Oncogene (2004) 23, 8731–8742 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc Rho/Rhotekin-mediated NF-jB activation confers resistance to apoptosis Ching-Ann Liu1, Mei-Jung Wang2, Chin-Wen Chi3, Chew-Wun Wu4 and Jeou-Yuan Chen*,2 1Graduate Institute of Life Sciences, National Defense Medical Center, Taiwan, ROC; 2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC; 3Department of Medical Research and Education, Taiwan, ROC; 4Department of Surgery, Veterans General Hospital, Taipei, Taiwan, ROC Rhotekin (RTKN), the gene coding for the Rho effector, Introduction RTKN, was shown to be overexpressed in human gastric cancer (GC). In this study, we further showed that RTKN The Rho GTPases are members of the Ras superfamily is expressed at a low level in normal cells and is of monomeric low molecular mass (approx. 21 kDa) overexpressed in many cancer-derived cell lines. The guanine nucleotide-binding proteins. By cycling between function of RTKN as an effector protein in Rho GTPase- an active (GTP-bound) and an inactive (GDP-bound) mediated pathways regulating apoptosis was investigated. state, Rho GTPases function as molecular switches to By transfection and expression of RTKN in cells that control signal transduction pathways in regulation of a expressed endogenous RTKN at a low basal level, we plethora of cellular processes, including cytoskeleton showed that RTKN overexpression conferred cell resis- reorganization, gene transcription, cell-cycle progres- tance to apoptosis induced by serum deprivation or sion, and survival (Bishop and Hall, 2000). The diverse treatment with sodium butyrate, and the increased function of Rho GTPases is mediated through interact- resistance correlated to the level of RTKN. Conversely, ing with, and modulating the activity of, respective reducing RTKN expression by small interfering RNAs effector proteins. Rho, Rac, and Cdc42 are the three greatly sensitized cells to apoptosis. The RTKN-mediated best-characterized members of the family for their roles antiapoptotic effect was blocked by the nuclear factor-jB in the reorganization of the actin cytoskeleton (Paterson (NF-jB) inhibitors, curcumin or parthenolide, but not et al., 1990; Ridley and Hall, 1992; Nobes and Hall, by the phosphatidylinositol 30-OH-kinase inhibitor, 1995). Rho induces the formation of focal adhesions and LY294002, or the MAP kinase inhibitor, PD98059. stress fibers, this effect being mediated through the Reporter gene assays and electrophoretic mobility shift cooperation of two downstream effector proteins, the assay confirmed that RTKN overexpression led to Rho-associated kinase (ROCK/ROK/Rho kinase) and constitutive activation of NF-jB through the phosphor- the mammalian orthologue of the Drosophila Diapha- ylation of IjB by IKKb. By using the RTKN truncation nous protein, mDia (Ishizaki et al., 1997; Watanabe mutants, we showed that RTKN mediated Rho activity et al., 1999; Sahai and Marshall, 2002). Cdc42 and Rac, eliciting signaling pathway to activate NF-jB, with a respectively, induce the formation of filopodia and concomitant induction of expression of the NF-jB lamellipodia (Ridley et al., 1992; Kozma et al., 1995), antiapoptotic genes, cIAP-2, BCl-xL, A1, and A20. and the effectors of these two GTPases, Wiskott– Consistent with these data, RTKN-expressing cells Aldrich-syndrome protein (WASP)/N-WASP (the N showed increased chemoresistance to 5-fluorouracil and isoform of WASP) and WASP-like Verprolin-homo- paclitaxol, and the resistance was greatly attenuated by logous protein (WAVE, also known as Scar), mediate NF-jB inhibitor. In conclusion, overactivated Rho/ actin nucleation by binding to the Arp2/3 complex RTKN/NF-jB signaling pathway through overexpression (Machesky and Insall, 1998; Rohatgi et al., 1999). More of RTKN may play a key role in gastric tumorigenesis than 30 potential effector proteins have been identified by conferring cells resistance to apoptosis, and this signaling for Rho, Rac, and CDC42, including protein kinases, pathway may serve as an important target for novel lipid kinases, phosphatase, lipases, and a number of therapeutic approaches to the treatment of human GC. scaffold proteins (Bishop and Hall, 2000). Much effort Oncogene (2004) 23, 8731–8742. doi:10.1038/sj.onc.1208106 has now been made in characterizing the biological Published online 11 October 2004 abilities of Rho GTPases in addition to actin organiza- tion and in identifying the individual effectors that Keywords: Rho GTPases; rhotekin; nuclear factor-kB; transit the distinct signal pathways linked to these antiapoptosis; gastric cancer cellular responses. Dysfunctional regulation of Rho GTPase family protein activation has been implicated in certain aspects of cancer development. Activated Rho protein mutants *Correspondence:J-Y Chen, Institute of Biomedical Sciences, can transform fibroblasts (Qiu et al., 1995). Rho protein Academia Sinica, 128 Section 2 Academia Road, Taipei 11529, Taiwan, ROC; E-mail:[email protected] can promote cell cycle progression by affecting CDK Received 20 May 2004; revised 10 August 2004; accepted 11 August activity through regulation of the levels of cyclin D1, 2004; published online 11 October 2004 p21WAF1, and p27KIP1 (Weber et al., 1997; Olson et al., Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8732 1998). Transcriptional upregulation of the levels of RTKN was associated with an increased survival (Liu particular Rho proteins has been described in many et al., 2004). To test whether RTKN-mediated signaling types of human cancers, including cancers of the colon, pathway may confer cell resistance to apoptotic insults breast, lung, and pancreas (Suwa et al., 1998; Fritz et al., and enables cells to evade apoptosis during the process 1999; Schnelzer et al., 2000; van Golen et al., 2000; Kleer of transformation, we first examined the expression of et al., 2002). In the case of breast cancer, increased RTKN in a serious of normal and transformed cells. As RhoA expression correlated with malignancy (Fritz shown in Figure 1, RTKN expression was low in all the et al., 2002), and RhoC expression was highly associated test normal cells, including several human fibroblasts with invasive tumors (Kleer et al., 2002). RhoC and embryonic intestine-derived 407 cells, but its expression also correlated significantly with tumor expression was readily detected in many of the cancer- progression in ductal adenocarcinoma of the pancreas derived cell lines. To explore the role of RTKN in cell (Suwa et al., 1998). In line with the role of Rho proteins survival, AGS gastric cells that express endogenous in tumor invasion and metastasis, reduced expression of RTKN at a low level were transfected with RTKN RhoGDI2 has recently been shown to correlate with expression vector. Cell clones stably expressing RTKN increasing stage and grade of tumors from several were established as AGS/RTKN cells, and compared common sites (Gildea et al., 2002). Other evidences also with AGS/Control cells, the same cells transfected with suggest that overactivated Rho-mediated cellular func- a control vector, for their ability to withstand apoptosis tions contribute to cancer. Members of the diffuse B cell induced by various stimuli. AGS, like many other cancer lymphoma-derived (Dbl) family of Rho GEFs have cell lines, shows a time-dependent increase in the transforming activity. Naturally occurring mutations number of apoptotic cells upon serum deprivation resulting in constitutive activation of Rho GEFs have (Kanai et al., 2001; Tovar Sepulveda et al., 2002). As been described in Dbl, LARG, and Tiam (Srivastava shown, marked apoptosis was observed in the AGS/ et al., 1986; Engers et al., 2000; Kourlas et al., 2000). Control cells when cultured in serum-free medium, with Overexpression or activation of Rho GTPase down- the sub-G1 apoptotic population increasing to 32% in stream effector proteins ROCK and protein kinase 48 h (Figure 2a). In contrast, AGS/RTKN cells were C-related kinase (PRK1) have recently been described in significantly more resistant to serum deprivation- several human cancers (Kaneko et al., 2002; Ikeda et al., induced apoptosis, with about 4–5% of the cells 2003; Kamai et al., 2003; Metzger et al., 2003; Zhou showing apoptosis after 48 h in serum-free medium. et al., 2003). RTKN-mediated survival was not unique to AGS cells, We have previously shown that RTKN, a Rho as similar results were also obtained with non-small-cell effector protein, is overexpressed in human gastric lung cancer H1299 cells. Figure 2b shows that two cancer (GC) (Liu et al., 2004). RTKN was initially H1299/RTKN clones were established and they also isolated as a scaffold protein interacting with GTP- displayed increased resistance to serum deprivation- bound form of Rho (Reid et al., 1996). Other than induced apoptosis, and the increase was correlated to interacting with TIP-1 (human T cell lymphotrophic the level of RTKN. Our data therefore showed that virus, type 1 Tax interacting protein-1) to facilitate Rho- RTKN overexpression protects cells from serum deple- mediated activation of serum response element tion-induced apoptosis. To test whether RTKN pro- (Reynaud et al., 2000), the role of RTKN involved in tected cells from other apoptotic insults, AGC/Control Rho-mediated signal transduction remains largely un- and AGS/RTKN cells were subjected to sodium known. In this study, we demonstrated that RTKN butyrate (NaBT) treatments, which are known to induce activation links