Validation of the p21-activated kinases as targets for inhibition in neurofibromatosis type 2

Abstract

Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate Rac1 signaling by inhibiting its downstream effector kinases, the p21-activated kinases (Pak). Given the implication of Paks in tumorigenesis, it is plausible that merlin's tumor suppressive function might be mediated, at least in part, via inhibition of the Paks. We present data indicating this is indeed the case. First, analysis of primary schwannoma samples derived from NF2 patients showed that in a significant fraction of the tumors, the activity of Pak1 was highly elevated. Second, we used shRNAs to knockdown Pak1, 2, and 3 in NIH3T3 cells expressing a dominant-negative form of merlin, NF2(BBA) (NIH3T3/NF2(BBA)), and find that simultaneous knockdown of Pak1-3 in these cells significantly reduced their growth rates in vitro and inhibited their ability to form tumors in vivo. Finally, while attempting to silence Pak1 in rat schwannoma cells, we found that these cells were unable to tolerate long-term Pak1 inhibition and rapidly moved to restore Pak1 levels by shutting down Pak1 shRNA expression through a methylation-dependent mechanism. These data suggest that inhibiting Pak could be a beneficial approach for the development of therapeutics toward NF2. In addition, the finding that the shRNA-mediated Pak1 suppression was silenced rapidly by methylation raises questions about the future application of such technologies for the treatment of diseases such as cancer.

Figure 1. Pak1 is activated in schwannomas from NF2 patients

A, analysis of Pak1 activation by 2-D gel electrophoresis coupled to western blot analysis. Arrowheads indicate the various phosphorylated forms of Pak1. B, summary of Pak1 phosphorylation status in primary schwannomas. All samples were normalized for variations in sample loading by comparison to actin as an internal standard.

Figure 2. Pak1 knockdown on NIH3T3 cells is insufficient to inhibit cell transformation due to impaired NF2 function

NIH3T3 (WT) or NIH3T3/NF2^BBA (NF2^BBA) cells were infected with empty lentiviruses (Lenti) or lentiviruses harboring Pak1 shRNAs (Lenti-Pak1sh). A, Pak1 expression levels were determined by western blot analysis. Actin was used as an internal standard. B, cellular proliferation kinetics. 3×10⁴ cells from each of the cell types above were plated in triplicate into 12-well plates. Cells were harvested and counted at day 0, 1, 2, 3 and 4. The data are representative of 3 independent experiments. Error bars show standard deviation (SD). C, tumor formation capability. 10⁶ cells were injected into the rear flanks of SCID mice. Four mice were used for each cell line. Tumors were measured 15 days after injections.

Figure 3. Knockdown of Pak1–3 is sufficient to inhibit cellular transformation due to impaired NF2 function

The effects of simultaneous Pak1+2+3 knockdown were examined in NIH3T3 (WT) or NIH3T3/NF2^BBA (NF2^BBA) cells infected with empty lentivirus (Lenti) or lentiviruses harboring Pak1+2+3 shRNAs (Lenti-pak1-3sh). A, western blot analysis of Pak1, 2 and 3 levels. Actin was used as an internal standard. B, assessment of cellular proliferation. 3×10⁴ cells were plated in triplicate into 12-well plates. Cells were harvested and counted at day 0, 1, 2, 3, 4, 5, and 6. The data is representative of 3 independent experiments. Error bars show standard deviation (SD). C, tumor size distribution in SCID mice. Tumor formation was assessed by injecting 5×10⁵ NIH3T3/NF2^BBA cells infected with empty virus (Lenti) to the left flanks and 5×10⁵ of NIH3T3/NF2^BBA -Lenti-PAK1-3sh into the right flanks of the same mice. Five mice were used in total. The size of tumors was measured 23 days after injection. Horizontal lines represent average tumor diameters. D, western blot analysis of tumors dissected from mice #7007, #7045, and #7049. L: tumors from left flank arising from NIH3T3/NF2^BBA-Lenti cells R: tumors from right flank arising from NIH3T3/NF2^BBA -Lenti-PAK1-3sh cells.

Figure 4. Rat schwannoma cells are dependent on Pak1 expression

A, western blot analysis of Pak1 expression in RT4 cells at various passages after infection with lentiviruses harboring a Pak1 specific shRNA. RT4 cells infected with empty lentivirus vector (Lenti) were used as control. Actin serves as an internal loading control. B, methylation analysis of Lenti-shPak1 promoter in RT4 cells. The genomic DNA was isolated from RT4 cells at passage 6 following infection with lentiviruses harboring a Pak1 specific shRNA and subjected to bisulphite conversion, methylation specific PCRs and direct sequencing. “*”s mark the cytosines in the original sequence that have been completely or partially converted to uracils, whereas arrowheads indicate unconverted/methylated cytosines. C, western blot analysis of Pak1 expression in RT4 cells infected with empty lenti vector (−) or Pak1shRNA (+) treated with 5-aza. The 5-aza was added to the culture medium at 5 ng/ml final concentration. The cells were harvested and analyzed at day 0, 1, 2, and 3 into the treatment. D, assessment of cellular proliferation. 4×10⁴ cells were plated in triplicate into 12-well plates and cells were harvested and counted at day 0, 1, 2, 3 and 4. The data is representative of 3 independent experiments. Error bars show standard deviation (SD). E, images of RT4 cells infected with vector or Pak1shRNA after 3 days of culture in the presence (+5-aza) or absence (−5-aza) of 5-aza. In the bottom panel, RT4 cells were first fixed and stained with X-gal. Images were taken using Nikon Coolpix 995 digital camera under Leica DM1L light microscope at 10x magnification.