Rho/Rhotekin-Mediated NF-Jb Activation Confers Resistance to Apoptosis

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 the Rho signal to nuclear factor-kB (NF- apoptosis in many cell types, including AGS cells. Our kB) activation, leading to increased cell survival by data showed that overexpression of RTKN also rescued transactivating antiapoptotic genes downstream of NF- kB, supporting an important role of Rho/RTKN/NF-kB signaling pathway in gastric tumorigenesis. In line with the prosurvival effect of Rho/RTKN/NF-kB signaling pathway, we also showed that overexpression of RTKN leads to increased chemoresistance in gastric AGS cells Detroit 551 HR SCM-1 AGS H3347 H1299 IMR90 NUGC-3 HT29 Colo205 Hs68 WI 38 SW480 H358 H322 H460 and blockade of NF-kB activity greatly attenuates the INT407 resistance to chemotherapeutic drug, suggesting that RTKN novel therapeutic regimens including NF-kB blockers may represent a promising approach for treating a subset of GCs with increased chemoresistance. β-actin

Results Figure 1 Western blot analysis of RTKN expression in cell lines. Total cell lysates were prepared from designated cell lines. Proteins were fractionated, probed against anti-RTKN and antiactin Overexpression of RTKN inhibits apoptosis induced antibodies, and detected by ECL. Cell lines examined include GC by various stimuli HR, NUGC-3, SCM-1, and AGS cells, colon cancer HT29, H3347, SW480, and Colo205 cells, lung cancer H322, H358, H460, and We have previously shown that RTKN was expressed at H1299 cells, and ﬁbroblast Hs68, WI 38, IMR90, and Detroit 551, an elevated level in human GC, and overexpression of and embryonic intestine-derived INT407 cells

Oncogene Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8733 AGS cells from NaBT-induced apoptosis (Liu et al., 2004).

Elimination of RTKN expression sensitizes cells to apoptotic insult To further conﬁrm the antiapoptotic role of RTKN, synthetic siRNA was used to knockdown the expression of RTKN in AGS cells. Two sets of small interfering RNA (siRNA) were employed:siRNA R, which was designed to target at RTKN to speciﬁcally inhibit RTKN expression, and siRNAC, the control siRNA that have no known homology with mammalian genes and should have no effect on RTKN expression. The introduction of siRNAR effectively abolished RTKN expression in the AGS/RTKN cells (Figure 3a), and the elimination of RTKN expression was accompanied with the loss of resistance to apoptosis (Figure 3b, panel 4). In fact, introduction of siRNAR into AGS/RTKN cells not only resulted in the loss of RTKN-mediated protection against apoptosis but also greatly sensitized the cells to serum depletion-induced apoptosis. A trace amount of endogenous RTKN detected in the AGS/ Control cells was also diminished by the treatment of siRNAR (Figure 3a). The ultrasensitivity to apoptotic insult was also observed in AGS/Control cells upon the treatment of siRNAR (Figure 3b, panel 2), probably due to the depletion and inhibition of endogenous RTKN expression by siRNAR. On the contrary, the introduction of siRNAC had little effect on RTKN expression, nor did it affect the sensitivity of either AGS/Control or AGS/RTKN cells to apoptosis. The antiapoptotic function of RTKN was further substantiated by transfection of siRNAR into colorectal cancer H3347 cells that expressed endogenous RTKN at a high level followed by monitoring their ability to withstand apoptosis. Upon UV irradiation at 5 mJ/cm2, most of the H3347 cells remained viable and about 3–4% of cells were observed in sub-G1 fraction (Figure 3c). After transfection of siRNAR to knockdown the expression of RTKN, the cells became more sensitive to UV-induced apoptosis that 38% of sub-G1 apoptotic population was observed, whereas transfection of cells with the control siRNA has little effect on the sensitivity toward UV- induced apoptosis (Figure 3c).

RTKN mediates Rho signaling to provide cell survival Figure 2 Expression of RTKN suppresses serum starvation- through activation of nuclear factor kB induced apoptosis. (a) Gastric AGS cells stably expressing RTKN (AGS/RTKN) or the same cells containing the control vector To study the signaling pathways that are involved in (AGS/Control) were incubated in serum-free medium for the RTKN-mediated antiapoptosis, cells were incubated in indicated time, then 106 cells were collected and subjected to FACS serum-free medium in the presence of speciﬁc inhibitors analysis as described in the ‘Materials and methods’. Percentage of of NF-kB, PI-3 kinase, or MAP kinase. As shown in apopototic cells in the sub-G1 fraction was shown. (b) Two non- small-cell lung cancer H1299 clones stably expressing RTKN Figure 4a, the NF-kB inhibitors, curcumin and parthe- (H1299/RTKN-90 and H1299/RTKN-55) were established, along nolide, caused only an insigniﬁcant change in the with the clone containing the control vector (H1299/Control), and percentage of apoptotic AGS/Control cells, but pro- subjected to serum starvation-induced apoptosis as described duced a dramatic increase in the percentage of apoptotic above. Immunoblotting was performed to determine RTKN AGS/RTKN cells, that reached the same level as in expression in these cells using anti-Flag M2 control cells. In contrast, the antiapoptotic effect of RTKN was not affected by the presence of the PI 3-kinase inhibitor, LY294002, or the MAP kinase

Oncogene Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8734

Figure 3 Elimination of RTKN expression sensitizes cells to apoptosis. AGS/RTKN and AGS/Control cells (a) and H3347 cells (b) were transfected with RTKN siRNA (siRNAR) or control siRNA (siRNAC) as described in the ‘Materials and methods’. After 24 h, cells were incubated in serum-free medium or treated with UV irradiation (5 mJ/cm2) as indicated. After incubation for additional 48 h, cells were harvested for immunoblot and FACS analysis. Anti-Flag M2 antibody and polyclonal anti-RTKN antibodies were used to determine the expression of RTKN in these cells. The percentage of apoptotic cells was determined as described above

inhibitor, PD98059. These data show that the prosurvi- tion, suggesting that Rho activity is involved in RTKN- val effect of RTKN is mediated through NF-kB induced NF-kB activation. The RTKN-mediated activation and that the PI 3-kinase/Akt and MEK/ NF-kB activation was also observed in cells of normal MAP kinase pathways are not involved. Reporter assays background. In fact, Rho/RTKN/NF-kB signaling were next performed to examine the effect of RTKN on pathway was observed in all cell lines examined, NF-kB activation in AGS cells. As shown in Figure 4b including the human GC AGS, NUGC-3, and SCM-1, (left panel), RTKN and active RhoA.V14 were both colon cancer SW480, and H3347, lung cancer H1299, strong inducers of NF-kB activation. Coexpression of H322, and H358, Hs68 fibroblast and INT407 (embryo- RTKN and active RhoA.V14 caused stronger activation nic intestine-derived epithelial) cells and mouse NIH- of NF-kB. Cotransfection and expression of C3 3T3 fibroblasts. The results of reporter assays performed transferase, a specific inhibitor to the small GTPase in NIH3T3 cells were also shown (Figure 4b, right Rho, greatly suppressed RTKN-induced NF-kB activa- panel).

Oncogene Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8735

Figure 4 RTKN-mediated cell survival is dependent on NF-kB activity. (a) NF-kB inhibitors block RTKN-mediated antiapoptosis. AGS/RTKN and AGS/Control cells were subjected to serum starvation for 48 h in the presence of curcumin (5 mM), parthenolide (10 mM), PD98059 (5 mM), or LY294002 (10 mM), then the cells were harvested, and the percentage of apoptotic cells determined by FACS analyses. (b) RTKN overexpression induces NF-kB activation. Reporter assays were performed in AGS cells and NIH3T3 cells, respectively, by transfection of a pNF-kB-Luc plasmid and the vectors encoding RTKN, RhoA.V14, or C3 transferase as indicated. After normalization to the internal control, the luciferase activity of RTKN-transfected cells relative to that of cells transfected with empty vector was taken as the fold induction. The data are presented as the means7s.d. for at least three independent experiments. (c) EMSA of NF-kB. Nuclear extracts were prepared from AGS/RTKN and AGS/Control cells and incubated for 30 min at room temperature with [a-32P]dCTP-labeled, double-stranded oligonucleotide containing the NF-kB binding site in the presence or absence of anti-p50 or anti-p65 antibodies. The protein–DNA interaction was analysed by electrophoresis on 5% polyacrylamide gel as described in the ‘Materials and methods’. Shifted and supershifted complexes are indicated by an arrow and asterisks. In some experiments, an excess of unlabeled oligonucleotide (cold oligo) was added as competitor. (d) Phosphorylation of IkBa. AGS/RTKN and AGS/Control cells were incubated in serum-free medium for 48 h. After incubation in the presence and absence of TNF-a 10 ng/ml) for 6 h, 106 cells were collected and total cell lysates prepared for Western blot analysis against antiphospho-IkBa (e) RTKN-induced NF-kB activation is mediated through IkBa and IKKb. Reporter assays were performed by cotransfection of AGS cells with the pNF- kB-Luc plasmid and either empty vector or expression vector encoding RTKN, with or without expression vectors encoding DN-IkBa, DN-IKKa, or DN-IKKb. Luciferase activity was measured and the fold induction was calculated as described above

NF-kB is present in its inactive form in the cytoplasm transactivates downstream target genes. An electro- as a heterotrimeric complex of p50, p65, and the phoretic mobility shift assay (EMSA) was performed to inhibitor, IkB. On activation, IkB is phosphorylated determine whether NF-kB translocation was increased by IkBa kinases (IKKs), dissociates from the complex, in RTKN-expressing cells. When nuclear extracts were and is degraded in the proteosome, while NF-kBis prepared and tested for their ability to interact with the translocated to the nucleus, binds to DNA, and consensus NF-kB site, increased NF-kB DNA binding

Oncogene Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8736 activity was seen in the nuclear extract from RTKN- overexpressing cells (Figure 4c). The shifted band represented the specific interaction between NF-kB and its consensus DNA sequence, since antibodies specific for RelA (p65) or NF-kB1 (p50) super-shifted the band to a higher molecular mass. These results also suggested that the major NF-kB activity in AGS cells overexpressing RTKN consisted of p50 and p65 subunits. The specificity of NF-kB binding was further confirmed by a competition study using a 100-fold excess of unlabeled oligonucleotide (Figure 4c). In line with this data, marked phosphorylation of IkBa was detected in AGS/RTKN cells but not in AGS/Control cells (Figure 4d). The role of IkBa in Rho/RTKN- mediated activation of NF-kB was further confirmed by cotransfection of AGS cells with RTKN and a supersuppressor IkBa mutant, IkBa(SS-AA), using the reporter assay. The supersuppressor IkBa(SS-AA) contains serine-to-alanine mutations at residues 32 and 36, which inhibit signal-induced phosphorylation and subsequent proteosome-mediated degradation of IkBa As shown in Figure 4e, RTKN-mediated activation of pNF-kB-Luc was completely blocked by the dominant- Figure 5 RTKN mediates Rho activity to confer cell survival negative mutant IkBa(SS-AA), indicating that NF-kB through activation of NF-kB. (a) Schematic diagram of the activation by RTKN is mediated through IkBa phos- structures of RTKN and truncation mutants. Bars represent the phorylation. Given that IkBa phosphorylation is region encoded in each protein, and numbers to the right indicate the amino-acid number at the NH2 and COOH terminals of each mediated by IKKa or IKKb, we investigated which protein. Rho-binding domain (RBD), pleckstrin homology (PH) IKK was responsible for NF-kB activation in RTKN- domain, proline-rich motifs (PR-I and PR-II), and the carboxyl- expressing cells by co-transfecting with the RTKN terminal QSPV-COOH sequence are shown by hatched, black, plasmid and plasmids encoding the dominant-negative cross-hatched, and gray boxes, respectively. (b) Survival effect of forms of IKKa or IKKb and found that the dominant- RTKN and its mutant proteins. Full-length RTKN, or the RTKN- N or RTKN-C mutant was stably expressed in AGS cells, and negative IKKb mutant blocked NF-kB activation, while serum starvation-induced apoptosis was measured as described the dominant-negative IKKa mutant had little effect above. (c) Induction of NF-kB by RTKN and its mutant proteins. (Figure 4e). Reporter assays were performed in AGS cells by transfection with a The most common mechanism of effector activation pNF-kB-Luc plasmid and expression vector encoding RTKN-N, or RTKN-C along with or without the vector encoding RhoA.V14 is by disruption of the intramolecular autoinhibitory or C3 transferase as indicated. Luciferase activity was measured interaction upon the binding of Rho GTPases, thus and the fold induction was calculated as described above exposing the functional regulatory domain. A mutant effector protein containing only the GTPase-binding domain has been shown to act as a dominant-negative molecule by sequestering the respective GTPase, while RTKN overexpression induces elevated expression removal of the GTPase-binding region is sufficient to of NF-kB antiapoptotic genes render the effector active (Tu and Wigler, 1999; Watanabe et al., 1999; Kim et al., 2000). We therefore NF-kB activation promotes cell survival by switching on tested the RTKN-N and RTKN-C mutant proteins for the transcription of a series of antiapoptotic genes their ability to protect cells from apoptosis. As shown in (Karin et al., 2002). We further investigated whether Figure 5b, AGS cells stably expressing the RTKN-N RTKN overexpression induces the expression of anti- mutant became supersensitive to serum depletion- apoptotic genes downstream of NF-kB in AGS cells and induced apoptosis. On the other hand, the RTKN-C gastric adenocarcinomas. As shown in Figure 6a, after mutant conferred cells highly resistant to serum incubation in serum-free medium for 48 h, expression of deprivation-induced apoptosis. Consistent with these the Bcl-xl, cIAP2, A1, and A20 transcripts was markedly observations, RTKN-C mutant strongly activated NF- increased in RTKN-expressing AGS cells, but not in kB in the reporter assays and this activity was refractory cells containing the control vector, in which it remained to the inhibition by C3 transferase, suggesting its role as at the same level as before serum deprivation. The a dominant active mutant, whereas RTKN-N mutant relative expression of the transcripts of these NF-kB was inactive to induce NF-kB activity and, furthermore, target genes determined by real-time PCR analysis is it worked as a dominant-negative mutant to block also shown (Figure 6b). As shown in Figure 6c (upper RhoA.V14-induced NF-kB activation (Figure 5c). These panel), overexpression of RTKN was seen in five of results suggest that RTKN mediates Rho signaling to seven (71%) primary GCs where RTKN was increased confer cells increased survival through the activation of in expression by 1.6 to 4.4-fold as compared to the NF-kB. nontumorous mucosa. A marked increase in transcrip-

Oncogene Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8737

Figure 6 RTKN overexpression stimulates the expression of NF-kB downstream antiapoptotic genes. (a) Expression of NF-kB antiapoptotic genes in AGS cells. Total RNA was prepared from AGS/RTKN and AGS/Control cells that were cultured in serum-free medium for the indicated time, and the levels of cIAP2, Bcl-xL, A1, A20, and cyclin D1 transcripts were determined by RT–PCR analyses. GAPDH expression was used as a control to monitor RNA quality and concentration. (b) Real-time PCR analysis of relative expression of NF-kB antiapoptotic genes in AGS cells. (c) RTKN expression and differential expression of NF-kB antiapoptotic genes in primary GC. Total RNA was prepared from tumor (T) and matched nontumor (N) tissue from GC patients (ID numbers given above panel), and subjected to RT–PCR analyses. Top panel shows PCR products ampliﬁed using primers speciﬁc for RTKN and GAPDH, respectively. Relative RTKN values were determined by densitometric analyses of RTKN-specifc products after normalization to that of GAPDH. GC that showed elevated expression of RTKN transcripts were subjected to real-time PCR analyses of the expression of NF-kB antiapoptotic genes

tion of NF-kB antiapoptotic genes cIAP2, A1, A20, and cancer cell lines, and blockade of this activity signiﬁ- Bcl-xL was observed in the RTKN-overexpressing cantly increases the chemosensitivity of the cells (Arlt primary gastric adenocarcinoma tissues (Figure 6c, et al., 2002). We therefore tested the chemosensitivity of lower panel). Although NF-kB drives mammary epithe- RTKN-expressing AGS cells in the WST-1 viability lial cell proliferation during pregnancy by turning on assay by incubating the control and RTKN-expressing expression of cyclin D1 (Cao et al., 2001), in our study, AGS cells with increasing concentrations (1–100 mg/ml) cyclin D1 expression in GC cells was not affected by of ﬂuorouracil (5-FU), one of the most effective clinical RTKN expression. antitumor agents available for the treatment of gastro- intestinal cancer. As shown in Figure 7 (upper trace), RTKN overexpression is associated with increased the cytotoxic effect of 5-FU on control cells was dose- chemoresistance dependent, cell viability being reduced to 20% after 72 h treatment with 100 mg/ml 5-FU, whereas, under the Resistance to apoptosis represents one of the major same conditions, more than 70% of RTKN-expressing mechanisms whereby tumors are able to resist chemo- cells remained viable. To verify that the increased and radiotherapy. Elevated NF-kB activity is reported chemoresistance of the RTKN-expressing AGS cells to be associated with chemoresistance in pancreatic was dependent on NF-kB activity, we tested the effect

Oncogene Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8738 Discussion

100 Our data support an important role of Rho/RTKN- 80 mediated signaling pathway in the pathogenic development of human GC. We demonstrated that RTKN 60 : AGS/Control mediates Rho signaling to activate NF-kB, with : AGS/Control + 5 µM curcumin 40 concomitant enhanced expression of NF-kB down- : AGS/RTKN stream antiapoptotic genes. These data suggest that Cell viability (%) 20 : AGS/RTKN + 5 µM curcumin activation of Rho/RTKN/NF-kB signaling pathway through RTKN overexpression renders cells resistant 0 vehicle 1 10 100 to apoptotic insults and enables them to evade apoptosis Fluorouracil (µg/ml) during the process of transformation, and may therefore represent an important molecular event in gastric tumorigenesis. In line with the high potential of RTKN 100 leading to the transactivation of NF-kB antiapoptotic genes, GC cells overexpressing RTKN displayed in- 80 creased resistance to antitumor drugs, further suggesting

60 that signaling pathways eliciting NF-kB activation may be important targets for novel therapeutic approaches in 40 the treatment of human GC.

Cell viability (%) 20 The Rho/RTKN-mediated cell survival was shown to be NF-kB-dependent, because treatment with curcumin 0 or parthenolide completely abolished the increased vehicle1 10 100 1000 10000 resistance to apoptotic insults (Figure 4a). Although Paclitaxel (nM) Rho GTPase promotes cell survival, partly via activa- Figure 7 RTKN overexpression desensitizes GC cells to the tion of PI-3 kinase and subsequent activation of Akt/ cytotoxic effect of antitumor drugs. AGS cells stably expressing PKB (Maeda et al., 2000), our data clearly showed that RTKN or containing the control vector were seeded at 5000 cells/ PI-3K/Akt pathway is not involved in Rho/RTKN- well in 96-well microtiter plates and treated for 72 h with increasing mediated cell survival. In addition, the Ras/MAP kinase concentrations of 5-FU (upper panel) or paclitaxol (lower panel) in the presence or absence of 5 mM curcumin. Surviving cells (J: pathway, which has been shown to block cell death in AGS/control cells; D:AGS/control cells in the presence of other systems, was not involved in RTKN-mediated curcumin K:AGS/RTKN cells; m:AGS/RTKN cells in the antiapoptosis. Elevated NF-kB activity has been found presence of curcumin) were measured by the WST-1 viability test. in a number of malignant tumors (Arlt et al., 2002; The viability of cells treated with antitumor drugs is shown as a percentage of that of vehicle-treated cells. The values represent the Bhat-Nakshatri et al., 2002; Chiao et al., 2002). means of a triplicate experiment7s.d. The experiments were Permanent amplification of NF-kB activity may involve repeated three times with essentially the same results mutations and/or over-representation of members of the Rel protein family (Romero and Humphries, 1995), IkBa gene, or IKKs (DiDonato et al., 1997; Romieu- of an NF-kB inhibitor, curcumin, and found that it Mourez et al., 2001; Sanlioglu et al., 2001). In certain increased the chemosensitivity of RTKN-expressing breast and pancreatic tumors and cell lines, the cells to a similar level to that of control AGS cells autocrine action of IL-1 and IL-1b has been described (Figure 7, upper trace), showing that the cytoprotective to induce NF-kB-dependent protection from apoptosis effect of RTKN was mediated mainly through NF-kB (Nakshatri et al., 1997; Arlt et al., 2002; Bhat-Nakshatri activation. Similar results were obtained using another et al., 2002). Complementary to these findings, our data chemotherapeutic agent, paclitaxol (1 nM–10 mM) have clearly demonstrated that, in a subset of GCs, NF- (Figure 7, lower trace), where less than 30% of control kB activity was elevated as a result of activated Rho AGS cells remained viable after treatment with 1 nM signaling pathway through overexpressed effector pro- paclitaxol for 72 h. In contrast, more than 80% of the tein RTKN. The Rho GTPases, Rho, Rac, and Cdc42, RTKN-expressing cells remained viable even when are also activated by the upstream GEF Dbl to induce treated with much higher concentrations of paclitaxol NF-kB activity; whereas Rac stimulates the activity of (10–10 000 nM). As expected, the cytoprotective effect of IKKb, Cdc42 and Rho activate NF-kB by IKK- RTKN was also significantly compromised if the cells independent pathways (Cammarano and Minden, were incubated with curcumin. The vector-containing 2001). In this study, we found that RTKN-mediated control clones were as sensitive to the cytotoxic effect of Rho signaling pathway leading to enhanced nuclear 5-FU and paclitaxol as the parental AGS cells, NF-kB activity involves activation of IKK-b, which indicating that transfection and selection did not affect phosphorylates, and thereby induces proteasomal de- the sensitivity of cells to these agents (data not shown). gradation of, IkBa (Figure 4c–e). In addition, our data These results show that constitutive expression of further suggested that the Rho/RTKN/NF-kB signaling RTKN in AGS cells renders them significantly more pathway appears to be present in many cell types. Using resistant to cytotoxic agents and the resistance can be reporter assays, RTKN-mediated NF-kB activation was attenuated by the incorporation of an NF-kB inhibitor. examined in 11 representative cell lines that expressed

Oncogene Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8739 endogenous RTKN at different levels. These cell lines Rho activity but does not completely abolish RTKN- included human GC AGS, NUGC-3, and SCM-1, colon induced NF-kB activation (Figure 4b). In agreement cancer SW480 and H3347, lung cancer H1299, H322, with these data, we also found that RTKN-mediated and H358, Hs68 fibroblast and INT407 (embryonic antiapoptotic effect was greatly but not completely intestine-derived epithelial) cells and mouse NIH-3T3 inhibited by C3 transferase (Supplementary Figure 2). fibroblasts. Our data showed that RTKN induced NF- Several possibilities exist for the observed phenomena. kB activation in all cell lines tested (the data from AGS Firstly, overexpression of RTKN may facilitate an and NIH3T3 were shown in Figure 4b). Interestingly, an intermolecular interaction between RTKN molecules inverse correlation of RTKN-induced NF-kB activation that relieves an intramolecular contact between the and endogenous RTKN expression level was observed GTPase binding domain and autoinhibitory domain. that NF-kB activation was markedly induced in cell Alternatively, RTKN may work as an effector for lines that expressed endogenous RTKN at undetectable proteins other than Rho. Further studies are required to or low level, whereas less strong induction was observed test these hypotheses. Nevertheless, our data suggest in cell lines that expressed endogenous RTKN at high that RTKN mediates Rho activity leading to increased level (Supplementary Figure 1). It may reflect that cell survival through the activation of NF-kBand RTKN-expressing cell lines exert high background of subsequent induction of its downstream antiapoptotic endogenous NF-kB activity and therefore limited fold genes. NF-kB is also known to induce the expression of of induction was achieved upon expression of ectopi- gene products that control proliferative responses cally introduced RTKN. These data suggest that RTKN (Karin et al., 2002). However, in the context of this mediates Rho signaling pathway leading to NF-kB study, AGS cells expressing RTKN at a high level did activation, and that dysregulation of this pathway not gain significant growth advantage over the parental through overexpression of the effector protein RTKN or vector-transfected control cells (data not shown). In may represent a common mechanism in the tumorigenic agreement with these observations, the expression of process. cyclin D1 transcripts remained unchanged in GC cells RTKN was initially isolated as a target molecule for that overexpressed RTKN (Figure 6). the small GTPase, Rho (Reid et al., 1996). It contains In view of that NF-kB activation suppresses apoptosis the Rho binding domain in its amino terminus, a PH by inducing the expression of certain antiapoptotic domain in the central region, two PXXP motifs, and the genes, NF-kB activity may play a detrimental role in carboxy terminal QSPV-COOH sequence, which recog- blocking the efficacy of cancer chemotherapies and nizes PSD-95/Discs-large/ZO-1 (PDZ) domain. Its Rho radiation. Given that sustained NF-kB activation has binding domain shows 30% amino-acid sequence been reported in a number of cancers, these tumors may identity with those of two other Rho target proteins, have developed a level of chemoresistance prior to the serine/threonine kinase, PKN, and rhophilin. This cancer therapy. In line with this notion, Chuang et al. class of Rho-binding domain, termed the Rho effector (2002) have recently reported that the basal level of motif class I (REM-1), shows no similarity to the NF-kB activity in a series of representative cancer cell binding motifs of the Rac/Cdc42 effectors, nor is it lines was heterogeneous and was inversely correlated present in the coil-bearing Rho effectors, such as ROCK with the sensitivity of these cell lines to several major and citron (Reid et al., 1996). Acting as a scaffold anticancer drugs. In this study, we showed that Rho/ protein, RTKN was recently shown to interact with RTKN-mediated NF-kB activation in GC cells was TIP-1 and greatly enhanced the Rho-mediated activa- associated with increased chemoresistance and blockade tion of serum response element (Reynaud et al., 2000). of NF-kB activity strongly attenuated the increased Other possible links downstream of RTKN that elicit resistance (Figure 7). These data demonstrated that the Rho-mediated signal transduction remain largely un- apoptotic response to genotoxic anticancer drugs is explored. In this study, we demonstrated that RTKN greatly desensitized in tumor cells where NF-kBis links Rho activity to induce NF-kB activity. Our data constitutively active, and inhibition of NF-kB activity in showed that the inhibition of Rho activity by coexpres- these cells may therefore tremendously enhance their sion of C3 transferase significantly suppresses RTKN- sensitivity to such treatment. mediated NF-kB activation (Figure 4b), suggesting that In conclusion, we have established in this study that RTKN works downstream to mediate Rho activity. This Rho/RTKN/NF-kB signal axis confers cell resistance to was further substantiated by the findings that RTKN-N apoptosis. We demonstrated that RTKN overexpression mutant protein, which contains the Rho binding leads to sustained NF-kB activity, with concomitant domain, acts as a dominant-negative molecule to block induction of the expression of NF-kB antiapoptotic genes. RhoA.V14-induced NF-kB activity, whereas the These data suggest that overactivated Rho/RTKN/NF-kB RTKN-C mutant protein acts as a dominant-active signaling pathway may play an important role in gastric molecule to induce robust NF-kB activity that is tumorigenesis. In addition, GCs with an overactivated independent of Rho activity (Figure 5c). Consistent Rho/RTKN/NF-kB signaling pathway displayed higher with these findings, expression of RTKN-C mutant resistance to antitumor drugs and the resistance was renders cells highly resistant to serum deprivation- significantly attenuated by the incorporation of NF-kB induced apoptosis and cells expressing RTKN-N inhibitor, suggesting that combining NF-kB blockers with mutant became supersensitive to apoptosis (Figure 5b). other chemotherapeutic agents may be of great benefit in It is noted that coexpression of C3 transferase inhibits the treatment of a subset of GC patients.

Oncogene Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8740 Materials and methods Expression of RTKN was detected by Western blot analysis. Cells were lysed in 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, Plasmids and materials 0.5% Triton X-100, 0.5% sodium deoxycholate, 10 mM Na P O ,50mM sodium fluoride, 20 mg/ml of leupeptin, The plasmid pFLAG-CMV2-RTKN encoding the full-length 4 2 7 20 mg/ml of aprotinin, and 1 mM phenylmethylsulfonyl fluor- RTKN was constructed as described (Liu et al., 2004). pFlag- ide, and the protein concentration was determined using a CMV-RTKN-N and pFlag-CMV-RTKN-C were constructed commercial kit based on the Bradford method (Bio-Rad by PCR amplification of the corresponding DNA fragments Laboratories, Richmond, CA, USA). Equal amounts of encoding the N-terminal region containing the Rho-binding protein were fractionated by SDS polyacrylamide gel electro- domain (amino acids 1–188) and the C-terminal region (amino phoresis and transferred onto a polyvinylidenedifluoride acids 295–551) of human RTKN, respectively, using the membrane, which was then incubated with anti-Flag M2 pFLAG-CMV2-RTKN as template, and subsequent subclon- antibody and polyclonal antibodies specifically recognizing ing of the PCR products into pFLAG-CMV2 vector. The RTKN, respectively, followed by peroxidase-conjugated sec- sequences of the plasmids were verified using an ABI Prismt ondary antibodies. Proteins were detected using the enhanced 370 autosequencer (Applied Biosystems, Foster City, CA, USA). chemiluminescence system (Amersham Pharmacia Biotech., The vectors encoding constitutively activated RhoA.V14 in Buckinghamshire, England). Polyclonal antibodies against pUHD and C3 transferase in pEF1a have been previously RTKN were generated by immunizing rabbit with a synthetic described (Lai et al., 2001). pCMV4-IkBa(S32A/S36A), peptide corresponding to RTKN sequence from amino acids encoding a dominant-negative IkBa (DN-IkBa), was provided 43–57 (RKLDHEIRMREGACK). Antibody against phos- by Dr DW Ballard (Scherer et al., 1995). pcDNA3-IKKa phor-IkB-a was purchased from Santa Cruz Biotechnology, (D144N), encoding DN-IKKa and pcDNA3-IKKb(K44A), Inc. (Santa Cruz, CA, USA). encoding DN-IKKb were gifts from Dr CV Paya (Asin et al., 1999). PD098059, curcumin, parthenolide, LY294002, and anti-Flag M2 antibody were purchased from Sigma Chemical Flow cytometric analysis Co. (St Louis, MO, USA). Cells were seeded at 1 Â 105 cells/well in six-well microplates and incubated overnight in medium containing 10% fetal calf serum. To induce apoptosis, cells were cultured in serum-free Mammalian cell transfection and NF-kB reporter assays RPMI-1640 medium and incubated for the indicated times, the The human gastric adenocarcinoma AGS, colorectal adeno- cells were harvested, treated with RNaseA, incubated with carcinoma H3347, and the non-small-cell lung cancer H1299 50 mg/ml of propidium iodide for 30 min at room temperature, cell lines, and mouse NIH3T3 cell line were purchased from then analysed immediately by flow cytometry (FACSCalibur, American Type Culture Collection. AGS cells were maintained Becton Dickinson, USA). in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum and 100 mg/ml of penicillin/ RNA interference streptomycin/gentamycin at 371C in a humidified atmosphere Annealed, purified, and desalted double-stranded RTKN of 5% CO2. NIH3T3 cells were maintained in DMEM siRNA (sense, 50-AGC AUC AGU AAC CAG UAU GTT- supplemented with 10% calf serum, and H3347 and H1299 30; and antisense, 50-CAU ACU GGU UAC UGA UGC UTT- cells were maintained in RPMI-1640 medium supplemented 30) were ordered from Qiagen. A pair of RNA oligonucleotides with 10% fetal calf serum and 100 mg/ml of penicillin/ that do not share sequence homology to any mammalian genes streptomycin/gentamycin under the same condition. At 24 h was also purchased from Qiagen to serve as control siRNA before transfection, the cells were transferred to serum-free (Xia et al., 2002). The day before transfection, 2 Â 105 cells medium. Transient transfection was performed with 1 mgof were seeded in 60-mm plate. Cells were transfected with 1 mM NF-kB luciferase plasmid (pNF-kB-Luc, Strategene USA, La RTKN siRNA or control siRNA in 100 ml EC-R buffer Jolla, CA, USA) and 300 ng of pFlag-CMV2, pFlag-CMV- (Qiagen) using LipofectAMINE 2000 (Invitrogen), according RTKN, pFlag-CMV-RTKN-N, or pFlag-CMV-RTKN-C to the protocol recommended by Qiagen. After 6 h, the cells using the LipofectAMINE-2000 method (Invitrogen Corp., were changed into medium containing 10% FBS. To induce Carlsbad, CA, USA), according to the manufacturer’s proto- apoptosis, cells were incubated in serum-free medium instead col. A CMV-driven b-galactosidase expression vector was also of the serum-containing medium. After incubation for an cotransfected to monitor transfection efficiency. Cells were additional 48 h, cells were harvested for the assessment of harvested 30 h after transfection under serum-free conditions, RTKN expression and apoptosis. and cell lysates were prepared and luciferase activities were measured. Luciferase activity was normalized to the b- Electrophoretic mobility shift assay galactosidase activity. In some experiments, a plasmid encoding RhoA.V14 or C3 transferase was also cotransfected. All Nuclear extracts were prepared as described previously experiments were performed in duplicate and at least three (Schreiber et al., 1993). Briefly, 6 Â 106 cells were collected by times. centrifugation at 110 g for 5 min, washed with ice-cold To generate AGS or H1299 cells stably expressing RTKN phosphate-buffered saline (PBS), suspended in 200 ml of buffer and its mutants, cells were cotransfected with pFlag-CMV2- containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM RTKN, pFlag-CMV2-RTKN-N, or pFlag-CMV2-RTKN-C EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 1 Â protease plasmid along with pcDNA3 vector (neor) at 10 :1 ratio using inhibitor (final concentrations 1 mM ABESF-HCl, 150 nM the electroporation method as described previously (Chen aprotinin, 15 mM E-64, and 20 mM leupeptin), and incubated et al., 1993). At 16 h post-transfection, cells were transferred to on ice for 15 min. After addition of 12.5 ml of 10% Nonidet P- fresh medium containing neomycin at a final concentration of 40, the sample was mixed thoroughly for 10 s using a Vortex 500 mg/ml. After 14 days, G418-resistant clones were estab- mixer, then centrifuged at 10 000 g at 41C for 3 min. The lished. AGS cells stably harboring the empty pFlag-CMV2 nuclear pellets thus obtained were resuspended in 25 ml of ice- plasmid DNA were also established by the same selection cold extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, procedure and were used as controls. 1mM EDTA, 1 mM EGTA, 1 mM dithiothreitol and 1 Â

Oncogene Rho/RTKN/NF-jB axis and antiapoptosis C-A Liu et al 8741 protease inhibitor) and kept on ice for 15 min with intermittent ATG AG-30; GenBank Accession # NM_001165), cyclin D1 agitation. After centrifugation at 10 000 g for 5 min at 41C, the transcripts (sense primer, 50-GCT GCG AAG TGG AAA supernatants were collected as nuclear protein extracts. A CCA TC-30; antisense primer 50-CAG GAC CTC CTT CTG double-stranded oligonucleotide containing the NF-kB con- CAC AC-30; GenBank Accession #NM_053056). In total, 25 sensus binding site, prepared by annealing two synthetic cycles of PCR (941C for 30 s, 551C for 30 s, and 721C for 1 min) oligonucleotides (50-CGC GAG TTG AGG GGA CTT TCC were performed using primers specific for GAPDH transcripts CAG GC-30 and 50-CGC GGC CTG GGA AAG TCC CCT (sense primer, 50-TGG TAT CGT GGA AGG ACT CAT CAA CT-30; NF-kB binding site underlined), was radiolabeled GAC-30; antisense primer, 50-ATG CCA GTG AGC TTC with Klenow enzyme in the presence of [a-32P]dCTP. Binding CCG TTC AGC-30; GenBank Accession # NM_002046). PCR was performed by incubating nuclear extracts (10 mg) for products were separated by agarose gel electrophoresis and 30 min at room temperature with the radiolabeled probe examined under UV after ethidium bromide staining. (1 Â 106 cpm) in 15 ml of buffer containing 10 mM HEPES, pH Quantitative real-time PCR was carried out using the DNA 7.6, 200 ng/ml poly(dI-dC), 0.1% NP-40, 10% glycerol, 1 mM Engine Opticons 2 System (MJ Research Inc., Reno, Nevada, DTT, 5 mM MgCl2, and 50 mM NaCl in the presence or USA) in a final volume of 20 ml with 1 Â FastStart DNA absence of antibodies against p50 or p65 (0.2 mg) (CHEMI- Master SYBR Green I mix (Roche Diagnostics GmBH, CON International Inc., Temecula, CA, USA). The protein– Germany) with 2 mM of primers, 2 ml of relevant sample DNA interaction was analysed by electrophoresis on a cDNA, and magnesium chloride adjusted to a final concentra- nondenaturing 5% polyacrylamide gel, followed by autora- tion of 1.5 mM. Primers used and cycling conditions were diography. described as above. The GAPDH primer set was used as the reference control for normalization. The threshold cycles (CT) RNA isolation, RT–PCR and real-time PCR were recorded for all samples for both the target gene and GAPDH. Melt curve analysis was performed for each run. Gastric adenocarcinoma tissue and matching nontumor Relative gene expression of the target gene was calculated as mucosal tissue were obtained from patients who underwent DCT, determined by subtracting the CT of reference gene from surgery at the Veterans General Hospital-Taipei. Informed the CT of target gene. Differential expression of the target gene consent was obtained from each patient. They consisted of in tumor vs nontumor mucosal samples was shown as DDCT, biopsies from five males and two females, representative of two determined by subtracting the DCT of tumor sample from the intestinal, one diffuse, and four mixed type GC. Tissues were DCT of the matching mucosal sample. snap-frozen immediately following resection. Total RNA was prepared from homogenized tissues using the guanidine Cell viability assay isothiocyanate method and pelleted through a 5.7 M CsCl cushion. Total RNA was prepared from cell lines using the The WST tetrazolium assay was used to measure the cell TRIZOL reagent (Invitrogen Corp., Carlsbad, CA, USA). viability (Mosmann, 1983). Briefly, approximately 5 Â 104 In total, 5 mg of total RNA was converted to cDNA using target cells/well were plated in triplicate in 96-well microtiter 200 U of MMLV reverse transcriptase (SuperscriptII, Invitro- plates and incubated for the indicated time at 371C in the gen Corp., Carlsbad, CA, USA) in 50 mlof50mM Tris-HCl, presence or absence of the indicated test samples in a final pH 8.3, 75 mM KCl, 3 mM MgCl2,10mM DTT, and 1 mM volume of 0.1 ml. In total, 10 ml of the cell proliferation dNTP containing 0.625 mg of random primers, following the reagent, WST-1, (Roche Molecular Biochemicals, Mannheim, manufacturer’s instructions. The polymerase chain reaction Germany) was added and incubation continued for 4 h. The (PCR) was carried out in a 10 ml reaction mixture containing formazan dye formed was quantified by scanning the plates on 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 3 mM MgCl2, 0.01% (w/ a microplate reader at a wavelength of 440 nm. Relative cell v) gelatin, 2 mM dNTP, 0.1 mg/ml of each primer, 1 mlof viability was calculated by comparing the OD of the test suitably diluted cDNA, and 2.5 U of Taq DNA polymerase. In samples to that of the medium control. total, 30 cycles of PCR (941C for 30 s, 651C for 30 s, and 721C for 1 min) were performed using the following primers: RTKN transcripts (sense primer, 50-GAG CGC ACT GAG CAG GCA AAG-30; antisense primer, 50-CAG GAT CCA TAC Abbreviations AGC TCT AAC CGC AGT TC-30; GenBank Accession # GC, gastric cancer; NF-kB, nuclear factor-kB; IkB, inhibitory BC004558), A1 transcripts (sense primer, 50-CAA GAC TTT subunit of NF-kB; IKK, IkBa kinase; MAPK, mitogen- GCT CTC CAC CA-30; antisense primer, 50-GGC AAT CGT activated protein kinase; PI 3-kinase, phosphatidylinositol 3- TTC CAT ATC AGT-30, GenBank Accession # NM_004049), kinase; RT–PCR, reverse transcription–polymerase chain A20 transcripts (sense primer, 50-GAG AGC ACA ATG GCT reaction; FACS, fluorescence-activated cell sorting. GAA CA-30; antisense primer, 50-CCA GTG TGT ATC GGT GCA T-30; GenBank Accession # NM_006290), BCL-xl Acknowledgements transcripts (sense primer 50-ACC CAT CCT GGC ACC We thank Dr Z-F Chang (Institute of Biochemistry TGG CA-30, and antisense primer 50-GGA TCC AAG GCT and Molecular Biology, National Taiwan University, CTA GGT GG-30; GenBank Accession # NM_138578), cIAP2 Taiwan) for constructive suggestions. This work is supported transcripts (sense primer, 50-CAA CCC CAA AGA GTT GTG in part by NSC91-3112-P-001-024-Y and Chen Shuyi Cancer TTC-30; antisense primer, 50-GAG GCA GTT TTG TGC Foundation.

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Supplementary Information accompanies the paper on Oncogene website (http://www.nature.com/onc).

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