Epithelial-mesenchymal Transition Gene Signature Research Articles

Comprehensive analyses and experimental verification of NETs and an EMT gene signature for prognostic prediction, immunotherapy, and chemotherapy in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is an aggressive malignancy with high mortality and poor prognosis. Neutrophil extracellular traps (NETs) and the epithelial-mesenchymal transition (EMT) significantly influence on the progression of various cancers. However, the underlying relevance of NETs- and EMT-associated genes on the outcomes of patients with PAAD remains to be elucidated. Transcriptome RNA sequencing data, together with clinical information and single-cell sequencing data of PAAD were collected from public databases. In the TCGA-PAAD cohort, ssGSEA was used to calculate NET and EMT scores. WGCNA was used to determine the key gene modules. A risk model with eight NET- and EMT-related genes (NERGs) was established using LASSO and multivariate Cox regression analysis. Patients in the reduced risk (RR) group showed better prognostic values compared with those in the elevated risk (ER) group. The prognostic model exhibited reliable and robust prediction when validated using an external database. The distributions of risk genes were explored in a single-cell sequencing data set. Immune infiltration, immune cycle, and immune checkpoints were compared between the RR and ER groups. Moreover, potential chemotherapeutic drugs were examined. DCBLD2 was identified as a key gene in PAAD cell lines by qRT-PCR, and was highly expressed in PAAD tissues. GSEA demonstrated that DCBLD2 induced the EMT. Transwell assays and western blotting showed that cell invasion and EMT induction were significantly reduced after DCBLD2 knockdown. Collectively, we constructed a prognosis model based on a NET and EMT gene signature, providing a valuable perspective for the prognostic evaluation and management of PAAD patient.

Systematic establishment and verification of an epithelial-mesenchymal transition gene signature for predicting prognosis of oral squamous cell carcinoma.

Objective: Epithelial-mesenchymal transition (EMT) is linked to an unfavorable prognosis in oral squamous cell carcinoma (OSCC). Here, we aimed to develop an EMT gene signature for OSCC prognosis. Methods: In TCGA dataset, prognosis-related EMT genes with p < 0.05 were screened in OSCC. An EMT gene signature was then conducted with LASSO method. The efficacy of this signature in predicting prognosis was externally verified in the GSE41613 dataset. Correlations between this signature and stromal/immune scores and immune cell infiltration were assessed by ESTIMATE and CIBERSORT algorithms. GSEA was applied for exploring significant signaling pathways activated in high- and low-risk phenotypes. Expression of each gene was validated in 40 paired OSCC and normal tissues via RT-qPCR. Results: A prognostic 9-EMT gene signature was constructed in OSCC. High risk score predicted poorer clinical outcomes than low risk score. ROCs confirmed the well performance on predicting 1-, 3- and 5-year survival. Multivariate cox analysis revealed that this signature was independently predictive of OSCC prognosis. The well predictive efficacy was validated in the GSE41613 dataset. Furthermore, this signature was distinctly related to stromal/immune scores and immune cell infiltration in OSCC. Distinct pathways were activated in two subgroups. After validation, AREG, COL5A3, DKK1, GAS1, GPX7 and PLOD2 were distinctly upregulated and SFRP1 was downregulated in OSCC than normal tissues. Conclusion: Our data identified and verified a robust EMT gene signature in OSCC, which provided a novel clinical tool for predicting prognosis and several targets against OSCC therapy.

Upregulation of SPI1 in Ectopic Endometrium Contributes to an Invasive Phenotype

Endometriosis is one of the most common gynecological diseases associated with chronic pelvic pain, infertility, and cancer. However, its molecular pathogenesis remains unclear. This study aimed to identify key genes involved in the pathogenesis of endometriosis. Bioinformatic analyses were perfomed to identify key differentially expressed genes (DEGs), transcription factors (TFs), and functionally enriched pathways. Effect of SPI1 on migration, invasion, expression of ADH1B, MYH11, and PLN were analyzed in human endometriotic cells. By screening three transcriptome datasets from the GEO for overlapping DEGs between eutopic and ectopic endometria in patients with endometriosis, we found that the expression of ADH1B, MYH11, and PLN was markedly upregulated in the ectopic endometrium. Knockdown of ADH1B, MYH11, and PLN significantly inhibited the migration and invasion of human endometriotic 12Z cells. Additionally, gene set enrichment analysis revealed that epithelial-mesenchymal transition gene signature was positively correlated with ADH1B, MYH11, and PLN expression. Notably, the TF SPI1 was found to regulate the expression of these three genes in the endometriotic tissues and 12TZ cells. Moreover, SPI1 expression was associated with the invasion of endometriotic cells and was increased in the ectopic endometrium of patients with endometriosis. These data suggest that SPI1 plays a key role in the progression of endometriosis by regulating ADH1B, MYH11, and PLN expression and may therefore serve as a potential prognostic and therapeutic factor for endometriosis.

Epithelial-Mesenchymal Transition Gene Signature Is Associated with Neoadjuvant Chemoradiotherapy Resistance and Prognosis of Esophageal Squamous Cell Carcinoma.

Background Neoadjuvant chemoradiotherapy (neo-CRT) in combination with surgery increases survival compared to surgery alone, as indicated by the esophageal squamous cell carcinoma (ESCC) treatment recommendations. However, the benefits of neo-CRT are diverse among patients. Consequently, the development of new biomarkers that correlate with neo-CRT might be important for the treatment of ESCC. Methods The differentially expressed genes (DEG) between responsive and resistant samples from the GSE45670 dataset were obtained. On the TCGA dataset, survival analysis was performed to identify prognosis-related-EMT-genes. For EMT score model construction, lasso regression analysis in the TCGA cohort was used to identify the genes. In the TCGA-ESCC cohort, age, stage, and EMT score were used to construct a nomogram. Results In total, 10 prognosis-related-EMT-genes were obtained. These 10 genes consisted of 6 risky genes and 4 protective genes. Based on the lasso analysis and univariate Cox regression, an EMT score model consisting of 7 genes (CLEC18A, PIR, KCNN4, MST1R, CAPG, ALDH5A1, and COX7B) was identified. ESCC patients with a high EMT score have a worse prognosis. These genes were differentially expressed between responsive and resistant patients and had a high accuracy for distinguishing resistant and responsive patients. Conclusions The identified genes have the potential to function as molecular biomarkers for predicting ESCC patients' resistance to neo-CRT. This research may aid in the elucidation of the molecular processes driving resistance and the identification of targets for improving the prognosis for ESCC.

A Novel Epithelial-Mesenchymal Transition Gene Signature Correlated With Prognosis, and Immune Infiltration in Hepatocellular Carcinoma

Background: Although many genes related to epithelial-mesenchymal transition (EMT) have been explored in hepatocellular carcinoma (HCC), their prognostic significance still needs further analysis. Methods: Differentially expressed EMT-related genes were obtained through the integrated analysis of 4 Gene expression omnibus (GEO) datasets. The univariate Cox regression and Lasso Cox regression models are utilized to determine the EMT-related gene signature. Based on the results of multivariate Cox regression, a predictive nomogram is established. Time-dependent ROC curve and calibration curve are used to show the distinguishing ability and consistency of the nomogram. Finally, we explored the correlation between EMT risk score and immune immunity. Results: We identified a nine EMT-related gene signature to predict the survival outcome of HCC patients. Based on the EMT risk score’s median, HCC patients in each dataset were divided into high and low-risk groups. The survival outcomes of HCC patients in the high-risk group were significantly worse than those in the low-risk group. The prediction nomogram based on the EMT risk score has better distinguishing ability and consistency. High EMT risk score was related to immune infiltration. Conclusion: The nomogram based on the EMT risk score can reliably predict the survival outcome of HCC patients, thereby providing benefits for medical decisions.

Bronchial epithelium epithelial-mesenchymal plasticity forms aberrant basaloid-like cells in vitro.

Although epithelial-mesenchymal transition (EMT) is a common feature of fibrotic lung disease, its role in fibrogenesis is controversial. Recently, aberrant basaloid cells were identified in fibrotic lung tissue as a novel epithelial cell type displaying a partial EMT phenotype. The developmental origin of these cells remains unknown. To elucidate the role of EMT in the development of aberrant basaloid cells from the bronchial epithelium, we mapped EMT-induced transcriptional changes at the population and single-cell levels. Human bronchial epithelial cells grown as submerged or air-liquid interface (ALI) cultures with or without EMT induction were analyzed by bulk and single-cell RNA-Sequencing. Comparison of submerged and ALI cultures revealed differential expression of 8,247 protein coding (PC) and 1,621 long noncoding RNA (lncRNA) genes and revealed epithelial cell-type-specific lncRNAs. Similarly, EMT induction in ALI cultures resulted in robust transcriptional reprogramming of 6,020 PC and 907 lncRNA genes. Although there was no evidence for fibroblast/myofibroblast conversion following EMT induction, cells displayed a partial EMT gene signature and an aberrant basaloid-like cell phenotype. The substantial transcriptional differences between submerged and ALI cultures highlight that care must be taken when interpreting data from submerged cultures. This work supports that lung epithelial EMT does not generate fibroblasts/myofibroblasts and confirms ALI cultures provide a physiologically relevant system to study aberrant basaloid-like cells and mechanisms of EMT. We provide a catalog of PC and lncRNA genes and an interactive browser (https://bronc-epi-in-vitro.cells.ucsc.edu/) of single-cell RNA-Seq data for further exploration of potential roles in the lung epithelium in health and lung disease.

A Prognostic Risk Score Based on Hypoxia-, Immunity-, and Epithelialto-Mesenchymal Transition-Related Genes for the Prognosis and Immunotherapy Response of Lung Adenocarcinoma.

Background: Lung adenocarcinoma (LUAD), the most common subtype of non-small cell lung cancer (NSCLC), is associated with poor prognosis. However, current stage-based clinical methods are insufficient for survival prediction and decision-making. This study aimed to establish a novel model for evaluating the risk of LUAD based on hypoxia, immunity, and epithelial-mesenchymal transition (EMT) gene signatures. Methods: In this study, we used data from TCGA-LUAD for the training cohort and GSE68465 and GSE72094 for the validation cohorts. Immunotherapy datasets GSE135222, GSE126044, and IMvigor210 were obtained from a previous study. Using bioinformatic and machine algorithms, we established a risk model based on hypoxia, immune, and EMT gene signatures, which was then used to divide patients into the high and low risk groups. We analyzed differences in enriched pathways between the two groups, following which we investigated whether the risk score was correlated with stemness scores, genes related to m6A, m5C, m1A and m7G modification, the immune microenvironment, immunotherapy response, and multiple anti-cancer drug sensitivity. Results: Overall survival differed significantly between the high-risk and low-risk groups (HR = 4.26). The AUCs for predicting 1-, 3-, and 5-year survival were 0.763, 0.766, and 0.728, respectively. In the GSE68465 dataset, the HR was 2.03, while the AUCs for predicting 1-, 3-, and 5-year survival were 0.69, 0.651, and 0.618, respectively. The corresponding values in the GSE72094 dataset were an HR of 2.36 and AUCs of 0.653, 0.662, and 0.749, respectively. The risk score model could independently predict OS in patients with LUAD, and highly correlated with stemness scores and numerous m6A, m5C, m1A and m7G modification-related genes. Furthermore, the risk model was significantly correlated with multiple immune microenvironment characteristics. In the GSE135222 dataset, the HR was 4.26 and the AUC was 0.702. Evaluation of the GSE126044 and IMvigor210 cohorts indicated that PD-1/PD-LI inhibitor treatment may be indicated in patients with low risk scores, while anti-cancer therapy with various drugs may be indicated in patients with high risk scores. Conclusion: Our novel risk model developed based on hypoxia, immune, and EMT gene signatures can aid in predicting clinical prognosis and guiding treatment in patients with LUAD.

Deep Learning-Based Classification of Epithelial-Mesenchymal Transition for Predicting Response to Therapy in Clear Cell Renal Cell Carcinoma.

Epithelial–mesenchymal transition (EMT) profoundly impacts prognosis and immunotherapy of clear cell renal cell carcinoma (ccRCC). However, not every patient is tested for EMT status because this requires additional genetic studies. In this study, we developed an EMT gene signature to classify the H&E-stained slides from The Cancer Genome Atlas (TCGA) into epithelial and mesenchymal subtypes, then we trained a deep convolutional neural network to classify ccRCC which according to our EMT subtypes accurately and automatically and to further predict genomic data and prognosis. The clinical significance and multiomics analysis of the EMT signature was investigated. Patient cohorts from TCGA (n = 252) and whole slide images were used for training, testing, and validation using an algorithm to predict the EMT subtype. Our approach can robustly distinguish features predictive of the EMT subtype in H&E slides. Visualization techniques also detected EMT-associated histopathological features. Moreover, EMT subtypes were characterized by distinctive genomes, metabolic states, and immune components. Deep learning convolutional neural networks could be an extremely useful tool for predicting the EMT molecular classification of ccRCC tissue. The underlying multiomics information can be crucial in applying the appropriate and tailored targeted therapy to the patient.

TGF-\u03b2-mediated epithelial\u2013mesenchymal transition and tumor-promoting effects in CMT64 cells are reflected in the transcriptomic signature of human lung adenocarcinoma

Epithelial–mesenchymal transition (EMT) is a cellular process during which epithelial cells acquire mesenchymal phenotypes. Cancer cells undergo EMT to acquire malignant features and TGF-β is a key regulator of EMT. Here, we demonstrate for the first time that TGF-β could elicit EMT in a mouse lung adenocarcinoma cell line. TGF-β signaling activation led to cell morphological changes corresponding to EMT and enhanced the expression of mesenchymal markers and EMT-associated transcription factors in CMT64 lung cancer cells. RNA-sequencing analyses revealed that TGF-β increases expression of Tead transcription factors and an array of Tead2 target genes. TGF-β stimulation also resulted in alternative splicing of several genes including Cd44, tight junction protein 1 (Tjp1), and Cortactin (Cttn). In parallel with EMT, TGF-β enhanced cell growth of CMT64 cells and promoted tumor formation in a syngeneic transplantation model. Of clinical importance, the expression of TGF-β-induced genes identified in CMT64 cells correlated with EMT gene signatures in human lung adenocarcinoma tissue samples. Furthermore, TGF-β-induced gene enrichment was related to poor prognosis, underscoring the tumor-promoting role of TGF-β signaling in lung adenocarcinoma. Our cellular and syngeneic transplantation model would provide a simple and useful experimental tool to study the significance of TGF-β signaling and EMT.

Single-Cell RNA-Seq Analysis Reveals the Acquisition of Cancer Stem Cell Traits and Increase of Cell-Cell Signaling during EMT Progression.

Simple SummaryPhenotypic plasticity is emerging as a crucial feature across multiple axes of cancer progression. Cells undergoing the epithelial–mesenchymal transition (EMT) can fall into intermediate or hybrid epithelial/mesenchymal (E/M) cell states. Moreover, cancer cells across the EMT spectrum can exhibit the traits of cancer stem cells (CSCs) and communicate through several cell-cell signaling pathways. By integrating multiple analytical tools into a single computational framework, we investigated several single-cell RNA-sequencing (scRNA-seq) datasets and identified the emerging relationship between EMT, acquisition of CSC traits, and cell–cell communication. Our integrated analysis shows that the increase of EM plasticity correlates with high expression of CSC markers as well as intensification of cell–cell signaling between cancer cells. Furthermore, these observations are consistent across different cancer types and anatomical locations. Overall, our results shine light onto the interconnected and multi-dimensional landscape of cancer progression.Intermediate cell states (ICSs) during the epithelial–mesenchymal transition (EMT) are emerging as a driving force of cancer invasion and metastasis. ICSs typically exhibit hybrid epithelial/mesenchymal characteristics as well as cancer stem cell (CSC) traits including proliferation and drug resistance. Here, we analyze several single-cell RNA-seq (scRNA-seq) datasets to investigate the relation between several axes of cancer progression including EMT, CSC traits, and cell–cell signaling. To accomplish this task, we integrate computational methods for clustering and trajectory inference with analysis of EMT gene signatures, CSC markers, and cell–cell signaling pathways, and highlight conserved and specific processes across the datasets. Our analysis reveals that “standard” measures of pluripotency often used in developmental contexts do not necessarily correlate with EMT progression and expression of CSC-related markers. Conversely, an EMT circuit energy that quantifies the co-expression of epithelial and mesenchymal genes consistently increases along EMT trajectories across different cancer types and anatomical locations. Moreover, despite the high context specificity of signal transduction across different cell types, cells undergoing EMT always increased their potential to send and receive signals from other cells.

Eosinophilic features in clear cell renal cell carcinoma correlate with outcomes of immune checkpoint and angiogenesis blockade

BackgroundClear cell renal cell carcinoma (ccRCC) displays heterogeneity in appearance—a distinctive pale clear to eosinophilic cytoplasm; however, little is known about the underlying mechanisms and clinical implications. We investigated the...

Identifying Molecular Signatures of Distinct Modes of Collective Migration in Response to the Microenvironment Using Three-Dimensional Breast Cancer Models

Simple SummaryThe objective of this study was to investigate the role of two microenvironmental factors, namely, tumor-intrinsic hypoxia and secretome in inducing collective migration. We utilized three-dimensional (3D) discrete-sized microtumor models, which recapitulate hallmarks of transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Tumor-intrinsic hypoxia induced directional migration in large hypoxic microtumors while secretome from large microtumors induced radial migration in non-hypoxic microtumors. This highlights the emergence phenotypic heterogeneity and plasticity in cancer cells in response to different microenvironmental stimuli. To unravel mechanisms underlying these two distinct modes of migration, we performed differential gene expression analysis of hypoxia- and secretome-induced migratory phenotypes using non-migratory, non-hypoxic microtumors as controls. We proposed unique gene signature sets related to tumor-intrinsic hypoxia, hypoxia-induced epithelial-mesenchymal transition (EMT), as well as hypoxia-induced directional migration and secretome-induced radial migration.Collective cell migration is a key feature of transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) among many other cancers, yet the microenvironmental factors and underlying mechanisms that trigger collective migration remain poorly understood. Here, we investigated two microenvironmental factors, tumor-intrinsic hypoxia and tumor-secreted factors (secretome), as triggers of collective migration using three-dimensional (3D) discrete-sized microtumor models that recapitulate hallmarks of DCIS-IDC transition. Interestingly, the two factors induced two distinct modes of collective migration: directional and radial migration in the 3D microtumors generated from the same breast cancer cell line model, T47D. Without external stimulus, large (600 µm) T47D microtumors exhibited tumor-intrinsic hypoxia and directional migration, while small (150 µm), non-hypoxic microtumors exhibited radial migration only when exposed to the secretome of large microtumors. To investigate the mechanisms underlying hypoxia- and secretome-induced directional vs. radial migration modes, we performed differential gene expression analysis of hypoxia- and secretome-induced migratory microtumors compared with non-hypoxic, non-migratory small microtumors as controls. We propose unique gene signature sets related to tumor-intrinsic hypoxia, hypoxia-induced epithelial-mesenchymal transition (EMT), as well as hypoxia-induced directional migration and secretome-induced radial migration. Gene Set Enrichment Analysis (GSEA) and protein-protein interaction (PPI) network analysis revealed enrichment and potential interaction between hypoxia, EMT, and migration gene signatures for the hypoxia-induced directional migration. In contrast, hypoxia and EMT were not enriched in the secretome-induced radial migration, suggesting that complete EMT may not be required for radial migration. Survival analysis identified unique genes associated with low survival rate and poor prognosis in TCGA-breast invasive carcinoma dataset from our tumor-intrinsic hypoxia gene signature (CXCR4, FOXO3, LDH, NDRG1), hypoxia-induced EMT gene signature (EFEMP2, MGP), and directional migration gene signature (MAP3K3, PI3K3R3). NOS3 was common between hypoxia and migration gene signature. Survival analysis from secretome-induced radial migration identified ATM, KCNMA1 (hypoxia gene signature), and KLF4, IFITM1, EFNA1, TGFBR1 (migration gene signature) to be associated with poor survival rate. In conclusion, our unique 3D cultures with controlled microenvironments respond to different microenvironmental factors, tumor-intrinsic hypoxia, and secretome by adopting distinct collective migration modes and their gene expression analysis highlights the phenotypic heterogeneity and plasticity of epithelial cancer cells.

Epithelial-mesenchymal transition gene signature is associated with prognosis and tumor microenvironment in head and neck squamous cell carcinoma

In this study we assessed the clinical significance of an epithelial-mesenchymal transition (EMT) gene signature and explored its association with the tumor microenvironment related to immunotherapy in patients with head and neck squamous cell carcinoma (HNSCC). Genes were selected when mRNA levels were positively or negatively correlated with at least one well-known EMT marker. We developed an EMT gene signature consisting of 82 genes. The patients were classified into epithelial or mesenchymal subgroups according to EMT signature. The clinical significance of the EMT signature was validated in three independent cohorts and its association with several immunotherapy-related signatures was investigated. The mesenchymal subgroup showed worse prognosis than the epithelial subgroup, and significantly elevated PD-1, PD-L1, and CTLA-4 levels, and increased interferon-gamma, cytolytic, T cell infiltration, overall immune infiltration, and immune signature scores. The relationship between PD-L1 expression and EMT status in HNSCC after treatment with TGF-β was validated in vitro. In conclusion, the EMT gene signature was associated with prognosis in HNSCC. Additionally, our results suggest that EMT is related to immune activity of the tumor microenvironment with elevated immune checkpoint molecules.

Abstract PD2-02: Activation of the EGFR/RAS/p42,44 MAPK axis as a convergent mechanism of resistance to CDK4/6 inhibitors in ER+ breast cancer

Abstract Background: In the PALOMA-3 trial, the combination of the CDK4/6 inhibitor palbociclib (P) with fulvestrant (F) led to a significant improvement in clinical outcomes, compared to F+Placebo, in endocrine-pretreated patients (pts) with ER+/HER2- metastatic breast cancer (BC). Not all pts, however, benefit from F+P, and virtually all ultimately progress. Elucidating the resistance mechanisms would assist in identifying biomarkers to select pts who may most benefit from F+P, as well as novel therapeutic targets for combating resistance. Materials and Methods: The ER+ MCF7 BC cell lines made resistant (R) to long-term estrogen deprivation (EDR) or tamoxifen (TamR), and the 600MPE de novo TamR cells were tested for sensitivity to F, P and F+P. MCF7 EDR and MCF7 TamR cells were exposed to increasing concentrations of F+P to generate R derivatives (FPR, cells grow in the presence of F 100nM + P 100nM). The transcriptomic and proteomic profiles of MCF7 EDR, TamR, EDR/FPR and TamR/FPR were determined by RNA-seq and reverse-phase protein arrays (RPPA), respectively. Pathway analysis was performed by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). mRNA and protein levels of select markers were assessed by RT-PCR and Western blot (WB), respectively. Cell growth, colony formation, and migratory and invasive potential were assessed by methylene blue staining, clonogenic assay, and in vitro migration and invasion assays. Results: Treatment of MCF7 EDR and TamR cells with P resulted in a dose-dependent inhibition of cell growth [IC50 = 149nM and 89nM, respectively] and colony formation [clonogenic inhibition at 100nM &amp;gt;85% and &amp;gt;95%, respectively]. In both MCF7 endocrine-R models, P (100nM) or F (100nM) monotherapy significantly inhibited cell growth and reduced phosphorylated (p)-Rb and the E2F target CDK2 protein levels compared to vehicle control. However, F+P resulted in an enhanced cell growth inhibition (&amp;gt;90%) and a more pronounced reduction in p-Rb and CDK2 expression compared to each agent alone. In the FPR lines, ‘oncogenic signatures’ representative of altered KRAS and MEK signaling were highly enriched compared to their sensitive counterparts, as revealed by GSEA analysis of the transcriptomic profiles. IPA analysis of the proteomic profiles confirmed the activation of the MAPK signaling pathway in FPR cells and identified EGFR as one of the top ranked upstream regulators. Increased EGFR mRNA and protein levels in FPR models were confirmed by RT-PCR and WB analysis. Additionally, gene signatures of cyclin D1 hyperactivation and increased cyclin D1 protein levels were enriched in FPR cell lines. Importantly, the EGFR inhibitor gefitinib (1µM), the pan-HER inhibitor neratinib (250nM), and the MEK1 inhibitor selumetinib (S) (1µM) selectively or more greatly inhibited the cell growth and reduced Cyclin D1 protein levels in FPR cells compared to their sensitive counterparts. Further, we found a significant enrichment of the ‘hallmark’ epithelial-mesenchymal transition gene signature in FPR models, which is in line with their increased migratory and invasive capabilities. Treatment with S reduced the migration and invasion of the FPR cells. Finally, the aggressive 600MPE de novo TamR cells harboring hyperactive MEK/MAPK signaling due to the KRAS and MAP2K4 activating mutations were significantly less sensitive to F, P, and F+P. However, the addition of S to F+P significantly increased the cell growth inhibition in this model. Conclusion: Acquired and intrinsic resistance to F+P is associated with hyperactivation of the EGFR/RAS/MEK signaling pathway, which sustains cell proliferation and promotes an aggressive phenotype. The EGFR/RAS/MEK pathway may serve as a predictive biomarker for F+P resistance and offer therapeutic targets to overcome resistance in pts with ER+/HER2- BC. Citation Format: Carmine De Angelis, Maria Letizia Cataldo, Agostina Nardone, Jamunarani Veeraraghavan, Xiaoyong Fu, Sarmistha Nanda, Lanfang Qin, Vidyalakshmi Sethunath, Resel Pereira, Britta Weigelt, Jorge S. Reis-Filho, Mothaffar F. Rimawi, Rinath Jeselsohn, Kent Osborne, Rachel Schiff. Activation of the EGFR/RAS/p42,44 MAPK axis as a convergent mechanism of resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-02.

EMT-related gene expression is positively correlated with immunity and may be derived from stromal cells in osteosarcoma.

BackgroundOsteosarcoma is the most common type of bone cancer in children and young adults. Recent studies have shown a correlation between epithelial–mesenchymal transition (EMT)-related gene expression and immunity in human cancers. Here, we investigated the relationship among EMT, immune activity, stromal activity and tumor purity in osteosarcoma.MethodsWe defined EMT gene signatures and evaluated immune activity and stromal activity based on the gene expression and clinical data from three independent microarray datasets. These factors were evaluated by single sample Gene Set Enrichment Analyses and the ESTIMATE tool. Finally, we analyzed the key source of EMT gene expression in osteosarcoma using microarray datasets from the Gene Expression Omnibus and human samples that we collected.ResultsEMT-related gene expression was positively correlated with immune and stromal activity in osteosarcoma. Tumor purity was negatively correlated with EMT, immune activity and stromal cells. We further demonstrated that high EMT gene expression could significantly predict poor overall survival (OS) and recurrence-free survival (RFS) in osteosarcoma patients, while high immune activity cannot. However, combining these factors could have further prognostic value for osteosarcoma patients in terms of OS and RFS. Finally, we found that stromal cells may serve as a key source of EMT gene expression in osteosarcoma.ConclusionThe results of this study reveal that the expression of EMT genes and immunity are positively correlated, but these signatures convey disparate prognostic information. Furthermore, the results indicate that EMT-related gene expression may be derived from stromal rather than epithelial cancer cells.

Prognostic value and association with epithelial-mesenchymal transition and molecular subtypes of the proteoglycan biglycan in advanced bladder cancer

ObjectiveDysregulation of the extracellular matrix molecule biglycan (BGN) predicts poor survival in several cancer entities. Our study investigated the prognostic impact of BGN in bladder cancer (BC) in 2 independent cohorts and assessed its role in epithelial-mesenchymal transition (EMT) and association with molecular BC subtypes. MethodsBGN protein expression was correlated with the oncological outcome of 162 patients with BC undergoing radical cystectomy (RC) in a single center and furthermore on gene expression level in the TCGA database. Cut-off values for BGN protein and RNA expression were tested with receiver operating characteristic (ROC) curves. BGN gene expression was correlated with established EMT and BC gene signatures in the TCGA database using gene set enrichment analysis (GSEA). Key EMT and basal/luminal molecular BC subtype markers were correlated with BGN expression and data were shown in a heat map. ResultsBGN upregulation in BC cells on the protein level predicted poor oncological survival in the institutional cohort for both univariate (P = 0.007) and multivariate (P = 0.040) analyses. BGN expression was not associated with other clinicopathological parameters. The prognostic value of BGN was validated on the mRNA level in the BC TCGA database (P = 0.002). Both EMT and BC core gene signatures (P < 0.001) correlated with BGN expression in GSEA. BGN gene expression was associated with key indicators of EMT. BGN was associated positively with the molecular basal BC subtype and negatively with the BC luminal subtype. ConclusionBGN is an independent prognosticator for poor survival in BC patients. BGN is associated with the basal molecular BC subtype. EMT might be a key player for BGN driven oncogenesis, as BGN expression correlates with EMT gene signatures.

Glucocorticoid receptor expression in Müllerian carcinosarcomas.

e17038 Background: Carcinosarcomas (CS) are aggressive gynecologic malignancies defined by a biphasic tumor histology of carcinomatous and sarcomatous components. High expression of glucocorticoid receptor (GR) is associated with aggressive tumor biology in triple-negative breast, ovarian, and endometrial cancers. GR activation results in transcriptional regulation of epithelial-mesenchymal transition (EMT) genes. Therefore, we hypothesized that the sarcomatous component of Müllerian CS may be associated with relatively higher GR expression compared to the carcinomatous portion. Methods: Müllerian CSs from the University of Chicago tissue bank diagnosed from 5/19/2009 – 8/25/2014 were identified and pathology confirmed (RL). In addition, we evaluated GR expression in a mouse PDX CS model. Immunohistochemical expression of nuclear estrogen (ER), progesterone (PR), androgen (AR), and GR was quantified using the H-score. Scores between the sarcomatous and carcinomatous components were compared using a paired t-test. Results: Mean GR H-score was relatively higher in the sarcomatous vs carcinomatous component, overall (144.44 vs 38.89, P = 0.0014). Mean H-score also differed between sarcomatous and carcinomatous components for ER (3.06 vs 63.06, P = 0.016), PR (1.67 vs 47.22, P = 0.004), and AR (0 vs 2.78, P = 0.056). The significantly higher sarcomatous GR expression was replicated in IHC evaluation of the mouse PDX model. Conclusions: GR expression is significantly higher in the sarcomatous than the carcinomatous component of Müllerian CS, while ER, PR, and AR expression is significantly lower. Recent data suggest that an EMT gene signature is more prominently expressed in the sarcomatous versus carcinomatous component. We hypothesize that GR expression and activity may mediate EMT gene expression and represent a therapeutic target for CS. [Table: see text]

Periostin blockade overcomes chemoresistance via restricting the expansion of mesenchymal tumor subpopulations in breast cancer

Recent studies suggest a functional involvement of Epithelial-Mesenchymal Transition (EMT) in tumor chemoresistance. Specifically, EMT is associated with chemoresistance and poor prognosis in triple-negative breast cancer. However, no effective therapy targeting EMT has been developed. Here, we report that periostin, an extracellular matrix protein, was induced upon chemotherapy and tightly correlated with the EMT gene signature and poor prognosis in breast cancer. In triple-negative breast cancer xenografts, chemotherapy upregulated periostin expression in tumor cells, triggered expansion of mesenchymal tumor cells and promoted invasion in residual tumors. Knockdown of periostin inhibited outgrowth and invasion of mesenchymal tumor cells upon chemotherapy. Furthermore, chemotherapy upregulated cancer-specific variants of periostin and application of a blocking antibody specifically targeting those variants overcame chemoresistance and halted disease progression without toxicity. Together, these data indicate that periostin plays a key role in EMT-dependent chemoresistance and is a promising target to overcome chemoresistance in triple-negative breast cancer.

Abstract A82: Epithelial-mesenchymal transition (EMT) genes are involved in the behavior and aggressiveness of neuroendocrine lung carcinomas (NELC) and non-small cell lung cancer (NSCLC): A comparative analysis

Abstract Background: Recurrence and metastasis are responsible for 90% of deaths of patients with lung cancer. Adenocarcinomas (ADc) primarily invade blood vessels with distant metastasis, whereas squamous cell carcinoma (SqCC) involves the mediastinal lymph nodes. Neuroendocrine carcinomas of low grade (typical and atypical carcinoid) are indolent, while high-grade NE carcinoma (large-cell NE and small-cell carcinomas) metastasize rapidly. Biomarkers of invasiveness in lung carcinomas still cannot be definitely determined. Epithelial to mesenchymal transition (EMT) genes profile have promise as indicator of invasion and metastasis. Our aim was to compare EMT gene expression in NELC, ADc, and SqCC and its impact on behavior of these tumors. Methods: EMT gene expression was quantified with a quantitative real-time (RT)- PCR carried out on StepOnePlus™ Real-Time PCR System (Applied Biosystems) with RT2 Profiler PCR Array System (Qiagen, Dusseldorf, Germany). Results: Younger patients expressed higher amount of AHNAK, MSN, and TGF-beta 3 than older (p&amp;lt;0.05), whereas SNAI3 and TGF-beta 2 were more expressed in smokers (p&amp;lt;0.05). ADc and SqCC presented significant higher expression of COL3A1, DSP, and MSN in tumor compared to normal tissue (p&amp;lt;0.05). 13-gene signature (AHNAK, COL3A1, DSP, IL1RN, MSN, PDGFRB, SNAI1, SNAI3, TCF3, TGF-beta 1, TGF-beta 2, TGF-beta 3, and VIM) was upregulated in tumor tissue from all NELC patients. In ADc and NELC, AHNAK, IL1RN, SNAI3, and VIM was significantly different (p&amp;lt;0.05). ADc and SqCC compared with high-grade NELC also presented differences in COL3A1, DSP, and PDGFR (p&amp;lt;0.01). Advanced tumors, usually with metastasis, showed higher expression of AHNAK, MSN, SNAI3, TGF-beta 3, and VIM (p&amp;lt;0.05), as well as association with poor outcome (p &amp;lt;0.01). Conclusion: Different expression of EMT gene signature in neuroendocrine lung carcinomas and non-small cell lung cancer, its relationship with histologic types, advanced stage, lymph node metastasis, and death suggest a possible role of these markers in this malignancy, but more importantly provide a potential biomolecular marker to predict outcome. The correlation between NELC, ADc, SqCC, and specific EMT genes involved in cell proliferation and motility provides a possible role of these genes in the development and aggressiveness in these tumors. Moreover, the difference of gene expression between NSCLC and NELC emerges as a promising biomarker of behavior. Further studies are needed to validate EMT gene expression to predict prognosis and tumoral aggressiveness. Citation Format: Tabatha Gutierrez Prieto, Vanessa Karen de Sá, Eloisa Helena Ribeiro Olivieri, Eduardo Caetano Albino da Silva, Rui Manuel Reis, Dirce Maria Carraro, Vera Luiza Capelozzi. Epithelial-mesenchymal transition (EMT) genes are involved in the behavior and aggressiveness of neuroendocrine lung carcinomas (NELC) and non-small cell lung cancer (NSCLC): A comparative analysis [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A82.

RNA G-quadruplex secondary structure promotes alternative splicing via the RNA-binding protein hnRNPF.

It is generally thought that splicing factors regulate alternative splicing through binding to RNA consensus sequences. In addition to these linear motifs, RNA secondary structure is emerging as an important layer in splicing regulation. Here we demonstrate that RNA elements with G-quadruplex-forming capacity promote exon inclusion. Destroying G-quadruplex-forming capacity while keeping G tracts intact abrogates exon inclusion. Analysis of RNA-binding protein footprints revealed that G quadruplexes are enriched in heterogeneous nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated alternatively spliced exons in the human transcriptome. Moreover, hnRNPF regulates an epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a G-quadruplex-dependent manner, which results in inhibition of EMT. Mining breast cancer TCGA (The Cancer Genome Atlas) data sets, we demonstrate that hnRNPF negatively correlates with an EMT gene signature and positively correlates with patient survival. These data suggest a critical role for RNA G quadruplexes in regulating alternative splicing. Modulation of G-quadruplex structural integrity may control cellular processes important for tumor progression.