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Validation of the p21-Activated Kinases as Targets for Inhibition in Neurofibromatosis Type 2

¹ Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania; ² Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts; and ³ Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts

Requests for reprints: Joseph L. Kissil, Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: 215-898-3874; Fax: 215-898-3792; E-mail: jkissil{at}wistar.org.

	Abstract

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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. [Cancer Res 2008;68(19):7932–7]

	Introduction

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The hallmark of neurofibromatosis type 2 (NF2), which affects 1 in 30,000 to 40,000 of the population, is the development of bilateral vestibular schwannomas (VS) at the eighth cranial nerve. Most NF2 patients go on to develop multiple cranial and spinal schwannomas, meningiomas, and less frequently, intraspinal ependymomas, with symptoms including auditory, visual, and motor impairments. Currently, risky surgical excision is the only treatment option, and the tumors will inevitably recur, leading to a high early mortality rate among NF2 patients (1).

Merlin, the protein encoded by the NF2 gene, is closely related to the ERM (ezrin, radixin, and moesin) proteins, containing an NH₂-terminal FERM domain followed by a coiled-coil domain. The carboxyl-terminal domain (CTD) of merlin is unique and lacks the conventional actin-binding motif found in ERM proteins. Merlin CTD can associate with its NH₂-terminal FERM domain, forming a "closed" and functionally active configuration. Phosphorylation of merlin at serine 518 disrupts this intramolecular interaction and suppresses its growth inhibitory activity. This phosphorylation is mediated by p21-activated kinases (Pak), which bind to activated (GTP bound) Rac/Cdc42 and function as their direct effectors (2, 3). In addition, it has been shown that merlin S518 can also be phosphorylated by the cyclic AMP-dependent protein kinase (PKA), whereas its dephosphorylation involves the myosin phosphatase MYPT-1-PP1 (4, 5).

Several lines of evidence indicate that merlin is not only regulated by the Rac-Pak axis but also functions as an inhibitor of Rac signaling. Specifically, merlin overexpression suppresses Rac1-induced activation of c-Jun-NH₂-terminal kinase and activator protein–1 transcriptional activity, whereas loss of merlin results in the opposite (6). In addition, merlin inhibition via dominant-negative mutants or siRNA enables Rac1 recruitment to plasma membrane and alleviates contact inhibition in confluent cells (7). Furthermore, increased levels of activated Rac with disrupted temporal control have been reported in primary NF2-mutant schwannoma cells (8, 9). Finally, merlin overexpression in Schwann cells suppresses Pak1 activity, whereas ablation of merlin in MEFs leads to Pak1 activation (7, 10, 11). The inhibitory activity of merlin toward Pak1 has been attributed to its direct interaction with the Rac-binding domain of Pak1, which interferes with the binding of activated Rac to Pak1 (10, 12).

In this study, we examine whether inhibiting Pak can reverse the transformed and cancer phenotypes that arise from NF2 deficiency. We find that Pak1 is hyperactive in primary schwannomas isolated from NF2 patients and that suppression of the Paks via shRNAs reduces the ability of NF2 mutant cells to grow in vitro and form tumors in a xenograft model of NF2.

	Materials and Methods

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Schwannoma biopsies. Frozen tissue samples from NF2 patients that underwent surgery in Massachusetts General Hospital (MGH), were received from the MGH Brain Tumor tissue repository, under the approval of the MGH Institutional Review Board.

Two-dimensional gel analysis. Frozen tumor samples were minced and protein was extracted in sample buffer (9.8 mol/L Urea, 2% CHAPS, 5 µl/mL 0.5% IPG buffer pH 4–7, DTT 15 mg/mL). All subsequent steps were performed as previously described (10).

Cell culture conditions. RT4 and NIH3T3 cells were grown in low-glucose DME, 10% FCS, 1x nonessential amino acids, and 100 IU/mL penicillin-streptomycin. Transfections were performed with Lipofectamine2000 (Invitrogen), following manufacturer's instructions. To inhibit DNA methylation, 5-aza-2'-deoxycytidine (5-aza; Sigma) was added to the media (5 ng/mL).

Knockdown of Pak1, 2, and 3 using lentiviral delivered shRNAs. For silencing endogenous Pak1, 2, and 3 in NIH3T3 and RT4 cells, several sequences were tested and the following targeting sequences were selected based on their knockdown efficiency: Pak1, "5'-GGATTCTGTGCACAGATAA" and "5'-GGAATGGATGGCTCTGTCA"; Pak2, "5'-GAGATTATGGAGAAATTAA"; Pak3, "5'-GGAATTAATTATTAATGAA." DNA oligos containing individual targeting sequences were cloned into pLenti-lox3.7 (pLL3.7) as previously described (13). The resulting vectors were cotransfected with packaging vectors VSVG and 8.9 into 293T cells and viral supernatant was collected 48 and 72 h posttransfection. For infection, cells were incubated with the viral supernatant overnight and infected cells [green fluorescent protein (GFP) positive] were selected by flow cytometry.

Western blot analysis. Protein extracts were prepared with radioimmunoprecipitation assay buffer [50 mmol/L TRIS-HCl (pH 7.5), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L sodium orthovanadate, and 1 mmol/L NaF]. The following antibodies were used according to manufactures' instructions: Pak1 (N-20), Pak3 (N-19), merlin (C-18; Santa Cruz); Pak2 (2608; Cell Signaling); and -tubulin (AC42) and β-actin (AC40; Sigma).

Cell proliferation assays. Thirty thousand cells per well were plated in 12-well dishes in triplicate for each cell line. At indicated time points, cells from individual wells were trypsinized and counted using a Coulter counter (Z1 series; Beckman Coulter). Cells were fed every 3 d throughout the assay. Statistical analysis was performed using Student's t test.

Tumorigenicity assays. Cells were trypsinized, washed, and resuspended at 10⁷ cells/mL in PBS. Cells (10⁶; 100 µL) were injected s.c. into the flanks of 5-wk-old nude mice (BALB/c nu/nu; The Jackson Laboratory). Mice were monitored and tumor diameters were measured weekly after injections.

Bisulfite sequencing. Genomic DNA was first digested with Sac I, precipitated, and dissolved in double-distilled water (ddH₂O). One microliter of 6N NaOH was added to 19 µL ddH₂O containing 2 µg of the digested DNA, and incubated at 37°C for 15'. Subsequently, 120 µL bisulfite conversion solution (107 µL 4.04M NaHSO₃, 7 µL 10 mmol/L Hydroquinone, and 6 µL 6N NaOH) was added to the DNA solution, and bisulfite conversion was carried out (30" at 95°C followed by 15' at 50°C for 15 cycles). Bisulfite-treated DNA was then desalted (Wizard DNA cleanup kit; Promega) and eluted in 50 µL TE buffer. 6N NaOH (2.5 µL) was added to the eluted DNA and incubated at RT for 5'. DNA was precipitated and resuspended in 30 µL ddH₂O. One microliter was used as template in a 20 µL nested PCR reaction with the following primers: "5'-TTTAAAAGAAAAGGGGGGATT" and "5'-TCTCAAAACACACAATTACT." One microliter of the first round was used as template for a second round of PCR using the nested primers: "5'-AGGGGAAAGAATAGTAGATATAA" and "5'-TTAACCRAAACAAAAAATAACC." The resulting PCR product was purified and sequenced using primer "5'-AGGGGAAAGAATAGTAGATATAA."

Senescence-associated β-Gal assay. Cells were washed thrice with PBS and fixed with 0.2% glutaraldehyde for 5'. After three washes with PBS, cells were incubated overnight with X-gal solution [1 mg/mL X-gal, 150 mmol/L NaCl, 2 mmol/L MgCl₂, 5 mmol/L K₃Fe(CN)₆, 5 mmol/L K₄Fe(CN)₆, and 40 mmol/L NaPi (pH 6.0) at 37°C].

	Results

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Elevated levels of Pak1 activity in primary schwannoma tumor samples. The activation of Pak1 involves its phosphorylation at multiple sites (10, 14, 15). We have previously established a two-dimensional gel approach to monitor the phosphorylation state of Pak1, which has been shown to accurately reflect the activation state of Pak1 in the cell (7, 10). By use of this approach, we examined the status of Pak1 in primary schwannoma samples isolated from NF2 patients. Myelinated nerve from a patient with wild-type NF2 was used as control. As shown in Fig. 1A , Pak1 migrates as a single spot toward the neutral pH range in "normal" myelinated nerve, indicating that Pak1 is mostly unphosophorylated/inactive. In contrast, with the exception of sample xt3224, 18 of the 19 schwannoma samples we analyzed displayed various forms of phosphorylated Pak1 (Fig. 1A and B), indicative of Pak1 activation. In 6 of the 19 samples, as exemplified by sample xt1441, the majority of the Pak1 proteins existed in highly acidic forms, suggestive of hyper-phosphorylation (Fig. 1A and B). In 12 of the 19 samples, multiple forms of hypophosphorylated or hyperphosphorylated Pak1 were present, as represented by sample xt1729. These data suggest that in primary tumors from NF2 patients, Pak1 exists in a phosphorylated/activated form. This is consistent with previous findings that merlin acts as a direct inhibitor of Pak1 (7, 10).

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Pak1 is activated in schwannomas from NF2 patients. A, analysis of Pak1 activation by two-dimensional gel electrophoresis coupled to Western blot analysis. Arrowheads, 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.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Pak1 knockdown on NIH3T3 cells is insufficient to inhibit cell transformation due to impaired NF2 function. NIH3T3 (WT) or NIH3T3/NF2BBA (NF2BBA) cells were infected with empty lentiviruses (Lenti) or lentiviruses harboring Pak1shRNAs (Lenti-Pak1sh). A, Pak1 expression levels were determined by Western blot analysis. Actin was used as an internal standard. B, cellular proliferation kinetics. Cells (3 x 104) 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 three independent experiments. Points, mean; bars, SD. C, tumor formation capability. Cells (106) were injected into the rear flanks of severe combined immunodeficient mice. Four mice were used for each cell line. Tumors were measured 15 d after injections.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   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 or NIH3T3/NF2BBA cells infected with empty lentivirus or lentiviruses harboring Pak1+2+3 shRNAs. A, Western blot analysis of Pak1, 2, and 3 levels. Actin was used as an internal standard. B, assessment of cellular proliferation. Cells (3 x 104) 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 are representative of three independent experiments. Points, mean; bars, SD. C, tumor size distribution in severe combined immunodeficient mice. Tumor formation was assessed by injecting 5 x 105 NIH3T3/NF2BBA cells infected with empty virus (Lenti) into the left flanks and 5 x 105 of NIH3T3/NF2BBA-Lenti-PAK1-3sh into the right flanks of the same mice. Five mice were used in total. The size of tumors was measured 23 d after injection. Horizontal lines, average tumor diameters. D, Western blot analysis of tumors dissected from mice #7007, #7045, and #7049. L, tumors from left flank arising from NIH3T3/NF2BBA-Lenti cells; R, tumors from right flank arising from NIH3T3/NF2BBA/Lenti-PAK1-3sh cells.

To investigate what caused the eventual development of small tumors from injected NIH3T3/NF2^BBA cells expressing Pak1-3 shRNAs in some of the mice, we isolated the tumors from both flanks of these mice and examined the expression levels of Pak1, 2, and 3. As shown in Fig. 3D, in two of the mice examined (#7007 and #7049), the expression of at least two of the Paks was partially restored to similar levels of those in tumors from control NIH3T3/NF2^BBA cells. This may explain why these cells did eventually form tumors. In a third mouse (#7045), the levels of all three Paks remained low in the tumor developed from Pak-shRNA expressing NIH3T3/NF2^BBA cells (Fig. 3D), indicating that additional mechanisms involved in offsetting the loss of Pak in these cells.

Schwannoma cells are dependent on Pak1 and silence Pak1 shRNA expression via de novo methylation. Because NF2 patients primarily develop Schwann cell tumors, we next examined the effect of Pak knockdown in a well-characterized rat schwannnoma cell line, RT4. Unlike NIH3T3 cells in which all three Paks are expressed, RT4 cells express predominantly Pak1 (Supplementary Fig. S2). We infected RT4 cells with lentiviruses carrying the same Pak1-specific shRNAs that were used in NIH3T3 cells (Figs. 2 and 3), and as expected, RT4 cells showed significantly reduced Pak1 expression after the infection (Fig. 4A ). However, the Pak1 levels in RT4 cells infected with Pak1 shRNAs were quickly restored, within two to three passages, to similar levels found in control RT4 cells (Fig. 4A). Correspondingly, the expression level of GFP, a positive selection marker in the lentiviral vector, pLenti-lox3.7, into which Pak1shRNAs were cloned, dropped rapidly during the first two to three passages of the infected RT4 cells (Fig. 4A).

View larger version (62K): [in this window] [in a new window] [Download PPT slide]   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 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 after infection with lentiviruses harboring a Pak1-specific shRNA and subjected to bisulfite conversion, methylation-specific PCRs, and direct sequencing. *, cytosines in the original sequence that have been completely or partially converted to uracils; arrowheads, 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. Cells (4 x 104) were plated in triplicate into 12-well plates and cells were harvested and counted at day 0, 1, 2, 3, and 4. The data are representative of three independent experiments. Points, mean; bars, SD. E, images of RT4 cells infected with vector or Pak1shRNA after 3 d 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 x10 magnification.

We then measured the growth rates of RT4 cells carrying empty vectors or Pak1 shRNAs in the presence or absence of 5-aza. In the absence of 5-aza, RT4 cells infected with Pak1 shRNAs showed only slightly reduced growth rate compared with control RT4 cells (Fig. 4D). In the presence of 5-aza, control RT4 cells grew relatively slower than untreated cells (Fig. 4D). This is not surprising, considering that 5-aza is a global methylation inhibitor. In sharp contrast, however, RT4 cells carrying Pak1 shRNAs completely failed to grow under culture conditions that included 5-aza (Fig. 4D). The difference is also obvious in the morphologies of these cells. Compared with the mostly spindle-shaped untreated or 5-aza–treated control RT4 cells, the Pak1-shRNA–infected RT4 cells grown in the presence of 5-aza are enlarged and flattened, displaying a morphology consistent with cellular senescence (Fig. 4E). These cells also stained positive for the senescence-associated expression of β-galactosidase activity, indicating that sustained expression of Pak1shRNAs in RT4 cells leads to inhibition of Pak1 expression, resulting in cellular senescence (Fig. 4E). Importantly, RT4 cells transiently transfected with Pak1 siRNAs, which should not be susceptible to methylation-mediated silencing, did not proliferate during the first few days post transfection (Supplementary Fig. S2A). Consistent with the notion that Pak1 is the dominant Pak in RT4 cells, cotransfection of Pak2 siRNAs with Pak1 siRNAs failed to further reduce the growth rate of RT4 cells (Supplementary Fig. S2B). Taken together, we concluded that RT4 cells require Pak1 for proliferation and quickly move to silence Pak1 shRNA expression through de novo methylation.

	Discussion

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We have shown for the first time that Pak1 is highly activated in primary schwannomas from NF2 patients. This is consistent with our hypothesis that merlin functions as a tumor suppressor by blocking Pak activation. Employing both cellular and animal models, we directly tested this hypothesis. We found that the simultaneous knockdown of all three Paks is required to counter the hyperproliferation and tumor-forming capacity of NIH3T3 cells harboring the NF2^BBA allele. Taken together, these results validate Paks as potential targets for the development of NF2 therapeutics.

Previous studies have linked Pak1 to cancer and transformation and use of Pak inhibitors to reverse cellular transformation have also been reported (17–19). However, these studies are inconclusive, as the inhibitors used are broad-range kinase inhibitors that do not specifically inhibit Paks. Therefore, the important next step is to develop specific Pak inhibitors and test them in cellular and animal models of NF2. Another interesting finding in this study is that different cell types respond differently to Pak1 inhibition. Although knockdown of Pak1 is inconsequential in NIH3T3 cells (Fig. 2), Pak1 ablation in RT4 cells leads to growth arrest (Fig. 4D; Supplementary Fig. S2). In addition, NIH3T3 cells maintained reduced Pak1 expression mediated by Pak1 shRNAs even after over 20 passages (data not shown), whereas RT4 cells quickly silenced Pak1 shRNA via de novo methylation (Fig. 4A and B). These differences suggest that although Pak1 is probably the dominant Pak in RT4 cells, it functions redundantly with Pak2 and Pak3 in NIH3T3 cells. However, because Pak1, 2, and 3 are highly homologous, especially within their catalytic domains, it is most likely that any Pak inhibitors will work equally well on all three Pak isoforms. Therefore, these cell type differences in Pak function should not affect the effectiveness of Pak inhibitors.

Finally, virus-delivered shRNA have been proposed as a potential therapeutic tool in treating human diseases such as cancer (20). As described in this study, an shRNA targeting an essential gene, in our case, Pak1 in RT4 cells, can be silenced directly by DNA methylation. Further research should be carried out to investigate whether other cell types can also use similar mechanisms to silence stably integrated foreign shRNA and how frequently such events can occur. Addressing these questions would be prudent before any future application of such technologies as a treatment modality.

	Disclosure of Potential Conflicts of Interest

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No potential conflicts of interest were disclosed.

	Acknowledgments

 
Grant support: Department of Defense Neurofibromatosis Research Program. C. Yi is a recipient of Ruth L. Kirschstein NRSA fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Joanne Thorvaldsen for advice on bisulfite sequencing.

	Footnotes

 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 3/ 7/08. Revised 7/14/08. Accepted 7/22/08.

	References

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This Article Abstract Full Text (PDF) Supplementary Data Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yi, C. Articles by Kissil, J. L. Search for Related Content PubMed PubMed Citation Articles by Yi, C. Articles by Kissil, J. L.