Modulation of p21-activated kinase 1 alters the behavior of renal cell carcinoma
Gerald C. O'Sullivan
Cork Cancer Research Center, Leslie C. Quick Jnr. Cancer Laboratories, Bioscience Institute and Mercy University Hospital, Cork, Ireland
Search for more papers by this authorMark Tangney
Cork Cancer Research Center, Leslie C. Quick Jnr. Cancer Laboratories, Bioscience Institute and Mercy University Hospital, Cork, Ireland
Search for more papers by this authorGarret Casey
Cork Cancer Research Center, Leslie C. Quick Jnr. Cancer Laboratories, Bioscience Institute and Mercy University Hospital, Cork, Ireland
Search for more papers by this authorMonica Ambrose
Department of Pharmacology and Therapeutics, Cork University Hospital and National University of Ireland, Cork, Ireland
Search for more papers by this authorAileen Houston
Department of Medicine, Cork University Hospital and National University of Ireland, Cork, Ireland
Search for more papers by this authorCorresponding Author
Orla P. Barry
Cork Cancer Research Center, Leslie C. Quick Jnr. Cancer Laboratories, Bioscience Institute and Mercy University Hospital, Cork, Ireland
Department of Pharmacology and Therapeutics, Cork University Hospital and National University of Ireland, Cork, Ireland
Fax: +353-21-4343211.
Department of Pharmacology and Therapeutics, Clinical Science Building, Cork University Hospital, Wilton Road, Cork, IrelandSearch for more papers by this authorGerald C. O'Sullivan
Cork Cancer Research Center, Leslie C. Quick Jnr. Cancer Laboratories, Bioscience Institute and Mercy University Hospital, Cork, Ireland
Search for more papers by this authorMark Tangney
Cork Cancer Research Center, Leslie C. Quick Jnr. Cancer Laboratories, Bioscience Institute and Mercy University Hospital, Cork, Ireland
Search for more papers by this authorGarret Casey
Cork Cancer Research Center, Leslie C. Quick Jnr. Cancer Laboratories, Bioscience Institute and Mercy University Hospital, Cork, Ireland
Search for more papers by this authorMonica Ambrose
Department of Pharmacology and Therapeutics, Cork University Hospital and National University of Ireland, Cork, Ireland
Search for more papers by this authorAileen Houston
Department of Medicine, Cork University Hospital and National University of Ireland, Cork, Ireland
Search for more papers by this authorCorresponding Author
Orla P. Barry
Cork Cancer Research Center, Leslie C. Quick Jnr. Cancer Laboratories, Bioscience Institute and Mercy University Hospital, Cork, Ireland
Department of Pharmacology and Therapeutics, Cork University Hospital and National University of Ireland, Cork, Ireland
Fax: +353-21-4343211.
Department of Pharmacology and Therapeutics, Clinical Science Building, Cork University Hospital, Wilton Road, Cork, IrelandSearch for more papers by this authorAbstract
The p21-activated kinase 1 (Pak1) is a serine/threonine kinase whose activity is regulated by both Rho GTPases and AGC kinase family members. It plays a role in cytoskeletal remodeling and cell motility as well as cell proliferation, angiogenesis, tumorigenesis and metastasis. An involvement of Pak1 in renal cell carcinoma (RCC), which remains highly refractory to chemotherapy and radiotherapy, remains to be investigated. Pak1 expression, phosphorylation and kinase activity were examined in RCC cell lines and human tissue from normal and renal carcinoma. We report increased Pak1 expression and constitutive activity in the membrane and nucleus but not the cytoplasm of resected human RCC. To study a role for Pak1 in RCC, we developed 786-0 clones that expressed either a kinase-active Pak1L83,L86 2 different Pak1 dominant negative mutants, Pak1R299 and Pak1L83,L86,R299 or Pak1 siRNA. The expression of Pak1L83,L86 increased 786-0 proliferation, motility and anchorage independent growth, while the dominant negative mutants and Pak1 siRNA abrogated these effects. In addition, Pak1L83,L86 conferred resistance to 5-fluorouracil with a 40% ± 10% increase in cell viability. Conversely, Pak1L83,L86,R299, Pak1R299 and Pak1 siRNA conferred sensitivity with a 65.2% ± 5.5%, 69.2% ± 3.3% and 73.0% ± 8.4% loss in viability, respectively. Finally, Pak1 plays a role in renal tumor growth in vivo. Only 33% of mice developed tumors in the Pak1L83,L86,R299 group and no tumors developed from Pak1R299 cell challenge. Together these findings point to Pak1 as an exciting target for therapy of renal cancer, which remains highly refractory to existing treatments. © 2007 Wiley-Liss, Inc.
References
- 1 Amato RJ. Renal cell carcinoma: review of novel single-agent therapeutics and combination regimens. Ann Oncol 2005; 16: 7–15.
- 2 Motzer RJ, Bacik J, Murphy BA, Russo P, Mazumdar M. Interferon-α as a comparative treatment for clinical trials of new therapies against advanced renal cell carcinoma. J Clin Oncol 2002; 20: 289–96.
- 3 Ratain MJ, Eisen T, Stadler WM, Flaherty KT, Kaye SB, Rosner Gl, Gore M, Desai AA, Patnaik A, Xiong HQ, Rowinsky E, Abbruzzese JL, et al. Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma. J Clin Oncol 2006; 24: 2505–12.
- 4 Motzer RJ, Michaelson MD, Redman BG, Hudes GR, Wilding G, Figlin RA, Ginsberg MS, Kim ST, Baum CM, DePrimo SE, Li JZ, Bello CL, et al. Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma. J Clin Oncol 2006; 24: 16–24.
- 5 Hampton T. Trials probe new agents for kidney cancer. JAMA 2006; 296: 155–7.
- 6 Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ, Topalian SL, Steinberg SM, Chen HX, Rosenberg SA. A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 2003; 349: 427–34.
- 7 Pasche B. A new strategy in the war on renal cell cancer. JAMA 2006; 295: 2537–8.
- 8 Rosenberg SA, Yang JC, Topalian SL, Schwartzentruber DJ, Weber JS, Parkinson DR, Seipp CA, Einhorn JH, White DE. Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin 2. JAMA 1994; 271: 907–13.
- 9 Mira J-P, Benard V, Groffen J, Sanders LC, Knaus UG. Endogenous hyperactive Rac3 controls proliferation of breast cancer by p21-activated kinase-dependent pathway. Proc Natl Acad Sci USA 2000; 97: 185–9.
- 10 Kumar R, Vadlamudi RK. Emerging functions of p21-activated kinases in human cancer cells. J Cell Physiol 2002; 193: 133–44.
- 11 Schurmann A, Mooney AF, Sanders LC, Sells MA, Wang HG, Reed JC, Bokoch GM. P21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis. Mol Cell Biol 2000; 20: 453–61.
- 12 Kumar R, Gururaj AE, Barnes CJ. P21-activated kinases in cancer. Nat Rev Cancer 2006; 6: 459–71.
- 13 Narumiya S, Ishizaki T, Watanabe N. Rho effectors and reorganization of actin cytoskeleton. Rho effectors and reorganization of actin cytoskeleton. FEBS Lett 1997; 410: 68–72.
- 14 Menard RE, Mattingly RR. Cell surface receptors activate p21-activated kinase 1 via multiple Ras and PI3-kinase-dependent pathways. Cell Signal 2003; 15: 1099–109.
- 15 Menard RE, Mattingly RR. Gβγ subunits stimulate p21-activated kinase 1 (PAK1) through activation of PI3-kinase and Akt but act independently of Rac1/Cdc42. FEBS Lett 2004; 556: 187–92.
- 16 Zhou GL, Zhuo Y, King CC, Fryer BH, Bokoch GM, Field J. Akt phosphorylation of serine 21 on Pak1 modulates Nck binding and cell migration. Mol Cell Biol 2003; 23: 8058–69.
- 17 King CC, Gardiner EM, Zenke FT, Bohl BP, Newton AC, Hemmings BA, Bokoch GM. p21-activated kinase (PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1 (PDK1). J Biol Chem 2000; 275: 41201–9.
- 18 Howe AK, Juliano RL. Regulation of anchorage-dependent signal transduction by protein kinase A and p21-activated kinase. Nat Cell Biol 2000; 2: 593–600.
- 19 Fryer BH, Wang C, Vedantam A, Zhou GL, Jin S, Fletcher L, Simon MC, Field J. cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak)1, inhibiting Pak/Nck binding and stimulating pak/vasodilator-stimulated phosphoprotein association. J Biol Chem 2006; 281: 11487–95.
- 20 Chong C, Tan L, Lim L, Manser E. The mechanism of Pak activation. Autophosphorylation events in both regulatory and kinase domains control activity. J Biol Chem 2001; 276: 17347–53.
- 21 Balasenthil S, Sahin AA, Barnes CJ, Wang RA, Pestell RG, Vadlamudi RK, Kumar R. p21-activated kinase-1 signaling mediates cyclin D1 expression in mammary epithelial and cancer cells. J Biol Chem 2004; 279: 1422–8.
- 22 Wang R-A, Zhang H, Balasenthil S, Medina D, Kumar R. Pak1 hyperactivation is sufficient for mammary gland tumor formation. Oncogene 2006; 25: 2931–6.
- 23 Schraml P, Schwerdtfeger G, Burkhalter F, Raggi A, Schmidt D, Ruffalo T, King W, Wilber K, Mihatsch MJ, Moch H. Combined array comparative genomic hybridization and tissue microarray analysis suggest PAK1 at 11q13.5-q14 as a critical oncogene target in ovarian carcinoma. Am J Pathol 2003; 163: 985–92.
- 24 Knowles HJ, Phillips RM. Identification of differentially expressed genes in experimental models of the tumor microenvironment using differential display. Anticancer Res 2001; 21: 2305–11.
- 25 Zhou Gl, Tucjer DF, Bae SS, Bhatheja K, Birnbaum MJ, Field J. Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration. J Biol Chem 2006; 281: 36443–53.
- 26 Gustafson B, Smith U. Cytokines promote Wnt signaling and inflammation and impair the normal differentiation and lipid accumulation in 3T3-L1 preadipocytes. J Biol Chem 2006; 281: 9507–16.
- 27 Katner AL, Gootam P, Hoang QB, Gnarra JR, Rayford W. A recombinant adenovirus expressing p27(Kip1) induces cell cycle arrest and apoptosis in human 786-0 renal carcinoma cells. J Urol 2002; 168: 766–73.
- 28 Barry OP, Mullan B, Sheehan D, Kazanietz MG, Shanahan F, Collins JK, O' Sullivan GC. Constitutive ERK1/2 activation in esophagogastric rib bone marrow micrometastatic cells is MEK-independent. J Biol Chem 2001; 276: 15537–46.
- 29 Holm C, Rayala S, Jirström, Stål O, Kumar R, Landberg G. Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients. J Natl Cancer Inst 2006; 98: 671–80.
- 30 Ambrose M, Ryan A, O'Sullivan GC, Dunne C, Barry OP. Induction of apoptosis in renal cell carcinoma by reactive oxygen species: involvement of ERK1/2, p38{δ}/{γ}, COX-2 downregulation and AIF translocation. Mol Pharmacol 2006; 69: 1879–90.
- 31 Cox AD, Der CJ. Biological assays for cell transformation. Methods Enzymol 1994; 238: 277–94.
- 32 Zenke FT, King CC, Bohl BP, Bokach GM. Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity. J Biol Chem 1999; 274: 32565–73.
- 33 Tang Y, Marwaha S, Rutkowski JL, Tennekoon GI, Phillips PC, Field J. A role for Pak protein kinases in Schwann cell transformation. Proc Natl Acad Sci USA 1998; 98: 5139–44.
- 34 Xiao GH, Beeser A, Chernoff A, Testa JR. p21-Activated kinase links Rac/Cdc42 signaling to merlin. J Biol Chem 2002; 277: 883–6.
- 35 Jaffer AM, Chernoff J. p21-Activated kinases: three more join the Pak. Int J Biochem Cell Biol 2002; 34: 713–17.
- 36 Vadlamudi RK, Adam L, Wang RA, Mandal M, Nguyen D, Sahin A, Chernoff J, Hung MC, Kumar R. Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells. J Biol Chem 2000; 275: 36238–44.
- 37
Natali PG,
Prat M,
Nicotra MR,
Bigotti A,
Olivero M,
Comoglio PM,
Di Renzo MF.
Overexpression of the met/HGF receptor in renal cell carcinomas.
Int J Cancer
1996;
69:
212–17.
10.1002/(SICI)1097-0215(19960621)69:3<212::AID-IJC11>3.0.CO;2-9 CAS PubMed Web of Science® Google Scholar
- 38 Otani N, Tsukamoto T, Masumori N, Saiki I, Yoneda J, Kumamoto Y. Influence of growth factors on in vitro invasiveness and type IV collagenolysis of human renal cell carcinoma cells. J Urol 1994; 151: 223–6.
- 39 Koochekpour S, Jeffers M, Wang PH, Gong C, Taylor GA, Roessler LM, Stearman R, Vasselli JR, Stetler-Stevenson WG, Kaelin WGJr, Linehan WM, Klausner RD, et al. The von Hippel-Lindau tumor suppressor gene inhibits hepatocyte growth factor/scatter factor-induced invasion and branching morphogenesis in renal carcinoma cells. Mol Cell Biol 1999; 19: 5902–12.
- 40 Vadlamudi RK, Bagheri-Yarmand R, Yang Z, Balasenthil S, Nguyen D, Sahin AA, den Hollander P, Kumar R. Dynein light chain 1, a p21-activated kinase 1-interacting substrate, promotes cancerous phenotypes. Cancer Cell 2004; 5: 575–85.
- 41 Shenghao J, Zhuo Y, Guo W, Field J. p21-Activated kinase 1 (Pak1)-dependent phosphorylation of Raf-1 regulates its mitochondrial localization, phosphorylation of BAD, and Bcl-2 association. J Biol Chem 2005; 26: 24698–705.
- 42 Mizutani Y, Wada H, Yoshida O, Fukushima M, Nonomura M, Nakao M, Miki T. Significance of thymidylate synthase activity in renal cell carcinoma. Clin Cancer Res 2003; 9: 1453–60.
- 43 Beasley D, Schwartz JH, Brenner BM. Interleukin 1 induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells. J Clin Invest 1991; 87: 602–8.
- 44 Schmidt MJ, Sawyer BD, Truex LL, Marshall WS, Fleisch JH. LY83583: an agent that lowers intracellular levels of cyclic guanosine 3′, 5′-monophosphate. J Pharmacol Exp Ther 1985; 232: 764–9.
- 45 Liou LS, Shi T, Duan ZH, Sadhukhan P, Der SD, Novick AA, Hissong J, Skacel M, Almasan A, DiDonato JA. Microarray gene expression profiling and analysis in renal cell carcinoma. BMC Urol 2004; 4: 1–11.
- 46 Kopper L, Timar J. Genomics of renal cell cancer—does it provide breakthrough? Pathol Oncol Res 2006; 12: 5–11.
- 47 Pavey S, Zuidervaart W, van Nieuwpoort F, Packer L, Jager M, Gruis N, Hayward N. Increased p21-activated kinase-1 expression is associated with invasive potential in uveal melanoma. Melanoma Res 2006; 16: 285–96.
- 48 Stofega MR, Sanders LC, Gardiner EM, Bokoch GM. Constitutive p21-activated kinase (Pak) activation in breast cells as a result of mislocalization of Pak to focal adhesions. Mol Biol Cell 2004; 15: 2965–77.
- 49 Bokoch GM, Wang Y, Bohl BP, Sells MA, Quilliam LA, Knaus UG. Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J Biol Chem 1996; 271: 25746–9.
- 50 Puto LA, Pestonjamasp K, King CC, Bokoch GM. p21-activated kinase 1 (PAK1) interacts with the Grb2 adapter protein to couple to growth factor signaling. J Biol Chem 2003; 287: 9388–93.
- 51 Stoletov KV, Ratcliffe KE, Terman BI. Fibroblast growth factor receptor substrate 2 participates in vascular endothelial growth factor-induced signaling. FASEB J 2002; 16: 1283–5.
- 52 Blancher C, Moore JW, Robertson N, Harris AL. Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1α, HIF-2α, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3′-kinase/Akt signaling pathway. Cancer Res 2001; 61: 7349–55.
- 53 Freyer BH, Field J. Rho, Rac, Pak and angiogenesis: old roles and newly identified responsibilities in endothelial cells. Cancer Lett 2005; 229: 13–23.
- 54 Rini Bl, Halabi S, Taylor J, Small EJ, Schilsky RL. Cancer and Leukemia Group B 90206: a randomized phase III trial of interferon-α or interferon-α plus anti-vascular endothelial growth factor antibody (bevacizumab) in metastatic renal cell carcinoma. Clin Cancer Res 2004; 10: 2584–6.
- 55
Kish JA,
Wolf M,
Crawford ED,
Leimert JT,
Bueschen A,
Neefe JR,
Flanigan RC.
Evaluation of low dose continuous infusion 5-fluorouracil in patients with advanced and recurrent renal cell carcinoma. A Southwest Oncology Group Study.
Cancer
1994;
74:
916–19.
10.1002/1097-0142(19940801)74:3<916::AID-CNCR2820740319>3.0.CO;2-Z CAS PubMed Web of Science® Google Scholar
- 56 Zaniboni A, Simoncini E, Marpicati P, Montini E, Ferrarri V, Marini G. Phase II trial of 5-fluorouracil and high-dose folinic acid in advanced renal cell cancer. J Chemother 1989; 1: 350–1.
- 57 Singh RR, Song C, Yang Z, Kumar R. Nuclear localization and chromatin targets of p21-activated kinase 1. J Biol Chem 2005; 280: 18130–7.
- 58 Frost JA, Steen H, Shapiro P, Lewis T, Ahn N, Shaw PE, Cobb MH. Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins. EMBO J 1997; 16: 6426–38.
- 59 Tang Y, Chen Z, Ambrose D, Liu J, Gibbs JB, Chernoff J, Field J. Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts. Mol Cell Biol 1997; 17: 4454–64.
- 60 Weber DS, Taniyama Y, Rocic P, Seshiah PN, Dechert MA, Gerthoffer WT. Phosphoinositide-dependent kinase 1 and p21-activated protein kinase mediate reactive oxygen species-dependent regulation of platelet-derived growth factor-induced smooth muscle cell migration. Circ Res 2004; 94: 1219–26.
Citing Literature
