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DLC1 Induces Expression of E-Cadherin in Prostate Cancer Cells Through Rho Pathway and Suppresses Invasion

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DLC1 Induces Expression of E-Cadherin in Prostate Cancer Cells Through Rho Pathway and Suppresses Invasion Oncogene (2014) 33, 724–733 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE DLC1 induces expression of E-cadherin in prostate cancer cells through Rho pathway and suppresses invasion V Tripathi, NC Popescu and DB Zimonjic E-cadherin is a cell–cell adhesion molecule that acts as a suppressor of cancer cell invasion and its expression is downregulated in many advanced, poorly differentiated, human cancers. In this study, we found that the expression of DLC1 (deleted in liver cancer 1) tumor-suppressor gene in metastatic prostate carcinoma (PCA) cells increased the expression of E-cadherin and resulted in an elevated rate of cell–cell aggregation as measured by aggregation assay. DLC1-mediated increase in E-cadherin expression was not dependent on a-catenin, a DLC1-binding protein associated with E-cadherin, and/or cellular density. The increase of E-cadherin expression occurred at mRNA level and relied on DLC1 RhoGAP function, leading to suppression of high level of RhoA-GTP and RhoC-GTP activity in metastatic PCA cells. Application of Rho/ROCK inhibitors produced the same effect as introduction of DLC1. Knocking down of RhoA produced a moderate increase in E-cadherin whereas knocking down of RhoC resulted in a significant increase of E-cadherin. Downregulation of E-cadherin caused by constitutively active RhoAV14 and RhoCV14 could not be reversed by expression of DLC1 in DLC1-negative cell line. DLC1-mediated suppression of metastatic PCA cells invasion was comparable with the one associated with ectopic E-cadherin expression, or caused by suppression of Rho pathway either by Rho/ROCK inhibitors, or by shRNA repression. This study demonstrates that DLC1 expression positively regulates E-cadherin and suppresses highly metastatic PCA cell invasion by modulating Rho pathway, which appears as a feasible therapeutic target in cancers with high activity of RhoGTPases. Oncogene (2014) 33, 724–733; doi:10.1038/onc.2013.7; published online 4 February 2013 Keywords: E-cadherin; DLC1; Rho; invasion; prostate cancer INTRODUCTION functionally interact with Rac1, RhoA and Cdc42, all of which have 15,16 Deleted in liver cancer 1 (DLC1) is a potent tumor-suppressor gene role in coupling cytoskeletal structure to cell–cell contacts. in several cancers and is increasingly recognized by experts in the Several studies have reported a strong correlation between field as a metastasis-suppressor gene.1–5 DLC1 is a multidomain reduced or lost expression of E-cadherin and tumor progression; protein that contains SAM (sterile alpha motif), GAP (GTPase- E-cadherin downregulation is generally a result of trans- 17–19 20,21 activating protein) and START (steroidogenic acute regulatory criptional repression, post-translational processing or its 22 protein-related lipid transfer) domains. It suppresses proliferation, endocytosis. A direct role of E-cadherin expression in migration and invasion and induces apoptosis of human liver, suppression of invasive phenotype was explored in transgenic 23 breast and prostate cancer cells,1,6,7 by acting mainly as a GAP for mouse model of pancreatic b-cell carcinogenesis, and was RhoA, RhoC and, to lesser extent, for Cdc42.8 shown that loss of E-cadherin facilitated a transition from well- DLC1 is critically involved in the control of Rho signaling and, differentiated adenoma to carcinoma and, in the case of forced therefore, affects actin cytoskeletal remodeling and cell–cell expression of a truncated dominant negative gene, increased contacts. DLC1 protein co localizes and binds with a-catenin9 incidence of carcinomas and of metastasis. Conversely, and interacts with tensin, talin and focal adhesion kinase,10–13 thus reintroduction of E-cadherin expression resulted in significant influencing molecular machineries of adherens junctions (AJs) and reduction in frequency of carcinomas vs well-differentiated focal adhesions, both of which are highly relevant in cancer cell adenomas, thus indicating that the loss of E-cadherin-mediated migration and invasion. High level of RhoGTPases has been cell adhesion is required for invasion in vivo. Recently, it was implicated in various steps during cellular transformation and reported that E-cadherin-mediated suppression of anchorage- tumor cell invasion.14 During tumor progression, loss of epithelial independent growth and cell migration is associated with reduced characteristics involves morphological changes that affect activity of RhoA or cdc42, apparently consequential to DLC1 and cytoskeletal network, cell–cell adhesion and cell–matrix adhesion. p190RhoGAP action.24 Inappropriate activation of Rho proteins, reported in highly In this study, we show that DLC1expression increases the invasive human cancer cells, could be responsible for destabiliza- expression of E-cadherin and, consequently, abrogate the inva- tion of cell–cell contacts and facilitation of invasion. siveness of a highly malignant prostate carcinoma (PCA) cell line. E-cadherin, on the other side, is a transmembrane protein that is The DLC1-mediated process apparently results from its RhoGAP a key component of cell–cell adhesion machinery. It forms a action; attenuation of Rho proteins activity by specific Rho/Rock complex with other cytoplasmic proteins like b-catenin, a-catenin inhibitors, or intervention with shRNA, restores E-cadherin and p120 catenin and stabilizes the cell–cell junctions. Cadherins expression. Expression and localization of E-cadherin, disrupted Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. Correspondence: Dr NC Popescu, Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Building 37, Room 4128B, 37 Convent Drive, MSC 4262, Bethesda, MD 20892-4262, USA. E-mail: [email protected] Received 21 August 2012; revised 29 November 2012; accepted 17 December 2012; published online 4 February 2013 DLC1 modulates E-cadherin and suppresses invasion V Tripathi et al 725 by constitutively active, GTP-bound, RhoAV14 and RhoCV14, shown was measured at 0 and 60 min in a calcium-containing buffer 2 þ to be resistant to GAP action, is not reversed by DLC1 expression. (2 mM Ca ). At the 60-min endpoint, DLC1-transduced cells with Reduction in invasiveness of highly malignant PCA cells mimics increased levels of E-cadherin expression showed increased the one observed in cases of E-cadherin overexpression or aggregation compared with their control counterparts (Figures downregulation of Rho activity. 1c and d). Treatment with either anti-E-cadherin antibody (HEDC-1) or with calcium chelating agent EGTA (ethylene glycol tetraacetic acid), both used to disrupt E-cadherin activity, abolished DLC1/E-cadherin-induced aggregation (Figure 1d). RESULTS DLC1 increases expression of E-cadherin in cancer cells and elevates the rate of cell–cell aggregation DLC1-mediated increase in E-cadherin expression is independent Androgen independent and metastatic prostate cancer cells C4-2- of a-catenin and culture density, but E-cadherin localization is B2 and PC-3 express lower levels of E-cadherin as compared with dependent on a-catenin RWPE-1, a normal immortalized prostate epithelial cell (Figure 1a). Alpha-catenin acts as a molecular link between DLC1 and Transduction of DLC1 in these cells and its expression to the E-cadherin. To check whether DLC-mediated increase in E-cad- physiological levels was associated with noticeable increase in the herin expression was dependent on a-catenin, shRNA was used to expression of E-cadherin as shown by western blot and knock down a-catenin in C4-2-B2 cells, and stable clone exhibiting immunofluorescence staining (Figures 1a and b). To examine the maximum effect was selected for the study.9 Transduction of effects of DLC1-mediated increased expression of E-cadherin on these cells with DLC1 resulted in increased expression of cell aggregation ability in vitro, Ad-LacZ- and DLC1-transduced E-cadherin compared with controls and proved that such C4-2-B2 and PC-3 cells were cultured, and their aggregation index increase was independent of a-catenin (Figure 2a). Yet, as shown Figure 1. DLC1 induces expression of E-cadherin. (a) E-cadherin expression in RWPE-1, C4-2-B2 and PC-3 cells (left) and of C4-2-B2 and PC-3 cells transduced with either Ad-Lac Z or DLC1 (right). (b) Immunofluorescence analysis of E-cadherin in Ad-Lac Z or DLC1 transduced C4-2-B2 and PC-3 cells. Bar ¼ 20 mm. (c) Aggregation of Ad-LacZ- or Ad-DLC1-transduced C4-2-B2 and PC-3 cells at time 0 and 60 mins. Bar ¼ 50 mm. (d) Effect of DLC1 expression on cell aggregation at various time points. C4-2-B2 and PC-3 cells were grown in calcium-negative (in the presence of EGTA) and calcium-positive medium. Each batch of cells was transduced with DLC1 or LacZ in the presence or absence of HECD-1. & 2014 Macmillan Publishers Limited Oncogene (2014) 724 – 733 DLC1 modulates E-cadherin and suppresses invasion V Tripathi et al 726 Figure 2. DLC1-mediated E-cadherin induction is independent of a-catenin and cell density. (a) E-cadherin expression in C4-2-B2 and PC-3 cells in the presence or absence of DLC1 and a-catenin. (b, c) Immunofluorescence analysis of E-cadherin in the above-mentioned cell lines. Bars ¼ 10 and 20 mm. (d) Expression of E-cadherin in Ad-LacZ- or Ad- DLC1-transduced C4-2-B2 cells in sparse, medium and confluent culture (right). Densitometric analysis by image quat of western blotted bands compared with GAPDH (glyceraldehyde 3-phosphate dehydrogenase; left). (e) E-cadherin expression in TritonX-100 soluble and insoluble fractions from Ad-LacZ- and Ad-DLC1-transduced C4-2-B2 cells. Bar ¼ 20 mm. DAPI, 40,6-diamidino-2-phenylindole. by Immunofluorescence analysis, localization of E-cadherin at AJs of cell membranes into TritonX-100-soluble and insoluble fractions required both DLC1 and a-catenin (Figure 2b). This observation and their subsequent probing for E-cadherin confirmed irrele- was confirmed in PC-3 cells negative for both DLC1 and a-catenin vance of cell density and showed that DLC1-mediated increase in (Figures 2a and c).
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