P21-activated Kinase 4 Expression Research Articles

PAK4 inhibition improves PD-1 blockade immunotherapy.

Lack of tumor infiltration by immune cells is the main mechanism of primary resistance to programmed cell death protein 1 (PD-1) blockade therapies for cancer. It has been postulated that cancer cell-intrinsic mechanisms may actively exclude T cells from tumors, suggesting that the finding of actionable molecules that could be inhibited to increase T cell infiltration may synergize with checkpoint inhibitor immunotherapy. Here, we show that p21-activated kinase 4 (PAK4) is enriched in non-responding tumor biopsies with low T cell and dendritic cell infiltration. In mouse models, genetic deletion of PAK4 increased T cell infiltration and reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, combination of anti-PD-1 with the PAK4 inhibitor KPT-9274 improved anti-tumor response compared with anti-PD-1 alone. Therefore, high PAK4 expression is correlated with low T cell and dendritic cell infiltration and a lack of response to PD-1 blockade, which could be reversed with PAK4 inhibition.

PAK4-NAMPT Dual Inhibition as a Novel Strategy for Therapy Resistant Pancreatic Neuroendocrine Tumors.

Pancreatic neuroendocrine tumors (PNET) remain an unmet clinical need. In this study, we show that targeting both nicotinamide phosphoribosyltransferase (NAMPT) and p21-activated kinase 4 (PAK4) could become a synthetic lethal strategy for PNET. The expression of PAK4 and NAMPT was found to be higher in PNET tissue compared to normal cells. PAK4-NAMPT dual RNAi suppressed proliferation of PNET cell lines. Treatment with KPT-9274 (currently in a Phase I trial or analogs, PF3758309 (the PAK4 selective inhibitor) or FK866 (the NAMPT inhibitor)) suppressed the growth of PNET cell lines and synergized with the mammalian target of rapamycin (mTOR) inhibitors everolimus and INK-128. Molecular analysis of the combination treatment showed down-regulation of known everolimus resistance drivers. KPT-9274 suppressed NAD pool and ATP levels in PNET cell lines. Metabolomic profiling showed a statistically significant alteration in cellular energetic pathways. KPT-9274 given orally at 150 mg/kg 5 days/week for 4 weeks dramatically reduced PNET sub-cutaneous tumor growth. Residual tumor analysis demonstrated target engagement in vivo and recapitulated in vitro results. Our investigations demonstrate that PAK4 and NAMPT are two viable therapeutic targets in the difficult to treat PNET that warrant further clinical investigation.

Effects of dienogest on breast : MCF cell line data

Dienogest (DNG) is a widely used progestin which is safe and effective for long-term management of endometriosis. However, its association to breast cells remains to be elucidated. We perform this study to investigate whether in vitro treatment of DNG can cause any biologic changes on MCF cell line (human estrogen receptor (ER)-positive breast cancer cell line) experiments. A laboratory study. Following in vitro culture of MCF cells, we treated those cells and compared cell viability and the expression of several markers have shown to be increased in breast cancer cells between with estradiol alone and estradiol with DNG. Cell viability was measured utilizing MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and the expression of PCNA (proliferating cell nuclear antigen) and PAK4 (p21 activated kinase 4) was measured by western blot analyses. VEGF (vascular endothelial growth factor) and IL (interleukin)-32 were analyzed by ELISA, and MMP2 (matrix metalloproteinase 2) activity was assayed by zymography. In vitro treatment of MCF7 cells led to an increased cell viability by estradiol alone and decreased by both estradiol and DNG after 24 and 48-hour culture. The expression of PCNA after 48 hours showed the same result. VEGF and IL-32 were also significantly increased with estradiol and decreased following DNG treatment. However, there was no significant changes in MMP2 activity and PAK4 expression. These findings suggest that DNG may have inhibitory effects on carcinogenesis of breast cells by suppressing specific biologic changes treated by estradiol. However, further study is necessary using normal human breast cells.

MicroRNA-140-5p inhibits the tumorigenesis of oral squamous cell carcinoma by targeting p21-activated kinase 4.

Oral squamous cell carcinoma (OSCC) is a serious global health problem. Recently, accumulating microRNA (miRNA) has emerged as crucial players in the development and progression of carcinomas including OSCC. Our study aimed to further investigate the roles of miR-140-5p in OSCC tumorigenesis and related molecular basis. In this study, OSCC tissues and adjacent normal tissues were isolated from 34 OSCC patients who suffered from surgical resection at our hospital. MiR-140-5p level was measured by reverse-transcription quantitative polymerase chain reaction assay. p21-activated kinase 4 (PAK4) protein level was determined by western blot assay in OSCC cells at 48 h posttransfection or OSCC xenograft tumors at day 35 after OSCC cell injection. The cell proliferative ability was assessed by cell counting kit-8 assay in OSCC cells at 0, 24, 48, 72 h after transfection. Cell apoptosis and cell-cycle analysis was conducted using a flow cytometry in OSCC cells at 48 h after transfection. The interaction between miR-140-5p and PAK4 3'-untranslated region was tested by bioinformatics analysis and luciferase reporter assay in OSCC cells at 48 h after transfection. Mouse xenograft models of OSCC were established to examine the influence of miR-140-5p on OSCC tumorigenesis in vivo during 35 days after OSCC cell injection. Our data showed that miR-140-5p expression was notably downregulated in OSCC tissues and cell lines. MiR-140-5p inhibited the expression of PAK4 by direct interaction in OSCC cells. Functional analysis disclosed that miR-140-5p overexpression or PAK4 knockdown suppressed cell proliferation, promoted cell apoptosis, and induced cell-cycle arrest in OSCC. Moreover, PAK4 upregulation rescued the detrimental effects of miR-140-5p on cell proliferation and cell-cycle progression and hampered cell apoptosis induced by miR-140-5p in OSCC. In vivo experiments demonstrated that miR-140-5p overexpression suppressed the growth of OSCC xenograft tumors by downregulating PAK4. In conclusion, our data revealed miR-140-5p suppressed OSCC tumorigenesis by targeting PAK4 in vitro and in vivo, deepening our understanding on the function and molecular basis of miR-140-5p in the development of OSCC.

PAK6- A novel kinase in the maintenance of T cell anergy

Abstract Costimulation of T cells through the antigen receptor and the CD28 receptor engages an ‘effector’ gene expression program and a burst of proliferation. Conversely, antigen receptor engagement in the absence of costimulation induces a hypo-responsive state, referred to as anergy, that is associated with active silencing of effector genes. Our ATAC-seq analysis of CD28-dependent chromatin remodeling in anergic and effector CD4+ T cells has identified p21-activated kinase 6 (PAK6) as a gene that becomes accessible in anergic but not effector T cells. PAK6 is a member of a family of kinases comprised of two groups, group I (PAK1-3) and group II (PAK4-6). PAK6 and other group II members lack a regulatory domain that is present in group I, causing it to function differently. Previous studies by others have shown that PAK2, a group I PAK, was a positive regulator of CD3/CD28 T cell activation. Here, we demonstrate that PAK6 has the opposite role in T cell activation as PAK2, where it inhibits IL2 secretion, a hallmark characteristic of T cell anergy. Knockdown of PAK6 in anergic and effector T cells, using a CRISPR/CAS9 approach, resulted in a 50% increase in IL2 secretion, and 5-fold more cell expansion in anergic cultures upon restimulation with CD3/CD28. This suggests that PAK6 may be involved in a signaling pathway which inhibits cellular proliferation or promotes apoptosis. The highest level of PAK6 expression was observed upon re-stimulation of anergic cells, suggesting that PAK6 may be involved in the maintenance of T cell anergy. This study identifies a novel role for PAK6 in T cell anergy and a potential new target for regulating T cell function in autoimmune diseases.

Dual PAK4-NAMPT Inhibition Impacts Growth and Survival, and Increases Sensitivity to DNA-Damaging Agents in Waldenström Macroglobulinemia.

p21-activated kinase 4 (PAK4) plays a significant biological and functional role in a number of malignancies, including multiple myeloma (MM). On the basis of our promising findings in MM, we here characterize PAK4 expression and role in WM cells, as well effect of dual PAK4-NAMPT inhibitor (KPT-9274) against WM cell growth and viability. We have analyzed mRNA and protein expression levels of PAK4 in WM cells, and used loss-of-function approach to investigate its contribution to WM cell viability. We have further tested the in vitro and in vivo effect of KPT-9274 against WM cell growth and viability. We report here high-level expression and functional role of PAK4 in WM, as demonstrated by shRNA-mediated knockdown; and significant impact of KPT-9274 on WM cell growth and viability. The growth inhibitory effect of KPT-9274 was associated with decreased PAK4 expression and NAMPT activity, as well as induction of apoptosis. Interestingly, in WM cell lines treated with KPT-9274, we detected a significant impact on DNA damage and repair genes. Moreover, we observed that apart from inducing DNA damage, KPT-9274 specifically decreased RAD51 and the double-strand break repair by the homologous recombination pathway. As a result, when combined with a DNA alkylating agents bendamustine and melphalan, KPT-9274 provided a synergistic inhibition of cell viability in WM cell lines and primary patient WM cells in vitro and in vivo. These results support the clinical investigation of KPT-9274 in combination with DNA-damaging agent for treatment of WM.

MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4.

Although microRNA (miR) 199a-3p functions as a tumor suppressor in multiple malignancies, its expression and role in esophageal cancer have not been studied. Based on our previous observation that miR-199a-3p is markedly downregulated in esophageal cancer cell lines relative to esophageal epithelial cells, we examined the function of miR-199a-3p in these cells. MiR-199a-3p is predicted to bind with high affinity to the mRNA of p21 activated kinase 4 (PAK4). This kinase has been shown to be overexpressed in several malignancies and to modulate proliferation and motility. The current study is designed to determine whether miR-199a-3p regulates the expression of PAK4 in esophageal cancer cells and to understand the functional consequences of this interaction. Herein, we demonstrate reduced expression of miR-199a-3p in human esophageal cancer specimens and cell lines compared to esophageal epithelial cells, with associated increased expression of PAK4. Forced expression of miR-199a-3p decreases expression of PAK4 in esophageal cancer cell lines. Mechanistic studies reveal that miR-199a-3p binds to the 3’UTR of PAK4 mRNA. This interaction results in reduced levels of PAK4 mRNA due to decreased mRNA stability. Downregulation of PAK4 leads to decreased cyclin D1 (CD1) transcription and protein expression, resulting in markedly impaired cellular proliferation. When PAK4 expression is rescued, both CD1 transcription and protein return to baseline levels. Our results show that miR-199a-3p functions as a tumor suppressor in esophageal cancer cells through repression of PAK4. These findings suggest that both miR-199a-3p and PAK4 may be novel therapeutic targets in the treatment of esophageal cancer.

Inhibition of neuroblastoma proliferation by PF-3758309, a small-molecule inhibitor that targets p21-activated kinase 4

Neuroblastoma is the most common extracranial solid childhood tumor. Despite the availability of advanced multimodal therapy, high-risk patients still have low survival rates. p21-activated kinase 4 (PAK4) has been shown to regulate many cellular processes in cancer cells, including migration, polarization and proliferation. However, the role of PAK4 in neuroblastoma remains unclear. In the present study, we demonstrated that PAK4 was overexpressed in neuroblastoma tissues and was correlated with tumor malignance and prognosis. To investigate the function of PAK4 in neuroblastoma, we used a small-molecule inhibitor that targets PAK4, that is, PF-3758309. Our results showed that PF-3758309 significantly induced cell cycle arrest at the G1 phase and apoptosis in neuroblastoma cell lines. Meanwhile, the inhibition of PAK4 by PF-3758309 increased the expression of CDKN1A, BAD and BAK1 and decreased the expression of Bcl-2 and Bax. In addition, we screened the target genes of PAK4 by PCR array and found that 23 genes were upregulated (including TP53I3, TBX3, EEF1A2, CDKN1A, IFNB1 and MAPK8IP2) and 20 genes were downregulated (including TNFSF8, Bcl2-A1, Bcl2L1, SOCS3, BIRC3 and NFKB1) after PAK4 inhibition by PF-3758309. Moreover, PAK4 was found to regulate the cell cycle and apoptosis via the ERK signaling pathway. In conclusion, the present study demonstrated, for the first time, the expression and function of PAK4 in neuroblastomas and the inhibitory effect of PF-3758309, which deserves further investigation as an alternative strategy for neuroblastoma treatment.

MicroRNA-485 inhibits malignant biological behaviour of glioblastoma cells by directly targeting PAK4.

Glioblastoma (GBM), which is characterised by rapid growth, cellular heterogeneity, angiogenesis, extensive invasion, hypoxia and necrosis, is the most common histological subtype of glioma in adults. MicroRNA(miRNA) dysregulation is a common feature of human cancers, including GBM. Previous studies have suggested that miRNAs are a novel class of regulatory molecules in various human cancers. Therefore, miRNAs may be investigated as a novel candidate and screening tool in the clinical diagnosis, therapy and prognosis of GBM. Recent accumulating evidence has demonstrated that miRNA‑485(miR‑485) is involved in the development and progression of several types of human cancers. However, the expression level, exact role and underlying mechanisms of miR‑485 in GBM remain unclear. In this study, miR‑485 was downregulated in both GBM tissue specimens and cell lines. miR‑485 overexpression inhibited GBM cell proliferation, colony formation, migration and invasion; increased apoptosis invitro; and reduced tumour growth invivo. In addition, p21‑activated kinase4 (PAK4) was demonstrated to be a direct and functional target of miR‑485 in GBM. Furthermore, PAK4 was upregulated in GBM tissues and negatively correlated with miR‑485 expression. Moreover, PAK4 knockdown exhibited a similar effect to miR‑485 overexpression in GBM cells. Enforced expression of PAK4 rescued miR‑485 tumour‑suppressor functions in GBM cells. miR‑485 inhibited the activation of the AKT and ERK signalling pathways in GBM. These results indicate that miR‑485 acts as a tumour suppressor in GBM by, at least partially, directly targeting PAK4 and regulating the AKT and ERK signalling pathways. Thus, miR‑485 may be a potential target for the treatment of patients with GBM.

Prognostic value of p21‐activated kinase 4 in resected pancreatic cancer

Resectable pancreatic cancer has a high recurrence rate after curative surgery. Biomarkers are needed for distinguishing patients who may benefit from curative resection. In this study, we sought to analyze the prognostic value of p21-activated kinase 1 (PAK1), p21-activated kinase 4 (PAK4), human equilibrative nucleoside transporter 1 (hENT1), and thymidylate synthase (TS) in surgically resected pancreatic ductal adenocarcinomas. A total of 160 pancreatic cancer patients who underwent surgery with curative intent were retrospectively reviewed. Tissue microarrays were constructed and immunohistochemical stains were performed for PAK1, PAK4, hENT1, and TS. The absence of PAK4 expression in pancreatic adenocarcinomas was associated with poorer histologic differentiation (p < 0.001), shorter overall survival [hazard ratio (HR) = 2.86, 95% confidence interval (CI) 1.43-5.71; p = 0.003], and disease-free survival (HR = 2.29, 95% CI: 1.11-4.74; p = 0.025) on univariate analyses. In addition, more frequent venous invasion and lymph node metastases were seen in PAK4-negative tumors although not statistically significant. PAK1, hENT1, and TS expression status did not have significant influences on patient survival. In conclusion, PAK4 of the markers tested in this study may be a potential prognostic biomarker for pancreatic adenocarcinomas.

PAK4 regulates G6PD activity by p53 degradation involving colon cancer cell growth

The p21-activated kinase 4 (PAK4) is overexpressed in different cancers and promotes proliferation of cancer cells. Reprogramming of glucose metabolism is found in most cancer cells which in turn supports rapid proliferation. However, the relationship between PAK4 and glucose metabolism in cancer cells has not been explored. In this study, we reported that PAK4 promoted glucose intake, NADPH production and lipid biosynthesis, leading to an increased proliferation of colon cancer cells. Mechanistically, PAK4 interacted with glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway and increased G6PD activity via enhancing Mdm2-mediated p53 ubiquitination degradation. In addition, we demonstrated a close positive correlation between PAK4 and G6PD expression in colon cancer specimens. Furthermore, expression of PAK4 or G6PD was positively correlated with an aggressive phenotype of clinical colon cancer. These findings revealed a novel glucose metabolism-related mechanism of PAK4 in promoting colon cancer cell growth, suggesting that PAK4 and/or G6PD blockage might be a potential therapeutic strategy for colon cancer.

P21 activated kinase 4 binds translation elongation factor eEF1A1 to promote gastric cancer cell migration and invasion.

P21 activated kinase 4 (PAK4), as an effector of Cdc42, playing important roles in regulating the processes of cytoskeleton organization. PAK4 has been considered to be an oncogenic protein, which has strong relationship with gastric cancer metastasis. However, the mechanism of PAK4 in regulating gastric cancer metastasis is still not fully understood. In this study, using yeast two-hybrid system, we identified that the eukaryotic elongation factor 1 α1 (eEF1A1) is a new binding partner of PAK4. The interaction between PAK4 and eEF1A1 was confirmed by GST pull-down and co-immunoprecipitation. PAK4 co-localized with eEF1A1 in the cytoplasm of gastric cancer cells. Overexpression of PAK4 enhanced the expression level of eEF1A1 and vice versa. PAK4 and eEF1A1 could cooperate to promote gastric cancer cell migration and invasion. Furthermore, the expression of PAK4 and eEF1A1 in clinical gastric cancer samples were examined by western blotting and immunohistochemistry. Statistical analysis indicated that there was positive correlation between the expression of PAK4 and eEF1A1. This study demonstrated for the first time that PAK4 interacted with eEF1A1 to promote migration and invasion of gastric cancer cells, thereby providing new insights into the function of PAK4 and eEF1A1 in the progression of gastric cancer.

PAK4 interacts with p85 alpha: implications for pancreatic cancer cell migration

It has been reported that p21-activated kinase 4 (PAK4) is amplified in pancreatic cancer tissue. PAK4 is a member of the PAK family of serine/threonine kinases, which act as effectors for several small GTPases, and has been specifically identified to function downstream of HGF-mediated c-Met activation in a PI3K dependent manner. However, the functionality of PAK4 in pancreatic cancer and the contribution made by HGF signalling to pancreatic cancer cell motility remain to be elucidated. We now find that elevated PAK4 expression is coincident with increased expression levels of c-Met and the p85α subunit of PI3K. Furthermore, we demonstrate that pancreatic cancer cells have a specific motility response to HGF both in 2D and 3D physiomimetic organotypic assays; which can be suppressed by inhibition of PI3K. Significantly, we report a specific interaction between PAK4 and p85α and find that PAK4 deficient cells exhibit a reduction in Akt phosphorylation downstream of HGF signalling. These results implicate a novel role for PAK4 within the PI3K pathway via interaction with p85α. Thus, PAK4 could be an essential player in PDAC progression representing an interesting therapeutic opportunity.

Abstract 1873: The role of p21-activated kinase 4 (PAK4) in cancer stemness and epithelial-to-mesenchymal transition

Abstract The p21-activated kinase 4 (PAK4) is a key downstream effector of the Rho family GTPases. PAK4 is found to be over-expressed in many oncogenic Ras-driven cancers and cell lines but not in their normal counterparts. Mesenchymal pancreatic ductal adenocarcinoma (PDAC) stem cells (CSCs) that are triple positive for stemness markers (CD133+, CD44+, and EpCam+) as well as Ras and snail transduced human mammary epithelial cells (HMLER-snail) showed enhanced expression of PAK4 along with activation of Rho, Rac1 and CDC42. PAK4 RNA interference resulted in disruption of PDAC CSC spheroids, reduction of mesenchymal markers (i.e. vimentin and snail) and suppression of CSCs potential to form subcutaneous tumors in mice. These findings point to a novel role of PAK4 in promoting oncogenic Ras-driven EMT and stemness. Using PDAC CSCs (MiaPaCa-2 and L3.6pl) and human mammary epithelial cells (HMECs) that are transduced with Ras and the EMT promoter snail (HMLE-Snail and HMLER-Snail), we investigated the impact of PAK4 allosteric modulators [PAMs (KPT-7189, KPT-7523, KPT-9274 and KPT-9307)] on the reversal of EMT and stemness. The toxicity and efficacy of PAMs were evaluated in vitro and in multiple sub-cutaneous xenograft models. PAMs show anti-proliferative activity in vitro against different PDAC CSCs and HMECs while sparing normal cells. Cell growth inhibition was concurrent with apoptosis induction, suppression of colony formation and reversal of mesenchymal morphology to epithelial (MET). PAMs not only reduced PAK4 RNA and protein steady state levels but also inhibited proliferative and anti-apoptotic signals downstream of PAK4. PAMs caused suppression of EMT markers (EpCAM, vimentin and snail) and re-expression of an epithelial promoter E-cadherin. PAM treatment inhibited RhoA, Rac1 and CDC42 activation in GLISA assays. KPT-9274 showed remarkable anti-tumor activity in sub-cutaneous xenograft models of CSCs and residual tumors showed suppression of EMT markers that was concurrent with inhibition of PAK4 signaling. These studies open a novel therapeutic opportunity against EMT and cancer stem cells through PAK4 inhibition. Citation Format: Asfar S. Azmi, Irfana Muqbil, Amro Aboukameel, William Senapedis, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Michael Kauffman, Philip A. Philip, Ramzi M. Mohammad. The role of p21-activated kinase 4 (PAK4) in cancer stemness and epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1873.

Activated-PAK4 predicts worse prognosis in breast cancer and promotes tumorigenesis through activation of PI3K/AKT signaling.

The p21-activated kinase 4 (PAK4) is sufficient to transform noncancerous mammary epithelial cells and to form tumors in the mammary glands of mice. The accumulated information suggests that PAK4 might be an oncogenic protein in breast cancer. In this study, we sought to identify the role for PAK4 in breast cancer progression. Immunohistochemical study revealed that high PAK4 expression is associated with larger tumor size, lymph node metastasis, and advanced stage cancer in 93 invasive breast carcinoma patients. Moreover, high PAK4 expression was significantly associated with poor overall and disease-free survival. PAK4 remained an independent adverse prognosticator after univariate and multivariate analysis. Ectopic expression of wild-type PAK4 in MDA-MB-231 cells activated PI3K/AKT signaling and resulted in the enhancement of the cell proliferation, migration, and invasion, whereas PAK4-induced effects were blocked by the PAK4 kinase inhibitor PF- 3758309, PAK4 siRNAs or the PI3K inhibitor LY294002. Furthermore, a kinase-active PAK4 (S474E) strongly induced PI3K/AKT activation, and promoted proliferation, migration and invasion in breast cancer cells. A kinase-inactive PAK4 KD (K350A/K351A) did partially upregulate PI3K/AKT, and promoted invasive phenotype. Taken together, these findings suggest that PAK4-activated PI3K/AKT signaling is both kinase-dependent and -independent, which contributes to breast cancer progression. Thus, our results imply that dual inhibition of PAK4 and PI3K/AKT signaling might be a potential therapeutic approach for breast cancer therapy.

Expression and role of PAK6 after spinal cord injury in adult rat

ObjectiveTo observe p21-activated kinase 6 (PAK6) expression and its possible role after spinal cord injury (SCI) in adult rat. MethodsSprague-Dawley rats were subjected to spinal cord injury. To explore the pathological and physiological significance of PAK6, the expression patterns and distribution of PAK6 were observed by Western blot, immuno-histochemistry and immunofluorescence. ResultsWestern blot analysis showed PAK6 protein level was significantly up-regulated on day 2 and day 4, then reduced and had no up-regulation till day 14. Immuno-histochemistry analysis showed that the expression of PAK6 was significantly increased on day 4 compared with the control group. Besides, double immunofluorescence staining showed PAK6 was primarily expressed in the neurons and astrocytes in the control group. While after injury, the expression of PAK6 was increased significantly in the as-trocytes and neurons, and the astrocytes were largely proliferated. We also examined the expression of proliferating cell nuclear antigen (PCNA) and found its change was correlated with the expression of PAK6. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many PAK6-express-ing cells on day 4 after injury. ConclusionThe up-regulation of PAK6 in the injured spinal cord may be associated with glial proliferation.

PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion.

P21-activated kinase 4 (PAK4) is a Cdc42 effector protein thought to regulate cell adhesion disassembly in a kinase-dependent manner. We found that PAK4 expression is significantly higher in high-grade human breast cancer patient samples, whereas depletion of PAK4 modifies cell adhesion dynamics of breast cancer cells. Surprisingly, systematic analysis of PAK4 functionality revealed that PAK4-driven adhesion turnover is neither dependent on Cdc42 binding nor kinase activity. Rather, reduced expression of PAK4 leads to a concomitant loss of RhoU expression. We report that RhoU is targeted for ubiquitination by the Rab40A-Cullin 5 complex and demonstrate that PAK4 protects RhoU from ubiquitination in a kinase-independent manner. Overexpression of RhoU rescues the PAK4 depletion phenotype, whereas loss of RhoU expression reduces cell adhesion turnover and migration. These data support a new kinase-independent mechanism for PAK4 function, where an important role of PAK4 in cellular adhesions is to stabilize RhoU protein levels. Thus, PAK4 and RhoU cooperate to drive adhesion turnover and promote cell migration.

P-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling

Pancreatic cancer (PC) remains a highly lethal malignancy due to its unusual chemoresistance and high aggressiveness. A subpopulation of pancreatic tumor cells, known as cancer stem cells (CSCs), is considered responsible not only for tumor-maintenance, but also for its widespread metastasis and therapeutic failure. Here we investigated the role of p-21 activated kinase 4 (PAK4) in driving PC stemness properties. Our data demonstrate that triple-positive (CD24+/CD44+/EpCAM+) subpopulation of pancreatic CSCs exhibits greater level of PAK4 as compared to triple-negative (CD24−/CD44−/EpCAM−) cells. Moreover, PAK4 silencing in PC cells leads to diminished fraction of CD24, CD44, and EpCAM positive cells. Furthermore, we show that PAK4-silenced PC cells exhibit decreased sphere-forming ability and increased chemosensitivity to gemcitabine toxicity. PAK4 expression is also associated with enhanced levels of stemness-associated transcription factors (Oct4/Nanog/Sox2 and KLF4). Furthermore, our data show decreased nuclear accumulation and transcriptional activity of STAT3 in PAK4-silenced PC cells and restitution of its activity leads to restoration of stem cell phenotypes. Together, our findings deliver first experimental evidence for the involvement of PAK4 in PC stemness and support its clinical utility as a novel therapeutic target in PC.

Abstract 3324: A novel role for PAK4 in the regulation of EMT and stemness in glioblastoma

Abstract Glioblastomas (GB) are the most aggressive of primary brain tumors with high rates of tumor recurrence and poor prognosis. Epithelial-mesenchymal transition (EMT) is the dynamic transdifferentiation process which leads to acquisition of invasiveness, stem-like characteristics and drug resistance in tumor cells. A small fraction of radio- and chemo-resistant cancer stem cells (CSC) with a capacity of self-renewal is considered to be crucial for tumor recurrence. Our previous studies indicated high levels of expression of p21-activated kinase 4 (PAK4) in GBs and confirmed its role in the regulation of proliferation, migration and invasion. Here, we aimed to determine whether PAK4-regulated invasiveness was associated with mesenchymal transition of glioma cells and to delineate the essential signaling events that governed EMT and maintenance of stemness in GBs. PAK4 overexpression in glioma cells resulted in loss of E-cadherin and Zo-1 and a significant increase in N-cadherin and vimentin levels. Conversely, shRNA-mediated PAK4 downregulation led to loss of mesenchymal phenotype and markers in glioma cell lines. Further, irradiation (IR, 8 Gy) treatments significantly elevated PAK4 expression and nuclear localization in correlation with increased EMT in glioma cells. FACS analysis and western blotting indicated an enrichment of CD133+ CSCs with high expression of CD133 and Nestin in IR-treated cells. In order to study the significance IR-induced EMT and stemness with PAK4 nuclear translocation, we performed a transcription factor (TF) interaction array with immunoprecipitated PAK4 as bait, which revealed nuclear-PAK4 association with PPARγ and other TFs. Moreover, immunoprecipitation, ChIP and re-ChIP experiments showed IR-induced PAK4 binding with PPARγ nuclear receptor complex and recruitment on the PPREs of NADPH oxidase-1 promoter and elevated Nox1 expression in IR-treated cells. PAK4-knockdown suppressed the IR-induced EMT and stemness in the glioma cells lines. In line with these in vitro results, mouse orthotopic intracranial xenograft experiments showed decreased tumor growth and N-cadherin expression in tumors from PAK4-shRNA expressing cells when compared with controls. These results strongly support a role for PAK4 in the regulation of IR-induced EMT and stemness via PPARγ/Nox1 signaling and provide a rationale for PAK4 as a new therapeutic target against glioblastoma and suggest a potential for overcoming radioresistance in these tumors. Citation Format: Divya Kesanakurti, Jihong Xu, Rao S. Jasti, Vinay K. Puduvalli. A novel role for PAK4 in the regulation of EMT and stemness in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3324. doi:10.1158/1538-7445.AM2015-3324

Overexpression of P21-activated kinase 4 is associated with poor prognosis in non-small cell lung cancer and promotes migration and invasion.

BackgroundP21-activated kinase 4 (PAK4), an effector of the Rho family protein Cdc42, is an important oncogene whose expression is increased in many human cancers and is generally positively correlated with advanced disease and decreased survival. However, little is known about the expression and biological function of PAK4 in human non-small cell lung cancer (NSCLC).MethodsPAK4 expression in NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry, real-time PCR, and western blotting. Prognostic value of PAK4 expression was evaluated by Kaplan-Meier analysis and Cox regression. siRNA-mediated gene silencing and protein kinase assay was applied to demonstrate the role and the mechanism of PAK4 in lung cancer cell migration, invasion.ResultsThe results showed that PAK4 was overexpressed in NSCLC cell lines and human NSCLC tissues. PAK4 expression was detected both in the membranes and cytoplasm of NSCLC cancer cells in vivo. Moreover, increased expression of PAK4 was associated with metastasis, shorter overall survival, advanced stage of NSCLC. Furthermore, PAK4 expression was positively correlated with phosphorylation of LIMK1 expression levels. Knockdown of PAK4 in NSCLC cell lines led to reduce the phosphorylation of LIMK1, which resulted in decrease of the cell migration and invasion. In addition, PAK4 bound to LIMK1 directly and activated it via phosphorylation.ConclusionsThese data demonstrate that PAK4 mediated LIMK1 phosphorylation regulates the migration and invasion in NSCLC. Therefore, PAK4 might be a significant prognostic marker and potential therapeutic molecular target in NSCLC.