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Rhoc Protein Stimulates Migration of Gastric Cancer Cells Through Interaction with Scaffold Protein IQGAP1

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Rhoc Protein Stimulates Migration of Gastric Cancer Cells Through Interaction with Scaffold Protein IQGAP1 MOLECULAR MEDICINE REPORTS 4: 697-703, 2011 RhoC protein stimulates migration of gastric cancer cells through interaction with scaffold protein IQGAP1 YAN WU, YONG-CHANG CHEN, JIAN-RONG SANG and WEN-RONG XU School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China Received February 28, 2011; Accepted April 18, 2011 DOI: 10.3892/mmr.2011.482 Abstract. The scaffold protein IQGAP1 is closely related to Introduction certain Rho GTPases. Research has revealed that IQGAP1 acts as an effector of Cdc42 and Rac1 in the regulation of Ras GTPase-activating-like proteins (IQGAPs) are evolu- cell activity such as proliferation and migration. However, tionary conserved multi-domain proteins that share a similar whether IQGAP1 is associated with RhoC, another important domain structure and a marked sequence homology. To date, Rho GTPase, is unclear. Previous results from our labora- three IQGAPs have been identified in humans. Among them, tory indicated that IQGAP1 and RhoC are highly expressed the best-characterized is IQGAP1, which contains a calponin- in gastric cancer tissues and cells. This study was designed homology domain, a tryptophan repeat motif, four centrally to investigate the possible interaction between IQGAP1 and located IQ motifs, a C-terminal RasGAP-related domain RhoC in the regulation of the migration of cancer cells. The and a RasGAP-C-terminal domain. As a scaffold protein, expression of IQGAP1 and RhoC in gastric cancer tissues and IQGAP1 binds multiple proteins and facilitates their interac- cell lines was detected by Western blotting. siRNAs targeting tion. Research has shown that IQGAP1 interacts directly IQGAP1 or RhoC were transfected into gastric cancer cells with calmodulin (1), actin (2), E-cadherin (3,4), β-catenin (5), to knock down the expression of the proteins. Adenoviral and the small GTPases Cdc42 (3), Rac1 (6) and Rap1 (7,8). constructs encoding full length IQGAP1, the C-terminal Through its interaction with these proteins, IQGAP1 regulates fragment of IQGAP1, and the constitutively active RhoC many cellular functions including cell-cell adhesion, migra- gene were used to infect gastric cancer cells to increase the tion (9), cytoskeletal architecture (10), transcription (11) and expression of the proteins. The migratory activity of a gastric selected signal transductions. cancer cell line was measured by a transwell migration IQGAP1 has been implicated in tumorigenesis due to the assay. Western blotting revealed that the IQGAP1 and RhoC observed overexpression and altered localization of IQGAP1 proteins were highly expressed in gastric cancer tissues and in neoplasia, particularly at the peripheral regions of more cells. Spearman's rank correlation analysis indicated that aggressive breast cancer (12), colorectal carcinoma (13) and the increases in the expression of IQGAP1 and RhoC were ovarian adenocarcinomas (14). A lack of IQGAP1 expression closely correlated. The transwell migration assay revealed that is associated with a favorable prognosis in gastric cancer (15). both IQGAP1 and RhoC stimulated the migration activity of Furthermore, IQGAP1 assists in various signal transductions the gastric cancer cell line AGS. The knockdown of IQGAP1 implicated in cancer. For example, IQGAP1 interacts with expression by siRNA blocked the migration-stimulating MEK, ERK (16) and B-Raf (1) to facilitate their sequential activity of RhoC, while the knockdown of RhoC expres- activation and to stimulate MAPK-mediated signaling (17), sion had no effect on the migration-stimulatory activity of and promotes proliferation or differentiation (3). IQGAP1 IQGAP1. Co-IP results showed that RhoC and IQGAP1 bound is also capable of directly associating with β-catenin (18), to each other. These results reveal a previously unrecognized an oncogenic protein and primary mediator of the Wnt interaction between IQGAP1 and RhoC, and demonstrate that pathway, to cause cell transformation, invasion and migration. IQGAP1 is a downstream effector of RhoC in the regulation Emerging evidence supports a potential role of IQGAP1 in of the migration activity of gastric cancer cells. regulating protein traffic (19), cell proliferation and division (20) via the CDC42-mTOR pathway. In another study, it was demonstrated that IQGAP1 facilitated mTOR and Akt inte- raction and promoted cell proliferation by PI3K/Akt signaling in hepatocellular carcinoma (21). However, although there Correspondence to: Professor Yong-Chang Chen, School of Medical are adequate data supporting the role of IQGAP1 in tumor Science and Laboratory Medicine, Jiangsu University, Xue Road, progression, what triggers its up-regulation and what key Zhenjiang, Jiangsu 212013, P.R. China binding partners augment its role in tumorigenicity remain to E-mail: [email protected] be determined. The Rho family of GTPase belongs to the Ras super-family Key words: RhoC, IQGAP1, gastric cancer cell, migration of small G proteins, which currently comprises more than 20 members. Among them, RhoA, RhoB, and RhoC are 698 WU et al: IQGAP1 IS AN EFFECTOR OF RhoC IN REGULATING CELL MIGRATION A C B D Figure 1. Expression of IQGAP1 and RhoC in gastric cancer tissues and cell lines. (A) Representative results showing alterations in the expression levels of IQGAP1 and RhoC. Proteins were extracted from adjacent normal mucosa and gastric cancer tissues, and Western blotting was used to detect the expression of the target proteins. Lane T, gastric cancer tissues; N, adjacent normal mucosa tissue. (B) Graphical presentation of (A); *p<0.05. (C) Western blot analysis of IQGAP1 and RhoC expression levels in GES-1, BGC-823, AGS and SGC-7901 cell lines. (D) Graphical presentation of (C). Values are presented as the means ± SD obtained from triplicate samples. Table Ⅰ. Correlation between RhoC and IQGAP1 expression in IQGAP1 is closely related to some GTPases of the 29 gastric cancer patients. Rho family. For example, both Cdc42 and Rac1, key members of the other two subclasses of the Rho family, interact No. of cases (%) with IQGAP1 to stimulate the motility of cells. Recently, Relative ------------------------------------------------ Statistical researchers also observed that Rap1b (7,8) was co-localized expression RhoC IQGAP1 analysis with IQGAP1, facilitated the sequential phosphorylation a of B-Raf, C-Raf, and ERK1/2, and aided IQGAP1 to form T/N ≤1 7 (75.9) 7 (75.9) r=0.521 a large signalosome in the perinuclear region. Among the a T/N 1-10 22 (24.1) 22 (24.1) p=0.004 members of the Rho subclass, RhoA (29) has been revealed to have no interaction with IQGAP1. However, it remains to T, gastric cancer tissue; N, adjacent non-neoplastic mucosa tissue. a be determined whether RhoC is associated with IQGAP1 or Spearman's rank correlation analysis confirmed the statistical signifi- not. Based on our previous observation that the expression cance of the association between RhoC and IQGAP1. of IQGAP1 and RhoC in gastric cancer tissues was markedly higher than that in non-neoplasia mucosa tissues, here we investigated the possible association between IQGAP1 and RhoC. grouped into the Rho subclass. Recent studies have revealed that RhoA and RhoB appear to be associated with the prolif- Materials and methods eration of cancer cells; nevertheless the overexpression of RhoC enhances the metastasis of cancer cells (22,23). RhoC Tissue samples. Gastric cancer tissue specimens (n=29) were is frequently overexpressed in a variety of human tumors, obtained from patients who had undergone surgical resec- and the degree of overexpression correlates with tumor tion at the Department of Surgery, Affiliated Hospital of progression and clinical outcome in some cases, such as Jiangsu University. None received preoperative chemotherapy breast cancer (24), pancreatic cancer (25), colon cancer (26), or radiotherapy. Fresh specimens were removed from the bladder cancer, hepatocellular carcinoma, non-small cell lung resected tissues and were immediately frozen at -80˚C and carcinoma (27) and primary gastric tumors (23). In brief, stored until use. The corresponding non-neoplasia mucosa research suggests that RhoC is correlated with tumorigenesis, tissues, resected at least 5 cm away from the tumor margin, development and the infiltration metastasis of certain tumor served as controls. All patients provided written informed types (28); however, the mechanisms of its action have yet to consent, and the study protocol and patient information sheets be elucidated. were approved by the Ethics Committee of Jiangsu University. MOLECULAR MEDICINE REPORTS 4: 697-703, 2011 699 A C B D Figure 2. IQGAP1 stimulates the migration of gastric cancer cells. (A) Western blot analysis of IQGAP1 expression in AGS cells infected with pAd-LacZ, pAd-IQGAP1-C or pAd-IQGAP1. (B) The relative migration activity of AGS cells infected with pAd-IQGAP1-C or pAd-IQGAP1 using the transwell migra- tion assay; *p<0.05 compared to the LacZ group. (C) Protein expression of IQGAP1 in the gastric cancer cell line AGS transfected with IQGAP1 siRNA. (D) Migration assay of AGS cells transfected with IQGAP1 siRNA; *p<0.05 compared to control siRNA group. Data are the means ± SD from three independent experiments, each performed in duplicate. Reagents, antibodies, siRNA and plasmids. Dulbecco's Cell culture, transfection and RNA interference. BGC-823, modified Eagle's medium (DMEM) and fetal bovine AGS, GES-1, SGC-7901 and COS-7 cells were cultured serum (FBS) were from Gibco (Grand Island, NY, USA). in 1X DMEM
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