Group I p21-activated kinases (PAKs) promote tumor cell proliferation and survival through the AKT1 and Raf-MAPK pathways

Abstract

Group I p21-activated kinases (PAK) are important effectors of the small GTPases Rac and Cdc42, which regulate cell motility/migration, survival, proliferation, and gene transcription. Hyperactivation of these kinases have been reported in many tumor types, making PAKs attractive targets for therapeutic intervention. PAKs are activated by growth factor-mediated signaling and are negatively regulated by the tumor suppressor neurofibromatosis type 2 (NF2)/Merlin. Thus, tumors characterized by NF2 inactivation would be expected to show hyperactivated PAK signaling. On the basis of this rationale, we evaluated the status of PAK signaling in malignant mesothelioma, an aggressive neoplasm that is resistant to current therapies and shows frequent inactivation of NF2. We show that group I PAKs are activated in most mesotheliomas and mesothelioma cell lines and that genetic or pharmacologic inhibition of PAKs is sufficient to inhibit mesothelioma cell proliferation and survival. We also identify downstream effectors and signaling pathways that may contribute mechanistically to PAK-related tumorigenesis. Specifically, we show that inhibition of PAK results in attenuation of AKT and Raf-MAPK signaling and decreased tumor cell viability. Collectively, these data suggest that pharmacologic inhibition of group I PAKs may have therapeutic efficacy in tumors characterized by PAK activation.

Figure 1

PAKs are activated in MM specimens and cell lines. A) Immunohistochemical-staining of human MM and normal lung pleura with a phospho-specific antibody against Ser141 of PAK1/2/3 (P-PAK1/2/3). B) Immunohistochemical staining of asbestos-induced MMs from wild-type and Nf2+/− mice using P-PAK1/2/ antibody. In figure 1B, NF2 should be mouse designation Nf2. C) Immunoblot analysis of P-PAK1/2/3, PAK1, and total PAK and β-actin in human MM cell lines (top panel) and P-PAK1/2/3, PAK PAK1/2/3 and β-actin MM cell lines from asbestos-treated wild-type and Nf2+/− mice (bottom panel). D) 2-dimensional immunoblot analysis of PAK1, PAK1/2/3 and P-PAK1/2/3 levels in human MM cell lines.

Figure 2

Dominant-negative PID expression inhibits cell viability and PAK phosphorylation in human MM cell lines. A) Immunoblot analysis of P-PAK1/2/3, total PAK1/2/3 and β-actin levels 72-hr post-nucleofection of pcDNA or pcDNA-PID in Me12 and Meso 22 cells. B) MTS cell viability assay 72-hr post-nucleofection of pcDNA or pcDNA-PID in Me12 and Meso 22 cells. C) G418-resistent colonies 2-weeks post-nucleofection of pcDNA or pcDNA-PID in Me12 and Meso 22 cells.

Figure 3

Stable knockdown of PAK1 or PAK2 inhibits MM cell proliferation. A) Immunoblot analysis demonstrating knockdown of PAK1 (top panel) and PAK2 (middle panel, bottom band). B) Plot of cell number (×10⁴) over time (days) for ME12 and Meso 22 cells infected with lentivirus expressing shGFP (control), shPAK1A, shPAK1B, shPAK2A, and shPAK2B. C) Clonogenic assay demonstrating colony formation in ME12 and Meso 22 cells infected with lentivirus expressing shGFP (control), shPAK1A, shPAK1B, shPAK2A and shPAK2B.

Figure 4

A selective group I PAK inhibitor (IPA-3) inhibits PAK phosphorylation and MM cell viability, proliferation and survival. A) MTS cell viability assay of Me12 and Meso 22 cells 72-hrs post treatment with varying concentrations of IPA-3. B) Immunoblot analysis of P-PAK1/2/3, total PAK and actin in protein lysates of Me12 and Meso 22 cells 72-hrs post-IPA-3 treatment. C) 2-dimensional immunoblot analysis of PAK1 and PAK1/2/3 in Me12 and Meso 22 after treatment for 24 hr with IPA-3. Arrows represent different phosphorylation states of PAKs. D) Cell cycle analysis of propidium iodide-stained Me12 and Meso 22 cells treated with DMSO (−) or IPA-3 (+) for 24 hr. E) Apoptosis as determined by the Cell Death Detection Elisa^PLUS Kit (Roche Applied Biosciences) in Me12 and Meso 22 cells treated with DMSO or IPA-3 for 48 hr.

Figure 4

A selective group I PAK inhibitor (IPA-3) inhibits PAK phosphorylation and MM cell viability, proliferation and survival. A) MTS cell viability assay of Me12 and Meso 22 cells 72-hrs post treatment with varying concentrations of IPA-3. B) Immunoblot analysis of P-PAK1/2/3, total PAK and actin in protein lysates of Me12 and Meso 22 cells 72-hrs post-IPA-3 treatment. C) 2-dimensional immunoblot analysis of PAK1 and PAK1/2/3 in Me12 and Meso 22 after treatment for 24 hr with IPA-3. Arrows represent different phosphorylation states of PAKs. D) Cell cycle analysis of propidium iodide-stained Me12 and Meso 22 cells treated with DMSO (−) or IPA-3 (+) for 24 hr. E) Apoptosis as determined by the Cell Death Detection Elisa^PLUS Kit (Roche Applied Biosciences) in Me12 and Meso 22 cells treated with DMSO or IPA-3 for 48 hr.

Figure 5

Identification of AKT and MAPK signaling as potentially important effector pathways in regulated tumor cell viability. A) Immunoblot analysis of P-PAK1/2/3, total PAK1/2/3, P-AKT, total AKT, P-ERK1/2, and β-actin in Me12 and Meso 22 treated with DMSO or IPA-3 for 24 hr. B) G418-resistent colonies 2-weeks post-nucleofection of pcDNA, pcDNA-PID, pcDNA-PID + pcDNA-HA-myr-AKT1, or pcDNA-PID + myc-Raf1(BXB) in Me12 and Meso 22 cells. C) MTS assay of ME12 or Meso 22 cells 72 hours post-treatment with the following drugs or drug combinations: DMSO (D), 25 uM IPA-3 (I), 15 uM LY294002 (L), 20 uM PD98509 (P) and various combinations labeled with the aforementioned letters. D) Schematic model of PAK involvement in MM cell survival and proliferation through effectors AKT1 and Raf1 and the inhibitors used to target each pathway.

Figure 5

Identification of AKT and MAPK signaling as potentially important effector pathways in regulated tumor cell viability. A) Immunoblot analysis of P-PAK1/2/3, total PAK1/2/3, P-AKT, total AKT, P-ERK1/2, and β-actin in Me12 and Meso 22 treated with DMSO or IPA-3 for 24 hr. B) G418-resistent colonies 2-weeks post-nucleofection of pcDNA, pcDNA-PID, pcDNA-PID + pcDNA-HA-myr-AKT1, or pcDNA-PID + myc-Raf1(BXB) in Me12 and Meso 22 cells. C) MTS assay of ME12 or Meso 22 cells 72 hours post-treatment with the following drugs or drug combinations: DMSO (D), 25 uM IPA-3 (I), 15 uM LY294002 (L), 20 uM PD98509 (P) and various combinations labeled with the aforementioned letters. D) Schematic model of PAK involvement in MM cell survival and proliferation through effectors AKT1 and Raf1 and the inhibitors used to target each pathway.