1499

CTNNB1

The nuclear p68 RNA helicase (referred to as p68) is a prototypical member of the DEAD box family of RNA helicases. The protein plays a very important role in early organ development. In the present study, we characterized the tyrosine phosphorylation of p68 under platelet-derived growth factor (PDGF) stimulation. We demonstrated that tyrosine phosphorylation of p68 at Y593 mediated PDGF-stimulated epithelial-mesenchymal transition (EMT). We showed that PDGF treatment led to phosphorylation of p68 at Y593 in the cell nucleus. The Y593-phosphorylated p68 (referred to as phosphor-p68) promotes beta-catenin nuclear translocation via a Wnt-independent pathway. The phosphor-p68 facilitates beta-catenin nuclear translocation by blocking phosphorylation of beta-catenin by GSK-3beta and displacing Axin from beta-catenin. The beta-catenin nuclear translocation and subsequent interaction with the LEF/TCF was required for the EMT process. These data demonstrated a novel mechanism of phosphor-p68 in mediating the growth factor-induced EMT and uncovered a new pathway to promote beta-catenin nuclear translocation.

OBJECTIVE: To observe the expressions of E-cadherin and beta-catenin in LNCaP and ARCaP cell lines and explore their relationship with the metastasis of human prostate cancer. METHODS: The expressions and distribution of E-cadherin and beta-catenin in LNCaP and ARCaP cell lines (IF11 and IA8) were detected by Western blot and immunofluorescent staining. RESULTS: The expression of E-cadherin was high in LNCaP, but absent in IF11 and IA8, while beta-catenin was expressed highly in IF11 and LA8, but lowly in LNCaP. Immunofluorescent staining showed that E-cadherin was mainly in the membrane of LNCaP, while beta-catenin both in the membrane of LNCaP and in the nuclei of IF11 and IA8. CONCLUSION: E-cadherin and beta-catenin are differently expressed and distributed in prostate cancer cell lines with different characteristics of epithelial-mesenchymal transition (EMT), and the abnormal activation of the beta-catenin signal pathway may be involved in the EMT of prostate cancer cells.

OBJECTIVE: epithelial-mesenchymal transition (EMT) is an important process in tumor development. Several studies suggest that the beta-catenin signal pathway may play an important role in EMT. However, there is no direct evidence showing that this pathway actually determines the EMT induced by exogenous signal. Our previous study has successfully proved that over-expression of HIF-1alpha could induce EMT in LNCaP cells, but not in PC-3. So the present study was intended to indicate that the signal of HIF-1alpha for inducing prostate cancer cell to undergo EMT might pass through the beta-catenin pathway. METHODS: Firstly, we analyzed the expression of HIF-1alpha and its target proteins in LNCaP/HIF1alpha and PC-3/HIF1alpha by Western blot. And then EMT-associated proteins were detected by Western blot. Furthermore the potency of invasiveness and proliferation of several cell lines were evaluated by transwell and MTT assay. Lastly the expressions of beta-catenin and GSK-3beta in these cells were analyzed by Western blot and RT-PCR. RESULTS: HIF-1alpha, Glut-1 and VEGF were highly expressed in LNCaP/HIF1alpha and PC-3/HIF1alpha. And PC-3, LNCaP and PC-3/HIF1alpha were EMT-negative cell lines whereas LNCaP/HIF1alpha and IA8 had undergone EMT process. LNCaP/HIF1alpha, IA8 and PC-3/HIF1alpha exhibited a much stronger potency of invasiveness and proliferation than those of PC-3 and LNCaP. The protein levels of total GSK-3beta and phospho-GSK-3beta in LNCaP/HIF1alpha, IA8 and PC-3/HIF1alpha cells significantly decreased; however, the relative ratios of p-GSK3beta/t-GSK3beta increased. Consistently, beta-catenin protein expression increased in LNCaP/HIF1alpha and IA8 cells, but not in PC-3/HIF1alpha; RT-PCR confirmed these results, except for an enhanced transcription activity of beta-catenin mRNA in PC-3/HIF1alpha. CONCLUSION: The activation of the beta-catenin signaling pathway correlates with the characteristic of EMT and potency of invasiveness and proliferation. And it may be one critical factor directly controlling the process of EMT induced by HIF-1alpha in prostate cancer.

OBJECTIVE: Wnt/beta-catenin signaling pathway regulates pattern formation during embryogenesis as well as tumor progression. Numbers of studies suggest that this signaling pathway may play an important role in epithelial-mesenchymal transition (EMT), however, there was no evidence that Wnt/beta-catenin signaling pathway directly controlled the EMT occurrence. Our previous research has successfully proved that overexpression of hypoxia inducible factor-1alpha (HIF-1alpha) could induce EMT in LNCaP cells, but not in PC-3. Consistently, the expression of beta-catenin protein increased in LNCaP/HIF-1alpha cells, but not in PC-3/HIF-1alpha. This study mainly aimed at exploring the essentiality and importance of Wnt/beta-catenin signaling pathway in HIF-1alpha-induced EMT. METHODS: Human prostate cancer cells (LNCaP) were stably transfected by recombinant plasmid pcDNA3.1(-)/HIF-1alpha. The positive clones were selected by G418 and confirmed through western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and indirect immunofluoesence. Then LNCaP/HIF-1alpha was transiently transfected with beta-catenin shRNA (shRNA1 and shRNA2) and negative shRNA (shRNA-scr). The epithelial markers, mesenchymal markers, and critical proteins in Wnt/beta-catenin signaling pathway were separately detected by western blot analysis. Finally, the invasive potency of cells in different transfection group was examined by Matrgel transwell assay. RESULT: We successfully established prostate cancer cell line LNCaP/HIF-1alpha and LNCaP/HIF-1alpha/beta-catenin(-). LNCaP/HIF-1alpha displayed high expression of mesenchymal markers and low expression of epithelial markers. However, compared with LNCaP/HIF-1alpha, the epithelial marker E-cadherin was increased in LNCaP/HIF-1alpha/beta-catenin(-), whereas the expression of mesenchymal marker N-cadherin, vimentin, MMP-2 were significantly decreased. Inhibition of Wnt signal activity through beta-catenin shRNA cause a reversal of EMT induced by HIF-1alpha in human prostate cancer. CONCLUSION: Overexpression of HIF-1alpha stimulates the invasion potency of human prostate carcinoma cells through EMT pathway and Wnt/beta-catenin signaling pathway played a vital role in this process. Wnt/beta-catenin signaling pathway might be a necessary endogenous signal that directly controlled the EMT occurrence induced by HIF-1alpha.

In most carcinomas, invasion of malignant cells into surrounding tissues involves their molecular reprogramming as part of an epithelial-to-mesenchymal transition (EMT). mutation of the APC gene in most colorectal carcinomas (CRCs) contributes to the nuclear translocation of the oncoprotein beta-catenin that upon binding to T-cell and lymphoid enhancer (TCF-LEF) factors triggers an EMT and a proinvasive gene expression profile. A key inducer of EMT is the ZEB1 transcription factor whose expression promotes tumorigenesis and metastasis in carcinomas. As inhibitor of the epithelial phenotype, ZEB1 is never present in the epithelium of normal colon or the tumor center of CRCs where beta-catenin remains membranous. We show here that ZEB1 is expressed by epithelial cells in intestinal tumors from human patients (familial adenomatous polyposis) and mouse models (APC(Min/+)) with germline mutations of APC that result in nuclear accumulation of beta-catenin. However, ZEB1 is not expressed in the epithelium of hereditary forms of CRCs that carry wild-type APC and where beta-catenin is excluded from the nucleus (Lynch syndrome). We found that beta-catenin/TCF4 binds directly to the ZEB1 promoter and activates its transcription. Knockdown of beta-catenin and TCF4 in APC-mutated CRC cells inhibited endogenous ZEB1, whereas forced translocation of beta-catenin to the nucleus in APC-wild-type CRC cells induced de novo expression of ZEB1. Upregulation of MT1-MMP and LAMC2 by beta-catenin/TCF4 has been linked to invasiveness in CRCs, and we show here that both proteins are activated by ZEB1 coexpressing with it in primary colorectal tumors with mutated APC. These results set ZEB1 as an effector of beta-catenin/TCF4 signaling in EMT and tumor progression.

We previously showed that methyl-F-anandamide, a stable analogue of the anandamide, inhibited the growth and the progression of cultured human breast cancer cells. As accumulating evidences indicate that the constitutive activation of the canonical Wnt pathway in human breast cancer may highlight a key role for aberrant activation of the beta-catenin-TCF cascade and tumour progression, we studied the anandamide effect on the key elements of Wnt pathway in breast cancer cells. In this study we described that the treatment of human breast cancer cells, MDA MB 231 cells, with methyl-F-anandamide reduced protein levels of beta-catenin in the cytoplasmic and nuclear fractions inhibiting the transcriptional activation of T Cell Factor (TCF) responsive element (marker for beta-catenin signalling). The anandamide treatment resulted in up-regulation of epithelial markers, like E-cadherin with a concomitant decrease in protein levels of mesenchymal markers, including vimentin and Snail1. We, furthermore, observed that the induction of experimental epithelial-mesenchymal transition by exposure to adriamycin in MCF7 human breast cancer cell line was inhibited by anandamide treatment. In the present study we reported a novel anticancer effect of anandamide involving the inhibition of epithelial-mesenchymal transition, a process triggered during progression of cancer to invasive state.CI - Copyright (c) 2012 Elsevier Ltd. All rights reserved.

BACKGROUND: Previous research has suggested a putative utility of pancreatic (pro)enzymes in cancer treatment. The aim of the present study was to investigate the in vitro effects of a mixture of two pancreatic pro-enzymes, i.e., Chymotrypsinogen and Trypsinogen, and the enzyme Amylase on three human cancer cell lines, i.e., OE33 (derived from an oesophageal carcinoma), Panc1 (derived from a pancreatic carcinoma) and Caco-2 (derived from a colon carcinoma). RESULTS: After treatment of the three cancer cell lines with different doses of the (pro)enzymes for up to 7 days, we observed (i) growth inhibition in a dose-dependent manner, (ii) enhanced expression of beta-catenin and E-cadherin and decreased expression of several epithelial-mesenchymal transition (EMT)-associated genes, such as Vimentin, Snail and Slug, (iii) differentiation of Caco-2 cells, including the appearance of cell-specific differentiated structures such as microvilli and tight junctions, the acquisition of a more regular polygonal morphology, and an increased expression of the intestinal differentiation markers alkaline phosphatase and cytokeratin 8, and (iv) differentiation of Panc1 cells, including the formation of cell aggregates, an increment on lamellar bodies and an increased expression of the pancreatic differentiation markers glucagon and insulin. CONCLUSIONS: Our results show that the treatment of three different human cancer cell lines with pancreatic (pro)enzymes results in an enhancement of cell adhesion, an attenuation of several EMT-associated markers, and an increase in the expression of several differentiation-associated markers, suggesting the acquisition of a less malignant phenotype and a decrease in proliferative capacity due to lineage-specific cellular differentiation.

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer deaths worldwide. In recent studies, a crucial link has been discovered between the acquisition of metastatic traits and tumour-initiating abilities in cancer cells during the epithelial-mesenchymal transition (EMT). Herein, we demonstrated that the ectopic expression of TWIST1, the EMT regulator, in HNSCC FaDu cells triggered EMT and resulted in the acquisition of a mesenchymal phenotype. Moreover, FaDu-pFLAG-TWIST1 cancer cell populations that were induced to EMT displayed an increased proportion of cells with the CD44 marker, which is associated with tumour initiation. Interestingly, we found that emodin treatment reduced the tumour-initiating abilities and inhibited cell migration and invasion in FaDu-pFLAG-TWIST1 cells. Emodin directly inhibited TWIST1 expression, upregulated E-cadherin mRNA and protein expression, and downregulated vimentin mRNA and protein expression. Moreover, we found that emodin inhibited TWIST1 binding to the E-cadherin promoter and repressed E-cadherin transcription activity. We also found that emodin inhibited TWIST1-induced EMT by inhibiting the beta-catenin and Akt pathways. More interestingly, emodin significantly inhibited TWIST1-induced invasion in vivo. Therefore, emodin might be applicable to anticancer therapy and could be a potential new therapeutic drug for HNSCC.CI - Copyright (c) 2013 Elsevier Ltd. All rights reserved.

Cisplatin is a first-line chemotherapeutic agent in the treatment of non-small cell lung cancer (NSCLC), but the therapeutic effect is disappointing, partly due to drug resistance. Emerging evidence showed that chemoresistance associates with acquisition of epithelial-mesenchymal transition (EMT) phenotype and cancer stem cell-like properties. However, the underlying mechanism is not entirely clear. In this study, we showed that cisplatin-resistant A549 cells (A549/CDDP) acquire EMT phenotype associated with migratory and invasive capability. A549/CDDP cells also displayed enhanced cancer stem cell-like properties. Increased expression of transcription factor Snail, but not ZEB1, Slug and Twist, was observed in A549/CDDP cells. Knockdown of Snail reversed EMT and significantly attenuated migration, invasion and cancer stem cell-like properties of A549/CDDP cells. Conversely, overexpressed Snail in A549 cells induced EMT and cancer stem cell-like properties. Finally, we demonstrated that activated AKT signal leads to increased beta-catenin expression and subsequently up-regulates Snail in A549/CDDP cells. Taken together, these results revealed that AKT/beta-catenin/Snail signaling pathway is mechanistically associated with cancer stem cell-like properties and EMT features of A549/CDDP cells, and thus, this pathway could be a novel target for the treatment of NSCLC.CI - (c) 2013 Published by Elsevier B.V.

BACKGROUND: beta-Catenin is a potent oncogenic protein in colorectal cancer (CRC), but the targets and regulation of this important signalling molecule are not completely understood. Hypoxia is a prominent feature of solid tumours that contributes to cancer progression. METHODS: Here, we analysed the regulation between Nur77 and beta-catenin under hypoxic conditions. Cell proliferation, migration, and invasion assays were performed to assess functional consequences. RESULTS: We showed that hypoxia stimulated co-upregulation of beta-catenin and Nur77 in a number of human CRC cell lines. Interestingly, expression of beta-catenin and Nur77 by hypoxia formed a mutual feedback regulation circuits that conferred aggressive growth of CRC. Overexpression of beta-catenin increased Nur77 transcription through hypoxia-inducible factor-1alpha rather than T-cell factor. Nur77-mediated activation of beta-catenin by hypoxia was independent of both DNA binding and transactivation. Further, we showed that hypoxic activation of beta-catenin was independent of the classical adenomatous polyposis coli and p53 pathways, but stimulated by phosphatidylinositol 3-kinase/Akt in a Nur77-dependent manner. Under hypoxic conditions, enhanced beta-catenin and Nur77 expression synergistically stimulated CRC cell migration, invasion, and epithelial-mesenchymal transition. CONCLUSION: These findings provide a novel molecular mechanism for hypoxic CRCs that may contribute to tumour progression, and its targeting may represent an effective therapeutic avenue.

SOX2 is a high-mobility group box containing transcription factor essential for the maintenance of embryonic stem cells. Recent evidence indicates that SOX2 overexpression correlates with metastasis and poor prognosis in patients with laryngeal squamous cell cancer. To investigate how SOX2 contributes to this aggressive phenotype, we introduced the human SOX2 gene into a low SOX2-expressing human laryngeal cancer cell line Hep-2. Cell migration and invasion were determined by the Transwell assay with or without Matrigel coating. The epithelial-mesenchymal transition (EMT)-related markers were assayed by Western blot analysis or immunofluorescence. Our results showed that exogenous expression of SOX2 in Hep-2 cells substantially promoted their migratory and invasive capabilities in culture. Moreover, Hep-2 cells stably overexpressing SOX2 underwent EMT phenotype, as evidenced by mesenchymal morphology, decreased expression of epithelial marker (E-cadherin), and increased expression of mesenchymal markers (N-cadherin, vimentin, fibronectin, and alpha-smooth muscle actin). Strikingly, Western blot analysis and immunofluorescence also showed that overexpression of SOX2 resulted in substantial increase and nuclear accumulation of beta-catenin in Hep-2 cells. However, small interfering RNA targeting beta-catenin significantly attenuated the reduced expression of E-cadherin and increased cell migration and invasion abilities in SOX2-overexpressing cells, suggesting that SOX2-induced EMT process, migration, and invasion are dependent on beta-catenin activation. Taken together, our findings underscore a novel role for SOX2 in laryngeal cancer migration and invasion.

Increased expression of the molecular chaperone Hsp27 is associated with the progression of prostate cancer (PCa) to castration-resistant disease, which is lethal due to metastatic spread of the prostate tumor. Metastasis requires epithelial to mesenchymal transition (EMT), which endows cancer cells with the ability to disseminate from the primary tumor and colonize new tissue sites. A wide variety of secreted factors promote EMT, and while overexpression and constitutive activation of epidermal growth factor (EGF) signaling is associated with poor prognosis of PCa, a precise role of EGF in PCa progression to metastasis remains unclear. Here, we show that Hsp27 is required for EGF-induced cell migration, invasion and MMPs activity as well as the expression of EMT markers including Fibronectin, Vimentin and Slug with concomitant decrease of E-cadherin. Mechanistically, we found that Hsp27 is required for EGF-induced AKT and GSK3beta phosphorylation and beta-catenin nuclear translocation. Moreover, silencing Hsp27 decreases EGF dependent phosphorylation of beta-catenin on tyrosine 142 and 654, enhances beta-catenin ubiquitination and degradation, prevents beta-catenin nuclear translocation and binding to the Slug promoter. These data suggest that Hsp27 is required for EGF-mediated EMT via modulation of the beta-catenin/Slug signaling pathway. Together, our findings underscore the importance of Hsp27 in EGF induced EMT in PCa and highlight the use of Hsp27 knockdown as a useful strategy for patients with advanced disease.CI - (c) 2014 UICC.

BACKGROUND: Distant metastasis is the major cause of cancer-related death, and epithelial-to-mesenchymal transition (EMT) has a critical role in this process. Accumulating evidence indicates that EMT can be regulated by microRNAs (miRNAs). miR-29c has been implicated as a tumor suppressor in several human cancers. However, the role of miR-29c in the progression of colorectal cancer (CRC) metastasis remains largely unknown. PATIENTS AND METHODS: The expression of miR-29c was examined by qRT-PCR in a cohort of primary CRC (PC) and distant liver metastasis (LM) tissues. A series of in vivo and in vitro assays were carried out in order to elucidate the functions of miR-29c and the molecular mechanisms underlying the pathogenesis of metastatic CRC. RESULTS: miR-29c was markedly downregulated in PCs with distant metastasis and determined to be an independent predictor of shortened patient survival. But LM tissues showed higher levels of miR-29c than that in PC tissues. In CRC cells, miR-29c dramatically suppressed cell migration and invasion abilities in vitro and cancer metastasis in vivo. In addition, miR-29c inhibited EMT and negatively regulated Wnt/beta-catenin signaling pathway. Guanine nucleotide binding protein alpha13 (GNA13) and protein tyrosine phosphatase type IVA (PTP4A) were identified as direct targets of miR-29c, which acted through ERK/GSK3beta/beta-catenin and AKT/GSK3beta/beta-catenin pathways, respectively, to regulate EMT. Furthermore, significant associations between miR-29c, its target genes (GNA13 and PTP4A) and EMT markers were validated in both PC and LM tissues. CONCLUSION: Our findings highlight the important role of miR-29c in regulating CRC EMT via GSK-3beta/beta-catenin signaling by targeting GNA13 and PTP4A and provide new insights into the metastatic basis of CRC.CI - (c) The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is suggested to be a crucial factor for the development of an invasive and metastatic cell phenotype, which is characterized by down-regulation of epithelial adhesive proteins (e.g. E-cadherin) and induction of mesenchymal proteins (e.g. vimentin). Therefore, there is a great clinical interest to specify this phenotype. Different growth factors induce EMT, such as epithelial growth factor (EGF) and transforming growth factor beta 1 (TGFbeta1). The role of EMT in human papilloma virus (HPV)-positive squamous cell carcinoma (SCC) is still not understood. The aim of this study was to investigate the expression pattern in p16-positive and -negative SCC cells of vimentin, beta-catenin and E-cadherin after stimulation with growth factors. MATERIALS AND METHODS: We incubated the p16-positive CERV196 and p16-negative HNSCC22B SCC cell lines with EGF and EGF/TGFbeta1 (10 ng/ml) and detected E-cadherin, vimentin and beta-catenin by immunocytochemistry and enzyme-linked immunosorbent assay after 5, 24 and 96 h. RESULTS: We found a low expression of vimentin in all studied tumor cell lines. The negative control of HNSCC22B cells showed a higher intrinsic level of membranous E-cadherin and beta-catenin. We found statistically significant EGF/TGFbeta1-induced expression of vimentin dependent on incubation time in p16-negative HNSCC22B cells. Particularly in the presence of EGF, we detected an increase of beta-catenin and vimentin expression in p16-positive SCC tumor cell lines in addition to induced cell scattering and unexpected expression of E-cadherin. CONCLUSION: In conclusion, E-cadherin, beta-catenin and vimentin expression are important features to characterize EMT-like events. We were able to show incomplete EGF-induced EMT with beta-catenin expression in p16-positive SCC. Extended studies are required to investigate the mechanistic role of EMT markers, especially in p16-positive SCC, in order to develop new anti-SCC therapies to block EMT progression.CI - Copyright(c) 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

BACKGROUND: Wnt/beta-catenin signaling pathway plays a key role in human breast cancer progression. In this study, we down regulated beta-catenin expression in human breast cancer MDA-MB-231 cells and investigated the effect of beta-catenin knockdown on the cell biological characteristics. MATERIALS AND METHODS: The recombinant plasmids of pSUPER-enhancement green fluorescent protein 1 (EGFP1)-scrabble-beta-catenin-short hairpin ribonucleic acid (shRNA) and pSUPER-EGFP1-beta-catenin-shRNA-1 were transfected into MDA-MB-231 cells, respectively, and the stably transfected cells were isolated from G418 selected clones. The beta-catenin gene silenced efficiency was measured by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) and Western blot. The biological characteristics of MDA-MB-231 cells with down regulated beta-catenin were evaluated by analyzing cell proliferation, clonogenicity, cell mobility and tumorigenicity. The expression of E-cadherin and Vimentin was concurrently detected by QRT-PCR. RESULTS: The beta-catenin-shRNA-1 stably transfected MDA-MB-231 cells significantly decreased beta-catenin expression, cell proliferation, clonogenicity, and tumorigenicity in Balb/c nude mice compared with the MDA-MB-231 cells transfected with pSUPER-EGFP1-scrabble-beta-catenin-shRNA. Interestingly, knockdown of beta-catenin led to the reduction of epithelial E-cadherin expression, the increase of cell mobility and mesenchymal vimentin expression in MDA-MB-231 cells, indicating an epithelial to mesenchymal transition. CONCLUSION: Knockdown of beta-catenin expression in human breast cancer MDA-MB-231 cells inhibits cell tumorigenicity in mice, but promotes cell epithelial-mesenchymal transition.

Adrenal carcinoma (ACC) is a rare neoplasm with a poor outcome. Aberrant expression of beta-catenin has been found in approximatively 30% of ACC. We herein studied its effects on the growth of the human ACC cell line H295R. The cells were infected with short hairpin RNA (shRNA)-mediated silencing beta-catenin. Two shRNAs used induced down-regulation of beta-catenin protein levels. The expression of these shRNAs decreased cell growth and increased H295R cells in S and G2/M phases. This cytostatic effect is due to a decrease of phosphorylated MAPK and to an up-regulation expression of the cyclin-dependent kinase inhibitors p57(KIP2), p21(CIP) and p27(KIP1). In addition, the knockdown of beta-catenin decreased phosphorylated Akt and increased apoptosis. Finally, loss of beta-catenin was sufficient to induce the reversal of the epithelial-to-mesenchymal transition. We then transplanted these genetically modified H295R cells in Scid mice. tumor growth suppression was achieved by the two shRNAs showing in vitro efficacy. Proliferation was not reduced in silenced tumors. In contrast, p57, p27 and p21 proteins were found expressed at high levels in silenced tumors along with an increase in apoptotic cells. These findings indicate that beta-catenin loss in H295R cells inhibits tumor growth by inducing transcriptional and functional changes.

The canonical Wnt/beta-catenin signaling pathway has been shown to promote the epithelial-mesenchymal transition (EMT), which is a crucial process in multiple embryonic developmental processes and the progression of carcinomas. We recently provided evidence that leucine-rich repeat flightless-1-interacting protein 1 (LRRFIP1) promotes cancer metastasis and invasion. In the present study, we identified the signaling elements targeted by LRRFIP1 for promotion of the EMT in pancreatic and lung cancer. LRRFIP1 silencing reversed the EMT, as shown by increased expression of E-cadherin (an epithelial marker) and decreased expression of vimentin (a mesenchymal marker). Silencing of LRRFIP1 up-regulated phosphorylation of beta-catenin and decreased its nuclear localization by targeting the beta-catenin destruction complex. The expression of beta-catenin and E-cadherin in the plasma membrane fraction was increased in LRRFIP1 silenced cancer cells, and the migration and invasion capabilities were strongly inhibited. In addition, this protein was highly expressed at the invasion front of malignant tissue collected from pancreatic cancer patients. Consequently, our data strongly suggested that LRRFIP1 played an important role in the invasion of carcinoma cells. Our data provide experimental evidence that LRRFIP1 is an attractive candidate for targeted therapy in human cancers.CI - Copyright (c) 2015 Elsevier Ireland Ltd. All rights reserved.

Wnt signaling contributes to the reprogramming and maintenance of cancer stem cell (CSC) states that are activated by epithelial-mesenchymal transition (EMT). However, the mechanistic relationship between EMT and the Wnt pathway in CSC is not entirely clear. Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) indicated that EMT induces a switch from the beta-catenin/E-cadherin/Sox15 complex to the beta-catenin/Twist1/TCF4 complex, the latter of which then binds to CSC-related gene promoters. Tandem coimmunoprecipitation and re-ChIP experiments with epithelial-type cells further revealed that Sox15 associates with the beta-catenin/E-cadherin complex, which then binds to the proximal promoter region of CASP3. Through this mechanism, Twist1 cleavage is triggered to regulate a beta-catenin-elicited promotion of the CSC phenotype. During EMT, we documented that Twist1 binding to beta-catenin enhanced the transcriptional activity of the beta-catenin/TCF4 complex, including by binding to the proximal promoter region of ABCG2, a CSC marker. In terms of clinical application, our definition of a five-gene CSC signature (nuclear beta-catenin(High)/nuclear Twist1(High)/E-cadherin(Low)/Sox15(Low)/CD133(High)) may provide a useful prognostic marker for human lung cancer.CI - (c)2015 American Association for cancer Research.

The epithelium of asthmatics is characterized by reduced expression of E-cadherin and increased expression of the basal cell markers ck-5 and p63 that is indicative of a relatively undifferentiated repairing epithelium. This phenotype correlates with increased proliferation, compromised wound healing and an enhanced capacity to undergo epithelial-mesenchymal transition (EMT). The transcription factor beta-catenin plays a vital role in epithelial cell differentiation and regeneration, depending on the co-factor recruited. Transcriptional programs driven by the beta-catenin/CBP axis are critical for maintaining an undifferentiated and proliferative state, whereas the beta-catenin/p300 axis is associated with cell differentiation. We hypothesized that disrupting the beta-catenin/CBP signaling axis would promote epithelial differentiation and inhibit EMT. We treated monolayer cultures of human airway epithelial cells with TGFbeta1 in the presence or absence of the selective small molecule ICG-001 to inhibit beta-catenin/CBP signaling. We used western blots to assess expression of an EMT signature, CBP, p300, beta-catenin, fibronectin and ITGbeta1 and scratch wound assays to assess epithelial cell migration. Snai-1 and -2 expressions were determined using q-PCR. Exposure to TGFbeta1 induced EMT, characterized by reduced E-cadherin expression with increased expression of alpha-smooth muscle actin and EDA-fibronectin. Either co-treatment or therapeutic administration of ICG-001 completely inhibited TGFbeta1-induced EMT. ICG-001 also reduced the expression of ck-5 and -19 independent of TGFbeta1. Exposure to ICG-001 significantly inhibited epithelial cell proliferation and migration, coincident with a down regulation of ITGbeta1 and fibronectin expression. These data support our hypothesis that modulating the beta-catenin/CBP signaling axis plays a key role in epithelial plasticity and function.CI - Copyright (c) 2015 Elsevier Ltd. All rights reserved.

The Wnt/beta-catenin signalling pathway is activated in pancreatic cancer initiation and progression. Dickkopf-related protein 3 (DKK3) is a member of the human Dickkopf family and an antagonist of Wnt ligand activity. However, the function of DKK3 in this pathway in pancreatic cancer is rarely known. We examined the expression of DKK3 in six human pancreatic cancer cell lines, 75 pancreatic cancer and 75 adjacent non-cancerous tissues. Dickkopf-related protein 3 was frequently silenced and methylation in pancreatic cancer cell lines (3/6). The expression of DKK3 was significantly lower in pancreatic cancer tissues than in adjacent normal pancreas tissues. Further, ectopic expression of DKK3 inhibits nuclear translocation of beta-catenin induced by hypoxia in pancreatic cancer Bxpc-3 cell. The forced expression of DKK3 markedly suppressed migration and the stem cell-like phenotype of pancreatic cancer Bxpc-3 cell in hypoxic conditions through reversing epithelial-mesenchymal transition (EMT). The stable expression of DKK3 sensitizes pancreatic cancer Bxpc-3 cell to gemcitabine, delays tumour growth and augments gemcitabine therapeutic effect in pancreatic cancer xenotransplantation model. Thus, we conclude from our finding that DKK3 is a tumour suppressor and improved gemcitabine therapeutic effect through inducing apoptosis and regulating beta-catenin/EMT signalling in pancreatic cancer Bxpc-3 cell.CI - (c) 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Autocrine vascular endothelial growth factor (VEGF) can regulate the survival and progression of cancers through its various receptors. But the mechanisms and mediators for these functions are largely uncovered, especially in breast cancer. We examined the potential roles and mechanisms of VEGF/neuropilin-1 (NRP-1) axis in regulating the tumorigenesis and metastasis of breast cancer and found the expression of VEGF and NRP-1 correlated with aggressiveness of breast cancer. Knockdown of VEGF or NRP-1 inhibited the proliferation, migration and invasion, but enhanced the apoptosis of MDA-MB-231 cells. In contrast, induction of NRP-1 over-expression promoted the proliferation, migration and invasion of MCF-7 cells. VEGF or NRP-1 silencing attenuated the epithelial-mesenchymal transition (EMT) process and the activation of NF-kappaBp65, but enhanced GSK-3beta expression in MDA-MB-231 cells while NRP-1 over-expression reversed the effects in MCF-7 cells. Treatment with hVEGF165 did not change the inhibition in NRP-1 silencing MDA-MB-231 cells, but enhanced the aggressiveness of NRP-1 over-expressing MCF-7 cells. In addition, VEGF-silencing inhibited the growth and metastasis of implanted MDA-MB-231 tumors in vivo. Our novel data suggest that the positive regulation of the VEGF/NRP-1 axis on the tumorigenesis and metastasis of breast cancer may be associated with enhancing the EMT process and the NF-kappaB and beta-catenin signaling. Hence, the VEGF/NRP-1 axis may be a valuable target for design of therapies for intervention of breast cancer.CI - Copyright (c) 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

epithelial to mesenchymal transition (EMT) has received considerable attention as a conceptual paradigm for explaining the invasive and metastatic behavior of cells during cancer progression. Our previous study showed that loss of expression of TSSC3 is positively associated with osteosarcoma malignancy and progression. However, whether TSSC3 mediates EMT in osteosarcoma is poorly understood. In the present study, we determined that TSSC3 downregulation induced cell migration and invasion ability and promoted mesenchymal transition of osteosarcoma cells by upregulating mesenchymal markers and inhibiting the epithelial markers. Furthermore, TSSC3 downregulation elicited a signaling cascade that included increased levels of Wnt3a and LRP5, inactivation of GSK-3beta, accumulation of nuclear beta-catenin and Snail, the augmented binding of beta-catenin to TCF-4, and accordingly increased the expression of Wnt target genes (CD44, MMP7). The gene knockdown of these signaling proteins could inhibit TSSC3 downregulation-promoted EMT, migration, and invasion in osteosarcoma. Finally, TSSC3 overexpression obviously inhibited cell migration, invasion, and repressed mesenchymal phenotypes, reducing lung metastasis through GSK-3beta activation. Collectively, TSSC3 downregulation promotes the EMT of osteosarcoma cells by regulating EMT markers via a signal transduction pathway that involves Snail, Wnt-beta-catenin/TCF, and GSK-3beta.CI - Copyright (c) 2016 Elsevier Ireland Ltd. All rights reserved.

Cytochrome P450 1B1 (CYP1B1) is a major E2 hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[alpha]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including beta-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/beta-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/beta-catenin signaling via Sp1 induction.

epithelial to mesenchymal transition has been suggested as a relevant contributor to pulmonary fibrosis, but how and where this complex process is triggered in idiopathic pulmonary fibrosis is not fully understood. Beta-tubulin-III (Tubbeta3), ZEB1, and beta-catenin are partially under the negative control of miR-200, a family of micro-RNAs playing a major role in epithelial to mesenchymal transition, that are reduced in experimental lung fibrosis and idiopathic pulmonary fibrosis. We wonder whether in situ expression of these proteins is increased in idiopathic pulmonary fibrosis, to better understand the significance of miR-200 feedback loop and epithelial to mesenchymal transition. We investigated the immunohistochemical and immunofluorescent expression and precise location of ZEB1, Tubbeta3, and beta-catenin in tissue samples from 34 idiopathic pulmonary fibrosis cases and 21 controls (5 normal lungs and 16 other interstitial lung diseases). In 100% idiopathic pulmonary fibrosis samples, the three proteins were concurrently expressed in fibroblastic foci, as well in damaged epithelial cells overlying these lesions and in pericytes within neo-angiogenesis areas. These results were also confirmed by immunofluorescence assay. In controls the abnormal expression of the three proteins was absent or limited. This is the first study that relates concurrent expression of Tubbeta3, ZEB1, and beta-catenin to abnormal epithelial and myofibroblast differentiation in idiopathic pulmonary fibrosis, providing indirect but robust evidence of miR-200 deregulation and epithelial to mesenchymal transition activation in idiopathic pulmonary fibrosis. The abnormal expression and localization of these proteins in bronchiolar fibro-proliferative lesions are unique for idiopathic pulmonary fibrosis, and might represent a disease-specific marker in challenging lung biopsies.

Oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), occur with high frequency in hepatocellular carcinoma (HCC). The protein kinase Akt is considered to be the primary effector of PI3K, but there is evidence to suggest that serum and glucocorticoid kinase 3 (SGK3) acts in an Akt-independent manner downstream of PI3K. In this report, we found that SGK3 promotes epithelial-mesenchymal transition (EMT) and reduces phosphorylation-dependent degradation of beta-catenin in HCC cells. We determined that miR-155, previously shown to promote EMT, stimulates the expression of SGK3 by targeting and repressing P85alpha, thereby removing its inhibitory effect on PI3K-AKT signaling. These findings suggest that miR-155 promotes EMT and metastatic properties in HCC cells through activation of PI3K/SGK3/beta-catenin signaling pathways.

Recently, beta-arrestin1 was indicated as a tumor promoter in prostate cancer, but its exact role in cancer metastasis still have not been well clarified. Here, our data revealed that beta-arrestin1 could promote the migration and invasion of prostate cancer cells via initiating epithelial-mesenchymal transition (EMT). Mechanically, beta-arrestin1 could increase the transcriptional activity and expression of beta-catenin, together with Akt activity, whereas decrease the activities of GSK-3beta and PP2A. In addition, beta-arrestin1 could function as a scaffold protein in modulating the interactions between PP2A, Akt, GSK-3beta and beta-catenin. These results reveal a novel mechanism of beta-arrestin1 in modulating EMT and GSK-3beta/beta-catenin signaling in prostate cancer, thereby suggest that assessment of beta-arrestin1 may provide a potential therapeutic target for prostate cancer.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

We previously demonstrated that fermitin family member 1 (FERMT1) was significantly overexpressed in colon cancer (CC) and associated with poor metastasis-free survival. This study aimed to investigate the precise role of FERMT1 in CC metastasis and the mechanism by which FERMT1 is involved in the epithelial-mesenchymal transition (EMT). Correlations between FERMT1 and EMT markers (E-cadherin, Slug, N-cadherin and beta-catenin) were examined via immunohistochemistry in a cohort of CC tissues and adjacent normal colon mucosae. A series of in vitro and in vivo assays were performed to elucidate the function of FERMT1 in CC metastasis and underlying mechanisms. The upregulated expression of FERMT1 in CC tissues correlated positively with that of Slug, N-cadherin and beta-catenin, but correlated inversely with E-cadherin expression. Altered FERMT1 expression led to marked changes in the proliferation, migration, invasion and EMT markers of CC cells both in vitro and in vivo. Investigations of underlying mechanisms found that FERMT1 interacted directly with beta-catenin and activated the Wnt/beta-catenin signaling pathway by decreasing the phosphorylation level of beta-catenin, enhancing beta-catenin nuclear translocation and increasing the transcriptional activity of beta-catenin/TCF/LEF. Activation of the Wnt/beta-catenin pathway by CHIR99021 reversed the effect of FERMT1 knockdown, whereas inhibition of the Wnt/beta-catenin pathway by XAV939 impaired the effect of FERMT1 overexpression on EMT and cell motility. In conclusion, findings of this study suggest that FERMT1 activates the beta-catenin transcriptional activity to promote EMT in CC metastasis.

Filamin A interacting protein 1-like (FILIP1L) is an inhibitor of the canonical WNT pathway. WNT/beta-catenin signaling and its downstream pathway, epithelial-to-mesenchymal transition (EMT), play a key role in ovarian cancer metastasis and chemoresistance. To study the clinical implications of FILIP1L in regulating the WNT/beta-catenin pathway, the expression of FILIP1L, beta-catenin, SNAIL and SLUG was analyzed by immunohistochemistry on tissue microarrays of 369 ovarian samples ranging from normal to metastatic. In addition, the results were validated in mouse model and in vitro cell culture. In the present study, we demonstrated that FILIP1L expression was inversely correlated with poor prognosis, stage and chemoresistance in ovarian cancer. Notably, low FILIP1L expression was independent negative prognostic factor with respect to overall and disease-free survival. FILIP1L inhibited peritoneal metastases in orthotopic mouse model. FILIP1L knockdown induced chemoresistance in ovarian cancer cells and this phenotype was rescued by simultaneous knockdown of FILIP1L and SLUG, an EMT activator. We also demonstrated that FILIP1L regulates beta-catenin degradation. FILIP1L co-localizes with phospho-beta-catenin and increases phospho-beta-catenin at the centrosomes, destined for proteosomal degradation. Finally, we showed that FILIP1L regulates EMT. Overall, these findings suggest that FILIP1L promotes beta-catenin degradation and suppresses EMT, thereby inhibiting metastases and chemoresistance. Our study provides the first clinical relevance of FILIP1L in human cancer, and suggests that FILIP1L may be a novel prognostic marker for chemotherapy in ovarian cancer patients. Further, the modulation of FILIP1L expression may have the potential to be a target for cancer therapy.

APC (80-90%) and K-Ras (40-50%) mutations frequently occur in human colorectal cancer (CRC) and these mutations cooperatively accelerate tumorigenesis including metastasis. In addition, both beta-catenin and Ras levels are highly increased in CRC, especially in metastatic CRC (mCRC). Therefore, targeting both the Wnt/beta-catenin and Ras pathways could be an ideal therapeutic approach for treating mCRC patients. In this study, we characterized the roles of KY1022, a small molecule that destabilizes both beta-catenin and Ras via targeting the Wnt/beta-catenin pathway, in inhibiting the cellular events, including EMT, an initial process of metastasis, and apoptosis. As shown by in vitro and in vivo studies using APCMin/+/K-RasG12DLA2 mice, KY1022 effectively suppressed the development of mCRC at an early stage of tumorigenesis. A small molecular approach degrading both beta-catenin and Ras via inhibition of the Wnt/beta-catenin signaling would be an ideal strategy for treatment of mCRC.

Melanoma, as one of the most highly metastatic types of cancer, is resistant to current treatment methods, including popular targeted molecular therapy. Consequently, it is essential to develop a deeper understanding of the mechanisms involved in melanoma progression so that alternative treatments may be identified. To date, accumulating evidence supports the use of calpains, including calpain small subunit 1 (also known as Capn4 or CAPNS1), which affect cancer progression through many pathways, such as epithelialmesenchymal transition (EMT), the Wnt/beta-catenin (beta-catenin) and the nuclear factor kappaB (NF-kappaB) signaling pathways. The EMT pathway is well known as one of the most important events in tumor metastasis. The present study observed cross-talk among the EMT, beta-catenin and NF-kappaB pathways. To identify the underlying mechanisms of Capn4 activity in melanoma cells, we determined Capn4 expression by gene chip and immunohistochemistral analyses in melanoma tissues and cells in vitro. The extent of apoptosis as determined by TUNEL assay, DAPI staining, and cleaved-caspase-3 assay was increased in human melanoma cells in which Capn4 expression had been knocked down when compared with untreated cells. Transwell assays and xenograft tumorigenicity studies were also performed to assess the effects of Capn4 on migration and invasion in vitro and tumor growth in vivo, respectively. The levels of beta-catenin, vimentin, E-cadherin and N-cadherin were altered in human melanoma cells as determined by western blot analysis assay. Our study demonstrated that Capn4 is an underlying target for melanoma treatment.

tumor progression is a complex process that also involves the restructuring of the actin cytoskeleton and the weakening of intercellular adhesive contacts due to the tumor cells that pass through the epithelial-mesenchymal transition (EMT). AIM: capital TE, Cyrillico identify correlations between clinical features, risk of progression and/or recurrence of human colon adenocarcinomas (CAC), and EMT-related tumor markers. MATERIAL AND METHODS: Descending colon and sigmoid colon adenocarcinoma samples were examined immunohistochemically. Formalin-fixed paraffin-embedded tissue sections were incubated with antigen-specific antibodies, then secondary antibodies labeled with fluorochromes, and the fluorescence intensity of microscopy images was analyzed. RESULTS: The cells of a tumor compared to those of intact colon tissue showed a weak staining of E-cadherin in the cell-cell contact areas. The reduced membrane staining and nuclear localization of beta-catenin were detected in moderately (G2) and poorly (G3) differentiated tumors. There were substantially decreased beta-actin levels in almost all tumor samples and increased gamma-actin ones, mainly in the samples belonging to stage IV disease. CONCLUSION: A correlation was found between stage, tumor differentiation grade, risk for relapse or progression of disease, and the impaired expression of different EMT markers: total or partial loss of E-cadherin expression, beta-catenin reorganization in cell-cell contacts, and a change in the ratio of cytoplasmic actin isoforms in the late stages of CAC development. We believe that these molecular markers may have a prognostic potential.

BACKGROUND: KIAA1199 was upregulated in diverse cancers, but the association of KIAA1199 with gastric cancer (GC), the biological role of KIAA1199 in GC cells and the related molecular mechanisms remain to be elucidated. METHODS: KIAA1199 expression was analysed by reverse transcription-polymerase chain reaction assay (RT-PCR) and immunohistochemistry (IHC) in GC patient tissue. The small hairpin RNA (shRNA) was applied for the knockdown of endogenous KIAA1199 in NCI-N87 and AGS cells. MTT, colony formation, scratch wounding migration, transwell chamber migration and invasion assays were employed respectively to investigate the role of KIAA1199 in GC cells. The potential signaling pathway of KIAA1199 induced migration and invasion was detected. RESULTS: KIAA1199 was upregulated in GC tissue and was an essential independent marker for poor prognosis. Knockdown KIAA1199 suppressed the proliferation, migration and invasion in GC cells. KIAA1199 stimulated the Wnt/beta-catenin signaling pathway and the enzymatic activity of matrix metalloproteinase (MMP) family members and thus accelerated the epithelial-to-mesenchymal transition (EMT) progression in GC cells. CONCLUSION: These findings demonstrated that KIAA1199 was upregulated in GC tissue and associated with worse clinical outcomes in GC, and KIAA1199 acted as an oncogene by promoting migration and invasion through the enhancement of Wnt/beta-catenin signaling pathway and MMPs mediated EMT progression in GC cells.

This study aimed to elucidate how microRNA27a-3p (miR-27a-3p) modulates the Wnt/beta-catenin signaling pathway to promote the epithelial-mesenchymal transition (EMT) in oral squamous carcinoma stem cells (OSCSCs) by targeting secreted frizzled-related protein 1 (SFRP1). Flow cytometry was used to sort OSCSCs from the SCC-9 and Tca8113 cell lines. The OSCSCs were randomly assigned into the miR-27a-3p inhibitors group, the miR-27a-3p inhibitors-NC group, the si-SFRP1 group, the si-SFRP1 + miR-27a-3p inhibitors group and the blank group. A luciferase reporter, immunofluorescence and Transwell assays were performed to detect luciferase activity, SFRP1, and cell migration and invasion, respectively. The mRNA expression of miR-27a-3p, SFRP1 and EMT markers (E-cadherin, N-cadherin, vimentin and ZEB1) were detected using qRT-PCR. The protein expression of SFRP1, EMT markers and the proteins of the Wnt/beta-catenin signaling pathway was detected by Western blotting. OSCSCs showed up-regulated miR-27a-3p, Wnt/beta-catenin signaling pathway-related proteins, vimentin, N-cadherin and ZEB1 and down-regulated SFRP1 and E-cadherin. MiR-27a-3p targeted SFRP1. Down-regulated miR-27a-3p resulted in increased E-cadherin and SFRP1 but decreased vimentin, N-cadherin, ZEB1, the Wnt/beta-catenin signaling pathway-related proteins, and invasive and migratory cells. Silenced SFRP1 reversed this effect. We found that miR-27a-3p modulated the Wnt/beta-catenin signaling pathway to promote EMT in OSCSCs by down-regulating SFRP1.

Annexin A2 is a member of the Annexin family that acts as a Ca2+-dependent phospholipid and membrane binding protein, which is associated with the survival and spread of multiple neoplasms. However, the function of Annexin A2 in ovarian cancer progression remains unclear. In this study, we aimed to investigate the role and underlying molecular mechanism of Annexin A2 in cell proliferation and invasion in ovarian cancer. We found that the mRNA expression of Annexin A2 was upregulated in ovarian cancer tissues and cell lines. In the loss-of-function of Annexin A2, beta-catenin was indicated to be significantly suppressed and EMT constrained. Moreover, cell proliferation and invasion were both markedly inhibited by the downregulation of Annexin A2. Additionally, the overexpression of beta-catenin obviously reversed the effect of Annexin A2 on EMT, and cell proliferation and invasion, indicating that Annexin A2 suppression regulated EMT through controlling beta-catenin. Taken together, this study showed that Annexin A2 inhibition suppresses proliferation and invasion in ovarian cancer via beta-catenin/EMT, proposing the potential role of Annexin A2 in the prevention and treatment of ovarian cancer.

epithelial-mesenchymal transition (EMT) plays a critical role in the process of cancer invasion and metastasis. The Wnt/beta-catenin signaling pathway is known as a stimulative factor, which may trigger EMT and metastasis of cancer cells. In addition, several microRNAs (miRNAs) have been proven to regulate the EMT process. Recent research revealed that miR148a is downregulated in pancreatic cancer. However, the definite role of miR-148a in EMT and invasion of pancreatic cancer is still unknown. The present study attempted to demonstrate the underlying mechanism of miR-148a in the regulation of EMT and invasion of pancreatic cancer cells. Our data revealed that the expression of miR-148a was markedly downregulated in human pancreatic ductal adenocarcinoma (PDAC) cell lines and tissues. In addition, the downregulation of miR-148a was associated with poor prognosis and EMT phenotype. Furthermore, restoration of miR-148a expression inhibited the EMT process, as well as the migration and invasion of BxPC-3 pancreatic cancer cells. Wnt10b, a promoting molecule of the Wnt/beta-catenin signaling pathway, was demonstrated by dualluciferase reporter assay to be a direct target of miR148a. Subsequently, we found that miR148a negatively regulated the protein expression of beta-catenin, cyclin D1 and MMP-9, which were important components of the Wnt/beta-catenin signaling pathway. In conclusion, these findings revealed that miR-148a suppresses EMT and invasion of pancreatic cancer cells by targeting Wnt10b and inhibiting the Wnt/beta-catenin signaling pathway, and thus, miR-148a may serve as a novel therapeutic target for pancreatic cancer.

Akt1 is essential for the oncogenic transformation and tumor growth in various cancers. However, the precise role of Akt1 in advanced cancers is conflicting. Using a neuroendocrine TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model, we first show that the genetic ablation or pharmacological inhibition of Akt1 in mice blunts oncogenic transformation and prostate cancer (PCa) growth. Intriguingly, triciribine (TCBN)-mediated Akt inhibition in 25-week old, tumor-bearing TRAMP mice and Akt1 gene silencing in aggressive PCa cells enhanced epithelial to mesenchymal transition (EMT) and promoted metastasis to the lungs. Mechanistically, Akt1 suppression leads to increased expression of EMT markers such as Snail1 and N-cadherin and decreased expression of epithelial marker E-cadherin in TRAMP prostate, and in PC3 and DU145 cells. Next, we identified that Akt1 knockdown in PCa cells results in increased production of TGFbeta1 and its receptor TGFbeta RII, associated with a decreased expression of beta-catenin. Furthermore, treatment of PCa cells with ICG001 that blocks nuclear translocation of beta-catenin promoted EMT and N-cadherin expression. Together, our study demonstrates a novel role of the Akt1-beta-catenin-TGFbeta1 pathway in advanced PCa.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.

Ormeloxifene is a clinically approved selective estrogen receptor modulator, which has also shown excellent anticancer activity, thus it can be an ideal repurposing pharmacophore. Herein, we report therapeutic effects of ormeloxifene on prostate cancer and elucidate a novel molecular mechanism of its anticancer activity. Ormeloxifene treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), beta-catenin/TCF-4 transcriptional activity, and induced the expression of pGSK3beta. In molecular docking analysis, ormeloxifene showed proficient docking with beta-catenin and GSK3beta. In addition, ormeloxifene induced apoptosis, inhibited growth and metastatic potential of prostate cancer cells and arrested cell cycle in G0-G1 phase via modulation of cell-cycle regulatory proteins (inhibition of Mcl-1, cyclin D1, and CDK4 and induction of p21 and p27). In functional assays, ormeloxifene remarkably reduced tumorigenic, migratory, and invasive potential of prostate cancer cells. In addition, ormeloxifene treatment significantly (P < 0.01) regressed the prostate tumor growth in the xenograft mouse model while administered through intraperitoneal route (250 mug/mouse, three times a week). These molecular effects of ormeloxifene were also observed in excised tumor tissues as shown by immunohistochemistry analysis. Our results, for the first time, demonstrate repurposing potential of ormeloxifene as an anticancer drug for the treatment of advanced stage metastatic prostate cancer through a novel molecular mechanism involving beta-catenin and EMT pathway. Mol cancer Ther; 16(10); 2267-80. (c)2017 AACR.CI - (c)2017 American Association for cancer Research.

In this study, we investigated the underlying mechanism of the phosphoinositide 3-kinase/Akt- and Wnt/beta-catenin-mediated promotion of epithelial-to-mesenchymal transition by epigenetic regulation of histone acetylation in gastric cancer. First, we used immunohistochemistry to detect the expression of phosphorylated Akt, phosphorylated glycogen synthase kinase 3 beta, and beta-catenin in gastric cancer tissues and adjacent tissues. In addition, we confirmed that the phosphoinositide 3-kinase/Akt and Wnt/beta-catenin signaling pathways were correlated with tumorigenesis, progression, and maintenance of gastric cancer using the phosphoinositide 3-kinase inhibitor LY294002 and an inhibitor of the beta-catenin/TCF4 complex, FH535. epithelial-to-mesenchymal transition-related gene expression was measured by western blotting and quantitative real-time polymerase chain reaction assays. Furthermore, we detected the acetylation of histone H3 lysine 4 and lysine 27 using the FH535 and LY294002 inhibitors at different concentrations for 24 and 48 h. Finally, chromatin immunoprecipitation-quantitative polymerase chain reaction was performed to detect the specific binding of H3K27ac to the promoter of the epithelial-to-mesenchymal transition-related factor, Twist. Taken together, abnormal activation of the phosphoinositide 3-kinase/Akt and Wnt/beta-catenin signaling pathway was correlated with the gastric cancer progression and contributed to epithelial-to-mesenchymal transition regulation by controlling histone acetylation.

Disregulation of dickkopf-related protein 1 (DKK1) has been reported in a variety of human cancers. However, how DKK1 functions in Non-small cell lung cancer has not been revealed. In the current study, DKK1 was knocked out by the lentivirus-mediated short hairpin RNA interference approach in H1299 and 95C non-small cell lung cancer cell lines. Subsequently, the migration and invasion ability were assessed by wound-healing and transwell assays. In addition, epithelial-mesenchymal transition markers and beta-catenin were examined by Western blot analysis. The signaling pathway downstream of DKK1 was characterized using the Wnt signaling pathway inhibitor, IWP2, and glycogen synthase kinase 3 beta inhibitor, LiCl. Immunofluorescence analysis investigated the subcellular localization of beta-catenin. The results suggested that knockdown of DKK1 caused reduced migration and invasion ability of H1299 and 95C cells. DKK1 silencing resulted in the downregulation of epithelial-mesenchymal transition-related proteins, such as Snail and zinc finger E-box binding homeobox 1. Besides, DKK1 silencing inhibited beta-catenin and promoted the phosphorylation of beta-catenin. Mechanism results indicated that the expression of beta-catenin was reduced in H1299 or 95C cells after being treated with Wnt signaling inhibitor, IWP2. In addition, the inhibition of beta-catenin phosphorylation by glycogen synthase kinase 3 beta inhibitor, LiCl, significantly enhanced the migration and invasion capacities in DKK1-knockdown cell lines. Furthermore, cell immunofluorescence revealed that nuclear beta-catenin was reduced when DKK1 was knocked down. Taken together, these findings suggest that DKK1 induces the occurrence of epithelial-mesenchymal transition and promotes migration and invasion in non-small cell lung cancer cells. Mechanically, beta-catenin plays a vital role in DKK1-induced non-small cell lung cancer cell migration and invasion, and DKK1 inhibits the phosphorylation of beta-catenin, resulting in the increased nuclear localization of beta-catenin.

Bladder cancer has a considerable morbidity and mortality impact with particularly poor prognosis. Curcumin has been recently noticed as a polyphenolic compound separated from turmeric to regulate tumor progression. However, the precise molecular mechanism by which curcumin inhibits the invasion and metastasis of bladder cancer cells is not fully elucidated. In this study, we investigate the effect of curcumin on the bladder cancer as well as possible mechanisms of curcumin. The expression of beta-catenin was detected by quantitative real-time polymerase chain reaction and immunohistochemical analysis in a series of bladder cancer tissues. In addition, bladder cancer cell lines T24 and 5637 cells were treated with different concentrations of curcumin. The cytotoxic effect of curcumin on cell proliferation of T24 and 5637 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The migration and invasion capacity of T24 and 5637 cells were measured by transwell assay. The effects of curcumin on expression levels of beta-catenin and epithelial-mesenchymal transition marker were determined by western blotting. The beta-catenin expression was significantly upregulated in bladder cancer tissues when compared with corresponding peri-tumor tissues. Furthermore, curcumin inhibited the cell proliferation of T24 and 5637 cells, and curcumin reduced the migration and invasive ability of T24 and 5637 cells via regulating beta-catenin expression and reversing epithelial-mesenchymal transition. Curcumin may be a new drug for bladder cancer.

This study explored the role of fibulin-3 in osteosarcoma progression and the possible signaling pathway involved. Fibulin-3 mRNA and protein expression in normal tissue, benign fibrous dysplasia, osteosarcoma, osteosarcoma cell lines (HOS and U-2OS), the normal osteoblastic cell line hFOB, and different invasive subclones was evaluated by immunohistochemistry (IHC) or immunocytochemistry (ICC) and real time reverse transcriptase-polymerase chain reaction (real time qRT-PCR). To assess the role of fibulin-3 in the invasion and metastasis of osteosarcoma cells, lentiviral vectors with fibulin-3 small hairpin RNA (shRNA) and pLVX-fibulin-3 were constructed and used to infect the highly invasive and low invasive subclones. The effects of fibulin-3 knockdown and upregulation on the biological behavior of osteosarcoma cells were investigated by functional in vitro and in vivo assays. The results revealed that fibulin-3 expression was upregulated in osteosarcoma, and was positively correlated with low differentiation, lymph node metastasis, and poor prognosis. Fibulin-3 could promote osteosarcoma cell invasion and metastasis by inducing EMT and activating the Wnt/beta-catenin signaling pathway. Collectively, our findings demonstrate that fibulin-3 is a promoter of osteosarcoma development and progression, and suggest a novel therapeutic target for future studies.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is a crucial event in the development of renal interstitial fibrosis (RIF). A growing body of evidence indicates that beta-catenin plays an important role in various types of fibrosis. Although members of the microRNA (miRNA)-200 family have been suggested to suppress EMT in cancer and fibrosis, the function of miRNA-200a in regulating the progression of RIF is unknown. We speculate that miRNA-200a may hinder this progression through the suppression of beta-catenin. METHODS: Transforming growth factor beta1 (TGF beta1) was used to induce EMT of proximal tubule epithelial (HK-2) cells in vitro, quantitative real time polymerase chain reaction (qPCR) and Western Blot analysis measured the miRNA-200a and beta-catenin expression. qPCR, Western Blot analysis, Migration Assay and cell immunofluorescence were applied to detect the influence of up- and downregulated miRNA-200a expression and beta-catenin siRNA on beta-catenin and EMT. Dual luciferase report plasmid (CTNNB1 UTR WT/MT) verifies the target relationship between miRNA-200a and CTNNB1 (beta-catenin gene). RESULTS: miRNA-200a is downregulated and beta-catenin is upregulated during TGF beta1-induced EMT. Upregulation of miRNA-200a inhibits beta-catenin and attenuates TGF beta1-induced EMT and cell migration, while its downregulation increases beta-catenin and induces EMT and HK-2 cell migration. Further, knocking down beta-catenin suppressed the EMT induced by miRNA-200a downregulation. miRNA-200a directly targets for CTNNB1. CONCLUSIONS: miRNA-200a inhibits TGF beta1-induced EMT by directly targeting beta-catenin in proximal tubule epithelial cells.CI - (c) 2017 S. Karger AG, Basel.

AIMS: MicroRNAs (miRNAs) plays important role in development and disease, especially in cancer including non-small cell lung cancer (NSCLC). However, the role of miR-770 in NSCLC remains unclear. In this study, we aimed to study the function and mechanism of miR-770 in tumorigenesis of NSCLC. MAIN METHODS: RT-qPCR was used to measure the expression levels of miR-770 in NSCLC tissues and cells. MTT assay, colony formation assay, flow cytometric analysis and transwell migration and invasion assays were performed to investigate the role of miR-770 in NSCLC cells. Bioinformatics and luciferase reporter analyses were used to demonstrate that whether the Jumonji domain-containing 6 (JMJD6) as a direct target of miR-770. The function of JMJD6 in NSCLC was also investigated. Finally, in vivo animal experiment was used to study whether miR-770 was capable of inhibiting tumor growth by inhibiting JMJD6. KEY FINDINGS: We first showed that miR-770 was downregulated in NSCLC tissues and cell lines, and the low expression of miR-770 was correlated with poor patient survival in NSCLC patients. miR-770 acted on a tumor suppressor by binding to the 3'UTR of JMJD6 and downregulated its expression in NSCLC cells. This study also demonstrated that JMJD6 played as an oncogene in NSCLC cells. miR-770 overexpression was capable of inhibiting NSCLC tumor growth by inhibiting JMJD6 and its downstream WNT/beta-catenin pathway both in vitro and in vivo. SIGNIFICANCE: The present study indicated that miR-770 functioned as a tumor suppressor and it might be a potential biomarker and therapeutic target in NSCLC.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

COPD is characterised by poorly reversible airflow obstruction usually due to cigarette smoking. The transcription factor clusters of beta-catenin/Snail1/Twist has been implicated in the process of epithelial mesenchymal transition (EMT), an intermediate between smoking and airway fibrosis, and indeed lung cancer. We have investigated expression of these transcription factors and their "cellular localization" in bronchoscopic airway biopsies from patients with COPD, and in smoking and non-smoking controls. An immune-histochemical study compared cellular protein expression of beta-catenin, Snail1 and Twist, in these subject groups in 3 large airways compartment: epithelium (basal region), reticular basement membrane (Rbm) and underlying lamina propria (LP). beta-catenin and Snail1 expression was generally high in all subjects throughout the airway wall with marked cytoplasmic to nuclear shift in COPD (P < 0.01). Twist expression was generalised in the epithelium in normal but become more basal and nuclear with smoking (P < 0.05). In addition, beta-catenin and Snail1 expression, and to lesser extent of Twist, was related to airflow obstruction and to expression of a canonical EMT biomarker (S100A4). The beta-catenin-Snail1-Twist transcription factor cluster is up-regulated and nuclear translocated in smokers and COPD, and their expression is closely related to both EMT activity and airway obstruction.

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 beta-catenin mRNA and protein was dose-dependently downregulated by baicalin in highly invasive breast cancer cell lines, and overexpression of beta-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 beta-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 beta-catentin expression.

The inhibitor of beta-catenin and TCF (ICAT) blocks the binding of TCF to beta-catenin and has been demonstrated as a suppressor of the Wnt/beta-catenin signaling pathway. It has been reported to exert a different function around a wide variety of cancers. However, its function and underlying mechanisms in human cervical cancer remains unknown. In the present study, the expression of ICAT in 41 human cervical cancer tissues and 30 normal cervical tissues was evaluated by immunohistochemical analysis. ICAT was found highly expressed in cancer tissues. ICAT overexpression significantly promoted SiHa cell proliferation in vitro by causing G1 arrest, and enhanced cell migration and invasion whereas, ICAT knockdown induced opposite effects in Caski cells which have higher expression of ICAT. Downregulation or overexpression of ICAT resulted in an altered expression of the epithelial-mesenchymal transition (EMT). Furthermore, immunoprecipitation assays revealed that ICAT pormoted cervical cancer EMT by competing in E-cadhenin binding to beta-caterin. Overexpression of ICAT in SiHa cells promoted tumor growth and EMT was also demonstrated by the xenograft mouse experiment. These results demonstrate that ICAT contributed to the progression of cervical cancer and may play a role in the regulation of EMT by distrupting the E-cadherin/beta-catenin complex. It may be a novel potential therapeutic target for therapy in human cervical cancer.

This study determines whether cullin 4B (CUL4B) promotes pancreatic cancer (PC) metastasis by inducing epithelial-mesenchymal transition (EMT) via the Wnt/beta-catenin signaling pathway. A total of 64 PC patients were enrolled in this study. Human PC cell lines were distributed into blank, negative control, shCUL4B, PLOC, PLOC-CUL4B, and PLOC-CUL4B + siRNA-beta-catenin groups. The expressions of CUL4B, Wnt/beta-catenin signaling pathway-related proteins, and EMT-related proteins were determined using RT-qPCR and Western blotting. The positive expressions of CUL4B and beta-catenin protein in tissues were detected by immunohistochemistry. MTT assay and flow cytometry was performed for cell proliferation and cell cycle, scratch test, and transwell assay for cell migration and invasion ability. CUL4B and beta-catenin were expressed at a higher level in PC tissues than in paracancerous tissues though paracancerous tissues had higher expressions of CUL4B and beta-catenin than normal tissues. The PLOC-CUL4B group showed increased CUL4B, Wnt, beta-catenin, LEF-1, c-Jun, Cyclin D1, N-cadherin, Vimentin, Snail, and ZEB1 expression; decreased E-cadherin expression; accelerated cell proliferation; increased S-phase cell percentages; increased cell migration ability; more liver metastases; and enlarged tumor than the PLOC and PLOC-CUL4B + siRNA-beta-catenin groups. The shCUL4B group showed decreased CUL4B, Wnt, beta-catenin, LEF-1, c-Jun, Cyclin D1, N-cadherin, Vimentin, Snail, and ZEB1 expression; increased E-cadherin expression; decelerated cell proliferation; decreased S-phase cell percentages; reduced cell migration ability; less liver metastases; and decreased tumor weight than the blank and negative control groups. We demonstrate that CUL4B promotes PC metastasis by inducing EMT via the Wnt/beta-catenin signaling pathway. Therefore, CUL4B might be the clinical target for treating PC.CI - (c) 2017 Wiley Periodicals, Inc.

This study investigated the effects of microRNA-135a (miR-135a) targeting of glycogen synthase kinase 3beta (GSK3beta) on the epithelial-mesenchymal transition (EMT), migration and invasion of bladder cancer (BC) cells by mediating the Wnt/beta-catenin signaling pathway. BC and adjacent normal tissues were collected from 165 BC patients. Western blotting and quantitative real-time PCR were used to detect the expression of GSK3beta, beta-catenin, cyclinD1, E-cadherin, vimentin and miR-135a in BC tissues and cells. Cells were assigned to blank, negative control (NC), miR-135a mimics, miR-135a inhibitors, small interfering RNA (siRNA)-GSK3beta or miR-135a inhibitors+siRNA-GSK3beta groups. miR-135a, beta-catenin, cyclinD1 and vimentin expression increased, while GSK3beta and E-cadherin expression decreased in BC tissues compared with adjacent normal tissues. Compared with the blank and NC groups, the expression of miR-135a, beta-catenin, cyclinD1 and vimentin was higher, and cell proliferation, migration, invasion and tumor growth were increased in the miR-135a mimics and siRNA-GSK3beta groups. These groups showed an opposite trend in GSK3beta and E-cadherin expression and cell apoptosis. The miR-135a inhibitors group was inversely correlated with the blank and NC groups. It was concluded that miR-135a accelerates the EMT, invasion and migration of BC cells by activating the Wnt/beta-catenin signaling pathway through the downregulation of GSK3beta expression.

MiR-3127-5p has been implicated as a tumor-suppressive microRNA (miRNA) in non-small-cell lung cancer (NSCLC) and its expression was associated with tumor recurrence and poor prognosis. The aim of this study was to determine whether miR-3127-5p regulates epithelial-mesenchymal transition (EMT) in NSCLC, and to investigate the underlying mechanisms. Using qRT-PCR, we examined the expression levels of miR-3127-5p in a cohort of primary NSCLC specimens with and without distant metastasis. We further performed a series of in vitro and in vivo experiments to investigate the effects and underlying mechanism of miR-3127-5p on EMT, cell migration, invasion, and adhesion in NSCLC. We found that metastatic NSCLC tissues showed markedly downregulated miR-3127-5p expression. Transforming growth factor-beta1 (TGF-beta1) treatment induced EMT in A549 and H1299 cells, and downregulation of miR-3127-5p could result in the similar effect. Mechanically, we demonstrated that frizzled-4 (FZD4) is a target gene and miR-3127-5p exerts its effects by regulating the Wnt/beta-catenin signaling. In addition, the expression levels of FZD4 and miR-3127-5p were also negatively associated in both clinical and xenografted tumors. Overall, these findings suggest that downregulation of miR-3127-5p promotes EMT through activating the Wnt/FZD4/beta-catenin signaling pathway and may represent a therapeutic target for NSCLC metastasis.CI - (c) 2018 Wiley Periodicals, Inc.

Despite the availability of a number of treatment options, certain cases of primary prostate cancer (PCa) will develop into metastatic PCa, in which epithelialmesenchymal transition (EMT) serves an important role. Recently, a natural flavonoid known as 2'hydroxyflavanone (2HF) exerts remarkable anticancer activity on various types of cancer. Our previous study demonstrated that 2HF could promote apoptosis and inhibit the proliferation of PCa cells, but whether 2HF is involved in the regulation of EMT, and cell migration and invasion in metastatic PCa remains unknown. The present study used two different metastatic PCa cell lines (PC3 and DU145) to investigate the effects of 2HF on EMT, and cell migration and invasion. The results demonstrated that 2HF could inhibit EMT, and cell migration and invasion through the Wnt/betacatenin signaling pathway by suppressing GSK3beta phosphorylation, betacatenin expression and transactivation. In conclusion, the present study revealed a novel function of 2HF, which may be used to prevent or treat PCa metastasis.

BACKGROUND/AIM: Recent studies implied a significant role of hypoxia-inducible factor-1alpha (HIF1alpha) in cell transformation. This study aimed to assess the effects of HIF1alpha on the epithelial-to-mesenchymal transition (EMT) and tumorigenesis of lung adenocarcinoma cells. MATERIALS AND METHODS: Invasion, migration and colony formation assays were used to evaluate cell transformation. expression of EMT-related markers were analyzed by western blot, reverse-transcription polymerase chain reaction or zymography. A luciferase assay was carried out to access the transcriptional activity of beta-catenin. RESULTS: Hypoxia enhanced migration, invasion and transformation of A549 lung adenocarcinoma cells. Hypoxic stimulation promoted the expression of EMT-related markers in lung cancer cells. The expression of HIF1alpha was found to be involved in hypoxia-mediated modulation of expression of snail family transcriptional repressors 1 (SNAI1) and 2 (SLUG). Hypoxia enhanced nuclear accumulation and transcriptional activity of beta-catenin. CONCLUSION: beta-Catenin promotes expression of EMT-related genes and eventually contributes to the metastatic process.CI - Copyright(c) 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Glioma is a type of malignant brain tumor. Forkhead box C1 (FOXC1) is a conserved transcription factor that is involved in tumorigenesis; however, the function of FOXC1 in glioma remains unclear. The present study aimed to investigate the effects of FOXC1 silencing on the epithelialtomesenchymal transition (EMT) of glioma cells. FOXC1specific small interfering RNAs were employed to downregulate the expression levels of FOXC1 in glioma cells. The proliferation, migration and invasion of glioma cells were assessed by MTT assay, wound healing assay and Transwell assay. Western blot analysis was performed to reveal the effects of FOXC1 on EMTassociated proteins and betacatenin signaling. The results revealed that, following FOXC1 silencing, the proliferation, migration and invasion of glioma cells were decreased. The expression levels of EMTassociated proteins were also affected. Further examination demonstrated that betacatenin signaling was involved in the effects of FOXC1 on glioma cells. Previous results suggested that overexpression of betacatenin reversed the effects of FOXC1 silencing on glioma cells. The present study demonstrated that FOXC1 may regulate the EMT of glioma cells, potentially via betacatenin signaling. Therefore, FOXC1 may be a potential therapeutic target for the treatment of glioma.

BACKGROUND: Early invasion and metastasis are responsible for the dismal prognosis of pancreatic ductal adenocarcinoma (PDAC), and epithelial-to-mesenchymal transition (EMT) is recognized as a crucial biological progress in driving tumor invasion and metastasis. The transcription factor FOXO3a is inactivated in various types of solid cancers and the loss of FOXO3a is associated with EMT and tumor metastasis. In this study, we sought to explore whether SPRY2, a regulator of receptor tyrosine kinase (RTK) signaling, is involved in FOXO3a-mediated EMT and metastasis in PDAC. METHODS: Immunohistochemistry was performed in 130 paired PDAC tissues and paracarcinomatous pancreatic tissues. Cell proliferation and apoptosis were assessed by cell counting kit and flow cytometry, while cell migration and invasion were evaluated with wound healing and transwell assays. The changes in mRNA and protein levels were estimated by qRT-PCR and western blot. BALB/c nude mice xenograft model was established to evaluate tumorigenesis and metastasis in vivo. RESULTS: FOXO3a expression was remarkably reduced in PDAC tissues, and correlated with metastasis-associated clinicopathologic characteristics and poor prognosis in patients with PDAC. In addition to the promotion of proliferation and suppression of apoptosis, knockdown of FOXO3a or SPRY2 induced EMT and promoted the migration and invasion of PDAC cells via activation of the beta-catenin/TCF4 pathway. Moreover, silencing of SPRY2 reversed the suppressor effects induced by FOXO3a overexpression on EMT-associated migration and invasion of PDAC cells, while blockade of beta-catenin reversed the effects of SPRY2 loss. FOXO3a knockdown decreased SPRY2 protein stability, whereas SPRY2 knockdown enhanced beta-catenin protein stability. In vivo, FOXO3a knockdown promoted the tumorigenic ability and metastasis of PDAC cells. CONCLUSIONS: Our study suggests that knockdown of FOXO3a induces EMT and promotes metastasis of PDAC by activation of the beta-catenin/TCF4 pathway through SPRY2. Thus, FOXO3a may represent a candidate therapeutic target in PDAC.

Several signalling pathways contribute to the regulation of epithelial to mesenchymal transition (EMT), either during developmentally regulated processes or in cancer progression and metastasis. Induction of EMT in fully polarized mouse mammary epithelial cells (EpH4) by an inducible c-fos estrogen receptor (FosER) oncoprotein involves loss of E-cadherin expression, nuclear translocation of beta-catenin, and autocrine production of TGFbeta. Reporter assays demonstrate that both beta-catenin/LEF-TCF- and TGFbeta-Smad-dependent signalling activities are upregulated, probably coregulating mesenchymal-specific gene expression during EMT. Stable expression of E-cadherin in mesenchymal FosER cells decreased beta-catenin activity and reduced cell proliferation. However, these cells still exhibited a defect in epithelial polarization and expressed E-cadherin/beta-catenin complexes in the entire plasma membrane. On the other hand, inhibition of TGFbeta-Smad signalling in mesenchymal FosER cells induced flat, cobblestone-like clusters of cells, which relocalized beta-catenin to the plasma membrane but still lacked detectable E-cadherin. Interestingly, inhibition of TGFbeta signalling in the E-cadherin-expressing mesenchymal FosER cells caused their reversion to a polarized epithelial phenotype, in which E-cadherin, beta-catenin, and ZO-1 were localized at their correct lateral plasma membrane domains. These results demonstrate that loss of E-cadherin can contribute to increased LEF/TCF-beta-catenin signalling, which in turn cooperates with autocrine TGFbeta signalling to maintain an undifferentiated mesenchymal phenotype.

An epithelial sheet, the epicardium, lines the surface of the heart. In the developing embryo, the epicardium expresses the transcriptional regulator Wilm's tumor Gene 1 (Wt1). Through incompletely understood mechanisms, Wt1 inactivation derails normal heart development. We investigated mechanisms by which Wt1 regulates heart development and epicardial epithelial to mesenchymal transition (EMT). We used genetic lineage tracing approaches to track and isolate epicardium and epicardium derivatives in hearts lacking Wt1 (Wt1(KO)). Wt1(KO) hearts had diminished proliferation of compact myocardium and impaired coronary plexus formation. Wt1(KO) epicardium failed to undergo EMT. Wt1(KO) epicardium expressed reduced Lef1 and Ctnnb1 (beta-catenin), key components of the canonical Wnt/beta-catenin signaling pathway. Wt1(KO) epicardium expressed decreased levels of canonical Wnt downstream targets Axin2, Cyclin D1, and Cyclin D2 and exhibited decreased activity of the Batgal Wnt/beta-catenin reporter transgene, suggestive of diminished canonical Wnt signaling. Hearts with epicardium-restricted Ctnnb1 loss of function resembled Wt1(KO) hearts and also failed to undergo epicardial EMT. However, Ctnnb1 inactivation did not alter WT1 expression, positioning Wt1 upstream of canonical Wnt/beta-catenin signaling. Wnt5a, a prototypic non-canonical Wnt with enriched epicardial expression, and Raldh2, a key regulator of retinoic acid signaling confined to the epicardium, were also markedly downregulated in Wt1(KO) epicardium. Hearts lacking Wnt5a or Raldh2 shared phenotypic features with Wt1(KO). Although Wt1 has been proposed to regulate EMT by repressing E-cadherin, we detected no change in E-cadherin in Wt1(KO) epicardium. Collectively, our study shows that Wt1 regulates epicardial EMT and heart development through canonical Wnt, non-canonical Wnt, and retinoic acid signaling pathways.CI - Copyright (c) 2011 Elsevier Inc. All rights reserved.

The differentiation of dental epithelia into enamel-producing ameloblasts or the root epithelial lineage compartmentalizes teeth into crowns and roots. Bmp signaling has been linked to enamel formation, but its role in root epithelial lineage differentiation is unclear. Here we show that cessation of epithelial Bmp signaling by Bmpr1a depletion during the differentiation stage switched differentiation of crown epithelia into the root lineage and led to formation of ectopic cementum-like structures. This phenotype is related to the upregulation of Wnt/beta-catenin signaling and epithelial-mesenchymal transition (EMT). Although epithelial beta-catenin depletion during the differentiation stage also led to variable enamel defect and precocious/ectopic formation of fragmented root epithelia in some teeth, it did not cause ectopic cementogenesis and inhibited EMT in cultured dental epithelia. Concomitant epithelial beta-catenin depletion rescued EMT and ectopic cementogenesis caused by Bmpr1a depletion. These data suggested that Bmp and Wnt/beta-catenin pathways interact antagonistically in dental epithelia to regulate the root lineage differentiation and EMT. These findings will aid in the design of new strategies to promote functional differentiation in the regeneration and tissue engineering of teeth and will provide new insights into the dynamic interactions between the Bmp and Wnt/beta-catenin pathways during cell fate decisions.

BACKGROUND: The formation of the primitive streak is the first visible sign of gastrulation, the process by which the three germ layers are formed from a single epithelium during early development. Embryonic stem cells (ESCs) provide a good system for understanding the molecular and cellular events associated with these processes. Previous work, both in embryos and in culture, has shown how converging signals from both nodal/TGFbetaR and Wnt/beta-catenin signalling pathways specify cells to adopt a primitive-streak-like fate and direct them to undertake an epithelial-to-mesenchymal transition (EMT). However, many of these approaches have relied on genetic analyses without taking into account the temporal progression of events within single cells. In addition, it is still unclear to what extent events in the embryo are able to be reproduced in culture. RESULTS: Here, we combine flow cytometry and a quantitative live single-cell imaging approach to demonstrate how the controlled differentiation of mouse ESCs towards a primitive streak fate in culture results in cells displaying many of the characteristics observed during early mouse development including transient brachyury expression, EMT and increased motility. We also find that the EMT initiates the process, and this is both fuelled and terminated by the action of brachyury, whose expression is dependent on the EMT and beta-catenin activity. CONCLUSIONS: As a consequence of our analysis, we propose that a major output of brachyury expression is in controlling the velocity of the cells that are transiting out of the primitive streak.

We have previously demonstrated that Cyclin A2 is involved in cytoskeletal dynamics, epithelial-mesenchymal transition (EMT) and metastasis. This phenotype was potentiated by activated oncogenic H-Ras. However, the mechanisms governing EMT in these cells have not yet been elucidated. Here, we dissected the pathways that are responsible for EMT in cells deficient for Cyclin A2. In Cyclin A2-depleted normal murine mammary gland (NMuMG) cells expressing RasV12, we found that beta-catenin was liberated from the cell membrane and cell-cell junctions and underwent nuclear translocation and activation. Components of the canonical wingless (WNT) pathway, including WNT8b, WNT10a, WNT10b, frizzled 1 and 2 and TCF4 were upregulated at the messenger RNA and protein levels following Cyclin A2 depletion. However, suppression of the WNT pathway using the acetyltransferase porcupine inhibitor C59 did not reverse EMT whereas a dominant negative form of TCF4 as well as inhibition of phospholipase C using U73122 were able to do so. This suggests that a WNT-independent mechanism of beta-catenin activation via phospholipase C is involved in the EMT induced by Cyclin A2 depletion. Our findings will broaden our knowledge on how Cyclin A2 contributes to EMT and metastasis.CI - (c) The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

OBJECTIVE: It is known that connective tissue growth factor (CTGF) and beta-catenin are involved in DN; however, the underlying molecular mechanisms remain unknown. Here we hypothesized that podocytes undergo epithelial-mesenchymal transition (EMT) in high-glucose condition and CTGF mediates high-glucose induced EMT by activating beta-catenin in podocytes. METHODS: The differentiated podocytes were cultured and divided into three groups: the normal glucose group (5 mmol/L glucose), the high-glucose group (30 mmol/L glucose), and the osmotic control group (5 mmol/L glucose supplemented with 25 mmol/L mannitol). The morphology of cultured podocytes was observed under phase contrast microscopy. To study the relevant markers of EMT, as well as CTGF and beta-catenin, the mRNA and protein expressions were analyzed by real-time PCR and western blotting, respectively. In addition, the effects of inhibition CTGF by anti-CTGF antibody on high-glucose-induced EMT and beta-catenin expression in podocytes were studied. RESULTS: High glucose not only induced phenotypic transition of podocytes but also increased the expression of CTGF and beta-catenin. Under high-glucose condition, podocytes underwent EMT, which were demonstrated by downregulation of nephrin and upregulation of desmin. Moreover, high-glucose-induced EMT and beta-catenin overexpression in podocytes were attenuated by anti-CTGF antibody. CONCLUSION: CTGF and beta-catenin are involved in the EMT of podocytes in diabetes. CTGF mediates high-glucose induced EMT through activation of beta-catenin in podocytes. CTGF inhibition may protect podocytes from EMT in diabetes.

We analyzed the molecular basis for carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-controlled inhibition of epithelial-mesenchymal transition (EMT) in a mouse model for mammary adenocarcinoma (WAP-T mice). We demonstrate that silencing of CEACAM1 in WAP-T tumor-derived G-2 cells induces epithelial-mesenchymal plasticity (EMP), as evidenced by typical changes of gene expression, morphology and increased invasion. In contrast, reintroduction of CEACAM1 into G-2 cells reversed up-regulation of genes imposing mesenchymal transition, as well as cellular invasion. We identified the Wnt-pathway as target for CEACAM1-mediated repression of EMT. Importantly, beta-catenin phosphorylation status and transcriptional activity strongly depend on CEACAM1 expression: CEACAM1high G-2 cells displayed enhanced phosphorylation of beta-catenin at S33/S37/T41 and decreased phosphorylation at Y86, thereby inhibiting canonical Wnt/beta-catenin signaling. We identified Src-homology 2 domain-containing phosphatase 2 (SHP-2) as a critical binding partner of CEACAM1 that could modulate beta-catenin Y86 phosphorylation. Hence, CEACAM1 serves as a scaffold that controls membrane proximal beta-catenin signaling. In vivo, mammary tumors of WAP-T/CEACAM1null mice displayed increased nuclear translocation of beta-catenin and a dramatically enhanced metastasis rate compared to WAP-T mice. Hence, CEACAM1 controls EMT in vitro and in vivo by site-specific regulation of beta-catenin phosphorylation. Survival analyses of human mammary carcinoma patients corroborated these data, indicating that CEACAM1 is a prognostic marker for breast cancer survival.

Paraquat (PQ) poisoning-induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia-inducible factor-1alpha (HIF-1alpha) and epithelial-mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and beta-catenin are two other factors involved in promoting EMT. However, the relationship among HIF-1alpha, Snail and beta-catenin in PQ poisoning-induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF-1alpha in EMT occurs via the Snail and beta-catenin pathways in PQ poisoning-induced pulmonary fibrosis. Sixty-six Sprague-Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE-6TN cell lines were transfected with HIF-1alpha siRNA for 48 hrs before being exposed to PQ. Western blotting, real-time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF-1alpha and alpha-SMA were increased at 2 hrs and the level of ZO-1 (Zonula Occluden-1) was reduced at 12 hrs. In vitro, the transient transfection of HIF-1alpha siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and beta-catenin were significantly reduced when HIF-alpha was silenced. These data demonstrate that EMT may be involved in PQ poisoning-induced pulmonary fibrosis and regulated by HIF-1alpha via the Snail and beta-catenin pathways. Hypoxia-inducible factor-1alpha may be a therapeutic target for the treatment of PQ poisoning-induced pulmonary fibrosis.CI - (c) 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.