Epithelial-mesenchymal Transition Signature Research Articles

Epithelial-to-Mesenchymal Stem Cell Transition in a Human Organ: Lessons from Lichen Planopilaris

Epithelial-to-mesenchymal transition (EMT) is critical for embryonic development and wound healing, and occurs in fibrotic disease and carcinoma. Here, we show that EMT also occurs within the bulge, the epithelial stem cell (eSC) niche of human scalp hair follicles, during the inflammatory permanent alopecia, lichen planopilaris. We show that a molecular EMT signature can be experimentally induced in healthy human eSCs in situ by antagonizing E-cadherin, combined with transforming growth factor-β1, epidermal growth factor, and IFN-γ administration, which to our knowledge has not been reported previously. Moreover, induction of EMT within primary human eSCs can be prevented and even partially reversed exvivo by peroxisome proliferator-activated receptor-γ agonists, likely through suppression of the transforming growth factor-β signaling pathway. Furthermore, we show that peroxisome proliferator-activated receptor-γ agonists also attenuates the EMT signature even in lesional lichen planopilaris hair follicles exvivo. We introduce lichen planopilaris as a model disease for pathological EMT in human adult eSCs, report a preclinical assay for therapeutically manipulating eSC EMT within a healthy human (mini-)organ, and show that peroxisome proliferator-activated receptor-γ agonists are promising agents for suppressing and partially reversing EMT in human hair follicles eSCs exvivo, including in lichen planopilaris.

Epigenetic landscape change analysis during human EMT sheds light on a key EMT mediator TRIM29.

Epithelial to mesenchymal transition (EMT) is a key trans-differentiation process, which plays a critical role in physiology and pathology. Although gene expression changes in EMT have been scrutinized, study of epigenome is in its infancy. To understand epigenetic changes during TWIST-driven EMT, we used the AcceSssIble assay to study DNA methylation and chromatin accessibility in human mammary epithelial cells (HMECs). The DNA methylation changes were found to have functional significance in EMT – i.e. methylated genes were enriched for E-box motifs that can be recognized by TWIST, at the promoters suggesting a potential targeting phenomenon, whereas the demethylated regions were enriched for pro-metastatic genes, supporting the role of EMT in metastasis. TWIST-induced EMT triggers alterations in chromatin accessibility both independent of and dependent on DNA methylation changes, primarily resulting in closed chromatin conformation. By overlapping the genes, whose chromatin structure is changed during early EMT and a known “core EMT signature”, we identified 18 driver candidate genes during EMT, 14 upregulated and 4 downregulated genes with corresponding chromatin structure changes. Among 18 genes, we focused on TRIM29 as a novel marker of EMT. Although loss of TRIM29 is insufficient to suppress CDH, it is enough to induce CDH2 and VIM. Gene functional annotation analysis shows the involvement of TRIM29 in epidermal development, cell differentiation and cell migration. Taken together, our results provide a robust snapshot of chromatin state during human EMT and identify TRIM29 as a core mediator of EMT.

Epithelial-to-Mesenchymal Transition and MicroRNAs in Lung Cancer.

Despite major advances, non-small cell lung cancer (NSCLC) remains the major cause of cancer-related death in developed countries. Metastasis and drug resistance are the main factors contributing to relapse and death. Epithelial-to-mesenchymal transition (EMT) is a complex molecular and cellular process involved in tissue remodelling that was extensively studied as an actor of tumour progression, metastasis and drug resistance in many cancer types and in lung cancers. Here we described with an emphasis on NSCLC how the changes in signalling pathways, transcription factors expression or microRNAs that occur in cancer promote EMT. Understanding the biology of EMT will help to define reversing process and treatment strategies. We will see that this complex mechanism is related to inflammation, cell mobility and stem cell features and that it is a dynamic process. The existence of intermediate phenotypes and tumour heterogeneity may be debated in the literature concerning EMT markers, EMT signatures and clinical consequences in NSCLC. However, given the role of EMT in metastasis and in drug resistance the development of EMT inhibitors is an interesting approach to counteract tumour progression and drug resistance. This review describes EMT involvement in cancer with an emphasis on NSCLC and microRNA regulation.

Abstract 852: Bromodomain and extraterminal proteins regulate the epithelial-mesenchymal transition in breast cancer

Abstract The epithelial-mesenchymal transition (EMT) is a developmental program that cancer cells often activate to acquire a highly plastic phenotype that promotes invasion, metastasis, but also chemoresistance and cancer stem cell generation. As readers of epigenetic marks, bromodomain and extra-terminal (BET) proteins BRD2, BRD3 and BRD4 participate in the regulation of multiple transcriptional programs implicated in cancer progression. We sought to unravel the roles of BET proteins in EMT in breast cancer. Despite their homology, we report that BET proteins differentially regulate EMT. Based on an EMT PCR array, we identify a BRD2-specific transcriptional profile that promotes EMT, whereas BRD3 and BRD4 signatures repress this program. These individual signatures are unidentifiable upon pan-BET inhibition using JQ1, reinforcing the necessity to target each BET member separately to better understand their functions. Upon BRD2 depletion, basal-like breast cancer cells, which present a mesenchymal phenotype, exhibit a reduced expression of mesenchymal markers (N-cadherin, vimentin) and re-express epithelial markers (E-cadherin, cytokeratins). Moreover, a large panel of EMT master transcription factors is downregulated in BRD2-depleted cells, including the Snail and ZEB families or Twist. Interestingly, we found that BRD3 or BRD4 depletion leads to the opposite phenotype: an increase of mesenchymal marker expression and repression of the epithelial markers. In luminal A breast cancer cells which present an epithelial phenotype, BRD2 overexpression leads to the expression of mesenchymal markers. Similar results were obtained by depleting BRD3 or BRD4 in these cells, confirming the differential roles of BET proteins in EMT regulation. Taken together, our results establish that BRD2 positively regulates EMT, whereas BRD3 and BRD4 repress this program. BET proteins possess separate and opposite biological functions, reinforcing the relevance of an individual targeting instead of a pan-BET inhibition using JQ1. We hypothesize that BET proteins modulate EMT through the regulation of its master transcription factors. We propose that the balance of BET proteins presence at the promoters of the EMT genes is a novel mechanism of regulation of this program in breast cancer cells. Citation Format: Guillaume Andrieu, Gerald V. Denis. Bromodomain and extraterminal proteins regulate the epithelial-mesenchymal transition in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 852. doi:10.1158/1538-7445.AM2017-852

Abstract 2443: Massively parallel single-cell RNA-Seq identifies diverse subpopulations displaying EMT and stem-like features

Abstract Single-cell transcriptome analysis enables a new paradigm for studying complex systems in cancer. As opposed to bulk sequencing, which averages genomic signals across thousands or millions of cells and obscures the presence of rare subtypes, single cell sequencing enables the interrogation of individual cells. In cancer, intratumoral heterogeneity is observed at both genomic and epigenomic levels, and its analysis enables the discovery of new actionable targets and treatment modalities tailored to individual subpopulations. As an example, many cancer cell lines and those derived from patients contain subpopulations marked by distinct patterns of surface markers such as CD44, and are linked to drug resistant and tumor initiating phenotypes. A complete characterization of such cellular populations ideally requires marker-free sampling, followed by clustering into distinct subgroups. In this study, we demonstrate the significant advantages of such an approach; we utilize a high-throughput single-cell RNA-Seq method to characterize the transcriptomic profiles of cellular populations. We performed single-cell RNA-Seq on thousands of cells in the matched SW480 (primary) and SW620 (metastatic) colorectal cell lines using a microfluidic droplet barcoding technology that enables the tracking of single cells during library preparation. By focusing on genes with high inter-cell variability, we discovered a small subpopulation of cells that displayed a distinct gene expression signature from the major subpopulation. Differential gene expression analysis of this subpopulation yielded genes virtually all enriched in the epithelial-to-mesenchymal transition (EMT) pathway. These cells showed significant increases in canonical mesenchymal marker genes such as VIM, CD44, and SOX9. Gene expression profiles of these subpopulations also correlated with established EMT signatures. Remarkably, this subpopulation did not display mutual exclusivity in gene expression with the epithelial marker EPCAM, which possibly indicates an intermediate mesenchymal phenotype. We also observed in the major population cluster a small subset of cells totaling less than 1% of the population that were significantly enriched for LGR5 expression, a common stem-like marker in colorectal cancer. Overall, we demonstrate the use of single-cell RNA-Seq to discover and characterize a diversity of cellular states that would otherwise be impossible from bulk analysis. Citation Format: Billy Lau, Jiamin Chen, Hanlee P. Ji. Massively parallel single-cell RNA-Seq identifies diverse subpopulations displaying EMT and stem-like features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2443. doi:10.1158/1538-7445.AM2017-2443

DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells

Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.

A Transcriptional Program for Detecting TGFβ-Induced EMT in Cancer.

Most cancer deaths are due to metastasis, and epithelial-to-mesenchymal transition (EMT) plays a central role in driving cancer cell metastasis. EMT is induced by different stimuli, leading to different signaling patterns and therapeutic responses. TGFβ is one of the best-studied drivers of EMT, and many drugs are available to target this signaling pathway. A comprehensive bioinformatics approach was employed to derive a signature for TGFβ-induced EMT which can be used to score TGFβ-driven EMT in cells and clinical specimens. Considering this signature in pan-cancer cell and tumor datasets, a number of cell lines (including basal B breast cancer and cancers of the central nervous system) show evidence for TGFβ-driven EMT and carry a low mutational burden across the TGFβ signaling pathway. Furthermore, significant variation is observed in the response of high scoring cell lines to some common cancer drugs. Finally, this signature was applied to pan-cancer data from The Cancer Genome Atlas to identify tumor types with evidence of TGFβ-induced EMT. Tumor types with high scores showed significantly lower survival rates than those with low scores and also carry a lower mutational burden in the TGFβ pathway. The current transcriptomic signature demonstrates reproducible results across independent cell line and cancer datasets and identifies samples with strong mesenchymal phenotypes likely to be driven by TGFβ.Implications: The TGFβ-induced EMT signature may be useful to identify patients with mesenchymal-like tumors who could benefit from targeted therapeutics to inhibit promesenchymal TGFβ signaling and disrupt the metastatic cascade. Mol Cancer Res; 15(5); 619-31. ©2017 AACR.

High prevalence of TP53 mutations is associated with poor survival and an EMT signature in gliosarcoma patients

Gliosarcoma (GS) is a rare variant (2%) of glioblastoma (GBM) that poses clinical genomic challenges because of its poor prognosis and limited genomic information. To gain a comprehensive view of the genomic alterations in GS and to understand the molecular etiology of GS, we applied whole-exome sequencing analyses for 28 GS cases (6 blood-matched fresh-frozen tissues for the discovery set, 22 formalin-fixed paraffin-embedded tissues for the validation set) and copy-number variation microarrays for 5 blood-matched fresh-frozen tissues. TP53 mutations were more prevalent in the GS cases (20/28, 70%) compared to the GBM cases (29/90, 32%), and the GS patients with TP53 mutations showed a significantly shorter survival (multivariate Cox analysis, hazard ratio=23.9, 95% confidence interval, 2.87–199.63, P=0.003). A pathway analysis showed recurrent alterations in MAPK signaling (EGFR, RASGRF2 and TP53), phosphatidylinositol/calcium signaling (CACNA1s, PLCs and ITPRs) and focal adhesion/tight junction (PTEN and PAK3) pathways. Genomic profiling of the matched recurrent GS cases detected the occurrence of TP53 mutations in two recurrent GS cases, which suggests that TP53 mutations play a role in treatment resistance. Functionally, we found that TP53 mutations are associated with the epithelial–mesenchymal transition (EMT) process of sarcomatous components of GS. We provide the first comprehensive genome-wide genetic alternation profiling of GS, which suggests novel prognostic subgroups in GS patients based on their TP53 mutation status and provides new insight in the pathogenesis and targeted treatment of GS.

Abstract P4-04-02: Bromodomain and extra-terminal proteins regulate the epithelial-mesenchymal transition in breast cancer

Abstract P4-04-02: Bromodomain and extra-terminal proteins regulate the epithelial-mesenchymal transition in breast cancer

Molecular and clinical characterization of a claudin (CLDN)-low subtype of gastric cancer (GC).

67 Background: A CLDN-low subtype has been identified in breast and bladder cancers and is characterized by low expression of tight junction proteins CLDN, enrichment for epithelial-to-mesenchymal transition (EMT) and tumor initiating cell (TIC) features. Given the genomically stable (GS) subtype of GC defined by TCGA has features suggestive of CLDN-low tumors, we evaluated whether the CLDN-low subtype also exists in GC. Methods: 415 tumors from TCGA GC mRNA dataset were clustered on the CLDN, EMT and TIC gene sets with significance testing using SigClust2 to identify CLDN-low GC. A minimal set of genes that could accurately classify CLDN-low GC was defined by prediction analysis of microarrays (PAM). Tumors identified by SigClust2 or the PAM were called CLDN-low GC regardless of the original subtype call. The 300 GCs in the Asian Cancer Research Group (ACRG) dataset [GSE62254] were used to validate the predictor. We characterized clinical and molecular (gene expression, mutation and copy number alteration) features of CLDN-low GC. Results: We identified 46 tumors that had consensus enrichment for CLDN-low features in TCGA. CLDN-low tumors were most commonly diffuse (35/42=83%, 4 tumors=mixed) and GS (36/46=78%). CLDN-low GC showed high expression of immune gene signatures including T and NK cell signatures, but not an immunosuppression signature. Compared to GS subtype, CLDN-low GC had increased frequency of CD44, GATA4, and GATA6 amplification. In ACRG, 28/300 GCs were CLDN-low using the PAM predictor. The CLDN-low GC in ACRG was phenotypically similar to the CLDN-low GC in TCGA based on the CLDN, EMT and TIC gene signatures. Clinically, CLDN-low GC was associated with the shortest overall survival of the 5 subtypes (CLDN-low plus TCGA defined 4 subtypes). Notably, a hazard ratio comparing CLDN-low GC vs GS was 2.10 (95%CI; 1.07-4.11) in TCGA and 2.32 (95%CI; 1.18-4.55) in ACRG cohort, adjusting for age and pathological stage. Conclusions: We identified a CLDN-low GC which has a poor prognosis likely related to the resistance to conventional chemotherapy due to its EMT and TIC-like properties. Further development of targeted therapies against these molecular features is warranted to improve the outcome of CLDN-low GC.

Fluid shear stress induces cancer stem cell-like phenotype in MCF7 breast cancer cell line without inducing epithelial to mesenchymal transition.

Metastasis is the leading cause of cancer deaths due to the spread of cancer cells through the blood vessels and the subsequent formation of secondary tumors. Metastasizing cancer cells in the human vasculature are called circulating tumor cells (CTCs) and are characterized to express the epithelial cell adhesion molecule (EpCAM). They are further known to survive physiological fluid shear stress (FSS) conditions. However, the effect of FSS on CTC molecular phenotype, such as the epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC) expression, has not been extensively studied. Here, CTCs in FSS are evaluated in an invitro model system. MCF7 and MDA-MB-231 breast cancer cell lines were grown in adherent and suspension culture media. The cell lines were tested for EMT and CSC genetic and protein markers using qRT-PCR and flow cytometry, respectively. Suspension cells showed a significantly increased EMT signature compared to adherent cells (p<0.05), suggesting that they model cells detaching from primary tumors invivo. Upon application of FSS, MCF7 and MDA-MB-231 cells did not show a significant change in EMT expression (p>0.05), but there was a statistically significant increase of the CSC population in MCF7 suspension cultures (p<0.05). These results with MCF7 suggest that CTCs can be modeled invitro as non-adherent cancer cells in FSS and that they show an increased CSC-like signature during circulation, providing new insights to the importance of CSC-targeting strategies when treating metastatic patients.

LGR5 Is a Gastric Cancer Stem Cell Marker Associated with Stemness and the EMT Signature Genes NANOG, NANOGP8, PRRX1, TWIST1, and BMI1.

BackgroundAccumulating evidence supports the hypothesis that cancer stem cells (CSCs) are essential for cancer initiation, metastasis and drug resistance. However, the functional association of gastric CSC markers with stemness and epithelial-mesenchymal transition (EMT) signature genes is unclear.MethodsqPCR was performed to measure the expression profiles of stemness and EMT signature genes and their association with putative CSC markers in gastric cancer tissues, cancer cell lines and sphere cells. Western blot analysis was used to confirm the results of the transcript analysis. Cell proliferation, cell migration, drug resistance and sphere cell growth assays were conducted to measure the expansion and invasion abilities of the cells. Tumor xenograft experiments were performed in NOD/SCID mice to test cell stemness in vivo. Flow cytometry and immunofluorescence staining were used to analyze cell subpopulations.ResultsThe expression of LGR5 was strikingly up-regulated in sphere cells but not in cancer tissues or parental adherent cells. The up-regulation of LGR5 was also positively associated with stemness regulators (NANOG, OCT4, SOX2, and AICDA) and EMT inducers (PRRX1, TWIST1, and BMI1). In addition, sphere cells exhibited up-regulated vimentin and down-regulated E-cadherin expression. Using gene-specific primers, we found that the NANOG expression primarily originates from the retrogene NANOGP8. Western blot analysis showed that the expression of both LGR5 and NANOG is significantly higher in sphere cells. LGR5 over-expression significantly enhanced sphere cell growth, cell proliferation, cell migration and drug resistance in MGC803 cells. Tumor xenografts in nude mice showed that sphere cells are at least 10 times more efficient at tumor initiation than adherent cells. Flow cytometry analysis showed that ~20% of sphere cells are LGR5+/CD54+, but only ~3% of adherent cells are Lgr5+/CD54+. Immunofluorescence staining supports the above results.ConclusionThe LGR5-expressing fraction of CD54+ cells represents gastric cancer CSCs, in which LGR5 is closely associated with stemness and EMT core genes, and NANOG expression is mainly contributed by the retrogene NANOGP8. Sphere cells are the best starting materials for the characterization of CSCs.

1551P - Epithelial to mesenchymal transition induces autophagy in renal cell carcinoma: Implications in cancer therapy

Epithelial to mesenchymal transition (EMT) contributes to the metastatic and invasive potential of tumors including renal-cell carcinoma (RCC). Various preclinical and clinical results have indicated that dysregulated elements leading to EMT can be a potential target in RCC. We assessed the expression profile of EMT associated genes in surgically resected tumor tissue and targeted the survival mechanism of EMT-induced cells. We studied the expression of epithelial marker (E-cadherin), mesenchymal markers (Snail, Slug, Vimentin, Twist) and cancer stem cell marker (ALDH1) in 71 patients of histologically proven RCC. These were analysed in both tumor section and the adjoining normal looking parenchyma by real time PCR and Western immunoblot. In vitro studies were carried out in primary cultures and RCC cell line (A498) where EMT was induced pharmacologically using tumor growth factor-beta (TGF-&bgr;, 10ng/ml). Autophagy was assessed in EMT induced cells by acridine orange staining. Tetrazolium dye, (MTT) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide (PI)-Annexin staining were done to evaluate post-treatment cell survival in EMT cells with temsirolimus and autophagy inhibitor, choloroquine. Epithelial marker, E-cadherin was significantly down-regulated in tumor tissue while expression of mesenchymal and cancer stem cell markers increased in tumor tissue as compared to adjoining normal tissue. EMT signature proteins showed an increase in in vitro culture of primary cells from tumor tissue as well as in EMT- induced A498 cells. We also observed increased invasiveness and autophagy in EMT induced A498 cells and primary tumor cells as compared to the normal cells. It was observed that addition of autophagy inhibitor (chloroquine) with temsirolimus to EMT-induced cells decrease their viability. Annexin-PI assay showed a significant cell death in combination of chloroquine and temsirolomus on EMT induced cells. Our study shows that the process of EMT is involved in the metastatic spread of RCC and autophagy helps in survival of the EMT-induced cells. Thus, inhibition of autophagy might represent a future therapeutic option.

Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition.

Mutations of the tricarboxylic acid cycle (TCA cycle) enzyme fumarate hydratase (FH) cause Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC)1. FH-deficient renal cancers are highly aggressive and metastasise even when small, leading to an abysmal clinical outcome2. Fumarate, a small molecule metabolite that accumulates in FH-deficient cells, plays a key role in cell transformation, making it a bona fide oncometabolite3. Fumarate was shown to inhibit α-ketoglutarate (aKG)-dependent dioxygenases involved in DNA and histone demethylation4,5. However, the link between fumarate accumulation, epigenetic changes, and tumorigenesis is unclear. Here we show that loss of FH and the subsequent accumulation of fumarate elicits an epithelial-to-mesenchymal-transition (EMT), a phenotypic switch associated with cancer initiation, invasion, and metastasis6. We demonstrate that fumarate inhibits Tet-mediated demethylation of a regulatory region of the antimetastatic miRNA cluster6 miR-200ba429, leading to the expression of EMT-related transcription factors and enhanced migratory properties. These epigenetic and phenotypic changes are recapitulated by the incubation of FH-proficient cells with cell-permeable fumarate. Loss of FH is associated with suppression of miR-200 and EMT signature in renal cancer patients, and is associated with poor clinical outcome. These results imply that loss of FH and fumarate accumulation contribute to the aggressive features of FH-deficient tumours.

Abstract LB-170: Unique microRNA signatures in small cell lung cancer correlate with distinct protein expression profiles and drug response

Abstract In the decades since the introduction of platinum-etoposide, treatment of small cell lung cancer (SCLC) remains virtually unchanged. Data from our lab and others have identified novel targets now being investigated in clinical trials. However, further characterization of the molecular heterogeneity within SCLCs will be critical to identify subsets with specific therapeutic vulnerabilities and change the treatment paradigm, similar to what has occurred in non-small cell lung cancer (NSCLC). Here we report expression analyses of 2578 microRNAs (miRs) in 62 SCLC cell lines, followed by unsupervised clustering to identify three distinct miR subtypes. Using ANOVA, we then identified differences in mRNA and protein expression (by reverse phase protein array), as well as drug response, which corresponded to miR expression profiles. The first subtype is characterized by low expression of miR-200, a family with known function in epithelial to mesenchymal transition (EMT) and previously implicated in cisplatin resistance. miR-200 low lines have significantly higher EMT scores (using our published EMT signature), as well as increased sensitivity to the polo-like kinase 1 (PLK-1) inhibitor GW843682X (p = 0.007), the sensitivity to which is associated with EMT in NSCLC. In addition, this subtype possesses increased expression of c-MYC (p = 1.03 e-05), a known repressor of miR-200. A second miR subtype is defined by reduced expression of miR-378, a family whose expression predicts cisplatin sensitivity in NSCLC. This subtype is also typified by increased expression of Wee1 (p = 5.91 e-04), a putative target in SCLC, as well as increased sensitivity to inhibitors of protein phosphatase 1D (PPM1D), tropomyosin receptor kinase A (TrkA) and B-Raf (p = 0.009, 0.011 and 0.032, respectively). The third subtype is distinguished by reduction in several miRs including miRs-216 and -217, a family with a putative role as tumor suppressors in pancreatic adenocarcinoma. Intriguingly, this subtype shows an increase in multiple proteins associated with immune checkpoint pathways, such as programmed death ligand 1 (PDL-1), cluster of differentiation 40 (CD40) and MHC class I polypeptide-related sequence A (MICA) (p = 6.38 e-04, 0.016 and 0.039, respectively) suggesting a group that may have enhanced response to immune-checkpoint inhibition. These subtypes are divided almost equally among the tested lines, constituting 34%, 32% and 34%, respectively. This is the first report of SCLC heterogeneity at the miR level and provides preliminary evidence regarding differences in mRNA and protein expression, as well as drug sensitivity between SCLC with distinct miR profiles. Integrated analysis across miRs, mRNA, protein and drug response represents a powerful tool for identifying candidate biomarkers for drug response, novel molecular targets and insights into the biological heterogeneity across SCLC. Citation Format: Carl M. Gay, Samrat Kundu, Lixia Diao, Bonnie S. Glisson, Jing Wang, John V. Heymach, Lauren A. Byers, Don L. Gibbons. Unique microRNA signatures in small cell lung cancer correlate with distinct protein expression profiles and drug response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-170.

Abstract 2320: CD70 immune checkpoint ligand is associated with the epithelial-to-mesenchymal transition in non-small cell lung cancer

Abstract Non-small-cell lung cancers (NSCLC) account for 85% of lung cancers and are mostly diagnosed at advanced stage. Drugs interrupting immune checkpoints, such as anti-CTLA-4, anti-PD-1, anti-PD-L1, have proven to unleash anti-tumor immunity and mediate durable cancer regressions in a portion of patients with NSCLC. Epithelial-to-mesenchymal transition (EMT) is a developmental process that enables reprogramming of polarized epithelial cells towards a mesenchymal phenotype with migratory and invasive properties. EMT has been involved in escape from oncogenic-induced senescence, resistance to chemotherapy and development of metastatic disease. Moreover, EMT promotes cancer cell plasticity and favors their adaptability to selective pressures. Here we made use of in silico approaches and further in vivo validation to evaluate the potential role of EMT as a major escape mechanism to tumor immunosurveillance in NSCLC. We performed an initial in silico analysis to assess the expression of immune checkpoint ligands (ICPLs) in 129 NSCLC cell lines (CCLE), in relation to their EMT status defined by a 72-gene EMT signature that was previously reported in NSCLC. Unsupervised hierarchical clustering analysis revealed that the expression of selected ICPLs was associated with the mesenchymal or epithelial phenotype. In particular, CD70, a member of the tumor necrosis factor superfamily, was significantly associated with the mesenchymal status (p &amp;lt; 0.001). We identified an E-box sequence (CANNTG) in CD70 gene promoter thus suggesting the possible regulation of CD70 by ZEB1 and/or SNAI. We extended the in silico analysis to a set of 488 adenocarcinomas (ADC) and 501 squamous cell carcinomas (SCC) from the TCGA and confirmed that CD70 expression was associated with a mesenchymal status (q-value &amp;lt;0.001). In a set of E-like (H441 and H1650) and M-like (H23 and HCC44) NSCLC cell lines we validated the association between an increased level of CD70 by flow cytometry and the mesenchymal status. Expression of CD70 by immunohistochemistry was observed in 5/51 lung ADC (10%), 6/45 (13%) lung SCC, 16/26 (63%) pulmonary sarcomatoid carcinomas and 2/10 (20%) small cell lung carcinomas. Interestingly, in pulmonary sarcomatoid carcinomas, CD70 expression was closely associated with the mesenchymal component. Our results suggest an association between the expression of CD70 and the mesenchymal phenotype, thus shedding light on the potential role of CD70 in immune escape of a subset of NSCLC. Citation Format: Sandra Ortiz-Cuaran, Aurélie Swalduz, Maria A. Albaret, Christine Menetrier-Caux, Véronique Haddad, Arnaud Paré, Geneviève De Souza, Anne P. Morel, Maurice Pérol, Christophe Caux, Sylvie Lantuejoul, Alain Puisieux, Pierre Saintigny. CD70 immune checkpoint ligand is associated with the epithelial-to-mesenchymal transition in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2320.

Abstract 3175: Identification of a unique subpopulation of mesenchymal-like colorectal cancers associated with better survival

Abstract The epithelial-mesenchymal transition (EMT) has been well-recognized as an important mechanism promoting cancer cell invasion and “stemness”, metastasis and therapeutic resistance. Various types of cancers with high-EMT (mesenchymal-like) features have been correlated with poorer survival. For example, recently, an international consortium (Guinney et al, Nature Medicine, 2015) has coalesced six independent classification systems of colorectal cancers into four consensus molecular subtypes (CMSs), among which only CMS4 (mesenchymal, 23%) was associated with worse overall survival and worse relapse free survival. Here we report an analysis of 458 colorectal tumors that identified a subpopulation (n = 31, 6.8%) of mesenchymal-like colorectal cancers with unique molecular features. Surprisingly, these tumors were significantly associated with better survival. All samples underwent targeted exome sequencing of 1,321 cancer-related genes, global gene expression profiling and microsatellite instability (MSI) analysis, and were evaluated for the Kaplan-Meier survival and various additional correlation analyses. We explored gene expression signatures to measure EMT, RAS/MAPK, growth factor signaling, and mucinous characteristics. While we recently found that colorectal tumors with wild-type APC (n = 151, 33.0%) were associated with worse outcome, we identified a mucinous subpopulation of these tumors (n = 31, 20.7%) which were associated with better overall survival (p = 0.004). This subpopulation of wt APC tumors were mesenchymal-like, as indicated by its significantly higher mRNA expression of the mesenchymal marker VIM (p = 0.0001) along with a robustly higher EMT signature scores (p&amp;lt;0.0001). This is in contrast to another mucinous colorectal subpopulation, MSI tumors that are epithelial-like, also with a good survival. Moreover, unlike hyper-mutated MSI tumors, the subpopulation identified appears to be correlated with the least-mutated tumors that lack common drivers (e.g. TP53, KRAS and BRAF). Compared to other wt APC tumors, this subpopulation had lower mRNA expression of a number of “canonical” Wnt/beta-catenin targeted genes including MYC (p = 0.009), MET (p&amp;lt;0.0001), CDKN2A (p = 0.03), LGR5 (p = 0.0015), HNF1A (p = 0.003), and SOX9 (p = 0.01), while expression of other targeted genes TCF4 (p&amp;lt;0.0001), TNC (p = 0.04) and NOTCH2 (p = 0.0006) were upregulated. Furthermore, these tumors were also characterized by attenuated activation of RAS/MAPK and growth factor-mediated signaling. Taken together, we have identified a specific subpopulation of colorectal cancers with distinct biological and prognostic features, indicating that a “fine-tune” classification of colorectal cancers is necessary to improve resolution of appropriate therapy. Citation Format: Mingli Yang, Michael J. Schell, Michael V. Nebozhyn, Andrey Loboda, Timothy J. Yeatman. Identification of a unique subpopulation of mesenchymal-like colorectal cancers associated with better survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3175.

A ZEB1-miR-375-YAP1 pathway regulates epithelial plasticity in prostate cancer.

MicroRNA-375 (miR-375) is frequently elevated in prostate tumors and cell-free fractions of patient blood, but its role in genesis and progression of prostate cancer is poorly understood. In this study, we demonstrated that miR-375 is inversely correlated with epithelial-mesenchymal transition signatures (EMT) in clinical samples and can drive mesenchymal-epithelial transition (MET) in model systems. Indeed, miR-375 potently inhibited invasion and migration of multiple prostate cancer lines. The transcription factor YAP1 was found to be a direct target of miR-375 in prostate cancer. Knockdown of YAP1 phenocopied miR-375 overexpression, and overexpression of YAP1 rescued anti-invasive effects mediated by miR-375. Furthermore, transcription of the miR-375 gene was shown to be directly repressed by the EMT transcription factor, ZEB1. Analysis of multiple patient cohorts provided evidence for this ZEB1-miR-375-YAP1 regulatory circuit in clinical samples. Despite its anti-invasive and anti-EMT capacities, plasma miR-375 was found to be correlated with circulating tumor cells in men with metastatic disease. Collectively, this study provides new insight into the function of miR-375 in prostate cancer, and more broadly identifies a novel pathway controlling epithelial plasticity and tumor cell invasion in this disease.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24− cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

PD-L1 expression is associated with epithelial-mesenchymal transition in head and neck squamous cell carcinoma.

Virus-associated malignancies and sarcomatoid cancers correlate with high PD-L1 expression, however, underlying mechanisms remain controversial. We evaluated the correlation between PD-L1 expression and epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinomas (HNSCC). Tumor tissues from 50 patients with HNSCC were evaluated for PD-L1 by immunohistochemistry, which showed 32 (64.0%) were PD-L1 positive (PD-L1+). Interestingly, PD-L1 expression was significantly associated with EMT (P = 0.010), as assessed by low E-cadherin and high vimentin expression. The overall survival of PD-L1+ patients with EMT features was significantly worse than those without EMT features (P = 0.007). In an independent validation cohort (N = 91), as well as in HNSCC cases of The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia, high PD-L1 expression was also associated with the high probability of an EMT signature, referred from the GEO dataset, GSE4824. Survival analysis confirmed PD-L1+/EMT+ patients had a poorer prognosis than PD-L1+/EMT- patients in the TCGA cohort. PD-L1 positivity can thus be divided into two categories according to the absence or presence of EMT. PD-L1 expression is also independently associated with EMT features in HNSCC.