P21-activated Kinase 4 Expression Research Articles

P21-Activated Kinase 4 (PAK4) as a Predictive Marker of Gemcitabine Sensitivity in Pancreatic Cancer Cell Lines.

Purposep21-activated kinases (PAKs) are involved in cytoskeletal reorganization, gene transcription, cell proliferation and survival, and oncogenic transformation. Therefore, we hypothesized that PAK expression levels could predict the sensitivity of pancreatic cancer cells to gemcitabine treatment, and PAKs could be therapeutic targets.Materials and MethodsCell viability inhibition by gemcitabine was evaluated in human pancreatic cancer cell lines (Capan-1, Capan-2, MIA PaCa-2, PANC-1, Aspc-1, SNU-213, and SNU-410). Protein expression and mRNA of molecules was detected by immunoblot analysis and reverse transcription polymerase chain reaction. To define the function of PAK4, PAK4 was controlled using PAK4 siRNA.ResultsCapan-2, PANC-1, and SNU-410 cells were resistant to gemcitabine treatment. Immunoblot analysis of signaling molecules reported to indicate gemcitabine sensitivity showed higher expression of PAK4 and lower expression of human equilibrative nucleoside transporter 1 (hENT1), a well-known predictive marker for gemcitabine activity, in the resistant cell lines. Knockdown of PAK4 using siRNA induced the upregulation of hENT1. In resistant cell lines (Capan-2, PANC-1, and SNU-410), knockdown of PAK4 by siRNA resulted in restoration of sensitivity to gemcitabine.ConclusionPAK4 could be a predictive marker of gemcitabine sensitivity and a potential therapeutic target to increase gemcitabine sensitivity in pancreatic cancer.

MicroRNA-433 inhibits cell proliferation in hepatocellular carcinoma by targeting p21 activated kinase (PAK4).

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. P21-activated kinase 4 (PAK4) has been identified as an oncogenic protein in a variety of cancers. However, the contribution and regulation of PAK4 in HCC remain poorly understood. In the present study, we found that inhibition of PAK4 expression by specific shRNA significantly attenuated HCC cell proliferation. Moreover, we show that microRNA-433 (miRNA-433) could directly target PAK4 through the miRNA-433 binding sequence at the 3'-UTR of PAK4 mRNA, and inhibit PAK4 protein expression. We further show that miRNA-433 expression was downregulated in HCC tissues and cell culture as well, which inversely correlated with PAK4 expression levels. Overexpression of miRNA-433 significantly suppressed the proliferation of HepG2 cells, while this effect was partially rescued by forced expression of PAK4 through restoring PI3K/AKT signaling in HepG2 cells. These findings will shed light on the roles and mechanisms of miRNA-433 in regulating HCC proliferation, and may benefit future development of therapeutics targeting miRNA-433 and PAK4 in HCC.

PAK6 increase chemoresistance and is a prognostic marker for stage II and III colon cancer patients undergoing 5-FU based chemotherapy.

p21-Activated kinase 6 (PAK6) has been implicated in radiotherapy and docetaxel resistance. We have further evaluated PAK6 as a predictor of 5-fluorouracil (5-FU) treatment response in colon cancer. Here we report that in colon cancer PAK6 promotes tumor progression and chemoresistance both in vitro and in vivo. In the clinical analysis, PAK6 was overexpressed in 104 of 147 (70.75%) stage II and III patients who received 5-FU based chemotherapy after surgery. Multivariate Cox regression analysis indicated that PAK6 was an independent prognostic factor for overall survival (P < 0.001) and disease-free survival (P < 0.001). Colon cancer cell lines showed increased PAK6 expression upon 5-FU treatment. In PAK6-knockdown cells treated with 5-FU, cell viability and phosphorylation of BAD decreased, and the number of apoptotic cells, levels of cleaved caspase 3 and PARP increased compared to control cells. The opposite was observed in PAK6 overexpressing cells. Short hairpin RNA knockdown of PAK6 blocked cells in G2-M phase. Furthermore, Animal experiments results in vivo are consistent with outcomes in vitro. This study demonstrates that PAK6 is an independent prognostic factor for adjuvant 5-FU-based chemotherapy in patients with stage II and stage III colon cancer.

Abstract 912: P21-activated kinase 4 (PAK4) as a predictive marker for gemcitabine in pancreatic cancer cell line

Abstract Pancreatic cancer is a highly lethal disease. Although gemcitabine is activelyused in pancreatic cancer, modest activity and relativeresistanceare revealed.P21-activated kinases(PAKs) are involved in cytoskeletal reorganization, gene transcription, cellproliferation and survival, and oncogenic transformation.The gene amplification and protein overexpression of PAK1 and PAK4were reported in pancreatic cancer, which were associated with K-ras mutation, MUC13 and Smad4. Therefore, we hypothesized that PAKs could predict the drug-sensitivity of gemcitabine in pancreatic cancer and could be a therapeutic target. Using cell viability assay of human pancreatic cancer cell lines (Capan1, Capan2, MIA-paca2, PANC1, Aspc1, SNU213, SNU410) for gemcitabine, Capan-2, PANC-1 and SNU-410 cell lines was identifiedas resistant cell lines for gemcitabine. Through western blots of molecules related with down-steam pathway and drug-sensitivity, higher expression of PAK4 and lower expression of human equilibrative nucleoside transporter 1 (hENT1) were associated with the resistance for gemcitabine. PAK4 down-regulation through siRNA induced the up-regulation of hENT1, which is well-known as predictive marker for gemcitabine activity. In resistance cell lines (Capan-2, PANC-1, SNU-410), down-regulation of PAK4 through siRNArestored the drug-sensitivity for gemcitabine. In conclusion, PAK4 can be a potential predictive marker and therapeutic target in gemcitabine chemotherapy for pancreatic cancer. Citation Format: Sung Ung Moon, Jin Won Kim, Ji Hea Sung, Mi Hyun Kang, Hyun Chang, Jeong Ok Lee, Yu Jung Kim, Keun Wook Lee, Jee Hyun Kim, Soo Mee Bang, Jong Seok Lee. P21-activated kinase 4 (PAK4) as a predictive marker for gemcitabine in pancreatic cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 912. doi:10.1158/1538-7445.AM2014-912

386P - Preclinical Evaluation of Novel Pak4 Allosteric Modulators Against Triple Negative Breast Cancer

ABSTRACT Aim: Triple Negative Breast Cancer (TNBC) is characterized by a lack of receptors for estrogen (ER), progesterone (PR), and hormone epidermal growth factor 2 (HER-2). TNBC occurs in ∼20% of diagnosed breast cancers. Hormone targeted therapies are ineffective thus making TNBC more aggressive and difficult to treat. Studies have shown that P21-Activated Kinase 4 (PAK4) is elevated in TNBC. PAK4 regulates cell survival, cell division and apoptosis. PAK4 overexpression in mammary epithelial cells leads to abnormal acini formation and drives tumorigenensis in mice. Methods: Tissue microarrays were used to evaluate PAK4 expression in normal and cancerous breast tissue. We identified and tested selective, orally bioavailable, small molecule PAK4 allosteric modulators (PAMs) against breast cancer cell lines. MTS assay was used to determine effects on proliferation and viability of 30 breast cancer (BC) cell lines including 13 TNBCs. mRNA was analyzed using the Genome Atlas (TCGA) and deep sequencing. Immunoblots were used to measure protein steady state levels and phosphorylation. TNBC xenograft models were used to test efficacy. Results: PAMs demonstrated anti-tumor activity against BC cell lines (IC50 0.04- > 10 µM) of which TNBC were the most sensitive: ∼80% of TNBC lines had IC50 Conclusions: Oral PAMs inactivate PAK4 by directly inducing PAK4 destabilization. This represents a novel mechanism of the protein kinase inactivation involving degradation of PAK4 rather than direct inhibition of the kinase activity. Oral PAMs have potent anti-tumor activity against BC cells including TNBC both in vitro and in vivo where they induce tumor cell growth arrest and apoptosis with excellent tolerability. Based on this anti-tumor activity and high tolerability, PAMs show promise for the treatment of TNBC. Disclosure: W. Senapedis, Y. Landesman, J. Ellis, R. Carlson and E. Baloglu: I am a current employee of Karyopharm Therapeutics, Inc.; O. Kalid: I currently am a consultant with Karyopharm Therapeutics, Inc.; D. McCauley: I am a current employee of Karyopharm Therapeutics, Inc. I own stock in Karyopharm Therapeutics, Inc.; S. Shacham: I am a current employee of Karyopharm Therapeutics, Inc. I am a stock holder of Karyopharm Therapeutics, Inc. I am on the scientific advisory board of Karyopharm Therapeutics, Inc.

Prognostic Significance of p21-activated Kinase 6 Expression in Patients with Clear Cell Renal Cell Carcinoma

To investigate prognostic values of intratumoral p21-activated kinase 6 (PAK6) expression in patients with clear cell renal cell carcinoma (ccRCC). Immunohistochemistry in tissue microarray was used to evaluate the expression of PAK6 in 104 patients who had undergone nephrectomy at Zhongshan Hospital of Fudan University. Prognostic value and clinical outcomes were evaluated. Intratumoral PAK6 expression was significantly lower than nontumoral tissues (P < 0.001). Moreover, low expression of PAK6 was associated with unfavorable overall survival (OS) (P = 0.001) and recurrence-free survival (RFS) (P = 0.001). In the subgroup of patients with tumor, node, metastasis classification system (TNM) stage I + II disease, those with low expression of PAK6 were prone to have poor OS (P = 0.014) and RFS (P = 0.037). Intratumoral PAK6 low expression was correlated with tumor size (P = 0.001) and TNM stage (P = 0.006), and was identified as an independent prognostic factor for OS (hazard ratio 4.109, P = 0.002) and RFS (hazard ratio 3.175, P = 0.002). Use of an extended TNM staging system with intratumoral PAK6 expression showed a better prognostic value for OS [area under the receiver operating characteristic curve (AUC) 0.790, P = 0.022] and RFS (AUC 0.769, P = 0.040). The c-index of a Cox-based model combined with Eastern Cooperative Oncology Group performance status, TNM stage, Fuhrman grade, and PAK6 was 0.83 for OS and 0.80 for RFS. Intratumoral PAK6 could be a potential prognosticator for OS and RFS in ccRCC patients after nephrectomy. Further external validation and functional analysis should be pursued to assess its potential prognostic and therapeutic values for ccRCC patients.

PAK4 confers cisplatin resistance in gastric cancer cells via PI3K/Akt- and MEK/ERK-dependent pathways.

CDDP [cisplatin or cis-diamminedichloroplatinum(II)] and CDDP-based combination chemotherapy have been confirmed effective against gastric cancer. However, CDDP efficiency is limited because of development of drug resistance. In this study, we found that PAK4 (p21-activated kinase 4) expression and activity were elevated in gastric cancer cells with acquired CDDP resistance (AGS/CDDP and MKN-45/CDDP) compared with their parental cells. Inhibition of PAK4 or knockdown of PAK4 expression by specific siRNA (small interfering RNA)-sensitized CDDP-resistant cells to CDDP and overcome CDDP resistance. Combination treatment of LY294002 [the inhibitor of PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B or PKB) pathway] or PD98509 {the inhibitor of MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] pathway} with PF-3758309 (the PAK4 inhibitor) resulted in increased CDDP efficacy compared with LY294002 or PD98509 alone. However, after the concomitant treatment of LY294002 and PD98509, PF-3758309 administration exerted no additional enhancement of CDDP cytotoxicity in CDDP-resistant cells. Inhibition of PAK4 by PF-3758309 could significantly suppress MEK/ERK and PI3K/Akt signalling in CDDP-resistant cells. Furthermore, inhibition of PI3K/Akt pathway while not MEK/ERK pathway could inhibit PAK4 activity in these cells. The in vivo results were similar with those of in vitro. In conclusion, these results indicate that PAK4 confers CDDP resistance via the activation of MEK/ERK and PI3K/Akt pathways. PAK4 and PI3K/Akt pathways can reciprocally activate each other. Therefore, PAK4 may be a potential target for overcoming CDDP resistance in gastric cancer.

Oncogenic PAK4 regulates Smad2/3 axis involving gastric tumorigenesis

The alteration of p21-activated kinase 4 (PAK4) and transforming growth factor-beta (TGF-β) signaling effector Smad2/3 was detected in several types of tumors, which acts as oncogenic factor and tumor suppressor, but the relationship between these events has not been explored. Here, we demonstrate that PAK4 interacts with and modulates phosphorylation of Smad2/3 via both kinase-dependent and kinase-independent mechanisms, which attenuate Smad2/3 axis transactivation and TGF-β-mediated growth inhibition in gastric cancer cells. First, PAK4 interaction with Smad2/3, which is independent of PAK4 kinase activity, blocks TGF-β1-induced phosphorylation of Smad2 Ser465/467 or Smad3 Ser423/425 and the consequent activation. In addition, PAK4 phosphorylates Smad2 on Ser465, leading to the degradation of Smad2 through ubiquitin-proteasome-dependent pathway under hepatocyte growth factor (HGF) stimulation. Interestingly, PAK4 expression correlates negatively with phospho-Ser465/467 Smad2 but positively with phospho-Ser465 Smad2 in gastric cancer tissues. Furthermore, the expressions of HGF, phospho-Ser474 PAK4 and phospho-Ser465 Smad2 are markedly increased in gastric cancer tissues, and the expression of Smad2 is decreased in gastric cancer tissues. Our results document an oncogenic role of PAK4 in repression of Smad2/3 transactivation that involved in tumorigenesis, and suggest PAK4 as a potential therapeutic target for gastric cancer.

PAK4 kinase-mediated SCG10 phosphorylation involved in gastric cancer metastasis

Superior cervical ganglia 10 (SCG10), as a microtubule (MT) destabilizer, maintains MT homeostasis and has a critical role in neuronal development, but its function in tumorigenesis has not been characterized. In the present study, we demonstrated that p21-activated kinase 4 (PAK4)-mediated SCG10 phosphorylation regulates MT homeostasis in metastatic gastric cancer. Our results indicate that SCG10 is a physiological substrate of PAK4, which is phosphorylated on serine 50 (Ser50) in a PAK4-dependent manner. Phosphorylated SCG10 regulated MT dynamics to promote gastric cancer cell migration and invasion in vitro and metastasis in a xenograft mouse models. Inhibiting PAK4, either by LCH-7749944 or RNA interference, resulted in the inhibition of Ser50 phosphorylation and a blockade to cell invasion, suggesting that PAK4-SCG10 signaling occurs in gastric cancer cell invasion. Moreover, we demonstrated a strong positive correlation between PAK4 and phospho-Ser50 SCG10 expression in gastric cancer samples. We also showed that high expression of SCG10 phospho-Ser50 is highly correlated to an aggressive phenotype of clinical gastric cancer. These findings revealed a novel function of SCG10 in promoting invasive potential of gastric cancer cells, suggesting that blocking PAK4-mediated SCG10 phosphorylation might be a potential therapeutic strategy for metastasis of gastric cancer.

P21-activated Kinase 6 (PAK6) Inhibits Prostate Cancer Growth via Phosphorylation of Androgen Receptor and Tumorigenic E3 Ligase Murine Double Minute-2 (Mdm2)

The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.

Impact of miRNA-23a-p21-activated kinase 6 signaling on the migration and invasion activities of prostate cancer cells

Objective To investigate the role of the p21-activated kinase 6 (PAK6) and its target microRNA (miRNA) on prostate cancer cells migration and invasion.Methods MiRNA candidates which potentially target PAK6 was predicted by target prediction programs.The expression of PAK6 were measured by real-time polymerase chain reaction ( Real-time PCR ) and Western blotting after PC-3 cells were transfected with miR-23a mimics or inhibitor oligonucleotides.Luciferase reporter assay was used to determine whether PAK6 was the direct target of miR-23a.Cell migration and invasion were detected hy matrigel invasion assay and transwell migration assay.Results MiRNA 23a was identified by target prediction programs. Exogenetic overexpression of miR-23a resulted in a remarkable decrease of PAK6 expression (79％),whereas miR-23a inhibitor oligonucleotides induced pronounced increase of PAK6 expression (40％).The luciferase activities were significantly inhibited 32％ in wild-type PAK6 group ( P＜0.05 ),while that were no significant difference in the mutation group (P＞0.05). PAK6 mRNA levels had no changed as detected by real-time PCR( P＞0.05). Matrigel invasion assay and transwell migration assay demonstrated that exogenetic overexpression of miR-23a markedly reduced migration and invasion of PC-3 cells (57％,91％ ).Conclusion MiRNA-23a inhibited prostate cancer cell migration and invasion by repressing PAK6. Key words: MicroRNA; p21-activated kinase 6; Prostate cancer; Migration; Invasion

Protein Kinase D Regulates Cofilin Activity through p21-activated Kinase 4

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.

Upregulation of p21-activated Kinase 6 in rat brain cortex after traumatic brain injury

p21-activated Kinase 6 (PAK6) is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family. PAK kinases are well-known regulators of a wide variety of cellular functions, including regulation of cytoskeleton rearrangement, cell survival, apoptosis and the mitogen-activated protein kinase signaling pathway. To elucidate the expressions and possible functions of PAK6 in central nervous system (CNS) lesion and repair, we performed a traumatic brain injury (TBI) model in adult rats. Western blot analysis revealed that PAK6 level significantly increased at day 3 after damage, and then declined during the following days. Besides, double immunofluorescence staining showed PAK6 was primarily expressed in the neurons and a few of glial cells in the normal group. While after injury, the expression of PAK6 was increased significantly in the astrocytes and neurons, and the astrocytes had largely proliferated. We also examined the expression of proliferating cell nuclear antigen (PCNA) whose change was correlated with the expression of PAK6. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many PAK6-expressing cells at day 3 after injury. In addition, injury-induced expression of PAK6 was co-labeled by active caspase-3 during neuronal apoptosis after injury. Collectively, we hypothesized PAK6 may play important roles in CNS pathophysiology after TBI and further research is needed to have a good understanding of its function and mechanism.

412 KNOCKDOWN OF P21-ACTIVATED KINASE 6 INHIBITS PROSTATE CANCER GROWTH AND ENHANCES CHEMOSENSITIVITY TO DOCETAXEL

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011412 KNOCKDOWN OF P21-ACTIVATED KINASE 6 INHIBITS PROSTATE CANCER GROWTH AND ENHANCES CHEMOSENSITIVITY TO DOCETAXEL Xingqiao Wen, Xiaojuan LI, Chunwei Ye, Baoyi Zhu, and Xin Gao Xingqiao WenXingqiao Wen Guangzhou, China, People's Republic of More articles by this author , Xiaojuan LIXiaojuan LI Guangzhou, China, People's Republic of More articles by this author , Chunwei YeChunwei Ye Guangzhou, China, People's Republic of More articles by this author , Baoyi ZhuBaoyi Zhu Guangzhou, China, People's Republic of More articles by this author , and Xin GaoXin Gao Guangzhou, China, People's Republic of More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.501AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To investigate whether silencing of p21-activated kinase 6 (PAK6) would inhibit prostate cancer growth and to assess the effect of PAK6 silencing when used in combination with docetaxel. PAK6 is a serine/threonine kinase belonging to the PAK family and has been implicated in cell motility, apoptosis, and transformation. METHODS Expression of PAK6 in PC-3, DU145, and LAPC4 prostate cancer cell lines was knocked down by small, interfering RNA (siRNA). Cellular proliferation, cell cycle distribution, the ability to undergo apoptosis, and the invasive capacity through matrigel were evaluated. Furthermore, in vivo growth of prostate cancer in nude mice treated with PAK6-siRNA alone or in combination with docetaxel was examined. RESULTS PAK6 was overexpressed in prostate cancer tissues. PAK6-siRNA efficiently and specifically downregulated the expression of PAK6 and inhibited cell growth of prostate cancer. It also suppressed invasive ability in matrigel and caused cell cycle arrest at the G(2)/M phase. Xenograft tumor growth in nude mice was inhibited significantly by PAK6-siRNA. The average weight of the harvested tumor was 46 +/− 8.5 mg in the PAK6-siRNA group, lower than the 451 +/− 22.3 mg in the control group (P < .05). Combined use of PAK6-siRNA and docetaxel produced a greater effect than docetaxel alone in both in vitro and in vivo models. CONCLUSIONS Knockdown of PAK6 expression produced an inhibitory effect on prostate cancer growth and enhanced chemosensitivity of docetaxel. PAK6 could be a valuable molecular target for the treatment of prostate cancer. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e166 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xingqiao Wen Guangzhou, China, People's Republic of More articles by this author Xiaojuan LI Guangzhou, China, People's Republic of More articles by this author Chunwei Ye Guangzhou, China, People's Republic of More articles by this author Baoyi Zhu Guangzhou, China, People's Republic of More articles by this author Xin Gao Guangzhou, China, People's Republic of More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

PAK4: a pluripotent kinase that regulates prostate cancer cell adhesion

Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Migration and invasion require coordinated reorganisation of the actin cytoskeleton and regulation of cell-adhesion dynamics. Rho-family GTPases orchestrate both of these cellular processes. p21-activated kinase 4 (PAK4), a specific effector of the Rho GTPase Cdc42, is activated by HGF, and we have previously shown that activated PAK4 induces a loss of both actin stress fibres and focal adhesions. We now report that DU145 human prostate cancer cells with reduced levels of PAK4 expression are unable to successfully migrate in response to HGF, have prominent actin stress fibres, and an increase in the size and number of focal adhesions. Moreover, these cells have a concomitant reduction in cell-adhesion turnover rates. We find that PAK4 is localised at focal adhesions, is immunoprecipitated with paxillin and phosphorylates paxillin on serine 272. Furthermore, we demonstrate that PAK4 can regulate RhoA activity via GEF-H1. Our results suggest that PAK4 is a pluripotent kinase that can regulate both actin cytoskeletal rearrangement and focal-adhesion dynamics.

1289 A MECHANISM FOR ANDROGEN-INDEPENDENT PROSTATE CANCER THROUGH MODULATION OF ANDROGEN RECEPTOR SIGNALING BY THE P21-ACTIVATED KINASE 6 (PAK6)

You have accessJournal of UrologyProstate Cancer: Basic Research VII1 Apr 20101289 A MECHANISM FOR ANDROGEN-INDEPENDENT PROSTATE CANCER THROUGH MODULATION OF ANDROGEN RECEPTOR SIGNALING BY THE P21-ACTIVATED KINASE 6 (PAK6) Min Zhang, Michael Siedow, Gregory Saia, and Arnab Chakravarti Min ZhangMin Zhang More articles by this author , Michael SiedowMichael Siedow More articles by this author , Gregory SaiaGregory Saia More articles by this author , and Arnab ChakravartiArnab Chakravarti More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.873AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES p21-activated kinase 6 (PAK6) is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family. PAK6 was first identified in a yeast two-hybrid screen for novel androgen receptor (AR) interactors, but its functions have not been established. In this study, we explored the role of PAK6 in mediation hormone resistance in prostate cancer cells. METHODS PAK6 expression was detected in androgen deprived prostate cancer cell line LNCaP. Short hairpin RNA (shRNA) was used to attenuate PAK6 expression in LNCaP cells, and apoptosis and cell cycle distribution were assessed in these cells. Given that AR can be phosphorylated by either the AKT or the mitogen-activated protein kinase (MAPK) pathway and becomes a ligand-independent AR, we investigated the effect of PAK6 on the cross-talks between AR signaling and AKT/MAPK pathways. RESULTS We found that PAK6 expression in LNCaP PCa cells was markedly increased when the cells were cultured for 8 weeks in steroid hormone depleted medium. Antagonism of PAK6 using short hairpin RNA (shRNA) significantly increased antiangogen bicalutamide-induced apoptosis and G1 arrest. These finds implied that PAK6 may play a role in the development of androgen independent prostate cancer. Knockdown of PAK6 in LNCaP cells diminished AKT phosporylation at Ser473 and Thr308 as well as p44/42 MAPK phophorylation at Thr202 and Tyr204, indicating that overexpression of PAK6 during androgen deprivation may activate AR signaling by phosporylation of both AKT and MAPK. CONCLUSIONS These findings extended the current vision of PAK6 functions, and indicated that PAK6 may be involved in androgen-independent prostate cancer development through modulation of AR signaling. Columbus, OH© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e499 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Min Zhang More articles by this author Michael Siedow More articles by this author Gregory Saia More articles by this author Arnab Chakravarti More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Knockdown of p21-activated Kinase 6 Inhibits Prostate Cancer Growth and Enhances Chemosensitivity to Docetaxel

To investigate whether silencing of p21-activated kinase 6 (PAK6) would inhibit prostate cancer growth and to assess the effect of PAK6 silencing when used in combination with docetaxel. PAK6 is a serine/threonine kinase belonging to the PAK family and has been implicated in cell motility, apoptosis, and transformation. Expression of PAK6 in PC-3, DU145, and LAPC4 prostate cancer cell lines was knocked down by small, interfering RNA (siRNA). Cellular proliferation, cell cycle distribution, the ability to undergo apoptosis, and the invasive capacity through matrigel were evaluated. Furthermore, in vivo growth of prostate cancer in nude mice treated with PAK6-siRNA alone or in combination with docetaxel was examined. PAK6 was overexpressed in prostate cancer tissues. PAK6-siRNA efficiently and specifically downregulated the expression of PAK6 and inhibited cell growth of prostate cancer. It also suppressed invasive ability in matrigel and caused cell cycle arrest at the G(2)/M phase. Xenograft tumor growth in nude mice was inhibited significantly by PAK6-siRNA. The average weight of the harvested tumor was 46 +/- 8.5 mg in the PAK6-siRNA group, lower than the 451 +/- 22.3 mg in the control group (P < .05). Combined use of PAK6-siRNA and docetaxel produced a greater effect than docetaxel alone in both in vitro and in vivo models. Knockdown of PAK6 expression produced an inhibitory effect on prostate cancer growth and enhanced chemosensitivity of docetaxel. PAK6 could be a valuable molecular target for the treatment of prostate cancer.

A PAK4–LIMK1 pathway drives prostate cancer cell migration downstream of HGF

Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Cell migration and invasion requires reorganisation of the actin cytoskeleton; processes mediated by the Rho family GTPases. p21 activated kinase 4 (PAK4), an effector of the Rho family protein Cdc42, is activated downstream of HGF. We report here the novel finding that in prostate cancer cells PAK4 binds to and phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner. We show for the first time that variations in the level of PAK4 expression change the level of cofilin phosphorylation in cells, a change we correlate with LIMK1 activity, cell morphology and migratory behaviour. We identify for the first time a direct and localised interaction between PAK4 and LIMK1 within cells using FRET: FLIM. Moreover we show here that HGF mediates this interaction which is concentrated in small foci at the cell periphery. PAK4 and LIMK1 act synergistically to increase cell migration speed, whilst a reduction in PAK4 expression decreases cell speed. It is well established that unphosphorylated (active) cofilin is a required to drive cell migration. Our results support a model whereby HGF-stimulated cell migration also requires a cofilin phosphorylation step that is mediated by PAK4.