Chemical Carcinogen Induced Rat Mammary Carcinogenesis is a Potential Model of p21-activated kinase (PAK1) Positive Breast Cancer

The p21 activated kinase 1(PAK1) gene encodes a serine/threonine kinase that is overexpressed in a subset of human breast carcinomas with poor prognosis. The laboratory rat (Rattus norvegicus)orthologous gene is located at Mammary carcinoma susceptibility 3 (Mcs3) QTL on rat chromosome 1. We used quantitative PCR to determine effects of Mcs3 genotype and 7,12-dimethylbenz(a)anthracene (DMBA) exposure on Pak1expression. There was no effect of Mcs3genotype; however,there was a 3.5-fold higher Pak1level in DMBA-exposed mammary glands compared to unexposed glands (p<0.05). Sequence variants in Pak1exons did not alter amino acid sequence between Mcs3susceptible and resistant strains. Protein expression of PAK1/Pak1 in human breast carcinomas and DMBA-exposed rat mammary glands was detected using immunohistochemistry (IHC). Rat mammary glands from 12-week-old females not exposed to DMBA were negative for Pak1, while 24% of carcinogen-exposed mammary glands from age-matched females stained positive for Pak1. The positive mammary glands exposed to carcinogen had no pathological signs of disease. Human breast carcinomas, used as comparative controls, had a 22% positivity rats. This was consistent with other human breast cancer studies of PAK1 expression. Similar frequencies of human/rat PAK1/Pak1 expression in female breast carcinomas and carcinogen-induced rat mammary glands, showing no visible pathogenesis of disease, suggests aberrant PAK1 expression is an early event in development of some breast cancers. Laboratory rats will be a useful experimental organism for comparative studies of Pak1-mediated mechanisms of breast carcinogenesis. Future studies of PAK1 as a diagnostic marker of early breast disease are warranted.

PAK1 as a Potential Therapeutic Target in Male Smokers with EGFR-Mutant Non-Small Cell Lung Cancer

P21-activated kinases (PAKs) are serine/threonine protein kinases that contribute to several cellular processes. Here, we aimed to determine the prognostic value of PAK1 and its correlation with the clinicopathological characteristics and five-year survival rates in patients with non-small cell lung cancer (NSCLC). We evaluated PAK1 mRNA and protein expression in NSCLC cells and resected tumor specimens, as well as in healthy human bronchial epithelial cells and adjacent healthy lung tissues, respectively, for effective comparison. Immunohistochemical tissue microarray analysis of 201 NSCLC specimens showed the correlation of PAK1 expression with clinicopathological characteristics. The mRNA and protein expression of PAK1 were 2.9- and 4.3-fold higher in six of seven NSCLC cell types and human tumors (both, p < 0.001) than in healthy human bronchial epithelial BEAS-2B cells and adjacent healthy lung tissues, respectively. Decreased survival was significantly associated with PAK1 overexpression in the entire cohort (χ2 = 8.48, p = 0.0036), men (χ2 = 17.1, p < 0.0001), and current and former smokers (χ2 = 19.2, p < 0.0001). Notably, epidermal growth factor receptor (EGFR) mutation-positive lung cancer patients with high PAK1 expression showed higher mortality rates than those with low PAK1 expression (91.3% vs. 62.5%, p = 0.02). Therefore, PAK1 overexpression could serve as a molecular target for the treatment of EGFR mutation-positive lung cancer, especially among male patients and current/former smokers.

Up-regulation of P21-activated kinase 1 in osteoarthritis chondrocytes is responsible for osteoarthritic cartilage destruction

Osteoarthritis is mainly caused by a degenerative joint disorder, which is characterized by the gradual degradation of articular cartilage and synovial inflammation. The chondrocyte, the unique resident cell type of articular cartilage, is crucial for the development of osteoarthritis. Previous studies revealed that P21-activated kinase-1 (PAK1) was responsible for the initiation of inflammation. The purpose of the present study was to determine the potential role of PAK1 in osteoarthritis. The level of PAK1 expression was measured by Western blot and quantitative real-time PCR in articular cartilage from osteoarthritis model rats and patients with osteoarthritis. In addition, the functional role of aberrant PAK1 expression was detected in the chondrocytes. We found that the expression of PAK1 was significantly increased in chondrocytes treated with osteoarthritis-related factors. Increased expression of PAK1 was also observed in knee articular cartilage samples from patients with osteoarthritis and osteoarthritis model rats. PAK1 was found to inhibit chondrocytes proliferation and to promote the production of inflammatory cytokines in cartilages chondrocytes. Furthermore, we found that PAK1 modulated the production of extracellular matrix and cartilage degrading enzymes in chondrocytes. Results of the present studies demonstrated that PAK1 might play an important role in the pathogenesis of osteoarthritis.

Tumor-infiltrating regulatory T lymphocytes orchestrate oncogenic PAK1-conferred immune evasion in clear-cell renal cell carcinoma

Background: Little is known about the associations between PAK1 and anti-tumor immunity in clear-cell renal cell carcinoma (ccRCC). This study aims to explore the prognostic value of PAK1 in ccRCC patients and investigated the molecular immune mechanism for its oncogenic role. Methods: We retrospectively enrolled 282 ccRCC patients undergoing nephrectomy between 2005 and 2007 in Zhongshan hospital. Immunohistochemistry evaluated PAK1, CCL22, FOXP3 and CD8 expression in clinical specimens. Fresh tumor tissues, para-tumor tissues and peripheral blood samples for RT-PCR, ELISA, flow cytometry analyses were collected from patients who underwent nephrectomy in Zhongshan Hospital from September 2017 to April 2018. We compared clinical outcomes by Kaplan-Meier survival analysis and Cox regression model. Bioinformatics analysis performed in TCGA KIRC cohort. Results: High PAK1 expression indicated poorer overall survival (OS) and recurrence free survival (RFS) (both p<0.001) in ccRCC patients. Multivariate analyses indicated PAK1 as an independent prognostic factor. In clinical samples, PAK1 clearly correlated with immunosuppressive microenvironment in ccRCC tissues. Significantly, PAK1 positively correlated with Tumor-infiltrating regulatory T lymphocytes (Tregs). Furthermore, IL-10+ and TGF-β+ tumor-infiltrating Tregs both increased in PAK1 high tumors. Additionally, CCL22 was highly secreted in PAK1 high tumors. After treated by IPA-3 (an PAK1 inhibitor), CCL22 secretion was clearly inhibited (p<0.001). Finally, we built a nomogram to predict overall survival for ccRCC patients with higher predictive accuracy. Conclusions: Increased PAK1 expression predicted dismal prognosis in ccRCC patients by inducing tumor immune escape. IL-10+ and TGF-β+ tumor-infiltrating Tregs recruited by CCL22 play dominant immunosuppressive roles in PAK1 high tumors.

PAK1 Expression in Pancreatic Cancer: Clinicopathological Characteristics and Prognostic Significance

Background: Improvement of the management of pancreatic cancer requires a better understanding of the genetic and molecular changes responsible for the development of the disease. The family of p21-activated kinases (PAKs) and especially PAK1 appears to mediate many cellular processes that contribute to the development and progression of pancreatic cancer, but the clinical relevance of PAK1 expression with the disease still remains unclear. Aim of the study was to assess the clinical value and the potential prognostic significance of PAK1 in pancreatic adenocarcinoma. Methods: We investigated the relationship between the PAK1 expression and the clinical and histopathologic characteristics of pancreatic cancer patients and the potential significance of PAK1 on survival. We examined tissue samples from 51 patients operated for pancreatic cancer. PAK1 expression was investigated with immunohistochemistry and correlated to clinicopathological parameters. Results: PAK1 was detected in all tumor samples and high expression was found in most patients. High PAK1 expression was also associated with younger age and well-differentiated tumors, but no association was found between PAK1 expression and Tumor-Node-Metastasis stage as well as deceased or alive status on follow-up. Moderate to high PAK1 expression favored higher 6-month and 1-year survival and low PAK1 expression 2-year survival but without statistical significance. Conclusions Our results indicate that PAK1 could potentially be used as a prognostic marker in pancreatic cancer. Further studies could clarify whether utilization of PAK1 in therapeutic protocols for the treatment of pancreatic cancer will render them more effective.

P21-Activated Kinase 1 Overactivates in Eutopic Endometrium of Adenomyosis

Adenomyosis is a common gynecological disease, characterized by the existence of endometrium in the myometrium. The pathogenesis of adenomyosis is not fully understood. P21-activated kinase 1 (PAK1) is an effector of small Rho GTPases including CDC42 and RAC1 and plays various roles in cellular biology, especially cytoskeletal remodeling. This study aimed to evaluate whether the expression and activation of PAK1 in adenomyosis were different from normal. Immunohistochemistry was performed to evaluate the expression of PAK1 and its active form phosphorylated-PAK1 (pPAK1) semi-quantitatively in women with and without adenomyosis. Immunofluorescence was performed to locate the distribution of pPAK1. This study found that PAK1 in eutopic endometrium of adenomyosis was overactivated compared to normal. Phosphorylated-PAK1 assembled along the apical surface of glandular cell membrane. In ectopic lesions, PAK1 expression decreased and its activation returned to the baseline. The expression of pPAK1 correlated with the frequency of reproduction. These findings suggest that PAK1 overactivation in the endometrium may be an important event during the development of adenomyosis, meanwhile, decreased phosphorylation may assist to form lesions.

PAK1 confers chemoresistance and poor outcome in non-small cell lung cancer via β-catenin-mediated stemness

PAK1 confers resistance to the estrogen antagonist tamoxifen in breast cancer. However, a role for PAK1 remains to be elucidated for chemoresistance and prognosis in non-small cell lung cancer (NSCLC). We provide evidence that PAK1 confers cisplatin resistance by increasing β-catenin expression through ERK/GSK3β signaling. The increased β-catenin expression promotes sphere cell formation and expression of stemness markers and this β-catenin-induced stemness is responsible for PAK1-mediated cisplatin resistance. We enrolled 87 NSCLC patients who had received cisplatin-based chemotherapy to confirm the association between PAK1 expression and response to chemotherapy and outcomes. PAK1 expression, evaluated by immunohistochemistry, was positively correlated with pERK and β-catenin expression in lung tumors. Patients with high-PAK1, high-pERK, and high-nuclear β-catenin tumors more frequently showed an unfavorable response to cisplatin-based chemotherapy when compared to their counterparts. Kaplan-Meier and Cox regression analysis also indicated a poorer overall survival (OS) and relapse free survival (RFS) in patients with high-PAK1, high-pERK, and high-nuclear β-catenin tumors. In conclusion, PAK1 confers cisplatin resistance in NSCLC via β-catenin-mediated stemness. Therefore, we suggest that clinical use of a combination of the MEK/ERK inhibitor AZD6244 and cisplatin might improve sensitivity to cisplatin-based chemotherapy and outcomes in NSCLC patients who harbor high-PAK1-expressing tumors.

PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression

Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway.