Epithelial-mesenchymal Transition Spectrum Research Articles

Abstract 3873: Understanding spatial organization of cellular plasticity in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is notorious for poor survival rates, underscoring the urgency of understanding factors that drive tumor progression. A key player in this process is epithelial-mesenchymal transition (EMT), known for its role in enhancing cancer cell adaptability, resistance to treatment, and metastasis. Our study focuses on EMT within PDAC, using single-cell spatial transcriptomics to explore how the tumor microenvironment influences changes in cellular states. We introduce a novel method involving Gaussian mixture models (GMM) to categorize EMT stages in single-cell data and utilize deep learning for label transfer of these tumor cell states, including intermediate cell states, onto spatial transcriptomic data with limited gene panels and sparse count profiles. We find strong spatial autocorrelation of classical cells and a dispersion of basal cells alongside other cells at intermediate stages of the EMT spectrum. By leveraging the spatial proximity of clonally related cells, we confirm that cells transition along the EMT spectrum in a gradient and infer transition rates. We next leverage density gradients of tumor cells in different states to identify distinct neighborhoods with varying EMT subtype composition. We observe significant variations in cell density among different EMT states. A focus on cancer-associated fibroblasts (CAFs) and macrophages reveals differences in these cells across density contours. We also examine differential ligand-receptor interactions across cell state gradients to better understand the cellular interactions that drive EMT transitions and alter the tumor environment. By leveraging spatial transcriptomics, this research aims to provide new biological insights into PDAC intratumoral heterogeneity, potentially leading to the identification of novel therapeutic targets to disrupt treatment resistance. Citation Format: Izabella Zamora, Milan Parikh, Bidish K. Patel, Lynn Bi, Doron Haviv, Jingyi Cao, Carina Shiau, Martin Hemberg, William L. Hwang, Nir Hacohen, David T. Ting, Arnav Mehta. Understanding spatial organization of cellular plasticity in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3873.

Cooperative RNA degradation stabilizes intermediate epithelial-mesenchymal states and supports a phenotypic continuum

SummaryMultiple intermediate epithelial-mesenchymal transition (EMT) states reflecting hybrid epithelial and mesenchymal phenotypes were observed in physiological and pathological conditions. Previous theoretical models explaining multiple EMT states rely on regulatory loops involving transcriptional feedback, which produce three or four attractors. This is incompatible with the observed continuum-like EMT spectrum. Here, we used mass-action-based models to describe post-transcriptional regulations, finding that cooperative RNA degradation via multiple microRNA binding sites can generate four-attractor systems without transcriptional feedback. Furthermore, the newly identified intermediates-enabling circuits are common in the EMT regulatory network, and they can synergize with transcriptional feedback to support phenotypic continuum. Finally, our model predicted a role of miR-101 in multistate EMT, and we identified evidence from single-cell RNA-sequencing data that support the prediction. Our work reveals a previously unknown role of cooperative RNA degradation and microRNAs in EMT, providing a framework that can bridge the gap between mechanistic models and single-cell experiments.

3D genome organization in the epithelial-mesenchymal transition spectrum

BackgroundThe plasticity along the epithelial-mesenchymal transition (EMT) spectrum has been shown to be regulated by various epigenetic repertoires. Emerging evidence of local chromatin conformation changes suggests that regulation of EMT may occur at a higher order of three-dimensional genome level.ResultsWe perform Hi-C analysis and combine ChIP-seq data across cancer cell lines representing different EMT states. We demonstrate that the epithelial and mesenchymal genes are regulated distinctively. We find that EMT genes are regulated within their topologically associated domains (TADs), with only a subset of mesenchymal genes being influenced by A/B compartment switches, indicating topological remodeling is required in the transcriptional regulation of these genes. At the TAD level, epithelial and mesenchymal genes are associated with different regulatory trajectories. The epithelial gene-residing TADs are enriched with H3K27me3 marks in the mesenchymal-like states. The mesenchymal gene-residing TADs, which do not show enrichment of H3K27me3 in epithelial-like states, exhibit increased interaction frequencies with regulatory elements in the mesenchymal-like states.ConclusionsWe propose a novel workflow coupling immunofluorescence and dielectrophoresis to unravel EMT heterogeneity at single-cell resolution. The predicted three-dimensional structures of chromosome 10, harboring Vimentin, identify cell clusters of different states. Our results pioneer a novel avenue to decipher the complexities underlying the regulation of EMT and may infer the barriers of plasticity in the 3D genome context.

Silencing of E-cadherin expression leads to increased chemosensitivity to irinotecan and oxaliplatin in colorectal cancer cell lines.

Colorectal carcinoma (CRC) is a leading malignant disease in most developed countries. In advanced stages it presents with metastatic dissemination and significant chemoresistance. Despite intensive studies, no convincing evidence has been published concerning the association of cadherins and epithelial-mesenchymal transition (EMT) as a direct cause of acquired chemoresistance in CRC. The present study was designed to investigate the role of E-cadherin in EMT and its associated chemosensitivity/chemoresistance in four immortalized CRC cell lines representing various stages of CRC development (i.e. HT29 and Caco-2-early, SW480 and SW620 late). The expression of E-cadherin gene CDH1 was downregulated by the specific siRNA. Cell proliferation and chemosensitivity to irinotecan (IT) and oxaliplatin (OPT) were detected using WST-1 and x-CELLigence Real Time analysis. Expression of selected EMT markers were tested and compared using RT-PCR and western blot analysis in both variants (E-cadherin silenced and non-silenced) of each cell line. We have discovered that downregulation of E-cadherin expression has a diverse effect on both cell proliferation as well as the expression of EMT markers in individual tested CRC cell lines, with Caco-2 cells being the most responsive. On the other hand, reduced E-cadherin expression resulted in increased sensitivity of all cell lines to IT and mostly to OPT which might be related to changes in intracellular metabolism of these drugs. These results suggest dichotomy of E-cadherin involvement in the phenotypic EMT spectrum of CRC and warrants further mechanistic studies.

SNAI1-Driven Sequential EMT Changes Attributed by Selective Chromatin Enrichment of RAD21 and GRHL2.

Over two decades of research on cancer-associated epithelial-mesenchymal transition (EMT) led us to ascertain the occurrence of transitional intermediate states (collectively referred to as the EMT spectrum). Among the molecular factors that drive EMT, SNAI1 plays an indispensable role in regulating other core transcription factors, and this regulation is highly context-dependent. However, molecular investigation on this context-dependent regulation is still lacking. Using two ovarian cancer cell lines, we show that SNAI1 regulation on other core EMT-TFs switches from a repressive control in highly epithelial cells to an activation signaling in intermediate epithelial cells. Upon further scrutiny, we identify that the expression of early epithelial genes PERP and ERBB3 are differentially regulated in SNAI1-induced sequential EMT changes. Mechanistically, we show that changes in PERP and ERBB3 transcript levels could be correlated to the selective enrichment loss of RAD21, a cohesin component, at the distal enhancer sites of PERP and ERBB3, which precedes that of the proximal promoter-associated sites. Furthermore, the RAD21 enrichment at the distal enhancer sites is dependent on GRHL2 expression. In a nutshell, the alteration of GRHL2-associated RAD21 enrichment in epithelial genes is crucial to redefine the transition of cellular states along the EMT spectrum.

Integrative Transcriptomic Analysis Reveals a Multiphasic Epithelial-Mesenchymal Spectrum in Cancer and Non-tumorigenic Cells.

Epithelial–mesenchymal transition (EMT), the conversion between rigid epithelial cells and motile mesenchymal cells, is a reversible cellular process involved in tumorigenesis, metastasis, and chemoresistance. Numerous studies have found that several types of tumor cells show a high degree of cell-to-cell heterogeneity in terms of their gene expression signatures and cellular phenotypes related to EMT. Recently, the prevalence and importance of partial or intermediate EMT states have been reported. It is unclear, however, whether there is a general pattern of cancer cell distribution in terms of the overall expression of epithelial-related genes and mesenchymal-related genes, and how this distribution is related to EMT process in normal cells. In this study, we performed integrative transcriptomic analysis that combines cancer cell transcriptomes, time course data of EMT in non-tumorigenic epithelial cells, and epithelial cells with perturbations of key EMT factors. Our statistical analysis shows that cancer cells are widely distributed in the EMT spectrum, and the majority of these cells can be described by an EMT path that connects the epithelial and the mesenchymal states via a hybrid expression region in which both epithelial genes and mesenchymal genes are highly expressed overall. We found that key patterns of this EMT path are observed in EMT progression in non-tumorigenic cells and that transcription factor ZEB1 plays a key role in defining this EMT path via diverse gene regulatory circuits connecting to epithelial genes. We performed Gene Set Variation Analysis to show that the cancer cells at hybrid EMT states also possess hybrid cellular phenotypes with both high migratory and high proliferative potentials. Our results reveal critical patterns of cancer cells in the EMT spectrum and their relationship to the EMT process in normal cells, and provide insights into the mechanistic basis of cancer cell heterogeneity and plasticity.

Quantifying Cancer Epithelial-Mesenchymal Plasticity and its Association with Stemness and Immune Response.

Cancer cells can acquire a spectrum of stable hybrid epithelial/mesenchymal (E/M) states during epithelial–mesenchymal transition (EMT). Cells in these hybrid E/M phenotypes often combine epithelial and mesenchymal features and tend to migrate collectively commonly as small clusters. Such collectively migrating cancer cells play a pivotal role in seeding metastases and their presence in cancer patients indicates an adverse prognostic factor. Moreover, cancer cells in hybrid E/M phenotypes tend to be more associated with stemness which endows them with tumor-initiation ability and therapy resistance. Most recently, cells undergoing EMT have been shown to promote immune suppression for better survival. A systematic understanding of the emergence of hybrid E/M phenotypes and the connection of EMT with stemness and immune suppression would contribute to more effective therapeutic strategies. In this review, we first discuss recent efforts combining theoretical and experimental approaches to elucidate mechanisms underlying EMT multi-stability (i.e., the existence of multiple stable phenotypes during EMT) and the properties of hybrid E/M phenotypes. Following we discuss non-cell-autonomous regulation of EMT by cell cooperation and extracellular matrix. Afterwards, we discuss various metrics that can be used to quantify EMT spectrum. We further describe possible mechanisms underlying the formation of clusters of circulating tumor cells. Last but not least, we summarize recent systems biology analysis of the role of EMT in the acquisition of stemness and immune suppression.

Testing the gene expression classification of the EMT spectrum

The epithelial-mesenchymal transition (EMT) plays a central role in cancer metastasis and drug resistance—two persistent clinical challenges. Epithelial cells can undergo a partial or full EMT, attaining either a hybrid epithelial/mesenchymal (E/M) or mesenchymal phenotype, respectively. Recent studies have emphasized that hybrid E/M cells may be more aggressive than their mesenchymal counterparts. However, mechanisms driving hybrid E/M phenotypes remain largely elusive. Here, to better characterize the hybrid E/M phenotype (s) and tumor aggressiveness, we integrate two computational methods—(a) RACIPE—to identify the robust gene expression patterns emerging from the dynamics of a given gene regulatory network, and (b) EMT scoring metric—to calculate the probability that a given gene expression profile displays a hybrid E/M phenotype. We apply the EMT scoring metric to RACIPE-generated gene expression data generated from a core EMT regulatory network and classify the gene expression profiles into relevant categories (epithelial, hybrid E/M, mesenchymal). This categorization is broadly consistent with hierarchical clustering readouts of RACIPE-generated gene expression data. We also show how the EMT scoring metric can be used to distinguish between samples composed of exclusively hybrid E/M cells and those containing mixtures of epithelial and mesenchymal subpopulations using the RACIPE-generated gene expression data.

Hybrid epithelial/mesenchymal phenotypes promote metastasis and therapy resistance across carcinomas.

Cancer metastasis and therapy resistance are the major unsolved clinical challenges, and account for nearly all cancer-related deaths. Both metastasis and therapy resistance are fueled by epithelial plasticity, the reversible phenotypic transitions between epithelial and mesenchymal phenotypes, including epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). EMT and MET have been largely considered as binary processes, where cells detach from the primary tumor as individual units with many, if not all, traits of a mesenchymal cell (EMT) and then convert back to being epithelial (MET). However, recent studies have demonstrated that cells can metastasize in ways alternative to traditional EMT paradigm; for example, they can detach as clusters, and/or occupy one or more stable hybrid epithelial/mesenchymal (E/M) phenotypes that can be the end point of a transition. Such hybrid E/M cells can integrate various epithelial and mesenchymal traits and markers, facilitating collective cell migration. Furthermore, these hybrid E/M cells may possess higher tumor-initiation and metastatic potential as compared to cells on either end of the EMT spectrum. Here, we review in silico, in vitro, in vivo and clinical evidence for the existence of one or more hybrid E/M phenotype(s) in multiple carcinomas, and discuss their implications in tumor-initiation, tumor relapse, therapy resistance, and metastasis. Together, these studies drive the emerging notion that cells in a hybrid E/M phenotype may occupy 'metastatic sweet spot' in multiple subtypes of carcinomas, and pathways linked to this (these) hybrid E/M state(s) may be relevant as prognostic biomarkers as well as a promising therapeutic targets.

Human trophoblast epithelial-mesenchymal transition in abnormally invasive placenta.

Differentiation of first trimester human placental cytotrophoblast (CTB) from an anchorage-dependent epithelial phenotype into the mesenchymal-like invasive extravillous trophoblast (EVT) is crucial in the development of the maternal-fetal interface. We showed previously that differentiation of first trimester CTB to EVT involves an epithelial-mesenchymal transition (EMT). Here we compare the epithelial-mesenchymal characteristics of CTB and EVT derived from normal third trimester placenta or placenta previa versus abnormally invasive placenta (AIP). CTB and EVT were isolated from normal term placenta or placenta previa following Caesarean section and EVT from AIP following Caesarean hysterectomy. Cell identity was validated by measurement of cytokeratin-7 and HLA-G. Comparing normal term CTB with EVT from normal term placenta or placenta previa for differential expression analysis of genes associated with the EMT showed changes in >70% of the genes probed. While demonstrating a mesenchymal phenotype relative to CTB, many of the gene expression changes in third trimester EVT were reduced relative to the first trimester EVT. We suggest that third trimester EVT are in a more constrained, metastable state compared to first trimester equivalents. By contrast, EVT from AIP demonstrate characteristics that are more mesenchymal than normal third trimester EVT, placing them closer to first trimester EVT on the EMT spectrum, consistent with a more invasive phenotype.

Loss of discoidin domain receptor 1 (DDR1) via CpG methylation during EMT in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) can be stratified according to the stages of epithelial-mesenchymal transition (EMT). Here, we aim to identify tyrosine kinases (TKs) in EOC that correlate with the EMT subtypes. By gene expression microarray, we analyzed the expression levels of tyrosine kinases in EOC cell lines in correlation with EMT. Among the candidate TKs identified, DDR1 was expressed mainly in EOC cells with an Epithelial phenotype. Its expression was validated by qPCR, ELISA and western blotting. Using Infinium HumanMethylation27K BeadChip array and pyrosequencing, we further analyzed the CpG methylation levels at the DDR1 promoter in EOC cells and found that the CpG methylation levels of DDR1 promoter correlated negatively with the expression of DDR1 along the EMT spectrum. Therefore, EMT stratification could be a potential biomarker to predict patient response to DDR1-targeting drugs.

New insights into the role of EMT in tumor immune escape

Novel immunotherapy approaches have provided durable remission in a significant number of cancer patients with cancers previously considered rapidly lethal. Nonetheless, the high degree of nonresponders, and in some cases the emergence of resistance in patients who do initially respond, represents a significant challenge in the field of cancer immunotherapy. These issues prompt much more extensive studies to better understand how cancer cells escape immune surveillance and resist immune attacks. Here, we review the current knowledge of how cellular heterogeneity and plasticity could be involved in shaping the tumor microenvironment (TME) and in controlling antitumor immunity. Indeed, recent findings have led to increased interest in the mechanisms by which cancer cells undergoing epithelial‐mesenchymal transition (EMT), or oscillating within the EMT spectrum, might contribute to immune escape through multiple routes. This includes shaping of the TME and decreased susceptibility to immune effector cells. Although much remains to be learned on the mechanisms at play, cancer cell clones with mesenchymal features emerging from the TME seem to be primed to face immune attacks by specialized killer cells of the immune system, the natural killer cells, and the cytotoxic T lymphocytes. Recent studies investigating patient tumors have suggested EMT as a candidate predictive marker to be explored for immunotherapy outcome. Promising data also exist on the potential utility of targeting these cancer cell populations to at least partly overcome such resistance. Research is now underway which may lead to considerable progress in optimization of treatments.

Acquisition of tumor cell phenotypic diversity along the EMT spectrum under hypoxic pressure: Consequences on susceptibility to cell-mediated cytotoxicity

ABSTRACTTumor escape to immunosurveillance and resistance to immune attacks present a major hurdle in cancer therapy, especially in the current era of new cancer immunotherapies. We report here that hypoxia, a hallmark of most solid tumors, orchestrates carcinoma cell heterogeneity through the induction of phenotypic diversity and the acquisition of distinct epithelial–mesenchymal transition (EMT) states. Using lung adenocarcinoma cells derived from a non-metastatic patient, we demonstrated that hypoxic stress induced phenotypic diversity along the EMT spectrum, with induction of EMT transcription factors (EMT-TFs) SNAI1, SNAI2, TWIST1, and ZEB2 in a hypoxia-inducible factor-1α (HIF1A)-dependent or -independent manner. Analysis of hypoxia-exposed tumor subclones, with pronounced epithelial or mesenchymal phenotypes, revealed that mesenchymal subclones exhibited an increased propensity to resist cytotoxic T lymphocytes (CTL), and natural killer (NK) cell-mediated lysis by a mechanism involving defective immune synapse signaling. Additionally, targeting EMT-TFs, or inhibition of TGF-β signaling, attenuated mesenchymal subclone susceptibility to immune attack. Together, these findings uncover hypoxia-induced EMT and heterogeneity as a novel driving escape mechanism to lymphocyte-mediated cytotoxicity, with the potential to provide new therapeutic opportunities for cancer patients.

Controlling Stochasticity in Epithelial-Mesenchymal Transition Through Multiple Intermediate Cellular States.

Epithelial-mesenchymal transition (EMT) is an instance of cellular plasticity that plays critical roles in development, regeneration and cancer progression. Recent studies indicate that the transition between epithelial and mesenchymal states is a multi-step and reversible process in which several intermediate phenotypes might coexist. These intermediate states correspond to various forms of stem-like cells in the EMT system, but the function of the multi-step transition or the multiple stem cell phenotypes is unclear. Here, we use mathematical models to show that multiple intermediate phenotypes in the EMT system help to attenuate the overall fluctuations of the cell population in terms of phenotypic compositions, thereby stabilizing a heterogeneous cell population in the EMT spectrum. We found that the ability of the system to attenuate noise on the intermediate states depends on the number of intermediate states, indicating the stem-cell population is more stable when it has more sub-states. Our study reveals a novel advantage of multiple intermediate EMT phenotypes in terms of systems design, and it sheds light on the general design principle of heterogeneous stem cell population.

Abstract POSTER-TECH-1112: Quantitate epithelial-mesenchymal transition in ovarian cancer

Abstract Purpose: Metastases account for more than 90% of cancer deaths and hence, inhibiting metastasis is an attractive therapeutic option. Accumulating evidences show that invasion and metastasis of ovarian carcinoma could be driven by Epithelial-Mesenchymal Transition (EMT), a developmental program being employed during carcinoma progression. As EMT is a reversible process, inhibiting or reversing EMT thus emerges as a potential anti-metastasis strategy. EMT is a spectrum consisting of the binary epithelial and mesenchymal states as well as transitional intermediate states. Therefore, reversing EMT from the mesenchymal state would result in the sliding along an EMT Spectrum. Currently, there is no quantitative tool available to assess the EMT spectrum. In this study, we describe a bioinformatics method for quantitating EMT. Experimental procedure: By using immunofluorescence staining of E-cadherin and N-cadherin, we defined the most epithelial and most mesenchymal cells in a panel of ovarian carcinoma cell lines. We derived an EMT signature from Affymetrix gene expression microarrays of these cell lines. Subsequently, with a Kolmogorov-Smirnov-based method, we computed EMT score that assesses the similarity of an ovarian carcinoma cell transcriptome with the derived EMT signature. This method scores ovarian carcinoma cell lines in the range of [-1.0,+1.0] which corresponded to the extreme epithelial and mesenchymal states respectively along the EMT spectrum. We thus applied the EMT scoring to estimate EMT status in ovarian carcinoma tissues. Results: The EMT signature derived included known EMT inducers and markers such as ZEB1, ZEB2, SNAI2 and VIM. Expressions of selected markers such as ZEB1, VIM and FN1 were confirmed by immunofluorescence staining. Ovarian carcinoma cell lines displaying a mesenchymal morphology were accurately reflected as having high EMT scores. Cell lines with high EMT scores were anoikis resistant. In addition, ovarian carcinoma tissues displaying high EMT scores had poorer overall survival than those of low EMT scores. Correlation analysis of EMT scores and pathways revealed that ovarian carcinoma with high EMT scores are associated with increased TGFβ, WNT, NOTCH, and SRC signaling pathways. This suggested that inhibitors targeting these pathways could potentially reverse EMT. We thus treated mesenchymal ovarian carcinoma cell lines SKOV3, HEY, DOV13 with a SRC inhibitor, AZD0530. We observed a partial reversal of EMT evident by decreased EMT scores (-0.11 to -0.17) in these cells. Conclusions: EMT scoring could reflect both the EMT status of a sample and the transitional changes along the EMT spectrum upon intervening with inhibitors. Thus, EMT scoring presents a promising tool to assess the effectiveness of anti-EMT or EMT reversing compounds. Citation Format: Tuan Zea Tan, Ruby Yun-Ju Huang, Qing Hao Miow, Jieru Ye, Meng Kang Wong, Jieying Amelia Lau, Seiichi Mori, Jean Paul Thiery. Quantitate epithelial-mesenchymal transition in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1112.

Abstract 1430: Transcriptional regulatory loops among SNAI1, TWIST1, ZEB1, and ZEB2 defines the epithelial-mesenchymal transition (EMT) spectrum in epithelial ovarian cancer (EOC)

Abstract Epithelial-mesenchymal transition (EMT) refers to a sequential phenotypic transformation process from epithelial to mesenchymal states, which is well defined in embryonic development. During cancer progression, cancer cells undergo EMT progressively loss epithelial characteristics and gain mesenchymal properties and are correlated with acquisition of increased capacity of metastasis and resistance to chemo- and immunotherapy. Although EMT is intensively studied in various types of cancers, its role in epithelial ovarian cancer (EOC), the most lethal gynaecological malignancy in the world, has yet to be elucidated. Previously, our group proposed the concept of an EMT Spectrum which encompasses the hybrid “intermediate” phenotype as well as the bona-fide epithelial and mesenchymal phenotypes. EOC cells harboring intermediate phenotypes showed distinct functional differences in in vitro aggressiveness. We hypothesize that EMT transcriptional drivers, SNAI1, TWIST1, ZEB1, and ZEB2, contribute to different biological consequences and show different hierarchy to induce step-wise phenotypic changes along the EMT Spectrum. EMT models were established by overexpressing classic EMT drivers in selected EOC cell lines with different EM phenotypes at different positions of EMT spectrum to test the intrinsic transcriptional regulation networks. Quantitative PCR (QPCR) was performed to examine the cross regulation among the EMT transcriptional drivers and their downstream targets. There was no cross regulation detected in the most epithelial cell line, PEO1, while a simple, repressive regulatory net was found in the OVCA420 cell line. The cell line with an intermediate phenotype, OVCA429, showed a complex double positive feedback loop among the EMT drivers. The divergence in the transcriptional regulatory loops correlated with the differences of in vitro EMT functions. These results indicate that there is a dynamic EMT transcriptional regulatory network to further define the EMT spectrum. In conclusion, this study demonstrates that cancer cells with an intermediate phenotype are more easily to be pushed along the EMT spectrum due to their ability to initiate a complex transcriptional feedback regulatory loop. Citation Format: Ming Tan, Vin Yee Chuang, Tuan Zea Tan, Jean Paul Thiery, Ruby Yun-Ju Huang. Transcriptional regulatory loops among SNAI1, TWIST1, ZEB1, and ZEB2 defines the epithelial-mesenchymal transition (EMT) spectrum in epithelial ovarian cancer (EOC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1430. doi:10.1158/1538-7445.AM2015-1430

Functional relevance of a six mesenchymal gene signature in epithelial-mesenchymal transition (EMT) reversal by the triple angiokinase inhibitor, nintedanib (BIBF1120)

Epithelial-mesenchymal transition (EMT), a crucial mechanism in carcinoma progression, describes the process whereby epithelial cells lose their apico-basal polarity and junctional complexes and acquire a mesenchymal-like morphology. Several markers are considered to be authentic indicators of an epithelial or mesenchymal status; however, there is currently no comprehensive or systematic method with which to determine their functional relevance. Previously, we identified a 33-gene EMT signature comprising 25 epithelial and 6 mesenchymal genes that best describe this concept of the EMT spectrum. Here, we designed small-scale siRNA screens targeting these six mesenchymal signature genes (CD99L2, EMP3, ITGA5, SYDE1, VIM, ZEB1) to explore their functional relevance and their roles during EMT reversal by nintedanib (BIBF1120) in a mesenchymal-like SKOV3 ovarian cancer cell line. We found that neither cell proliferation nor cytotoxicity was affected by silencing any of these genes. SKOV3 cells expressing siRNA against mesenchymal genes (ZEB1, EMP3, CD99L2, ITGA5, and SYDE1) showed enhanced colony compaction (reduced inter-nuclear distance). Inductions of E-cadherin expression were only observed in SYDE1- and ZEB1-silenced SKOV3 cells. In addition, only SYDE1-silenced SKOV3 cells showed increased anoikis. Finally, we identified that SYDE1 and ZEB1 were down-regulated in nintedanib-treated SKOV3 cells and SYDE1- and ZEB1-silenced SKOV3 cells showed enhanced nintedanib-induced up-regulation of E-cadherin. Nintedanib-treated SKOV3 cells also showed colony compaction and decreases in EMT scores both in vitro and in vivo. We conclude that SYDE1 and ZEB1 are functionally relevant in EMT reversal. This study thus provides a proof-of-concept for the use of in vitro siRNA screening to explore the EMT-related functions of selected genes and their potential relevance in the discovery of EMT reversing drugs.

Crosstalk of Oncogenic Signaling Pathways during Epithelial-Mesenchymal Transition.

Epithelial–mesenchymal transition (EMT) and cell transformation have been well-documented in multiple cancer cell models and are believed to be one of the earliest events in tumor progression. Genetic and epigenetic modifications shift cells toward either end of the EMT spectrum, and can be influenced by the microenvironment surrounding a tumor. EMT and mesenchymal–epithelial transition are critical to normal function and development and an intricate network of transcription factors and transcriptional regulators tightly regulates these processes. As evidenced in normal and transformed cell lines, many signaling pathways trigger EMT during development and differentiation. The signaling pathways include those triggered by different members of the transforming growth factor superfamily, epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, hypoxia-inducible factor, Wnt, Notch, and many others. Functional redundancies allow cells to undergo EMT even if these key transcriptional regulators are lacking, but these same redundancies also make these pathways particularly susceptible to gain-of-function mutations or constitutive signal activation; the “forced” transition toward either a mesenchymal or epithelial phenotype.

Epithelial-mesenchymal transition spectrum quantification and its efficacy in deciphering survival and drug responses of cancer patients.

Epithelial-mesenchymal transition (EMT) is a reversible and dynamic process hypothesized to be co-opted by carcinoma during invasion and metastasis. Yet, there is still no quantitative measure to assess the interplay between EMT and cancer progression. Here, we derived a method for universal EMT scoring from cancer-specific transcriptomic EMT signatures of ovarian, breast, bladder, lung, colorectal and gastric cancers. We show that EMT scoring exhibits good correlation with previously published, cancer-specific EMT signatures. This universal and quantitative EMT scoring was used to establish an EMT spectrum across various cancers, with good correlation noted between cell lines and tumours. We show correlations between EMT and poorer disease-free survival in ovarian and colorectal, but not breast, carcinomas, despite previous notions. Importantly, we found distinct responses between epithelial- and mesenchymal-like ovarian cancers to therapeutic regimes administered with or without paclitaxelin vivo and demonstrated that mesenchymal-like tumours do not always show resistance to chemotherapy. EMT scoring is thus a promising, versatile tool for the objective and systematic investigation of EMT roles and dynamics in cancer progression, treatment response and survival.

An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor, saracatinib (AZD0530)

The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial–mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial–mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.