2-6 induced the release of cytochrome c
and HtrA2 from mitochondria into the cytosol (Fig. 3C). Further-
more, EpAb2-6 treatment induced the dissipation of the mitochon-
drial membrane potential (Fig. 3D).
HtrA2 mRNA levels were increased after EpAb2-6 treatment,
implying transcriptional regulation of HtrA2 gene was induced by
EpAb2-6. To examine this hypothesis, HCT116 cells were transiently
transfected with a reporter for the HtrA2 promoter; a region encom-
passing 1,704 bases upstream of the HtrA2 translational start site was
used to drive the expression of the firefly luciferase gene (pGL4.18-
HtrA2-1;Fig. 3E). EpAb2-6 treatment led to an approximate 4-fold
induction in HtrA2 promoter activity, compared with the IgG control,
indicating that the HtrA2 gene is indeed a transcriptional target of
EpAb2-6–induced signaling (Fig. 3E). To further elucidate the molec-
ular mechanism controlling HtrA2 gene transcription, the HtrA2
promoter sequence was analyzed using the PROMO virtual laboratory
website. This analysis uncovered putative cis-acting response elements
(1283 to 1299) for FOXO transcription factors. Interestingly, to
our knowledge, there is no previously published evidence showing that
FOXO3a controls HtrA2 promoter activity. Thus, we examined
whether HtrA2 promoter activity was regulated by FOXO3a by
generating a reporter construct without the FOXO3a response element
(pGL4.18-HtrA2-2), as depicted in Fig. 3E. Indeed, the EpAb2-6–
induced increase in luciferase activity was abolished in the absence of a
FOXO3a response element (Fig. 3E).
To further elucidate whether FOXO3a directly regulates the HtrA2
promoter, chromatin immunoprecipitation (ChIP) analysis was per-
formed using primers flanking the putative FOXO3a response ele-
ments (1283 to 1299). FOXO3a occupied the HtrA2 promoter after
EpAb2-6 treatment, which was not observed in FOXO3a-knockdown
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Figure 2.
EpEX prevents cell apoptosis via inhibition of FOXO3a in colon cancer cells. A, HCT116 cells were starved and then treated with 2.5 mg/mL EpEX-Fc for the indicated
times. Total cell lysate was examined by Western blotting. B, After treating with AG1478 (5 mmol/L) or wortmannin (1 mmol/L) for 1 hour, cells were exposed to EpEX
(2.5 mg/mL) for 1 hour. The phosphorylation of FOXO3a was examined by Western blotting. C, The level of FOXO3a in nuclear fractions of EpEX-treated cells was
probed (top). Serum-starved HCT116 cells were treated with EpEX-Fc for 2 hours and the location of FOXO3a was assessed by immunofluorescence (bottom).
D, HCT116 cells were starved, pretreated with 20 mg/mL EpAb2-6, and then treated with EpEX for 15 minutes. The phosphorylation of AKT and FOXO3 awas analyz ed
by Western blotting. E, HCT116 cells were treated with 20 mg/mL EpAb2-6 for 6 hours, and b-catenin and FOXO3a were detected in the nuclear fraction of HCT116
cells. F, qPCR analysis of FOXO3a-related gene expression (BIM, p21,andFasL) in control IgG- or EpAb2-6–treated HCT116 cells. Gand H, HCT116 cell lines were
treated with EpAb2-6 (in vitro;G) and mice bearing HCT116 subcutaneous xenograft were treated with EpAb2-6 (in vivo;H). Arrows, cellular localization of FOXO3a
in the nucleus. Quantification of the number of cells with nuclear FOXO3a and b-catenin is shown when treated with EpAb2-6 in vitro (G)andin vivo (H; mean SD,
N¼200–300 cells in each group; ,P<0.05). Bar graphs show mean SEM. ,P<0.05; ,P<0.01.
EpCAM Signaling Regulates Tumorigenesis
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cells (Fig. 3F). These results indicate that FOXO3a increased HtrA2
transcription through direct binding to the HtrA2 promoter. We next
investigated whether HtrA2 plays an important role in EpAb2-6–
induced cell death. In this experiment, we used shRNA to knockdown
HtrA2 expression in colon cancer cells. In HtrA2-knockdown cells,
EpAb2-6 treatment induced fewer apoptotic cells (Fig. 3G) and less
disruption of mitochondrial membrane potential (Fig. 3H) than shLuc
(control knockdown) cells. Hence, FOXO3a-mediated HtrA2 tran-
scription appears to be involved in EpAb2-6–induced apoptosis.
EpCAM expression is positively correlated with PD-L1
stabilization
EGFR activation was reportedly associated with increased PD-L1
expression in mouse models of EGFR-driven lung cancer and bron-
chial epithelial cells with mutant EGFR expression, suggesting that
EGFR signaling is crucial for triggering immune escape (16). Further
investigations then demonstrated that increased EGFR signaling
upregulates PD-L1 expression by various mechanisms (17, 19, 21).
Moreover, EpCAM plays a crucial role in EGFR activation (5, 8, 10).
Figure 3.
EpEX prevents apoptosis by inhibiting FOXO3a-mediated HtrA2 expression. A, HCT116 cells were treated with control IgG or EpAb2-6 for 6 hours, and apoptosis-
related proteins were probed by the human apoptosis array kit. B, Expression of HtrA2 was examined by immunoblotting and qPCR analysis. C, EpAb2-6 induced the
release of mitochondrial apoptogenic proteins, HtrA2 and cytochrome c, in colon cancer cells. D, The percentage of cells with mitochondrial membrane potential
(Dcm) depolarization was measured after EpAb2-6 treatment. E, HCT116 cells with transfection of human HtrA2 proximal promoter–driven luciferas e reporter were
treated with control IgG or EpAb2-6. Luciferase activity assays were performed to evaluate the activity of the HtrA2 promoter. Results are expres sed as fold induction
compared with the IgG control. Statistical differences were determined by one-way ANOVA and Bonferroni multiple comparison test. F, Quantitative ChIP analysis of
FOXO3a binding to HtrA2 promoters was performed. G, HtrA2-knockdown HCT116 cells were treated with EpAb2-6, and the apoptotic cells were quantified by
fluorescein Annexin V-FITC/propidium iodide double labeling. Statistical differences were determined by one-way ANOVA and Bonferroni multiple comparison test.
H, HtrA2-knockdown HCT116 cells were treated with EpAb2-6, and the percentage of cells with mitochondrial membrane potential (Dcm) depolarization was
measured. Statistical differences were determined by one-way ANOVA and Bonferr oni multiple comparison test. N¼3 independent experiments. Graphical data are
shown as mean SEM. ,P<0.05; ,P<0.01; ,P<0.001. TSS, transcription start site.
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On the basis of these findings, we decided to investigate whether
EpCAM regulates PD-L1 expression via EGFR activation. Because
HCT116 cells expression of PD-L1 is extremely low, H441 cells with
high expression of PD-L1 were substituted for HCT116 cells as an
experimental model for further studies.
First, we wanted to clarify whether EpCAM is involved in escape
from immune surveillance. Immunodeficient NSG mice carrying
shLuc H441 xenografts in the left side and shEpCAM H441 xenografts
in the right side were intravenously injected with PBMCs to recon-
stitute immune cells. EpCAM-knockdown cells exhibited decreased
tumor weight, confirming that EpCAM promotes tumor progression
(Fig. 4A and B). Interestingly, shLuc tumors in mice with PBMC
injection were not different from shLuc tumors in mice without
PBMCs. However, shEpCAM tumors with PBMCs were smaller than
those without PBMCs, implying that EpCAM might inhibit antitumor
immunity (Fig. 4A and B). By flow cytometry analysis, we found that
CD8
þ
T cells were enriched in shEpCAM tumor tissue (Fig. 4C), and
Western blot analysis demonstrated that PD-L1 expression was
diminished in shEpCAM tumor tissue (Fig. 4D). These results indicate
that EpCAM promotes PD-L1 expression and reduces tumor-
associated CD8
þ
T cells in vivo.
To investigate the correlation between EpCAM and PD-L1 expres-
sion, we analyzed PD-L1 mRNA expression in two NSCLC cell lines,
H441 (high EpCAM expression) and H460 (no EpCAM expression).
Compared with H441 cells, H460 cells exhibited a higher level of PD-
L1 mRNA, but a lower level of PD-L1 protein (Fig. 4E). Flow
cytometry analysis of PD-L1 and EpCAM confirmed this finding
(Fig. 4F). Because many previous studies had indicated that the
protein stability of PD-L1 plays an important role in its
expression (17, 30–32), and, on the basis of our results, we suspected
that stability of PD-L1 might serve as a key regulator of protein level.
Indeed, a cycloheximide chase assay indicated that the half-life of PD-
L1 in H460 cells was reduced compared with that in H441 cells
(Fig. 4G). These findings suggest that the low PD-L1 protein level
in H460 cells results from reduced stability of PD-L1 protein.
To investigate whether EpCAM influences PD-L1 protein stability,
we overexpressed EpCAM in H460 cells (low endogenous PD-L1
expression and no EpCAM expression). We found EpCAM expression
increased PD-L1 protein level, but did not change its mRNA level
(Supplementary Fig. S4A and S4B). A cycloheximide chase assay
confirmed that PD-L1 protein half-life was increased by EpCAM
overexpression (Supplementary Fig. S4C). In addition, knockdown
of EpCAM in H441 cells (high endogenous EpCAM expression)
decreased PD-L1 protein level and protein half-life, but did not change
mRNA level (Fig. 4H and I). Consistent with this idea, H441 cells with
cytomegalovirus promoter–driven overexpression of PD-L1-Myc,
which lack endogenous regulatory elements, such as the PD-L1
promoter, 30untranslated region (UTR), and 50UTR, also exhibited
decreased PD-L1 protein when EpCAM was knocked down (Fig. 4J).
Furthermore, to validate our findings in samples from human patients
with cancer, PD-L1 and EpCAM protein levels were examined in both
lung and colon tumor specimens. Similar to our results in lung cancer
cell lines, the PD-L1 protein level was highly correlated with EpCAM
expression in a lung tumor tissue array (Fig. 4K). On the other hand,
most of the specimens in the colon tissue array showed only weak
staining for PD-L1, which prevented us from making a clear conclu-
sion, even though the signal for EpCAM was strong (Supplementary
Fig. S5A and S5B). Furthermore, GSEA indicated the related genes of
T-cell activation and proliferation enrichment in low EpCAM expres-
sion of lung cancer specimen (Fig. 4L). Accordingly, we concluded that
EpCAM expression is positively correlated to PD-L1 protein stability.
EpEX stabilizes PD-L1 through the EGFR–ERK pathway
Regulated intramembrane proteolysis triggers EpCAM-mediated
signal transduction through the dual actions of EpEX shedding by
ADAM17 and EpICD release by presenilin 2–containing g-secretase
complex (6). On the basis of the previous studies, EpEX is known to
function as an EGF-like growth factor, triggering EGFR signal-
ing (5, 8, 10). Moreover, activation of the EGFR signaling pathway
can increase PD-L1 expression through various mechan-
isms (17, 19, 21). Thus, we suspected that EpEX might regulate
PD-L1 expression through EGFR signaling. Moreover, EpICD associ-
ates with FHL2 and b-catenin in a complex that translocates to the
nucleus and transcribes downstream genes. Thus, we tested whether
EpEX or EpICD is sufficient to cause PD-L1 stabilization. To test
whether endogenous EpEX or EpICD is necessary for PD-L1 protein
stabilization, ADAM17 inhibitor (TAPI) and g-secretase inhibitor
(DAPT) were utilized to prevent the generation of endogenous EpEX
and EpICD, respectively. Blocking the shedding of endogenous EpEX,
but not EpICD, caused a reduction in PD-L1 protein level. Notably, no
changes in PD-L1 mRNA levels were observed, suggesting that
endogenous EpEX was crucial for PD-L1 protein stability (Fig. 5A).
Moreover, our results show that PD-L1 protein level was increased in a
dose- and time-dependent manner after H441 cells were treated with
EpEX or EGF (Fig. 5B and C). PD-L1 mRNA levels did not increase
after EpEX or EGF treatment (Fig. 5C). These results suggest that
EpEX can increase PD-L1 protein level, but EpICD cannot.
IFNgis known to induce PD-L1 expression through STAT family–
mediated transcription (33). We next wondered whether endogenous
EpEX and EpICD also participate in IFNg-mediated PD-L1 upregula-
tion. PD-L1 mRNA was upregulated in H441 cells treated with IFNg
and DMSO, IFNgand TAPI, and IFNgand DAPT (Supplementary
Fig. S6A). Surprisingly, the PD-L1 protein level was not increased as
much in the presence of TAPI as it was in the IFNgand DMSO–treated
cells (Supplementary Fig. S6B). In other words, despite the fact that
PD-L1 mRNA expression was elevated by IFNgstimulation, the
protein level could not be fully upregulated without generation of
EpEX. In addition, PD-L1 protein expression was not impaired in
IFNgand DAPT–treated cells (Supplementary Fig. S6B). While PD-L1
mRNA level was increased in all cells receiving IFNgtreatment, the
protein level was only upregulated in those cells with IFNgand EpEX
or IFNgand EGF treatment (Supplementary Fig. S6C and S6D). Thus,
EpEX appears to be an essential factor for PD-L1 protein upregulation.
The EGFR signaling pathway is important for PD-L1 expression in
cancer cells (16), and our previous study showed that EpEX can induce
EGFR signaling (5). Therefore, we speculated that EpEX might act
through EGFR signaling to prevent PD-L1 degradation. Indeed,
shRNA knockdown of EGFR in H441 cells attenuated EpEX- or
EGF-augmented PD-L1 levels (Fig. 5D). We next investigated which
EGFR downstream signaling pathway was involved in EpEX- and
EGF-mediated upregulation of PD-L1. Treatment with gefitinib abro-
gated both EpEX- and EGF-induced upregulation of PD-L1 protein
level. Importantly, U0126, which is an MEK inhibitor, but not an AKT
inhibitor, abolished EpEX-mediated upregulation of PD-L1 as well
(Fig. 5E). These results imply that, like EGF, EpEX-mediated upre-
gulation of PD-L1 requires signaling through the MAPK pathway.
Previous studies have shown that both ERK and AKT signaling
could regulate PD-L1 expression through different mechan-
isms (17, 19, 21). Hence, we wanted to know which signaling pathway
was more important for PD-L1 expression in our system. Inhibition of
ERK signaling, but not AKT signaling, decreased PD-L1 expression in
a time-dependent manner (Supplementary Fig. S7A). We then sought
to verify that the MAPK pathway affects PD-L1 protein stability. In the
EpCAM Signaling Regulates Tumorigenesis
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Figure 4.
EpCAM is correlated with PD-L1 expression. A, The tumor growth of shLuc or shEpCAM H441 cells in NSG mice with or without PBMC injection. B, Relative tumor size
was evaluated, and weights are shown in the graph. C, Evaluation of CD8
þ
T cells in tumor tissue. Dand E, Western blotting (D) and qRT-PCR analysis (E) of PD-L1 in
H460 and H441 cells. F, Flow cytometry analysis of PD-L1 and EpCAM in H460 and H441 cells. G, H460 and H441 cells were treated with 50 mmol/L cycloheximide
(CHX) for the indicated intervals and analyzed by Western blotting. Protein expression over time is shown in the gra ph. H, The protein and mRNA levels of PD-L1 in
EpCAM-knockdown H441 cells were analyzed by Western blotting and qRT-PCR , respectively. I, EpCAM-knockdown H441 cells were treated with 50 mmol/L
cycloheximide for the indicated intervals, and PD-L1 expression was analyzed by Western blotting. Protein expression over time is shown in the graph. J, The
expression of exogenous PD-L1 in EpCAM-knockdown H441 cells was analyzed by Western blotting. K, IHC staining for EpCAM and PD-L1 was performed in the lung
cancer tissue array, and the positive correlation between EpCAM and PD-L1 expression in patients with lung cancer is shown. L, GSEA enrichment profile of T-cell
activation and proliferation in the lung cancer specimen. Statistical differences were determined by two-tailed Student ttest. N¼6 independent experiments.
Graphical data are shown as mean SEM. ,P<0.01; ,P<0.001.
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Figure 5.
EpEX stabilizes PD-L1 proteinvia EGFR–MEK signaling. A, H441 cells were treated with TAPI (ADAM17 inhibitor)or DAPT (g-secretase inhibitor) for 24 hours, and PD-L1
expression was analyzedby Western blotting (left) and qRT-PCR (right). B, H441 cells were treatedwith the indicated concentrationsof EpEX or EGF for 1 hour and the
PD-L1 expression was analyzed by Western blotting.C, After treatment with EpEX (45 nmol/L) or EGF (16.5 nmol/L), for the indicated intervals, PD-L1 expression was
analyzed by Western blotting (left) or qRT-PCR (right).D, EGFR-knockdown H441 cells were treatedwith EpEX or EGF for 1 hour, and PD-L1 expressionwas analyzed
by Western blotting. E, PD-L1 wasdetermined by Western blotting in H441 cells pretreated with gefitinib, U1026,or wortmannin for 1 hour, followed by treatment with
EpEX or EGF for 1 hour. F, H441 cells were treated with EpAb2-6 or isotype for 16 hours, and PD-L1 expression was analyzed by Western blotting (top). PD-L1
expression was analyzed in EpAb2-6- or isotype-treated H441-derived xenograft tumors (bottom). G, After incubating cells with EpAb2-6 for 16 hours, the protein
half-life of PD-L1 was measured by treatment with 50 mmol/L cycloheximide (CHX) for theindicated intervals andWestern blotting analysis. Protein expression over
time is shown in the graph. H, H441 cells expressingPD-L1-eGFP-Flag were treated with EpAb2-6 for 16 hours and MG132 for 5 hours. The polyubiquitination of PD-L1-
eGFP-Flag was analyzed by Western blotting after immunoprecipitation (IP) with anti-Flag. I, MCF7, BT474, and Cal27 cells were treated with EpAb2-6or isotype for
16 hours, and PD-L1 expression was analyzed by Western blotting. J, After treatment with EpAb2-6 or isotype for 16 hours, H441 cells were cocultured with T cells for
16 hours and the apoptotic cells were quantifiedby fluorescein Annexin V-FITC/DAPI double staining. IB, immunoblotting. Statistical differences were determined by
two-tailed Student ttest. N¼6 independent experiments. Graphical data are shown as mean SEM. ,P<0.05; ,P<0.01; ,P<0.001.
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presence of U0126 and cycloheximide, the turnover rate of PD-L1 was
higher than it was in the DMSO control (Supplementary Fig. S7B).
Different inhibitors were then used to block the MAPK pathway to
prevent misinterpretation of effects from nonspecific targeting.
Indeed, the different MEK inhibitors, trametinib and U0126, and
ERK inhibitor, SCH772984, all decreased the PD-L1 protein level and
increased polyubiquitination of PD-L1 (Supplementary Fig. S7C and
S7D).
Previous studies have demonstrated that glycosylation of PD-L1 at
N192, N200, and N219 antagonizes GSK3bbinding, which normally
induces phosphorylation-dependent proteasome degradation of PD-
L1 by b-TrCP. As such, nonglycosylated PD-L1 is unstable compared
with glycosylated PD-L1 (17). Consistent with this mechanism, PD-L1
was upregulated in GSK3b-knockdown cells (Supplementary
Fig. S7E). However, EpEX and EGF could both induce PD-L1 upre-
gulation in GSK3b-knockdown cells (Supplementary Fig. S7F), sug-
gesting that GSK3bis dispensable for EpEX- or EGF-mediated PD-L1
stabilization.
We next tested whether EpAb2-6 could attenuate PD-L1 upregula-
tion and EpEX production. After EpAb2-6 treatment, PD-L1 protein
level was downregulated both in vitro and in vivo (Fig. 5F). Consistent
with our other findings, EpAb2-6 also decreased PD-L1 protein
stability and increased polyubiquitination of PD-L1 (Fig. 5G
and H). Notably, EpAb2-6 treatment inhibited PD-L1 protein level
in lung cancer cells and in cell lines derived from other EpCAM-
positive cancer types, including breast cancer (BT474 and MCF7) and
oral cancer (Cal27; Fig. 5I). Using an apoptosis assay, we further found
that coculturing cancer cells with either EpAb2-6 or T cells could
induce cancer cell apoptosis, and coculturing cancer cells in the
presence of both EpAb2-6 and T cells was more effective in inducing
apoptosis (Fig. 5J).
EpAb2-6 prolongs survival in metastatic and orthotopic mouse
models and improves the efficacy of anti-PD-L1 therapy in the
PBMC CDX model
We next used an animal model of metastatic colon carcinoma to test
whether EpAb2-6 treatment could increase the median overall survival
of metastatic tumor-bearing mice. EpAb2-6 reduced the fluorescence
intensity of mouse blood bearing circulating HCT116-GFP cells,
suggesting that EpAb2-6 decreased the number of HCT116-GFP cells
in mouse blood vessels in vivo (Fig. 6A). NOD/SCID mice were
intravenously injected with SW620 cells and then intravenously
treated with EpAb2-6, or an equivalent volume of control IgG,
at 24 and 96 hours after cell injection (antibody was delivered at
20 mg/kg/dose for a total dose of 40 mg/kg). The median survival time
of the EpAb2-6 treatment group was significantly increased compared
with the control IgG group (Fig. 6B;P<0.005 by the log-rank test).
Because the subcutaneous model may not accurately reproduce
human colon cancer biology (34), an orthotopic mouse model of
colorectal cancer was also established to study the efficacy of our
therapeutic antibody in the colorectal tumor microenvironment. We
investigated the antitumor potential of EpAb2-6 in an orthotopic
model with HCT116-Luc tumors that stably express firefly luciferase.
Before the first therapeutic injection (8 days after tumor cell implan-
tation), growing orthotopic tumors were monitored by biolumines-
cence imaging (Fig. 6C). Mice were treated with either control IgG or
EpAb2-6 (20 mg/kg) every 2 days for 16 days. Notably, the EpAb2-6–
treated group showed metastasis at 55 days, while the control group did
so at 45 days (Fig. 6C). Moreover, no significant changes in body
weight were observed during the treatment period (Supplementary
Fig. S8). At the end of the study, the median survival times for control
IgG and EpAb2-6 treatment groups were 50 and 116.5 days, respec-
tively (Fig. 6D). From these results, we conclude that EpAb2-6
treatment can prolong survival of tumor-bearing mice.
On the basis of our observations that EpAb2-6 treatment decreases
PD-L1 expression both in vitro and in vivo, we further wanted to
evaluate whether EpAb2-6 could improve the therapeutic efficacy of
anti-PD-L1 treatment in vivo. Because EpAb2-6 is not equipped with
cross-reaction with mouse EpCAM, using syngeneic mouse model fails
to evaluate the therapeutic efficacy of EpAb2-6 for immunotherapy. As
illustrated in Fig. 6E, H441 cells were subcutaneously injected into
NSG mice to establish the PBMC-H441–xenografted mice model. Two
weeks later, mice were intravenously injected with 10
7
PBMCs and
treated with atezolizumab, an anti-PD-L1 antibody, and EpAb2-6
twice weekly for 1 month. Treatment with atezolizumab or EpAb2-6
alone inhibited tumor growth in PBMC-H441 mice, and combination
therapy produced a much better tumor inhibitory effect (Fig. 6F
and G). At the end of treatment, tumor tissues were collected, and
CD8
þ
T cells were analyzed by flow cytometry. The CD8
þ
T-cell
population was increased in mice receiving combination therapy
(Fig. 6H). Collectively, these data indicate that inhibition of EpCAM
by EpAb2-6 may enhance the efficacy of anti-PD-L1 therapeutics in
PBMC-H441–xenografted mice.
Discussion
EpCAM-overexpressing carcinoma cells possess stem cell–like
features, and their presence results in high rates of recurrence,
metastasis, and drug resistance (35, 36). We have previously reported
that the EpEX induces EGFR/ERK signaling, which activates ADAM17
and g-secretase to stimulate shedding of EpEX and EpICD. Soluble
EpEX subsequently activates EGFR to form a positive feedback loop,
causing more cleavage of EpCAM. Moreover, the importance of EGFR
signaling on PD-L1 expression has been conclusively shown, and
clinical data demonstrate that PD-L1 expression in the tumor micro-
environment is a determinant of responses to EGFR-targeted thera-
pies. Although EpCAM expression correlates with cancer malignancy,
the molecular mechanism by which EpCAM prevents apoptosis and
suppresses immune response remains unclear.
In this study, we report that soluble EpEX and membrane-bound
EpCAM can directly bind to EGFR through the EGF-like domain I in
EpEX and induce EGFR signaling (Fig. 1). Similarly, recent studies
have reported that EGF-like domains on growth factors could bind to
members of the ErbB/HER family (37) and that membrane-bound
tenascin-C, a matrix component containing EGF-like repeats, could
directly bind to EGFR with very low affinity (38). In addition, because
both EpEX and EpCAM triggered EGFR signaling, the signal may be
transduced in either an autocrine or paracrine manner.
We have previously demonstrated that anti-EpCAM neutralizing
antibody, EpAb2-6, could bind to positions Y95 and D96 of EpCAM to
directly induce cancer cell apoptosis in vitro (23). Inhibition of
EpCAM cleavage or EpEX binding to EGFR through EpAb2-6 might
induce apoptosis both in vitro and in vivo to reduce survival of cancer
stem cells. Notably, we also found that HtrA2 is a direct transcriptional
target of FOXO3a (Fig. 3), based on ChIP evidence indicating that
FOXO3a binds to the HtrA2 promoter upon EpAb2-6 stimulation
(Fig. 3E and F). Interestingly, the expression of XIAP might promote
resistance of colon cancer cells to EpAb2-6 (39), but the functional
outcome of EpAb2-6 treatment was increased apoptosis of cancer cells.
HtrA2 promotes or induces cell death through two different mechan-
isms. One is by directly binding to and inhibiting inhibitor of apoptosis
proteins (IAP), an action that is accompanied by a significant increase
Chen et al.
Cancer Res; 80(22) November 15, 2020 CANCER RESEARCH
5046
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in caspase activity. The other mechanism is through a relatively
uncharacterized IAP inhibition–independent, caspase-independent,
and HtrA2 serine protease activity–dependent mechanism (40).
Previous studies have shown that p53 induces the activation of
HtrA2 after oncogenic Ras transformation (41). Oncogenic Ras
increases cytoplasmic p53 accumulation, which promotes p38 MAPK
translocation to mitochondria and phosphorylation of HtrA2 (41).
The phosphorylated HtrA2 releases from mitochondria into the
cytosol and cleaves F-actin to downregulate lamellipodia forma-
tion (41). Proapoptotic protein, Bax, increases endoplasmic reticulum
Ca
þ2
-ATPase inhibitor thapsigargin-induced HtrA2 release from
mitochondria to induce apoptosis in HCT116 cells (42). However,
whether EpAb2-6 regulation of HtrA2 expression involves p53 or Bax
remains poorly understood. To the best of our knowledge, this is the
first study to demonstrate that inhibition of EpCAM increases HtrA2
gene expression, mitochondria release, and the HtrA2-induced intrin-
sic pathway of apoptosis.
Among all antibody-based immune therapies, anti-PD-1/PD-L1
therapies have the most beneficial outcomes in the treatment of various
malignancies. This fact underscores the importance of deeply under-
standing the processes that control PD-L1 expression. We found that
EpCAM-mediated PD-L1 stabilization gives rise to escape from
Figure 6.
EpAb2-6 prolongs median survival in
metastasis and orthotopic colorectal
cancer models and improves the effi-
cacy of anti-PD-L1 therapy in a PBMC
CDX model. A, HCT116 cells transiently
expressing GFP were injected into the
tail veins of SCID mice with EpAb2-6.
Mice in the control group (without
EpAb2-6 treatment) were injected
with the same dose of control IgG.
N¼3 independent experiments. Data
are shown as mean SD. ,P<0.01.
B, NOD/SCID mice were intravenously
injected with 1 10
6
SW620 cells,
followed by treatment with either
control IgG or EpAb2-6 (N¼6).
According to the survival curves, mice
treated with EpAb2-6 exhibited a
greater survival rate than those trea-
ted with control IgG. C, NSG mice were
orthotopically implanted with HCT116-
Luc cells and then treated with control
IgG or EpAb2-6 starting at 8 days after
tumor inoculation. Antibody injections
were performed every 2 days for
16 days, via the tail vein. Tumor growth
was monitored by examining biolumi-
nescence with the IVIS 200 imaging
system. D, Kaplan–Meier survival plots
and median survival are shown for
both treatment groups (N¼8per
group; P<0.005, log-rank test).
E, A schema for the experiment
to evaluate treatment efficacy of
EpAb2-6 and/or atezolizumab in the
PBMC CDX model. F, The tumor
growth of H441 cells in EpAb2-6-
and/or atezolizumab-treated PBMC
CDX mice. G, Tumor weight was eval-
uated and is shown in the graph.
H, CD8
þ
T cells were quantified in
tumor tissue. Statistical differences
were determined by two-tailed Stu-
dent ttest. N¼6 independent experi-
ments. Graphical data are shown as
mean SEM. ,P<0.05; ,P<0.01;
,P<0.001.
EpCAM Signaling Regulates Tumorigenesis
AACRJournals.org Cancer Res; 80(22) November 15, 2020 5047
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immune surveillance through the EpEX–EGFR–ERK signaling axis.
According to these findings, EpCAM might be an excellent option for
combination with anti-PD-1/PD-L1 therapy. Indeed, EpAb2-6
decreases PD-L1 protein level and improves the therapeutic efficacy
of atezolizumab. Thus, our finding reveals a novel action of EpCAM in
the regulation of PD-L1 protein stability and suggests a new strategy of
EpCAM/PD-L1–targeted combination therapy. However, we did not
test EpCAM-mediated PD-L1 stabilization or the correlation between
EpCAM and PD-L1 in colon cancer, due to the low PD-L1 expression
in both colon cancer cell lines and tumor specimens. In our model, we
propose that EpCAM can stabilize PD-L1 protein, instead of increas-
ing its transcriptional expression. Therefore, if expression of endog-
enous PD-L1 is already low in a tumor, more EpCAM expression
would not increase its protein level to any major extent. Nevertheless,
more colon cancer cell lines or tumor specimens should be examined to
further test whether EpCAM-mediated PD-L1 stabilization and
EpCAM/PD-L1–targeted combination therapy might be beneficial in
colon cancer.
Activating EGFR mutations were reported to be associated with
increased PD-L1 expression, and EGFR inhibitors can reduce PD-L1
expression in NSCLC cell lines, suggesting that EGFR signaling can
trigger immune escape (16). Further investigations demonstrated that
EGF induces PD-L1 expression at a posttranslational level, but does
not affect PD-L1 mRNA expression. The authors also found that EGF
Figure 7.
EpCAM signaling regulates tumor progression.
Chen et al.
Cancer Res; 80(22) November 15, 2020 CANCER RESEARCH
5048
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stabilizes PD-L1 by inducing its glycosylation and inactivating GSK3b,
which promotes degradation of nonglycosylated PD-L1 (17). How-
ever, we found that EpEX-mediated stabilization of glycosylated PD-
L1 occurs through the MAPK pathway. This mechanism differs from
GSK3b-mediated degradation of nonglycosylated PD-L1. Whether the
MAPK pathway regulates stability of nonglycosylated PD-L1 needs
further examination.
While the crystal structure of EpEX has been published previous-
ly (43), the crystal structure of the EpAb2-6–EpEX complex is worth
exploring. A crystal structure for EpAb2-6–EpEX would reveal the
detailed EpAb2-6 binding sites and conformational changes in EpEX
that induce apoptosis. Previous studies have demonstrated that mAbs
bind to receptors and induce conformational changes in the receptor,
which modulates the expression, recycling, and function of the recep-
tor as well as ligand affinity (44, 45). Previous studies have demon-
strated that structure of EpEX forms a cis-dimer and endocytosis of
EpCAM into acidic compartments dissociates the dimer to permit
cleavage (43, 46, 47). EpAb2-6 might affect the conformation and the
endocytosis of EpCAM to disrupt acidification, dissociation, and/or
cleavage.
To the best of our knowledge, this is the first study to demonstrate
that inhibition of EpCAM is correlated with an increase in HtrA2 gene
expression. Moreover, this upregulation occurs through FOXO3a and
induces apoptosis. EpEX increases PD-L1 protein stability and the
combination immunotherapy of anti-EpCAM and anti-PD-L1 anti-
bodies provides a novel strategy for cancer therapy (Fig. 7). Most
importantly, our data indicate that the development of therapeutic
antibodies targeting EpCAM may hold great potential in promoting
immune surveillance and eradicating cancer stem cells in the cancer
microenvironment.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors’Contributions
H.-N. Chen: Investigation, methodology, writing-original draft, writing-review
and editing. K.-H. Liang: Investigation, methodology, writing-original draft, writing-
review and editing. J.-K. Lai: Investigation, methodology. C.-H. Lan: Investigation,
methodology. M.-Y. Liao: Investigation, methodology. S.-H. Hung: Investigation,
methodology. Y.-T. Chuang: Investigation, methodology. K.-C. Chen: Investigation,
methodology. W.W.-F. Tsuei: Investigation, methodology. H.-C. Wu:
Conceptualization, data curation, supervision, funding acquisition, validation,
writing-original draft, project administration, writing-review and editing.
Acknowledgments
We thank the Core Facility of the Institute of Cellular and Organismic Biology
(Nankang, Taipei, Taiwan) and National RNAi Core Facility, Academia Sinica
(Nankang, Taipei, Taiwan) for technical support. This research was supported by
Academia Sinica (AS-SUMMIT-108) and the Ministry of Science and Technology
(MOST-108-3114-Y-001-002 and MOST-108-2823-8-001-001 to H.-C. Wu).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Received April 18, 2020; revised July 17, 2020; accepted September 22, 2020;
published first September 25, 2020.
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