Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity

Journal of Translational Medicine

Seyedhossein Aharinejad¹  Dietmar Abraham¹  Trevor Lucas¹  Sandun Gunawardhana¹  Karin Zins¹ 

[1] Laboratory for Molecular Cellular Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Waehringerstrasse 13, A-1090 Vienna, Austria

关键词: Xenograft model;    Cancer therapy;    Cdc42;    Rho GTPases;    Small molecule inhibitor;    Colon cancer;   

Others  :  824453
DOI  :  10.1186/1479-5876-11-295

 received in 2013-06-12, accepted in 2013-11-22,  发布年份 2013

【 摘 要 】

Background

Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential.

Methods

We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197.

Results

In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo.

Conclusions

These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth.

【 授权许可】

   
2013 Zins et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Fritz G, Kaina B: Rho GTPases: promising cellular targets for novel anticancer drugs. Curr Cancer Drug Targets 2006, 6:1-14.
• [2]Walker K, Olson MF: Targeting Ras and Rho GTPases as opportunities for cancer therapeutics. Curr Opin Genet Dev 2005, 15:62-68.
• [3]Karlsson R, Pedersen ED, Wang Z, Brakebusch C: Rho GTPase function in tumorigenesis. Biochim Biophys Acta 2009, 1796:91-98.
• [4]Vega FM, Ridley AJ: Rho GTPases in cancer cell biology. FEBS Lett 2008, 582:2093-2101.
• [5]Onesto C, Shutes A, Picard V, Schweighoffer F, Der CJ: Characterization of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases. Methods Enzymol 2008, 439:111-129.
• [6]Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y: Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci USA 2004, 101:7618-7623.
• [7]Mardilovich K, Olson MF, Baugh M: Targeting Rho GTPase signaling for cancer therapy. Future Oncol 2012, 8:165-177.
• [8]Gomez del Pulgar T, Bandres E, Espina C, Valdes-Mora F, Perez-Palacios R, Garcia-Amigot F, Garcia-Foncillas J, Lacal JC: Differential expression of Rac1 identifies its target genes and its contribution to progression of colorectal cancer. Int J Biochem Cell Biol 2007, 39:2289-2302.
• [9]Espina C, Cespedes MV, Garcia-Cabezas MA, Gomez del Pulgar MT, Boluda A, Oroz LG, Benitah SA, Cejas P, Nistal M, Mangues R, Lacal JC: A critical role for Rac1 in tumor progression of human colorectal adenocarcinoma cells. Am J Pathol 2008, 172:156-166.
• [10]Fritz G, Just I, Kaina B: Rho GTPases are over-expressed in human tumors. Int J Cancer 1999, 81:682-687.
• [11]Cleverley SC, Costello PS, Henning SW, Cantrell DA: Loss of Rho function in the thymus is accompanied by the development of thymic lymphoma. Oncogene 2000, 19:13-20.
• [12]Gomez del Pulgar T, Benitah SA, Valeron PF, Espina C, Lacal JC: Rho GTPase expression in tumourigenesis: evidence for a significant link. Bioessays 2005, 27:602-613.
• [13]Braga VM, Yap AS: The challenges of abundance: epithelial junctions and small GTPase signalling. Curr Opin Cell Biol 2005, 17:466-474.
• [14]van Hengel J, D’Hooge P, Hooghe B, Wu X, Libbrecht L, De Vos R, Quondamatteo F, Klempt M, Brakebusch C, van Roy F: Continuous cell injury promotes hepatic tumorigenesis in cdc42-deficient mouse liver. Gastroenterology 2008, 134:781-792.
• [15]Stengel K, Zheng Y: Cdc42 in oncogenic transformation, invasion, and tumorigenesis. Cell Signal 2011, 23:1415-1423.
• [16]Ridley AJ: Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol 2006, 16:522-529.
• [17]Li LH, Zheng MH, Luo Q, Ye Q, Feng B, Lu AG, Wang ML, Chen XH, Su LP, Liu BY: P21-activated protein kinase 1 induces colorectal cancer metastasis involving ERK activation and phosphorylation of FAK at Ser-910. Int J Oncol 2010, 37:951-962.
• [18]Gomez Del Pulgar T, Valdes-Mora F, Bandres E, Perez-Palacios R, Espina C, Cejas P, Garcia-Cabezas MA, Nistal M, Casado E, Gonzalez-Baron M, et al.: Cdc42 is highly expressed in colorectal adenocarcinoma and downregulates ID4 through an epigenetic mechanism. Int J Oncol 2008, 33:185-193.
• [19]Ferri N, Corsini A, Bottino P, Clerici F, Contini A: Virtual screening approach for the identification of new Rac1 inhibitors. J Med Chem 2009, 52:4087-4090.
• [20]Zins K, Abraham D, Sioud M, Aharinejad S: Colon cancer cell-derived tumor necrosis factor-alpha mediates the tumor growth-promoting response in macrophages by up-regulating the colony-stimulating factor-1 pathway. Cancer Res 2007, 67:1038-1045.
• [21]Aharinejad S, Abraham D, Paulus P, Abri H, Hofmann M, Grossschmidt K, Schafer R, Stanley ER, Hofbauer R: Colony-stimulating factor-1 antisense treatment suppresses growth of human tumor xenografts in mice. Cancer Res 2002, 62:5317-5324.
• [22]Aharinejad S, Paulus P, Sioud M, Hofmann M, Zins K, Schafer R, Stanley ER, Abraham D: Colony-stimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice. Cancer Res 2004, 64:5378-5384.
• [23]Shi HY, Stafford LJ, Liu Z, Liu M, Zhang M: Maspin controls mammary tumor cell migration through inhibiting Rac1 and Cdc42, but not the RhoA GTPase. Cell Motil Cytoskeleton 2007, 64:338-346.
• [24]Nobes CD, Hall A: Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995, 81:53-62.
• [25]Ye DZ, Field J: PAK signaling in cancer. Cell Logist 2012, 2:105-116.
• [26]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, 276:17347-17353.
• [27]Huynh N, Liu KH, Baldwin GS, He H: P21-activated kinase 1 stimulates colon cancer cell growth and migration/invasion via ERK- and AKT-dependent pathways. Biochim Biophys Acta 1803, 2010:1106-1113.
• [28]Nheu T, He H, Hirokawa Y, Walker F, Wood J, Maruta H: PAK is essential for RAS-induced upregulation of cyclin D1 during the G1 to S transition. Cell Cycle 2004, 3:71-74.
• [29]McCormick F: Success and failure on the ras pathway. Cancer Biol Ther 2007, 6:1654-1659.
• [30]Young A, Lyons J, Miller AL, Phan VT, Alarcon IR, McCormick F: Ras signaling and therapies. Adv Cancer Res 2009, 102:1-17.
• [31]Bokemeyer C, Bondarenko I, Makhson A, Hartmann JT, Aparicio J, de Braud F, Donea S, Ludwig H, Schuch G, Stroh C, et al.: Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol 2009, 27:663-671.
• [32]de Toledo M, Anguille C, Roger L, Roux P, Gadea G: Cooperative anti-invasive effect of Cdc42/Rac1 activation and ROCK inhibition in SW620 colorectal cancer cells with elevated blebbing activity. PLoS One 2012, 7:e48344.
• [33]Ebi H, Corcoran RB, Singh A, Chen Z, Song Y, Lifshits E, Ryan DP, Meyerhardt JA, Benes C, Settleman J, et al.: Receptor tyrosine kinases exert dominant control over PI3K signaling in human KRAS mutant colorectal cancers. J Clin Invest 2011, 121:4311-4321.
• [34]Surviladze Z, Waller A, Strouse JJ, Bologa C, Ursu O, Salas V, Parkinson JF, Phillips GK, Romero E, Wandinger-Ness A, et al.: A Potent and Selective Inhibitor of Cdc42 GTPase. Bethesda (MD): Probe Reports from the NIH Molecular Libraries Program; 2010.
• [35]Pelish HE, Peterson JR, Salvarezza SB, Rodriguez-Boulan E, Chen JL, Stamnes M, Macia E, Feng Y, Shair MD, Kirchhausen T: Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro. Nat Chem Biol 2006, 2:39-46.
• [36]Friesland A, Zhao Y, Chen YH, Wang L, Zhou H, Lu Q: Small molecule targeting Cdc42-intersectin interaction disrupts Golgi organization and suppresses cell motility. Proc Natl Acad Sci USA 2013, 110:1261-1266.
• [37]Li XR, Ji F, Ouyang J, Wu W, Qian LY, Yang KY: Overexpression of RhoA is associated with poor prognosis in hepatocellular carcinoma. Eur J Surg Oncol 2006, 32:1130-1134.
• [38]Huang M, Prendergast GC: RhoB in cancer suppression. Histol Histopathol 2006, 21:213-218.
• [39]Gao L, Bai L, Nan Q: Activation of Rho GTPase Cdc42 promotes adhesion and invasion in colorectal cancer cells. Med Sci Monit Basic Res 2013, 19:201-207.
• [40]Gadea G, Sanz-Moreno V, Self A, Godi A, Marshall CJ: DOCK10-mediated Cdc42 activation is necessary for amoeboid invasion of melanoma cells. Curr Biol 2008, 18:1456-1465.
• [41]Kodama A, Takaishi K, Nakano K, Nishioka H, Takai Y: Involvement of Cdc42 small G protein in cell-cell adhesion, migration and morphology of MDCK cells. Oncogene 1999, 18:3996-4006.
• [42]Zhao ZS, Manser E: PAK family kinases: physiological roles and regulation. Cell Logist 2012, 2:59-68.
• [43]Carter JH, Douglass LE, Deddens JA, Colligan BM, Bhatt TR, Pemberton JO, Konicek S, Hom J, Marshall M, Graff JR: Pak-1 expression increases with progression of colorectal carcinomas to metastasis. Clin Cancer Res 2004, 10:3448-3456.
• [44]Qing H, Gong W, Che Y, Wang X, Peng L, Liang Y, Wang W, Deng Q, Zhang H, Jiang B: PAK1-dependent MAPK pathway activation is required for colorectal cancer cell proliferation. Tumour Biol 2012, 33:985-994.