Deleted in Liver Cancer 1 Controls Cell Migration Through a Dia1-Dependent Signaling Pathway
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Research Article Deleted in Liver Cancer 1 Controls Cell Migration through a Dia1-Dependent Signaling Pathway Gerlinde Holeiter, Johanna Heering, Patrik Erlmann, Simone Schmid, Ruth Ja¨hne, and Monilola A. Olayioye Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany Abstract Rho proteins cycle between an inactive GDP-bound state and an active GTP-bound state. When bound to GTP, they interact Deleted in liver cancer (DLC) 1 and 2 are Rho GTPase- with effector proteins, modulating their activity and localization. activating proteins that are frequently down-regulated in Signaling of growth factor receptors and integrins can induce various types of cancer. Ectopic expression in carcinoma cell exchange of GDP for GTP on Rho proteins. This activation of Rho lines lacking these proteins has been shown to inhibit cell proteins is controlled by the guanine nucleotide exchange factors migration and invasion. However, whether the loss of DLC1 or (GEF), which promote the release of bound GDP and facilitate DLC2 is the cause of aberrant Rho signaling in transformed GTP binding, and the GTPase-activating protein (GAP) proteins, cells has not been investigated. Here, we have down-regulated which increase the intrinsic GTPase activity of Rho GTPases to DLC1 and DLC2 expression in breast cancer cells using a RNA accelerate the return to the inactive state (2). The structurally interference approach. Silencing of DLC1 led to the stabiliza- related proteins deleted in liver cancer (DLC) 1 and 2 belong to tion of stress fibers and focal adhesions and enhanced cell the GAP family and display in vitro specificity for Rho and to a motility in wound-healing as well as chemotactic Transwell lesser extent for Cdc42 (4–6). In addition to their GAP domain, assays. We provide evidence that enhanced migration of cells DLC1 and DLC2 further contain a sterile a motif and a StAR- lacking DLC1 is dependent on the Rho effector protein Dia1 related lipid transfer (START) domain, which may have regulatory but does not require the activity of Rho kinase. By contrast, roles that remain to be defined. DLC2 knockdown failed to affect the migratory behavior of The DLC1 gene was originally isolated as a candidate tumor cells, suggesting that the two proteins have distinct functions. suppressor gene in primary human hepatocellular carcinoma This is most likely due to their differential subcellular located on chromosome 8p22 (7). Loss of expression due to localizations, with DLC1 found in focal adhesions and DLC2 chromosomal deletion or promoter hypermethylation has subse- being mainly cytosolic. Collectively, our data showthat DLC1 quently been shown in other tumor types, including breast, colon, is critically involved in the control of Rho signaling and actin prostate, and lung (8). Transfection of the DLC1 cDNA into cytoskeleton remodeling and that its cellular loss is sufficient carcinoma cell lines lacking DLC1 expression inhibited cell growth for the acquisition of a more migratory phenotype of breast and tumorigenicity in nude mice (9–12). Microinjection of rat DLC1 cancer cells. [Cancer Res 2008;68(21):8743–51] suppressed the formation of lysophosphatidic acid–induced stress fibers and focal adhesions (13). Furthermore, stable expression of Introduction human DLC1 in hepatocellular and breast carcinoma cell lines Cell migration is a biological process involved in development, was shown to reduce cell motility and invasiveness, consistent with inflammation, wound healing, and tumor metastasis. The migration the inhibition of Rho signaling (14, 15). The DLC2 gene whose of cells is a multistep cycle starting with the formation of membrane expression is similarly down-regulated in various tumor types is protrusions driven by actin polymerization at the cell front. These located on chromosome 13q13 and encodes a protein that is f60% protrusions are anchored to the extracellular matrix by integrin identical to DLC1 (5). Reminiscent of DLC1, cellular reexpression receptors forming focal adhesions. The migratory cycle is continued disrupted the actin cytoskeleton and inhibited cell proliferation by actomyosin-driven contraction of the cell body at the back and is and migration (16). It thus seems that DLC1 and DLC2 may be completed by the subsequent detachment of the cell rear. Therefore, functionally redundant in suppressing Rho signaling and Rho- cell migration requires coordinated changes in cytoskeletal mediated cellular processes. dynamics (1). The Rho GTPases Rho, Rac, and Cdc42 are key Rho is required for Ras-mediated oncogenic transformation and players in the control of cell migration and have defined functions activated mutants were shown to be weakly transforming in with regard to cytoskeletal remodeling. Active Rho is known to murine fibroblasts (17, 18). However, no constitutively active Rho induce the assembly of stress fibers and focal adhesions, whereas mutants have been identified in human tumors; instead, Rho Rac and Cdc42 promote the formation of specialized membrane proteins are rather found to be overexpressed. In mammalian cells, protrusions called lamellipodia and filopodia, respectively (2, 3). there are three structurally related Rho proteins: RhoA, RhoB, and RhoC. In breast cancer and testicular germ cell tumors, RhoA expression levels correlated positively with the tumor stage (19, 20), and overexpression of RhoC was shown to be causally linked to Note: Supplementary data for this article are available at Cancer Research Online inflammatory breast cancers (21). In in vivo models of tumor (http://cancerres.aacrjournals.org/). Requests for reprints: Monilola A. Olayioye, Institute of Cell Biology and dissemination, RhoC has been identified to enhance the metastatic Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. potential of melanoma cells (22). Phone: 49-711-685-69301; Fax: 49-711-685-67484; E-mail: [email protected] An alternative mechanism by which Rho activation can be stuttgart.de. I2008 American Association for Cancer Research. achieved is the deregulation of GEFs or the loss of its GAPs. In this doi:10.1158/0008-5472.CAN-08-0984 study, we analyzed the consequences of DLC1 and DLC2 www.aacrjournals.org 8743 Cancer Res 2008; 68: (21). November 1, 2008 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Cancer Research knockdown at the molecular and cellular level. We show that phosphate dehydrogenase (GAPDH)-F (5¶-CCCCTTCATTGACCTCAAC- silencing of DLC1 in breast cancer cells augments cellular RhoA TA-3¶) and GAPDH-R (5¶-CGCTCCTGGAAGATGGTGAT-3¶). levels and enhances cell motility, whereas down-regulation of DLC2 Cell lysis, SDS-PAGE, and Western blotting. Cells were lysed in had no effect on the migratory behavior of cells. Our results further radioimmunoprecipitation assay buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, shed light onto the underlying molecular mechanisms by 1 mmol/L sodium orthovanadate, 10 mmol/L sodium fluoride, 20 mmol/L identifying Dia1 as the Rho effector involved in DLC1-mediated h-glycerophosphate plus Complete protease inhibitors (Roche)]. Lysates control of breast cancer cell migration. were clarified by centrifugation at 16,000 Â g for 10 min. Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Roth). The membrane was blocked with 0.5% Materials and Methods blocking reagent (Roche) in PBS containing 0.1% Tween 20 and then Antibodies and reagents. Antibodies used were mouse anti-DLC1, incubated with primary antibodies followed by HRP-conjugated secondary mouse anti-Dia1, and mouse anti-paxillin monoclonal antibodies (mAb; antibodies. Visualization was with the enhanced chemiluminescence Becton Dickinson); mouse anti-RhoA mAb, rabbit anti-RhoA (119) detection system (Pierce). polyclonal antibody (pAb), rabbit anti-Cdc42 pAb, and goat anti-Dia1 pAb RBD pull-downs. BL21 bacteria were transformed with a pGEX vector (Santa Cruz Biotechnology); and mouse anti-tubulin mAb (Sigma). The encoding the RBD of rhotekin and expression was induced with 0.1 mmol/L anti-DLC2 antiserum was raised by immunizing rabbits with DLC2 peptide isopropyl-h-D-1-thiogalactopyranoside for 4 h at 37jC. Bacteria were (C-373TALPDAGDQSRMHEFH388) coupled to keyhole limpet hemocyanin harvested, resuspended in PBS containing Complete protease inhibitors, (Pineda). Antibodies were affinity purified with the SulfoLink Immobiliza- and sonicated. Triton X-100 was added (1% final) and the lysate was tion kit for Peptides (Pierce). Elution was with 100 mmol/L glycine buffer centrifuged for 10 min at 8,000 Â g. GST-RBD was purified with glutathione (pH 2.7), and neutralized antibody fractions were pooled and dialyzed resin (GE Healthcare). For pull-downs, cells were lysed in RBD extraction against PBS. Horseradish peroxidase (HRP)-labeled secondary anti-mouse buffer [50 mmol/L Tris (pH 7.5), 500 mmol/L NaCl, 10 mmol/L MgCl2,1% and anti-rabbit IgG antibodies were from GE Healthcare; HRP-labeled Triton X-100, 1 mmol/L sodium orthovanadate, 10 mmol/L sodium fluoride, secondary anti-goat IgG antibody was from Santa Cruz Biotechnology; and 20 mmol/L h-glycerophosphate, 0.5 mmol/L phenylmethylsulfonyl fluoride Alexa Fluor 488–labeled and Alexa Fluor 546–labeled secondary anti-mouse plus Complete protease inhibitors without EDTA (Roche)]. Equal amounts IgG antibodies and Alexa Fluor 546–labeled phalloidin were from Molecular