EMT-associated Transcription Factors Research Articles

Abstract 6977: ETV6 is an unexplored partner of the Chr.8 q24.3 amplicon-embedded immortalizing oncogene TONSL in tumorigenesis

Abstract Approximately 20% of breast cancers exhibit recurring genomic amplification involving chromosome 8q24.3. In our previous study, we identified the Tonsoku-like DNA repair protein (TONSL) within this amplicon as an immortalizing oncogene of breast epithelial cells, that can promote Estrogen Receptor-positive (ER+) breast adenocarcinomas when combined with other breast cancer-associated oncogenes. Our findings indicated that TONSL-amplified breast cancer cells are dependent on TONSL for tumor growth and exhibited deregulated homologous recombination and cell cycle pathways. High levels of TONSL in primary breast cancers, particularly ER+ breast cancers, were associated with poor outcomes. Although TONSL itself is not druggable, owing to its lack of enzymatic activity, molecules that disrupt the TONSL interactome can be developed. Towards this goal, we performed immunoprecipitation (IP) with TONSL antibody using protein lysates from TONSL-immortalized primary breast epithelial cells and subjected the immunoprecipitates to mass spectrometry. We identified several proteins selectively enriched with the TONSL antibody, the most significant of which was ETS variant transcription factor 6 (ETV6). ETV6 is a modular protein consisting of a C-terminal DNA-binding ETS domain and N-terminal point (PNT) domain and is known to play a role as a transcriptional repressor during embryonic development and hematopoiesis. We confirmed these data by IP followed by western blot analysis of primary breast epithelial cells and TONSL amplified MDA-MB-436. Furthermore, we utilized ClusPro to model the TONSL-ETV6 interaction and predicted that the TONSL’s ankyrin repeats docks onto ETV6’s PNT in a favorable manner. To investigate the functional significance of this interaction, we evaluated ETV6-regulated genes using RNA-seq. Our analysis revealed that ETV6-regulated genes were downregulated following TONSL knockdown and vice versa. Most ETV6 target genes affected upon TONSL manipulation are involved in Epithelial-Mesenchymal Transition (EMT), particularly TWIST1, SNAI2 and ZEB1. Previous studies have demonstrated a role of ETV6 in limiting EMT by repressing the expression of EMT-associated transcription factors. Our findings revealed that TONSL overexpression in primary breast epithelial cells led to a significant increase in these EMT gene expression, whereas TONSL knockdown in MDA-MB-436 cells resulted in a moderate decrease. These findings suggest that TONSL binds to ETV6 and restricts its ability to bind to the regulatory regions of EMT-associated genes, leading to a transcriptional surge of EMT-associated genes leading to tumor progression. Further studies will contribute to our understanding of the role of the chr.8q24.3 amplicon, specifically the TONSL gene, in initiating/promoting breast tumorigenesis and the development of novel drugs that target the TONSL interactome. Citation Format: Aditi S. Khatpe, Emma Doud, Amber Mosely, Kathy D. Miller, Harikrishna Nakshatri. ETV6 is an unexplored partner of the Chr.8 q24.3 amplicon-embedded immortalizing oncogene TONSL in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6977.

The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma.

Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through nonmutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated. Here, we evaluated the corepressor for element 1-silencing transcription factor (CoREST) epigenetic repressor complex and the recently developed bivalent inhibitor corin within the context of melanoma phenotype plasticity and therapeutic resistance. We found that CoREST was a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells led to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin revealed specific effects on histone marks connected to epithelial-mesenchymal transition-associated (EMT-associated) transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor-resistant (BRAFi-R) melanomas with corin promoted resensitization to BRAFi therapy. DUSP1 was consistently downregulated in BRAFi-R melanomas, which was reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity was recapitulated by the p38 MAPK inhibitor BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial for patients with BRAFi-resistant melanoma.

Identification of a core EMT signature that separates basal-like breast cancers into partial- and post-EMT subtypes.

Epithelial-mesenchymal transition (EMT) is a cellular plasticity program critical for embryonic development and tissue regeneration, and aberrant EMT is associated with disease including cancer. The high degree of plasticity in the mammary epithelium is reflected in extensive heterogeneity among breast cancers. Here, we have analyzed RNA-sequencing data from three different mammary epithelial cell line-derived EMT models and identified a robust mammary EMT gene expression signature that separates breast cancers into distinct subgroups. Most strikingly, the basal-like breast cancers form two subgroups displaying partial-EMT and post-EMT gene expression patterns. We present evidence that key EMT-associated transcription factors play distinct roles at different stages of EMT in mammary epithelial cells.

Downregulation of EZH2 inhibits epithelial-mesenchymal transition in enzalutamide-resistant prostate cancer.

Androgen signaling inhibitors (ASI) have been approved for treatment of metastatic castration-resistant prostate cancer (mCRPC). However, the limited success of ASI in clinic justifies an urgent need to identify new targets and develop novel approaches for treatment. EZH2 significantly increases in prostate cancer (PCa). Little is understood, however, regarding the roles of EZH2 in Enzalutamide-resistant (EnzR) mCRPC. We firstly investigated the levels of EZH2 and the altered pathways in public database which was comprised with primary and metastatic PCa patient tumors. To elucidate the roles of EZH2 in mCRPC, we manipulated EZH2 in EnzR PCa cell lines to examine epithelial-mesenchymal transition (EMT). To dissect the underlying mechanisms, we measured the transcription levels of EMT-associated transcription factors (TFs). We found that EZH2 was highly expressed in mCRPC than that of primary PCa tumors and that EnzR PCa cells gained more EMT characteristics than those of enzalutamide-sensitive counterparts. Further, loss of EZH2-induced inhibition of EMT is independent of polycomb repressive complex 2 (PRC2). Mechanistically, downregulation of EZH2 inhibits transcription of EMT-associated TFs by repressing formation of H3K4me3 to the promotor regions of the TFs. We identified the novel roles of EZH2 in EnzR mCRPC. EnzR PCa gains more EMT properties than that of enzalutamide-sensitive PCa. Loss of EZH2-assocaited inhibition of EMT is PRC2 independent. Downregulation of EZH2 suppresses EMT by impairing formation of H3K4me3 at the promotor regions, thus repressing expression of EMT-associated TFs.

LncRNA SNHG5 Suppresses Cell Migration and Invasion of Human Lung Adenocarcinoma via Regulation of Epithelial-Mesenchymal Transition.

Long noncoding RNAs (lncRNAs) are gradually being annotated as important regulators of multiple cellular processes. The goal of our study was to investigate the effects of the lncRNA small nucleolar RNA host gene 5 (SNHG5) in lung adenocarcinoma (LAD) and its underlying mechanisms. The findings revealed a substantial drop in SNHG5 expression in LAD tissues, which correlated with clinical-pathological parameters. Transcriptome sequencing analysis demonstrated that the inhibitory effect of SNHG5 was associated with cell adhesion molecules. Moreover, the expression of SNHG5 was shown to be correlated with epithelial-mesenchymal transition (EMT) markers in western blots and immunofluorescence. SNHG5 also had significant effects of antimigration and anti-invasion on LAD cells in vitro. Furthermore, the migration and invasion of A549 cells were suppressed by overexpressed SNHG5 in the EMT progress induced by transforming growth factor β1 (TGF-β1), and this might be due to the inhibition of the expression of EMT-associated transcription factors involving Snail, SLUG, and ZEB1. In LAD tissues, the expression of SNHG5 exhibited a positive association with E-cadherin protein expression but a negative correlation with N-cadherin and vimentin, according to the results of quantitative real-time PCR (qRT-PCR). In summary, the current work demonstrated that the lncRNA SNHG5 might limit cell migration and invasion of LAD cancer via decreasing the EMT process, indicating that SNHG5 might be used as a target for LAD therapeutic methods.

SIRT2 promotes the viability, invasion and metastasis of osteosarcoma cells by inhibiting the degradation of Snail

Osteosarcomas (OS) are highly metastatic and usually lead to poor outcomes. Epithelial-mesenchymal transition (EMT) is reported to be a critical event in metastasis. SIRT2 exerts dual functions in many different tumors. However, the underlying molecular mechanisms of SIRT2 in osteosarcoma cell metastasis and the question of whether SIRT2 regulates EMT have not been fully explored. In this study, we confirmed that SIRT2 was highly-expressed in human osteosarcoma MG63 and Saos-2 cell lines. The viability, migration and invasion of osteosarcoma cells were inhibited by knockdown of SIRT2 and were enhanced by overexpression of SIRT2. Moreover, SIRT2 positively regulated EMT and upregulated the protein levels of the mesenchymal markers N-cadherin and Vimentin and the levels of MMP2 and MMP9. A xenograft mouse model showed that SIRT2 knockdown in osteosarcoma cells led to reduced tumor growth, decreased expression of mesenchymal markers and impaired lung and liver metastasis in vivo. Furthermore, we showed that SIRT2 interacted with and upregulated the protein level of the EMT-associated transcription factor Snail. SIRT2 inhibited Snail degradation via its deacetylase activity. Knockdown of Snail abrogated the promoting effects of SIRT2 on migration and invasion of osteosarcoma cells. In conclusion, SIRT2 plays a crucial role in osteosarcoma metastasis by inhibiting Snail degradation and may serve as a novel therapeutic target to manage osteosarcoma.

A Hybrid Epithelial to Mesenchymal Transition in Ex Vivo Cutaneous Squamous Cell Carcinoma Tissues.

While most cases of cutaneous squamous cell carcinoma (cSCC) are benign, invasive cSCC is associated with higher mortality and is often more difficult to treat. As such, understanding the factors that influence the progression of cSCC are important. Aggressive cancers metastasize through a series of evolutionary changes, collectively called the epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells transition to a highly mobile mesenchymal cell type with metastatic capacities. While changes in expression of TGF-β, ZEB1, SNAI1, MMPs, vimentin, and E-cadherin are hallmarks of an EMT process occurring within cancer cells, including cSCC cells, EMT within tissues is not an “all or none” process. Using patient-derived cSCC and adjacent normal tissues, we show that cells within individual cSCC tumors are undergoing a hybrid EMT process, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. Interestingly, cells along the outer edges of a tumor mass exhibit a more mesenchymal phenotype, with reduced E-cadherin, β-catenin, and cytokeratin expression and increased vimentin expression. Conversely, cells in the center of a tumor mass retain a higher expression of the epithelial markers E-cadherin and cytokeratin and little to no expression of vimentin, a mesenchymal marker. We also detected inverse expression changes in the miR-200 family and the EMT-associated transcription factors ZEB1 and SNAI1, suggesting that cSCC EMT dynamics are regulated in a miRNA-dependent manner. These novel findings in cSCC tumors provide evidence of phenotypic plasticity of the EMT process occurring within patient tissues, and extend the characterization of a hybrid EMT program occurring within a tumor mass. This hybrid EMT program may be promoting both survival and invasiveness of the tumors. A better understanding of this hybrid EMT process may influence therapeutic strategies in more invasive disease.

Abstract LB022: A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues

Abstract While most cases of cutaneous squamous cell carcinoma (cSCC) are benign, invasive cSCC is associated with higher mortality and is often more difficult to treat. As such, understanding the factors that influence the progression of cSCC are important. Aggressive cancers metastasize through a series of evolutionary changes, collectively called the epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells transition to a highly mobile mesenchymal cell type with metastatic capacities. While changes in expression of TGF-β, vimentin, and E-cadherin are hallmarks, EMT is not an “all or none” process. Using patient-derived cSCC and adjacent normal tissues (ANT), we observed a significant increase in mRNA expression of TGF-β (p<0.05), TGF-βR2 (p<0.001), and E-cadherin (p<0.005) in cSCC tissues by qPCR. Immunoblots performed on homogenized tissues yielded significant and parallel increases in protein expression of TGF-β (p<0.05) and E-cadherin (p<0.05). We also observed increased expression of the EMT-associated transcription factors Zeb1 and Snai1 in cSCC tissue sections using immunohistochemistry. Interestingly, we observed a significant increase in expression of vimentin (p<0.01) by immunoblot and differential expression of four oncogenic and EMT-associated miRNAs; miR-31 expression was significantly higher in cSCC tissues (p<0.001), while we observed a significant decrease in expression of miR-34a (p<0.05), miR-200b, and miR-200c in cSCC tissues. Examination of ANT and cSCC tissue sections using immunofluorescence and immunohistochemistry showed differential, and reverse, expression of E-cadherin and vimentin among cells within an individual tumor. Collectively, our data support a hypothesis that cSCC tumors are undergoing a hybrid EMT program, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. This hybrid EMT program may be promoting both survival and invasiveness of the tumors. A better understanding of this hybrid EMT process may influence therapeutic strategies in more invasive disease. Citation Format: Christopher S. Pulford, Chandana K. Uppalapati, McKale R. Montgomery, Elizabeth E. Hull, Kathryn J. Leyva. A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues [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 LB022.

Role of EMT in the DNA damage response, double-strand break repair pathway choice and its implications in cancer treatment.

Numerous epithelial–mesenchymal transition (EMT) characteristics have now been demonstrated to participate in tumor development. Indeed, EMT is involved in invasion, acquisition of stem cell properties, and therapy‐associated resistance of cancer cells. Together, these mechanisms offer advantages in adapting to changes in the tumor microenvironment. However, recent findings have shown that EMT‐associated transcription factors (EMT‐TFs) may also be involved in DNA repair. A better understanding of the coordination between the DNA repair pathways and the role played by some EMT‐TFs in the DNA damage response (DDR) should pave the way for new treatments targeting tumor‐specific molecular vulnerabilities, which result in selective destruction of cancer cells. Here we review recent advances, providing novel insights into the role of EMT in the DDR and repair pathways, with a particular focus on the influence of EMT on cellular sensitivity to damage, as well as the implications of these relationships for improving the efficacy of cancer treatments.

Cooperative interaction between ER\u03b1 and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

The epithelial to mesenchymal transition (EMT) has been proposed to contribute to the metastatic spread of breast cancer cells. EMT-promoting transcription factors determine a continuum of different EMT states. In contrast, estrogen receptor α (ERα) helps to maintain the epithelial phenotype of breast cancer cells and its expression is crucial for effective endocrine therapies. Determining whether and how EMT-associated transcription factors such as ZEB1 modulate ERα signaling during early stages of EMT could promote the discovery of therapeutic approaches to suppress metastasis. Here we show that, shortly after induction of EMT and while cells are still epithelial, ZEB1 modulates ERα-mediated transcription induced by estrogen or cAMP signaling in breast cancer cells. Based on these findings and our ex vivo and xenograft results, we suggest that the functional interaction between ZEB1 and ERα may alter the tissue tropism of metastatic breast cancer cells towards bone.

Role of various non-coding RNAs in EMT, cancer, and metastasis: Recent trends and future perspective

Epithelial-to-mesenchymal transition (EMT) is a key cellular process involved in the various biological processes ranging from embryonic development, wound healing, and cancer metastasis. EMT is the key player in cancer progression to a metastatic state. The transformed tumor cells acquire the migratory property and invade surrounding tissues, which ultimately leads to cancer metastasis via EMT. Epithelial cells undergo certain phenotypic changes to acquire mesenchymal status. At a molecular level, this transition process is elicited by several signaling cascades that include cytokines and a group of transcription factors like Twist, snail, and Zeb1/2. Emerging evidence suggests that various non-coding RNAs (ncRNAs) play a significant role in modulating EMT-associated transcription factors (EMT-TFs) and molecular signaling at the transcriptional and post-transcriptional level and thus, regulating the EMT process. Various noncoding-RNAs like miRNAs, piRNAs, tsRNAs, lncRNAs, T-UCRs, CircRNAs, eRNAs, snoRNAs can either promote or can inhibit the process of EMT and thus modulates cancer progression. Here we review the recent research advances in delineating the role of various non-coding RNAs in the EMT, cancer, and metastasis process as well as their potential roles as biomarkers and therapeutic targets.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

CCR9 initiates epithelial\u2013mesenchymal transition by activating Wnt/\u03b2-catenin pathways to promote osteosarcoma metastasis

BackgroundOsteosarcoma (OS) patients with lung metastasis have poor prognoses, and effective therapeutic strategies for delaying or inhibiting the spread of lung metastasis from the primary OS site are lacking. Hence, it is critical to elucidate the underlying mechanisms of OS metastasis and to identify additional new effective treatment strategies for patients.MethodsDifferential expression and functional analyses were performed to identify key genes and relevant signaling pathways associated with OS lung metastasis. The expression of CCR9 in OS cell lines and tissues was measured by RT-qPCR, western blotting and immunohistochemistry. Cell migration and invasion were assessed by wound healing and Transwell Matrigel invasion assays, respectively. The regulatory relationship between CCR9 and the Wnt/β-catenin signaling pathway was further evaluated by rescue experiments.ResultsThe expression of CCR9 was elevated in OS cell lines and patients with lung metastasis. CCR9 promoted MG63 and HOS cell migration and invasion by activating the Wnt/β-catenin signaling pathway. Furthermore, knockdown of CCR9 repressed epithelial–mesenchymal transition (EMT) by downregulating mesenchymal markers (N-cadherin and Vimentin) and EMT-associated transcription factors (twist and snail) and upregulating an epithelial marker (E-cadherin).ConclusionsOur findings suggest that CCR9 promotes EMT by activating Wnt/β-catenin pathways to promote OS metastasis. CCR9 may be a promising therapeutic target to inhibit lung metastasis and serve as a novel prognostic marker for OS.

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.

MiR-144-3p inhibited the growth, metastasis and epithelial-mesenchymal transition of colorectal adenocarcinoma by targeting ZEB1/2.

miR-144-3p is aberrantly expressed in several types of human cancer and functions as a tumor suppressor by inhibiting metastasis. However, the clinical significance and biological function of miR-144-3p in colorectal adenocarcinoma (CRA) have yet to be elucidated. Here we reported that miR-144-3p expression level was significantly down-regulated in CRA tissues compared with matched noncancerous colorectal mucosae tissues. Low miR-144-3p expression was correlated with adverse clinicopathologic characteristics and poor prognosis of CRA patients. Cox regression analysis showed that low miR-144-3p expression was an independent risk factor for DFS and OS in CRA. In vitro and in vivo assays showed that miR-144-3p significantly inhibited proliferation, migration and invasion of CRA cells. In particular, miR-144-3p could suppress EMT process of CRA cells by regulating the cytoskeleton and EMT markers. Bioinformatics analysis indicated that EMT associated transcription factors ZEB1 and ZEB2 were potential targets of miR-144-3p, and miR-144-3p inhibited ZEB1 and ZEB2 expression and was negatively correlated with their expression in CRA. Finally, we confirmed that ZEB1 and ZEB2 down-regulation collaboratively mediated the inhibitory effect of miR-144-3p on proliferation, invasion and EMT of CRA cells. In conclusion, our study provided evidence that miR-144-3p could inhibit CRA cell proliferation, invasion and EMT by targeting ZEB1/2.

Abstract 2851: Trop-2 inactivates E-cadherin for metastatic diffusion in the absence of EMT

Abstract Trop-2 is a type I transmembrane glycoprotein, which is overexpressed in the majority of carcinomas, where it drives tumor cell proliferation. In its mature, glycosylated/functionally-competent form, Trop-2 associates with worse prognosis. A global quest was conducted for identifying decisive drivers of cancer metastasis. Cancer cell spheroids, wound healing and cell aggregation assays were utilized to assess cell-cell adhesion capacity. Immunofluorescence confocal microscopy and flow cytometry analysis were utilized to quantify expression of target signaling proteins, IHC analysis quantified the expression of target molecules in primary tumors and metastases. Pre-clinical models of orthotopic growth of colon cancer and metastatic diffusion to the liver were utilized. Xenotransplant transcriptome profiling analyzed transcription of EMT-related determinants. This led us to identify Trop-2 as a unique upregulated gene in colon cancer metastasis. Trop-2 was here shown to bind highly-expressed E-cadherin at the cell membrane. Trop-2 binding released E-cadherin from the cytoskeleton, inhibited cell-cell adhesion functions and activated signaling through β-catenin. This occurred in the absence of transcriptional downregulation of the CDH1 gene by EMT-associated transcription factors. The Trop-2/E-cadherin/β-catenin program was shown to lead to anti-apoptotic signaling, increased cell migration and enhanced cancer cell survival. In an orthotopic model of colon cancer, Trop-2 caused metastatic diffusion to the liver. The Trop-2-led E-cadherin-inactivation metastasis program was then found to induce metastatic relapse and shorter overall survival of colon cancer patients. Corresponding impact was indicated on breast, colon, ovary, uterus, stomach cancer metastatic diffusion, for broad impact on metastatic diffusion in human cancer. Acknowledgments:Support was provided by grants of Italian Ministry of Development (MI01_00424) and of University and Research (SCN_00558). M.T. was supported by the Programma Per Giovani Ricercatori “Rita Levi Montalcini” (Grant PGR12I7N1Z). Citation Format: Saverio Alberti, Emanuela Guerra, Valeria Relli, Rossano Lattanzio, Martina Ceci, Khouloud Boujnah, Valeria Garbo, Antonino Moschella, donato F. Altomare, Raffaella Depalo, Marco Trerotola. Trop-2 inactivates E-cadherin for metastatic diffusion in the absence of EMT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2851.

Role of epithelial-to-mesenchymal transition in the pulmonary fibrosis induced by paraquat in rats.

This study aims to explore the characteristics of the epithelial-to-mesenchymal transition (EMT) process and its underlying molecular mechanisms in the period of paraquat (PQ)-induced pulmonary fibrosis (PF). Picrosirius red staining and collagen volume fraction were utilized to evaluate the pathological changes of PQ-induced PF in rats. Immunohistochemistry, Western blot, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to measure the protein and gene expression of EMT markers, EMT-associated transcription factors, and regulators of EMT-related pathways, respectively. The collagen deposition in the alveolar septum and increased PF markers were characteristics of pathological changes in PQ-induced PF, reached a peak on day 14 after PQ poisoning, and then decreased on day 21. The protein and gene expression of the fibrosis marker, EMT markers, transcription factors, and regulators of EMT-related signaling pathways significantly increased at different time points after PQ poisoning compared with corresponding controls (P<0.05), and most of them reached a peak on day 14, followed by a decrease on day 21. The gene expression of EMT markers was significantly correlated with PF markers, transcription factors, and regulators of EMT-related signaling pathways (P<0.05). The mRNA expression of transcription factors was significantly correlated with that of TGF-β1 and Smad2 (P<0.05 or P<0.01), instead of Wnt2 and β-catenin (P>0.05). EMT process plays a role in the PQ-induced PF, in which most PF and EMT markers have a peak phenomenon, and its underlying molecular mechanisms might be determined by further studies.

Cytochalasin H isolated from mangrove-derived endophytic fungus inhibits epithelial-mesenchymal transition and cancer stemness via YAP/TAZ signaling pathway in non-small cell lung cancer cells.

Our previous studies have isolated cytochalasin H (CyH) from endophytic fungus derived from mangrove and found that CyH induced apoptosis and inhibited migration and angiogenesis in non-small cell lung cancer (NSCLC) cells. In this study, we further investigated the effect of CyH on epithelial-mesenchymal transition (EMT) and cancer stemness of A549 and NCI-H460 NSCLC cells and the underlying mechanisms, especially the role of YAP/ TAZ signaling pathway in the process. Our results showed that CyH significantly inhibited invasive ability and the sphere formation of NSCLC cells. The expression of E-cadherin, an EMT epithelial marker, was obviously up-regulated, while the expression of Vimentin and N-cadherin, the EMT mesenchymal markers, was dramatically down-regulated by CyH treatment in NSCLC cells. Moreover, the expression of EMT-associated transcription factors including Slug, Twist1, and Snail1 and stemness markers including Nanog, Sox-2, and Oct-4 was significantly down-regulated by CyH treatment in NSCLC cells. Additionally, CyH significantly down-regulated YAP and TAZ expression and up-regulated LAST1/2 and MST1/2 expression, and CyH inhibited the interaction between YAP and TEAD. Furthermore, YAP knockdown abolished the effect of CyH on the expression of EMT- and stemness-related markers in NSCLC cells. Taken together, these results suggest that CyH inhibits EMT and cancer stemness of NSCLC cells via the regulation of YAP/TAZ signaling pathway.

Monosomy 3 Influences Epithelial-Mesenchymal Transition Gene Expression in Uveal Melanoma Patients; Consequences for Liquid Biopsy.

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCβ4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.

Fibroblast growth factor 2 accelerates the epithelial\u2013mesenchymal transition in keratinocytes during wound healing process

In the wound healing process, the morphology of keratinocytes at the wound edge temporarily changes to a spindle morphology, which is thought to occur due to an epithelial–mesenchymal transition (EMT). Fibroblast growth factor (FGF) 2, also called basic FGF, has the potential to accelerate wound closure by activating vascular endothelial cells and fibroblasts. We examined the effects of FGF2 on keratinocyte morphology and EMT in wounded skin. Histological examination of murine wounds treated with FGF2 revealed that wound edge keratinocytes formed thickened and multilayered epithelia. In addition, we detected wound edge keratinocytes migrating individually toward the wound center. These migrating keratinocytes exhibited not only spindle morphology but also down-regulated E-cadherin and up-regulated vimentin expression, which is characteristic of EMT. In FGF2-treated wounds, a PCR array revealed the upregulation of genes related to EMT, including transforming growth factor (TGF) signaling. Further, FGF2-treated wound edge keratinocytes expressed EMT-associated transcription factors, including Snai2, and showed translocation of β-catenin from the cell membrane to the cytoplasm/nucleus. However, in vitro examination of keratinocytes revealed that FGF2 alone did not activate EMT in keratinocytes, but that FGF2 might promote EMT in combination with TGFβ1. These findings suggest that FGF2 treatment of wounds could promote keratinocyte EMT, accelerating wound closure.