Epithelial-mesenchymal Transition Transcription Factors Research Articles

Regulating the Expression of HIF-1α or lncRNA: Potential Directions for Cancer Therapy.

Previous studies have shown that tumors under a hypoxic environment can induce an important hypoxia-responsive element, hypoxia-induced factor-1α (HIF-1α), which can increase tumor migration, invasion, and metastatic ability by promoting epithelial-to-mesenchymal transition (EMT) in tumor cells. Currently, with the deeper knowledge of long noncoding RNAs (lncRNAs), more and more functions of lncRNAs have been discovered. HIF-1α can regulate hypoxia-responsive lncRNAs under hypoxic conditions, and changes in the expression level of lncRNAs can regulate the production of EMT transcription factors and signaling pathway transduction, thus promoting EMT progress. In conclusion, this review summarizes the regulation of the EMT process by HIF-1α and lncRNAs and discusses their relationship with tumorigenesis. Since HIF-1α plays an important role in tumor progression, we also summarize the current drugs that inhibit tumor progression by modulating HIF-1α.

An investigation of bacteriocin nisin anti-cancer effects and FZD7 protein interactions in liver cancer cells

The bacteriocin, nisin, produced by Lactococcus and Streptococcus species during fermentation, is widely used for bio preservatives in a wide variety of foods. Liver cancer has a high mortality rate and is the fourth leading cause of cancer-related deaths worldwide. Recently, researchers have shown the anti-cancer effects of nisin through in vitro and in vivo studies. This study aimed to investigate the effect of nisin on liver cancer cell lines, which represented two subgroups of the disease model. Nisin exhibited significant growth inhibition and apoptosis in both cell lines, HuH-7, and SNU182. Drug resistance is the main problem in liver cancer and the epithelial-to-mesenchymal transition has a role in the development of drug resistance in hepatocellular carcinoma. The expression of EMT transcription factors ZEB1, SNAI1, and TWIST1 were analyzed depending on nisin treatment, TWIST1 expression was down-regulated after nisin treatment compared to the untreated SNU182 and HuH-7 cell lines. Besides, due to the reported correlation between the overexpression of Frizzled (FZD) proteins, specifically FZD7, in primary hepatocellular carcinomas (HCCs), molecular docking was assessed for Nisin A in the binding site of FZD7. Results confirmed that Nisin A was able to form important hydrogen bonding with key residues. This research not only determined the role of nisin in different liver cancer cell models but it also provided the first result of FZD7 and nisin interaction.

Nrf2 Downregulation Contributes to Epithelial-to-Mesenchymal Transition in Helicobacter pylori-Infected Cells.

Simple SummaryGastric cancer is mainly linked to Helicobacter pylori infection. It is therefore important to decipher the mechanisms involved in H. pylori-induced gastric carcinogenesis, and especially the early events. We have previously demonstrated that the infection leads to an epithelial-to-mesenchymal transition (EMT) favoring gastric carcinogenesis. H. pylori infection is also associated with high levels of oxidative stress. In this work, we aimed at investigating the modulation of Nrf2, a major regulator of cellular antioxidant response to oxidative stress, upon infection with H. pylori and to decipher its implication in EMT. We demonstrated that H. pylori-induced Nrf2 downregulation may participate in gastric cells’ EMT, one crucial tumorigenic event in gastric cancer. These results could pave the way for new therapeutic strategies using Nrf2 modulators to reduce gastric carcinogenesis associated with H. pylori infection.Background: Gastric cancer, the fifth most common cancer worldwide, is mainly linked to Helicobacter pylori infection. H. pylori induces chronic inflammation of the gastric mucosa associated with high oxidative stress. Our study aimed at assessing the implication of Nrf2, a major regulator of cellular redox homeostasis, in H. pylori-induced gastric carcinogenesis. Methods: Using three different gastric epithelial cell lines, a non-cancerous (HFE-145) and two different subtypes of gastric cancer (AGS and MKN74), we analyzed the modulation of Nrf2 expression over time. After invalidation of Nrf2 by CRISPR-cas9, we assessed its role in H. pylori-induced epithelial-to-mesenchymal transition (EMT). Finally, we evaluated the expression of Nrf2 and ZEB1, a central EMT transcription factor, in human gastric tissues. Results: We first demonstrated that the Nrf2 signaling pathway is differentially regulated depending on the infection stage. Rapidly and transiently activated, Nrf2 was downregulated 24 h post-infection in a VacA-dependent manner. We then demonstrated that Nrf2 invalidation leads to increased EMT, which is even exacerbated after H. pylori infection. Finally, Nrf2 expression tended to decrease in human patients’ gastric mucosa infected with H. pylori. Conclusions: Our work supports the hypothesis that Nrf2 downregulation upon H. pylori infection participates in EMT, one of the most important events in gastric carcinogenesis.

Epithelial–Mesenchymal Transition by Synergy between Transforming Growth Factor-β and Growth Factors in Cancer Progression

Epithelial–mesenchymal transition (EMT) plays a crucial role in appropriate embryonic development, as well as wound healing, organ fibrosis, and cancer progression. During cancer progression, EMT is associated with the invasion, metastasis, and generation of circulating tumor cells and cancer stem cells, as well as resistance to chemo- and radiation therapy. EMT is induced by several transcription factors, known as EMT transcription factors (EMT-TFs). In nearly all cases, EMT-TFs appear to be regulated by growth factors or cytokines and extracellular matrix components. Among these factors, transforming growth factor (TGF)-β acts as the key mediator for EMT during physiological and pathological processes. TGF-β can initiate and maintain EMT by activating intracellular/intercellular signaling pathways and transcriptional factors. Recent studies have provided new insights into the molecular mechanisms underlying sustained EMT in aggressive cancer cells, EMT induced by TGF-β, and crosstalk between TGF-β and growth factors.

Molecular mechanism of CD163+ tumor-associated macrophage (TAM)-derived exosome-induced cisplatin resistance in ovarian cancer ascites

BackgroundStudies have found that tumor-associated macrophages (TAMs) in the malignant ascites of patients with serous ovarian cancer exhibit a mixed polarization phenotype and highly variable expression of the surface marker CD163. The exosomes secreted by mesenchymal cells can be taken up by tumor cells, affecting the malignant biological behavior of them.MethodsUsing reverse transcription-polymerase chain reaction (RT-PCR) to detect the genes expression of drug resistance related factors cancer stem cells (CSCs)/multidrug resistance (MDR)/epithelial-mesenchymal transition (EMT) after CD163+ TAMs exosomes in ovarian cancer ascites co-cultured with A2780 and A2780/cis-diamminedichloroplatinum (DDP). Differences of the level of miR-221-3p expression between CD163+ TAMs cells (M2) and peripheral blood mononuclear cells M0 detect by microarray screening, and bioinformatics analysis predicts that its target gene is ADAMTS6. Western blot (WB) and immunohistochemical detection of ADAMTS6 expression level in epithelial ovarian cancer (EOC) clinical sample tissues, and analysis of the ADAMTS6 expression in ovarian cancer tissues and its correlation with clinicopathological factors. WB was used to detect the expression of AKT pathway and downstream EMT-related molecules [SNAIL1, ZEB1, Vimentin (VIM)] after co-culture of CD163+ TAMs exosomes with ovarian cancer cell lines A2780, A2780/DDP. WB detects the expression of EGFR and TGF-β1 upstream molecules of the AKT signaling pathway (AKT-pAKT) and downstream EMT molecules (SNAIL1, ZEB1, VIM) after overexpression of ADAMTS6 in A2780 and A2780/DDP.ResultsWe demonstrated that after CD163+ TAM exosomes were taken up by EOC cells, the highly expressed miR-221-3p could downregulate the level of ADAMTS6, further activate the AKT signaling pathway, and increase the expression of EMT transcription factors SNAIL1 and ZEB1 and the mesothelial marker VIM. Decreased expression of the epithelial marker E-cadherin induced EMT, triggering a switch to a CSC-like phenotype and MDR, thereby promoting EOC cell proliferation, adhesion, migration, and resistance. Compared with benign ovarian tumors, ADAMTS6 expression was low in EOC tissues and was closely related to the clinical stage, age, and survival curve of ovarian cancer patients.ConclusionsOverexpression of ADAMTS6 reduced the IC50 of cisplatin in ovarian cancer cells. The mechanism may be related to the inhibition of EMT mediated by the EGFR/TGF-β/AKT pathway of EOC cells, which has potential value in the treatment of ovarian cancer.

Triple-negative breast cancer metastasis involves complex epithelial-mesenchymal transition dynamics and requires vimentin.

Triple-negative breast cancer (TNBC) is an aggressive subtype associated with early metastatic recurrence and worse patient outcomes. TNBC tumors express molecular markers of the epithelial-mesenchymal transition (EMT), but its requirement during spontaneous TNBC metastasis in vivo remains incompletely understood. We demonstrated that spontaneous TNBC tumors from a genetically engineered mouse model (GEMM), multiple patient-derived xenografts, and archival patient samples exhibited large populations in vivo of hybrid E/M cells that lead invasion ex vivo while expressing both epithelial and mesenchymal characteristics. The mesenchymal marker vimentin promoted invasion and repressed metastatic outgrowth. We next tested the requirement for five EMT transcription factors and observed distinct patterns of utilization during invasion and colony formation. These differences suggested a sequential activation of multiple EMT molecular programs during the metastatic cascade. Consistent with this model, our longitudinal single-cell RNA analysis detected three different EMT-related molecular patterns. We observed cancer cells progressing from epithelial to hybrid E/M and strongly mesenchymal patterns during invasion and from epithelial to a hybrid E/M pattern during colony formation. We next investigated the relative epithelial versus mesenchymal state of cancer cells in both GEMM and patient metastases. In both contexts, we observed heterogeneity between and within metastases in the same individual. We observed a complex spectrum of epithelial, hybrid E/M, and mesenchymal cell states within metastases, suggesting that there are multiple successful molecular strategies for distant organ colonization. Together, our results demonstrate an important and complex role for EMT programs during TNBC metastasis.

Glutamine deficiency promotes recurrence and metastasis in colorectal cancer through enhancing epithelial–mesenchymal transition

BackgroundGlutamine is the most abundant amino acid in the body and plays a vital role in colorectal cancer (CRC) cell metabolism. However, limited studies have investigated the clinical and prognostic significance of preoperative serum glutamine levels in patients with colorectal cancer, and the underlying mechanism has not been explored.MethodsA total of 121 newly diagnosed CRC patients between 2012 and 2016 were enrolled in this study. Serum glutamine levels were detected, and their associations with clinicopathological characteristics, systemic inflammation markers, carcinoembryonic antigen (CEA) and prognosis were analysed. In addition, the effect of glutamine depletion on recurrence and metastasis was examined in SW480 and DLD1 human CRC cell lines, and epithelial–mesenchymal transition (EMT)-related markers were detected to reveal the possible mechanism.ResultsA decreased preoperative serum level of glutamine was associated with a higher T-class and lymph node metastasis (P < 0.05). A higher serum level of glutamine correlated with a lower CEA level (r = − 0.25, P = 0.02). Low glutamine levels were correlated with shorter overall survival (OS) and disease-free survival (DFS). Multivariate Cox regression analysis showed that serum glutamine was an independent prognostic factor for DFS (P = 0.018), and a nomogram predicting the probability of 1-, 3- and 5-year DFS after radical surgery was built. In addition, glutamine deficiency promoted the migration and invasion of CRC cells. E-cadherin, a vital marker of EMT, was decreased, and EMT transcription factors, including zeb1and zeb2, were upregulated in this process.ConclusionsThis study elucidated that preoperative serum glutamine is an independent prognostic biomarker to predict CRC progression and suggested that glutamine deprivation might promote migration and invasion in CRC cells by inducing the EMT process.

PARP1-SNAI2 transcription axis drives resistance to PARP inhibitor, Talazoparib

The synthetic lethal association between BRCA deficiency and poly (ADP-ribose) polymerase (PARP) inhibition supports PARP inhibitor (PARPi) clinical efficacy in BRCA-mutated tumors. PARPis also demonstrate activity in non-BRCA mutated tumors presumably through induction of PARP1-DNA trapping. Despite pronounced clinical response, therapeutic resistance to PARPis inevitably develops. An abundance of knowledge has been built around resistance mechanisms in BRCA-mutated tumors, however, parallel understanding in non-BRCA mutated settings remains insufficient. In this study, we find a strong correlation between the epithelial-mesenchymal transition (EMT) signature and resistance to a clinical PARPi, Talazoparib, in non-BRCA mutated tumor cells. Genetic profiling demonstrates that SNAI2, a master EMT transcription factor, is transcriptionally induced by Talazoparib treatment or PARP1 depletion and this induction is partially responsible for the emerging resistance. Mechanistically, we find that the PARP1 protein directly binds to SNAI2 gene promoter and suppresses its transcription. Talazoparib treatment or PARP1 depletion lifts PARP1-mediated suppression and increases chromatin accessibility around SNAI2 promoters, thus driving SNAI2 transcription and drug resistance. We also find that depletion of the chromatin remodeler CHD1L suppresses SNAI2 expression and reverts acquired resistance to Talazoparib. The PARP1/CHD1L/SNAI2 transcription axis might be therapeutically targeted to re-sensitize Talazoparib in non-BRCA mutated tumors.

Stanniocalcin 2 drives malignant transformation of human glioblastoma cells by targeting SNAI2 and Matrix Metalloproteinases

Glioblastoma multiforme (GBM) is the most malignant brain tumor and is refractory to conventional therapies. Although previous studies have proposed that the interaction between gene mutations and the external environment leads to the occurrence of GBM, the pathogenesis of GBM is still unclear and much remains to be studied. Herein, we show an association between human glycoprotein stanniocalcin-2 (STC2) and aggressive GBM progression, and demonstrate the underlying mechanism. Elevated STC2 expression and secretion greatly increase GBM cell growth and invasive phenotypes. Mechanistically, both, conditioned media (CM) containing STC2 and recombinant STC2, can induce the transformation of GBM cells into more malignant phenotypes by upregulating the expression of the epithelial-mesenchymal transition transcription factor, snail family transcription repressor 2 (SNAI2) as well as matrix metalloproteinases (MMPs). Moreover, we further demonstrate that the oncogenic function of STC2 in GBM is mediated through the MAPK signaling pathway. Collectively, these results identify the mechanism of STC2 targeting SNAI2 and MMPs through the MAPK pathway in GBM, and provide insights into a potential therapeutic strategy for GBM.

GREM1 is required to maintain cellular heterogeneity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) shows pronounced epithelial and mesenchymal cancer cell populations1-4. Cellular heterogeneity in PDAC is an important feature in disease subtype specification3-5, but how distinct PDAC subpopulations interact, and the molecular mechanisms that underlie PDAC cell fate decisions, are incompletely understood. Here we identify the BMP inhibitor GREM16,7 as a key regulator of cellular heterogeneity in pancreatic cancer in human and mouse. Grem1 inactivation in established PDAC in miceresulted in a direct conversion of epithelial into mesenchymal PDAC cells within days, suggesting that persistent GREM1 activity is required to maintain the epithelial PDAC subpopulations. By contrast, Grem1 overexpression caused an almost complete 'epithelialization' of highly mesenchymal PDAC, indicating that high GREM1 activity is sufficient to revert the mesenchymal fate of PDAC cells. Mechanistically, Grem1 was highly expressed in mesenchymal PDAC cells and inhibited the expression of the epithelial-mesenchymal transition transcription factors Snai1 (also known as Snail) and Snai2 (also known as Slug) in the epithelial cell compartment, therefore restricting epithelial-mesenchymal plasticity. Thus, constant suppression of BMP activity is essential to maintain epithelial PDAC cells, indicating that the maintenance of the cellular heterogeneity of pancreatic cancer requires continuous paracrine signalling elicited by a single soluble factor.

Abstract 2442: Seminal fluid enhances cervical squamous carcinoma cell proliferation, epithelial mesenchymal transition (EMT), migration &amp; invasion

Abstract Cervical cancer is the leading cause of cancer related deaths &amp; the second most common cancer amongst South African women. Though there are various risk factors, the most common risk factor for cervical cancer development is sexual transmission &amp; persistent infection with high-risk Human Papillomavirus (HPV). Although most HPV infections are cleared naturally in women, persistent infection with high-risk HPV types promotes neoplastic transformation &amp; tumorigenesis. Furthermore, exposure of neoplastic cervical epithelial cells to inflammatory mediators may drive the progression of cervical cancer. Seminal fluid (SF), a biological fluid rich in inflammatory mediators &amp; growth factors, has been implicated in cervical cancer progression. SF has been shown to induce the expression of proinflammatory mediators which promotes cell proliferation in culture &amp; growth of explants in mice injected with HeLa cervical adenocarcinoma cells. Since majority of cervical cancer cases are squamous cell carcinoma, in this study, we examined the effect of SF on cell proliferation, EMT, cell migration &amp; invasion employing SiHa and Me180 squamous cell carcinoma cell lines. Our results showed that SF significantly enhanced cell proliferation in both cell lines. Using Confocal microscopy and phalloidin staining we further showed that SF caused morphological changes and stress fibre formation. RT-qPCR showed that SF up regulated EMT transcription factors Snail, ZEB1 &amp; TWIST mRNA expression. EMT induction was further confirmed by the increase of N-cadherin &amp; a subsequent decrease in E-cadherin protein expression. Additionally, we showed that the induction of EMT transcription factors by SF occurs via the EGF/ERK/COX pathway. To investigate the effect of SF on migration and invasion, transwell migration assays were used. Results showed that SF significantly enhanced cell migration &amp; invasion of the SiHa and Me180 cells. Our results show that SF plays a role in enhancing the migratory &amp; invasive potential of squamous cell carcinoma cells &amp; that this associates with increased expression of EMT transcription factors. These findings together implicate SF as a possible factor that may promote cervical cancer progression. Citation Format: Nonkululeko Mkwanazi, Virna D. Leaner, Arieh A. Katz. Seminal fluid enhances cervical squamous carcinoma cell proliferation, epithelial mesenchymal transition (EMT), migration &amp; invasion [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 2442.

Abstract 2620: Combination of growth hormone receptor antagonist and gemcitabine leads to highly efficacious tumor clearance in a mouse model of pancreatic ductal adenocarcinoma

Abstract Pancreatic cancer is responsible for more than 48,000 deaths and 60,000 new cancer cases every year in the United States while the overall 5-year survival rate is only 10%. These numbers point towards an urgent need for new therapeutic approaches wherein therapy resistance poses a tremendous challenge. We and others have identified the role of growth hormone (GH) and its cognate receptor (GHR), overexpressed in pancreatic ductal adenocarcinoma patients (PDAC; &amp;gt;90% of pancreatic cancer cases), in driving therapy resistance by directly upregulating multidrug efflux, phenotype switch via epithelial-to-mesenchymal transition (EMT) and inhibition of apoptosis. Our laboratory had pioneered the discovery of pharmacologic GHR antagonists including the only approved and prescribed GHR antagonist, pegvisomant, and a new antagonist candidate named compound-G. Here, we used in vitro, in vivo and in silico analyses to assess the feasibility and efficacy of a treatment regimen including GHR antagonist and chemotherapy to manage PDAC. Both pegvisomant and compound-G exhibit significant suppression of cancer cellular viability, invasive potential, drug efflux rate and apoptosis in PDAC cell cultures. In male and female Nude mice with human PDAC xenografts, GHR inhibition by either pegvisomant or compound-G markedly re-sensitized the tumors towards both low and high doses of gemcitabine treatment. Compound G plus high doses of gemcitabine lead to almost complete tumor clearance in 40% of the mice. Also, tissue analyses reveal increased production of GH in tumors treated with chemotherapy. In the GHR antagonist treated tumors, significantly lower expression of ABC transporter (Abcb1, Abcg5, Abcc2, Abcg2), EMT mediator (Cdh2, Zeb1, Snai2, Epas1) and apoptosis marker (Bcl2) genes compared to control were observed - accounting for the observed sensitization to gemcitabine. In the human PDAC patient datasets, increased expression of GHR correlates strongly with increased expression of multidrug efflux transporters, anti-apoptotic markers, and EMT transcription factors. Therefore, we provide robust pre-clinical validation for a treatment regimen combining GHR antagonist and chemotherapy leading to highly efficacious tumor clearance. Acknowledgement: This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, and Ohio University’s Student Enhancement Award and Edison Biotechnology Institute. Citation Format: Reetobrata Basu, Prateek Kulkarni, Emily Davis, Silvana Duran, John J. Kopchick. Combination of growth hormone receptor antagonist and gemcitabine leads to highly efficacious tumor clearance in a mouse model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2620.

Abstract 953: MEK/ERK induces the EMT transcription factor ZEB1 that is regulated by USP10 and promotes colorectal cancer metastasis that can be inhibited by BRAFV600E-directed therapy

Abstract Background: Constitutive MEK-ERK activation induced by BRAFV600E or RAS mutation promotes tumor metastasis in CRC that is mediated by transcription factors regulating epithelial-to-mesenchymal transition (EMT). We determined the role of ZEB1, an EMT master transcription factor, and its potential regulation by the ubiquitin specific peptidase 10 (USP10), in tumor metastasis mediated by MEK-ERK. Materials and Methods: Isogenic RKO [BRAFV600E or wild-type (wt)] and Colo320 (BRAF/KRAS wt/wt) human CRC cells were utilized. Colo320 cells were transfected with mutant KRASG12D. In addition to parental cells, we generated USP10 shRNA expressing RKO and Colo320 cells. Cells were treated with the MEK1/2 inhibitor cobimetinib or BRAF inhibitor encorafenib. Cell migration was analyzed by wound healing and transwell assays. Protein/protein interaction was analyzed by immunoprecipitation. Protein ubiquitination level was determined by ubiquitination assay. In an orthotopic model, lung metastases in mice were generated by tail vein injection of RKO cells. Results: We demonstrate MEK-ERK activation, induced by BRAFV600E, promotes CRC metastasis through inhibition of the USP10-ZEB1 axis. USP10 is shown to interact with ZEB1 and to remove its K27 linkage ubiquitination chain, thus increasing K48 ubiquitination of ZEB1 protein that results in proteosome-mediated degradation of ZEB1. MEK-ERK induced the phosphorylation of USP10 that is shown to attenuate the USP10-ZEB1 interaction and results in ZEB1 stabilization in RKO and Colo320 cell lines. Increased ZEB1 expression promotes tumor cell migration in vitro and metastasis in a murine xenograft model that could be suppressed by cobimetinib or BRAFV600E-directed therapy with encorafenib and cetuximab. Conclusions: Targeting RAF/RAS-driven MEK/ERK signaling can suppress ZEB1 stabilization to inhibit tumor metastasis. Suppression of metastasis occurs by a novel mechanism of ZEB1 stabilization that is regulated by an atypical USP10 deubiquitinase activity. Citation Format: Bo Qin, Lei Sun, Frank A. Sinicrope. MEK/ERK induces the EMT transcription factor ZEB1 that is regulated by USP10 and promotes colorectal cancer metastasis that can be inhibited by BRAFV600E-directed therapy [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 953.

Abstract 3325: ERK inhibitor increases cancer stem cell population in NSCLC through Slug-Mediated epithelial-to-mesenchymal transition

Abstract The aberrant activation of the Mitogen-activated protein kinase pathway (MAPK/ERK pathway) has been found to play an important role in many cancers including non-small-cell lung cancer (NSCLC). Multiple inhibitors targeting this pathway have been developed to treat cancer patients harboring the aberrant MAPK pathway genes. ERK inhibitors (ERKi) have been shown to be effective in a variety of solid-tumor malignancies, but like other MAPK inhibitors, they rarely confer complete and durable responses. Therefore, it is important to define the mechanism underlying potential tumor relapse following ERKi treatment. In this study, we found that ERKi treatment expanded the cancer stem cell (CSC) population in various NSCLC cell lines and a NSCLC patient-derived xenograft (PDX) harboring aberrant MAPK signaling. Such expansion of the CSC population is mainly caused by enhanced epithelial-to-mesenchymal transition (EMT)-mediated cancer cell dedifferentiation. Mechanistically, we revealed that ERK inhibition induces EMT via pSTAT3-mediated upregulation of EMT-transcription factor Slug. Specifically, we found that ERK inhibition increased phosphorylation of STAT3 at Y705, which further increased the expression of miR-204. Increased miR-204 then downregulated expression of SPDEF, a transcriptional repressor of Slug, thereby derepressing Slug expression. Moreover, we found that a low expression of Slug is necessary for maintaining stemness properties of CSCs. Thus, ERKi treatment-increased Slug must be downregulated to allow these cells to fully acquire stemness, and this process is mediated by a Slug-miR-200c negative feedback loop. Finally, we demonstrated that the JAK/STAT pathway inhibitor Ruxolitinib can block the ERK inhibitor-induced EMT and CSCs expansion, providing a feasible combination inhibitor approach for treating NSCLC patients. Overall, these studies provide a better understanding of the tumor relapse following ERKi treatment, and define the role of Slug in de novo production of CSCs and their maintenance. Citation Format: Shurui Cai, Na Li, Xuetao Bai, Ananya Banerjee, Lavudi Kousalya, Qi-en Wang. ERK inhibitor increases cancer stem cell population in NSCLC through Slug-Mediated epithelial-to-mesenchymal transition [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 3325.

Abstract 1091: TWIST1 inhibition overcomes resistance to tyrosine kinase inhibitors in MET driven non-small cell lung cancer

Abstract Background: The mesenchymal-epidermal transition (cMET/MET) tyrosine kinase receptor and its ligand, the hepatocyte growth factor (HGF) are overexpressed in a large percentage of non-small cell lung cancers (NSCLCs) and MET mutation and/or amplification leads to oncogene addiction in small subset of NSCLC. In addition, MET amplification is an established mechanism of resistance to EGFR and other oncogenic targeted TKIs. Several MET-inhibitors have been developed and two MET TKIs have been approved for MET exon 14 skipping mutant NSCLC and have shown activity against MET amplified NSCLC. However, long-term efficacy of MET TKIs is limited as acquired resistance is inevitable. HGF overexpression has been identified as one of the mechanisms of resistance to MET TKIs in MET-altered NSCLC, but the mechanism(s) by which the HGF-MET pathway causes resistance are poorly understood. Here, we investigated the requirement of the EMT-transcription factor, TWIST1 in HGF-mediated resistance to MET TKIs and the role of TWIST1 in de-novo and acquired resistance to MET TKIs. Methods: TWIST1 expression was measured in wild type and TWIST1 over expressing cell lines in presence and absence of HGF. TWIST1 was inhibited with shRNA and harmine. Apoptosis was assessed via immunoblotting and cleaved caspase 3 staining. We utilized MET altered (mutant/amplified) NSCLC cell lines, patient derived xenografts and a novel transgenic mouse model of Hgf, Twist1 overexpressing lung cancer to evaluate TWIST1 as a driver of MET TKI resistance. Results: We found that HGF induced TWIST1 expression and MET TKI treatment decreased TWIST1 expression through a post-translational mechanism. Re-expression of TWIST1 led to MET TKI resistance in MET amplified or MET mutant cell lines. Conversely, genetic and pharmacological inhibition of TWIST1 sensitizes to MET inhibition and overcame HGF-mediated MET TKI resistance and MET amplification mediated EGFR TKI resistance in vitro and in vivo. Furthermore, the role of TWIST1 in HGF/MET lung tumorigenesis was evaluated both in human NSCLC xenografts and an Hgf-driven NSCLC mouse model. Genetic inhibition of TWIST1 in MET mutant or amplified cell lines prevented tumor growth in vivo. Furthermore, TWIST cooperated with Hgf in a CCSP-Hgf (CH) mice model that constitutively overexpresses Hgf in the lung and develops NSCLC after treatment with the tobacco carcinogen, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) was utilized. We demonstrated that the Twist1 overexpressing CHT (CCSP-rtTA/CCSP-Hgf/Twist1-tetO-luc) mice developed significantly larger and more aggressive tumors as compared to CH and CCSP-rtTA/Twist1-tetO-luc (CT) mice and that continued TWIST1 expression was required for these tumors. Conclusions: Our findings suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome HGF-MET-driven resistance in MET-driven NSCLC. Citation Format: Vinod Kumar, Zachary A. Yochum, Princey Devadassan, Eric Huang, Ethan Miller, Vasavi Ayyala, Laura P. Stabile, Timothy F. Burns, Purva H. Rumde. TWIST1 inhibition overcomes resistance to tyrosine kinase inhibitors in MET driven non-small cell lung cancer [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 1091.

CD44v3,8-10 is essential for Slug-dependent vimentin gene expression to acquire TGF-β1-induced tumor cell motility.

CD44 is a widely expressed polymorphic adhesion molecule that has pleiotropic functions in development and tumor progression. Its mRNA undergoes alternative splicing to generate multiple variant (CD44v) isoforms, although the function of each CD44v isoform is not fully elucidated. Here, we show that CD44v plays an important role in the induction of vimentin expression upon transforming growth factor‐β1 (TGF‐β1)‐induced epithelial–mesenchymal transition (EMT). Among multiple CD44v isoforms expressed in NUGC3 gastric cancer cells, CD44v8‐10 and CD44v3,8‐10 are involved in the acquisition of migratory and invasive properties associated with TGF‐β1‐induced EMT, and only CD44v3,8‐10 induces the transcription of vimentin mediated by the EMT transcription factor Slug. In primary tumor specimens obtained from patients with gastric cancer, CD44‐containing variant exon 9 (CD44v9) expression and EMT features [E‐cadherin(−)vimentin(+)] were significantly correlated, and EMT features in the cells expressing CD44v9 were associated with tumor invasion depth, lymph node metastasis, and pStage, which indicate invasive and metastatic properties, and poor prognosis. These results indicate that certain CD44v isoforms promote tumor cell motility and metastasis in gastric cancer in association with EMT features, and CD44v3,8‐10 may contribute to these clinical characteristics.

368-OR: Activation of a Yap1-Dependent Mesenchymal Program Promotes ß-Cell Failure

A critical contributor to the pathogenesis of type 2 diabetes is the failure of insulin-secreting β-cells due to loss of cell number or identity. However, the signaling pathways that trigger these events remain unclear. While β-cells are of endodermal origin, recent studies show endocrine cells that coexpress the mesenchymal marker vimentin in the pancreas with diabetes, although the functional features of these cells are not known. Here, we identify an epithelial-to-mesenchymal (EMT) -like program that is activated in islets of mice and humans with type 2 diabetes. Characteristics of this EMT-like program include increased protein and mRNA abundance of the EMT transcription factor Snail and the mesenchymal adhesion molecule N-cadherin. Lineage-tracing techniques demonstrate that this mesenchymal program is initiated in β-cells that become multi-hormonal, a hallmark of dysfunctional endocrine cells. Interestingly, EMT-like cells minimally express Aldh1a3, a marker of dedifferentiated β-cells. To determine the mechanism driving EMT-like activation in β-cells, we tested stressors that phenocopy the effects of diabetes in islets. Treatment of islets with mitochondrial decouplers as well as oxidative stress increased expression of EMT markers. The transcription factor Yap1 is a known inducer of EMT. While Yap1 is expressed at minimal levels in β-cells, its abundance increased in db/db islets and in primary cells with impaired mitochondrial function. Importantly, chemical inhibition of Yap1 in db/db islets reversed activation of EMT-like factors. Yap1 inhibition also improved insulin secretion. Taken together, we found activation of an EMT-like program in the endocrine pancreas of mice and humans with type 2 diabetes. This program is dependent on increases in Yap1 due to oxidative stress and impaired mitochondrial function, and results in disrupted β-cell function. Disclosure W.Mckimpson: n/a. N.Hoque: None. J.Son: None. A.Y.Lee: None. D.Accili: Advisory Panel; Lilly Diabetes. Funding NIDDK (1K01DK121873)

Hepatitis B virus X protein promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells by regulating SOCS1

Hepatocellular carcinoma (HCC), a primary type of liver cancer, is one of the leading causes of cancer related deaths worldwide. HCC patients have poor prognosis due to intrahepatic and extrahepatic metastasis. Hepatitis B virus (HBV) infection is one of the major causes of various liver diseases including HCC. Among HBV gene products, HBV X protein (HBx) plays an important role in the development and metastasis of HCC. However, the mechanism of HCC metastasis induced by HBx has not been elucidated yet. In this study, for the first time, we report that HBx interacts with the suppressor of cytokine signaling 1 (SOCS1) which negatively controls NF-κB by degrading p65, a subunit of NF-κB. NF-κB activates the transcription of factors associated with epithelial-mesenchymal transition (EMT), a crucial cellular process associated with invasiveness and migration of cancer cells. Here, we report that HBx physically binds to SOCS1, subsequently prevents the ubiquitination of p65, activates the transcription of EMT transcription factors and enhance cell migration and invasiveness, suggesting a new mechanism of HBV-associated HCC metastasis.

SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, is a matricellular protein that modulates interactions between cells and their microenvironment. SPARC is expressed during extracellular matrix remodeling and is abundant in bone marrow and high-grade prostate cancer (PCa). In PCa, SPARC induces changes associated with epithelial–mesenchymal transition (EMT), enhancing migration and invasion and increasing the expression of EMT transcriptional factor Zinc finger E-box-binding homeobox 1 (ZEB1), but not Zinc finger protein SNAI1 (Snail) or Zinc finger protein SNAI2 (Slug). It is unknown whether the SPARC-induced downregulation of E-cadherin in PCa cells depends on ZEB1. Several integrins are mediators of SPARC effects in cancer cells. Because integrin signaling can induce EMT programs, we hypothesize that SPARC induces E-cadherin repression through the activation of integrins and ZEB1. Through stable knockdown and the overexpression of SPARC in PCa cells, we demonstrate that SPARC downregulates E-cadherin and increases vimentin, ZEB1, and integrin β3 expression. Knocking down SPARC in PCa cells decreases the tyrosine-925 phosphorylation of FAK and impairs focal adhesion formation. Blocking integrin αvβ3 and silencing ZEB1 revert both the SPARC-induced downregulation of E-cadherin and cell migration enhancement. We conclude that SPARC induces E-cadherin repression and enhances PCa cell migration through the integrin αvβ3/ZEB1 signaling pathway.

Autophagy regulates transforming growth factor β signaling and receptor trafficking

Transforming growth factor beta (TGFβ) stimulates tumorigenesis by inducing epithelial to mesenchymal transition (EMT) and cell migration. TGFβ signaling is regulated by the endocytosis of cell surface receptors and their subcellular trafficking into the endo-lysosomal system. Here we investigated how autophagy, a cellular quality control network that delivers material to lysosomes, regulates TGFβ signaling pathways that induce EMT and cell migration. We impaired autophagy in non-small cell lung cancer cells using chloroquine, spautin-1, ULK-101, or small interfering RNA (siRNA) targeting autophagy-related gene (ATG)5 and ATG7 and observed that inhibiting autophagy results in a decrease in TGFβ1-dependent EMT transcription factor and cell marker expression, as well as attenuated stress fiber formation and cell migration. This correlated with decreased internalization of cell surface TGFβ receptors and their trafficking to early/late endosomal and lysosomal compartments. The effects of autophagy inhibition on TGFβ signaling were investigated by Smad2/Smad3 phosphorylation and cellular localization using western blotting, subcellular fractionation, and immunofluorescence microscopy. We observed that inhibiting autophagy decreased the amount and timeframe of Smad2/Smad3 signaling. Taken together, our results suggest that inhibiting autophagy attenuates pro-tumorigenic TGFβ signaling by regulating receptor trafficking, resulting in impaired Smad2/Smad3 phosphorylation and nuclear accumulation.