Cytosolic EpCAM cooperates with H-Ras to regulate epithelial to mesenchymal transition through ZEB1

Next generation sequencing of human cancer mutations has identified novel therapeutic targets. Activating Ras oncogene mutations play a central role in oncogenesis, and Ras-driven tumorigenesis upregulates an array of genes and signaling cascades that can transform normal cells into tumor cells. In this study, we investigated the role of altered localization of epithelial cell adhesion molecule (EpCAM) in Ras-expressing cells. Analysis of microarray data demonstrated that Ras expression induced EpCAM expression in normal breast epithelial cells. Fluorescent and confocal microscopy showed that H-Ras mediated transformation also promoted epithelial-to-mesenchymal transition (EMT) together with EpCAM. To consistently localize EpCAM in the cytosol, we generated a cancer-associated EpCAM mutant (EpCAM-L240A) that is retained in the cytosol compartment. Normal MCF-10A cells were transduced with H-Ras together with EpCAM wild-type (WT) or EpCAM-L240A. WT-EpCAM marginally effected invasion, proliferation, and soft agar growth. EpCAM-L240A, however, markedly altered cells and transformed to mesenchymal phenotype. Ras-EpCAM-L240A expression also promoted expression of EMT factors FRA1, ZEB1 with inflammatory cytokines IL-6, IL-8, and IL1. This altered morphology was reversed using MEK-specific inhibitors and to some extent JNK inhibition. Furthermore, these transformed cells were sensitized to apoptosis using paclitaxel and quercetin, but not other therapies. For the first time, we have demonstrated that EpCAM mutations can cooperate with H-Ras and promote EMT. Collectively, our results highlight future therapeutic opportunities in EpCAM and Ras mutated cancers.


Retroviral and lentiviral transduction
Phoenix-AMPHO (ATCC) packaging cells (2x10 6 ) were transfected when nearly confluent with 2.5 μg of pBABE-H-Ras-Puro using FuGENE-HD (Promega, Madison, WI, USA). Twenty-four hours after transfection, the medium was replaced with 10% fetal bovine serum. Forty-eight hours after transfection, viral supernatants were collected, filtered through 0.45-micron filters, and then added to MCF-10A cells in media containing 8 μg/ml protamine sulfate. After two successive retroviral infections, cells were grown for 48 hours and selected in puromycin for 2 weeks. EpCAM-EGFP fused DNA was transiently transfected into HEK-293 T cells using FuGENE6 or stably transduced as described earlier [5].

Immunofluorescence and cell staining
For immunofluorescence assay 2X10 5 cells were grown on 8-well microscope slides. Following growth factor treatment, cells were washed twice with phosphate-buffered saline (PBS) and fixed at room temperature using 4% formaldehyde. Following fixation, cells were washed four times with PBS, permeabilized for 10 min at room temperature with 0.2% triton X-100 in PBS (v/v), washed 4 additional times and then blocked with 5% goat serum for 1 h. After blocking, cells were incubated with primary antibody overnight at 4˚C or 2 h at room temperature in PBS containing 5 mg/ml bovine serum albumin. Cells were washed 5 times and incubated with appropriate secondary antibody conjugated with Alexa-flour 488 or 555 (Invitrogen, Waltham, MA, USA) for 1 h at room temperature. Nuclear staining was carried out using 4,6-diamidino-2-phenylindole (Invitrogen) for 5 min. The cells were then dehydrated and mounted and visualized/captured with a fluorescence microscope (EVOS digital inverted microscope at 20× or 40× magnification).

Flow cytometry
Cell surface EpCAM expression levels were measured by flow cytometry using FITC or phycoerythrin-labeled EpCAM antibodies. Briefly, 1X10 6 cells were stained with EpCAM-PE antibody as recommended by manufacturer. Expression was quantified as mean fluorescence intensity (MFI) using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA).

Confocal microscopy
Fixed or living cells (2X10 5 cells per assay) were plated and grown on coverslips. imaged on a confocal laser scanning microscope TCS SP5 (Leica Microsystems, Wetzlar, Germany) using a 63× oil immersion objective. A helium/neon (λ543 nm) laser was used for excitation of Rhodamine. Living cells were imaged in imaging medium (Dulbecco's modified Eagle's medium without phenyl red). For fixation, cells were washed in PBS once, fixed in 4% paraformaldehyde for 10 min on ice, and then returned to PBS for imaging. To quantify rhodamine-EGF signal, the sum of pixel values was calculated from the raw images. For this, an ImageJ plugin was written that is available upon request. For normalization and comparability with flow cytometry data, the average pixel value obtained at each time point was divided by the maximal signal obtained at 30 min of stimulation.

RNA extraction and real-time PCR
For total RNA extraction, 2x10 6 cells were plated for 24 h and purified using RNAeasy (Qiagen, Valencia, CA, USA). Two micrograms of RNA were reverse transcribed using a High Capacity cDNA Synthesis Kit (Ambion, Austin, TX, USA). mRNA expression was quantified using SYBR green chemistry and an ABI Prism 7700 Sequence Detector (Life Technologies). Primer sequences are detailed in S1 Table. Each reaction was performed in triplicate, and the data represents from 2 independent RNA preparations.

Cytokine array
Cytokine antibody array was performed with a human cytokine array kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. Briefly, 2-mL conditioned media in Opti-Mem from 2x10 6 cell culture was collected after 72h. After centrifugation, the membranes pre-coated with capture antibodies were incubated with supernatants. After washing with wash buffer detection antibody was added followed by streptavidin-HRP. Chemi Reagent Mix were added to the membranes to develop the array blots. The immunoblot images were captured and visualized using the BioSpectrum Imaging System (Ultra-Violet Products Ltd., Cambridge, UK). The intensity of each spot in the captured images was analyzed using the ImageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA, USA).

Cell proliferation and viability assay
For the cell proliferation and viability assay, 5.0×10 3 cells per well were plated in 96-well plates and cultured in a 5% CO 2 incubator. After 24 h, compounds were added as IC50 or concentrations reported in literature. Compounds were dissolved in DMSO and added to the culture, CI-1040 (20nM), SB203580 (100nM), SP600125 (1uM), AKT inhibitor LY29004 (2uM), BAY117082 (10uM), Dasatinib (100nM), doxorubicin (2uM), etoposide (5uM), paclitaxol (100nM), quercetin (5uM). After 72 to 96 h later, cell proliferation was assessed using the Cell Titer-Glo1 Luminescent assay (Promega) following the protocols provided by the manufacturers. Cell viability was determined using MTT assay (Millipore Sigma, Burlington, MA, USA). The viable cells reduced the MTT to formazan through oxidoreductase enzymes. The insoluble formazan crystals were solubilized resulting in a colored solution which is quantified by measuring absorbance at 590 nm. The measured absorbance at OD 590 nm is proportional to the number of viable cells.

Invasion, migration and soft agar assay
Stably transduced 4×10 4 cells were added to matrigel transwell invasion chambers or control transwell chambers (BD Biosciences) and incubated for 72 h with chemoattractant media (Clonetics, Walkersville, MD, USA) supplemented with growth factors. Cells invading through the matrigel or control membranes were fixed using 70% ethanol, stained with 0.1% crystal violet, and photographed in 4 fields to cover the entire area. Cells were counted from all fields by a scientist blinded to the experimental conditions. For colony formation assay, 1.0×10 4 cells were plated into 6-cm dishes in 2 ml of medium containing 0.3% agarose, overlaid with 2 ml of 0.5% agarose. Dulbecco's Modified Eagle's Medium (Thermo Fisher Scientific, Waltham, MA, USA) was added on top, and dishes were maintained in a humidified atmosphere with 5% CO 2 at 37˚C for 12 days. Then colonies were stained with crystal violet, and the number of colonies was counted.

Statistical analysis
All experiments were performed at least three times in triplicate. All statistical analyses were performed using GraphPad Prism 9.4 (GraphPad, La Jolla, CA). Numerical data are presented as mean ± sd. Single comparisons were performed by unpaired Student's t tests and multiple comparisons were performed by ANOVA. P-values <0.05 were considered to be statistically significant.

H-Ras expression induces EpCAM expression
To establish the possible involvement of EpCAM in Ras signaling, we first analyzed genes correlated to E-cadherin. Microarray data from analysis of cancer cell lines revealed 121 genes, including EpCAM, closely correlated (R>0.6) to the bonafide epithelial marker Ecadherin [14]. Of these 121 epithelial genes, 35 genes were closely correlated (R>0.7) with EpCAM ( Fig 1A). We subsequently analyzed expression of top 14 highly EpCAM correlated genes from Ras-transduced normal cells (Fig 1A, right panel). We extended our analysis to NCI-60 cell lines database with Ras mutations (S1B Fig). Data revealed that EpCAM correlated gene (nearest neighbors) list from Figs 1A and S1B are enriched for epithelial specific markers. Two independent microarray data sets were analyzed from immortalized MCF- 10A cells transduced with H-Ras [15,16]. Gene expression data revealed that H-Ras-transduced cells upregulated EpCAM expression (Fig 1B and 1C). Further analysis and immunoblotting confirmed that compared to expression of a control vector or other protooncogenes, including myc, Src, β-catenin, E2F3, and MEK, Ras-transduced cells had a 5-fold increase in EpCAM expression ( Fig 1D). H-Ras transduced cells validated this observation ( Fig 1E) Altogether, this data suggests Ras overexpression induced expression of several epithelial/membrane proteins including EpCAM, which may play important role in Ras signaling. This is in agreement with a previous observation that K-Ras addiction is restricted in epithelial cells expressing EpCAM, but not in mesenchymal cells [17]. Based on previous studies, it is highly likely EpCAM plays a role in Ras-addicted cells.

EpCAM and H-Ras transforms the epithelial cells
To establish a functional model to study the role of EpCAM in Ras cells, MCF-10A cells were transduced using H-Ras retrovirus followed by WT-EpCAM and EpCAM-L240A. We have repeatedly observed that EpCAM expression varies in a panel of epithelial tumor cell lines [9]. Normal, immortalized MCF-10A cells require EGF and insulin to proliferate. Growth factor supplementation suppresses EpCAM expression through ERK/SLUG signaling [9]. However, withdrawal of growth factor re-express EpCAM (S1A To generate Ras and EpCAM stable lines, MCF10A cells were transduced with retrovirus harboring control vector (pBabe) or H-Ras. After selection in puromycin, both cell lines were further transduced with pBabe, EpCAM-WT, or EpCAM-L240A. During our previous study [5], we generated several cancer-associated EpCAM mutations to test the effect of cytosolic EpCAM expression on cathepsin-L protease activity. Compared to other mutations, EpCAM-L240A was exclusively and widely localized to the cytosol and maintained its expression. After subsequent transduction and selection of a uniform population, all 6 established cell lines were passaged for 2 months prior to functional investigation.
Compared to the other cell lines, H-Ras-EpCAM-L240A-transduced cells showed a markedly altered phenotype (Figs 2A and S2). Since cells were grown in growth factor media, endogenous EpCAM expression remained marginal as expected (S1A Fig). H-Ras-EpCAM-L240A cells overgrew in colonies, clustered heavily together, and later released as spheroids in culture media. These spheroids, when re-plated, regrew as a monolayer with mesenchymal morphology. Thin sheet-like membrane protrusions consistent with lamellipodia formation were seen at the leading edge of the cells in H-Ras-pBabe and WT-EpCAM cells. In contrast, only filopodia were seen in H-Ras-EpCAM-L240A cells. Lamellipodia and filopodia appearing on the leading edges of cell movement are often observed in migratory cells. Tumor cells often evolve to become invasive and migratory allowing them to spread to distant sites.
Boyden chambers were used to investigate the migratory/invasive ability of the tested cell lines. Cells were plated onto inserts coated with a layer of extracellular matrix. After 72 h of invasion, cells were fixed, stained with crystal violet, and quantified. As shown in Fig 2B, H-Ras-transformed cells were partially invasive compared those transformed with control

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vector alone. Compared to H-Ras control cells, EpCAM-WT and EpCAM-L240A cells showed a 2-and 3-fold increase in invasion, respectively. Additionally, H-Ras cells expressing EpCAM WT or L240A mutant showed increased proliferation (Fig 2C) in 72h assay. The tumorigenic potential of transformed cells was also investigated using soft-agar growth assays to confirm cellular anchorage-independent growth. As described in the methods section, cells were cultured on soft agar with complete growth medium. As shown in Fig 2D, Ras-expressing cells started forming colonies. There was not a statistical difference between the growth of control vector and EpCAM-WT cells; however, H-Ras-EpCAM-L240A cells showed a robust 4-fold increase in colony numbers.

Cytoplasmic localization of EpCAM in Ras-and TGF1β1-induced EMT
Based on the previous findings, we hypothesized that the morphological, invasive, and growth properties observed in H-Ras-EpCAM-L240A cells could be the result of EMT (Fig 2B-2D). To test this hypothesis, cells were cultured on cover slips and immuno-stained for epithelial markers E-cadherin and β-catenin and mesenchymal markers vimentin and ZEB1. As shown in Fig 3A, compared to H-Ras-EpCAM-WT cells, H-Ras-EpCAM-L240A cells lost E-cadherin expression, with increased expression of mesenchymal markers vimentin and ZEB1.
β-catenin is a critical component of cadherin-based intercellular adhesions and also plays a central role in Wnt signaling. β-catenin can be located at the cell membrane, the cytoplasm, or the nucleus. At the cell membrane, it is bound to the cytoplasmic domain of type-I cadherins and regulates and maintains the structural organization of the cytoskeleton [18]. When released from cadherins, β-catenin participates in downstream signaling and cytoplasmic localization of β-catenin and can serve as a predictive marker for poor outcomes in cancer patients [19]. Compared to normal and H-Ras-transduced cells, H-Ras-EpCAM-L240A cells show strong staining of β-catenin in the cytosol. Interestingly, EpCAM-WT is localized at the membrane, but H-Ras-transduced cells show moderate EpCAM localization in the cytosolic compartments ( Fig 3A).
It is known that Ras transduction in normal epithelial cells results in a partial EMT phenotype mediated by SNAI1/2 transcription factors [20]; however, ZEB1 expression is exclusively observed in mesenchymal cells. We have shown that in vitro cultured cells treated with TGF1β1 can induce EMT [9]. To investigate if EMT-transitioning cells express EpCAM in the cytosol, HaCAT cells (EpCAM MFI 500) were treated with TGF1β1 for 36 h, permeabilized, and stained with EpCAM antibody. As shown in Fig 3B, EMT-transitioning cells show EpCAM localization on the membrane and in the cytosol. Cytosolic localization of EpCAM in EMT transitioning cells may degrade or play a role in EMT initiation. Altogether, our results suggest that that EMT transition drives EpCAM expression to the cytosol and EpCAM-L240A-induced EMT in Ras cells.

Ras and EpCAM induces EMT
In light of the cytosolic localization of EpCAM and the altered phenotype in H-Ras-EpCAM-L240A cells, we hypothesized that EMT transcription factors together with other cancer-associated factors may be playing an important role. The EMT stage of epithelial cells has been well characterized by gene expression signatures [9,21]. Differential gene expression of a panel of epithelial and mesenchymal markers as well as MMP protease family members and cytokines involved in EMT was determined using qRT-PCR. As shown in Fig 4A, transduction with H-Ras-EpCAM-WT did not significantly alter the expression mesenchymal genes. H-Ras-EpCAM-L240A cells, however, showed decreased expression of epithelial markers and robust overexpression of Snail, Twist1, ZEB1/2, Fra1/2 and Foxc2. These transcription factors  Ras, EpCAM-WT and L240A were serum starved overnight and treated with growth media for 2 h. RNA extraction followed by cDNA synthesis and real-time PCR data is presented in a heat-plot. Results shows expression of epithelial, mesenchymal markers and cytokines. B. Quantitative relative ddCT data is presented for E-cadherin, vimentin and ZEB1. List of qRT-PCR primers used are listed in S1 Table. C. Cells were plated in 6CM plate and cultured for 48h followed by are known to induce EMT in normal cells. ZEB1, a master regulator of EMT, was expressed together with vimentin (Fig 4B and 4C). This finding suggests that cytosolic EpCAM with H-Ras expression may have induced EMT through ZEB1 expression. To prove this, we generated EpCAM-WT and EpCAM-L240A lentiviral particles and added to H-Ras-transduced cells. As shown in Fig 4D, infection with 5 MOI of EpCAM-WT lentiviral particles did not affect E-cadherin expression; however, dose dependent treatments of 2, 5, and 10 MOI of EpCAM-L240A lentiviral particles induced ZEB1 and vimentin expression and suppressed Ecadherin expression. These results show that normal cells transduced with H-Ras together with cytosolic EpCAM-L240A can induce EMT through ZEB1. We validated this inverse expression correlation in a panel of cell lines with an EMT gene signature. Hierarchical cluster data analysis showed that the epithelial gene cluster is different from mesenchymal genes and EpCAM negative cells were positive for ZEB1 expression (S3 Fig). We have also shown that ZEB1 shRNA treatment induced EpCAM expression [9]. This result agrees with one independent study demonstrating that ZEB1 negatively correlated to 324 genes with EpCAM at the top of the list [22].

EpCAM-transformed cells are sensitized to paclitaxel and quercetin
We sought to assess the efficacy of inhibiting common pathways that are likely activated in H-Ras-EpCAM-L240A cells. Transformed H-Ras-EpCAM-L240A cells were treated for 72h with most common signaling pathway inhibitors. Efficacy of signaling pathway inhibition was accessed immunoblotting phosphorylated and total proteins of ERK1/2, JNK, P38, AKT, NF-kB and Src (Fig 6A and 6B). MEK inhibitor (Ci-1040) treatment reversed cell morphology from a fibroblast-like to an epithelial-like morphology without significantly affecting cell death or proliferation (Fig 6A and 6C). Fig 6B-6D shows that treatment of cells with the JNK inhibitor SP600125 also did not cause significant changes in cell death or proliferation. The small lysate preparation and immunoblotting against epithelial marker, E-cadherin and mesenchymal markers ZEB1, FRA1, SNAIL and Vimentin. Results shows ZEB1, Snail and vimentin expressed in Ras-EpCAM-L240A cells. D. Ras-transduced cells were treated with EpCAM lentiviral particles EpCAM-WT (5 MOI) and EpCAM-L240A (2,5,10 MOI). Cell lysates were subjected to immunoblotting with EpCAM, E-cadherin, ZEB1, vimentin and actin antibodies. Results show that EpCAM-L240A induced ZEB1 expression in a dose-dependent manner.
https://doi.org/10.1371/journal.pone.0285707.g004 molecular inhibitor for P38 MAPK (SB203580) and NF-kB-AKTi (BAY117082) induced about a 20% cell death rate. Dasatinib, an SRC family and Abl inhibitor, suppressed lamellipodia and filopodia formation necessary for cell migration. Doxorubicin and etoposide treatment did not alter significant cell death or proliferation. The most dramatic cell death (more than 90%) was observed after treatment with paclitaxel and the plant flavonoid quercetin. Paclitaxel is a chemotherapy drug for several cancers which targets microtubules. Quercetin is known to induce cell cycle arrest and apoptosis in some cancer cells; it prevents cell division, arrests cells in the  , paclitaxol (100 nm), and quercetin (5 μM) to assay drug sensitivity. B. Efficacy of inhibitors used were analyzed by western blotting. Cells were harvested after 36h of drugs treatment, lysates were immunoblotted to common pathways, ERK1/2, JNK, P38, AKT, NF-kB and SRC. C. The cell proliferation assay was performed after 72 of plating using CellTiter-Glo Luminescent Cell Viability Assay. D. Cell viability was determined using MTT assay (see methods). Results shows paclitaxel and quercetin reduced cell growth significantly. E. MCF10A-Ras-EpCAM-L240A cells cultured until subconfluent and treated for 36-h with Vehicle, Doxorubicin, Etoposide, Paclitaxel, and quercetin. Cell lysates were subjected for immunoblotting to assay cleaved caspase-3 and PARP.
G2/M-phase of the cell cycle, inhibits replication, and eventually causes apoptosis [26,27]. In order to examine type of cell death induced by chemotherapy drugs, cells were treated with Doxorubicin, Etoposide, Paclitaxel and Quercetin for 72 hrs. We observed paclitaxel, quercetin affected cell proliferation within 24h of cell growth (Fig 6C). It is known that paclitaxel treatment results in cell death by expressing apoptosis-related proteins such as cleaved caspase-3 and cleaved PARP-1 [28]. Treated cells were harvested at 36h and analyzed for cleaved-PARP along with Caspase-3 (Fig 6E).Results demonstrated that paclitaxel and quercetin treated cell lysates detected cleaved caspase-3 and cleaved PARP. PARP is substrate for caspases, and caspase mediated cleavage of PARP is considered to be hallmark of apoptosis [29].

Discussion
EpCAM plays an important role in developmental biology and the maintenance of intercellular adhesions. Knockout of EpCAM in mice can be embryonically lethal due to placental malformation and also the development of congenital tufting enteropathy [30]. Other knockout murine models demonstrate that EpCAM affects intercellular junction formation through aberrant recruitment and/or regulation of claudin proteins, E-cadherin, and β-catenin [31,32]. In cancer, the prognostic significance of EpCAM expression is dependent on the tumor type. EpCAM overexpression is frequently associated with shorter patient survival in epithelial cancers [33]. In other cancer types, EpCAM expression is associated with improved outcomes. For example, high EpCAM expression in primary renal cell carcinoma tumors was associated with improved patient survival [34,35], and in gastric cancer, loss of EpCAM expression was associated with aggressive tumor behavior [36]. In patients with esophageal squamous cell carcinoma, high EpCAM expression conferred a significantly higher survival rate [37]. EpCAM interacts with multiple signaling pathways, which can affect cancer growth and progression. EpCAM was reported to promote tumorigenesis by modulating PI3K/AKT/mTOR pathway activity [38,39]. Protease-mediated cleavage of EpCAM generates EpICD fragments, which in turn activate Wnt-β-catenin signaling [40]. Our recent work shows that EpCAM expression suppresses ERK1/2 activation [9] and the protease cathepsin-L [5]. Both of these pathways are dominantly involved in promoting EMT, cancer cell invasion, migration, tumor metastasis, and drug resistance. We recently catalogued cancer associated EpCAM mutations and identified 115 unique cancer-associated somatic/missense mutations in the EpCAM coding region [5].
In the current study, we investigated the role of cell surface and cytosolic EpCAM expression in H-Ras-transduced epithelial cells. This work builds upon the insights of our prior study that examined the effects of cancer-associated EpCAM mutations on protein function [5]. Studies have revealed that overexpression of H-Ras is sufficient to initiate proliferation of cells in the absence of exogenous growth factors. H-Ras induces expression of growth factors HB-EGF, TGFα, and AREG [41,42]. Our finding that cytosolic EpCAM (EpCAM-L240A) has the ability to further transform H-Ras-positive cells suggests cooperation between Ras and cytosolic EpCAM to promote tumorigenesis. The transformation of H-Ras-EpCAM-L240A cells into a mesenchymal cells (Figs 2 and 3) can be explained by at least 2 mechanisms. First, mutated EpCAM may generate endoplasmic reticulum stress due to protein misfolding. Since endoplasmic reticulum stress interferes with the normal physiological function of the cell, this may trigger survival pathway signaling and promote EMT. Secondly, bioinformatics analyses from scansite (https://scansite4.mit.edu/) predicts that EpCAM may be phosphorylated at serine, threonine, or tyrosine by cam and polo like kinases. Ras pathway signaling causes downstream protein phosphorylation and activates the mitogen-activated protein kinase (MAPK) cascade and the PI3K/AKT/mTOR pathway. Thus, cytosolic localization of EpCAM may increase the rate of EpCAM phosphorylation and alter EpCAM function. As shown in S2 Fig,  MCF-10A cells transduced with pBabe vector and EpCAM-WT maintained an epithelial phenotype. H-Ras-and EpCAM-L240A-transformed cells acquired mesenchymal characteristics. This observation is in agreement with a recent study of MCF10A-Ras cell transformation and altered morphology [43]. Matjaz et al. concluded that these morphological alterations can be mediated by several changes in epithelial architecture including: i) cell growth in two layers with varying E-cadherin expression and localization in the top layer and bottom layer, ii) decreased cellular adhesion due to loss of adhesion-like proteins, and iii) redistributed F-actin altering cellular tension. Based on this observation, we anticipate that cancer-associated EpCAM mutations that cause loss of membrane localization may a play significant role in contributing to cancer cell growth and EMT.
We have previously shown that the loss of EpCAM expression activated ERK1/2 signaling [9]. In this situation, it's highly possible that additional oncogene signaling as demonstrated here, can induce EMT through MAPKs. The loss of EpCAM through EMT [9], proteolytic cleavage [44], mutations [5], and aberrant glycosylation [45] are known to induce varied cellular phenotypes. Since EMT phenomena can be reversed to MET, it is important to understand mechanism involved in transitions. Our data suggested that EpCAM mutations together with Ras can transform cells, which suggests that EpCAM may play important role initiating EMT. EpCAM has 3 di-sulfide linkages which are generally present in secreted and membrane proteins for their role to stabilize the protein. EpCAM is also glycosylated at N74, N111, and N198 and dimerizes as an active molecule. Thyroglobulin type-I domains of EpCAM are conserved and are present in Trop2, p41 invariant chain, and insulin growth factor-like binding proteins. In these situations, structural changes or mis-folding through cancer-associated mutations may introduce detrimental role to designate as a driver mutations.
It is necessary to understand the cell signaling pathways modulated by EpCAM mutations in different cancer types. In the current study, we have shown Ras-EpCAM cells expressed inflammatory cytokines, IL-1α, IL-1β, IL-6, and IL-8 (Figs 4 and 5). These cytokines play crucial roles in tumor initiation, development, progression, and drug resistance. IL-8 expression contributes to cancer stem cell-like properties and poor prognosis in pancreatic cancer patients [46]. Ras-induced IL-8 expression plays a critical role in tumor growth and angiogenesis [25]. A potent cytokine, IL-6, plays an important role in lung cancer development [47]. Ras-EpCAM cells also expressed ZEB1. ZEB1 mediated IL-1β expression and promoted colon cancer cell stemness and invasiveness [48]. ZEB1 also served as a target gene of inflammatory cytokines [49]. Our findings here suggest that dysregulated mutant EpCAM expression may also generate stem cell phenotypes, since tumor-initiating cells are enriched with EpCAM [50,51]. In line with these observations, the presence of EpCAM+/CD44+ cancer stem cells in tissue from colorectal cancer patients is significantly correlated with a more aggressive and higher grade tumor [52]. Only paclitaxel and quercetin treatment significantly affected cell proliferation and viability of MCF10A-Ras-EpCAM-240A cells (Fig 6). This suggests that tumors with EpCAM mutations can sensitize cancer cells to unique therapy regimens. EpCAM-based antibody therapies are still potential therapeutics; however, screening strategies based on EpCAM expression and localization are not available. Since, the Ras proteins have been termed "undruggable" [53], Ras-regulated genes such as EpCAM can serve as a better therapeutic target. Our data suggest that MEK inhibition reversed the mesenchymal morphology (Fig 6A). This is in agreement with reports in which MEK inhibitor monotherapy demonstrated modest efficacy due to transcription of negative feedback genes and the activation of PI3K-AKT survival pathway [54]. We anticipate that combined treatment strategy with survival pathway such as AKT may also induce cell death. EpCAM mutational data is being catalogued to a public TCGA database, and studying the role of these mutations in Ras-and RTKdriven tumorigenesis will help to develop future personalized therapies.