Role Of P21-activated Kinase 1 Research Articles

Perspectives on the Role of P21-Activated Kinase 1 (PAK1) in the Intestinal Anti-inflammatory and Antitumor Potential of Artepillin C.

The Brazilian biodiversity may bring new perspectives to the therapy of Inflammatory Bowel Diseases (IBD) and intestinal cancer. The effect of Brazilian Green Propolis in reducing ulcerative colitis in mice has already been described, as well as high amounts of the prenylated compound Artepellin C (ARC). The search for new pharmacological targets for IBD is also advancing. Among possibilities is the p21-activated kinase (PAK1), overexpressed and activated in the intestinal mucosa during IBD and colitis-associated colorectal cancer (CAC). PAK 1 contributes to tissue inflammation by reducing the expression of peroxisome proliferator-activated receptor type γ (PPAR47) and increasing activation of nuclear factor (NF)-κB. At least in vitro, inhibition of PAK1 has been reported to mitigate NF-κB-mediated inflammation in intestinal cells and ARC inhibits PAK1 activation. Given this pharmacological potential of ARC and the role of PAK1 in IBD and CAC, this perspective collected information that encourages future research to test the hypothesis that ARC can maintain intestinal integrity under the inflammatory and neoplastic stimulus and that inhibition of PAK1/NF-κB signaling and favoring PPAR-γ activity is pivotal in this action. Therefore, future studies employing in vitro and in vivo steps, using murine and human enterocytes and rodents submitted to ulcerative colitis and CAC models are incentivized by the data gathered here, favor retirar essas palavras: mostly in vitro studies, before clinical trials. Therefore, the perspective presented here points to an interesting path in the search for a drug useful in inflammatory and neoplastic intestinal diseases, which may have ARC as a prototype, acting on a target not yet explored clinically.

Targeting p21-activated kinase 1 for development of a novel anti-arrhythmic drug class.

Evidence accumulated over the past decade suggests that p21-activated kinase 1 (PAK1) is a critical cardiac-protective signalling molecule. The present article provides an updated review of recent findings regarding the role of PAK1 in maintaining normal cardiac electrophysiological function through its regulation of membrane and Ca2+ clocks. We first overviewed the PAK1 activation mechanism. We then discussed recent updated results showing the action mechanisms of PAK1 signalling on Cav1.2/Cav1.3 (ICaL)-mediated Ca2+ entry, ryanodine receptor type 2-mediated sarcoplasmic reticulum (SR) Ca2+ release, transcriptional regulation of SR Ca2+-ATPase 2a, Na+/Ca2+ exchangers, and Ca2+/calmodulin-dependent protein kinase II. Finally, we proposed a new and exciting route for developing a PAK1-based therapeutic strategy for cardiac arrhythmias. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Abstract P3-06-08: Pak1 as a novel mediator of resistance to endocrine therapy and CDK4/6 inhibitors in ER+/PAK-1amplified breast cancer

Abstract Background: CDK4/6 inhibitors have been approved in combination with endocrine therapy for the treatment of estrogen receptor positive (ER+) metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. Results: p21-activated kinase 1 (PAK1) is a serine/threonine kinase amplified in several human cancer types. Specifically, the survey of TCGA for copy number alterations and/or expression showed PAK1 amplification and/or overexpression in ~23% of ER+ breast cancer. To test the hypothesis that PAK1-amplification is related to CDK4/6 inhibitors resistance, we generated two ER+ breast cancer cell lines T47D (harbouring PAK1 amplification) and MCF7 (non PAK1-amplified) resistant to the combination of fulvestrant (ER downregulator) and abemaciclib (CDK4/6 inhibitor), namely T47DFAR and MCF7FAR. T47DFAR and MCF7FAR were obtained, exposing the parental cells to increasing doses of drugs. Interestingly, only the T47DFAR but not the MCF7FAR showed higher level of PAK1 expression and activation compared to the parental cell line. PAK1 pathway activation was evaluated measuring by immunoblot the levels of phosphorylation of PAK1, MEK, ERK and Beta-Catenin. Moreover, the genomic knockdown of PAK1 through RNAi causes a decrease in cell proliferation of T47DFAR, but not of MCF7FAR and parental cells, suggesting the role of PAK1 as a possible mediator of drug resistance. Conversely, overexpression of PAK1 in MCF7 and T47D parental cells was able to generate resistance to the drug combination. PAK-1 has shown to have a different role in the cytosol as pathways mediator and in the nucleus as transcriptional factor. Our data obtained by subcellular fractionation showed that after treatment with fulvestrant-abemaciclib, PAK1 protein accumulates into the cytosol in T47D-FAR compared to parental T47D, probably mediating an enhancement in cytoplasmatic cell signalling and pathway activation. To univocally demonstrate that PAK1 mediates tumor resistance to fulvestrant-abemaciclib combination, we are generating data from RNA-Seq and whole exome sequencing from T47DFAR and MCF7FAR and the relative parental cell lines. Also, we will assess the efficacy of PAK pharmacological inhibition in overcoming such resistance. Conclusions: In conclusion, our data suggest the role of PAK1 as a novel therapeutic target in ER+/PAK1-amplified breast cancer model resistant to endocrine therapy and CDK4/6 inhibitors. Citation Format: Stefania Belli, Alberto Servetto, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Fabiana Napolitano, Antonio Santaniello, Pietro De Placido, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Roberto Bianco, Luigi Formisano. Pak1 as a novel mediator of resistance to endocrine therapy and CDK4/6 inhibitors in ER+/PAK-1amplified breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-08.

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.

The Role of PAK1 in the Maturation of Invadopodia During Transient Mechanical Stimulation.

Cancer cells are affected by a wide range of mechanical forces within their extracellular environment. It has been widely shown that these forces can lead to increased metastatic activity of these cells. One such force is a transient tugging-like force that results from contractile forces generated by cells within the tumor microenvironment. When this force is simulated in vitro with a mechano-invasion assay, human fibrosarcoma cells exhibit enhanced cell invasion in a 3D collagen-fibronectin matrix by downregulating the expression of integrin β3. Furthermore, this force stimulates the maturation of invadopodia in an integrin β3-dependent manner that includes an increase in the active form of cofilin and MMP-2 secretion. In the present study we discovered that the decrease in integrin β3 signaling in response to mechanical stimulation is coupled to the activity of p21-activated kinase 1 (PAK1). It was found that PAK1 has decreased activity, as detected by a decrease in Ser144 phosphorylation, with mechanical stimulation. However, this loss in phosphorylation can be reversed if integrin β3 is overexpressed. Furthermore, PAK1 mutants show a correlated response in MMP-2 enzyme expression and activity, in addition to the lengthening of invadopodia, in response to stimulation. These results identify a novel mechano-sensitive response in human fibrosarcoma that utilizes PAK1 as a signaling player positioned downstream of integrin β3.

PAK1 overexpression promotes cell proliferation in cutaneous T cell lymphoma via suppression of PUMA and p21

BackgroundCutaneous T cell lymphoma (CTCL) comprises a heterogeneous group of skin-homing T cell tumors. The small guanosine triphosphate effector p21-activated kinase 1 (PAK1) plays an important role in many fundamental cellular functions, including cell motility, proliferation, and apoptosis. The expression of PAK1 is up-regulated in several types of human cancers. However, little is known about the role of PAK1 in the pathogenesis of CTCL. ObjectiveThe aim of this study was to evaluate the expression pattern and underlying mechanism of PAK1 in CTCL. MethodsQuantitative real-time polymerase chain reaction(qRT-PCR) was used to detect PAK1 mRNA expression in the peripheral blood mononuclear cells (PBMCs) of patients with CTCL. The expression of PAK1 protein in CTCL tumor tissues was determined by immunohistochemistry. CTCL cell lines were treated with a small molecule inhibitor of PAK1, p21-activated kinase inhibitor III (IPA3), at concentrations of 2, 3.5 and 5 μM for 24 h. Hut 78 and HH CTCL cells were transfected with lentiviral-based PAK1 gene knockdown vectors. We determined the effects of PAK1 knockdown on cell proliferation and apoptosis in CTCL cells by MTS assay and flow cytometry. Animal experiments were performed to investigate the effects of PAK1 knockdown on the growth of tumors in vivo. Transcriptomic sequencing was performed to detect the direct downstream targets of PAK1 silencing. Reverse transcription polymerase chain reaction and western blot analysis were applied to verify the results of the transcriptomic analysis. ResultsWe detected PAK1 overexpression in PBMCs and skin lesions from patients with CTCL compared with benign inflammatory dermatoses (BID). Knockdown of PAK1 inhibited cell proliferation and promoted spontaneous apoptosis. In addition, the inhibitory effect of IPA3 was validated in the CTCL cell lines. Additionally, mice injected with PAK1-silenced cells presented with a decreased rate of tumor growth compared with the control groups. Moreover, the mRNA and protein expression of PUMA (BBC3) and p21 (CDKN1A) were increased in PAK1-silenced Hut 78 and HH cells. ConclusionsOur data indicated that PAK1 is upregulated in CTCL. PAK1 silencing induced apoptosis and inhibited cell growth by stimulating the expression of PUMA and p21. Thus, PAK1 may be a potential tumor marker and therapeutic target of CTCL.

Up-regulation of stem cell markers by P21-activated kinase 1 contributes to 5-fluorouracil resistance of colorectal cancer

ABSTRACTCancer stem cells (CSC) are tumorigenic and resistant to chemotherapy. In colorectal cancer (CRC), CSCs have been identified by the expression of specific markers, including CD44, Bmi1 and Nanog. Although p21-activated kinase 1 (PAK1), acting downstream of Ras, stimulates Wnt/β-catenin signaling and is known to play an important role in CRC development and progression, the role of PAK1 in the expression of CSC markers has not previously been investigated. The effect of PAK1 over-expression, knockdown or inhibition on the expression or alteration (in the case of CD44) of CSC markers in human CRC cell lines was measured by immunofluorescence and Western blotting. The effect of PAK1 modulation on tumorigenesis, and on resistance to treatment with 5-fluorouracil (5-FU), was measured by sphere formation in vitro and by growth of xenografted tumors in vivo. The results show that PAK1 activity correlated with the expression of CSC markers and the CD44 isoform profile, and with tumor growth both in vitro and in vivo. Furthermore PAK overexpression partially overcame the inhibition of CRC growth by 5-FU, and PAK inhibition was synergistic with 5-FU treatment. Our findings lay the foundation for a combination therapy in which PAK1 inhibitors targeting CSCs may be combined with conventional 5-FU-based chemotherapy for the treatment of CRC.

Knockout of p21-activated kinase-1 attenuates exercise-induced cardiac remodelling through altered calcineurin signalling.

Despite its known cardiovascular benefits, the intracellular signalling mechanisms underlying physiological cardiac growth remain poorly understood. Therefore, the purpose of this study was to investigate a novel role of p21-activated kinase-1 (Pak1) in the regulation of exercise-induced cardiac hypertrophy. Wild-type (WT) and Pak1 KO mice were subjected to 6 weeks of treadmill endurance exercise training (ex-training). Cardiac function was assessed via echocardiography, in situ haemodynamics, and the pCa-force relations in skinned fibre preparations at baseline and at the end of the training regimen. Post-translational modifications to the sarcomeric proteins and expression levels of calcium-regulating proteins were also assessed following ex-training. Heart weight/tibia length and echocardiography data revealed that there was marked hypertrophy following ex-training in the WT mice, which was not evident in the KO mice. Additionally, following ex-training, WT mice demonstrated an increase in cardiac contractility, myofilament calcium sensitivity, and phosphorylation of cardiac myosin-binding protein C, cardiac TnT, and tropomyosin compared with KO mice. With ex-training in WT mice, there were also increased protein levels of calcineurin and increased phosphorylation of phospholamban. Our data suggest that Pak1 is essential for adaptive physiological cardiac remodelling and support previous evidence that demonstrates Pak1 signalling is important for cardiac growth and survival.

Abstract 1024: Targeting PAK1 activity in breast cancer: Inhibition of cell growth, survival, motility, and signaling

Abstract Breast cancer (BrCa) is one of the most common cancers diagnosed and is the second leading cause of cancer-related deaths of women in the United States. BrCa is divided into four major molecular subtypes; these categories are based upon the expression of hormone receptors (i.e., estrogen receptor (ER) and progesterone receptor (PR)), HER2/neu receptor, and proliferation rate. Of these subtypes triple negative (ER-, PR-, HER2-) BrCa are among the most lethal, are highly aggressive, have poor prognosis, limited treatment options, and lack effective targeted therapies. Our lab has shown p-21 activated kinase 1 (PAK1) protein expression increases with BrCa progression in human tumor samples. PAK1 is highly expressed in invasive tumor tissues in comparison to normal tissues. The role of PAK1 in BrCa development and progression is through its activity as a serine/threonine kinase involved in the regulation of several key oncogenic pathways (i.e., WNT/β-catenin and MAPK/ERK/JNK). Although PAK1 expression is increased in invasive tumor tissues, our studies have found that in human BrCa cell cultures PAK1 expression is independent of hormonal receptor status. Our hypothesis for this study is that triple negative BrCa cell lines are more reliant upon PAK1's activity for cell growth, survival, and motility. To test our hypothesis two triple negative BrCa cell lines, MDA-MB-231 and MDA-MB-468, and two ER+ BrCa cell lines, T47D and ZR75-1, were used. Our results demonstrate there are significantly higher levels of PAK1 protein in T47D and MDA-MB-468, compared to ZR75-1 and MDA-MB-231. To determine if the cells were reliant on the activity of PAK1 for cell viability, a trypan blue exclusion assay was performed. The PAK1 inhibitor 1,1′-Dithiodi-2-napthol (IPA-3) significantly inhibited all four BrCa cell lines, cell growth and viability in a dose-dependent manner. IPA-3 further inhibited cell proliferation of MDA-MB-468 (IC50 = 11.5μM), MDA-MB-231 (IC50 = 14.5μM), ZR75-1 (IC50 = 13.3μM), and T47D (IC50 = 17.1μM). IPA-3 also inhibited the phosphorylation and nuclear translocation of PAK1 in a dose-dependent manner in MDA-MB-231 cells. The inhibition of PAK1 by IPA-3 also significantly inhibited the migration of MDA-MB-231 cells in a time- and dose-dependent manner. The regulation of PAK1 activation by the WNT/β-catenin pathway was further explored in ER+ and triple negative BrCa cell lines. Additional studies are currently underway investigating the role of PAK1 activation on the MAPK/ERK/JNK pathway and its effects on cell migration and invasion. These studies explore the role of PAK-1 in triple negative BrCa cell growth, survival, motility, and oncogenic signaling. Further investigation of PAK1's targets within these signaling pathways will provide opportunities for the development of novel prevention and/or therapeutic strategies. Citation Format: Alexandra M. Fajardo, Tristan Browne, Hannah Graff, Kelly Kleier, Kyle Neltner, Courtney McCall, Brad Meyer, Larry Douglass, Julia Carter. Targeting PAK1 activity in breast cancer: Inhibition of cell growth, survival, motility, and signaling. [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 1024. doi:10.1158/1538-7445.AM2015-1024

PAK1 is a novel cardiac protective signaling molecule.

We review here the novel cardiac protective effects of the multifunctional enzyme, p21-activated kinase 1 (PAK1), a member of a serine/threonine protein kinase family. Despite the large body of evidence from studies in noncardiac tissue indicating that PAK1 activity is key in the regulation of a number of cellular functions, the role of PAK1 in the heart has only been revealed over the past few years. In this review, we assemble an overview of the recent findings on PAK1 signaling in the heart, particularly its cardiac protective effects. We present a model for PAK1 signaling that provides a mechanism for specifically affecting cardiac cellular processes in which regulation of protein phosphorylation states by protein phosphatase 2A (PP2A) predominates.We discuss the anti-adrenergic and antihypertrophic cardiac protective effects of PAK1, as well as its role in maintaining ventricular Ca(2+) homeostasis and electrophysiological stability under physiological, β-adrenergic and hypertrophic stress conditions.

The role of protein kinase PAK1 in the regulation of estrogen-independent growth of breast cancer

The main goal of this work was to study the intracellular signaling pathways responsible for the development of hormone resistance and maintaining the autonomous growth of breast cancer cells. In particular, the role of PAK1 (p21-activated kinase 1), the key mitogenic signaling protein, in the development of cell resistance to estrogens was analyzed. In vitro studies were performed on cultured breast cancer cell lines: estrogen-dependent estrogen receptor (ER)-positive MCF-7 cells and estrogen-resistant ER-negative HBL-100 cells. We found that the resistant HBL-100 cells were characterized by a higher level of PAK1 and demonstrated PAK1 involvement in the maintaining of estrogen-independent cell growth. We have also shown PAK1 ability to up-regulate Snail1, one of the epithelial-mesenchymal transition proteins, and obtained experimental evidence for Snail1 importance in the regulation of cell proliferation. In general, the results obtained in this study demonstrate involvement of PAK1 and Snail1 in the formation of estrogen-independent phenotype of breast cancer cells showing the potential role of both proteins as markers of hormone resistance of breast tumors.

Abstract 3712: p21-activated kinase-1 in the prevention of inflammation-driven colon cancer.

Abstract Chronic gut inflammation in inflammatory bowel diseases (IBD) predisposes to the development of colorectal cancer, causing increased mortality. Use of mesalamine in the treatment and remittance of ulcerative colitis (UC) modulates the risk of neoplastic progression. We have previously reported (Khare V, et al. Biochem Pharm 2012) that p-21 activated kinase-1 (PAK1) mediates mesalamine activity by orchestrating MAPK signaling; Wnt-β catenin pathway and cell adhesion; all implicated in the colon carcinogenesis. Since mesalamine has primarily an anti-inflammatory function, we evaluated the role of PAK1, its target, in IBD and in colitis-associated cancer (CAC). PAK1 expression was scored by immunohistochemistry in human samples from IBD, CAC and normal mucosa. Compared to normal mucosa and Crohn's disease, a higher PAK1 expression was detected in UC which further increased in CAC. PAK1 expression was also elevated in the inflamed mucosa and carcinomas in IL-10−/− mouse model of colitis. The efficacy of mesalamine in reducing inflammation and tumor incidence in these mice has been previously reported (Brown JB et al, Gastroenetrology 2010). The consequence of PAK1 overexpression was further investigated in vitro. Normal diploid colon epithelial cells (HCEC-1CT) showed higher proliferation (46 ± 3.1% increase) upon overexpression of wildtype (wt) PAK1 while dominant negative (dn) PAK1 overexpression reduced proliferation (25% ± 2.5 decrease) compared to control. Apoptosis (Annexin V staining) was reduced in HCEC-1CT overexpressing wtPAK1 (1.2% ± 0.40) compared to dnPAK1 (11.6% ± 0.3) and control (16% ± 0.83). Mesalamine treatment (20mM) was effective in counteracting the effect of PAK1 overexpression by reducing cell proliferation and inducing apoptosis. Treatment of cells with specific inhibitors (PD98059/LY294002/Rapamycin) of signaling pathways (MEK/PI3K/mTOR) demonstrated that mesalamine regulated PAK1 expression by the MEK, PI3K and mTOR signaling pathways. Our data indicate that (1) PAK1 is upregulated in UC and in CAC, (2) PAK1 may be utilized as a predictive marker for the development of CAC, and (3) PAK1 is a viable target for chemoprevention in the setting of colitis. Mesalamine counteracts PAK1 expression and its effect on cell proliferation and apoptosis. In the longterm management of UC mesalamine may protect from CAC through inhibition of PAK1. Citation Format: Vineeta Khare, Kyle Dammann, Manuela Jambrich, Andres Roig, Jerry Shay, Christoph Gasche. p21-activated kinase-1 in the prevention of inflammation-driven colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3712. doi:10.1158/1538-7445.AM2013-3712

LKB1 Suppresses p21-activated Kinase-1 (PAK1) by Phosphorylation of Thr109 in the p21-binding Domain

The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr(109) in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr(109) by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/-) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.