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SmgGDS Regulates Cell Proliferation, Migration, and NF-κB Transcriptional Activity in Non-small Cell Lung Carcinoma

2007; Elsevier BV; Volume: 283; Issue: 2 Linguagem: Inglês

10.1074/jbc.m707526200

ISSN

1083-351X

Autores

Gaik W. Tew, Ellen Lorimer, Tracy J. Berg, Huiying Zhi, Rongshan Li, Carol L. Williams,

Tópico(s)

Bone Metabolism and Diseases

Resumo

Non-small cell lung carcinoma (NSCLC) is promoted by the increased activities of several small GTPases, including K-Ras4B, Rap1A, Rap1B, RhoC, and Rac1. SmgGDS is an unusual guanine nucleotide exchange factor that activates many of these small GTPases, and thus may promote NSCLC development or progression. We report here that SmgGDS protein levels are elevated in NSCLC tumors, compared with normal lung tissue from the same patients or from individuals without cancer. To characterize SmgGDS functions in NSCLC, we tested the effects of silencing SmgGDS expression by transfecting cultured NSCLC cells with SmgGDS small interfering RNA (siRNA). Cells with silenced SmgGDS expression form fewer colonies in soft agar, do not proliferate in culture due to an arrest in G1 phase, and exhibit disrupted myosin organization and reduced cell migration. The transcriptional activity of NF-κB in NSCLC cells is diminished by transfecting the cells with SmgGDS siRNA, and enhanced by transfecting the cells with a cDNA encoding SmgGDS. Because RhoA is a major substrate for SmgGDS, we investigated whether diminished RhoA expression mimics the effects of diminished SmgGDS expression. Silencing RhoA expression with RhoA siRNA disrupts myosin organization, but only moderately decreases cell proliferation and does not inhibit migration. Our finding that the aggressive NSCLC phenotype is more effectively suppressed by silencing SmgGDS than by silencing RhoA is consistent with the ability of SmgGDS to regulate multiple small GTPases in addition to RhoA. These results demonstrate that SmgGDS promotes the malignant NSCLC phenotype and is an intriguing therapeutic target in NSCLC. Non-small cell lung carcinoma (NSCLC) is promoted by the increased activities of several small GTPases, including K-Ras4B, Rap1A, Rap1B, RhoC, and Rac1. SmgGDS is an unusual guanine nucleotide exchange factor that activates many of these small GTPases, and thus may promote NSCLC development or progression. We report here that SmgGDS protein levels are elevated in NSCLC tumors, compared with normal lung tissue from the same patients or from individuals without cancer. To characterize SmgGDS functions in NSCLC, we tested the effects of silencing SmgGDS expression by transfecting cultured NSCLC cells with SmgGDS small interfering RNA (siRNA). Cells with silenced SmgGDS expression form fewer colonies in soft agar, do not proliferate in culture due to an arrest in G1 phase, and exhibit disrupted myosin organization and reduced cell migration. The transcriptional activity of NF-κB in NSCLC cells is diminished by transfecting the cells with SmgGDS siRNA, and enhanced by transfecting the cells with a cDNA encoding SmgGDS. Because RhoA is a major substrate for SmgGDS, we investigated whether diminished RhoA expression mimics the effects of diminished SmgGDS expression. Silencing RhoA expression with RhoA siRNA disrupts myosin organization, but only moderately decreases cell proliferation and does not inhibit migration. Our finding that the aggressive NSCLC phenotype is more effectively suppressed by silencing SmgGDS than by silencing RhoA is consistent with the ability of SmgGDS to regulate multiple small GTPases in addition to RhoA. These results demonstrate that SmgGDS promotes the malignant NSCLC phenotype and is an intriguing therapeutic target in NSCLC. Lung cancer is the leading cause of cancer death for both men and women in the United States (1Jemal, A., Siegel, R., Ward, E., Murray, T., Xu, J., Smigal, C., and Thun, M. J. (2006) CA-Cancer J. Clin. 56, 106-130Google Scholar). More than 80% of all cases of lung cancer are caused by non-small cell lung carcinoma (NSCLC), 3The abbreviations used are:NSCLCnon-small cell lung carcinomaGEFguanine nucleotide exchange factorGFPgreen fluorescent proteinNHBEnormal human bronchial epithelialsiRNAsmall interfering RNATNF-αtumor necrosis factor-αGAPDHglyceraldehyde-3-phosphate dehydrogenase. which is represented mainly by squamous cell lung carcinoma and adenocarcinoma of the lung. NSCLC is the major cause of lung cancer mortality (1Jemal, A., Siegel, R., Ward, E., Murray, T., Xu, J., Smigal, C., and Thun, M. J. (2006) CA-Cancer J. Clin. 56, 106-130Google Scholar). non-small cell lung carcinoma guanine nucleotide exchange factor green fluorescent protein normal human bronchial epithelial small interfering RNA tumor necrosis factor-α glyceraldehyde-3-phosphate dehydrogenase. Numerous members of the Ras and Rho families of small GTPases have been implicated in the development or progression of lung cancer (2Mazieres J. 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The small GTPase then binds GTP, dissociates from the GEF, and interacts with its specific effectors to elicit a variety of cellular responses. Signaling by the GTP-bound small GTPase is terminated when the small GTPase hydrolyzes the bound GTP to GDP. Abnormalities in this GDP/GTP exchange cycle in NSCLC may generate high levels of activated, GTP-bound Ras and Rho family members, resulting in excessive signaling by the activated small GTPases. The increased activity of several Ras family members, including K-Ras4B, Rap1A, and Rap1B, is believed to contribute to NSCLC development or progression. NSCLC cells often express K-Ras4B that is mutated at codon 12, which slows the ability of the mutant K-Ras4B to hydrolyze GTP (reviewed in Refs. 8Mascaux C. Iannino N. Martin B. Paesmans M. Berghmans T. Dusart M. Haller A. Lothaire P. Meert A.P. Noel S. Lafitte J.J. Sculier J.P. Br. J. Cancer. 2005; 92: 131-139Crossref PubMed Scopus (517) Google Scholar and 9Johnson B.E. Heymach J.V. Clin. Cancer Res. 2004; 10: 4254-4257Crossref PubMed Scopus (52) Google Scholar). When this mutant K-Ras4B is activated by a GEF, the mutant K-Ras4B stays in the active GTP-bound form longer than usual due to its diminished ability to hydrolyze the bound GTP to GDP. This hyperactive K-Ras4B may promote NSCLC by stimulating the proliferation of NSCLC progenitor cells (12Tuveson D.A. Shaw A.T. Willis N.A. Silver D.P. Jackson E.L. Chang S. Mercer K.L. Grochow R. Hock H. Crowley D. Hingorani S.R. Zaks T. King C. Jacobetz M.A. Wang L. Bronson R.T. Orkin S.H. DePinho R.A. Jacks T. Cancer Cell. 2004; 5: 375-387Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 13Kim C.F. Jackson E.L. Kirsch D.G. Grimm J. Shaw A.T. Lane K. Kissil J. Olive K.P. Sweet-Cordero A. Weissleder R. Jacks T. Cold Spring Harbor Symp. Quant. Biol. 2005; 25 (2005): 241-250Crossref Scopus (57) Google Scholar, 14Kim C.F. Jackson E.L. Woolfenden A.E. Lawrence S. Babar I. Vogel S. Crowley D. Bronson R.T. Jacks T. Cell. 2005; 121: 823-835Abstract Full Text Full Text PDF PubMed Scopus (1859) Google Scholar, 15Berns A. Cell. 2005; 121: 811-813Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The small GTPases Rap1A and Rap1B may be abnormally activated in NSCLC due to overexpression of C3G, which is a GEF that activates both Rap1A and Rap1B (6Hirata, T., Nagai, H., Koizumi, K., Okino, K., Harada, A., Onda, M., Nagahata, T., Mikami, I., Hirai, K., Haraguchi, S., Jin, E., Kawanami, O., Shimizu, K., and Emi, M. (2204) J. Hum. Genet. 49, 290-295Google Scholar). It was recently reported that over 50% of NSCLC tumors tested express abnormally high levels of C3G (6Hirata, T., Nagai, H., Koizumi, K., Okino, K., Harada, A., Onda, M., Nagahata, T., Mikami, I., Hirai, K., Haraguchi, S., Jin, E., Kawanami, O., Shimizu, K., and Emi, M. (2204) J. Hum. Genet. 49, 290-295Google Scholar). Activated Rap1A and Rap1B may promote NSCLC metastasis by regulating cell spreading (16Hattori M. Minato N. J. Biochem. (Tokyo). 2003; 134: 479-484Crossref PubMed Scopus (155) Google Scholar), cell-cell adhesion (17Hogan C. Sepente N. Cogram P. Hosking C. Bialucha C. Feller M. Braga V. Birchmeier W. Fujita Y. Mol. Cell. Biol. 2004; 24: 6690-6700Crossref PubMed Scopus (221) Google Scholar), and cell migration (18Fujita H. Fukuhara S. Sakurai A. Yamagishi A. Kamioka Y. Nakaoka Y. Masuda M. Mochizuki N. J. Biol. Chem. 2005; 280: 5022-5031Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 19Wittchen E.S. van Buul J.D. Burridge K. Worthylake R.A. Curr. Opin. Hematol. 2005; 12: 14-21Crossref PubMed Scopus (71) Google Scholar). Rho family members reported to have a role in NSCLC include RhoB (2Mazieres J. Antonia T. Daste G. Muro-Cacho C. Berchery D. Tillement V. Pradines A. Sebti S. Favre G. Clin. Cancer Res. 2004; 10: 2742-2750Crossref PubMed Scopus (153) Google Scholar, 3Wang S. Yan-Neale Y. Fischer D. Zeremski M. Cai R. Zhu J. Asselbergs F. Hampton G. Cohen D. Oncogene. 2003; 22: 6204-6213Crossref PubMed Scopus (67) Google Scholar), RhoC (4Shikada Y. Yoshino I. Okamoto T. Fukuyama S. Kameyama T. Maehara Y. Clin. Cancer Res. 2003; 9: 5282-5286PubMed Google Scholar, 5Ikoma T. Takahashi T. Nagano S. Li Y.M. Ohno Y. Ando K. Fujiwara T. Fujiwara H. Kosai K. Clin. Cancer Res. 2004; 10: 1192-1200Crossref PubMed Scopus (96) Google Scholar), and Rac1 (10Regala R.P. Weems C. Jamieson L. Copland J.A. Thompson E.A. Fields A.P. J. Biol. Chem. 2005; 280: 31109-31115Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Lang W. Wang H. Ding L. Xiao L. Cell. Signal. 2004; 16: 457-467Crossref PubMed Scopus (50) Google Scholar). RhoB suppresses the aggressive NSCLC phenotype (2Mazieres J. Antonia T. Daste G. Muro-Cacho C. Berchery D. Tillement V. Pradines A. Sebti S. Favre G. Clin. Cancer Res. 2004; 10: 2742-2750Crossref PubMed Scopus (153) Google Scholar, 3Wang S. Yan-Neale Y. Fischer D. Zeremski M. Cai R. Zhu J. Asselbergs F. Hampton G. Cohen D. Oncogene. 2003; 22: 6204-6213Crossref PubMed Scopus (67) Google Scholar), whereas RhoC enhances the malignant characteristics of NSCLC cells (4Shikada Y. Yoshino I. Okamoto T. Fukuyama S. Kameyama T. Maehara Y. Clin. Cancer Res. 2003; 9: 5282-5286PubMed Google Scholar, 5Ikoma T. Takahashi T. Nagano S. Li Y.M. Ohno Y. Ando K. Fujiwara T. Fujiwara H. Kosai K. Clin. Cancer Res. 2004; 10: 1192-1200Crossref PubMed Scopus (96) Google Scholar). Rac1 activates unique c-Jun NH2-terminal kinase(JNK)-dependent signaling pathways that stimulate the proliferation of NSCLC cells (10Regala R.P. Weems C. Jamieson L. Copland J.A. Thompson E.A. Fields A.P. J. Biol. Chem. 2005; 280: 31109-31115Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Lang W. Wang H. Ding L. Xiao L. Cell. Signal. 2004; 16: 457-467Crossref PubMed Scopus (50) Google Scholar). Surprisingly, the specific functions of RhoA in NSCLC have not been previously reported, although RhoA is known to promote tumorigenesis and metastasis in other forms of cancer (reviewed in Refs. 20Ridley A.J. Breast Cancer Res. Treat. 2004; 84: 13-19Crossref PubMed Scopus (158) Google Scholar and 21Aznar S. Lacal J.C. Cancer Lett. 2001; 165: 1-10Crossref PubMed Scopus (289) Google Scholar). RhoA and other Rho family members may promote cancer cell invasion and migration by regulating the organization of the actin/myosin cytoskeleton (reviewed in Refs. 20Ridley A.J. Breast Cancer Res. Treat. 2004; 84: 13-19Crossref PubMed Scopus (158) Google Scholar and 21Aznar S. Lacal J.C. Cancer Lett. 2001; 165: 1-10Crossref PubMed Scopus (289) Google Scholar). Several Ras and Rho family members that participate in the development or promotion of NSCLC are able to regulate the activity of the transcription factor NF-κB (22Perona R. Montaner S. Saniger L. Sanchez-Perez I. Bravo R. Lacal J.C. 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Activation of NF-κB transcriptional activity occurs when the p65 RelA subunit of NF-κB translocates to the nucleus and binds DNA in association with the appropriate co-activators and DNA-binding partners (reviewed in Refs. 33Campbell K.J. Perkins N.D. Cell Cycle. 2004; 3: 869-872Crossref PubMed Scopus (16) Google Scholar and 34Vermeulen L. Vanden Berghe W. Haegeman G. Cancer Treat. Res. 2006; 130: 89-102Crossref PubMed Scopus (42) Google Scholar). These functions of RelA are modulated by post-translational modifications of RelA and its protein partners, including phosphorylation and acetylation (reviewed in Refs. 33Campbell K.J. Perkins N.D. Cell Cycle. 2004; 3: 869-872Crossref PubMed Scopus (16) Google Scholar and 34Vermeulen L. Vanden Berghe W. Haegeman G. Cancer Treat. Res. 2006; 130: 89-102Crossref PubMed Scopus (42) Google Scholar). Signals that induce these modifications in RelA and its protein partners in a coordinated and synchronized manner stimulate RelA-dependent transcription. In contrast, signals that promote these events in an uncoordinated and asynchronous manner can actually repress RelA-dependent transcription (Refs. 27Berman K.S. Verma U.N. Harburg G. Minna J.D. Cobb M.H. Gaynor R.B. Clin. Cancer Res. 2002; 8: 354-360PubMed Google Scholar, 28Mijatovic T. Op De Beeck A. Van Quaquebeke E. Dewelle J. Darro F. de Launoit Y. Kiss R. Mol. Cancer Ther. 2006; 5: 391-399Crossref PubMed Scopus (99) Google Scholar, 29Wang X. Ju W. Renouard J. Aden J. Belinsky S.A. Lin Y. Cancer Res. 2006; 66: 1089-1095Crossref PubMed Scopus (101) Google Scholar and 35Shumilla J.A. Broderick R.J. Wang Y. Barchowsky A. J. Biol. Chem. 1999; 274: 36207-36212Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 36Qanungo S. Wang M. Nieminen A.L. J. Biol. 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Due to this limitation, NSCLC cell proliferation and metastasis may best be controlled by suppressing the activities of multiple small GTPases simultaneously in NSCLC. The GEF SmgGDS (also known as smg GDS, RAP1GDS1, and GDS) is an excellent candidate for such a strategy, because SmgGDS is the only known GEF that activates multiple small GTPases in both the Ras and Rho families (reviewed in Refs. 38Lanning C. Ruiz-Velasco R. Williams C.L. J. Biol. Chem. 2003; 278: 12495-12506Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar and 39Williams C.L. Cell. Signal. 2003; 15: 1071-1080Crossref PubMed Scopus (158) Google Scholar). SmgGDS increases GTP binding by many small GTPases, including RhoA (40Mizuno T. Kaibuchi K. Yamamoto T. Kawamura M. Sakoda T. Fujioka H. Matsuura Y. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6442-6446Crossref PubMed Scopus (169) Google Scholar, 41Chuang T.H. Xu X. Quilliam L.A. Bokoch G.M. Biochem. 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Chem. 2004; 279: 44197-44210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). SmgGDS enhances DNA synthesis induced by Rap1A (47Yoshida Y. Kawata M. Miura Y. Musha T. Sasaki T. Kikuchi A. Takai Y. Mol. Cell. Biol. 1992; 12: 3407-3414Crossref PubMed Scopus (102) Google Scholar) and enhances the transforming activity of K-Ras4B (48Fujioka H. Kaibuchi K. Kishi K. Yamamoto T. Kawamura M. Sakoda T. Mizuno T. Takai Y. J. Biol. Chem. 1992; 267: 926-930Abstract Full Text PDF PubMed Google Scholar) in several cell types. The ability of SmgGDS to act as a master regulator of multiple small GTPases makes SmgGDS a very unique and promising candidate to regulate the malignant phenotype. Despite the obvious rationale for investigating the participation of SmgGDS in the development or progression of NSCLC, the functions of SmgGDS in NSCLC or any other cancer have not been reported. A previous study examined mRNA transcripts that are up-regulated in squamous cell lung carcinoma, and identified mRNA for SmgGDS as one of the transcripts that are overexpressed in this type of lung cancer (7Sun W. Zhang K. Zhang X. Lei W. Xiao T. Ma J. Guo S. Shao S. Zhang H. Liu Y. Yuan J. Hu Z. Ma Y. Feng X. Hu S. Zhou J. Cheng S. Gao Y. Cancer Lett. 2004; 212: 83-93Crossref PubMed Scopus (80) Google Scholar). Although this finding supports a role for SmgGDS in NSCLC, this previous study did not characterize SmgGDS protein levels in the lung tumors, nor did it test the functional significance of SmgGDS expression in NSCLC (7Sun W. Zhang K. Zhang X. Lei W. Xiao T. Ma J. Guo S. Shao S. Zhang H. Liu Y. Yuan J. Hu Z. Ma Y. Feng X. Hu S. Zhou J. Cheng S. Gao Y. Cancer Lett. 2004; 212: 83-93Crossref PubMed Scopus (80) Google Scholar). To test the expression and function of SmgGDS in NSCLC, we examined SmgGDS protein levels in NSCLC tumors, and determined the functional consequences of silencing SmgGDS expression in NSCLC cell lines. Because SmgGDS preferentially activates RhoA in comparison to other small GTPases that interact with SmgGDS (42Yaku H. Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1994; 198: 811-817Crossref PubMed Scopus (74) Google Scholar, 43Hutchinson J.P. Eccleston J.F. Biochemistry. 2000; 39: 11348-11359Crossref PubMed Scopus (37) Google Scholar), we also examined the effects of silencing RhoA expression in NSCLC cell lines. Diminished RhoA expression should mimic the effects of diminished SmgGDS expression if the phenotypic effects induced by silencing SmgGDS expression are mainly due to reduced RhoA activity. Our results demonstrate that SmgGDS protein expression is higher in NSCLC tumors than it is in normal lung tissue. We found that the proliferation and migration of cultured NSCLC cells are inhibited significantly by silencing SmgGDS expression, but not to the same extent by silencing RhoA expression. This finding indicates that SmgGDS most likely mediates its effects by regulating multiple small GTPases in addition to RhoA. Silencing SmgGDS induces nuclear translocation of RelA, but profoundly inhibits NF-κB transcriptional activity, suggesting that SmgGDS is needed to coordinate the signaling pathways that maintain NF-κB activity in NSCLC. This study indicates that SmgGDS expression may promote NSCLC tumor progression and metastasis, and identifies SmgGDS as a new therapeutic target in NSCLC. Cell Culture and siRNA Transfection—The NSCLC cell lines NCI-H23, NCI-H520, NCI-H522, and NCI-H1703 were obtained from the American Type Tissue Collection (Bethesda, MD), and cultured in complete NSCLC medium (RPMI 1640, 10% heat-inactivated fetal bovine serum, and antibiotics). The normal human bronchial epithelial (NHBE) cells and the medium in which they were cultured (Bronchial Epithelial Cell Medium) were purchased from Cambrex (San Diego, CA). All siRNA duplexes were purchased from Dharmacon (Lafayette, CO). Two siRNAs targeting different sequences in SmgGDS were used in the study. The first siRNA targeting SmgGDS is designated as siRNA I1, and targets the SmgGDS mRNA sequence 5′-GCAAAGAUGUUAUCAGCUG-3′. The second siRNA targeting SmgGDS is designated as siRNA I2, and targets the SmgGDS mRNA sequence 5′-GUUAAUAGAUGCACAAGAA-3′. The siRNA used to silence RhoA expression targets the RhoA mRNA sequence 5′-AUGGAAAGCAGGUAGAGUU-3′. A control siRNA, designated as Scramble siRNA, was designed by the manufacturer to not target any human genes (Dharmacon siControl number 1 siRNA). The cells were transfected with the different siRNAs at the indicated concentrations using Dharmafect-3 transfection reagent (Dharmacon) according to the manufacturer's instruction. Immunohistochemistry—Multiple arrays of formalin-fixed, paraffin-embedded lung tumors and matched or unmatched normal lung tissue were obtained from U.S. Biomax, Inc. (Rockville, MD). The arrays consisted of product number LC1001, which is an array of 45 NSCLC tumors with matched normal lung tissue and additional samples of unmatched normal lung tissue, product number LC801, which is an array of 78 lung tumors (including 30 squamous cell carcinoma, 23 adenocarcinoma, and 9 small cell carcinoma samples) and unmatched normal lung tissue, and product BS04011, which is an array of 63 squamous cell lung carcinoma tumors. The tissue arrays were deparaffinized, incubated in peroxidase blocking buffer (3% H2O2 in methanol) and heated in citrate buffer (pH 6.0, 95 °C). After incubating in protein blocking buffer (3% bovine serum albumin in phosphate-buffered saline), the sections were incubated with SmgGDS antibody (BD Transduction Laboratories 612511) followed by incubation with the peroxidase labeling system (Dakocytomation Envision + Dual Link Peroxidase System). The sections were incubated in peroxidase substrate solution (Dakocytomation Liquid DAB + Substrate Chromagen System) and counterstained with hematoxylin (Dakocytomation). The specimens were dehydrated and cleared in xylene. For negative controls, the tissue sections were stained in the same manner except that the primary antibody to SmgGDS was omitted. All specimens were mounted in aqueous medium and examined using an Olympus BX41 microscope and Olympus DP70 camera. Antibody reactivity was assessed independently by two investigators without knowledge of the identities of the tissues being examined. The relative immunohistochemical staining of each tissue specimen was ranked by the examiners using the following scale: 0 = undetectable staining, +1 = weak staining, +2 = moderate staining, +3 = strong staining (Fig. 1). To examine the immunohistochemical reactivity of SmgGDS antibody with cultured NSCLC cells, NCI-H1703 cells were transfected in the absence or presence of the indicated siRNAs and cultured for 72 h. The cells were then fixed with 10% buffered formalin, mixed with preheated Histogel (Richard-Allen Scientific, Kalamazoo, MI), and embedded in a paraffin block using our previously described techniques (49Li R. Ni J. Bourne P.A. Yeh S. Yao J. di Sant'Agnese P.A. Huang J. Appl. Immunohistochem. Mol. Morphol. 2005; 13: 85-90Crossref PubMed Scopus (11) Google Scholar). The paraffin block containing the embedded cells was cut with a microtome to generate 4-μm thick sections. The sections of NCI-H1703 cells embedded in paraffin were deparaffinized, immunohistochemically stained with the SmgGDS antibody, and counterstained with hematoxylin using the

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