Epithelial-to-mesenchymal Transition Scores Research Articles

Abstract 205: Adaptive membrane trafficking activated by epithelial-to-mesenchymal transition drives lung adenocarcinoma progression

Abstract Cancer cells can become metastatic and develop resistance to targeted therapies through epithelial-to-mesenchymal transition (EMT), which is regulated by epigenetic factors. Our ultimate objective is to understand the molecular mechanisms behind EMT-driven cancer progression and identify potential targets for preventing cancer metastasis. Recently, we made groundbreaking discoveries indicating that EMT can modify membrane trafficking machinery to coordinate cancer cell invasion and immunosuppression in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD). To dissect the “adaptive” membrane trafficking program, we initiated a CRISPRi in vivo screen to assess more than 2000 trafficking-related genes in syngeneic mouse LUAD models for their requirements for tumorigenesis both with and without immune selective pressure. We identified a panel of membrane trafficking regulators that specifically “dropped out” in immunocompetent mice, suggesting their essential roles in the immunosuppressive TME. Of particular interest is REEP2. REEPs are an evolutionarily conserved protein family that is critical to the endoplasmic reticulum (ER) function and potentially regulates secretory trafficking. We discovered that REEP2 is specifically linked to poor prognosis in LUAD patients and is highly correlated with a z-normalized, 16-gene EMT score (P = 1.32e-27) that was previously reported. Our previous studies demonstrated that the EMT activator, ZEB1, accelerates membrane trafficking dynamics by silencing a microRNA network that targets multiple membrane trafficking components. Analysis of REEP2 3’-untranslated regions (3’-UTRs) identified predicted binding sites for several microRNAs, which are silenced by ZEB1. Thus, we hypothesize that ZEB1 coordinates a microRNA network to activate REEP2-driven ER trafficking. We show that REEP2 depletion significantly reduces the invasion of cancer cells but does not affect their migration and proliferation. These findings suggest that REEP2 plays a significant role in extracellular matrix (ECM) modification to enhance the invasion of metastatic cells, conferring a vulnerability in EMT-driven LUAD metastasis. Citation Format: Oluwafunminiyi E. Obaleye, Kevin L. Fulp, Guan-Yu Xiao. Adaptive membrane trafficking activated by epithelial-to-mesenchymal transition drives lung adenocarcinoma progression [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 205.

CSRP1 expression is associated with a mesenchymal, stroma-rich tumor profile and poor prognosis in colon cancer.

Cysteine and glycine-rich protein 1 (CSRP1) is involved in the cysteine-rich protein family and is a marker of smooth muscle lineages. In colon cancer, the expression of this gene is associated with poor prognosis. In this study, the aim was to reevaluate its prognostic relationship in independent cohorts and explore potential underlying biological processes that are linked to aggressive behavior in tumors with high CSRP1 expression, such as epithelial-to-mesenchymal transition (EMT), stromal fractions in the tumor microenvironment, and consensus molecular subtypes (CMSs). RNA sequencing (RNAseq)-, microarray-, and single-cell RNAseq (scRNAseq)-based transcriptomic data were obtained from public databases. The EMT score was calculated based on the expression of E-cadherin and vimentin genes using a previously published method. The stromal score generated by the ESTIMATE method was utilized for the analysis of correlation with the CSRP1 expression. The scRNAseq data were analyzed via the Seurat R package. The immunohistochemistry-based protein level expression of CSRP1 was evaluated using the Human Protein Atlas database. Lower CSRP1 expression was noted in colon tumors compared to normal colon tissue. Patients with a high CSRP1 expression had shorter recurrence-free, overall, and disease-specific survivals in the GSE39582 and GSE17536 datasets (p < 0.05). The methylation level of the CSRP1 gene was negatively correlated (r = -0.57, p < 0.0001) with CSRP1 expression in The Cancer Genome Atlas colon adenocarcinoma dataset. CSRP1 expression was positively correlated with the expression of mesenchymal markers, EMT score, and stromal score obtained via the ESTIMATE method. CMS4 colon tumors had a significantly higher CSRP1 expression compared to other CMSs. Analysis of the scRNAseq data revealed that CSRP1 was expressed by epithelial cells and cancer-associated fibroblasts in the colorectal tumor microenvironment, which was also confirmed by the protein expression data from the Human Protein Atlas database. CSRP1 expression is associated with CMS4, a more mesenchymal stroma-rich molecular profile, and poor prognosis in colon cancer.

Abstract 6206: Combined inhibition of AXL and ATR enhances replication stress, cell death and immune response in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor. Despite high initial responses to frontline chemo-immunotherapy, therapeutic resistance develops rapidly. There are limited treatment options in the relapsed setting, where the prognosis remains dismal. SCLC tumors experience continuous and high levels of replication stress (RS) due to ubiquitous loss of key cell cycle checkpoints, RB1 and TP53. Frequent amplification and high expression of the transcription factor cMYC further contribute to increased RS. Thus, high levels of RS expose a potential SCLC vulnerability and provide a therapeutic opportunity. Our group and others have shown that AXL, a TAM family receptor tyrosine kinase that is highly expressed in mesenchymal tumors, mediates resistance to chemotherapy, radiation and targeted therapies in SCLC, non-small cell lung cancer and other cancers, through its role in driving epithelial to mesenchymal transition (EMT). More recently, a novel role for AXL in DNA damage repair and tolerance has emerged. Therefore, we hypothesize that AXL targeting may be a potential therapeutic approach in SCLC. We first investigated the transcriptomic expression profile of AXL in SCLC clinical cohorts. AXL-high tumors were seen in a subset of treatment-naïve SCLC tumors, frequently among, but not limited to, the inflamed SCLC subtype. AXL expression was also seen in many relapsed SCLC tumors. As expected, tumors with high AXL expression also expressed several mesenchymal genes and higher EMT scores. Interestingly, among the treatment-naïve SCLC tumors, AXL expression was inversely correlated with a RS signature (rho=-0.54, p&amp;lt;0.001). Next, we tested the effects of AXL inhibition in SCLC in vitro and in vivo models. In a panel of 30 SCLC cell lines, bemcentinib, a selective AXL inhibitor in clinical trials for various advanced solid tumors, exhibited a range of antiproliferative activity, with IC50 values ranging from 41 nM to 10 µM (median IC50 3.1 µM). Bemcentinib also significantly delayed tumor growth in in vivo SCLC models. Biomarkers associated with sensitivity to bemcentinib in SCLC cell lines included markers of RS (cMYC, replication stress score) and DNA damage response (phospho CHK1S345, phospho CHK2T68). Bemcentinib also induced RS, indicated by the activation of ATR/CHK1-mediated RS response pathway, and DNA damage, and the combination with an ATR inhibitor (ceralasertib) showed a greater than additive effect. In a syngeneic model of SCLC, the combination of bemcentinib, ceralasertib and an anti-PDL1 antibody induced significant tumor regression. Together, these promising findings demonstrate that AXL inhibition may be an effective strategy to target the RS vulnerability common in SCLC. Citation Format: Kavya Ramkumar, C. Allison Stewart, Azusa Tanimoto, Qi Wang, Yuanxin Xi, Benjamin B. Morris, Runsheng Wang, Li Shen, Robert J. Cardnell, Jing Wang, Carl M. Gay, Lauren A. Byers. Combined inhibition of AXL and ATR enhances replication stress, cell death and immune response in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6206.

MiR-455-5p suppresses PDZK1IP1 to promote the motility of oral squamous cell carcinoma and accelerate clinical cancer invasion by regulating partial epithelial-to-mesenchymal transition

BackgroundLymph node and distant metastasis contribute to poor outcomes in patients with oral squamous cell carcinoma (OSCC). The mechanisms regulating cancer migration and invasion play a key role in OSCC.MethodsWe determined migration and invasion ability of OSCC by wound-healing assay, two-chamber transwell invasion assay and cell mobility tracking and evaluated tumor metastasis in vivo. Western blot (WB), qRT-PCR, RNA-seq, dual-luciferase reporter assays and nuclear/cytoplasmic fractionation were performed to investigate the potential mechanism. Immunohistochimical (IHC) staining determined vimentin and PDZK1IP1 expression in OSCC tissues.Results and conclusionIn this study, we determined that miR-455-5p was associated with lymph node metastasis and clinical invasion, leading to poor outcomes in patients with OSCC. MiR-455-5p promoted oral cancer cell migration and invasion and induced epithelial-to-mesenchymal transition (EMT). We also identified a new biomarker, PDZK1IP1 (MAP17), that was targeted by miR-455-5p. PDZK1IP1 knockdown led to migration, metastasis, EMT, and increased transforming growth factor-β signaling in OSCC. In addition, miR-455-5p overexpression and PDZK1IP1 inhibition promoted collective OSCC cell migration. According to data from the Cancer Genome Atlas database and the NCKU-OrCA-40TN data set, miR-455-5p and PDZK1IP1 are positively and negatively correlated, respectively, with partial EMT score. High miR-455-5p expression was associated with high vimentin levels and low MAP17 H-scores. The patients with low MAP17 expression had higher rates of disease recurrence than did patients with high MAP17 expression, especially for patients with clinical invasion risk factors and low MAP17 expression. These results suggest that miR-455-5p suppresses PDZK1IP1 expression and mediates OSCC progression. MiR-455-5p and PDZK1IP1 may therefore serve as key biomarkers and be involved in regulating partial EMT in OSCC cells. PDZK1IP1 expression may also serve as an independent factor that impacts outcomes in patients with clinical risk factors for recurrence.

Integrated analysis reveals the protective mechanism and therapeutic potential of hyperbaric oxygen against pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a dreadful, chronic, and irreversibly progressive disease leading to death with a few effective treatments. Our previous study suggested that repetitive hyperbaric oxygen (HBO) treatment alleviates bleomycin-induced pulmonary fibrosis in mice. Here, we investigated the protective mechanism of HBO treatment against pulmonary fibrosis using an integrated approach. Analyzing publicly available expression data from the mouse model of bleomycin-induced pulmonary fibrosis as well as IPF patients, several potential mechanisms of relevance to IPF pathology were identified, including increased epithelial-to-mesenchymal transition (EMT) and glycolysis. High EMT or glycolysis scores in bronchoalveolar lavage were strong independent predictors of mortality in multivariate analysis. These processes were potentially driven by hypoxia and blocked by HBO treatment. Together, these data support HBO treatment as a viable strategy against pulmonary fibrosis.

An epithelial-mesenchymal plasticity signature identifies two novel LncRNAs with the opposite regulation.

The epithelial to mesenchymal transition (EMT) is crucial for cancer progression and chemoresistance. EMT is a dynamic process with multiple phases that change cell migration and invasion activity. We used pan-cancer expression data to find 14-LncRNAs that had a high correlation with the EMT markers VIM, CDH1, FN1, SNAI1, and SNAI2. The expression of 14 EMT-associated LncRNA, which also showed high cancer specificity, was used to calculate the pan-cancer EMT score. The EMT score was then applied to the 32 cancer types to classify them as epithelial, epithelial-mesenchymal, mesenchymal-epithelial, or mesenchymal tumors. We discovered that the EMT score is a poor prognostic predictor and that as tumor mesenchymal nature increased, patient survival decreased. We also showed that the cell of origin did not influence the EMT nature of tumors. Pathway analysis employing protein expression data revealed that the PI3K pathway is the most crucial in determining the EMTness of tumors. Further, we divided CCLE-cell lines into EMT classes and discovered that mesenchymal cells, which exhibited higher PI3K pathway activation, were more sensitive to PI3K inhibitors than epithelial cells. We identified Linc01615 as a mesenchymal LncRNA whose expression significantly correlated with survival in several cancer types. We showed that Linc01615 is regulated by the TGFβ-STAT3 pathway in a feedback loop. Knockdown of Linc01615 inhibited cell proliferation and migration by regulating the PI3K pathway and mesenchymal markers. We also identified RP4-568C11.4 as an epithelial cancer marker. We showed that knocking down RP4-568C11.4 decreased cell growth but not migration. In addition, we discovered that ESR1 regulates RP4-5681C11.4 in breast cancer. Taken together, we have developed a pan-cancer EMT signature. Also, we found two new LncRNAs that have different effects on cancer development and EMT.

Abstract 1561: Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation

Abstract Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides lacking protein-coding potential, which are involved in diverse pathophysiologic conditions including cancer progression and metastasis. Metastasis is initiated by a cellular process, epithelial-to-mesenchymal transition (EMT), and many lncRNAs can be affected by and participate in the EMT process. To find out lncRNAs regulated by the EMT signals, we performed an NGS-based RNA sequencing in murine lung cancer cells, and finally selected lnc-NR2F1 that is upregulated by ZEB1, an EMT-inducing transcription factor. lnc-NR2F1 expression levels showed a positive correlation with ZEB1 mRNA levels and EMT scores in 13 mouse lung cancer cells or in lung adenocarcinoma data from TCGA. The migration and invasion of lung cancer cells were decreased after lnc-NR2F1 knockdown and increased after lnc-NR2F1 overexpression. lnc-NR2F1 was transcriptionally up-regulated by NR2F1, a transcription factor transcribed from the antisense strand. Interestingly, NR2F1 was transcriptionally induced by ZEB1, and promoted the migration and invasion of lung cancer cells. Through the analysis of public ChIRP-seq data, we found that lnc-NR2F1 can modulate the expression of TWIST2, and add-back of TWIST2 restored the migration and invasion of lung cancer cells suppressed by lnc-NR2F1 knockdown. In conclusion, lnc-NR2F1 is induced by a ZEB1-NR2F1 axis and stimulates the migration and invasion of lung cancer cells via transcriptional regulation of TWIST2. Citation Format: Eun Ju Kim, Young-Ho Ahn. Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1561.

Abstract 2616: Stemness factors nanog and oct4 contribute to epithelial-to-mesenchymal transition and are predictive for outcome in esophageal adenocarcinoma

Abstract Background and Aims: The incidence of esophageal adenocarcinoma (EAC) has increased six-fold in Western countries over the last decades, and 5-year survival rates remain low at 5-20%. While multimodality treatment strategies for curative treatment of esophageal cancer, including the CROSS regimen (chemoradiotherapy followed by surgery) have increased median overall survival, the majority of patients develop recurrences after several months. Epithelial to mesenchymal transition (EMT) has been recently shown by our group to be one of the major underlying mechanisms of resistance to therapy. Paradoxically, therapeutic pressure of effective therapies such as the CROSS regimen are found to instruct a mesenchymal, resistant phenotype in models for EAC. In this study, the aim was to delineate the heterogeneity for the propensity to undergo EMT after chemoradiation and which mechanisms underpin this propensity. Methods: A panel of 8 EAC cell lines (5 primary and 3 ATCC cell lines) were treated with chemoradiotherapy and ranked by their propensity to undergo EMT, based on morphology when EMT occurred and protein marker expression. Next, the cell line panel as well as 44 pre-treated esophageal biopsies were RNA-sequenced. Expression data of the cell line panel were linked to their ranked in vitro EMT response. By means of Leave-one-out cross validation with Ridge Regression, EMT score prediction in pre-treated biopsies was validated. Gene expression profiles were related to clinical outcome data to identify markers that associated with propensity for EMT in patients. Results: In the panel of in vitro EAC models, a strong heterogeneity was observed for the propensity to EMT after chemoradiation. For each marker, Ridge regression analysis identified the top 50 highly correlating genes. Combining all positively correlating genes of days to EMT, NCAD and ZEB1, known key transcription factors of pluripotency including NANOG and OCT4 emerged. Expression of NANOG and OCT4 in pre-treatment biopsies was highly predictive for response to neoadjuvant chemoradiation, occurrence of recurrences, and survival in patients. Genetic perturbation by knockout and inhibition of NANOG and OCT4 reduced the onset of EMT and sensitized cells for chemoradiation. Conclusions: In conclusion, we were able to identify patients who are disproportionally prone to develop EMT in response to chemoradiation. Moreover, stemness factors NANOG and OCT4 are crucial regulators in plasticity of EAC and are promising predictive markers in pre-treatment biopsies of patients. By targeting NANOG and OCT4 in vitro, cells were sensitized to chemoradiation, holding promise for stemness inhibition to prevent therapy resistance in EAC. Citation Format: Amber Perenna van der Zalm, Mark P. Dings, Reimer Janssen, Peter Bailey, Jan Koster, Danny Zwijnenburg, Richard Volckmann, Cynthia Waasdorp, Jeroen Blokhuis, César Oyarce, Gerrit Hooijer, Sybren L. Meijer, Jan Paul Medema, Hanneke W. van Laarhoven, Maarten F. Bijlsma. Stemness factors nanog and oct4 contribute to epithelial-to-mesenchymal transition and are predictive for outcome in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2616.

Changes in Triple-Negative Breast Cancer Molecular Subtypes in Patients Without Pathologic Complete Response After Neoadjuvant Systemic Chemotherapy.

PURPOSELehmann et al have identified four molecular subtypes of triple-negative breast cancer (TNBC)—basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor—and an immunomodulatory (IM) gene expression signature modifier. Our group previously showed that the response of TNBC to neoadjuvant systemic chemotherapy (NST) differs by molecular subtype, but whether NST affects the subtype was unknown. Here, we tested the hypothesis that in patients without pathologic complete response, TNBC subtypes can change after NST. Moreover, in cases with the changed subtype, we determined whether epithelial-to-mesenchymal transition (EMT) had occurred.MATERIALS AND METHODSFrom the Pan-Pacific TNBC Consortium data set containing TNBC patient samples from four countries, we examined 64 formalin-fixed, paraffin-embedded pairs of matched pre- and post-NST tumor samples. The TNBC subtype was determined using the TNBCtype-IM assay. We analyzed a partial EMT gene expression scoring metric using mRNA data.RESULTSOf the 64 matched pairs, 36 (56%) showed a change in the TNBC subtype after NST. The most frequent change was from BL1 to M subtypes (38%). No tumors changed from M to BL1. The IM signature was positive in 14 (22%) patients before NST and eight (12.5%) patients after NST. The EMT score increased after NST in 28 (78%) of the 36 patients with the changed subtype (v 39% of the 28 patients without change; P = .002254).CONCLUSIONWe report, to our knowledge, for the first time that the TNBC molecular subtype and IM signature frequently change after NST. Our results also suggest that EMT is promoted by NST. Our findings may lead to innovative adjuvant therapy strategies in TNBC cases with residual tumor after NST.

Abstract P4-07-19: Quantifying epithelial-mesenchymal tumor heterogeneity for prediction of patient prognosis based on EMT state

Abstract Background: Triple Negative Breast Cancer (TNBC) is an aggressive and heterogeneous subtype characterized by ER/PR/HER2 negative status. Much of the disease potential and aggressive nature of this subtype derives from inter- and intra-tumoral heterogeneity, which makes developing targeted therapies challenging. A key contributor to both heterogeneity in TNBC and later stage chemo-resistance and metastasis is the Epithelial-to-Mesenchymal transition (EMT). This developmental program is frequently exploited in the context of cancer to increase migratory abilities, invasiveness, metastatic potential, and resistance to chemotherapy. Indeed, EMT has been demonstrated and linked to poor prognosis and decreased survival in many solid cancer types. Cells have been found to reside in multiple stable intermediate states along the EMT spectrum, which confer increased aggressive, metastatic, and chemoresistance attributes to a heterogeneous tumor through increased stem-like characteristics. Identifying and targeting this disease-potentiating population in patient tumors is a major hurdle in overcoming metastatic disease. Knowledge gap: Despite major advances in our understanding, the contributions of EMT research to improvements in diagnostic pathology or cancer therapy have been minimal. One reason for this gap stems from our inability to accurately detect and quantify epithelial-mesenchymal heterogeneity in primary tumor specimens. Secondly, the significance of recently identified intermediate or partial EMT states to predicting tumor prognosis and therapy response are unclear. Approach &amp; Results: To study the role of various states within the EMT spectrum and their regulatory networks, the heterogeneous breast cancer cell line, SUM149PT, was used to derive six single cell clones encompassing the spectrum of EMT states, from epithelial to mesenchymal. Interrogation of this model system in vivo has revealed increased tumor growth and metastatic potential in the intermediate EMT states when compared to the extreme epithelial and mesenchymal states. To further elucidate EMT states in vivo, we employ a 6-marker multi-round immunofluorescence-based staining approach to identify cells that reside in various states along the EMT spectrum. We subsequently used an entropy-based approach and nearest-neighbor analysis on these tumors with the purpose of scoring heterogeneity and overall EMT state. Notably, this analysis segregated stromal infiltrates and their contributions to aggregate EMT scoring, which has been a major hurdle in using EMT as a scoring metric in patient samples. Overall, SUM149 clone-derived tumors held true to the relative EMT states of the starting cell populations; intermediate-derived tumors displayed high heterogeneity while epithelial and mesenchymal clone-derived tumors had lower levels of heterogeneity, despite retaining different EMT scores. Decoupling of heterogeneity and EMT state in this way provides two metrics to assess potential metastatic ability of a tumor. This staining method and analysis has been successfully applied in a preliminary set of patient tumors, showing promise for these two factors, E-M Heterogeneity and EMT score, as a tumor prognostic indicator to inform therapeutic decision-making. Conclusions: EMT tumor states and EMT-derived intra-tumoral heterogeneity play an important role in tumor metastasis and disease progression. Here, we have validated a multiplexed staining approach to quantify these metrics within a tumor, while segregating out stromal infiltrating cells. In the future, this staining and quantification shows promise as a means of predicting patient prognosis and informing potential treatment options based on targeting EMT states Citation Format: Meredith S Brown, Behnaz Abdollahi, Nevena Ognjenovic, Kristen E Muller, Saeed Hassanpour, Diwakar R Pattabiraman. Quantifying epithelial-mesenchymal tumor heterogeneity for prediction of patient prognosis based on EMT state [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-19.

Loss of circadian gene Timeless induces EMT and tumor progression in colorectal cancer via Zeb1-dependent mechanism.

The circadian gene Timeless (TIM) provides a molecular bridge between circadian and cell cycle/DNA replication regulatory systems and has been recently involved in human cancer development and progression. However, its functional role in colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide, has not been fully clarified yet. Here, the analysis of two independent CRC patient cohorts (total 1159 samples) reveals that loss of TIM expression is an unfavorable prognostic factor significantly correlated with advanced tumor stage, metastatic spreading, and microsatellite stability status. Genome-wide expression profiling, in vitro and in vivo experiments, revealed that TIM knockdown induces the activation of the epithelial-to-mesenchymal transition (EMT) program. Accordingly, the analysis of a large set of human samples showed that TIM expression inversely correlated with a previously established gene signature of canonical EMT markers (EMT score), and its ectopic silencing promotes migration, invasion, and acquisition of stem-like phenotype in CRC cells. Mechanistically, we found that loss of TIM expression unleashes ZEB1 expression that in turn drives the EMT program and enhances the aggressive behavior of CRC cells. Besides, the deranged TIM-ZEB1 axis sets off the accumulation of DNA damage and delays DNA damage recovery. Furthermore, we show that the aggressive and genetically unstable 'CMS4 colorectal cancer molecular subtype' is characterized by a lower expression of TIM and that patients with the combination of low-TIM/high-ZEB1 expression have a poorer outcome. In conclusion, our results as a whole suggest the engagement of an unedited TIM-ZEB1 axis in key pathological processes driving malignant phenotype acquisition in colorectal carcinogenesis. Thus, TIM-ZEB1 expression profiling could provide a robust prognostic biomarker in CRC patients, supporting targeted therapeutic strategies with better treatment selection and patients' outcomes.

Lung Cancer Models Reveal Severe Acute Respiratory Syndrome Coronavirus 2–Induced Epithelial-to-Mesenchymal Transition Contributes to Coronavirus Disease 2019 Pathophysiology

IntroductionCoronavirus disease 2019 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which enters host cells through the cell surface proteins ACE2 and TMPRSS2. MethodsUsing a variety of normal and malignant models and tissues from the aerodigestive and respiratory tracts, we investigated the expression and regulation of ACE2 and TMPRSS2. ResultsWe find that ACE2 expression is restricted to a select population of epithelial cells. Notably, infection with SARS-CoV-2 in cancer cell lines, bronchial organoids, and patient nasal epithelium induces metabolic and transcriptional changes consistent with epithelial-to-mesenchymal transition (EMT), including up-regulation of ZEB1 and AXL, resulting in an increased EMT score. In addition, a transcriptional loss of genes associated with tight junction function occurs with SARS-CoV-2 infection. The SARS-CoV-2 receptor, ACE2, is repressed by EMT through the transforming growth factor-β, ZEB1 overexpression, and onset of EGFR tyrosine kinase inhibitor resistance. This suggests a novel model of SARS-CoV-2 pathogenesis in which infected cells shift toward an increasingly mesenchymal state, associated with a loss of tight junction components with acute respiratory distress syndrome-protective effects. AXL inhibition and ZEB1 reduction, as with bemcentinib, offer a potential strategy to reverse this effect. ConclusionsThese observations highlight the use of aerodigestive and, especially, lung cancer model systems in exploring the pathogenesis of SARS-CoV-2 and other respiratory viruses and offer important insights into the potential mechanisms underlying the morbidity and mortality of coronavirus disease 2019 in healthy patients and patients with cancer alike.

Epithelial-Mesenchymal Transition Inversely Associates With Immune Activity in Breast Cancer Tumour Immune Microenvironment

The epithelial to mesenchymal transition (EMT) is a major mechanism of resistance to cancer therapy, including immunotherapy in breast cancer. Recent studies highlighted the role of the EMT program in regulating immune checkpoints; however, the overall immune activity during EMT of carcinoma remains to be shown. Using in-silico data analysis on a large cohort of breast cancer samples from The Cancer Genome Atlas (TCGA) and measuring immune activities across various EMT scores, we found that an increasing EMT score reduces immune cells infiltration and activity, turning an immune infiltrated ‘hot’ tumor microenvironment (TME) to a ‘cold’ and less immune infiltrated TME. Remarkably, the high EMT score and expression of EMT related genes negatively associated with infiltration and activity of various immune cells particularly CD8+ T cells, while increased intratumoral heterogeneity level, infiltration of Treg cells, and M2 macrophages in TME. These data revealed the crucial role of the EMT program in regulating immune-suppression in the tumor-immune microenvironment (TIME) by reducing immune activity and highlighting the potential application of EMT score as a biomarker for the responsiveness of breast cancer patients to immunotherapy.

Epithelial to Mesenchymal Transition Regulates Surface PD-L1 via CMTM6 and CMTM7 Induction in Breast Cancer.

Simple SummaryEpithelial to mesenchymal transition (EMT) is a mechanism endowing tumor cells with aggressive properties and escape immune surveillance. Here, we reported that activating EMT process in breast cancer cells upregulates CMTM6, an essential protein required for cell surface expression of PD-L1. In addition to CMTM6, our in silico data on triple negative breast cancer patients showed a positive correlation between EMT markers and two other members of CMTM family (CMTM3 and CMTM7). These results were validated in EMT-inducible MCF-7 model that exhibited a downregulation of cell surface expression of PD-L1 by dual targeting CMTM6 and CMTM7. Considering the prominent role of PD-L1 in tumor immune evasion, our study provides an additional clue on how PD-L1 is regulated in aggressive breast cancer cells and pave the way to assess the therapeutic benefit of EMT inhibitors in combination with anti PD-L1 blockade in highly aggressive breast cancer patients.CMTM6 is a critical regulator of cell surface expression of PD-L1 in tumor cells, but little is known about the transcriptional regulation of CMTM6. Here we report that the expression of CMTM6 positively correlates with the epithelial to mesenchymal transition (EMT) score in breast cancer cell lines and with the major EMT marker Vimentin in triple-negative breast cancers (TNBC). We showed that CMTM6 is concomitantly overexpressed with PD-L1 in breast mesenchymal compared with the epithelial cells. Driving a mesenchymal phenotype in SNAI1-inducible MCF-7 cells (MCF-7Mes cells) increased both PD-L1 and CMTM6. CMTM6 silencing in MCF-7Mes cells partially reduced cell surface expression of PD-L1, indicating that a proportion of the PD-L1 on the surface of MCF-7Mes cells depends on CMTM6. We also found a positive correlation between CMTM3 and CMTM7 expression with EMT score in breast cancer cells, and with Vimentin in TNBC patients. Dual knockdown of CMTM6 and CMTM7 significantly decreased PD-L1 surface expression in MCF-7Mes cells, indicating that both CMTM6 and CMTM7 regulate the expression of PD-L1. This study highlights the importance of CMTM6 and CMTM7 in EMT-induced PD-L1 and suggests that EMT, CMTM6 or CMTM7 modulators can be combined with anti-PD-L1 in patients with highly aggressive breast cancer.

HYPERTENSIVE NEPHROPATHY

Objective: We aimed at investigating to which degree hypertensive and diabetic nephropathy show evidence of concurrent inflammation and partial epithelial-to-mesenchymal transition (EMT), including elevated expression of EMT promoting AXL receptor tyrosine kinase. Design and method: Formalin-fixed and paraffin-embedded (FFPE) kidney biopsies from adult patients with hypertensive nephropathy (HN, n = 6), type 2 diabetic nephropathy (DN, n = 6), and normal appearing tissue (n = 6) were recruited from the Norwegian Kidney Biopsy Registry. Total RNA was extracted from whole tissue sections using High Pure FFPE-tissue RNA Isolation kit (Roche). Total RNA inputs of 37.8–1324 ng per sample were used for library preparation and sequenced as 75 bp paired end on Illumina NextSeq500. Differentially expressed genes were analysed with Limma-Voom (Bioconductor) and formed the basis for calculation of a generic EMT score, principal component analysis (PCA) and ingenuity pathway analysis (IPA). Soluble AXL (sAXL) in serum samples (n = 14/group) from patients with HN and healthy controls was quantified by ELISA (Human Axl Duoset, R&D). Results: PCA separated diseased from normal tissue with a considerable overlap in expression profiles of HN and DN. The generic EMT score was higher in both HN and DN as compared to normal tissue. Expression of the EMT promoting receptor tyrosine kinase AXL was increased in both diseases. Immunohistochemistry confirmed increased AXL and vimentin, while E-cadherin was decreased in diseased tissues. Accordingly, sAXL levels were elevated in serum samples from HN patients compared to controls. IPA showed all top five canonical pathways to be involved in inflammation. Accordingly, expression deconvolution analyses provided evidence of eosinophils, natural-killer-T-cells, granulocytes and CD8+ effector-memory T-cells and myeloid-dendritic cells in HN. E.g. the CCL5-CCR5 axis represents a therapeutic target for seven existing drugs. Top scoring pathways common for both DN and HN included the iCOS-iCOSL signalling in T helper cells, Th1 & Th2 activation pathway and the Th1/Th2 pathway. Higher presence of the phagosome formation pathway in DN and the T cell receptor signalling pathway in HN differentiated the two pathologies. Conclusions: HN and DN show an increased mesenchymal phenotype and inflammation. AXL receptor tyrosine kinase represents a novel therapeutic target, especially in HN.

CHST11 Promotes Lung Cancer Metastasis through Changing Intracellular Iron Metabolism

Non‐small‐cell lung cancer (NSCLC) is one of the most lethal cancers, for it often develops metastasis after therapy. Cancer metastasis usually begins from epithelial‐to‐mesenchymal transition (EMT), and metabolic disorder is a key mediator of lung cancer EMT. The linkage between metabolic disorder and NSCLC EMT is still unclear. Here, we compared gene expression signatures from both TGFb induction EMT model of NSCLC cells and the EMT score from clinical NSCLC patients' cohort. We found several chondroitin sulfate (CS) biosynthesis genes were highly correlated to NSCLC EMT status. CHST11, the key enzyme of this pathway, was selected for further functional studies. Enforced CHST11 expression promoted NSCLC cells migration, invasion and increasing M‐type markers, such as Slug, N‐cadherin and Vimentin expression and vise versa. Deplete the final product of CS biosynthesis by chondroitinase ABC or enzyme‐dead CHST11 could abolish the NSCLC EMT ability from TGFb and CHST11, respectively. Moreover, we found the CHST11 expression changing the iron metabolic status in NSCLC cells and promoting the NSCLC EMT.In the end, immunohistochemistry showed that CHST11 was a poor prognostic marker of overall survival and disease‐free survival in clinical NSCLC patients. In conclusion, our findings implicated that 1) The chondroitin sulfate biosynthesis pathways is crucial for NSCLC EMT; 2) A novel CHST11‐CP‐Fe3+ pathway is involved in NSCLC EMT. 3) CHST11 is a poor prognostic marker in NSCLC patients. These results might help us to understand the role of aberrant cancer metabolism in NSCLC EMT.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

EMT Markers in Locally-Advanced Prostate Cancer: Predicting Recurrence?

Background: Prostate cancer (PCa) is the second most frequent cause of cancer-related death in men worldwide. It is a heterogeneous disease at molecular and clinical levels which makes its prognosis and treatment outcome hard to predict. The epithelial-to-mesenchymal transition (EMT) marks a key step in the invasion and malignant progression of PCa. We sought to assess the co-expression of epithelial cytokeratin 8 (CK8) and mesenchymal vimentin (Vim) in locally-advanced PCa as indicators of EMT and consequently predictors of the progression status of the disease.Methods: Co-expression of CK8 and Vim was evaluated by immunofluorescence (IF) on paraffin-embedded tissue sections of 122 patients with PCa who underwent radical prostatectomies between 1998 and 2016 at the American University of Beirut Medical Center (AUBMC). EMT score was calculated accordingly and then correlated with the patients' clinicopathological parameters and PSA failure.Results: The co-expression of CK8/Vim (EMT score), was associated with increasing Gleason group. A highly significant linear association was detected wherein higher Gleason group was associated with higher mean EMT score. In addition, the median estimated biochemical recurrence-free survival for patients with < 25% EMT score was almost double that of patients with more than 25%. The validity of this score for prediction of prognosis was further demonstrated using cox regression model. Our data also confirmed that the EMT score can predict PSA failure irrespective of Gleason group, pathological stage, or surgical margins.Conclusion: This study suggests that assessment of molecular markers of EMT, particularly CK8 and Vim, in radical prostatectomy specimens, in addition to conventional clinicopathological prognostic parameters, can aid in the development of a novel system for predicting the prognosis of locally-advanced PCa.

Abstract P5-07-12: Circular RNA NCAPG promotes breast cancer metastasis through acting as the sponge of miR-200s

Abstract Background Metastatic breast cancer remains incurable and underlying mechanisms are poorly understood. Tumor-associated macrophages (TAMs) represent a major component of the tumor microenvironment that supports breast cancer metastasis. Circular RNAs are new class of endogenous noncoding RNAs. Unlike linear RNAs terminated with 5'caps and 3'tails, they uniquely have a covalently closed loop structure, making them stable and ideal candidate biomarkers. Here, we identified a circular RNA and explored for the first time how it played a dual role in epithelial to mesenchymal transition (EMT) and macrophage recruitment. Methods We first analyzed TCGA database about whether mesenchymal tumors could be more likely to recruit TAMs. ELISA assay was performed for determining expression level of chemokines (CSF-1, CCL2, CCL5, C5a, VEGFA, IL-34) after cell line MCF-7 finishing EMT. Next, we predicted microRNAs which could regulate CSF-1 expression and inhibit EMT using Targetscan database. Circular RNA was identified via comparing expression levels between mesenchymal-like and epithelial-like cells using circRNA microarrays. The specific mechanisms were explored via series of molecular biology assays in vitro and in vivo. Results Correlation analysis of relationship between EMT score and expression of all classical chemokines that could recruit macrophage revealed a positive association (r2&amp;gt;0.3, P&amp;lt;0.05) among 1079 breast cancer cases. Increased expression level of CSF-1 was found the most significant after EMT. miR-200b and miR-200c that inhibiting breast cancer EMT were confirmed targeting 3'UTR of CSF-1 mRNA and decreasing migration of TAMs. Circular RNA NCAPG (circNCAPG) was found highly expressed in mesenchymal-like cells and could promote breast cancer metastasis in vitro and in vivo. Poor clinical outcome was found in circNCAPG overexpression group among 141 breast cancer cases. CircNCAPG was proven to be located in cytoplasm and found could act as the sponge of miR-200b and miR-200c. Moreover, expression level of CSF-1 and ZEB-1 (also targeted by miR-200s and regulating key process of EMT) was found decreasing significantly after knocking down circNCAPG both in vitro and in vivo, leading to breast cancer metastasis inhibition. Conclusion Mesenchymal tumors have the ability to recruit more TAMs, therefore further promoting breast cancer metastasis. A new molecule: circNCAPG participates in this process via acting as the sponge of miR-200s and regulates CSF-1 and ZEB-1 expression, which warrants further study in clinical settings. Citation Format: He Y, Tang J. Circular RNA NCAPG promotes breast cancer metastasis through acting as the sponge of miR-200s [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-12.

Epithelial-to-mesenchymal transition signature assessment in colorectal cancer quantifies tumour stromal content rather than true transition.

The process of epithelial‐to‐mesenchymal transition (EMT) in cancer is a well‐described process whereby epithelial tumour cells undergo molecular/phenotypic changes and transition to a mesenchymal biology. To aid in the transcriptional characterisation of this process, gene expression signatures have been developed that attribute a relative EMT score to samples in a given cohort. We demonstrate how such EMT signatures can identify epithelial cell line models with high levels of transition but also highlight that, unsurprisingly, fibroblast cell lines, which are inherently mesenchymal, have a higher EMT score relative to any epithelial cell line studied. In line with these data, we demonstrate how increased tumour stromal composition, and reduced epithelial cellularity, significantly correlates with increasing EMT signature score, which is evident using either in silico subtyping analysis (p < 0.00001) or in situ histopathological characterisation (p < 0.001). Considered together, these results reinforce the importance not only of interdisciplinary research to correctly define the nature of EMT biology but also the requirement for a cadre of multidisciplinary researchers who can analyse and interpret the underlying pathological, bioinformatic and molecular data that are essential for advancing our understanding of the malignant process. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Molecular classification and subtype-specific drug sensitivity research of uterine carcinosarcoma under multi-omics framework

ABSTRACTObjective: Uterine carcinosarcomas (UCSs) are aggressive rare tumors recognized as malignancies composed of metaplastic transformation of epithelial elements. Nay no comprehensive molecular classification has been applied to UCS to guide targeted therapies so far, which motivated us to subtyping UCS by aggregating multiple genomic platform data.Methods: We classified UCS into three distinct subtypes with different clinicopathologic and molecular characterization by using similarity network fusion under consensus clustering framework (SNFCC+) to aggregate four genomic data platforms of 55 UCS patients. Differences across subtypes were extracted by functional enrichment, gene mutations and clinical features. Subtypes were further distinguished by putative biomarkers. We also determined associations between individual oncogenes and chemotherapeutics to discuss subtype-specific drug sensitivity.Results: Functional enrichment analysis of the subtype-specific differential expression genes endowed three subtypes new designation: Myo, Cell and Hormone. Mutations in PTEN, PIK3CA, ARID1A and PPP2R1A altered across subtypes. The epithelial-to-mesenchymal transition (EMT) score distinguished Myo from another two subtypes whereby a high EMT scores prevalently existed and each case was judged as M (mesenchymal) phenotype in Myo subtype. Through the drug sensitivity analysis, we found that the response to – tinib drugs is quite different across subtypes according to expression level. Additionally, different subtypes’ response to broad-spectrum anti-cancer drug paclitaxel may be also different.Conclusions: In this study, we identified three distinct molecular subtypes of UCS with different features. Subtypes were also revealed to have different sensitivity to existing chemotherapy drugs, which may support in-depth study of subtype-specific dosing regimens.