As p21-activated kinase 1 (Pak1) has been shown to regulate both these signaling pathways and is itself upregulated in many human cancers, we assessed the role of Pak1 in Ras-driven skin cancer. Akt activity. Tumor regression was also seen in mice treated with a specific Mek inhibitor, but not with an Akt inhibitor. These findings establish Pak1 as a new target in – represent one of the most important oncogene families in human cancer, with activating mutations seen in approximately 30% of solid tumors (1). Ras proteins act as switch molecules by transmitting mitogenic signals in response CGB to variety of extracellular stimuli by binding and hydrolyzing GTP, as well as regulating diverse cellular processes such as proliferation, migration, senescence, differentiation, and survival. In human cancer, activating mutations in promote cell proliferation and result in tumorigenesis that generally correlates with poor prognosis and poor therapeutic response (2). Since the oncogenic role of the Ras protein is well-established, numerous attempts have been made to target this GTPase for the treatment of human cancers. Strategies for blocking activated Ras have included attempts to reduce its expression, interfere with its subcellular localization, and inhibit its downstream effectors (3, 4). With regard to the latter, more than twenty proteins have been reported as effectors of Ras, and many of these provide potentially suitable drug targets (5, 6). The phosphoinositol-3 kinase (PI3K)/Akt/mTOR and Raf/Erk signaling modules are among the best-studied Ras effector pathways. A growing body of evidence indicates that 6-OAU members of the p21-activated kinase (Pak) family, in particular Pak1, are required for the activation of both these pathways. Paks are serine-threonine-specific protein kinases that act downstream of the small GTPases Cdc42 and Rac in a variety of signaling pathways (7C9). Mammalian cells encode six Pak isoforms – group A (Pak1, -2, and -3) and group B (Pak4, -5, and -6) C with partly overlapping but also clearly distinct signaling properties (10). In Erk signaling, Pak1 phosphorylates c-Raf at S338 and Mek1 at S298, sites that are required for full activation of these proteins in some cell types (10, 11). In the Akt pathway, Pak1 is thought to act in a non-catalytic fashion, acting as a scaffold to bridge PDK1 to Akt (12, 13). Inhibition or loss of Pak1 might therefore be expected to interfere with the oncogenic potential of proteins such as Ras that induce transformation at least in part by activation of these pathways. A wealth of data support this 6-OAU view, as expression of dominant negative alleles of expression by RNAi, and small molecule inhibitors of Pak1 have all been shown to interfere with expression levels are associated with more aggressive grades and poorer differentiation of squamous cell carcinoma (SCC). Functional data were then obtained by crossing an inducible driven mouse model of skin cancer to wild-type, heterozygous, or knock-out mice. In such mice, we found that gene dosage was positively correlated with tumor initiation and progression. mice lacking showed marked reduction in both Erk and Akt activation, indicating that Pak1 function is required for activation of these signaling pathways by mice were treated with either of two distinct small molecule Pak inhibitors (PF03758309 or FRAX597) or a Mek inhibitor, but not with an Akt inhibitor. These findings establish Pak1 as 6-OAU a new target in Kknockout mice (FVB-N) (17) separately to generate and colonies. Progeny from these colonies were subsequently bred to generate K5-mice that were wild-type, heterozygous, or knockout for tumors. (ACC) Immunoblot analyses of Erk and Akt-mTOR signaling pathways from tumor lysates. Tumors were excised from newly sacrificed animals and protein lysates obtained and probed with the indicated antibodies. As tumors in mice were usually small (50 mm3 or less), equal sized tumors were also used for analysis of signaling from mice. (D) Papillomas (P) or large ( 75 mm3) carcinomas (C) from mice were excised and analyzed by immunoblot with the indicated antibodies. c-Raf was immunoprecipitated prior to analysis by immunoblot. Numbers at the top of each column indicate specific individual mice used for these experiments. Tissue preparation, histology, immunohistochemistry, and immunoblotting All tumor lesions, control tissues and internal organs were fixed overnight in 4% paraformaldehyde, dehydrated and embedded in paraffin. Hematoxylin and eosin (H&E) stained sections were used for diagnostic purposes and unstained sections for immunohistochemical (IHC) studies. IHC was performed with the following antibodies: rabbit polyclonal antibody for Pak1 (1:50), Pak2 (1:50), phospho-Erk1/2 (pThr202/pTyr204) (1:100), phospho-Akt (pThr308) (1:30), phospho-S6 (pSer235/p236) (1:5000), anti-cleaved caspase.