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CDH1

Down-regulation of E-cadherin marks the initiation of the epithelial-mesenchymal transition, a process exploited by invasive cancer cells. The zinc finger transcription factor, Snail, functions as a potent repressor of E-cadherin expression that can, acting alone or in concert with the Wnt/beta-catenin/T cell factor axis, induce an epithelial-mesenchymal transition. Although mechanisms that coordinate signaling events initiated by Snail and Wnt remain undefined, we demonstrate that Snail displays beta-catenin-like canonical motifs that support its GSK3beta-dependent phosphorylation, beta-TrCP-directed ubiquitination, and proteasomal degradation. Accordingly, Wnt signaling inhibits Snail phosphorylation and consequently increases Snail protein levels and activity while driving an in vivo epithelial-mesenchymal transition that is suppressed following Snail knockdown. These findings define a potential mechanism whereby Wnt signaling stabilizes Snail and beta-catenin proteins in tandem fashion so as to cooperatively engage transcriptional programs that control an epithelial-mesenchymal transition.

Using genome-wide expression profiling of a panel of 27 human mammary cell lines with different mechanisms of E-cadherin inactivation, we evaluated the relationship between E-cadherin status and gene expression levels. expression profiles of cell lines with E-cadherin (CDH1) promoter methylation were significantly different from those with CDH1 expression or, surprisingly, those with CDH1 truncating mutations. Furthermore, we found no significant differentially expressed genes between cell lines with wild-type and mutated CDH1. The expression profile complied with the fibroblastic morphology of the cell lines with promoter methylation, suggestive of epithelial-mesenchymal transition (EMT). All other lines, also the cases with CDH1 mutations, had epithelial features. Three non-tumorigenic mammary cell lines derived from normal breast epithelium also showed CDH1 promoter methylation, a fibroblastic phenotype and expression profile. We suggest that CDH1 promoter methylation, but not mutational inactivation, is part of an entire programme, resulting in EMT and increased invasiveness in breast cancer. The molecular events that are part of this programme can be inferred from the differentially expressed genes and include genes from the TGFbeta pathway, transcription factors involved in CDH1 regulation (i.e. ZFHX1B, SNAI2, but not SNAI1, TWIST), annexins, AP1/2 transcription factors and members of the actin and intermediate filament cytoskeleton organisation.

The estrogen receptor alpha (ER alpha) is key in regulating normal breast development and function and is closely involved in the onset and progress of cancers. ER alpha transcriptional activity is mediated through two activation functions, AF1 and AF2, whose activity is tightly regulated in a cell-specific manner through yet unknown processes. Here, we demonstrate that cell-cell junctions generate cell permissiveness to AF1 through an up-regulation of the activity of an AF1 sub-region termed box 1. Moreover, the loss of E-cadherin expression is shown to silence the AF1 activity of ER alpha, allowing the receptor to mainly act through its AF2. This switch from an AF1 to an AF2 cell permissiveness also consequently results in the attenuation of ER alpha activity. Therefore, a loss of cell-cell junctions, a key process that occurs during the epithelial-mesenchymal transition, should have a broad impact on ER alpha transcriptional functions.

BACKGROUND: epithelial-mesenchymal transformations (EMT) are critical for the invasion, progression, and metastasis of epithelial carcinogenesis. The role of EMT in head and neck squamous carcinoma (HNSC) tumorigenesis remains unexplored. In the current study, the expressions of several factors associated with the induction of EMT in HNSC cell lines and tumor specimens were investigated to define their functional and pathologic role in HNSC. METHODS: Eleven HNSC cell lines and 50 primary tumor tissue specimens formed the materials of this study. Western blot analysis as well as immunohistochemical, and functional techniques were used to assess the status of activated Src (p-Src), E-cadherin, and vimentin in both cell lines and tumor tissues and the results were correlated with patients' clinicopathologic parameters. RESULTS: The results demonstrated the inverse expression of p-Src and E-cadherin in the majority of cell lines and in primary tumor tissues compared with normal squamous mucosa. Elevated levels of p-Src were accompanied by down-regulation of E-cadherin and the expression of vimentin in epithelial tumor cells. In vitro inhibition of Src led to E-cadherin reexpression and increased cell contact in squamous carcinoma cell lines. Immunophenotypic analysis of these markers in primary tumor tissues demonstrated a significant correlation between increased p-Src, decreased E-cadherin, and vimentin expression and aggressive tumor features including penetrating invasive fronts, high-grade sarcomatoid transformation, and lymph node metastasis. CONCLUSIONS: The results of the current study indicate that Src and E-cadherin may play an important role in EMT, invasion, and aggressive clinicopathologic features of HNSC. These proteins may be targeted for the therapeutic intervention of patients with HNSC.

The loss of E-cadherin gene expression can cause the dysfunction of the cell-cell junction to trigger tumor metastasis. Members of the Snail family of transcription factors are repressors of the expression of the E-cadherin gene. In this study, we showed that the activated androgen receptor (AR) is a novel repressor of E-cadherin gene expression and can promote metastasis. Our results demonstrated that the activated AR could bind to the E-cadherin promoter in vitro and in vivo. The activated AR and HDAC1 had synergistic effects in downregulating E-cadherin gene expression. Treating cells with the AR ligand, dihydrotestosterone (DHT), triggered the reduction of E-cadherin expression and induced changes in cell morphology from an epithelial-like to a mesenchymal-like appearance. When nonmetastatic breast cancer cells expressing cytoplasmic AR were transplanted into mice and the mice were treated with DHT, tumors were detected at metastatic sites, whereas no tumors were detected in transplanted mice without DHT treatment. Furthermore, clinical data from breast cancer patients with invasive ductal carcinomas showed high levels of AR expression in the nuclei and low levels of E-cadherin expression. These results suggest that, similarly to Snail and Twist, the activated AR can downregulate E-cadherin expression to promote the activation of epithelial-mesenchymal transition and tumor metastasis.

AIMS: Endometrial endometrioid adenocarcinomas (EEC) may show a distinctive morphological alteration characterized by the presence of microcystic, elongated and fragmented ('MELF') glands. These changes share features of epithelial-mesenchymal transition (EMT) in carcinomas arising at other sites. The aim was to compare the immunophenotypic profile of MELF-type epithelium with conventional glandular areas of EEC. METHODS AND RESULTS: Twenty-one EEC were stained immunohistochemically for cytokeratin (CK) AE1/AE3, CK7, vimentin, oestrogen receptor, progesterone receptor and E-cadherin. Conventional tumour glands usually showed preserved membranous E-cadherin immunoreactivity with peripheral accentuation of vimentin and hormone receptor expression. MELF-type invasion was characterized by strong CK7 expression, sometimes in contrast to adjacent unstained tumour glands. MELF areas were usually negative for hormone receptors and showed reduced E-cadherin expression. CONCLUSIONS: The expression of hormone receptors and intermediate filaments shows specific distribution patterns within EEC. MELF pattern invasion shows an altered immunophenotype compared with conventional glandular tumour areas. These findings suggest that MELF-type invasion represents a specific tumour alteration, and the reduction in hormone receptor and E-cadherin expression would be consistent with EMT. Immunohistochemical studies of EEC should consider micro anatomical variations in immunoreactivity, since these may be relevant to tumour invasion and progression.

SNAI1, ZEB1, E-cadherin (CDH1), and vitamin D receptor (VDR) genes regulate the epithelial-mesenchymal transition (EMT) that initiates the invasion process of many tumor cells. We hypothesized that this process could also affect the behavior of normal cells adjacent to the tumor. To verify this hypothesis, the expression level of these genes was determined by quantitative RT-PCR in tumor, normal adjacent, and normal distant tissues from 32 colorectal cancer (CC) patients. In addition, we extended the study to human HaCaT normal keratinocytes and SW480-ADH colon cancer cells co-cultured with SW480-ADH cells overexpressing the mouse Snai1 gene. Of 18 CC cases with SNAI1 expression in tumor tissue, five also had SNAI1 in normal adjacent tissue (NAT). expression of SNAI1 in tumor tissue correlated with downregulation of CDH1 and VDR genes in both tumor (P=0.047 and P=0.014, respectively) and NAT lacking SNAI1 expression (P=0.054 and P=0.003). ZEB1 expression was directly related to VDR expression in tumor tissue (r=0.39; P=0.027) and inversely to CDH1 in NAT (r=-0.46; P=0.010). CDH1 and VDR were also downregulated in SW480-ADH and MaCaT cells, respectively, when they were co-cultured with Snai1-expressing cells. Furthermore, cytokine analysis showed differences in the conditioned media obtained from the two cell types. These results indicate that histologically normal tissue adjacent to tumor tissue expressing the EMT-inducing gene SNAI1 shows alterations in the expression of epithelial differentiation genes such as CDH1 and VDR.

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.

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.

MicroRNAs (miRNAs) are increasingly implicated in regulating the malignant progression of cancer. Here we show that miR-9, which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness. miR-9-mediated E-cadherin downregulation results in the activation of beta-catenin signalling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumour angiogenesis. Overexpression of miR-9 in otherwise non-metastatic breast tumour cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 by using a 'miRNA sponge' in highly malignant cells inhibits metastasis formation. expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumour grade and metastatic status. These findings uncover a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin.

The Kruppel-like factor 4 (KLF4) is a transcriptional regulator of proliferation and differentiation in epithelial cells, both during development and tumorigenesis. Although KLF4 functions as a tumor suppressor in several tissues, including the colon, the role of KLF4 in breast cancer is less clear. Here, we show that KLF4 is necessary for maintenance of the epithelial phenotype in non-transformed MCF-10A mammary epithelial cells. KLF4 silencing led to alterations in epithelial cell morphology and migration, indicative of an epithelial-to-mesenchymal transition. Consistent with these changes, decreased levels of KLF4 also resulted in the loss of E-cadherin protein and mRNA. Promoter/reporter analyses revealed decreased E-cadherin promoter activity with KLF4 silencing, while chromatin immunoprecipitation identified endogenous KLF4 binding to the GC-rich/E-box region of this promoter. Furthermore, forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cell line was sufficient to restore E-cadherin expression and suppress migration and invasion. These findings identify E-cadherin as a novel transcriptional target of KLF4. The clear requirement for KLF4 to maintain E-cadherin expression and prevent epithelial-to-mesenchymal transition in mammary epithelial cells supports a metastasis suppressive role for KLF4 in breast cancer.

OBJECTIVE: Metastasis is thought to be governed partially by induction of epithelial-mesenchymal transition. Combination of proteasome and histone deacetylase inhibitors has shown significant promise, but no studies have investigated this in esophageal cancer. This study investigated effects of vorinostat (histone deacetylase inhibitor) and bortezomib (proteasome inhibitor) on esophageal cancer epithelial-mesenchymal transition. METHODS: Three-dimensional tumor spheroids mimicking tumor architecture were created with esophageal squamous and adenocarcinoma cancer cells. Cells were treated with tumor necrosis factor alpha (to simulate proinflammatory tumor milieu) and transforming growth factor beta (cytokine critical for induction of epithelial-mesenchymal transition). tumor models were then treated with vorinostat, bortezomib, or both. Cytotoxic assays assessed cell death. Messenger RNA and protein expressions of metastasis suppressor genes were assessed. After treatment, Boyden chamber invasion assays were performed. RESULTS: Combined therapy resulted in 3.7-fold decrease in adenocarcinoma cell invasion (P = .002) and 2.8-fold decrease in squamous cell invasion (P = .003). Three-dimensional invasion assays demonstrated significant decrease in epithelial-mesenchymal transition after combined therapy. Quantitative reverse transcriptase polymerase chain reaction and Western blot analyses revealed robust rescue of E-cadherin transcription and protein expression after combined therapy. Importantly, inhibition of the E-cadherin gene resulted in abolition of the salutary benefits of combined therapy, highlighting the importance of this metastasis suppressor gene in the epithelial-mesenchymal transition process. CONCLUSIONS: Combined vorinostat and bortezomib therapy significantly decreased esophageal cancer epithelial-mesenchymal transition. This combined therapeutic effect on esophageal cancer epithelial-mesenchymal transition was associated with upregulation of E-cadherin protein expression.CI - 2010 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.

epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells is a pathological process that occurs during peritoneal dialysis. EMT leads to peritoneal fibrosis, ultrafiltration failure and eventually to the discontinuation of therapy. Signaling pathways involved in mesothelial EMT are thus of great interest, but are mostly unknown. We used primary mesothelial cells from human omentum to analyze the role of the p38 MAPK signaling pathway in the induction of EMT. The use of specific inhibitors, a dominant-negative p38 mutant and lentiviral silencing of p38alpha demonstrated that p38 promotes E-cadherin expression both in untreated cells and in cells co-stimulated with the EMT-inducing stimuli transforming growth factor (TGF)-beta1 and interleukin (IL)-1beta. p38 inhibition also led to disorganization and downregulation of cytokeratin filaments and zonula occludens (ZO)-1, whereas expression of vimentin was increased. Analysis of transcription factors that repress E-cadherin expression showed that p38 blockade inhibited expression of Snail1 while increasing expression of Twist. Nuclear translocation and transcriptional activity of p65 NF-kappaB, an important inducer of EMT, was increased by p38 inhibition. Moreover, p38 inhibition increased the phosphorylation of TGF-beta-activated kinase 1 (TAK1), NF-kappaB and IkappaBalpha. The effect of p38 inhibition on E-cadherin expression was rescued by modulating the TAK1-NF-kappaB pathway. Our results demonstrate that p38 maintains E-cadherin expression by suppressing TAK1-NF-kappaB signaling, thus impeding the induction of EMT in human primary mesothelial cells. This represents a novel role of p38 as a brake or 'gatekeeper' of EMT induction by maintaining E-cadherin levels.

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.CI - Copyright (c) 2011 Wiley-Liss, Inc.

Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-beta (TGF-beta) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-beta. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated beta1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating beta1 integrin expression necessary for metastatic outgrowth.

Detachment of epithelial cells from matrix or attachment to an inappropriate matrix engages an apoptotic response known as anoikis, which prevents metastasis. Cellular sensitivity to anoikis is compromised during the oncogenic epithelial-to-mesenchymal transition (EMT), through unknown mechanisms. We report here a pathway through which EMT confers anoikis resistance. NRAGE (neurotrophin receptor-interacting melanoma antigen) interacted with a component of the E-cadherin complex, ankyrin-G, maintaining NRAGE in the cytoplasm. Oncogenic EMT downregulated ankyrin-G, enhancing the nuclear localization of NRAGE. The oncogenic transcriptional repressor protein TBX2 interacted with NRAGE, repressing the tumor suppressor gene p14ARF. P14ARF sensitized cells to anoikis; conversely, the TBX2/NRAGE complex protected cells against anoikis by downregulating this gene. This represents a novel pathway for the regulation of anoikis by EMT and E-cadherin.

Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-beta-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-beta-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers.CI - (c)2011 AACR.

The progression of colorectal carcinoma (CRC) to invasive and metastatic disease may involve localized occurrences of epithelial-mesenchymal transition (EMT). However, mechanisms of the EMT process in CRC progression are not fully understood. We previously showed that knockdown of signal transducer and activator of transcription 3 (STAT3) up-regulated E-cadherin (a key component in EMT progression) in CRC. In this study, we examined the roles of STAT3 in CRC EMT and ZEB1, an EMT inducer, in STAT3-induced down-regulation of E-cadherin. Knockdown of STAT3 significantly increased E-cadherin and decreased N-cadherin and vimentin expressions in highly invasive LoVo CRC cells. Meanwhile, overexpression of STAT3 significantly reduced E-cadherin and enhanced N-cadherin and vimentin expressions in weakly invasive SW1116 CRC cells. Activation of STAT3 significantly increased CRC cell invasiveness and resistance to apoptosis. Knockdown of STAT3 dramatically enhanced chemosensitivity of CRC cells to fluorouracil. STAT3 regulated ZEB1 expression in CRC cells, and the STAT3-induced decrease in E-cadherin and cell invasion depended on activation of ZEB1 in CRC cells. Additionally, pSTAT3(Tyr-705) and ZEB1 expressions were significantly correlated with TNM (tumor, lymph node, and metastasis stages) (p < 0.01). In conclusion, STAT3 may directly mediate EMT progression and regulate ZEB1 expression in CRC. ZEB1 may participate in STAT3-induced cell invasion and E-cadherin down-regulation in CRC cells. The expressions of pSTAT3(Tyr-705) and ZEB1 may be positively associated with CRC metastasis. Our data may provide potential targets to prevent and/or treat CRC invasion and metastasis.

Transforming growth factor-beta (TGF-beta)-induced epithelial-mesenchymal transition (EMT) has been shown to be related to the pathogenesis of various diseases including lung cancer. Recently, microRNAs (miRNA) have been recognized as a new class of genes involved in human tumorigenesis. MiR-23a/24/27a is a miRNA cluster located in chromosome 19p13.12, which can function as an oncogene in several human cancers. In this study, we analyzed miR-23a/24/27a expression in 10 non-small cell cancer (NSCLC) cell lines by real-time PCR analysis. Correlation between expression of these miRNAs and TGF-beta/Smad signaling was evaluated. We found that miR-23a could be regulated by TGF-beta1 in a Smad-dependent manner in A549 lung adenocarcinoma cells showing the EMT phenomenon. Knockdown of miR-23a partially restored E-cadherin expression under conditions of TGF-beta1 stimulation. In contrast, overexpression of miR-23a could suppress E-cadherin expression and stimulate EMT. Furthermore, A549 cells with overexpressed miR-23a were more resistant to gefitinib compared to the parental cells. These findings suggest that miR-23a regulates TGF-beta-induced EMT by targeting E-cadherin in lung cancer cells and may be useful as a new therapeutic target in NSCLC.

The evolution of the cancer cell into a metastatic entity is the major cause of death in patients with cancer. Activation of the epithelial-to-mesenchymal transition (EMT) endows invasive and metastatic properties upon cancer cells that favor successful colonization of distal target organs. The observation that in many cancers distant metastases resemble the epithelial phenotype of primary tumors has led to speculation that the disseminated tumor cells recruited to the target organs undergo mesenchymal-to-epithelial transition (MET). However, the MET cascade has not been recapitulated in vivo, and the cellular and molecular regulators that promote MET remain unknown. In a recent report, using a model of spontaneous breast cancer, we have shown that bone marrow-derived myeloid progenitor cells in the premetastatic lung secrete the proteoglycan versican, which induces MET of metastatic tumor cells and accelerates metastases. This review summarizes recent progress in MET research, outlines a unique paracrine cross-talk between the microenvironment and the cancer cells, which promotes tumor outgrowth in the metastatic organ, and discusses opportunities for novel antimetastatic approaches for cancer therapy.CI - (c)2012 AACR.

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.

MicroRNAs (miRNAs) are important regulators of gastric cancer development and progression. miR-148a is one of the most frequently and highly downregulated miRNAs in gastric cancer and is associated with advanced clinical stage and poor prognosis. In this study, we investigated the role of miR-148a in gastric cancer metastasis. Levels of miR-148a were determined by qRT-PCR in 60 gastric cancer samples. Cell migration and invasion assays were performed in a stably expressing miRNA-148a gastric cancer cell line established using a lentivirus expression system. epithelial-mesenchymal transition (EMT) was evaluated using qRT-PCR and Western Blots to detect epithelial marker E-cadherin and mesenchymal marker, vimentin. Luciferase reporter assays were used to identify downstream targets and biological function of miR-148a. Gastric cancer tissue had significantly lower expression of miR-148a compared to non-tumor tissue. Low miR-148a levels were associated with lymph node metastasis, N stage, and blood vessel invasion. miR-148a overexpression inhibited metastasis of gastric cancer cells. miR-148a overexpression also downregulated vimentin expression and upregulated E-cadherin expression, suggesting that miR-148a inhibited EMT. Finally, the SMAD2 gene was identified as the direct and functional target of miR-148a. MiR-148a suppresses gastric cancer metastasis and EMT, likely via SMAD2. Restoration of miR-148a expression could have important implications in gastric cancer therapy.

In melanoma cells, high expression of the transcription factor GLI2 is associated with increased invasive potential and loss of E-cadherin expression, an event reminiscent of the epithelial-to-mesenchymal transition (EMT). Herein, we provide evidence that GLI2 represses E-cadherin gene (CDH1) expression in melanoma cells via distinct mechanisms, enhancing transcription of the EMT-activator ZEB1 and cooperative repression of CDH1 gene transcription via direct binding of both GLI2 and ZEB1 to two closely positioned Kruppel-like factor-binding sites within the CDH1 promoter. GLI2 silencing rescued CDH1 expression except in melanoma cell lines in which the CDH1 promoter was hypermethylated. Proximity ligation assays identified GLI2-ZEB1 complexes in melanoma cell nuclei, proportional to endogenous GLI2 and ZEB1 expression, and whose accumulation was enhanced by the classical EMT inducer TGF-beta. These data identify GLI2 as a critical modulator of the cadherin switch in melanoma, a molecular process that is critical for metastatic spread of the disease.CI - (c) 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The gold standard for determining the tumorigenic potential of human cancer cells is a xenotransplantation into immunodeficient mice. Higher tumorigenicity of cells is associated with earlier tumor onset. Here, we used xenotransplantation to assess the tumorigenic potential of human breast cancer cells following RNA interference-mediated inhibition of over 5000 genes. We identify 16 candidate tumor suppressors, one of which is the zinc-finger transcription factor SALL1. Analyzing this particular molecule in more detail, we show that inhibition of SALL1 correlates with reduced levels of CDH1, an important contributor to epithelial-to-mesenchymal transition. Furthermore, SALL1 expression led to an increased migration and more than twice as many cells expressing a cancer stem cell signature. Also, SALL1 expression correlates with the survival of breast cancer patients. These findings cast new light on a gene that has previously been described to be relevant during embryogenesis, but not carcinogenesis.

Recent research into the mechanisms of tumour cell invasiveness has highlighted the parallels between carcinogenesis and epithelial-mesenchymal transition (EMT), originally described as a developmental transdifferentiation program but also implicated in fibrosis and cancer. In a model system for mammary carcinogenesis, we previously observed that induced signalling from a homodimer of the c-erbB2 (HER2) receptor tyrosine kinase in an initially non-malignant mammary cell line caused EMT where i) cell scattering occurred before downregulation of the cell-cell adhesion molecule E-cadherin and ii) the progress of EMT was dramatically delayed when cells were grown at high density. Here, we have further analysed these phenomena. Ectopic expression of E-cadherin concomitant with c-erbB2 signalling was unable to impede the progression of EMT, suggesting that E-cadherin downregulation is not required for EMT. Furthermore, fibroblast-like cells isolated after EMT induced in the presence or absence of ectopic E-cadherin expression showed highly similar morphology and vimentin expression. E-cadherin expressed in these fibroblastic cells had a subcellular localisation similar to that found in epithelial cells, but it exhibited a much weaker attachment to the cytoskeleton, suggesting cytoskeletal rearrangements as an important mechanism in EMT-associated cell scattering. We also investigated whether density-dependent inhibition of EMT is mediated by E-cadherin as a sensor for cell-cell contact, by expressing dominant-negative E-cadherin. While expression of this mutant weakened cell-cell adhesion, it failed to facilitate EMT at high cell densities. These results indicate that loss of E-cadherin expression is a consequence rather than a cause of c-erbB2-induced EMT and that densitydependent inhibition of EMT is not mediated by E-cadherin signalling.

BACKGROUND: The epithelial-to-mesenchymal transition (EMT) accompanied by the downregulation of E-cadherin has been thought to promote metastasis. Cyclooxygenase-2 (Cox-2) is presumed to contribute to cancer progression through its multifaceted function, and recently its inverse relationship with E-cadherin was suggested. The aim of the present study was to investigate whether selective Cox-2 inhibitors restore the expression of E-cadherin in head and neck squamous cell carcinoma (HNSCC) cells, and to examine the possible correlations of the expression levels of EMT-related molecules with clinicopathological factors in HNSCC. METHODS: We used quantitative real-time PCR to examine the effects of three selective Cox-2 inhibitors, i.e., celecoxib, NS-398, and SC-791 on the gene expressions of E-cadherin (CDH-1) and its transcriptional repressors (SIP1, Snail, Twist) in the human HNSCC cell lines HSC-2 and HSC-4. To evaluate the changes in E-cadherin expression on the cell surface, we used a flowcytometer and immunofluorescent staining in addition to Western blotting. We evaluated and statistically analyzed the clinicopathological factors and mRNA expressions of Cox-2, CDH-1 and its repressors in surgical specimens of 40 patients with tongue squamous cell carcinoma (TSCC). RESULTS: The selective Cox-2 inhibitors upregulated the E-cadherin expression on the cell surface of the HNSCC cells through the downregulation of its transcriptional repressors. The extent of this effect depended on the baseline expression levels of both E-cadherin and Cox-2 in each cell line. A univariate analysis showed that higher Cox-2 mRNA expression (p = 0.037), lower CDH-1 mRNA expression (p = 0.020), and advanced T-classification (p = 0.036) were significantly correlated with lymph node metastasis in TSCC. A multivariate logistic regression revealed that lower CDH-1 mRNA expression was the independent risk factor affecting lymph node metastasis (p = 0.041). CONCLUSIONS: These findings suggest that the appropriately selective administration of certain Cox-2 inhibitors may have an anti-metastatic effect through suppression of the EMT by restoring E-cadherin expression. In addition, the downregulation of CDH-1 resulting from the EMT may be closely involved in lymph node metastasis in TSCC.

BACKGROUND: E-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT). METHODS: To improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4 bp deletion in CDH1 exon 11. RESULTS: The E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed. CONCLUSIONS: Overall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling.

The expression of pseudopodium-enriched atypical kinase 1(PEAK1) has been studied in human cancers. However, their roles in gastric cancer are still unknown. In this study, gastric cancer tissue microarrays were constructed with 159 gastric cancer tissue samples, 150 non-neoplastic gastric epithelium specimens and 152 lymph node samples. Immunohistochemical staining for PEAK1 and E-cadherin was performed. Our study found negative expression of PEAK1 in 113 of 159 (71.1%) gastric cancers, in 46 of 150 (30.7%) non-neoplastic gastric epithelium tissues and in 69 of 94 (73.4%) metastatic lymph nodes. Negative expression of PEAK1 and E-cadherin associated with tumor grading, depth of invasion, lymph node metastases, pTNM stage and macroscopic type. Patients with either positive PEAK1 or E-cadherin expression had a significantly higher survival than those with negative expression. When combined, PEAK1(-)/E-cadherin(-) had a significantly poor prognosis than the rest of the patients. The expression of PEAK1 protein was positively correlated with E-cadherin in cancer tissues. Cox regression analyses showed that PEAK1, E-cadherin and PEAK1(-)/E-cadherin(-) were independent predictors of overall survival. In conclusion, our findings suggest that loss of PEAK1 may play an important role in carcinogenesis and development of gastric cancer through activating epithelial to mesenchymal transition.CI - Copyright (c) 2014 Elsevier GmbH. All rights reserved.

The Groucho transcriptional corepressor TLE1 protein has recently been shown to be a putative lung specific oncogene, but its underlying oncogenic activity in lung cancer has not been fully elucidated. In this report, we investigated whether TLE1 regulates lung cancer aggressiveness using the human lung adenocarcinoma cell line A549 as a model system. Through a combination of genetic approaches, we found that TLE1 potentiates epithelial-to-mesenchymal transition (EMT) in A549 cells in part through suppression of the tumor suppressor gene E-cadherin. Exogenous expression of TLE1 in A549 cells resulted in heightened EMT phenotypes (enhanced fibroblastoid morphology and increased cell migratory potential) and in molecular alterations characteristic of EMT (downregulation of the epithelial marker E-cadherin and upregulation of the mesenchymal marker Vimentin). Conversely, downregulation of endogenous TLE1 expression in these cells resulted in reversal of basal EMT characterized by a cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Mechanistic studies showed that TLE1 suppresses E-cadherin expression at the transcriptional level in part by recruiting histone deacetylase (HDAC) activity to the E-cadherin promoter. Consistently, the HDAC inhibitor TSA partially reversed the TLE1-induced E-cadherin downregulation and cell migration, suggesting a role for HDACs in TLE1-mediated transcriptional repression of E-cadherin and EMT function. These findings uncover a novel role of TLE1 in regulating EMT in A549 cells through its repressive effect on E-cadherin and provide a mechanism for TLE1 oncogenic activity in lung cancer.CI - Published by Elsevier Inc.

Oral cancer was ranked as the sixth worldwide most common cancer, but recent studies noticed an overall downward trend in its incidence. However, for tumors localized on the tongue, the incidence seems to increase. The malignant transformation of many carcinomas is associated with loss of epithelial differentiation and gain of a mesenchymal phenotype, a process known as epithelial-mesenchymal transition (EMT) which for oral squamous cell carcinomas (OSCC) could be a predictor and a prognostic factor. The aim of our study was to investigate immunohistochemically the E-cadherin/N-cadherin "switch" and vimentin expression as markers of EMT process in tongue OSCC. Thus, we analyzed 15 cases of tongue OSCC by enzymatic double immunohistochemistry using the following double pairs of antibodies: E-cadherin/vimentin and N-cadherin/E-cadherin. E-cadherin reactivity was recorded in all the investigated cases, the pattern of expression being both membranous and cytoplasmic, with the membrane pattern decreasing simultaneously with the decrease of the differentiation degree and with the increase of invasion phenotype, while the cytoplasmic pattern had an opposite behavior. tumor parenchyma reactivity for vimentin was noticed in 73.3% and its expression was more obvious in tumor cells from the periphery of proliferative islands and in acantholytic carcinomatous cells. N-cadherin reactivity was restricted to only 33.33% of the investigated cases and its expression was prevalent in poorly differentiated forms. In conclusion, in tongue squamous cell carcinomas at the invasion front the E-cadherin reactivity decreases while vimentin expression increases, with a cytoplasmic N-cadherin reactivity in a few of the observed cases. This EMT phenotype was correlated with the decrease of differentiation degree, with the increase of the type of invasion pattern and with increasing disease' stages and thus these EMT markers could be used for prognostic stratification of such patients.

Decreased expression of epithelial cadherin (E-cadherin) has been noted to associate with aggressiveness and metastasis of breast cancer. The aim of this study was to examine the effect of C-Terminal EH domain-containing protein 2 (EHD2) expression on E-cadherin and related mechanism in the metastasis of breast cancer. Immunohistochemical analysis was performed in 96 human breast carcinoma samples and the data were correlated with clinicopathologic characteristics. Furthermore, Western blot analysis was performed for EHD2 and E-cadherin in breast carcinoma samples and cell lines to evaluate their protein levels and molecular interaction. We found that the expression of EHD2 was positively related with E-cadherin expression (P < 0.01), moreover, EHD2 expression was significantly correlated with histologic grade (P < 0.01). Meanwhile, E-cadherin expression obtained similar results. Kaplan-Meier survival analysis showed that decreased expression of EHD2 and E-cadherin exhibited a significant correlation with poor prognosis in human breast cancer (P < 0.01). While in vitro, we employed siRNA technique to knock down EHD2 expressions and observed their effects on breast cancer cells growth. EHD2 depletion by siRNA promoted PCNA expression, and it was concurrent with the decreased expression of epithelial marker E-cadherin and the increased expression of N-cadherin by Western blot analysis. Consistent with these observations, the suppression of EHD2 in breast cancer cells remarkably promoted cellular proliferation and migration. On the basis of these results, we suggested that EHD2 can inhibit the metastasis of human breast cancer by regulating the EMT key markers E-cadherin and N-cadherin.

The alteration of epithelial stability, which includes changes in the expression of E-cadherin and CD44, is one of the complex biomolecular mechanisms involved in the tumoral epithelial-mesenchymal transition (EMT) process. We followed the E-cadherin/CD44 immunophenotype by single and double detections in 25 cases of bladder urothelial carcinomas. Our study investigated simultaneously the differences in expression of the two markers, in different tumoral compartments, according to the prognostic parameters of the lesions. The study indicated significant differences in the expression of E-cadherin in relation to tumor grade, depth of invasion, tumor stage and Ki-67 proliferation index (PI), both intratumoral and at the advancing edge. For CD44, expression differences were found between the tumor grades in intratumoral sites, while for both intratumoral and advancing edge compartments the differences occurred for the depth of tumor invasion, tumor stage and Ki-67 PI. The only differences in the expression of the two markers in relation with the presence of lymph node metastasis were for E-cadherin at the advancing edge. In this study, the intratumoral E-caderin-/CD44- immunophenotype, respectively E-caderin-/CD44+ at the advancing edge were associated with the tumor aggressiveness analyzed parameters. The maintenance of CD44 expression at the advancing edge represents a negative prognostic factor for bladder urothelial carcinoma and supports the implication of EMT process, through the existence at this level of a cell population with particular properties.

As a critical developmental process, epithelial-mesenchymal transition (EMT) involves complex transcriptional reprogramming and has been closely linked to malignant progression. Although various epigenetic modifications, such as histone deacetylation and H3K9 methylation, have been implicated in this process, how they are coordinated remains elusive. We recently revealed that MPP8 couples H3K9 methylation and DNA methylation for E-cadherin gene silencing and promotes tumor cell migration, invasion, and EMT. Here, we show that MPP8 cooperates with the class III HDAC SIRT1 in this process through their physical interaction. SIRT1 antagonizes PCAF-catalyzed MPP8-K439 acetylation to protect MPP8 from ubiquitin-proteasome-mediated proteolysis. Conversely, MPP8 recruits SIRT1 for H4K16 deacetylation after binding to methyl-H3K9 on target promoters. Consequently, disabling either MPP8 methyl-H3K9 binding or SIRT1 interaction de-represses E-cadherin and reduces EMT phenotypes, as does knockdown of MPP8 or SIRT1 in prostate cancer cells. These results illustrate how SIRT1 and MPP8 reciprocally promote each other's function and coordinate epithelial gene silencing and EMT.CI - (c) 2015 The Authors.

Laryngeal carcinoma is the second most common malignancy of the head and neck squamous cell carcinoma. Therefore, there is an urgent need to understand the molecular mechanism of its metastasis. The present study was designed to investigate effects of miR-10b on the invasion and migration of laryngeal Hep-2 cells. We found that miR-10b had limited effects on cell proliferation; however, it can significantly promote the migration and invasion of Hep-2 cells. Further studies revealed that overexpression of miR-10b can induce the epithelial-mesenchymal transition (EMT) of Hep-2 cells by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin (N-Cad) with a concomitant decrease of E-cadherin (E-Cad). However, the messenger RNA (mRNA) and protein level of transcription factors such as Snail, Slug, Twist and ZEB was not changed during this process. Bioinformatic analysis revealed that miR-10b can directly target CDH1 (E-Cad gene) at nucleotides 461 and 481 within the 3'-UTR. This was confirmed by the results that miR-10 downregulated the protein and mRNA levels of E-Cad via a time-dependent manner and luciferase analysis by use of four-nucleotide substitution in the core binding sites. The present study provided a better understanding of laryngeal carcinoma metastasis and the roles of miR-10b during this process.

Previous reports have demonstrated that hepatitis B virus (HBV) X protein (HBx) represses E-cadherin expression to induce epithelial-mesenchymal transition (EMT), an essential component of cancer progression to more aggressive phenotypes characterized by tumour invasion, migration and metastasis; however, the underlying mechanism for this phenomenon is still unclear. In this study, we found that ectopic expression of HBx in human hepatocytes using overexpression and 1.2-mer WT HBV replicon systems upregulated levels of the transcriptional repressors E12 and E47, resulting in inactivation of the E-cadherin promoter, containing three E-box motifs, and subsequent repression of its expression. E12/E47 knockdown using a specific small interfering RNA almost completely abolished the potential of HBx to repress E-cadherin expression. HBx inhibited the ubiquitin-dependent proteasomal degradation of E12/E47 without affecting their expression at the transcriptional level. Upregulation of E12/E47 by HBx ultimately led to EMT in human hepatocytes, as demonstrated by morphological changes, altered protein levels of EMT markers, including E-cadherin, plakoglobin, fibronectin, vimentin and N-cadherin, and increased capacity for cell detachment and migration.

In human cancer, high telomerase expression is correlated with tumor aggressiveness and metastatic potential. Telomerase activation occurs through telomerase reverse transcriptase (hTERT) induction, which contributes to malignant transformation by stabilizing telomeres. Previous studies have shown that hTERT can promote tumor invasion and metastasis of gastric cancer, liver cancer and esophageal cancer. epithelial-to-mesenchymal transition (EMT), a requirement for tumor invasion and metastasis, plays a key role in cancer progression. Although hTERT promotes EMT through Wnt signaling in several cancers, it is unknown if other signaling pathways are involved. In the present study, we found that hTERT and ZEB1 form a complex, which directly binds to the E-cadherin promoter, and then inhibits E-cadherin expression and promots EMT in colorectal cancer cells. hTERT overexpression in HCT116 and SW480 cells could induce E-cadherin down-regulation. However, E-cadherin expression was recovered when ZEB1 function was impaired even during hTERT overexpression. Taken together, our findings suggest that hTERT can promote cancer metastasis by stimulating EMT through the ZEB1 pathway and therefore inhibiting them may prevent cancer progression.

Ubiquitinlike with plant homeodomain (PHD) and RINGfinger domain 1 (UHRF1) maintains methylation patterns following DNA replication and is expressed at high levels in various types of human cancer. UHRF1 has been identified as a novel oncogene involved in the pathogenesis of hepatocellular carcinoma. Previous studies have demonstrated that inhibition of the expression of UHRF1 suppresses the proliferation of cancer cells. However, the role of UHRF1 in human osteosarcoma has not been investigated. The present study examined the expression levels of UHRF1 and retinoblastoma 1 (Rb1) in human osteosarcoma cell lines by western blot analysis. Stable overexpression of UHRF1 or knockdown of Rb1 was achieved by lentiviral transfection. Subsequently, a Cell Counting Kit-8 assay and a cell invasion assay were performed to detect the biological functions of UHRF1 in vitro. The results of the present study demonstrated that UHRF1 promoted the proliferation of human osteosarcoma cells. The present study also reported that UHRF1 was able to enhance the invasion of osteosarcoma cells in a retinoblastoma 1 (Rb1)dependent manner. UHRF1 promoted invasion in Rb1positive osteosarcoma cells, but not in Saos2 cells with homozygous loss of Rb1. Similarly, knockdown of Rb1 in Rb1positive osteosarcoma cells enhanced levels of invasion and eliminated the regulation of invasion by UHRF1. UHRF1 was found to inhibit the mRNA and protein expression levels of Rb1. Furthermore, deletion of Rb1 was found to suppress the expression of Ecadherin and promote epithelialtomesenchymal transition (EMT). In addition, the overexpression of UHRF1 inhibited the expression of Ecadherin and promoted EMT via the suppression of Rb1. These data demonstrated that UHRF1 promotes osteosarcoma cell invasion by downregulating the expression of Ecadherin and increasing EMT in an Rb1dependent manner.

Downregulation of E-cadherin by the transcriptional repressor Snail is associated with acquisition of metastatic potential. Although hepatitis C virus (HCV) core protein has been implicated in hepatocarcinogenesis, it is unclear whether Snail is involved in HCV core-induced dysregulation of E-cadherin. Herein, we investigated the mechanism by which HCV core induces E-cadherin repression and the role of Snail in HCV core-mediated invasiveness and metastasis. We found that HCV infection, especially HCV core expression, effectively induced the epithelial-mesenchymal transition (EMT) in hepatoma cells by repressing E-cadherin. HCV core interacted with Snail and enhanced its binding to the E-box in the promoter region of E-cadherin, leading to decreased E-cadherin promoter activity. We found that HCV core, Snail, and the histone deacetylases HDAC1/HDAC2 formed a co-repressor complex at the E-cadherin promoter. Moreover, HCV core was shown to stabilize Snail through activation of the PI3K/Akt/GSK3beta pathway. Silencing Snail expression restored E-cadherin expression and inhibited HCV core-promoted tumor growth and distant lung metastasis in vivo. Collectively, these results demonstrated that HCV core induced EMT by interacting with the transcriptional repressor complex Snail/HDACs at the E-cadherin promoter, which led to E-cadherin repression and increased invasiveness of hepatoma cells. These findings increase understanding of factors regulating metastasis in hepatoma and may ultimately lead to the development of novel treatment strategies for HCV-associated hepatocellular carcinoma.

Acquired radioresistance during radiotherapy has significantly affected the treatment efficacy in esophageal cancer. Many of radioresistant cancer cells demonstrated epithelial-mesenchymal transition (EMT).We found in previous study that a radioresistant cell line (KYSE-150R) possessed EMT characteristic with cyclin D1 overexpression. Cyclin D1 has been demonstrated to affect the radiation sensitivity in cancer cells. To elucidate the molecular functions of cyclin D1 on EMT phenotypes and esophageal cancer radiosensitivity, we treated the radioresistant esophageal cancer cells (KYSE-150R) and parental cells (KYSE-150) with cyclin D1 small interfering RNA (siRNA). The cell proliferation rate of KYSE-150R and the radiation survival fraction were significantly decreased in cyclin D1 siRNA treatment group. Knocking down cyclin D1 resulted in G0/G1 arrest in KYSE-150R cells. The average number of irradiation-induced gamma-H2AX foci increased in the cells treated with cyclin D1 siRNA, indicating impaired DNA double-strand break (DSB) repair in KYSE-150R cells. Cyclin D1 also reversed EMT phenotypes with significantly increased expression of E-cadherin in KYSE-150R cells. However, cyclin D1 siRNA have no radiosensitizing effects on KYSE-150 cells, with no obvious change in EMT marker expression .Our work showed that EMT phenotypes can be reduced and the radiosensitivity of esophageal cancer cells can be enhanced by inhibiting cyclin D1.

BACKGROUND: Metastasis-associated gene 1(MTA1) has been identified as an oncogene in many tumors, and aberrant MTA1 expression has been linked to carcinogenesis and metastasis. We aim to investigate the mechanism of MTA1 and metastasis in malignant pleural mesothelioma (MPM). METHODS: Real-time polymerase chain reaction (PCR) and immunohistochemical staining were employed to detect MTA1 and E-cadherin expression in MPM tissues and corresponding adjacent tissues. Stable clone with knock-down of MTA1 was generated with shRNA via lentivirus technology in MPM cell lines. Wound-healing assay, transwell assay and PCR array were carried out for detecting invasion and migration of MPM cells. Luciferase reporter assay was performed to validate the effect of MTA1 on E-cadherin. RESULTS: MTA1 expression is up-regulated in MPM and shown a negative correlation with E-cadherin expression. MTA1 could enhance the invasion and migration of MPM cells via suppressing the expression of E-cadherin. MTA1 overexpression is associated with pathology, metastasis and survival rate of MPM patients. CONCLUSIONS: MTA1 plays an important role in epithelial-to-mesenchymal transition (EMT) to promote metastasis via suppressing E-cadherin expression, resulting in a poor prognosis in MPM. MTA1 is a novel biomarker and indicative of a poor prognosis in MPM patients.

BACKGROUND: Previously, we reported that polyploid giant cancer cells (PGCCs) induced by cobalt chloride (CoCl2) could have generated daughter cells with strong invasiveness and migration capabilities via asymmetric divisions. This study compared the expression of epithelial-mesenchymal transition (EMT)-related proteins, including E-cadherin, N-cadherin, and vimentin, between PGCCs and their daughter cells, and control breast cancer cell lines MCF-7 and MDA-MB-231. The clinicopathological significance of EMT-related protein expression in human breast cancer was analyzed. METHODS: Western blot was used to compare the expression levels of E-cadherin, N-cadherin, and vimentin in breast cancer lines MCF-7 and MDA-MB-231, between PGCCs with budding daughter cells and control breast cancer cells. Furthermore, 167 paraffin-embedded breast tumor tissue samples were analyzed, including samples obtained from 52 patients with primary breast cancer with lymph node metastasis (group I) and their corresponding lymph node metastatic tumors (group II), 52 patients with primary breast cancer without metastasis (group III), and 11 patients with benign breast lesions (group IV). The number of PGCCs was compared among these four groups. RESULTS: The number of PGCCs increased with the malignant grade of breast tumor. Group IIhad the highest number of PGCCs and the differences among group I, II, III and IV had statistically significance (P =0.000). In addition, the expression of E-cadherin (P = 0.000), N-cadherin (P = 0.000), and vimentin (P = 0.000) was significantly different among the four groups. Group II exhibited the highest expression levels of N-cadherin and vimentin and the lowest expression levels of E-cadherin. CONCLUSIONS: These data suggest that the number of PGCCs and the EMT-related proteins E-cadherin, N-cadherin, and vimentin may be valuable biomarkers to assess metastasis in patients with breast cancer.

The aim of the present study was to determine the role of interleukin-6 (IL-6) in the epithelial-mesenchymal transition (EMT) of human intrahepatic biliary epithelial cell (HIBEC) lines in vitro. HIBECs were stimulated with IL-6 at concentrations of 0, 10, 20, 50 and 100 microg/l for 24 h. A wound healing and Transwell assay were performed to determine the migratory and invasive capacity of HIBECs, respectively. Following 24 h of incubation, IL-6 at 10 and 20 microg/l significantly increased the number of migrated and invaded cells (P<0.05), while stimulation with 50 and 100 microg/l IL-6 resulted in a further increase of the migratory and invasive capacity compared to that in all other groups (P<0.05). Furthermore, reverse-transcription quantitative polymerase chain reaction and western blot analyses were used to detect the mRNA and protein expression of EMT markers E-cadherin and vimentin in HIBECs. Decreased mRNA levels of E-cadherin accompanied by higher mRNA levels of vimentin were observed in the 10, 20, 50, 100 microg/l IL-6 groups compared to those in the 0 microg/l group (all P<0.05). Furthermore, the protein expression of E-cadherin was decreased, while that of vimentin was increased in the 50 and 100 microg/l IL-6 groups compared to those in the 0, 10 and 20 microg/l IL-6 groups (all P<0.05). The present study therefore indicated that IL-6 promoted the process of EMT in HIBECs as characterized by increased migration and invasion of HIBECs and the typical changes in mRNA and protein expression of the EMT markers E-cadherin and vimentin.

AIM: Presenilin-1 (PS1), the main component of gamma-secretase activity support a key role during epithelial-mesenchymal transition (EMT) and chemoresistance acquisition by triggering a complex sequence of molecular events, including E-cadherin down-regulation. However, we hypothesize that EMT and chemoresistance should be deemed separate processes in HCT-8 colon cancer cells. MAIN METHODS: HCT-8 and HCT-8FUres invasion was evaluated by trans-well assay. uPA activity was detected by zymography. Prostaglandin E2 levels were quantified using an ELISA kit. E-cadherin FL and CTF2, PS1, Notch1, Cyclin D1, COX2, SNAI1 and alpha-SMA expression were determined using Western blot technique. beta-Catenin localization was observed by confocal microscopy. Cell apoptosis was evaluated by cytofluorimetric assay, and measurement of caspase-3 and cl-PARP. gamma-Secretase activity was inhibited by DAPT, a gamma-secretase inhibitor. KEY FINDINGS: Chemoresistant HCT-8 underwent EMT that can be efficiently reversed by inhibiting PS1 activity, leading thus to a normalization of mostly of the pivotal features showed by the invasive cancer phenotype. Indeed, we observed decreased SNAI1 and Notch 1 activation, altogether with reduced E-cadherin cleavage. Concomitantly, resistant HCT-8 invasiveness was almost completely abolished. However, such reversion was not followed by any increase in apoptotic rate, not by changes in E-cadherin levels. Indeed, despite HCT-8FUres underwent an undeniable EMT, full-length E-cadherin levels were found remarkably higher than those observed in wild HCT-8. SIGNIFICANCE: High E-cadherin concentration in presence of enhanced gamma-secretase activity is incontestably a paradoxically result, highlighting that E-cadherin loss is not a pre-requisite for EMT. Additionally, EMT and chemoresistance acquisition in HCT-8 should be considered as distinct processes.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

BACKGROUND: FoxC2 is an epithelial-mesenchymal transition (EMT) regulator which induces metastasis. The purpose of this study is to assess the prognostic value of FoxC2 expression in non-small cell lung cancer (NSCLC), alone or in combination with E-cadherin expression. METHODS: A retrospective study was conducted using immunohistochemistry to investigate FoxC2 and E-cadherin expression in a cohort of 309 patients with surgically resected NSCLCs. The prognostic value of FoxC2 and E-cadherin on overall survival (OS) and recurrence-free survival (RFS) was determined by Kaplan-Meier analysis and Cox proportional hazard models. RESULTS: High FoxC2 expression was detected in 26.5% of tumors, and significantly correlated with tobacco use (p = 0.047), adenocarcinoma (p = 0.008) and nodal involvement (p < 0.001). Univariate analysis revealed its association with OS (p = 0.036) and RFS (p = 0.011). By multivariate analysis, high FoxC2 expression lost its significance as an independent predictor of recurrence (p = 0.077), while TNM stage, nodal status and the presence of high FoxC2 and impaired E-cadherin expression retained independent prognostic significance in relation to both OS and RFS. Subset analyses indicated that high FoxC2 expression was significantly associated with disease outcome in node-positive, but not in node-negative patients. CONCLUSION: Evaluation of FoxC2 expression, alone or in combination with E-cadherin expression, may help to stratify NSCLC patients for risk of disease progression, pointing to this EMT regulator as a potential prognostic marker.

BACKGROUND: epithelial-mesenchymal transition (EMT) is a dedifferentiation process that mainly involves in mesenchymal marker upregulation, epithelial maker downregulation and cell polarity loss. Related hypoxia factors play a crucial role in EMT, however, it remains few evidence to clarify the role of HIF-2alpha in EMT in pancreatic cancer. METHOD: In this study, we investigated the expression of HIF-2alpha and E-cadherin by immunohistochemistry in 70 pancreatic cancer patients, as well as the correlation to the clinicopathologic characteristics. Then we regulated the expression of HIF-2alpha in pancreatic cancer cells to examine the role of HIF-2alpha on invasion and migration in vitro. Finally, we tested the relation of HIF-2alpha and EMT related proteins by Western blot and determined whether HIF-2alpha regulated EMT through Twist regulating the expression of E-cadherin by Chromatin immunoprecipitation (ChIP) assay. RESULTS: We found that HIF-2alpha protein was expressed positively in 67.1% (47/70) of pancreatic cancer tissues and 11.4% (8/70) of adjacent non-tumor pancreatic tissues, and there was a significant difference in the positive rate of HIF-2alpha protein between two groups (chi2 = 45.549, P < 0.05). In addition, the staining for HIF-2alpha was correlated with tumor differentiation (P < 0.05), clinical stage (P < 0.05) and lymph node metastasis (P < 0.05), while E-cadherin expression was only correlated with lymph node metastasis (P < 0.05). HIF-2alpha promoted cell migration, invasion in vitro, and regulated the expression of E-cadherin and MMPs, which are critical to EMT. Our further ChIP assay suggested that only Twist2 could bind to the promoter of E-cadherin in -714 bp region site, but there is no positive binding capacity in -295 bp promoter region site of E-cadherin. Clinical tissues IHC staining showed that Twist2 and E-cadherin expression had an obviously negative correlation in pancreatic cancer. Nevertheless, it had no obvious correlation between Twist1 and E-cadherin. CONCLUSION: These findings indicated that HIF-2alpha promotes EMT in pancreatic cancer by regulating Twist2 binding to the promoter of E-cadherin, which meant that HIF-2alpha and this pathway may be effective therapeutic targets for pancreatic cancer.

In vitro studies in melanoma indicate that up-regulation of the transcriptional repressor Snail occurs with a concomitant decrease of its target E-cadherin, both hallmarks of epithelial-mesenchymal transition-an association not established in vivo. We sought to elucidate the relationship between BRAF, Snail, E-cadherin, and established histopathologic prognosticators in primary cutaneous melanoma. Archived annotated samples with a diagnosis of primary cutaneous melanoma were retrieved (n = 68 cases; 34 BRAF mutant and 34 BRAF wild type) and immunohistochemically stained for Snail and E-cadherin protein expression. A semiquantitative scoring system was used. Multivariate logistic analysis was used to control confounders of BRAF. Snail expression was significantly associated only with ulceration (42% versus 13%; P = .02). E-cadherin expression was present in 26% of BRAF mutant and 71% of BRAF wild-type cases (P = .0003). Loss of E-cadherin expression was associated with female sex (60% versus 34%; P = .05), BRAF mutation (74% versus 29%; P = .0003), thickness greater than or equal to 1 mm (68% versus 32%; P = .004), mitosis (63% versus 25%; P = .007), and ulceration (75% versus 44%; P = .05). BRAF mutation was associated with male sex (60% versus 30%; P = .02), Breslow thickness (P = .007), thickness greater than or equal to 1 mm (68% versus 29%; P = .002), and ulceration (75% versus 42%; P = .02). Snail expression did not correlate with loss of E-cadherin expression (47% versus 53%; P = .79). After controlling for potential confounding, BRAF mutation was associated with loss of E-cadherin (adjusted odds ratio, 8.332; 95% confidence interval, 2.257-30.757; P = .0015) and Breslow thickness greater than 1 mm (adjusted odds ratio, 7.360; 95% confidence interval, 1.534-35.318; P = .0126). Our findings, indicating that mutant BRAF represses E-cadherin expression, implicating a catalytic role for BRAF in epithelial-mesenchymal transition.CI - Published by Elsevier Inc.

UNLABELLED: Globally, most patients are at late-stage when they have been diagnosed with ovarian cancer. Investigating the potential mechanisms involved in tumor progression and prognosis is essential for improving treatment options, outcomes, and survival. OBJECTIVE: This study elucidated the clinico-pathological significance of TWIST2 and the relationship of TWIST2, E-cadherin, and Vimentin expression in the progression and prognosis of epithelial ovarian cancer (EOC). MATERIALS AND METHODS: Immunohistochemical staining was used to quantify the expression and relevance of TWIST2, E-cadherin, and Vimentin in 103 ovarian specimens, including 30 cases of benign ovarian tumors, 30 cases of borderline ovarian tumors, and 43 cases of EOC. RESULTS: The expression of TWIST2 in the cytoplasm may help to maintain characteristics of epithelial cancer cells with E-cadherin normal membranous expression, while nuclear TWIST2 induces tumor translation front with membranous expression of Vimentin, which eventually promotes cancer metastasis. Moreover, the upregulation of TWIST2 was also related to the aberrant expression of E-cadherin and the increased expression of Vimentin, which were reported as important indicators of epithelial-mesenchymal transition (EMT). DISCUSSION: The data suggested that co-expression of TWIST2/Vimentin was an independent prognostic indicator for both overall survival and disease-free survival by multivariate Cox proportional hazards model. TWIST2 regulates EMT by depriving the epithelial cell phenotype of E-cadherin and endowing the mesenchymal cell phenotype with Vimentin, which may be involved in the progression and prognosis of ovarian cancer, and TWIST2/Vimentin co-expression might be a novel indicator with prognostic potential in EOC patients.

Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a "vascular protection god". In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

OBJECTIVES: To explore the effects of the competitive endogenous RNA (ceRNA) network between TP53INP1 and E-cadherin on the invasion and migration of glioma. RESULTS: TP53INP1 and E-cadherin mRNA and protein were significantly overexpressed in normal brain tissues compared with glioma tissue specimens and correlated with the grades of glioma negatively. The expression of TP53INP1 and E-cadherin were correlated positively. Patients with higher TP53INP1 or E-cadherin expression had longer overall survival. Moreover, TP53INP1 3'-UTR inhibited glioma cell proliferation, invasion and proliferation; Furthermore, the 3'-UTRs of TP53INP1 and E-cadherin harboured seven identical miRNAs binding sites, and TP53INP1 3'-UTR could increase the expression of E-cadherin and decrease the expression of vimentin thus repressing the epithelial-mesenchymal transition (EMT). However, the coding sequence of TP53INP1 could not increase the expression of E-cadherin and the inhibitory effect on EMT of TP53INP1 3'-UTR was reversed by the siRNA against Dicer. CONCLUSIONS: TP53INP1 3'-UTR could inhibit the EMT, thus hindering the migration and invasion of glioma via acting as a ceRNA for E-cadherin.

epithelial-mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti-fibrotic effect in various diseases. However, the pathophysiological role of isoform-specific HDACs or class-selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK-2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor beta1 (TGF-beta1)-induced HK2 cells. However, restoration of TGF-beta1-induced E-cadherin down-regulation was only seen in HK-2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF-beta1-induced N-cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E-cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective.CI - (c) 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Several members of the Poly(ADP-ribose) polymerase (PARP) family are essential regulators of genome integrity, actively prospected as drug targets for cancer therapy. Among them, PARP3 is well characterized for its functions in double-strand break repair and mitotis. Here we report that PARP3 also plays an integral role in TGFbeta and reactive oxygen species (ROS) dependent epithelial-to-mesenchymal transition (EMT) and stem-like cell properties in human mammary epithelial and breast cancer cells. PARP3 expression is higher in breast cancer cells of the mesenchymal phenotype and correlates with the expression of the mesenchymal marker Vimentin while being in inverse correlation with the epithelial marker E-cadherin. Furthermore, PARP3 expression is significantly upregulated during TGFbeta-induced EMT in various human epithelial cells. In line with this observation, PARP3 depletion alters TGFbeta-dependent EMT of mammary epithelial cells by preventing the induction of the Snail-E-cadherin axis, the dissolution of cell junctions, the acquisition of cell motility and chemoresistance. PARP3 responds to TGFbeta-induced ROS to promote a TG2-Snail-E-cadherin axis during EMT. Considering the link between EMT and cancer stem cells, we show that PARP3 promotes stem-like cell properties in mammary epithelial and breast cancer cells by inducing the expression of the stem cell markers SOX2 and OCT4, by increasing the proportion of tumor initiating CD44high/CD24low population and the formation of tumor spheroid bodies, and by promoting stem cell self-renewal. These findings point to a novel role of PARP3 in the control of TGFbeta-induced EMT and acquisition of stem-like cell features and further motivate efforts to identify PARP3 specific inhibitors.

The mitochondrial Bcl-2 inhibitor of transcription 1 (Bit1) protein is part of an anoikis-regulating pathway that is selectively dependent on integrins. We previously demonstrated that the caspase-independent apoptotic effector Bit1 exerts tumor suppressive function in lung cancer in part by inhibiting anoikis resistance and anchorage-independent growth in vitro and tumorigenicity in vivo. Herein we show a novel function of Bit1 as an inhibitor cell migration and epithelial-mesenchymal transition (EMT) in the human lung adenocarcinoma A549 cell line. Suppression of endogenous Bit1 expression via siRNA and shRNA strategies promoted mesenchymal phenotypes, including enhanced fibroblastoid morphology and cell migratory potential with concomitant downregulation of the epithelial marker E-cadherin expression. Conversely, ectopic Bit1 expression in A549 cells promoted epithelial transition characterized by cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Specific downregulation of E-cadherin in Bit1-transfected cells was sufficient to block Bit1-mediated inhibition of cell motility while forced expression of E-cadherin alone attenuated the enhanced migration of Bit1 knockdown cells, indicating that E-cadherin is a downstream target of Bit1 in regulating cell motility. Furthermore, quantitative real-time PCR and reporter analyses revealed that Bit1 upregulates E-cadherin expression at the transcriptional level through the transcriptional regulator Amino-terminal Enhancer of Split (AES) protein. Importantly, the Bit1/AES pathway induction of E-cadherin expression involves inhibition of the TLE1-mediated repression of E-cadherin, by decreasing TLE1 corepressor occupancy at the E-cadherin promoter as revealed by chromatin immunoprecipitation assays. Consistent with its EMT inhibitory function, exogenous Bit1 expression significantly suppressed the formation of lung metastases of A549 cells in an in vivo experimental metastasis model. Taken together, our studies indicate Bit1 is an inhibitor of EMT and metastasis in lung cancer and hence can serve as a molecular target in curbing lung cancer aggressiveness.

Vasculogenic mimicry (VM) plays an important role in colorectal cancer (CRC) metastasis, and both hypoxia and the epithelial-mesenchymal transition (EMT) are necessary for VM. In this study, HIF-1alpha expression was upregulated in the VM-positive CRC cell line HCT-116 and thereby affected the expression of the EMT-related markers Claudin-4, E-cadherin (E-cd) and Vimentin(VIM). SB431542 and U0126EtOH, which can inhibit of EMT were used to treat HCT-116 and HCT-8 in these experiments. Both of the inhibitors had significant effect on EMT markers and the formations of VM in CRC cells. In addition, knockdown of HIF-1alpha in the HCT-116 cells inhibited their capacity for VM. Our study reveals a regulatory role for HIF-1alpha in VM and suggests that targeting either HIF-1alpha or EMT may be a valuable strategy for the elimination of CRC metastasis.

epithelial mesenchymal transition (EMT), the transition of epithelial cells into motile mesenchymal cells, plays an important role in embryogenesis, cancer invasion, and metastasis. Ameloblastomas are common epithelial odontogenic tumors, occurring exclusively in the mandible with locally invasive growth. Thirty-seven ameloblastoma cases were evaluated for the involvement of EMT by immunohistochemical staining and western blotting using antibodies against Slug, Snail, Twist, TGF-beta, and E-cadherin. Double immunostaining was also performed. Slug and TGF-beta were expressed in the nuclei of peripheral and stellate reticulum cells of ameloblastoma nests. Twenty cases of Snail, 36 of Slug, 8 of Twist, and 19 of TGF-beta showed strong expression in tumor cells in follicular and plexiform patterns. expression of Slug and TGF-beta increased in regions where the expression of E-cadherin was reduced. EMT was found to be associated with the local invasive growth of ameloblastoma. These data suggest that reduced expression of E-cadherin and over-expression of Slug, Snail, and TGF-beta induce EMT. Given that ameloblastomas are characterized by local invasiveness, EMT might be related to their development. Thus, strong expression of Slug and TGF-beta and reduced expression of E-cadherin might be related to the local invasiveness of ameloblastoma.

Atrial natriuretic peptide (ANP) is increasingly expressed on airway and inhibits pulmonary arterial remodeling. However, the role of ANP in remodeling of respiratory system is still unclear. The role of ANP on airway remodeling and the possible mechanism was explored in this study. Both human bronchial epithelial 16HBE-14o cells and alveolar epithelial A549 cells were stimulated by TGF-beta1, ANP, cGMP inhibitor, PKG inhibitor, and cGMP analogue. The expressions of epithelial markers, mesenchymal markers, and Smad3 were assessed by quantitative real-time PCR and western blotting. Immunohistochemical staining was employed to assess Smad3 expression once it was silenced by siRNA in 16HBE-14o or A549 cells. Our results showed that the mRNA and protein expressions of E-Cadherin were decreased, whereas alpha-SMA expressions were increased after induction by TGF-beta1 in 16HBE-14o and A549 cells. The E-Cadherin expressions were increased and alpha-SMA expressions were decreased after ANP stimulation. Inhibition of cGMP or PKG decreased E-Cadherin expression but increased alpha-SMA expression, which could be reversed by cGMP analogue. Moreover, the phosphorylated Smad3 expression was consistent with alpha-SMA expression. After smad3 was silenced, Smad3 was mostly expressed in cytoplasm instead of nucleus as non-silenced cells during epithelial-mesenchymal transition (EMT). In conclusion, ANP inhibits TGF-beta1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-beta1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment on pulmonary diseases with airway remodeling.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

JPJD was an ideal alternative traditional Chinese medicine compound in the prevention and treatment of CRC, but its underlying mechanisms has not been fully elucidated. In this study, we demonstrated in vitro that TGF-beta-induced EMT promoted the invasion and metastasis of CRC cells, reduced the expression of E-cadherin, and elevated the expression of Vimentin. However, JPJD could inhibit the invasive and migratory ability of TGF-beta-stimulated CRC cells in a concentration-dependent manner through increasing the expression of E-cadherin and repressing the expression of Vimentin, as well as the inhibition of TGF-beta/Smad signaling pathway. Meanwhile, JPJD reduced the transcriptional activities of EMT-associated factors Snail and E-cadherin during the initiation of TGF-beta-induced EMT. In vivo, the results demonstrated that JPJD can significantly inhibit the liver and lung metastasis of orthotopic CRC tumor in nude mice, as well as significantly prolonging the survival time of tumor-bearing in a dose-dependent manner. Additionally, JPJD can upregulate the expression of E-cadherin and Smad2/3 in the cytoplasm and downregulate the expression of Vimentin, p-Smad2/3, and Snail in the orthotopic CRC tumor tissues. In conclusions, our new findings provided evidence that JPJD could inhibit TGF-beta-induced EMT in CRC through TGF-beta/Smad mediated Snail/E-cadherin expression.

BACKGROUND: The chemoresistance of prostate cancer (PCa) is invariably associated with the aggressiveness and metastasis of this disease. New emerging evidence indicates that the epithelial-to-mesenchymal transition (EMT) may play pivotal roles in the development of chemoresistance and metastasis. As a hallmark of EMT, E-cadherin is suggested to be a key marker in the development of chemoresistance. However, the molecular mechanisms underlying PCa chemoresistance remain unclear. The current study aimed to explore the association between EMT and chemoresistance in PCa as well as whether changing the expression of E-cadherin would affect PCa chemoresistance. METHODS: Parental PC3 and DU145 cells and their chemoresistant PC3-TxR and DU145-TxR cells were analyzed. PC3-TxR and DU145-TxR cells were transfected with E-cadherin-expressing lentivirus to overexpress E-cadherin; PC3 and DU145 cells were transfected with small interfering RNA to silence E-cadherin. Changes of EMT phenotype-related markers and signaling pathways were assessed by Western blotting and quantitative real-time polymerase chain reaction. tumor cell migration, invasion, and colony formation were then evaluated by wound healing, transwell, and colony formation assays, respectively. The drug sensitivity was evaluated using MTS assay. RESULTS: Chemoresistant PC3-TxR and DU145-TxR cells exhibited an invasive and metastatic phenotype that associated with EMT, including the down-regulation of E-cadherin and up-regulation of Vimentin, Snail, and N-cadherin, comparing with that of parental PC3 and DU145 cells. When E-cadherin was overexpressed in PC3-TxR and DU145-TxR cells, the expression of Vimentin and Claudin-1 was down-regulated, and tumor cell migration and invasion were inhibited. In particular, the sensitivity to paclitaxel was reactivated in E-cadherin-overexpressing PC3-TxR and DU145-TxR cells. When E-cadherin expression was silenced in parental PC3 and DU145 cells, the expression of Vimentin and Snail was up-regulated, and, particularly, the sensitivity to paclitaxel was decreased. Interestingly, Notch-1 expression was up-regulated in PC3-TxR and DU145-TxR cells, whereas the E-cadherin expression was down-regulated in these cells comparing with their parental cells. The use of gamma-secretase inhibitor, a Notch signaling pathway inhibitor, significantly increased the sensitivity of chemoresistant cells to paclitaxel. CONCLUSION: The down-regulation of E-cadherin enhances PCa chemoresistance via Notch signaling, and inhibiting the Notch signaling pathway may reverse PCa chemoresistance.

Signal transduction through the transforming growth factor-beta 1 (TGF-beta1) pathway affects epithelial to mesenchymal transition (EMT), partly by modulation of E-Cadherin expression. The concurrent impact of extracellular matrix driven regulation of integrin signaling on EMT has not been well characterized. We assessed the cumulative effect and molecular mechanisms of TGF-beta1 and integrin signal transduction on E-Cadherin in a renal cell cancer (RCC) model. Stimulation of RCC cells with TGF-beta1 demonstrated a three-fold increased expression of integrin alphav. A ligand of integrin alphav-beta3, (cyclopentapeptide containing Arginyl-Glycyl-Aspartic acid motif, RGD), was used to mimic integrin signaling. Treatment of cells with RGD and TGF-beta1 demonstrated significantly greater E-cadherin depletion than either ligand alone. This cooperative action on E-Cadherin expression is regulated by transcription factor Snai1 and is followed on a cellular level by increased cellular mobility as evidenced in a wound healing assay. Subsequent silencing of potential downstream mediators of the cumulative action of RGD and TGF-beta1 was carried out by small interfering RNA transfection and confirmed by Western blotting and/or RT-PCR. SiRNA mediated silencing of FAK and PINCH1 independently abrogated the cumulative effect of RGD and TGF-beta1 on E-Cadherin expression. We have identified a novel mechanism through which extracellular matrix event transduction by integrins further augments TGF-beta1 related effects on EMT. Molecular machinery involved in the integrin alphav-TGF-beta1 interplay may represent a therapeutic target in RCC.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

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.

Enhanced migratory potential and invasiveness of cancer cells contribute crucially to cancer progression. These phenotypes are achieved by precise alteration of invasion-associated genes through local epigenetic modifications which are recognized by a class of proteins termed a chromatin reader. ZMYND8 [zinc finger MYND (myeloid, Nervy and DEAF-1)-type containing 8], a key component of the transcription regulatory network, has recently been shown to be a novel reader of H3.1K36Me2/H4K16Ac marks. Through differential gene expression analysis upon silencing this chromatin reader, we identified a subset of genes involved in cell proliferation and invasion/migration regulated by ZMYND8. Detailed analysis uncovered its antiproliferative activity through BrdU incorporation, alteration in the expression of proliferation markers, and cell cycle regulating genes and cell viability assays. In addition, performing wound healing and invasion/migration assays, its anti-invasive nature is evident. Interestingly, epithelial-mesenchymal transition (EMT), a key mechanism of cellular invasion, is regulated by ZMYND8 where we identified its selective enrichment on promoters of CLDN1/CDH1 genes, rich in H3K36Me2/H4K16Ac marks, leading to their up-regulation. Thus, the presence of ZMYND8 could be implicated in maintaining the epithelial phenotype of cells. Furthermore, syngeneic mice, injected with ZMYND8-overexpressed invasive breast cancer cells, showed reduction in tumor volume and weight. In concert with this, we observed a significant down-regulation of ZMYND8 in invasive ductal and lobular breast cancer tissues compared with normal tissue. Taken together, our study elucidates a novel function of ZMYND8 in regulating EMT and invasion of cancer cells, possibly through its chromatin reader function.CI - (c) 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

In cancer biology, the architectural concept "form follows function" is reflected by cell morphology, migration, and epithelial-mesenchymal transition protein pattern. In vivo, features of epithelial-mesenchymal transition have been associated with tumor budding, which correlates significantly with patient outcome. Hereby, the majority of tumor buds are not truly detached but still connected to a major tumor mass. For detailed insights into the different tumor bud types and the process of tumor budding, we quantified tumor cells according to histomorphological and immunohistological epithelial-mesenchymal transition characteristics. Three-dimensional reconstruction from adenocarcinomas (pancreatic, colorectal, lung, and ductal breast cancers) was performed as published. tumor cell morphology and epithelial-mesenchymal transition characteristics (represented by zinc finger E-box-binding homeobox 1 and E-Cadherin) were analyzed qualitatively and quantitatively in a three-dimensional context. tumor buds were classified into main tumor mass, connected tumor bud, and isolated tumor bud. Cell morphology and epithelial-mesenchymal transition marker expression were assessed for each tumor cell. epithelial-mesenchymal transition characteristics between isolated tumor bud and connected tumor bud demonstrated no significant differences or trends. tumor cell count correlated significantly with epithelial-mesenchymal transition and histomorphological characteristics. Regression curve analysis revealed initially a loss of membranous E-Cadherin, followed by expression of cytoplasmic E-Cadherin and subsequent expression of nuclear zinc finger E-box-binding homeobox 1. Morphologic changes followed later in this sequence. Our data demonstrate that connected and isolated tumor buds are equal concerning immunohistochemical epithelial-mesenchymal transition characteristics and histomorphology. Our data also give an insight in the process of tumor budding. While there is a notion that the epithelial-mesenchymal transition zinc finger E-box-binding homeobox 1-E-Cadherin cascade is initiated by zinc finger E-box-binding homeobox 1, our results are contrary and outline other possible pathways influencing the regulation of E-Cadherin.

Lung cancer has been a major threat to human health worldwide. Vimentin (VIM) and E-cadherin (E-cad) are molecular markers that play important roles in epithelial-mesenchymal transition (EMT), which has been shown to be correlated with tumor progression. Herein, we investigated the associations between clinicopathological parameters and VIM/E-cad expression in primary T1 stage non-small cell lung cancer (NSCLC). Real-time PCR was performed on RNA extracted from tumor tissue isolated from 54 patients with T1 stage NSCLC. Immunohistochemistry was used to measure the protein levels of VIM and E-cad. The paired-samples t-test, independent-samples t- test, Kappa test, and nonparametric test were used to perform statistical analyses. The expression of VIM were lower (P<0.001) and that of E-cad was higher (P<0.001) in tumor tissue when compared with the normal tissue at the transcriptional level. The RNA of VIM in adenocarcinomas was significantly higher than that found in squamous cell carcinomas (P<0.01). Moreover, the level of metastasis observed in the lymph node was significantly higher than that observed in metastasis outside of the lymph node (P<0.05). Vimentin protein had a lower profile in the tumor tissue, and the protein level of E-cad was higher in the tumor tissue. The IHC score of VIM in tumor tissue was found associated with both the pathological type and smoke index, and the score of E-cad was found to be related to visceral pleura involvement. In conclusions, VIM is downregulated and E-cad is upregulated in T1 stage NSCLC. Our study results suggest that a mesenchymal-epithelial transition may take place in the early-stage of tumor development, and that EMT occurs when the tumor develops into a certain stage.

During epithelial ovarian cancer (EOC) progression, intraperitoneally disseminating tumor cells and multicellular aggregates (MCAs) present in ascites fluid adhere to the peritoneum and induce retraction of the peritoneal mesothelial monolayer prior to invasion of the collagen-rich submesothelial matrix and proliferation into macro-metastases. Clinical studies have shown heterogeneity among EOC metastatic units with respect to cadherin expression profiles and invasive behavior; however, the impact of distinct cadherin profiles on peritoneal anchoring of metastatic lesions remains poorly understood. In the current study, we demonstrate that metastasis-associated behaviors of ovarian cancer cells and MCAs are influenced by cellular cadherin composition. Our results show that mesenchymal N-cadherin-expressing (Ncad+) cells and MCAs invade much more efficiently than E-cadherin-expressing (Ecad+) cells. Ncad+ MCAs exhibit rapid lateral dispersal prior to penetration of three-dimensional collagen matrices. When seeded as individual cells, lateral migration and cell-cell junction formation precede matrix invasion. Neutralizing the Ncad extracellular domain with the monoclonal antibody GC-4 suppresses lateral dispersal and cell penetration of collagen gels. In contrast, use of a broad-spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) to block endogenous membrane type 1 matrix metalloproteinase (MT1-MMP) activity does not fully inhibit cell invasion. Using intact tissue explants, Ncad+ MCAs were also shown to efficiently rupture peritoneal mesothelial cells, exposing the submesothelial collagen matrix. Acquisition of Ncad by Ecad+ cells increased mesothelial clearance activity but was not sufficient to induce matrix invasion. Furthermore, co-culture of Ncad+ with Ecad+ cells did not promote a 'leader-follower' mode of collective cell invasion, demonstrating that matrix remodeling and creation of invasive micro-tracks are not sufficient for cell penetration of collagen matrices in the absence of Ncad. Collectively, our data emphasize the role of Ncad in intraperitoneal seeding of EOC and provide the rationale for future studies targeting Ncad in preclinical models of EOC metastasis.

Long non-coding RNAs play important roles in the regulation of cellular processes including cell proliferation, differentiation, and metastasis. The dysregulation of long non-coding RNAs, such as the SPRY4-IT1 (SPRY4 intronic transcript 1), has been associated with various types of malignancies. However, the functional roles and regulatory mechanism of SPRY4-IT1 in ovarian cancer remain to be elucidated. Here, we quantified the expression level of SPRY4-IT1 in ovarian cancer patients and found its downregulation in ovarian cancer tissues compared to the adjacent normal tissues. Patients with lower SPRY4-IT1 expression were associated with a relatively poor prognosis. In consistency, the expression of SPRY4-IT1 was found to be reduced in four human ovarian cancer cell lines compared to normal ovarian epithelial cells. Next, two ovarian cancer cell lines SKOV3 and HO8910 were employed in vitro assays to investigate biological functions of SPRY4-IT1 in ovarian cancer. The cell proliferation was reduced following SPRY4-IT1 overexpression in SKOV3/HO8910 cells based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. The SPRY4-IT1 overexpression also dramatically arrested cell cycle and promoted cell apoptosis. Both wound-healing and transwell-based assays demonstrated that cell migration and invasion were inhibited following SPRY4-IT1 overexpression. Meanwhile, overexpression of SPRY4-IT1 increased E-cadherin and decreased N-cadherin and vimentin protein levels, indicating that SPRY4-IT1 may regulate ovarian cancer cell metastasis through the inhibition of epithelial-mesenchymal transition. Taken together, our findings suggest that SPRY4-IT1 regulates various cellular processes of ovarian cancer cells and its downregulation may contribute to ovarian cancer progression and metastasis partly via affecting the epithelial-mesenchymal transition.

BACKGROUND: epithelial-mesenchymal transition (EMT), principally involving an E-cadherin to N-cadherin shift, linked to tumor invasion or metastasis, and therapeutic resistance in various human cancer. A growing body of recent evidence has supported the hypothesis that EMT play a crucial role in the invasive phenotype of gliomas. To evaluate the prognostic connotation of EMT traits in glioma, expression of E-cadherin and N-cadherin was explored in a large series of glioma patients in relation to patient survival rate. METHODS: expressions of E- and N-cadherin were examined using immunohistochemical analysis in 92 glioma cases diagnosed at our hospital. These markers expressions were also explored in 21 cases of fresh frozen glioma samples and in glioma cell lines by Western blot analysis. RESULTS: expression of E-cadherin was observed in eight cases (8.7%) with weak staining intensity in the majority of the immunoreactive cases (7/8). expression of N-cadherin was identified in 81 cases (88.0%) with high expression in 64 cases (69.5%). Fresh frozen tissue samples and glioma cell lines showed similar results by Western blot analysis. There was no significant difference in either overall survival (OS) or progression-free survival (PFS) according to E-cadherin expression (P > 0.05). Although the OS rates were not affected by N-cadherin expression levels (P = 0.138), PFS increased in the low N-cadherin expression group with marginal significance (P = 0.058). The survival gains based on N-cadherin expression levels were significantly augmented in a larger series of publicly available REMBRANDT data (P < 0.001). CONCLUSIONS: E- and N-cadherin, as representative EMT markers, have limited prognostic value in glioma. Nonetheless, the EMT process in gliomas may be compounded by enhanced N-cadherin expression supported by unfavorable prognostic outcomes.

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.

Background: TGF-beta1 plays an important role in the epithelial-mesenchymal transition (EMT) of epithelial cancers, including non-small cell lung cancer (NSCLC). While the full underlying mechanism remains unclear, miR-9 is known to play a critical role in the regulation of NSCLC cell invasion. We tested whether miR-9 targets E-cadherin and thus affects TGF-beta1-induced EMT in NSCLC cells by assessing the expression levels of miR-9 and E-cadherin for NSCLC patients and then verifying the targeting of E-cadherin by miR-9 using the dual luciferase reporter system. Results: MiR-9 was significantly upregulated in NSCLC tissues compared with its level in adjacent normal tissues. The expression of E-cadherin in NSCLC tissues was significantly decreased. In addition, we found that TGF-beta1 significantly upregulated the expression of miR-9 and downregulated the expression of E-cadherin. E-cadherin was confirmed as a direct target gene of miR-9. Using an miR-9 inhibitor reversed the TGF-beta1-mediated inhibition of E-cadherin expression and upregulation of the mesenchymal marker alpha-SMA. TGF-beta1 significantly induced cell invasion, and this effect was significantly inhibited by miR-9 inhibitors. Conclusions: TGF-beta1 induced EMT in NSCLC cells by upregulating miR-9 and downregulating miR-9's target, E-cadherin.

The epithelial-to-mesenchymal transition (EMT) induced by chronic hypoxia is one of the critical causes of renal fibrosis. Previous work reported that the transcription factors Twist plays an important role in hypoxia-induced EMT and renal fibrosis. Recent evidence indicates that miR-214 was regulated by Twist in many fibrotic diseases, but their role in hypoxia-induced EMT and renal fibrosis remains unknown. Here, we found that hypoxia significantly upregulated the expression of miR-214-3p in HK-2cells, unilateral ureteral obstruction (UUO) nephropathy and patients with chronic kidney disease. Knockdown of miR-214-3p reversed the EMT of renal tubular epithelial cells (TECs) and alleviated fibrosis in the UUO mouse in vivo, while the overexpression of miR-214-3p promoted EMT phenotype and expression of fibrotic factors in TECs under hypoxic condition. In addition, Twist was also observed increased gradually with the prolongation of hypoxia, and it positively correlated with the expression of miR-214-3p in HK-2cells transfected with Twist-overexpression or Twist-siRNA plasmid. Moreover, miR-214-3p negatively regulated the expression of epithelial cadherin (E-cadherin) by binding the E-cadherin 3' UTR under hypoxic condition. Overall, hypoxia-induced activation of Twist/miR-214/E-cadherin axis is involved in the EMT of TECs, and anti-miR-214 may be an attractive strategy to ameliorate the progression of renal fibrosis.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

Triple-negative breast cancer (TNBC) is a subtype of cancer with aggressive behaviors (high recurrence and metastasis rate) and poor prognosis. Therefore, studying the determining factors that lead to malignant TNBCs is necessary to develop personalized therapy and improve survival rates. In this study, we first analyzed levels of chromodomain helicase DNA binding protein 4 (CHD4) in 60 TNBC patients by immunohistochemical staining. We then clarified the role of CHD4 in TNBC and non-TNBC cell lines. Our clinical data indicated that higher CHD4 expression is positively correlated with metastatic stage, tumor recurrence, and survival status. Consistent with the clinical analytical data, our in vitro data also indicated that high level of CHD4 is positively correlated with malignant behaviors in TNBC cells, such as cell motility and mortality. For further analyses, we found that E-cadherin, N-cadherin and fibronetin are involved in CHD4-mediated epithelial-mesenchymal transition (EMT). Silencing of CHD4 also increased drug sensitivity to cisplatin and PARP1 inhibitor, especially in TNBC cells. Altogether, our findings showed that CHD4 is not only a potential prognostic biomarker for TNBC patient survival, but is also a powerful candidate in the development of new anti-cancer agents in TNBC.CI - Copyright (c) 2018 Elsevier Inc. All rights reserved.

BACKGROUND: Metastases play a role in about 90% of cancer deaths. Markers of epithelial-mesenchymal transition (EMT) measured in primary tumor cancer cells might provide diagnostic information about the likelihood that cancer cells have detached from the primary tumor. Used together with established diagnostic tests of detachment-lymph node evaluation and radiologic imaging-EMT marker measurements might improve the ability of clinicians to assess the patient's risk of metastatic disease. Translation of EMT markers to clinical use has been hampered by a lack of valid analyses of clinically-informative parameters. Here, we demonstrate a rigorous approach to estimating the sensitivity, specificity, and prediction increment of an EMT marker to assess cancer cell detachment from primary tumors. METHODS: We illustrate the approach using immunohistochemical measurements of the EMT marker E-cadherin in a set of colorectal primary tumors from a population-based prospective cohort in North Carolina. Bayesian latent class analysis was used to estimate sensitivity and specificity in a setting of multiple imperfect diagnostic tests and no gold standard. Risk reclassification analysis was used to assess the extent to which addition of the marker to the panel of established diagnostic tests would improve mortality prediction. We explored how changing the latent class conditional dependence assumptions and definition of marker positivity would impact the results. RESULTS: All diagnostic accuracy and prediction increment statistics varied with the choice of cut point to define marker positivity. When comparing different definitions of marker positivity to each other, numerous trade-offs were observed in terms of sensitivity, specificity, predictive discrimination, and prediction model calibration. We then discussed several implementation considerations and the plausibility of analytic assumptions. CONCLUSIONS: The approaches presented here can be extended to any EMT marker, to most forms of cancer, and to different kinds of EMT marker measurements, such as RNA or gene methylation data. These methods provide valid, clinically-informative assessment of whether and how to use a given EMT marker to refine tumor staging and consequent treatment decisions.

We have earlier found abnormal expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2, S18-2) in endometrial cancer, compared to the expression in hyperplasia and in normal endometrium. Here we report that expression of S18-2 was increased with disease progression in clinical specimens of prostate cancer (PCa). The level of induction of epithelial to mesenchymal cell transition (EMT) correlated with the expression level of S18-2 in PCa cell lines. Moreover, cells acquired increased ability of migration upon S18-2 overexpression, as was evaluated in zebrafish embryo model and in trans-well assay. We found that this is due to increased CXCR4 cell surface expression. Neutralizing CXCR4 protein or abrogating S18-2 expression in cells significantly reduced their migratory ability directed toward CXCL12. The mRNA expression of TWIST2, encoding one of transcription factors that induce EMT upon CXCR4 increase, positively correlated with the S18-2 protein level. Together, these data suggest that the S18-2 protein induces EMT through the TWIST2/E-cadherin signalling and, consequently, CXCR4-mediated migration of PCa cells.

epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. During EMT, tumor cells acquire the capacity to migrate and invade the stroma. Activation of the transforming growth factor-b (TGF-b) signaling pathway is of major importance for the initiation of EMT. Smad4, an essential protein of this pathway, is known to complex with multiple transcription factors (e.g. Snail-1, Slug, Twist-1), in various types of cancer, promoting the repression or activation of target genes. The role of Smad4 in colorectal cancer (CRC) is not straightforward so far. In the present study forty eight resected CRC tumor specimens were immunohistochemically examined in order to assess the expression of Smad4 and its association with E-cadherin, Snail-1, Slug, Twist-1 protein expression and with various pathological parameters. Smad4 was found to be positively correlated with Snail-1, Slug and Twist-1 expression (p < 0.001). On the other hand it was negatively correlated with the expression of E-cadherin (p < 0.001). Furthermore, lymphatic invasion could be clearly associated with Smad4 expression, a finding complying with the metastatic ability of EMT cells. In conclusion, Smad4 could be considered as a central component of EMT transition in human colorectal cancer that combines with transcriptional factors to reduce E-cadherin and alter the expression of the epithelial phenotype.

BACKGROUND/AIMS: Gastric cancer (GC) is a common malignancy with a global incidence that ranks fourth among all tumor types. epithelial-to-mesenchymal transition (EMT) is a tumor biological process with a role in GC cell metastasis. Long non-coding RNAs (lncRNAs) and microRNAs possess important regulatory functions at the cellular level and in diverse pathophysiological processes. This study was conducted to investigate whether lncRNA RP11-789C1.1 regulates EMT in GC by mediating the miR-5003/E-cadherin pathway. METHODS: RP11-789C1.1 and miR-5003 expression was detected in GC specimens and cell lines by quantitative real-time PCR. Western blotting and immunohistochemistry were performed to detect EMT markers in GC. Cell Counting Kit 8 assays were carried out to explore cell proliferation. Wound healing and Transwell assays were conducted to determine the migration and invasion of GC cells. To clarify the correlation between RP11-789C1.1, miR-5003, and E-cadherin, dual-luciferase reporter assays were applied. RESULTS: LncRNA RP11-789C1.1 was significantly down-regulated in GC patients and cell lines, along with the concomitant up-regulation of miR-5003. Silencing RP11-789C1.1 and over-expressing miR-5003 significantly promoted the tumor behavior of GC cells. Dual-luciferase reporter assays confirmed that miR-5003 was the target of both RP11-789C1.1 and E-cadherin. Furthermore, at both the mRNA and protein level, silencing RP11-789C1.1 remarkably reduced the expression of E-cadherin and promoted EMT, which were reversed by knocking down miR-5003. CONCLUSIONS: LncRNA RP11-789C1.1 inhibited EMT in GC through the RP11-789C1.1/miR-5003/E-cadherin axis, which could be a promising therapeutic target for GC.CI - (c) 2018 The Author(s). Published by S. Karger AG, Basel.

BACKGROUND: Plant homeodomain finger protein 8 (PHF8) serves an activator of epithelial-mesenchymal transition (EMT) and is implicated in various tumors. However, little is known about PHF8 roles in hepatocellular carcinoma (HCC) and regulating E-cadherin expression. METHODS: PHF8 expression pattern was investigated by informatic analysis and verified by RT-qPCR and immunochemistry in HCC tissues and cell lines. CCK8, xenograft tumor model, transwell assay, and tandem mCherry-GFP-LC3 fusion protein assay were utilized to assess the effects of PHF8 on proliferation, metastasis and autophagy of HCC cells in vitro and in vivo. ChIP, immunoblot analysis, rescue experiments and inhibitor treatment were used to clarify the mechanism by which PHF8 facilitated EMT, metastasis and autophagy. RESULTS: PHF8 upregulation was quite prevalent in HCC tissues and closely correlated with worse overall survival and disease-relapse free survival. Furthermore, PHF8-knockdown dramatically suppressed cell growth, migration, invasion and autophagy, and the expression of SNAI1, VIM, N-cadherin and FIP200, and increased E-cadherin level, while PHF8-overexpression led to the opposite results. Additionally, FIP200 augmentation reversed the inhibited effects of PHF8-siliencing on tumor migration, invasion and autophagy. Mechanistically, PHF8 was involved in transcriptionally regulating the expression of SNAI1, VIM and FIP200, rather than N-cadherin and E-cadherin. Noticeably, E-cadherin degradation could be accelerated by PHF8-mediated FIP200-dependent autophagy, a crucial pathway complementary to transcriptional repression of E-cadherin by SNAI1 activation. CONCLUSION: These findings suggested that PHF8 played an oncogenic role in facilitating FIP200-dependent autophagic degradation of E-cadherin, EMT and metastasis in HCC. PHF8 might be a promising target for prevention, treatment and prognostic prediction of HCC.

Gastric cancer is a common malignancy worldwide. The prognosis of early stage gastric cancer patients has significantly improved in recent years. However, in progressive stage gastric cancer patients, the prognosis remains relatively poor due to tumor metastases. In our previous study, we showed that the expression of miR711 in gastric cancer tissues is low, and restoration of miR711 inhibited the invasion and migration and the occurrence of epithelialmesenchymal transition (EMT) in gastric cancer cells. Yet, the mechanisms involved in these processes remain unknown. In the present study, we demonstrated that miR711mediated downregulation of CD44 expression inhibited EMT of gastric cancer cells in vitro and in vivo by downregulating vimentin protein expression and upregulating Ecadherin protein expression through transfection, qRTPCR and western blotting. Therefore, miR711 may provide a promising target for EMTrelated therapy for gastric cancer.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It has been reported that HCC has a poor prognosis. In the majority of cases, once metastatic, HCC is incurable. To identify an effective treatment for HCC, it is important to understand the underlying molecular mechanisms of HCCassociated occurrence, proliferation, metastasis and carcinogenesis. In the present study, the role of Upframeshift 1 (UPF1), a potential tumor suppressor, was investigated in the HCC cell lines. The expression levels of UPF1 in an HCC cell line were examined by reverse transcriptionquantitative polymerase chain reaction. The expression levels of 19 key proteins in numerous signaling pathways were detected via protein array analysis in the presence of UPF1 overexpression. The present study further investigated the effects of UPF1 expression levels on the epithelialmesenchymal transition (EMT) process by targeting Ecadherin, Ncadherin, Vimentin and Twistrelated protein 1 (Twist). The results of the present study revealed that UPF1 was significantly downregulated in an HCC cell line. The majority of the proteins exhibited upregulated expression levels in the presence of UPF1 overexpression in the HCC cell line, Huh7. Key proteins, including cluster of differentiation (CD)31 (platelet endothelial cell adhesion molecule1), Vimentin, CD44, PCNA, Ki67, NCadherin, Survivin, P53, Met and retinoblastoma exhibited a significant association with UPF1. Furthermore, western blotting indicated that the expression levels of Ncadherin, Vimentin and Twist were notably upregulated while UPF1 was overexpressed; however, Ecadherin was downregulated and opposing observations were reported with protein array analysis. In summary, Ecadherin expression levels were regulated by the manifold, and UPF1, a potential tumor suppressor, may promote the EMT process in Huh7 HCC cells. The findings of the present study suggested that UPF1 expression levels affected the EMT process by targeting Ecadherin, Ncadherin, Vimentin and Twist.

epithelial-mesenchymal transition (EMT) plays an important role in organ fibrosis, including that of the kidney. Loss of E-cadherin expression is a hallmark of EMT; however, whether the loss of E-cadherin is a consequence or a cause of EMT remains unknown, especially in the renal system. In this study, we show that transforming growth factor (TGF)-beta1-induced EMT in renal tubular epithelial cells is dependent on proteolysis. Matrix metalloproteinase-mediated E-cadherin disruption led directly to tubular epithelial cell EMT via Slug. TGF-beta1 induced the proteolytic shedding of E-cadherin, which caused the nuclear translocation of beta-catenin, the transcriptional induction of Slug, and the repression of E-cadherin transcription in tubular epithelial cells. These findings reveal a direct role for E-cadherin and for matrix metalloproteinases in causing EMT downstream of TGF-beta1 in fibrotic disease. Specific inhibition rather than activation of matrix metalloproteinases may offer a novel approach for treatment of fibrotic disease.

Hepatic differentiation of embryonic stem cells (ESCs) usually results in a single cell lineage, and the formation of liver tissues remains difficult. Here, we examine the role of epithelial-mesenchymal transition (EMT) that is regulated by epithelial cadherin (E-cad) expression in hepatic tissue formation from ESCs. E-cad was transfected into mouse ESCs to enable a stable expression of E-cad. Hepatic differentiation of ESCs was then induced by hepatic growth factors. Wnt/beta-catenin signaling and EMT speed were examined to determine the differentiation process. Hepatic and angiogenesis markers, as well as differentiated cell-adhesive force were also examined to identify the hepatic tissue differentiation. In our results, E-cad expression gradually decreased in normal ESC (N-ESC) differentiation, but remained stable in the E-cad transfected ESC (EC-ESC) group. In EC-ESC differentiation, expressions of cytoplastic beta-catenin and EMT were much lower and significantly prolonged. Angiogenesis markers vascular endothelial growth factor receptor-1 (VEGFR-1) and CD31/PECAM-1 were expressed only on day 5-13 in N-ESC differentiation, whereas VEGFR-1 and CD31/PECAM-1 were expressed prolonged on day 5-17 in the EC-ESC group and were coincident with the expression of hepatic markers. Finally, EC-ESC differentiation maintained multilayer-growth patterns, and abundant vascular network structures appeared and migrated in albumin-positive cell areas. The cellular adhesion forces between embryonic body cells in EC-ESC differentiation during day 13-17 were similar to those of mouse liver tissue. In conclusion, accelerated EMT due to the decreased E-cad expression may partially contribute to the failure of hepatic tissue formation in N-ESC differentiation. E-cad can act in synergy with hepatic growth factors and facilitate the early-stage formation of hepatic tissues through down-regulating Wnt/beta-catenin signaling and delaying EMT. This work provides a new insight into hepatic tissue differentiation that is mediated by E-cad from ESC.

Snail1 (Snail) and Snail2 (Slug) are transcription factors that share a similar DNA binding structure of four and five C2H2 zinc finger motifs (ZF), respectively. Both factors bind specifically to a subset of E-box motifs (E2-box: CAGGTG/CACCTG) in target promoters like the E-cadherin promoter and are key mediators of epithelial-to-mesenchymal transition (EMT). However, there are differences in the biological actions, in binding affinities to E-cadherin promoter, and in the target genes of Snail1 and Snail2, although the molecular bases are presently unknown. In particular, the role of each Snail1 and Snail2 ZF in the binding to E-boxes and in EMT induction has not been previously explored. We have approached this question by modeling Snail1 and Snail2 protein-DNA interactions and through mutational and functional assays of different ZFs. Results show that Snail1 efficient repression and binding to human and mouse E-cadherin promoter as well as EMT-inducing ability require intact ZF1 and ZF2, while for Snail2, either ZF3 or ZF4 is essential for those functions. Furthermore, the differential distribution of E2-boxes in mouse and human E-cadherin promoters also contributes to the differential Snail factor activity. These data indicate a non-equivalent role of Snail1 and Snail2 ZFs in gene repression, contributing to the elucidation of the molecular differences between these important EMT regulators.

Substantial experimental evidence has shown that dedifferentiation from an epithelial state to a mesenchymal-like state (EMT) drives tumor cell metastasis. This transition facilitates tumor cells to acquire motility and invasive features. Intriguingly, tumor cells at the metastatic site are primarily epithelial, and it is believed that they differentiate back to an epithelial state by a process called mesenchymal to epithelial transition (MET). However, there is little in vivo evidence to support the MET process. To investigate EMT and MET in vivo, we generated two epithelial (E) and two mesenchymal (M) primary clonal cell lines from a spontaneous mouse mammary tumor (Tg MMTV/neu). These cells were labeled with reporters (GFP and luciferase), and tracked in vivo during primary tumor growth and subsequent secondary metastasis. Once E cells were implanted into the mammary fat pad, E-cadherin expression progressively decreased and continued to decrease as the primary tumor enlarged over time. A greater percentage of E tumor cells expressed E-cadherin at the secondary metastatic site as compared to the corresponding primary tumor site. Collectively, these data provide direct in vivo evidence that epithelial tumor cells have metastatic potential, undergo EMT at the primary tumor site, and MET at the metastatic site.