Epithelial-mesenchymal Transition Molecules Research Articles

The differences between pure and mixed invasive micropapillary breast cancer: the epithelial-mesenchymal transition molecules and prognosis.

Invasive micropapillary carcinoma (IMPC) of the breast is known for its high metastatic potential, but the definition of pure and mixed IMPC remains unclear. This retrospective cohort study aims to investigate the prognostic significance of the micropapillary component ratio and the expression of critical molecules of epithelial-mesenchymal transition (EMT), including E-cadherin (E-cad), N-cadherin (N-cad), CD44s, and β-catenin (β-cat), in distinguishing between pure and mixed IMPCs. We analyzed 100 cases of locally advanced IMPC between 2000 and 2018 and excluded patients who received neoadjuvant chemotherapy. Pure IMPC was defined as having a micropapillary component of over 90%. A comprehensive recording of prognostic parameters was conducted. The IMPC areas were analyzed using the immunohistochemical (IHC) staining method on the microarray set for pure and mixed IMPC patients. Pearson's chi-square, Fisher's exact tests, Kaplan-Meier analysis, and Cox proportional hazards analysis were employed. The comparative survival analysis of the entire group, based on overall survival (OS) and disease-free survival (DFS), revealed no significant difference between the pure and mixed groups (P = 0.480, HR = 1.474 [0.502-4.325] and P = 0.390, HR = 1.587 [0.550-4.640], respectively). However, in the pure IMPC group, certain factors were found to be associated with a higher risk of short survival. These factors included skin involvement (P = 0.050), pT3&4 category (P = 0.006), a ratio of intraductal component (> 5%) (P = 0.032), and high-level expression of N-cad (P = 0.020). Notably, none of the risk factors identified for short OS in pure IMPC cases were observed as significant risks in mixed cases and vice versa. Furthermore, N-cad was identified as a poor prognostic marker for OS in pure IMPCs (P = 0.002). The selection of a 90% ratio for classifying pure IMPCs revealed significant differences in certain molecular and prognostic parameters between pure and mixed groups. Notably, the involvement of N-cadherin in the epithelial-mesenchymal transition (EMT) process provided crucial insights for predicting OS and DFS while also distinguishing between the two groups. These findings strongly support the notion that the pure IMPC subgroup represents a distinct entity characterized by unique molecular characteristics and behavioral patterns.

Decidual stromal cell–derived exosomes deliver miR-22-5p_R-1 to suppress trophoblast metabolic switching from mitochondrial respiration to glycolysis by targeting PDK4 in unexplained recurrent spontaneous abortion

IntroductionStudies have shown that EMT (epithelial-mesenchymal transition) and energy metabolism influence each other, and it is unclear whether the trophoblast energy metabolism phenotype is dominated by glycolysis or mitochondrial respiration, and the relationship between trophoblast energy metabolism and EMT is still unclear. MethodsExosomes were isolated from the DSC of URSA patients and their miRNA profile was characterized by miRNA sequencing. Wound healing assays and transwell assays were used to assess the invasion and migration ability of trophoblasts. Mitochondrial stress and glycolysis stress test were used to evaluate energy metabolism phenotype of trophoblast. Luciferase reporter assays, qRT-PCR and WB were conducted to uncover the underlying mechanism. Finally, animal experiments were employed to explore the effect of DSC-exos on embryo absorption in mice. ResultsOur results showed that URSA-DSC-exos suppressed trophoblast EMT to reduce their migration and invasion, miR-22-5p_R-1 was the most upregulated miRNAs. URSA-DSC-exos can suppress trophoblast MGS (metabolic switch from mitochondrial respiration to glycolysis) and inhibit trophoblast migration and invasion by transferring miR-22-5p_R-1. Mechanistically, miR-22-5p_R-1 suppress trophoblast MGS and inhibit trophoblast EMT by directly suppressing PDK4 expression at the post-transcriptional level. Furthermore, in vivo experiment suggested that URSA-DSC-exos aggravated embryo absorption in mice. Clinically, PDK4 and EMT molecule were aberrant in villous of URSA patients, and negative correlations were found between miR-22-5p_R-1 and PDK4. DiscussionOur findings indicated that URSA-DSC-exos induced MGS obstacle playing an important role in intercellular communication between trophoblast and DSC, illuminating a novel mechanism in DSC regulation of trophoblasts and their role in URSA.

ANP promotes HTR-8/SVneo cell invasion by upregulating protein kinase N 3 via autophagy inhibition.

Preeclampsia is a gestational disease characterized by two major pathological changes-shallow trophoblast invasion and impaired spiral artery remodeling. Atrial natriuretic peptide (ANP) is a kind of peptide hormone that regulates blood pressure, while the lack of active ANP participates in preeclampsia pathogenesis. However, the underlying mechanism of how ANP modulates trophoblasts function remains unclarified. Here, we performed isobaric tags for relative and absolute quantification (iTRAQ) in ANP-treated HTR-8/SVneo cells and identified Protein Kinase 3 (PKN3) as the downstream factor of ANP, which was downregulated in preeclamptic placenta. Chromatin immunoprecipitation analysis and luciferase assays showed that NFYA was one of the transcription factors for the PKN3 promoter, which was also regulated by ANP treatment in HTR-8/SVneo cells. Transmission electron microscopy and Western Blotting in HTR-8/SVneo cells indicated that ANP inhibited autophagy via AMPK-mTORC1 signaling, while excess autophagy was observed in preeclamptic placenta. The increased expression of PKN3 and enhanced cell invasion ability in HTR-8/SVneo cells induced by ANP could be abolished by autophagy activation or transfection with PKN3 shRNA or NFYA shRNA or NPR-A shRNA via regulating the invasion-related genes and the epithelial mesenchymal transition molecules. Our results demonstrated that ANP could enhance trophoblast invasion by upregulating PKN3 via NFYA promotion through autophagy inhibition in an AMPK/mTORC1 signaling-dependent manner.

EMT Molecular Signatures of Pancreatic Neuroendocrine Neoplasms.

Neuroendocrine neoplasms (NENs) are relatively rare neoplasms occurring predominantly in the gastrointestinal tract and pancreas. Their heterogeneity poses challenges for diagnosis and treatment. There is a paucity of markers for characterisation of NEN tumours. For routine diagnosis, immunohistochemistry of the NEN-specific markers CgA and synaptophysin and the proliferation marker Ki-67 are used. These parameters, however, are qualitative and lack the capacity to fully define the tumour phenotype. Molecules of epithelial-mesenchymal transition (EMT) are potential candidates for improved tumour characterisation. Using qRT-PCR, we measured mRNA levels of 27 tumour markers, including 25 EMT-associated markers, in tumour tissue and matched non-tumour tissues for 13 patients with pancreatic NENs. Tissue from patients with three different grades of tumour had distinctly different mRNA profiles. Of the 25 EMT-associated markers analysed, 17 were higher in G3 tissue relative to matched non-tumour tissue, including CD14, CD24, CD31, CD44, CD45, CD56, CK6, CK7, CK13, CK20, NSE, CDX2, CgA, DAXX, PCNA, laminin and Ki-67. The differences in levels of seven EMT-associated markers, Ki-67, DAXX, CD24, CD44, vimentin, laminin and PDX1 plus CgA and NSE (neuroendocrine markers) enabled a distinct molecular signature for each tumour grade to be generated. EMT molecules differentially expressed in three tumour grades have potential for use in tumour stratification and prognostication and as therapeutic targets for treatment of neuroendocrine cancers, following validation with additional samples.

Identification of BGN and THBS2 as metastasis-specific biomarkers and poor survival key regulators in human colon cancer by integrated analysis.

Colon cancer is the second leading cause of death worldwide. Exploring key regulators in colon cancer metastatic progression could lead to better outcomes for patients. Initially, the transcriptional profiles of 681 colonrectal cancer (CRC) cases were used to discover signature genes that were significantly correlated with colon cancer metastasis. These signature genes were then validated using another independent 210 CRC cases' transcriptomics and proteomics profiles, and Kaplan-Meier regression analyses were used to screen the key regulators with patients' survival. Immunohistochemical staining was used to confirm the biomarkers, and transit knockdown was used to explore their implications on colon cancer cells migration and invasion abilities. The impact on the key signalling molecules in epithelial-mesenchymal transition (EMT) process that drive tumour metastasis was tested using Western blot. The response to clinical standard therapeutic drugs was compared to clinical prognosis of key regulators using an ROC plotter. Five genes (BGN, THBS2, SPARC, CDH11 and SPP1) were initially identified as potential biomarkers and therapeutic targets of colon cancer metastasis. The most significant signatures associated with colon cancer metastasis were determined to be BGN and THBS2. Furthermore, highly expression of BGN and THBS2 in tumours was linked to a worse survival rate. BGN and THBS2 knockdown significantly reduced colon cancer cells migration and invasion, as well as down-regulating three EMT-related proteins (Snail, Vimentin and N-cadherin), and increasing the proliferation inhibitory effect of 5-fluorouracil, irinotecan and oxaliplatin treatment. CRC metastatic progression, EMT phenotypic transition and poor survival time have been linked to BGN and THBS2. They could be utilized as potential diagnostic and therapeutic targets for colon cancer metastatic patients with a better prognosis.

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.

Drugs targeting epithelial-mesenchymal transition molecules for treatment of lichen planopilaris.

Primary cicatricial alopecia (PCA), also known as scarring alopecia, comprises a diverse group of hair disorders that cause permanent destruction of the pilosebaceous unit, resulting in disappearance of the follicular ostia. Lichen planopilaris (LPP) is a subtype of primary lymphocytic cicatricial alopecia. There is an urgent need to identify novel molecules that successfully target specific pathogenic pathways in LPP to inhibit and reverse disease progression. Recent studies into LPP pathogenesis have discovered that follicular stem cells undergo epithelial-mesenchymal transition (EMT). We sought to identify drugs that target molecules involved in EMT to repurpose these drugs for treatment of LPP. We identified 8 molecules and 15 drugs that target these EMT molecules. Only four of these drugs (pioglitazone, tofacitinib, barcitinib and apremilast) have been reported in individual cases or case series of patients with LPP and controlled studies are missing. We describe each drug and mechanism of action target EMT in detail. Although previous studies have demonstrated the efficacy of EMT inhibitors in anticancer therapy, there are, to our knowledge, no studies using EMT-attenuating drugs for the treatment of LPP. The treatment molecules discussed in this paper provide a new platform for clinical studies and controlled trials in LPP.

Propolin G-Suppressed Epithelial-to-Mesenchymal Transition in Triple-Negative Breast Cancer Cells via Glycogen Synthase Kinase 3β-Mediated Snail and HDAC6-Regulated Vimentin Degradation.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer with a poor prognosis. The incidence and mortality rate of TNBC are frequently found in younger women. Due to the absence of a good therapeutic strategy, effective remedies for inhibiting TNBC have been developed for improving the cure rate. Epithelial-to-mesenchymal transition (EMT) is a critical mechanism to regulate cancer cell motility and invasion. Furthermore, ectopic expression of EMT molecules correlates with the metastasis and poor prognosis of TNBC. Targeting EMT might be a strategy for the therapy and prevention of TNBC. Propolin G, an active c-prenylflavanone in Taiwanese propolis, has been shown to possess anti-cancer activity in many cancers. However, the anti-metastasis activity of propolin G on TNBC is still unclear. The present study showed that the migration and invasion activities of TNBC cells was suppressed by propolin G. Down-regulated expression of Snail and vimentin and up-regulated expression of E-cadherin were dose- and time-dependently observed in propolin G-treated MDA-MB-231 cells. Propolin G inhibited Snail and vimentin expressions via the signaling pathways associated with post-translational modification. The activation of glycogen synthase kinase 3β (GSK-3β) by propolin G resulted in increasing GSK-3β interaction with Snail. Consequently, the nuclear localization and stability of Snail was disrupted resulting in promoting the degradation. Propolin G-inhibited Snail expression and the activities of migration and invasion were reversed by GSK-3β inhibitor pretreatment. Meanwhile, the outcomes also revealed that histone deacetylase 6 (HDAC6) activity was dose-dependently suppressed by propolin G. Correspondently, the amounts of acetyl-α-tubulin, a down-stream substrate of HDAC6, were increased. Dissociation of HDAC6/Hsp90 with vimentin leading to increased vimentin acetylation and degradation was perceived in the cells with the addition of propolin G. Moreover, up-regulated expression of acetyl-α-tubulin by propolin G was attenuated by HDAC6 overexpression. On the contrary, down-regulated expression of vimentin, cell migration and invasion by propolin G were overturned by HDAC6 overexpression. Conclusively, restraint cell migration and invasion of TNBC by propolin G were activated by the expression of GSK-3β-suppressed Snail and the interruption of HDAC6-mediated vimentin protein stability. Aiming at EMT, propolin G might be a potential candidate for TNBC therapy.

Dual Targeting of Sorafenib-Resistant HCC-Derived Cancer Stem Cells.

Sorafenib, an oral multi-tyrosine kinase inhibitor, has been the first-line therapy for the treatment of patients with advanced HCC, providing a survival benefit of only three months in approximately 30% of patients. Cancer stem cells (CSCs) are a rare tumour subpopulation with self-renewal and differentiation capabilities, and have been implicated in tumour growth, recurrence and drug resistance. The process of epithelial-to-mesenchymal transition (EMT) contributes to the generation and maintenance of the CSC population, resulting in immune evasion and therapy resistance in several cancers, including HCC. The aim of this study is to target the chemoresistant CSC population in HCC by assessing the effectiveness of a combination treatment approach with Sorafenib, an EMT inhibitor and an immune checkpoint inhibitor (ICI). A stem-cell-conditioned serum-free medium was utilised to enrich the CSC population from the human HCC cell lines Hep3B, PLC/PRF/5 and HepG2. The anchorage independent spheres were characterised for CSC features. The human HCC-derived spheres were assessed for EMT status and expression of immune checkpoint molecules. The effect of combination treatment with SB431542, an EMT inhibitor, and siRNA-mediated knockdown of programmed cell death protein ligand-1 (PD-L1) or CD73 along with Sorafenib on human HCC-derived CSCs was examined with cell viability and apoptosis assays. The three-dimensional spheres enriched from human HCC cell lines demonstrated CSC-like features. The human HCC-derived CSCs also exhibited the EMT phenotype along with the upregulation of immune checkpoint molecules. The combined treatment with SB431542 and siRNA-mediated PD-L1 or CD73 knockdown effectively enhanced the cytotoxicity of Sorafenib against the CSC population compared to Sorafenib alone, as evidenced by the reduced size and proliferation of spheres. Furthermore, the combination treatment of Sorafenib with SB431542 and PD-L1 or CD73 siRNA resulted in an increased proportion of an apoptotic population, as evidenced by flow cytometry analysis. In conclusion, the combined targeting of EMT and immune checkpoint molecules with Sorafenib can effectively target the CSC tumour subpopulation.

Combined Inhibition of TGF-β1-Induced EMT and PD-L1 Silencing Re-Sensitizes Hepatocellular Carcinoma to Sorafenib Treatment.

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic malignancy. HCC is one of the leading causes of cancer deaths worldwide. The oral multi-tyrosine kinase inhibitor Sorafenib is the standard first-line therapy in patients with advanced unresectable HCC. Despite the significant survival benefit in HCC patients post treatment with Sorafenib, many patients had progressive disease as a result of acquiring drug resistance. Circumventing resistance to Sorafenib by exploring and targeting possible molecular mechanisms and pathways is an area of active investigation worldwide. Epithelial-to-mesenchymal transition (EMT) is a cellular process allowing epithelial cells to assume mesenchymal traits. HCC tumour cells undergo EMT to become immune evasive and develop resistance to Sorafenib treatment. Immune checkpoint molecules control immune escape in many tumours, including HCC. The aim of this study is to investigate whether combined inhibition of EMT and immune checkpoints can re-sensitise HCC to Sorafenib treatment. Post treatment with Sorafenib, HCC cells PLC/PRF/5 and Hep3B were monitored for induction of EMT and immune checkpoint molecules using quantitative reverse transcriptase (qRT)- PCR, western blot, immunofluorescence, and motility assays. The effect of combination treatment with SB431542, a specific inhibitor of the transforming growth factor (TGF)-β receptor kinase, and siRNA mediated knockdown of programmed cell death protein ligand-1 (PD-L1) on Sorafenib resistance was examined using a cell viability assay. We found that three days of Sorafenib treatment activated EMT with overexpression of TGF-β1 in both HCC cell lines. Following Sorafenib exposure, increase in the expression of PD-L1 and other immune checkpoints was observed. SB431542 blocked the TGF-β1-mediated EMT in HCC cells and also repressed PD-L1 expression. Likewise, knockdown of PD-L1 inhibited EMT. Moreover, the sensitivity of HCC cells to Sorafenib was enhanced by combining a blockade of EMT with SB431542 and knockdown of PD-L1 expression. Sorafenib-induced motility was attenuated with the combined treatment of SB431542 and PD-L1 knockdown. Our findings indicate that treatment with Sorafenib induces EMT and expression of immune checkpoint molecules, which contributes to Sorafenib resistance in HCC cells. Thus, the combination treatment strategy of inhibiting EMT and immune checkpoint molecules can re-sensitise HCC cells to Sorafenib.

Immune checkpoint molecules are regulated by transforming growth factor (TGF)-β1-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a high mortality rate. Epithelial-to-mesenchymal transition (EMT) confers cancer cells with immune evasive ability by modulating the expression of immune checkpoints in many cancers. Thus, the aim of our study is to examine the interplay between EMT and immune checkpoint molecules in HCC. A reversible EMT model was utilised with transforming growth factor (TGF)-β1 as an EMT inducer for HCC cell lines Hep3B and PLC/PRF/5. HCC cells were treated with TGF-β1 for 72 h and the EMT status and immune checkpoint expression were examined. In addition, the migratory ability of HCC cells were examined using wound healing and transwell migration assays in the reversible EMT model. siRNA-mediated knockdown of immune checkpoint molecule, B7-H3, was further utilised to validate the association between TGF-β1-mediated EMT and immune checkpoint expression in HCC. In addition, a web-based platform, SurvExpress, was utilised to evaluate the association between expression of TGF-β1 in combination with immune checkpoint molecules and overall survival in HCC patients. We observed induction of EMT upon treatment of HCC cells with TGF-β1 revealed by reduced expression of epithelial markers along with increased expression of mesenchymal markers. Withdrawal of TGF-β1 reversed the process of EMT with elevated expression of epithelial markers and reduced expression of mesenchymal markers. TGF-β1 treatment elevated the migratory potential of HCC cells which was reversed following reversal assay. Notably, during TGF-β1-induced EMT, there was upregulation of immune checkpoint molecules PD-L1 and B7-H3. However, the reversal of EMT decreased the expression of PD-L1 and B7-H3. In addition, TGF-β1 driven EMT was reversed following knockdown of B7-H3 in both HCC cells further validating the interplay between TGF-β1-mediated EMT and immune checkpoint expression in HCC. Furthermore, the coordinate expression of TGF-β1 with PD-L1 (p=0.01487) and B7-H3 (p=0.009687) was correlated with poor overall survival in 422 HCC patients. Our study has demonstrated a close association between TGF-β1-mediated EMT and regulation of immune checkpoints in HCC.

Diosgenin inhibits TGF-β1/Smad signaling and regulates epithelial mesenchymal transition in experimental pulmonary fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a grave disease characterized by abnormal wound healing associated with chronic, progressive, irreversible fatal lung disease, leading to persistent injuries to the alveolar epithelium. A consequent disturbance of fibroblast proliferation and apoptosis results in subsequent release of pro-inflammatory and pro-fibrotic mediators coupled with accumulation of extracellular matrix within the interstitium. Inexorable distortion of lung alveolar architecture leads to respiratory failure with a median survival rate of 3–5 years. Currently available drugs can only slowdown the progression of fibrosis and novel drugs are warranted to treat this disease. In this study, we demonstrate the fibro-protective effect of diosgenin in experimental lung fibrosis through regulation of Epithelial Mesenchymal Transition (EMT). A single dose of 3 U/kg body weight (b.wt) Bleomycin (BLM) was administered intratracheally in Wistar male albino rats and fibrotic animals were treated with diosgenin (100 mg/kg b.wt) orally for 28 days. BLM administered rat show histological alteration with increased mast cell and collagen accumulation. BLM induced abnormalities were significantly reduced upon treatment with diosgenin. Western blot analysis revealed an increased level of pro-inflammatory and pro-fibrotic molecules such as IL-1β and TGF-β in BLM induced rats. Rats supplemented with diosgenin showed a decreased expression of inflammatory and pro-fibrotic mediators. Markers of EMT molecules were evaluated by immunoblot. The results of immunoblot demonstrate that diosgenin regulated the expression of TGF-β mediated EMT. Hence, from the overall study, administration of diosgenin prevents pulmonary fibrosis by restraint inflammation and EMT.

A TGF-β-MTA1-SOX4-EZH2 signaling axis drives epithelial-mesenchymal transition in tumor metastasis.

MTA1, SOX4, EZH2, and TGF-β are all potent inducers of epithelial-mesenchymal transition (EMT) in cancer; however, the signaling relationship among these molecules in EMT is poorly understood. Here, we investigated the function of MTA1 in cancer cells and demonstrated that MTA1 overexpression efficiently activates EMT. This activation resulted in a significant increase in the migratory and invasive properties of three different cancer cell lines through a common mechanism involving SOX4 activation, screened from a gene expression profiling analysis. We showed that both SOX4 and MTA1 are induced by TGF-β and both are indispensable for TGF-β-mediated EMT. Further investigation identified that MTA1 acts upstream of SOX4 in the TGF-β pathway, emphasizing a TGF-β-MTA1-SOX4 signaling axis in EMT induction. The histone methyltransferase EZH2, a component of the polycomb (PcG) repressive complex 2 (PRC2), was identified as a critical responsive gene of the TGF-β-MTA1-SOX4 signaling in three different epithelial cancer cell lines, suggesting that this signaling acts broadly in cancer cells in vitro. The MTA1-SOX4-EZH2 signaling cascade was further verified in TCGA pan-cancer patient samples and in a colon cancer cDNA microarray, and activation of genes in this signaling pathway predicted an unfavorable prognosis in colon cancer patients. Collectively, our data uncover a SOX4-dependent EMT-inducing mechanism underlying MTA1-driven cancer metastasis and suggest a widespread TGF-β-MTA1-SOX4-EZH2 signaling axis that drives EMT in various cancers. We propose that this signaling may be used as a common therapeutic target to control epithelial cancer metastasis.

Expression and significance of c-kit and epithelial-mesenchymal transition (EMT) molecules in thymic epithelial tumors (TETs).

To investigate the expression and significance of c-kit and epithelial-mesenchymal transition (EMT) molecules (E-cadherin, N-cadherin, Twist, Snail) in thymic epithelial tumors (TETs). The tissue microarray technology and immunohistochemistry MaxVisionTM-use kit were used to detect the expression of c-kit and EMT molecular markers in 150 cases of paraffin sections of TET tissue and analysis the correlation between c-kit and EMT molecules and explore the malignancy and the relationship of clinicopathological parameters between c-kit, EMT molecules and TETs. The expression difference of c-kit and EMT molecular markers (E-cadherin, N-cadherin, Snail, Twist) in TETs subtypes was statistically significant (P<0.01) and their positive expression rate of thymic carcinoma was significantly higher than that in thymoma, and the difference was statistically significant, respectively (P<0.01). There is a negative correlation between the expression of c-kit and E-cadherin as well as a positive correlation between the expression level of c-kit, N-cadherin, Twist, and Snail. Furthermore, E-cadherin was negatively correlated with N-cadherin, Twist, and Snail while N-cadherin expression was positively correlated with Twist, Snail. Five indicators (c-kit, E-cadherin, N-cadherin, Twist, and Snail) may determine the malignancy of TETs, especially for distinguishing thymoma and thymic carcinoma.

MiR-15a-3p Suppresses Prostate Cancer Cell Proliferation and Invasion by Targeting SLC39A7 Via Downregulating Wnt/β-Catenin Signaling Pathway.

Background: MiR-15a-3p has been reported as a tumor suppressor in several kinds of cancer, including cervical cancer and gastric cancer. However, the precise molecular mechanisms underlying its role in prostate cancer (PCa) remain largely unknown. Methods: The expression of miR-15a-3p was determined in PCa tissues and cell lines using quantitative real time PCR. The biological function of miR-15a-3p in PCa cells was investigated using a MTT assay, Edu staining and transwell assay. Moreover, luciferase reporter assay, quantitative real time PCR and western blotting were used to identify and verify the direct downstream target of miR-15a-3p. Results: We found that the expression of miR-15a-3p was down-regulated in both PCa tissues and cell lines. The in vitro results showed that miR-15a-3p overexpression suppressed cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) via down-regulating Wnt/β-catenin signaling in PCa cells. Moreover, SLC39A7 was a direct downstream target of miR-15a-3p. Furthermore, SLC39A7 overexpression attenuated the effects of miR-15a-3p on cell proliferation, invasion, Wnt/β-catenin pathway and EMT molecules. Conclusions: In summary, our study indicated that miR-15a-3p inhibited the proliferation, invasion, and EMT process of PCa cells via targeting SLC39A7 and suppressing Wnt/β-catenin signaling pathway, which may represent a new therapeutic objective for PCa treatment.

Abstract P2-01-14: Adipocytes and cancer cell interactions promote leptin receptor expression and drive β-catenin-mediated progression in triple negative breast cancer

Abstract Background Triple negative breast cancer (TNBC) patients suffer from poor prognosis due to short-term recurrence and/or metastasis. Obesity is reported to predict poor prognosis of TNBC. However, the mechanisms connecting obesity to progression of TNBC remain unclarified. Methods We co-cultured TNBC model cells with adipocytes differentiated from human adipose-derived mesenchymal stem cells (hMSC-ad) and TNBC patients derived primary adipocytes to examine migration, invasion and metastasis abilities using Transwell assays and tail vein injection xenograft mouse model, respectively. Expression levels of leptin receptor (LEPR) , epithelial-mesenchymal transition (EMT) markers and β-catenin signaling pathway related molecules in the control and co-cultured TNBC model were analyzed by real time quantitative polymerase chain reaction (RT-qPCR) and western blot assays. Downregulation of LEPR by LEPR specific short hairpin RNA (shRNA) and upregulation of the downstream pathway with continuous activated β-catenin were applied to access their roles in adipocytes and cancer cell interactions. Degree of adipocytes infiltration in tumor tissue was evaluated by histopathology. LEPR mRNA expression level in breast cancer and corresponding patients' information was retrieved from The Cancer Genome Atlas (TCGA) database. Results Here we first reported that both hMSC-ad- and patients-derived adipocytes promoted migration and invasion of TNBC model cells in vitro and lung metastasis in vivo. Adipocytes were then confirmed to induce upregulation of LEPR in each TNBC model cell compared to the control groups (p&amp;lt;0.0001), resulting in higher expression of EMT markers and molecules related to β-catenin signaling pathway.Afterwards, LEPR knockdown led to decreased migration, invasion and metastasis capacities, as well as reduced expression of EMT markers and molecules related to ?-catenin signaling pathway, while activation of ?-catenin could restore robust EMT and metastatic abilities of TNBC model cells. Last, clinical specimen analyses showed that infiltration of adipocytes to tumor tissue was significantly associated with shorter DFS of TNBC patients (p&amp;lt;0.05) and upregulated LEPR in breast cancer tissue indicated poor prognosis (median OS of 7.43 vs 10.81 years of the LEPR-low group, p &amp;lt; 0.05). Conclusions To our knowledge, adipocytes are first shown to promote progression of TNBC via previously uncharacterized LEPR-β-catenin signaling pathway. Citation Format: Feng Z, Tao Z, Hu X. Adipocytes and cancer cell interactions promote leptin receptor expression and drive β-catenin-mediated progression in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-14.

Fisetin Inhibited Growth and Metastasis of Triple-Negative Breast Cancer by Reversing Epithelial-to-Mesenchymal Transition via PTEN/Akt/GSK3β Signal Pathway.

Triple negative breast cancer (TNBC), characterized by its highly aggressive and metastatic features, is associated with poor prognosis and high mortality partly due to lack of effective treatment. Fisetin, a natural flavonoid compound, has been demonstrated to possess anti-cancer effects in various cancers. However, the effects and mechanisms of fisetin on metastasis of TNBC remain uncovered. In this study, we found that fisetin dose-dependently inhibited cell proliferation, migration and invasion in TNBC cell lines MDA-MB-231 and BT549 cells. In addition, fisetin reversed epithelial to mesenchymal transition (EMT) as evaluated by cell morphology and EMT markers in MDA-MB-231 and BT549 cells. Furthermore, fisetin suppressed phosphoinositol 3-kinase (PI3K)-Akt-GSK-3β signaling pathway but upregulated the expression of PTEN mRNA and protein in a concentration-dependent manner. Further, silence of PTEN by siRNA abolished these benefits of fisetin on proliferation and metastasis of TNBCs. In vivo, using the metastatic breast cancer xenograft model bearing MDA-MB-231 cells, we found that fisetin dramatically inhibited growth of primary breast tumor and reduced lung metastasis, meanwhile, the expression of EMT molecules and PTEN/Akt/GSK-3β in primary and metastatic tissues changed in the same way as those in vitro experiments. In conclusion, all these results indicated that fisetin could effectively suppress proliferation and metastasis of TNBC and reverse EMT process, which might be mediated by PTEN/Akt/GSK-3β signaling pathway.

The Correlation Between the Immune and Epithelial-Mesenchymal Transition Signatures Suggests Potential Therapeutic Targets and Prognosis Prediction Approaches in Kidney Cancer

Both epithelial-mesenchymal transition (EMT) and immune regulation are important biological process in malignant tumours. The current research aims to comprehensively explore the potential association between the epithelial-mesenchymal transition (EMT) signature and immune checkpoint signature and its role in predicting the prognosis of clear-cell renal cell carcinoma (ccRCC) patients. EMT-related genes were collected from an experiment-based study and then were investigated using data from the Cancer Genome Atlas. A total of 357 genes were included, and 23 of them that were upregulated and correlated with prognosis were analysed further as core EMT genes in ccRCC. Interestingly, the emerging immune checkpoints CD276, OX40 and TGFB1 were found to be significantly co-expressed with core EMT genes, and TGFB1, CXCR4, IL10, and IL6 were the most important molecules potentially interacting with EMT molecules in our model, as determined from mRNA co-expression and protein-protein interaction network analysis. Additionally, an integrated scoring model based on FOXM1, TIMP1 and IL6 was successfully established to distinguish ccRCC patients with different clinical risks. Our results identified core genes in the EMT-immunophenotyping correlation and evaluated their risk assessment capabilities, providing more potential therapeutic targets and prediction approaches regarding the translational research of treatment and prognosis in ccRCC.

Zoledronic Acid Suppresses Epithelial-to-Mesenchymal Transition and Invasion via Degradation of Ubiquitinated NEDD9 in PC-3 Prostate Cancer Cells

Objective: Zoledronic acid (ZA) is highly effective in the treatment of castration-resistant prostate cancer (CRPC) patients with bone metastases. It is one of bone modifying agents (BMAs) that has been shown to exert not only inhibiting the activation of osteoclasts but also preventing the tumor cell growth, invasion and migration in some cancers. Neural precursor cell-expressed developmentally downregulated protein 9 (NEDD9) is a key regulator of tumor aggressiveness including invasion, epithelial-to-mesenchymal transition (EMT), dedifferentiation and resistance to chemo-drugs. However, research into a biological mechanism in the inhibitory effects of ZA on prostate cancer (PCa) metastasis is still limited. In this study, we examined its effects on tumor cell invasion and EMT via the ubiquitin-proteasomal system for NEDD9 in PC-3 cells.Methods: We assessed the expression of NEDD9 and its down-stream molecules associated with EMT in PC-3 cells exposure to ZA under the condition with/without TGF-β. By a boyden chamber assay, the suppressive effect of ZA on PC-3 cell invasion triggered by TGF-β was measured. We measured the expression levels of NEDD9 in PC-3 cells exposure to a proteasome inhibitor, MG132. In addition, we detected the effect of ZA on ubiquitinated NEDD9 using an immunoprecipitation method.Results: ZA markedly inhibited the expression of NEDD9 and its down-stream EMT molecules. Both the invasion and expression of EMT markers of PC-3 cells triggered by TGF-β were significantly suppressed by the exposure to ZA. The exposure to MG132 inhibited the degradation of NEDD9 in PC-3 cells. The further add-on of ZA enhanced the polyubiquitination of NEDD9 in PC-3 cells.Conclusion: The results from a current study indicate that ZA inhibited the invasion and expression of NEDD9 and its EMT markers, along with the enhanced degradation of ubiquitinated NEDD9 in PC-3 cells.

Baicalin inhibits the metastasis of highly aggressive breast cancer cells by reversing epithelial-to-mesenchymal transition by targeting β-catenin signaling.

Metastasis is the main cause of death in breast cancer patients, which is due partly to the lack of effective treatment. Baicalin, a flavonoid compound isolated from the roots of Scutellaria lateriflora Georgi (Huang Qin), has recently been confirmed as an effective agent for the treatment of a variety of cancers. Yet, the effects and underlying molecular mechanisms of baicalin in regards to the metastasis of breast cancer remain unclear. In the present study, we found that baicalin had the potential to suppress the migration and invasion of highly aggressive breast cancer cells in a dose-dependent manner but had no impact on the viability of these cancer cells. Additionally, baicalin reversed the epithelial-to-mesenchymal transition (EMT) process, as evaluated by EMT markers in breast cancer cell lines with a change from a mesenchymal feature to an epithelial type. At the same time, the expression of β-catenin mRNA and protein was dose-dependently downregulated by baicalin in highly invasive breast cancer cell lines, and overexpression of β-catenin by adenoviruses abolished these beneficial effects of baicalin in regards to the migration and invasion, and EMT of breast cancer cells. Furthermore, using a xenograft mouse model, baicalin markedly reduced liver and lung metastasis of breast cancer, inhibited expression of β-catenin, and degraded the EMT molecules vimentin and Slug in the orthotopic tumor tissues. Taken together, all these results indicate that baicalin effectively suppresses the metastasis of breast cancer by reversing EMT, which may be mediated by downregulation of β-catentin expression.