Mesenchymal-like Cancer Cells Research Articles

Aspirin prevents colorectal cancer metastasis in mice by splitting the crosstalk between platelets and tumor cells.

We investigated whether platelets prime colon cancer cells for metastasis and whether pharmacological inhibition of platelet function may prevent it. Coculturing HT29 human colon carcinoma cells with human platelets led to the induction of mesenchymal-like cancer cells characterized by downregulation of E-cadherin and upregulation of Twist1, enhanced cell mobility and a proaggregatory action on platelets. These changes were prevented by different antiplatelet agents, aspirin[an inhibitor of cyclooxygenase(COX)-1], DG-041[an antagonist of prostaglandin(PG)E2 EP3 receptor] and ticagrelor (a P2Y12 receptor antagonist). The injection of HT29 cells, exposed to platelets in vitro, into the tail vein of humanized immunodeficient mice led to higher incidence of lung metastasis compared to the injection of untreated HT29 cells. This effect was associated with enhanced systemic biosynthesis of thromboxane(TX)A2 and PGE2 in vivo. Platelet COX-1 inhibition by aspirin administration to mice prevented the increased rate of metastasis as well as the enhanced production of TXA2 and PGE2 induced by the in vitro priming of HT29 cells by platelets. In conclusion, targeting platelet COX-1 with low-dose aspirin exerts an antimetastatic action by averting the stem cell mimicry of cancer cells associated with enhanced proaggregatory effects induced by platelet-tumor cell interactions. These effects may be shared by other antiplatelet drugs.

MUC1 upregulation promotes immune resistance in tumor cells undergoing brachyury-mediated epithelial-mesenchymal transition

ABSTRACT Epithelial-mesenchymal transition (EMT) is a molecular and cellular program in which epithelial cells lose their well-differentiated phenotype and adopt mesenchymal characteristics. This process, which occurs naturally during embryogenesis, has also been shown to be associated with cancer progression and with tumor recurrence following conventional therapies. Brachyury is a transcription factor that mediates EMT during development, and is aberrantly expressed in various human cancers where it promotes tumor cell EMT, metastatic dissemination, and resistance to conventional therapies. We have recently shown that very high expression of brachyury can protect tumor cells against immune cell-mediated cytotoxicity. In seeking to elucidate mechanisms of immunotherapy resistance, we have discovered a novel positive association between brachyury and mucin-1 (MUC1). MUC1 is overexpressed in the majority of carcinomas, and it has been shown to mediate oncogenic signaling and confer resistance to genotoxic agents. We found that MUC1 is concomitantly upregulated in tumor cell lines that highly express brachyury due to an enhancement of MUC1 mRNA stability. Analysis of patient lung tumor tissues also identified a positive association between these two proteins in the majority of samples. Inhibition of MUC1 by siRNA-based gene silencing markedly enhanced the susceptibility of brachyury-expressing cancer cells to killing by tumor necrosis-related apoptosis-inducing ligand (TRAIL) and to perforin/granzyme-dependent lysis by immune cytotoxic cells. These studies confirm a protective role for MUC1 in brachyury-expressing cancer cells, and suggest that inhibition of MUC1 can restore the susceptibility of mesenchymal-like cancer cells to immune attack.

Inflammasome-independent NLRP3 is required for epithelial-mesenchymal transition in colon cancer cells

Inflammasome NLRP3 plays a crucial role in the process of colitis and colitis­-associated colon cancer. Even though much is known regarding the NLRP3 inflammasome that regulates pro-inflammatory cytokine release in innate immune cells, the role of NLRP3 in non-immune cells is still unclear. In this study, we showed that NLRP3 was highly expressed in mesenchymal-like colon cancer cells (SW620), and was upregulated by tumor necrosis factors-α (TNF-α) and transforming growth factor-β1 (TGF-β1) respectively, during EMT in colon cancer epithelial cells HCT116 and HT29. Knockdown of NLRP3 retained epithelial spindle-like morphology of HCT116 and HT29 cells and reversed the mesenchymal characteristic of SW620 cells, indicated by the decreased expression of vimentin and MMP9 and increased expression of E-cadherin. In addition, knockdown of NLRP3 in colorectal carcinoma cells displayed diminished cell migration and invasion. Interestingly, during the EMT process induced by TNF-α or TGF-β1, the cleaved caspase-1 and ASC speck were not detected, indicating that NLRP3 functions in an inflammasome-independent way. Further studies demonstrated that NLRP3 protein expression was regulated by NF-κB signaling in TNF-α or TGF-β1-induced EMT, as verified by the NF-κB inhibitor Bay 11-7082. Moreover, NLRP3 knockdown reduced the expression of Snail1, indicating that NLRP3 may promote EMT through regulating Snail1. In summary, our results showed that the NLRP3 expression, not the inflammasome activation, was required for EMT in colorectal cancer cells.

Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells.

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy.

Abstract A42: MK-1775, a WEE1 inhibitor alleviates resistance to immune attack of tumor cells undergoing an epithelial-mesenchymal transition

Abstract The phenomenon of epithelial-mesenchymal transition (EMT) is a phenotypic switch that allows epithelial tumor cells to acquire features of mesenchymal cells, including the ability to migrate and invade and, therefore, to disseminate from the primary site. In addition to promoting tumor dissemination, various reports have demonstrated that acquisition of features of EMT associates with tumor resistance to the cytotoxic effects of chemotherapy, radiation, and some targeted therapies. It is not well understood, however, the contribution of EMT to the escape of tumors from host immune-surveillance and immune-mediated rejection. Our laboratory has characterized the T-box transcription factor brachyury as a tumor-associated antigen and a regulator of EMT in human carcinomas. By generating isogenic cancer cell lines with various levels of brachyury expression, we demonstrated that high levels of brachyury significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer cells, and render tumor cells less responsive to a cancer vaccine in syngeneic murine tumor models. This resistance was due to inefficient caspase-dependent apoptosis, manifested as inefficient nuclear lamin degradation in the presence of activated effector caspases. We correlated this phenomenon to loss of cell cycle kinase CDK1, which mediates lamin phosphorylation. In support of a causal connection, pre-treatment of tumor cells with MK-1775, a specific inhibitor of WEE1, which is known to be a negative regulator kinase of CDK1 activity, could counter the defective apoptosis of tumor cells expressing high levels of brachyury. Thus, our findings suggest reconstituting CDK1 activity to threshold levels might be sufficient to elevate immune-mediated lysis of mesenchymal-like cancer cells, and improve the efficacy of anti-tumor vaccines. Citation Format: Duane H. Hamilton, Romaine I. Fernando, Kwong-Yok Tsang, Claudia Palena. MK-1775, a WEE1 inhibitor alleviates resistance to immune attack of tumor cells undergoing an epithelial-mesenchymal transition. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A42.

MiR-155, identified as anti-metastatic by global miRNA profiling of a metastasis model, inhibits cancer cell extravasation and colonizationin vivoand causes significant signaling alterations

To gain insight into miRNA regulation in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using global miRNA profiling, 28 miRNAs were found to exhibit significantly altered expression between isogenic metastasizing and non-metastasizing cancer cells, with miR-155 being the most differentially expressed. Highly metastatic mesenchymal-like CL16 cancer cells showed very low miR-155 expression, and miR-155 overexpression in these cells lead to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. Our experiments addressing the underlying mechanism of the altered tumor burden revealed that miR-155-overexpressing CL16 cells were less invasive than CL16 control cells in vitro, while miR-155 overexpression had no effect on cancer cell proliferation or apoptosis in established lung tumors. To identify proteins regulated by miR-155 and thus delineate its function in our cell model, we compared the proteome of xenograft tumors derived from miR-155-overexpressing CL16 cells and CL16 control cells using mass spectrometry-based proteomics. >4,000 proteins were identified, of which 92 were consistently differentially expressed. Network analysis revealed that the altered proteins were associated with cellular functions such as movement, growth and survival as well as cell-to-cell signaling and interaction. Downregulation of the three metastasis-associated proteins ALDH1A1, PIR and PDCD4 in miR-155-overexpressing tumors was validated by immunohistochemistry. Our results demonstrate that miR-155 inhibits the ability of cancer cells to extravasate and/or colonize at distant organs and brings additional insight into the complexity of miR-155 regulation in metastatic seeding.

MicroRNA-146a inhibits epithelial mesenchymal transition in non-small cell lung cancer by targeting insulin receptor substrate 2.

During cancer progression, some tumor cells show changes in their plasticity by morphological and phenotypical conversions, as an expression of mesenchymal markers and loss of epithelial markers, collectively referred to as epithelial-mesenchymal transition (EMT). EMT has been increasingly recognized as a critical phenomenon in lung cancer progression. The goal of this study was to identify microRNAs involved in lung cancer progression. A microarray and qRT-PCR were performed to investigate the miRNA expression profiles in mesenchymal-like lung cancer cells. The role of miR‑146a in lung cancer progression was measured by invasion and migration assays in vitro. Bioinformatics and luciferase report assays were used to identify the target of miR‑146a. The expression of miR‑146a was reduced in mesenchymal-like lung cancer cell lines. The overexpression of miR‑146a induced a marked reduction of the mesenchymal marker and increase the epithelial marker in lung cancer cell lines. Moreover, the overexpression of miR‑146a suppressed lung cancer cell migration and invasion. Co-treatment with miR‑146a and gefitinib treatment showed a significant reduction of invasion in the resistant lung cancer cells induced by EMT. The expression of miR‑146a was downregulated in advanced lung cancer tissues. Insulin receptor substrate 2 (IRS2), an adaptor protein that modulates normal growth, metabolism, survival, and differentiation, was identified as a target of miR‑146a. miR‑146a regulated the expression of IRS2 at the mRNA and protein levels. These data demonstrate for the first time that miR‑146a suppresses lung cancer progression by repressing IRS2 expression. This provides new insight into the post-transcriptional regulation of lung cancer progression by miRNAs, a potential approach for the treatment of lung cancer.

Tracking and Functional Characterization of Epithelial-Mesenchymal Transition and Mesenchymal Tumor Cells during Prostate Cancer Metastasis.

The epithelial-mesenchymal transition (EMT) has been postulated as a mechanism by which cancer cells acquire the invasive and stem-like traits necessary for distant metastasis. However, direct in vivo evidence for the role of EMT in the formation of cancer stem-like cells (CSC) and the metastatic cascade remains lacking. Here we report the first isolation and characterization of mesenchymal-like and EMT tumor cells, which harbor both epithelial and mesenchymal characteristics, in an autochthonous murine model of prostate cancer. By crossing the established Pb-Cre(+/-);Pten(L/L);Kras(G12D) (/+) prostate cancer model with a vimentin-GFP reporter strain, generating CPKV mice, we were able to isolate epithelial, EMT, and mesenchymal-like cancer cells based on expression of vimentin and EpCAM. CPKV mice (but not mice with Pten deletion alone) exhibited expansion of cells with EMT (EpCAM(+)/Vim-GFP(+)) and mesenchymal-like (EpCAM(-)/Vim-GFP(+)) characteristics at the primary tumor site and in circulation. These EMT and mesenchymal-like tumor cells displayed enhanced stemness and invasive character compared with epithelial tumor cells. Moreover, they displayed an enriched tumor-initiating capacity and could regenerate epithelial glandular structures in vivo, indicative of epithelia-mesenchyme plasticity. Interestingly, while mesenchymal-like tumor cells could persist in circulation and survive in the lung following intravenous injection, only epithelial and EMT tumor cells could form macrometastases. Our work extends the evidence that mesenchymal and epithelial states in cancer cells contribute differentially to their capacities for tumor initiation and metastatic seeding, respectively, and that EMT tumor cells exist with plasticity that can contribute to multiple stages of the metastatic cascade.

The lncRNA H19 promotes epithelial to mesenchymal transition by functioning as miRNA sponges in colorectal cancer.

Recently, the long non-coding RNA (lncRNA) H19 has been identified as an oncogenic gene in multiple cancer types and elevated expression of H19 was tightly linked to tumorigenesis and cancer progression. However, the molecular basis for this observation has not been characterized in colorectal cancer (CRC) especially during epithelial to mesenchymal transition (EMT) progression. In our studies, H19 was characterized as a novel regulator of EMT in CRC. We found that H19 was highly expressed in mesenchymal-like cancer cells and primary CRC tissues. Stable expression of H19 significantly promotes EMT progression and accelerates in vivo and in vitro tumor growth. Furthermore, by using bioinformatics study and RNA immunoprecipitation combined with luciferase reporter assays, we demonstrated that H19 functioned as a competing endogenous RNA (ceRNA) for miR-138 and miR-200a, antagonized their functions and led to the de-repression of their endogenous targets Vimentin, ZEB1, and ZEB2, all of which were core marker genes for mesenchymal cells. Taken together, these observations imply that the lncRNA H19 modulated the expression of multiple genes involved in EMT by acting as a competing endogenous RNA, which may build up the missing link between the regulatory miRNA network and EMT progression.

CRIPTO overexpression promotes mesenchymal differentiation in prostate carcinoma cells through parallel regulation of AKT and FGFR activities.

Members of the EGF-CFC (Cripto, FRL-1, Cryptic) protein family are increasingly recognized as key mediators of cell movement and cell differentiation during vertebrate embryogenesis. The founding member of this protein family, CRIPTO, is overexpressed in various human carcinomas. Yet, the biological role of CRIPTO in this setting remains unclear. Here, we find CRIPTO expression as especially high in a subgroup of primary prostate carcinomas with poorer outcome, wherein resides cancer cell clones with mesenchymal traits. Experimental studies in PCa models showed that one notable function of CRIPTO expression in prostate carcinoma cells may be to augment PI3K/AKT and FGFR1 signaling, which promotes epithelial-mesenchymal transition and sustains a mesenchymal state. In the observed signaling events, FGFR1 appears to function parallel to AKT, and the two pathways act cooperatively to enhance migratory, invasive and transformation properties specifically in the CRIPTO overexpressing cells. Collectively, these findings suggest a novel molecular network, involving CRIPTO, AKT, and FGFR signaling, in favor of the emergence of mesenchymal-like cancer cells during the development of aggressive prostate tumors.

Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion.

The cholesterol reducing drugs, statins, exhibit anti-tumor effects against cancer stem cells and various cancer cell lines, exert potent additivity or synergy with existing chemotherapeutics in animal models of cancer and may reduce cancer incidence and cancer related mortality in humans. However, not all tumor cell lines are sensitive to statins, and clinical trials have demonstrated mixed outcomes regarding statins as anticancer agents. Here, we show that statin-induced reduction in intracellular cholesterol levels correlate with the growth inhibition of cancer cell lines upon statin treatment. Moreover, statin sensitivity segregates with abundant cytosolic vimentin expression and absent cell surface E-cadherin expression, a pattern characteristic of mesenchymal-like cells. Exogenous expression of cell surface E-cadherin converts statin- sensitive cells to a partially resistant state implying that statin resistance is in part dependent on the tumor cells attaining an epithelial phenotype. As metastasizing tumor cells undergo epithelial to mesenchymal transition during the initiation of the metastatic cascade, statin therapy may represent an effective approach to targeting the cells most likely to disseminate.

Mesenchymal-like pancreatic cancer cells harbor specific genomic alterations more frequently than their epithelial-like counterparts

The aggressiveness of pancreatic cancer is associated with the acquisition of mesenchymal characteristics by a subset of pancreatic cancer cells. The factors driving the development of this subset are not well understood. In this study, we tested the hypothesis that acquisition of a mesenchymal phenotype occurs selectively in tumor cells that harbor specific enabling genetic alterations. We obtained whole-genome comparative genomic hybridization (CGH) measurements on pancreatic cancer cell lines that have either an epithelial-like (17 cell lines) or a mesenchymal-like (9 cell lines) phenotype in vitro. The total amounts of amplifications and deletions were equivalent between the epithelial and mesenchymal groups, but 20 genes showed a major difference between the groups in prevalence of alterations. All 20 alterations (18 deletions and 2 amplifications) were more prevalent in the mesenchymal group, confirming the advanced nature of this cellular subtype. CDKN2A was altered in more than 50% of both groups, but co-deletions in neighboring genes, and concomitant loss of gene expression, were more prevalent in the mesenchymal group, suggesting that the size of the loss around CDKN2A affects cell phenotype. Whole-genome CGH on 11 primary cancer tissues revealed that the 20 genes were altered at a higher prevalence (up to 55% of the cases for certain genes) than randomly selected sets of 20 genes, with the same direction of alteration as in the cell lines. These findings support the concept that specific genetic alterations enable phenotype plasticity and provide promising candidate genes for further research.

A Positive Feedback Loop between Mesenchymal-like Cancer Cells and Macrophages Is Essential to Breast Cancer Metastasis

The close vicinity of cancer cells undergoing epithelial-mesenchymal transition (EMT) and tumor-associated macrophages (TAMs) at the invasive front of tumors suggests that these two cell type may mutually interact. We show that mesenchymal-like breast cancer cells activate macrophages to a TAM-like phenotype by GM-CSF. Reciprocally, CCL18 from TAMs induces cancer cell EMT, forming a positive feedback loop, in coculture systems and humanized mice. Inhibition of GM-CSF or CCL18 breaks this loop and reduces cancer metastasis. High GM-CSF expression in breast cancer samples is associated with more CCL18(+) macrophages, cancer cell EMT, enhanced metastasis, and reduced patient survival. These findings suggest that a positive feedback loop between GM-CSF and CCL18 is important in breast cancer metastasis.

WEE1 Inhibition Alleviates Resistance to Immune Attack of Tumor Cells Undergoing Epithelial–Mesenchymal Transition

Aberrant expression of the T-box transcription factor brachyury in human carcinomas drives the phenomenon of epithelial-mesenchymal transition (EMT), a phenotypic modulation that facilitates tumor dissemination and resistance to conventional therapies, including chemotherapy and radiotherapy. By generating isogenic cancer cell lines with various levels of brachyury expression, we demonstrate that high levels of brachyury also significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer cells. Our results indicated that resistance of brachyury-high tumor cells to immune-mediated attack was due to inefficient caspase-dependent apoptosis, manifested as inefficient nuclear lamin degradation in the presence of activated effector caspases. We correlated this phenomenon with loss of cell-cycle-dependent kinase 1 (CDK1), which mediates lamin phosphorylation. In support of a causal connection, pretreatment of tumor cells with a specific inhibitor of WEE1, a negative regulator kinase of CDK1, could counter the defective apoptosis of tumor cells expressing high levels of brachyury. Thus, our findings suggested that reconstituting CDK1 activity to threshold levels may be sufficient to restore immunosurveillance of mesenchymal-like cancer cells that have escaped previous immune detection or eradication.

MiR-655 Is an EMT-Suppressive MicroRNA Targeting ZEB1 and TGFBR2

Recently, the epithelial-to-mesenchymal transition (EMT) has been demonstrated to contribute to normal and disease processes including cancer progression. To explore EMT-suppressive microRNAs (miRNAs), we established a cell-based reporter system using a stable clone derived from a pancreatic cancer cell line, Panc1, transfected with a reporter construct containing a promoter sequence of CDH1/E-cadherin in the 5′ upstream region of the ZsGreen1 reporter gene. Then, we performed function-based screening with 470 synthetic double-stranded RNAs (dsRNAs) mimicking human mature miRNAs using the system and identified miR-655 as a novel EMT-suppressive miRNA. Overexpression of miR-655 not only induced the upregulation of E-cadherin and downregulation of typical EMT-inducers but also suppressed migration and invasion of mesenchymal-like cancer cells accompanied by a morphological shift toward the epithelial phenotype. In addition, we found a significant correlation between miR-655 expression and a better prognosis in esophageal squamous cell carcinoma (ESCC). Moreover, ZEB1 and TGFBR2, which are essential components of the TGF-b signaling pathway, were identified as direct targets of miR-655, suggesting that the activation of the TGF-b-ZEB1-E-cadherin axis by aberrant downregulation of miR-655 may accelerate cancer progression.

Cancer Cells Induced to Express Mesenchymal Phenotype Release Exosome-like Extracellular Vesicles Carrying Tissue Factor

Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes has not been studied. EVs are heterogeneous membrane structures that originate either at the surface (microparticles), or within (exosomes) activated or transformed cells, and are involved in intercellular trafficking of bioactive molecules. Here, we show that epithelial cancer cells (A431, DLD-1) adopt mesenchymal features (epithelial-to-mesenchymal transition-like state) upon activation of epidermal growth factor receptor (EGFR) coupled with blockade of E-cadherin. This treatment leads to a coordinated loss of EGFR and tissue factor (TF) from the plasma membrane and coincides with a surge in emission of small, exosome-like EVs containing both receptors. TF (but not EGFR) is selectively up-regulated in EVs produced by mesenchymal-like cancer cells and can be transferred to cultured endothelial cells rendering them highly procoagulant. We postulate that epithelial-to-mesenchymal transition-like changes may alter cancer cell interactions with the vascular systems through altered vesiculation and TF shedding.

Differential Regulation of Smad3 and of the Type II Transforming Growth Factor-β Receptor in Mitosis: Implications for Signaling

The response to transforming growth factor-β (TGF-β) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-β receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-β stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-β receptor (TβRII). Moreover, following the stimulation of mitotic cells with TGF-β, the proteasome-mediated attenuation of TGF-β receptor activity, the degradation and clearance of TβRII from the plasma membrane, and the clearance of the TGF-β ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-β receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-β stimuli in mitosis.

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs.

Abstract LB-246: Epithelial-mesenchymal transition (EMT) does not necessarily decrease the sensitivity of cancer cells to chemotherapeutic agents

Abstract Epithelial to mesenchymal transition (EMT) has been recently associated with metastatic competence and therapy resistance of cancer cells and the need for development of EMT-targeted therapies has been emphasized. In this report we address the differential responses of breast cancer cell lines with epithelial and mesenchymal phenotypes to anticancer agents using pharmacoinformatics and experimental approaches. First, we identified breast cancer cell lines with epithelial-like and mesenchymal-like phenotypes among the National Cancer Institute (NCI) panel of 60 cell lines and analyzed their differential responses to 10,081 anticancer agents. Second, we evaluated chemotherapeutic response of a mesenchymal-like isogenic breast cancer cell line (MCF-7-Snail) stably transfected to express the EMT-inducing Snail transcription factor and its epithelial-like precursor cell line transfected with the control plasmid (MCF-7-neo). The results of both approaches are inconsistent with the claim that mesenchymal-like cancer cells are generally more resistant to anticancer agents. We found that most of the 10,081 agents examined did not display significantly different potencies between epithelial-like and mesenchymal-like cells. Of the 10,081 anti-cancer agents examined, only 78 displayed consistent differences in effect between epithelial-like and mesenchymal-like cells and all of these agents displayed a more potent effect against mesenchymal-like cell lines. MCF-7-Snail cells were found to be more sensitive to the cytotoxic effects of doxorubicin, 5-FU and methotrexate, while MCF-7-neo cells were more sensitive to gemcitabine and docetaxel and both of these isogenic cell lines were equally responsive to mitomycin C and vincristine. The relatively lower sensitivity of MCF-Snail cells to docetaxel and gemcitabine can be explained by their overexpression of TUBB4 and RRM1, respectively, as demonstrated by gene expression analysis (U133 Plus 2.0 Affymetrix microarrays). In contrast, the higher sensitivity of MCF-7-Snail cells to 5-FU may be due to the down-regulation of the DPYD gene in these cells. Compared to isogenic epithelial cells, MCF-7-Snail cells display down-regulation of the anti-apoptotic genes XIAP, BCL2L1 and NAIP. Collectively, our results indicate that EMT per se does not generally result in a greater resistance of cancer cells to chemotherapeutic agents, but it may, in some instances, result in increased sensitivity of cancer cells to specific chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-246. doi:1538-7445.AM2012-LB-246

Abstract 69: Genomic deletions clustered on chromosomes 8 and 9 are associated with pancreatic cancer progression

Abstract Pancreatic cancer cells that have mesenchymal traits can be particularly migratory and resistant to chemotherapeutics. These cells are presumed to represent a more advanced and deadly population of cells relative to cancer cells with more differentiated, epithelial traits. The factors leading to the acquisition of a mesenchymal phenotype in pancreatic cancer are not well characterized. In this study, we tested the hypothesis that the development of a mesenchymal phenotype occurs selectively in tumor cells that harbor specific enabling genomic alterations. As a model system, we used pancreatic cancer cell lines that had either an epithelial-like or a mesenchymal-like phenotype when cultured in vitro, as defined by morphology and key molecular indicators. We used whole-genome comparative genomic hybridization to compare the genomic amplifications and deletions between 17 epithelial-like cell lines and 9 mesenchymal-like cell lines. An unbiased search revealed that 18 genes (each comprising at least three contiguous probes in a coding region) had alterations in a significantly greater percentage of the mesenchymal-like cells than the epithelial-like cells, whereas no genes had more frequent alterations in the epithelial-like cells. Sixteen of the 18 alterations were deletions, and 14 of the deletions were clustered in specific regions on chromosomes 8 and 9, suggesting the importance of those regions for suppressing de-differentiation. Some of the 18 genes were previously identified in studies of pancreatic cancer, such as SGCZ, KIAA1797 and SMAD4, while others were not. Certain genes showed a good correlation between DNA copy number and mRNA expression, providing evidence for the functional importance of those alterations. The known functions of the 18 genes include cell cycle control, cell membrane homeostasis, immune system regulation, and TGF-β pathway transduction, the deletion of which might provide improved survival advantage and cell structure modification required for metastasis. These findings support the concept that mesenchymal-like cancer cells are genetically more progressed than their epithelial-like counterparts and provide leads on the genomic hits that enable such progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 69. doi:1538-7445.AM2012-69