Epithelial-mesenchymal Transition Scores Research Articles

A novel subtype based on driver methylation–transcription in lung adenocarcinoma

AimsTo identify driver methylation genes and a novel subtype of lung adenocarcinoma (LUAD) by multi-omics and elucidate its molecular features and clinical significance.MethodsWe collected LUAD patients from public databases, and identified driver methylation genes (DMGs) by MethSig and MethylMix algrothms. And novel driver methylation multi-omics subtypes were identified by similarity network fusion (SNF). Furthermore, the prognosis, tumor microenvironment (TME), molecular features and therapy efficiency among subtypes were comprehensively evaluated.Results147 overlapped driver methylation were identified and validated. By integrating the mRNA expression and methylation of DMGs using SNF, four distinct patterns, termed as S1-S4, were characterized by differences in prognosis, biological features, and TME. The S2 subtype showed unfavorable prognosis. By comparing the characteristics of the DMGs subtypes with the traditional subtypes, S3 was concentrated in proximal-inflammatory (PI) subtype, and S4 was consisted of terminal respiratory unit (TRU) subtype and PI subtype. By analyzing TME and epithelial mesenchymal transition (EMT) features, increased immune infiltration and higher expression of immune checkpoint genes were found in S3 and S4. While S4 showed higher EMT score and expression of EMT associated genes, indicating S4 may not be as immunosensitive as the S3. Additionally, S3 had lower TIDE and higher IPS score, indicating its increased sensitivity to immunotherapy.ConclusionThe driver methylation-related subtypes of LUAD demonstrate prognostic predictive ability that could help inform treatment response and provide complementary information to the existing subtypes.

Mechanistic Insights into Yifei Sanjie Pill's Regulation of EMT to Enhance Gefitinib Treatment effect in NSCLC by in silico analysis and experimental validation

Ethnopharmacological relevanceThe Yi-Fei San-Jie pill (YFSJ) is a well-known Chinese medicine that has been used to treat non-small cell lung cancer in China for decades. Aim of the studyPrevious studies have shown that YFSJ combined with gefitinib can effectively inhibit the proliferation of gefitinib-resistant non-small cell lung cancer (NSCLC) cell lines by promoting apoptosis and autophagy, but the molecular biological mechanisms involved and whether YFSJ combined with gefitinib can have synergistic effects still need to be further explored. Thus, the present study aimed to establish an in silico and experimental framework to decipher the underlying mechanism by which YFSJ augments the efficacy of gefitinib in treating NSCLC. Materials and methodsIntegrated approaches, including microarray analysis, network pharmacology, RNA sequencing, bioinformatics algorithm analysis and in vivo and in vitro experiments, were applied to elucidate the underlying mechanism. ResultsAnalysis of microarray datasets indicated that gefitinib may play a role in the regulation of the epithelial–mesenchymal transition (EMT) of PC9 cells. EMT-related Gene Ontology (GO) terms and the MAPK pathway were found to be enriched in the differentially expressed genes (DEGs), and a decreasing trend was observed in the EMT score. Network pharmacology analysis revealed that the potential NSCLC-related targets of YFSJ also showed enrichment in EMT-related GO terms and the MAPK pathway. Experimental findings demonstrated that combined YFSJ-treated serum and gefitinib treatment significantly inhibited PC9 cell migration and invasion. In addition, the combined treatment dramatically reduced the tumour volume in an animal model. The effectiveness of the combination treatment surpassed that of gefitinib alone in both cell and animal experiments. RNA sequencing analysis revealed significant enrichment of DEGs in EMT-related GO terms for the gefitinib treatment group, YFSJ treatment group, and combination treatment group compared to the control group. Notably, the negative regulation of EMT showed significant enrichment in the DEGs of the combination treatment group. The MAPK pathway was significantly enriched among the different groups. Moreover, combined treatment with YFSJ and gefitinib may exert synergistic anti-NSCLC effects by inhibiting the p-p38 MAPK/GSK3β signalling axis, subsequently suppressing downstream EMT processes. ConclusionCombined treatment with YFSJ and gefitinib could enhance the sensitivity of NSCLC cells to gefitinib by suppressing EMT through the EGFR/p-p38 MAPK/GSK3β signalling axis. YFSJ may serve as an important adjunctive medication for NSCLC patients receiving gefitinib treatment in clinical practice

Enhanced epithelial-mesenchymal transition signatures are linked with adverse tumor microenvironment, angiogenesis and worse survival in gastric cancer.

Epithelial-mesenchymal transition (EMT) is a crucial mechanism that facilitates cancer cell metastasis. Despite its importance, the clinical significance of EMT in gastric cancer (GC) patients has yet to be clearly demonstrated. For gauging the extent of EMT in GC, we employed gene set variation analysis to score 807 patient samples from two large cohorts: TCGA and GSE84437. In both cohorts, EMT high GC showed a significant association with worse overall survival (hazard ratio (HR) = 1.74, p = 0.011 and HR = 2.01, p < 0.001, respectively). This association was stronger when considering the EMT signature score compared to the individual expressions of EMT-related genes (CDH1, CDH2, VIM, and FN1). While the EMT signature level did not differ among various cancers, high EMT signature specifically correlated with survival in GC alone. Mucinous and diffuse histological types exhibited higher EMT levels compared to others (p < 0.001), and the EMT signature level was correlated with tumor depth and AJCC stage (all p < 0.001). Interestingly, the EMT score was an independent factor for overall and disease-specific survival (multivariate; p = 0.006 and 0.032, respectively). EMT high GC displayed a lower fraction of Th1 cells and a higher fraction of dendritic cells, M1 macrophages and several stromal cells. EMT high GC exhibited an inverse correlation with cell proliferation-related gene sets. While they significantly enriched multiple pro-cancerous gene sets, such as TGF-β signaling, hypoxia, and angiogenesis. The presence of EMT signature in a bulk tumor was linked to TGF-β signaling, hypoxia, and angiogenesis, and was also associated with poorer survival outcomes in GC patients.

RAB22A as a predictor of exosome secretion in the progression and relapse of multiple myeloma.

Multiple myeloma (MM) is an incurable malignant plasma cell disease. We explored the role of RAB22A in exosome secretion, epithelial-mesenchymal transition (EMT) and immune regulation. We obtained MM samples from Gene Expression Omnibus (GEO) data sets. We downloaded the "IOBR" package, and used the "PCA" and "ssGSEA" algorithms to calculate the EMT scores and exosome scores. The "CIBERSORT" package was used to analyze the infiltration of immune cells. We extracted the exosomes of mesenchymal stem cell (MSC) to verify the biological function of RAB22A. The expression level of RAB22A in smoldering multiple myeloma (SMM) and MM patients was significantly higher than that in normal people and monoclonal gammopathy of undetermined significance (MGUS) patients, and the expression level of RAB22A in relapse MM patients was significantly higher than that in newly diagnosed patients. The EMT scores and exosome scores of high RAB22A group were significantly higher than those of low RAB22A group, and the exosome scores of MSC in recurrent patients were significantly higher than those of newly diagnosed patients. In addition, the infiltration levels of monocyte, NK cells resting, eosinophils, T cells regulatory and T cells CD4 memory activated were positively correlated with RAB22A. After down-regulating the expression of RAB22A in MM-MSC, the secretion of exosomes decreased. Compared with the exosomes of MSC in si-RAB22A group, the exosomes in control group significantly promoted the proliferation of MM. RAB22A is a potential therapeutic target to improve the prognosis of MM, which is closely related to exosome secretion, EMT and immune cell infiltration.

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.

RAB22A As a Predictor of Exosome Secretion in the Progression and Relapse of Multiple Myeloma

Background: Multiple myeloma（MM）is an incurable malignant plasma cell disease in the blood system. After remission, patients will still have multiple recurrences, and the remission time after each recurrence will gradually become shorter. RAB22A is a member of RAS oncogene family, and there is no relevant research in MM at present. In this study, we explored the role of RAB22A in exosome secretion, Epithelial-mesenchymal transition（EMT）and immune regulation. Methods: We obtained MM samples from GEO data sets (GSE5900, GSE4581 and GSE146649), and analyzed the difference of expression of RAB22A. We downloaded the “IOBR” package, and used the “PCA” and “ssGSEA” algorithms to calculate the EMT scores and exosome scores of the high/low RAB22A expression groups. The “CIBERSORT” package was used to analyze the infiltration of immune cells. RT-PCR and Western-blot were used to verify the expression level of RAB22A in normal people and MM patients. After regulating the expression of RAB22A, the exosomes of MSC were collected for immunofluorescence staining and CCK-8 experiment. Results: The expression level of RAB22A in SMM and MM patients was significantly higher than that in normal people and MGUS patients, and the expression level of RAB22A in relapse MM patients was significantly higher than that in newly diagnosed patients. We calculated the EMT score and exosomes score by “IOBR” package. The results showed that the EMT score and exosomes score of high RAB22A group were significantly higher than those of low RAB22A group, and the exosomes score of MSC in recurrent patients was significantly higher than that of newly diagnosed patients. In addition, the infiltration levels of Monocyte, NK cells resting, Eosinophils, T cells regulatory (Treg) and T cells CD4 memory activated were positively correlated with RAB22A, while plasma cells and T cells gamma delta were negatively correlated with RAB22A. The expression levels of m6A genes (METTL14, VIRMA, RBM15 and FMR1) in the high RAB22A group were significantly higher than those in the low RAB22A group. We constructed a nomogram based on m6A genes significantly related to RAB22A to predict the risk value of high RAB22A expression, and the calibration curve showed that the nomogram had good prediction ability. We screened sensitive drugs according to the difference of IC50 values between high and low RAB22A groups, and found that cisplatin, Doxorubicin and bcl-2 inhibitors had higher sensitivity in low RAB22A group. Finally, we verified the above results in the samples of our center. The results showed that the expression level of RAB22A in MM patients was significantly higher than that of donors. After down-regulating the expression of RAB22A in MM-MSC, the secretion of exosomes decreased（Figure 1). Down-regulation of RAB22A reduced the proliferation of MM cells, and the addition of exosomes of MM-MSC partially reversed this effect. Conclusion: RAB22A is a potential therapeutic target to improve the prognosis of MM, which is closely related to exosome secretion, EMT and immune cell infiltration.

Comparative Analysis of Driver Mutations and Transcriptomes in Papillary Thyroid Cancer by Region of Residence in South Korea.

Radiation exposure is a well-known risk factor for papillary thyroid cancer (PTC). South Korea has 24 nuclear reactors in operation; however, no molecular biological analysis has been performed on patients with PTC living near nuclear power plants. We retrospectively included patients with PTC (n=512) divided into three groups according to their place of residence at the time of operation: inland areas (n=300), coastal areas far from nuclear power plants (n=134), and nuclear power plant areas (n=78). After propensity score matching (1:1:1) by age, sex, and surgical procedure, the frequency of representative driver mutations and gene expression profiles were compared (n=50 per group). Epithelial-mesenchymal transition (EMT), BRAF, thyroid differentiation, and radiation scores were calculated and compared. No significant difference was observed in clinicopathological characteristics, including radiation exposure history and the frequency of incidentally discovered thyroid cancer, among the three groups. BRAFV600E mutation was most frequently detected in the groups, with no difference among the three groups. Furthermore, gene expression profiles showed no statistically significant difference. EMT and BRAF scores were higher in our cohort than in cohorts from Chernobyl tissue bank and The Cancer Genome Atlas Thyroid Cancer; however, there was no difference according to the place of residence. Radiation scores were highest in the Chernobyl tissue bank but exhibited no difference according to the place of residence. Differences in clinicopathological characteristics, frequency of representative driver mutations, and gene expression profiles were not observed according to patients' region of residence in South Korea.

Aberrations in ion channels interacting with lipid metabolism and epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most prevalent malignant gastrointestinal tumor. Ion channels contribute to tumor growth and progression through interactions with their neighboring molecules including lipids. The dysregulation of membrane ion channels and lipid metabolism may contribute to the epithelial-mesenchymal transition (EMT), leading to metastatic progression. Herein, transcriptome profiles of patients with ESCC were analyzed by performing differential gene expression and weighted gene co-expression network analysis to identify the altered ion channels, lipid metabolism- and EMT-related genes in ESCC. A total of 1,081 differentially expressed genes, including 113 ion channels, 487 lipid metabolism-related, and 537 EMT-related genes, were identified in patients with ESCC. Thereafter, EMT scores were correlated with altered co-expressed genes. The altered co-expressed genes indicated a correlation with EMT signatures. Interactions among 22 ion channels with 3 hub lipid metabolism- and 13 hub EMT-related proteins were determined using protein-protein interaction networks. A pathway map was generated to depict deregulated signaling pathways including insulin resistance and the estrogen receptor-Ca2+ signaling pathway in ESCC. The relationship between potential ion channels and 5-year survival rates in ESCC was determined using Kaplan-Meier plots and Cox proportional hazard regression analysis. Inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) was found to be associated with poor prognosis of patients with ESCC. Additionally, drugs interacting with potential ion channels, including GJA1 and ITPR3, were identified. Understanding alterations in ion channels with lipid metabolism and EMT in ESCC pathophysiology would most likely provide potential targets for the better treatment of patients with ESCC.

Association of enhanced epithelial-mesenchymal transition signature with tumor microenvironment, angiogenesis, and survival in gastric cancer.

e16002 Background: Among the hallmarks of cancer, epithelial-mesenchymal transition (EMT) that epithelial cancer cells gain the characteristics of mesenchymal cells, is one of the key mechanisms of cancer cell metastasis. On the other hand, the clinical relevance of EMT that occurs in gastric cancer patients has not been clearly shown. Methods: To measure the level of EMT in gastric cancer, gene set variation analysis was used to score total of 807 gastric cancer patient samples from two independent large cohorts; TCGA and GSE84437. Results: EMT high gastric cancer was significantly associated with worse overall survival (hazard ratio (HR)=1.74, p=0.011 in the TCGA, and HR=2.01, p&lt;0.001 in the GSE84437), and the association was stronger with EMT signature score compared to expressions of each EMT-related genes, CDH1, CDH2, VIM, or FN1. EMT signature levels were not significantly different among gastric cancer, hepatocellular carcinoma, colorectal cancer, breast cancer, and pancreatic ductal adenocarcinoma, and high level of EMT signature was associated with survival only in gastric cancer. Among the histological types, mucinous and diffuse type had high EMT level compared to tubular, papillary, or signet gastric cancer ( p&lt;0.001). EMT signature level was significantly correlated with tumor depth and AJCC stage (all p&lt;0.001), but not consistently with lymphatic metastasis. Interestingly, the EMT score was independent factor for patient survival including clinicopathological features, not only for overall survival but also disease-specific survival in the TCGA cohort (multivariate; p=0.006 and 0.032, respectively). EMT high gastric cancer was associated with low fraction of Th1 cells and high fraction of dendritic cells and M1 macrophages (all p&lt;0.001 in both cohort), but not with interferon-gamma immune response. EMT high gastric cancer tended to be inversely correlated with cell proliferation-related gene sets, and significantly associated with high infiltration fraction of stromal cells, including fibroblasts, adipocytes (all p&lt;0.001). On the other hand, they significantly enriched multiple pro-cancerous gene sets, such as TGF-β signaling, hypoxia, and angiogenesis, consistent with infiltration of angiogenesis-related stromal cells such as microvascular and lymphatic endothelial cells, and pericytes (all p&lt;0.001). These results were shown consistently in two large cohort; TCGA and GSE84437. Conclusions: EMT signature in a bulk tumor was associated with cancer aggravating pathways such as TGF-β signaling, hypoxia, and angiogenesis, as well as with worse survival in gastric cancer patients.

Distinct molecular features and potential therapeutic strategies for spinal cord gliomas.

2056 Background: Spinal cord gliomas (SCG) are rare primary CNS tumors with varied prognosis. Standard treatment remains poorly defined, as no clear survival advantage exists with extensive resection, radiation, or chemotherapy. Mounting evidence suggests that tumors from different regions of the CNS harbor unique molecular signatures. In a retrospective study, we comprehensively characterized molecular alterations to identify potential therapeutic strategies in the largest cohort of SCG reported to date. Methods: We performed centralized pathology review and analyzed SCG with next-generation sequencing of DNA (592 gene NextSeq or WES, Novaseq), RNA (WTS, NovaSeq) and pyrosequencing (MGMT promoter methylation, MGMTme). We estimated tumor microenvironment cell infiltration by quanTIseq &amp; Epithelial-Mesenchymal Transition (EMT) by RNAseq. We used X2/Fisher’s-exact/Mann-Whitney U tests for comparison &amp; determined significance (p &lt; 0.05), adjusting for multiple comparison by the Benjamini-Hochberg method (q &lt; 0.05). Results: We analyzed 39 surgically accessible SCG (19 high grade, 17 low grade, 3 NOS); ependymomas were excluded. The most common alterations were mutations in H3F3A (31%), TP53 (25%), ATRX (21%), NF1 (18%) &amp; BRAF fusion (19%). H3F3A mutations occurred exclusively in high grade SCG (63%, q &lt; 0.05) while BRAF fusions occurred exclusively in low grade SCG (60%, p &lt; 0.05). TP53 mutations were more prevalent in high grade SCG (50% vs. 8%, p &lt; 0.05). Compared to intracranial gliomas (n = 6732), SCG harbor significantly more frequent H3F3A (31% vs. 1.6%), KRAS mutations (10% vs. 0.7%) &amp; BRAF fusions (19% vs. 0.7%) but less frequent TERT (4.3% vs. 66%) &amp; PTEN (0 vs. 27%) mutations or EGFR amplification (0 vs. 28%) (all q &lt; 0.05). SCG rarely harbor the canonical intracranial alterations IDH1 (2.6% vs. 17%), EGFRvIII (0 vs. 17%) or MGMTme (19% vs. 47%) (p &lt; 0.05). SCG have greater penetration of immune infiltrates of DCs (median cell fraction/MCF: 13% vs. 9%), T regs (positive percent: 47% vs. 26%) &amp; B cells (MCF: 9% vs. 7%), with less penetration of monocytes (MCF: 0 vs. 2%) (q &lt; 0.05). EMT score associates with high grade glioma in intracranial disease (q &lt; 0.05) but does not recapitulate in SCG (p &gt; 0.05). Conclusions: Clinical management of SCG is currently drawn from experience with intracranial gliomas. Our results identify unique molecular features of SCG suggesting an underlying biology distinct from intracranial gliomas. In SCG, H3F3A mutations are exclusive to high grade while BRAF fusions are exclusive to low grade; SCG rarely harbor canonical intracranial alterations such as IDH1, EGFR or MGMTme; and SCG have greater penetration of DCs with less penetration of monocytes. We provide a biological explanation for limited effectiveness of current therapies in SCG with potential implications for chemotherapy, targeted and immunotherapy. Our work underscores the need for investigations dedicated uniquely to SCG.

Abstract P4-02-03: Transcriptomic signature score of Epithelial-Mesenchymal Transition (EMT) of a bulk tumor may not reflect that of cancer cells

Abstract Background: Epithelial-mesenchymal transition (EMT) is a well-known multistep process of cancer cell invasion and metastasis, as well as treatment resistance. Our group has been reporting the predictive role of scores generated using Gene Set Variation Analysis (GSVA) of Hallmark pathways in breast cancer. In this study, we hypothesized that EMT score high breast cancer is aggressive and is associated with poor clinical outcome. Material and Methods: The clinicopathological data and transcriptome data of breast cancer patients from three independent large publicly available databases, The Cancer Genome Atlas (TCGA, n = 1077), The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC, n = 1904), and GSE96058 (n=3069) were utilized. Survival analyses; Overall survival (OS), Disease-specific survival (DSS) and Disease-free survival (DFS) were performed by comparing the high and low score groups. Tumor immune microenvironment was analyzed utilizing the values reported by Thorsson et al. Also, single sample Gene Set Enrichment analysis (ssGSEA) was performed between EMT high and low expression groups utilizing singe cell sequence cohorts. Results: EMT score was generated by Gene Set Variation Analysis of a Hallmark gene set and we divided each cohort into EMT score high and low groups by utilizing median as the cutoff. To our surprise, EMT score of the primary tumor was not associated with metastasis (N and M categories of cancer staging), Nottingham histological grade, nor MKI67 expression levels consistently in TCGA, METABRIC, and GSE96058. EMT score high tumors were not associated with worse DFS, DSS, OS in TCGA and METABRIC and OS in GSE96058. Analyses using xCell demonstrated that EMT score high tumors were associated with high infiltration of stromal cells such as adipocyte (p&amp;lt; 0.001, p&amp;lt; 0.001 and p&amp;lt; 0.001, respectively) and fibroblasts (p&amp;lt; 0.001, p&amp;lt; 0.001 and p&amp;lt; 0.001, respectively) in all three cohorts, TCGA, METABRIC, and GSE96058. Also, myeloid cells such as macrophages (p&amp;lt; 0.001, p&amp;lt; 0.001 and p&amp;lt; 0.001, respectively) and dendritic cells (p&amp;lt; 0.001, p&amp;lt; 0.001 and p&amp;lt; 0.001, respectively) were highly infiltrated with EMT score high tumors. Result of ssGSEA of single cell sequence cohorts revealed that cancer associated fibroblasts demonstrated highest EMT scores compared with the other cell types such as cancer cells, T-cells, B-cells, or myeloid cells. In other words, EMT score of a bulk tumor may reflect the signature from fibroblasts rather than cancer cells. Conclusion: We found that cancer associated fibroblasts rather than cancer cells are the major source of the transcriptomic signatures of EMT in the bulk tumor, which cautions us to be careful with the interpretation of the results of EMT signature from a bulk tumor. Citation Format: Yoshihisa Tokumaru, Rongrong Wu, Junko Ukai, Masanori Oshi, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. Transcriptomic signature score of Epithelial-Mesenchymal Transition (EMT) of a bulk tumor may not reflect that of cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-03.

Abstract P2-19-02: Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors

Abstract Background: Epithelial-mesenchymal transition (EMT) phenotype is a complex process and plays a central role in tumor progression, aggression, invasion, metastasis, and resistance to therapy. Role of androgen receptor (AR) and androgens in inducing EMT has been well established in experimental systems of prostate cancer. AR is widely expressed in all subtypes of breast cancer (BC) and is considered to have context dependent role based on the steroid hormone microenvironment. Previous studies have shown AR as the only sex steroid receptor detectable in BC metastases and in metastatic lesion. We investigated the role of androgens in driving the EMT phenotype and association of AR induced genes with EMT in BC tumors using public dataset. Methods: We treated AR positive breast cancer cell line MDA-MB-453 with the androgen, dihydrotestosterone (DHT) followed by AR antagonist bicalutamide (Bic) and examined the expression levels of AR induced genes by quantitative real time PCR (qPCR) and AR induced protein GCDFP-15 by western blot. AR driven genes (SEC14L2, C1orf116, FKBP5, UGT2B11, UGT2B28, KCNMA1, PIP and ABCC11) were identified by bioinformatic pipeline using a systematic bioinformatic approach from public microarray data sets derived from AR positive breast cancer cell lines (S Rajarajan et al, SABCS 2021). Mesenchymal phenotype of the cell lines under DHT/Bic was evaluated morphologically and expression levels of EMT markers (ZEB1, Slug, Vimentin) and E-cadherin was estimated by qPCR, western blot and immunofluorescence. Migratory potential of the cell line under treatment was evaluated by wound healing assay and proliferation was examined by the MTT assay. Association of AR induced genes and levels of enzymes involved in intracrinal testosterone metabolism (SRD5A1 &amp; SRD5A3) with EMT in breast tumors was evaluated using transcript data from METABRIC database. EMT scores were derived from previous publications (Byers et al, 2013, Mak et al, 2016) and correlation was tested with AR induced genes. Results: The MDAMB-453 cell line was treated with 10nM DHT for 72hrs and this increased the expression of AR downstream protein GCDFP-15 by 31.47% (p=0.022) when compared to the control. Further, a significant increase in the expression levels of AR driven genes with a fold change &amp;gt;2 was observed in the DHT treated cells (p&amp;lt; 0.05). The expression of GCDP15 protein (p=0.028) and the AR downstream genes (p&amp;lt; 0.05) were significantly repressed upon treatment with bicalutamide. DHT treatment also showed a significant higher expression of EMT master regulator, SLUG (fold change&amp;gt;3, p=0.045) and a wound healing assay showed a 22% increase in migration (p=0.027). The expression of SLUG was significantly repressed (p=0.003) and the migratory ability of the DHT treated cells reduced upon treatment with bicalutamide (p=0.145). We also observed a significant loss of E-cadherin protein (by 23.65%; p=0.024) with DHT treatment and this was completely reversed upon treatment with bicalutamide (p=0.047). No significant change in proliferation was observed among the different treatment conditions. A significant positive correlation was observed between AR induced genes and the EMT scores (p&amp;lt; 0.05). Expression level of SRD5A3 was also positively correlated with EMT score (p=0.043) and AR induced genes (p&amp;lt; 0.05), except SEC14L2. Conclusion: Androgen receptor plays an important role in the biology of breast cancer and is considered a useful marker for prognosis. However, antiandrogen therapies haven’t seen the expected success in clinical trial settings. Our results support the controversial role played by AR in higher androgenic environment within breast cancer and warrant the consideration of the intratumoral levels of sex steroid hormones. Citation Format: Savitha Rajarajan, Snijesh VP, Madhumathy G Nair, Apoorva D, Chandrakala M, Vidya P Nimbalkar, Maalavika Pillai, Mohit Kumar Jolly, Jyothi S Prabhu. Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-02.

Expression of EMT-related genes in lymph node metastasis in endometrial cancer: a TCGA-based study

BackgroundEndometrial cancer (EC) with metastasis in pelvic/para-aortic lymph nodes suggests an unsatisfactory prognosis. Nevertheless, there is still rare literature focusing on the role of epithelial-mesenchymal transition (EMT) in lymph node metastasis (LNM) in EC.MethodsTranscriptional data were derived from the TCGA database. Patients with stage IA–IIIC2 EC were included, constituting the LN-positive and LN-negative groups. To evaluate the extent of EMT, an EMT signature composed of 315 genes was adopted. The EMT-related genes (ERGs) were obtained from the dbEMT2 database, and the differentially expressed ERGs (DEERGs) between these two groups were screened. On the basis of DEERGs, pathway analysis was carried out. We eventually adopted the logistic regression model to build an ERG-based gene signature with predictive value for LNM in EC.ResultsA total of 498 patients were included, with 75 in the LN-positive group. Median EMT score of tumor tissues from LN-negative group was − 0.369, while that from the LN-positive group was − 0.296 (P < 0.001), which clearly exhibited a more mesenchymal phenotype for LNM cases on the EMT continuum. By comparing expression profiles, 266 genes were identified as DEERGs, in which 184 were upregulated and 82 were downregulated. In pathway analysis, various EMT-related pathways were enriched. DEERGs shared between molecular subtypes were comparatively few. The ROC curve and logistic regression analysis screened 7 genes with the best performance to distinguish between the LN-positive and LN-negative group, i.e., CIRBP, DDR1, F2RL2, HOXA10, PPARGC1A, SEMA3E, and TGFB1. A logistic regression model including the 7-gene-based risk score, age, grade, myometrial invasion, and histological subtype was built, with an AUC of 0.850 and a favorite calibration (P = 0.074). In the validation dataset composed of 83 EC patients, the model exhibited a satisfactory predictive value and was well-calibrated (P = 0.42).ConclusionThe EMT status and expression of ERGs varied in LNM and non-LNM EC tissues, involving multiple EMT-related signaling pathways. Aside from that, the distribution of DEERGs differed among molecular subtypes. An ERG-based gene signature including 7 DEERGs exhibited a desirable predictive value for LNM in EC, which required further validation based upon clinical specimens in the future.

Interleukin-6 and Hypoxia Synergistically Promote EMT-Mediated Invasion in Epithelial Ovarian Cancer via the IL-6/STAT3/HIF-1α Feedback Loop.

Extensive peritoneal spread and capacity for distant metastasis account for the majority of mortality from epithelial ovarian cancer (EOC). Accumulating evidence shows that interleukin-6 (IL-6) promotes tumor invasion and migration in EOC, although the molecular mechanisms remain to be fully elucidated. Meanwhile, the hypoxic microenvironment has been recognized to cause metastasis by triggering epithelial-mesenchymal transition (EMT) in several types of cancers. Here, we studied the synergy between IL-6 and hypoxia in inducing EMT in two EOC cell lines, A2780 cells and SKOV3 cells. Exogenous recombination of IL-6 and autocrine production of IL-6 regulated by plasmids both induced EMT phenotype in EOC cells characterized by downregulated E-cadherin as well as upregulated expression of vimentin and EMT-related transcription factors. The combined effects of IL-6 and hypoxia were more significant than those of either one treatment on EMT. Suppression of hypoxia-inducible factor-1α (HIF-1α) before IL-6 treatment inhibited the EMT phenotype and invasion ability of EOC cells, indicating that HIF-1α occupies a key position in the regulatory pathway of EMT associated with IL-6. EMT score was found positively correlated with mRNA levels of IL-6, signal transducer and activator of transcription 3 (STAT3), and HIF-1α, respectively, in 489 ovarian samples from The Cancer Genome Atlas dataset. Next, blockade of the abovementioned molecules by chemical inhibitors reversed the alteration in the protein levels of EMT markers induced by either exogenous or endogenous IL-6. These findings indicate a positive feedback loop between IL-6 and HIF-1α, and induce and maintain EMT phenotype through STAT3 signaling, which might provide a novel rationale for prognostic prediction and therapeutic targets in EOC.

Immune micro-environment and drug analysis of peritoneal endometriosis based on epithelial-mesenchymal transition classification.

Epithelial-mesenchymal transition (EMT) is a complex event that drives polar epithelial cells transform from adherent cells to motile mesenchymal cells, in which are involved immune cells and stroma cells. EMT plays crucial roles in migration and invasion of endometriosis. The interaction of endometrial implants with the surrounding peritoneal micro-environment probably affects the development of peritoneal endometriosis. To date, very few studies have been carried out on peritoneal endometriosis sub-type classification and micro-environment analysis based on EMT. The purpose of this study is to investigate the potential application of EMT-based classification in precise diagnosis and treatment of peritoneal endometriosis. Based on EMT hallmark genes, 76 peritoneal endometriosis samples were classified into two clusters by consistent cluster classification. EMT scores, which calculated by Z score of 8 epithelial cell marker genes and 8 mesenchymal cell marker genes, were compared in two clusters. Then, immune scores and the abundances of corresponding immune cells, stroma scores and the abundances of corresponding stroma cells were analyzed by the "xCell" package. Futhermore, a diagnostic model was constructed based on 9 diagnostic markers which related to immune score and stroma score by Lasso-Logistic regression analysis. Finally, based on EMT classification, a total of 8 targeted drugs against two clusters were screened out by drug susceptibility analysis via "pRRophetic" package. Hallmark epithelial-mesenchymal transition was the mainly enriched pathway of differentially expressed genes between peritoneal endometriosis tissues and endometrium tissues. Compared with cluster 2, EMT score and the abundances of most infiltrating stroma cell were significantly higher, while the abundances of most infiltrating immune cells were dramatically less. The diagnostic model could accurately distinguish cluster 1 from cluster 2. Pathway analysis showed drug candidates targeting cluster 1 mainly act on the IGF-1 signaling pathway, and drug candidates targeting cluster 2 mainly block the EGFR signaling pathway. In peritoneal endometriosis, EMT was probably promoted by stroma cell infiltration and inhibited by immune cell infiltration. Besides, our study highlighted the potential uses of the EMT classification in the precise diagnosis and treatment of peritoneal endometriosis.

Triple blockade of Ido-1, PD-L1 and MEK as a potential therapeutic strategy in NSCLC

BackgroundDespite the recent progress in the treatment and outcome of Non Small Cell Lung Cancer (NSCLC), immunotherapy has still significant limitations reporting a significant proportion of patients not benefiting from therapy, even in patients with high PD-L1 expression. We have previously demonstrated that the combined inhibition of MEK and PD-L1 in NSCLC patients derived three dimensional cultures exerted significant synergistic effect in terms of immune-dependent cancer cell death. However, subsequent experiments analyzing the expression of Indoleamine 2,3-dioxygenase-1 (Ido-1) gene expression demonstrated that Ido-1 resulted unaffected by the MEK inhibition and even increased after the combined inhibition of MEK and PD-L1 thus representing a potential escape mechanism to this combination.MethodsWe analyzed transcriptomic profile of NSCLC lung adenocarcinoma cohort of TCGA (The Cancer Genome Atlas), stratifying tumors based on EMT (Epithelial mesenchymal Transition) score; in parallel, we investigated the activation of Ido-1 pathway and modulation of immune cytokines productions both in NSCLC cells lines, in peripheral blood mononuclear cells (PBMCs) and in ex-vivo NSCLC spheroids induced by triple inhibition with an anti-PD-L1 monoclonal antibody, the MEK inhibitor and the Ido-1 inhibitor.ResultsIn NSCLC lung adenocarcinoma patient cohort (from TCGA) Ido-1 gene expression was significantly higher in samples classified as mesenchymal according EMT score. Similarly, on a selected panel of NSCLC cell lines higher expression of MEK and Ido-1 related genes was detected in cells with mesenchymal phenotype according EMT score, thus suggesting a potential correlation of co-activation of these two pathways in the context of EMT, with cancer cells sustaining an immune-suppressive microenvironment. While exerting an antitumor activity, the dual blockade of MEK and PD-L1 enhances the secretion of pro-inflammatory cytokines (IFNγ, TNFα, IL-12 and IL-6) and, consequently, the expression of new immune checkpoints such as Ido-1. The triple inhibition with an anti-PD-L1 monoclonal antibody, the MEK inhibitor and the Ido-1 inhibitor demonstrated significant antiproliferative and proapoptotic activity on ex-vivo NSCLC samples; at the same time the triple combination kept increased the levels of pro-inflammatory cytokines produced by both PBMCs and tumor spheroids in order to sustain the immune response and simultaneously decreased the expression of other checkpoint (such as CTLA-4, Ido-1 and TIM-3) thus promoting an immune-reactive and inflamed micro-environment.ConclusionsWe show that Ido-1 activation is a possible escape mechanism to immune-mediated cell death induced by combination of PD-L1 and MEK inhibitors: also, we show that triple combination of anti-PD-L1, anti-MEK and anti-Ido-1 drugs may overcome this negative feedback and restore anti-tumor immune response in NSCLC patients’ derived three dimensional cultures.

TRPV1 Is a Potential Tumor Suppressor for Its Negative Association with Tumor Proliferation and Positive Association with Antitumor Immune Responses in Pan-Cancer.

Background Although numerous studies have shown that the expression and activation of TRPV1 have an important role in cancer development, a comprehensive exploration of associations between TRPV1 expression and tumor proliferation, microenvironment, and clinical outcomes in pan-cancer remains insufficient. Methods From The Cancer Genome Atlas (TCGA) program, we downloaded multiomics data of ten cancer cohorts and investigated the correlations between TRPV1 expression and immune signatures' enrichment, stromal content, genomic features, oncogenic signaling, and clinical features in these cancer cohorts and pan-cancer. Results Elevated expression of TRPV1 correlated with better clinical outcomes in pan-cancer and diverse cancer types. In multiple cancer types, TRPV1 expression correlated negatively with the expression of tumor proliferation marker genes (MKI67 and RACGAP1), proliferation scores, cell cycle scores, stemness scores, epithelial-mesenchymal transition scores, oncogenic pathways' enrichment, tumor immunosuppressive signals, intratumor heterogeneity, homologous recombination deficiency, tumor mutation burden, and stromal content. Moreover, TRPV1 expression was downregulated in late-stage versus early-stage tumors. In breast cancer, bladder cancer, and low-grade glioma, TRPV1 expression was more inferior in invasive than in noninvasive subtypes. Pathway analysis showed that the enrichment of cancer-associated pathways correlated inversely with TRPV1 expression levels. Conclusion TRPV1 upregulation correlates with decreased tumor proliferation, tumor driver gene expression, genomic instability, and tumor immunosuppressive signals in various cancers. Our results provide new understanding of the role of TRPV1 in both cancer biology and clinical practice.

Abstract 80: Genomic characterization of PDX models from rare cancer patients in the NCI Patient-Derived Models Repository

Abstract Background: The National Cancer Institute’s Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) has developed a large number of patient-derived xenograft (PDX) models from a diverse set of rare cancers. These models have been genomically characterized using whole-exome sequencing (WES) and RNAseq. The resource provides a unique opportunity to explore the genomic features of rare tumor models in NCI PDMR and to understand the oncogenic processes in pre-clinical models to identify biomarkers associated with therapeutic responses. Methods: Genomic characterization was done in 4-6 PDX samples across multiple passages and lineages from each model. As the samples exhibited a high level of genomic stability within each model, consensus mutation and copy number variation (CNV), microsatellite instability (MSI), genomic loss of heterozygosity (LOH), homologous recombination deficiency score (scarHRD), and mutational signature data were generated from WES. Fusions were identified from RNASeq data using Star-Fusion and FusionInspector. Gene set enrichment analysis was conducted from the gene expression data obtained from RNAseq. Results: 1) 233 PDX models have been developed and characterized from more than 45 different rare malignancies. Most frequent cancer types are different sarcomas (n=63), head &amp; neck squamous cell carcinoma (n=61), and malignant fibrous histiocytoma (MFH) (n=11); 2) TP53 was the most frequently altered gene, mutated in 51% of models, followed by NOTCH1 (16%) and PIK3CA (11%). In terms of CNVs, ovarian epithelial cancer (OVT) showed relatively high chromosomal instability, while uterine endometrioid carcinoma (UEC) and synovial sarcoma (SYNS) had low instability; 3) MSI-H was observed in only 7 models. Esophageal adenocarcinoma (ESCA), OVT, and cervical squamous cell carcinoma (CESC) had high scarHRD and genomic LOH scores, while both scores were low in UEC and anal squamous cell carcinoma (ANSC). COSMIC v2 mutational signature 3 is significantly associated with a high scarHRD score (p-value &amp;lt; 0.01, Wilcoxon rank-sum test); 4) Characteristic fusions were observed in certain sarcoma models: SS18-SSX1 and ASPSCR1-TFE3 fusions were observed in SYNS and alveolar soft part sarcoma (ASPS) models respectively. EWSR1-FLI1 fusion was present in 2 out of 3 Ewing sarcoma (ES) models. 5) Gene set enrichment analysis from RNASeq data showed that epithelial-mesenchymal transition score could accurately distinguish carcinoma from sarcoma models, confirming the divergent gene expression programs. Conclusion: Comprehensive genomic characterization of NCI PDMR models generated from rare cancers solves an unmet need in the community. It will serve as a valuable resource for translational researchers interested in pre-clinical drug development and discovery. Citation Format: Li Chen, Rini Pauly, Ting-Chia Chang, Biswajit Das, Yvonne A. Evrard, Chris A. Karlovich, Tomas Vilimas, Alyssa Chapman, Nikitha Nair, Luis Romero, Anna Lee Fong, Amanda Peach, Shahanawaz Jiwani, Nastaran Neishaboori, Lindsay Dutko, Kelly Benauer, Gloryvee Rivera, Erin Cantu, Corinne Camalier, Thomas Forbes, Michelle Gottholm-Ahalt, John Carter, Suzanne Borgel, Chelsea McGlynn, Candace Mallow, Emily Delaney, Tiffanie Miner, Michelle A. Eugeni, Dianne Newton, Melinda G. Hollingshead, P. Mickey Williams, James H. Doroshow. Genomic characterization of PDX models from rare cancer patients in the NCI Patient-Derived Models Repository [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 80.

MP47-04 VALIDATION OF A 22-GENE EPITHELIAL-MESENCHYMAL TRANSITION (EMT) PROGNOSTIC SIGNATURE IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 May 2022MP47-04 VALIDATION OF A 22-GENE EPITHELIAL-MESENCHYMAL TRANSITION (EMT) PROGNOSTIC SIGNATURE IN CLEAR CELL RENAL CELL CARCINOMA Brittney Cotta, Srinivas Nallandhighal, Judith Stangl-Kremser, Trinh Pham, Jeffrey Tosoian, Kathryn Marchetti, Joel Berends, Mahir Maruf, Scott Tomlins, Todd Morgan, Ganesh Palapattu, Eman Abdulfatah, Aaron Udager, and Simpa Salami Brittney CottaBrittney Cotta More articles by this author , Srinivas NallandhighalSrinivas Nallandhighal More articles by this author , Judith Stangl-KremserJudith Stangl-Kremser More articles by this author , Trinh PhamTrinh Pham More articles by this author , Jeffrey TosoianJeffrey Tosoian More articles by this author , Kathryn MarchettiKathryn Marchetti More articles by this author , Joel BerendsJoel Berends More articles by this author , Mahir MarufMahir Maruf More articles by this author , Scott TomlinsScott Tomlins More articles by this author , Todd MorganTodd Morgan More articles by this author , Ganesh PalapattuGanesh Palapattu More articles by this author , Eman AbdulfatahEman Abdulfatah More articles by this author , Aaron UdagerAaron Udager More articles by this author , and Simpa SalamiSimpa Salami More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002618.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Current predictors of metastatic progression after radical nephrectomy for localized clear cell renal cell carcinoma (ccRCC) include clinicopathologic features such as tumor stage and grade. The addition of molecular tumor characteristics in a prognostic score may improve risk stratification and patient selection for enhanced follow up strategies or adjuvant therapies. METHODS: We retrospectively identified consecutive patients with ccRCC who underwent radical nephrectomy (RNx) for localized disease. Those who developed metastasis were identified. Whole-transcriptome mRNA sequencing of primary tumors was performed followed by gene set enrichment analysis (GSEA) for the most significant cancer hallmark pathways enriched in patients who did or did not develop metastasis. For each patient, the 22-gene epithelial mesenchymal transition (EMT) score was calculated (high vs. low), using cut-offs from a prior study using TCGA data. The prognostic impact of the EMT score was evaluated by performing multivariable cox-proportional hazard testing and Kaplan-Meier (KM) survival analysis. RESULTS: We analyzed 82 patients with median age 62 years and median tumor size 6 ±2.9 cm. The median time to metastasis after radical nephrectomy for patients who developed metastasis (n=12) was 18.2 months and median follow up 31.1 months for patients who did not develop metastasis (n=70). We observed a significant enrichment of EMT, myogenesis, inflammatory response and hypoxia hallmark pathways in patients with metastasis vs. those without metastasis. Kaplan-Meier survival analysis showed a significant decrease in metastasis-free survival in patients with a high EMT score (p=0.018). Multivariable analysis controlling for relevant clinicopathologic features such as age, sex, tumor size, tumor stage etc. revealed high EMT score to be significantly associated with development of metastasis [hazard ratio (HR) 7.2; 95% CI 1.15-44.8; p=0.035]. CONCLUSIONS: We validated a prognostic 22-gene epithelial mesenchymal transition (EMT) score in patients treated with radical nephrectomy for localized ccRCC. Pending further studies, the EMT score may improve risk stratification and select patients for adjuvant therapy. Source of Funding: UM-Peking University Joint Institute, NCI T32 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e810 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Brittney Cotta More articles by this author Srinivas Nallandhighal More articles by this author Judith Stangl-Kremser More articles by this author Trinh Pham More articles by this author Jeffrey Tosoian More articles by this author Kathryn Marchetti More articles by this author Joel Berends More articles by this author Mahir Maruf More articles by this author Scott Tomlins More articles by this author Todd Morgan More articles by this author Ganesh Palapattu More articles by this author Eman Abdulfatah More articles by this author Aaron Udager More articles by this author Simpa Salami More articles by this author Expand All Advertisement PDF DownloadLoading ...

The clinical relevance of epithelial-mesenchymal transition and its correlations with tumorigenic immune infiltrates in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a cancer with extremely high mortality. Epithelial-mesenchymal transition (EMT) may play an important role in the occurrence, invasion and prognosis of HCC; however, its relationship with immunity in HCC has not yet been studied. Therefore, we investigated the diagnostic and prognostic values of EMT and explored its potential connections with tumorigenic immune infiltrates in HCC. We first proposed a quantitative metric of EMT activity, the EMT score. After applying this metric to 20 datasets from the Integrative Molecular Database of Hepatocellular Carcinoma, the Cancer Genome Atlas, and the Gene Expression Omnibus, we explored the ability of the EMT score to stratify across sample types. We then applied the EMT score for survival analysis and to differentiate patients with/without vascular invasion to test its prognostic value. We also collected and calculated data on the abundance of immune cells and immune cell markers in HCC and investigated their correlations with EMT scores. Finally, we synthesized and analyzed 20 datasets and constructed an EMT-gene-immune linkage network. The results showed higher EMT scores in HCC samples than in cirrhotic and normal livers. The cases with higher EMT scores also showed poorer performance in terms of prognostic factors such as vascular invasion and overall survival time. Our research demonstrated a broad correlation between EMT and the tumor immune microenvironment, and we uncovered multiple potential linkers associated with both EMT and immunity. Studying EMT has clinical relevance and high diagnostic and prognostic value for HCC.