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Integrated proteomics and genomics analysis reveals a novel mesenchymal to epithelial reverting transition in leiomyosarcoma through regulation of slug.

ABSTRACT: Leiomyosarcoma is one of the most common mesenchymal tumors. Proteomics profiling analysis by reverse-phase protein lysate array surprisingly revealed that expression of the epithelial marker E-cadherin (encoded by CDH1) was significantly elevated in a subset of leiomyosarcomas. In contrast, E-cadherin was rarely expressed in the gastrointestinal stromal tumors, another major mesenchymal tumor type. We further sought to 1) validate this finding, 2) determine whether there is a mesenchymal to epithelial reverting transition (MErT) in leiomyosarcoma, and if so 3) elucidate the regulatory mechanism responsible for this MErT. Our data showed that the epithelial cell markers E-cadherin, epithelial membrane antigen, cytokeratin AE1/AE3, and pan-cytokeratin were often detected immunohistochemically in leiomyosarcoma tumor cells on tissue microarray. Interestingly, the E-cadherin protein expression was correlated with better survival in leiomyosarcoma patients. Whole genome microarray was used for transcriptomics analysis, and the epithelial gene expression signature was also associated with better survival. Bioinformatics analysis of transcriptome data showed an inverse correlation between E-cadherin and E-cadherin repressor Slug (SNAI2) expression in leiomyosarcoma, and this inverse correlation was validated on tissue microarray by immunohistochemical staining of E-cadherin and Slug. Knockdown of Slug expression in SK-LMS-1 leiomyosarcoma cells by siRNA significantly increased E-cadherin; decreased the mesenchymal markers vimentin and N-cadherin (encoded by CDH2); and significantly decreased cell proliferation, invasion, and migration. An increase in Slug expression by pCMV6-XL5-Slug transfection decreased E-cadherin and increased vimentin and N-cadherin. Thus, MErT, which is mediated through regulation of Slug, is a clinically significant phenotype in leiomyosarcoma.

SUBMITTER: Yang J

PROVIDER: S-EPMC2984227 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Integrated proteomics and genomics analysis reveals a novel mesenchymal to epithelial reverting transition in leiomyosarcoma through regulation of slug.

Yang Jilong J Eddy James A JA Pan Yuan Y Hategan Andrea A Tabus Ioan I Wang Yingmei Y Cogdell David D Price Nathan D ND Pollock Raphael E RE Lazar Alexander J F AJ Hunt Kelly K KK Trent Jonathan C JC Zhang Wei W

Molecular & cellular proteomics : MCP 20100722 11

Leiomyosarcoma is one of the most common mesenchymal tumors. Proteomics profiling analysis by reverse-phase protein lysate array surprisingly revealed that expression of the epithelial marker E-cadherin (encoded by CDH1) was significantly elevated in a subset of leiomyosarcomas. In contrast, E-cadherin was rarely expressed in the gastrointestinal stromal tumors, another major mesenchymal tumor type. We further sought to 1) validate this finding, 2) determine whether there is a mesenchymal to epi ...[more]

PMID: 20651304

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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: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, β‑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 3β (GSK3β) 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 GSK3β inhibitor, induces MET in vitro, suggesting that LiCl and GSK3β could represent, respectively, interesting drug, and target for CRC therapy.

Project description:Rationale: The role of SLUG in epithelial-mesenchymal transition during tumor progression has been thoroughly studied, but its precise regulation remains poorly explored. Methods: The affinity purification, mass spectrometry and CO-IP were performed to identify the interaction between SLUG and ubiquitin-specific protease 5 (USP5). Cycloheximide chase assays and deubiquitination assays confirmed that the effect of USP5 on the deubiquitin of SLUG. The dual-luciferase reporter and chromatin immunoprecipitation assays were employed to observe the direct transcriptional regulation of E-cadherin by SLUG effected by USP5. EMT related markers was detected by western blotting and immunofluorescence. Molecular docking, SPR sensor (biacore) and co-location were detected to prove Formononetin targets USP5. Bioinformatics analysis was used to study the relation of USP5 and SLUG to malignancy degree of HCC. Cell migration, invasion in HCC cells and xenografts model in nude mouse were conducted to detect the promotion of USP5 and the inhibition of Formononetin on EMT. Results: USP5 interacts with and stabilizes SLUG to regulate its abundance through USP5 deubiquitination activities in epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC). USP5 is highly expressed and positively correlated with SLUG expression in HCC with high malignancy. Knockdown of USP5 inhibits SLUG deubiquitination and inhibits HCC cells proliferation, metastasis, and invasion, while overexpression of USP5 promotes SLUG stability and EMT in vitro and in vivo. Through virtual screening, we found that Formononetin exhibits excellent binding to USP5. Moreover, Formononetin inhibits deubiquitinating activities of USP5 to SLUG and consequently impedes the EMT and malignant progression of HCC. Conclusion: Our findings reveal that USP5 serve as a potential target for tumor intervention and provide a preliminary antitumor therapy for inhibit EMT by targeting USP5 or its interaction with SLUG in HCC.

Dataset Information

Integrated proteomics and genomics analysis reveals a novel mesenchymal to epithelial reverting transition in leiomyosarcoma through regulation of slug.

Publications

Integrated proteomics and genomics analysis reveals a novel mesenchymal to epithelial reverting transition in leiomyosarcoma through regulation of slug.

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