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Lithium chloride induces mesenchymal?to?epithelial reverting transition in primary colon cancer cell cultures.

ABSTRACT: Epithelial?to?mesenchymal transition (EMT) confers stem cell?like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E?cadherin decreases, resulting in loss of cell?cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approaches. Western blot assay, quantitative real?time PCR and immunofluorescence were performed to investigate the expression of E?cadherin, vimentin, ??catenin, cytokeratin?20 and ?18, Twist1, Snail, CD44, cyclooxygenase?2 (COX2), Sox2, Oct4 and Nanog. Moreover, cell differentiation was induced by incubation with LiCl?containing medium for 10 days. We observed that these primary colorectal cancer (CRC) cells lost expression of the E?cadherin epithelial marker, which was instead expressed in cancer and normal colon mucosa of the same patient, while overexpressed vimentin (mesenchymal marker), Twist1, Snail (EMT markers) and COX2. Cytokeratin?18 was expressed both in tissues and cell cultures. Expression of stem cell markers, such as CD44, Oct4 and Nanog, were also observed. Following differentiation with the glycogen synthase kinase 3? (GSK3?) inhibitor LiCl, the cells began to express E?cadherin and, at once, Twist1 and Snail expression was strongly downregulated, suggesting a MET?reverting process. In conclusion, we established primary colon mesenchymal cancer cell cultures expressing mesenchymal and epithelial biomarkers together with high level of EMT transcription factors. We propose that they could represent a good model for studying EMT and its reverting mechanism, the mesenchymal?to?epithelial transition (MET). Our observation indicates that LiCl, a GSK3? inhibitor, induces MET in vitro, suggesting that LiCl and GSK3? could represent, respectively, interesting drug, and target for CRC therapy.

SUBMITTER: Costabile V

PROVIDER: S-EPMC4383027 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Lithium chloride induces mesenchymal‑to‑epithelial reverting transition in primary colon cancer cell cultures.

Costabile Valeria V Duraturo Francesca F Delrio Paolo P Rega Daniela D Pace Ugo U Liccardo Raffaella R Rossi Giovanni Battista GB Genesio Rita R Nitsch Lucio L Izzo Paola P De Rosa Marina M

International journal of oncology 20150302 5

Epithelial‑to‑mesenchymal transition (EMT) confers stem cell‑like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E‑cadherin decreases, resulting in loss of cell‑cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approache ...[more]

PMID: 25738332

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Project description:BackgroundEpithelial to mesenchymal transition (EMT), implicated as a mechanism for tumor dissemination, is marked by loss of E-cadherin, disruption of cell adhesion, and induction of cell motility and invasion. In most intraductal breast carcinomas E-cadherin is regulated epigenetically via methylation of the promoter. E-cadherin expression is therefore dynamic and open to modulation by the microenvironment. In addition, it has been observed that metastatic foci commonly appear more differentiated than the primary tumor, suggesting that cancer cells may further undergo a mesenchymal to epithelial reverting transition (MErT) in the secondary organ environment following the EMT that allows for escape.ResultsWe first examined E-cadherin expression in primary breast tumors and their corresponding metastases to liver, lung and brain and discovered that 62% (10/16) of cases showed increased E-cadherin expression in the metastases compared to the primaries. These observations led to the question of whether the positive metastatic foci arose from expansion of E-cadherin-positive cells or from MErT of originally E-cadherin-negative disseminated cells. Thus, we aimed to determine whether it was possible for the mesenchymal-like MDA-MB-231 breast cancer cells to undergo an MErT through the re-expression of E-cadherin, either through exogenous introduction or induction by the microenvironment. Ectopic expression of full-length E-cadherin in MDA-MB-231 cells resulted in a morphological and functional reversion of the epithelial phenotype, with even just the cytosolic domain of E-cadherin yielding a partial phenotype. Introduction of MDA-MB-231 cells or primary explants into a secondary organ environment simulated by a hepatocyte coculture system induced E-cadherin re-expression through passive loss of methylation of the promoter. Furthermore, detection of E-cadherin-positive metastatic foci following the spontaneous metastasis of MDA-MB-231 cells injected into the mammary fat pad of mice suggests that this re-expression is functional.ConclusionsOur clinical observations and experimental data indicate that the secondary organ microenvironment can induce the re-expression of E-cadherin and consequently MErT. This phenotypic change is reflected in altered cell behavior and thus may be a critical step in cell survival at metastatic sites.

Project description:Aberrant T-cell factor (TCF) transcription is implicated in the majority of colorectal cancers (CRCs). TCF transcription induces epithelial-mesenchymal transition (EMT), promoting a tumor-initiating cell (TIC) phenotype characterized by increased proliferation, multidrug resistance (MDR), invasion and metastasis. The data presented herein characterize topoisomerase II? (TopoII?) as a required component of TCF transcription promoting EMT. Using chromatin immunoprecipitation (ChIP) and protein co-immunoprecipitation (co-IP) studies, we show that TopoII? forms protein-protein interactions with ?-catentin and TCF4 and interacts with Wnt response elements (WREs) and promoters of direct target genes of TCF transcription, including: MYC, vimentin, AXIN2 and LEF1. Moreover, both TopoII? and TCF4 ChIP with the N-cadherin promoter, which is a new discovery indicating that TCF transcription may directly regulate N-cadherin expression. TopoII? N-terminal ATP-competitive inhibitors, exemplified by the marine alkaloid neoamphimedine (neo), block TCF activity in vitro and in vivo. Neo effectively inhibits TopoII? and TCF4 from binding WREs/promoter sites, whereas protein-protein interactions remain intact. Neo inhibition of TopoII?-dependent TCF transcription also correlates with significant antitumor effects in vitro and in vivo, including the reversion of EMT, the loss of TIC-mediated clonogenic colony formation, and the loss of cell motility and invasion. Interestingly, non-ATP-competitive inhibitors of TopoII?, etoposide and merbarone, were ineffective at preventing TopoII?-dependent TCF transcription. Thus, we propose that TopoII? participation in TCF transcription may convey a mechanism of MDR to conventional TopoII? inhibitors. However, our results indicate that TopoII? N-terminal ATP-binding sites remain conserved and available for drug targeting. This article defines a new strategy for targeted inhibition of TCF transcription that may lead to effective therapies for the treatment of CRC and potentially other Wnt-dependent cancers.

Dataset Information

Lithium chloride induces mesenchymal?to?epithelial reverting transition in primary colon cancer cell cultures.

Publications

Lithium chloride induces mesenchymal‑to‑epithelial reverting transition in primary colon cancer cell cultures.

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