p21-Activated kinase 1 is required for efficient tumor formation and progression in a Ras-mediated skin cancer model

Abstract

The RAS genes are the most commonly mutated oncogenes in human cancer and present a particular therapeutic dilemma, as direct targeting of Ras proteins by small molecules has proved difficult. Signaling pathways downstream of Ras, in particular Raf/Mek/Erk and PI3K/Akt/mTOR, are dominated by lipid and protein kinases that provide attractive alternate targets in Ras-driven tumors. 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. In human squamous cell carcinoma (SCC), we found a strong positive correlation between advanced stage and grade and PAK1 expression. Using a mouse model of Kras-driven SCC, we showed that deletion of the mouse Pak1 gene led to markedly decreased tumorigenesis and progression, accompanied by near total loss of Erk and Akt activity. Treatment of Kras(G12D) mice with either of two distinct small molecule Pak inhibitors (PF3758309 and FRAX597) caused tumor regression and loss of Erk and 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 KRAS-driven tumors and suggest a mechanism of action through the Erk, but not the Akt, signaling pathway.

Figure 1

Pak1 immunohistochemistry in human skin cancer. (A) Pak1 immunohistochemistry showing basal expression in keratinocytes and Langerhans cells in normal human skin. (B) In a basal cell carcinoma and a (C) poorly differentiated SCC, diffuse cytoplasmic Pak1 expression is observed. Nuclear Pak1 expression is also seen. (D) In well-differentiated SCC, Pak1 expression is either negative or restricted to keratinocytes adjacent to the stroma.

Figure 2

Effect of Pak1 on Kras-driven tumorigenesis. (A) The effect of doxycycline administration on Kras, Pak1, and Pak2 protein levels in single transgenic (K5-tTR or tet-Kras) and doubly transgenic mice (K5-tTR::tet-Kras), fed a control or a doxycycline-containing diet. Kras, Pak1, and Pak2 expression in skin was analyzed by immunoblot. Numerals represent relative expression to GAPDH. (B) Kaplan-Meier tumor-free survival curves showing effects on latency of tumor formation in Pak1^−/− mice versus Pak1^+/+ and Pak1^+/− mice. (C) Median days until onset of tumor initiation, (D) total number of tumors and (E) tumor volume. Abbreviations: SCC, squamous cell carcinoma; ND, not detected. * Median treatment days to detect tumor.

Figure 3

Effect of Pak1 gene dosage on histology and proliferative capacity of Ras-driven SCC. (A) Tumors in Pak1^+/+, Pak1^+/− and Pak1^−/− mice stained with H&E and Ki-67. Note reduced Ki67-positive staining cells in tumors of Pak1^−/− mice. Magnifications ×10 and ×40 for H&E staining and Ki-67 respectively. Scale bar =100 µm. (B) Histogram showing Ki-67 labeling index (i.e. percentage of positively stained cells) of tumors from Pak1^+/+, Pak1^+/− and Pak1^−/− mice. (C) Percentage of mice with papillomas or SCC in K5-tTR::tet-Kras animals.

Figure 4

Erk and Akt-mTOR signaling in Pak1^+/+, Pak1^+/−, and Pak1^−/− tumors. (A–C) 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 Pak1^−/− mice were usually small (50 mm³ or less), equal sized tumors were also used for analysis of signaling from Pak1^+/+, and Pak1^+/− mice. (D) Papillomas (P) or large (>75 mm³) carcinomas (C) from Pak1^+/+::K5-tTR::tet-Kras^G12D, Pak1^+/−::K5-tTR::tet-Kras^G12D, or Pak1^−/−::K5-tTR::tet-Kras^G12D 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.

Figure 5

Tumor regression in mice treated with Pak inhibitors. (A) Specificity of Pak inhibitors. 1 µM FRAX597 or PF3758309 was tested for inhibitory activity against a panel of >300 protein kinases (Invitrogen). Inhibition of >50% is represented by yellow spheres, and >75% by red spheres, mapped onto a kinome tree diagram. Inset shows protein kinase family groups plotted on tree diagram. Unique and common targets of the two kinase inhibitors (>75% inhibition) are shown on a Venn diagram. (B) Structure of Pak inhibitor FRAX-597. (C–F) Volumetric changes in tumor size between untreated mice (vehicle) and mice treated with inhibitors for seven (FRAX597) or ten (PF03758309, PD0325901, GSK690693) days. (G–J) Immunoblot analysis of tumor lysates from animals treated with inhibitors or vehicle.