PAK1 Overexpression Research Articles

Overexpression of the Signaling Integrator Gab2 Accelerates AML Development in Mice with Dnmt3a R878Hand Npm1 cA Mutations

NPM1 and DNMT3A are among the most commonly mutated genes in Acute Myeloid Leukemia (AML), affecting 25-30% of patients. Importantly, these mutations co-occur much more frequently than expected by chance, with ~15% of AML samples containing mutations in both genes.This synergy is recapitulated in mouse models, where mice with either Dnmt3aR878H or Npm1cA mutations develop leukemia with a long latency and low penetrance, whereas mice with both Dnmt3aR878H and Npm1cA mutations develop leukemia in 8-12 months with nearly 100% penetrance (Dovey et al., Blood, 2017; Loberg et al., Leukemia, 2019).To better understand the mechanisms underlying this synergy, and the process of leukemic transformation, we have characterized pre-leukemic bone marrow and 9 independent spontaneous AMLs that have arisen in these mice. These spontaneous AMLs rapidly cause fatal leukemias in secondary recipients; whole genome sequencing of all tumors revealed one or more human AML-like cooperating mutations in each, including canonical mutations in Ptpn11 (E69K, A72V and S506L), an activating KitD818Y mutation, a Cbl exon 9 deletion, a CblY369H mutation,and an amplification of Flt3 associated with >20 fold overexpression of WT Flt3. Characterization of the pre-leukemic transcriptomes and methylomes demonstrated little change in the pre-leukemic state for several months, followed by dramatic and canonical shifts in transcription and methylation after transformation. Remarkably, 8 of the 9 AMLs contained an amplification of the entirety of murine chromosome 7 as the sole structural variant in each genome. We analyzed panel sequencing data from a set of published AMLs arising in this model (Loberg et al., Leukemia, 2019), and also exome sequencing data from a different Npm1 cA driven tumor model (Dovey et al., Blood, 2017); these AMLs also developed complete or partial amplification of chromosome 7 in 7 of 8 and 10 of 15 cases, respectively. Through integration of these datasets, we identified a minimally amplified 8.9 Mbp region on chromosome 7, containing 209 genes, including two candidate genes ( Gab2, Pak1) that show overexpression in both murine and human AMLs with DNMT3A and NPM1 mutations. Retroviral constructs expressing each cDNA (with an IRES-GFP tag) were transduced into lineage depleted bone marrow cells from preleukemic WT, Dnmt3aR878H, Npm1cA or Dnmt3aR878H x Npm1cA mice, and transplanted into recipient mice. Overexpression of GAB2 (but not “Empty Vector” or PAK1) induced significant expansion of hematopoietic cells from the Dnmt3aR878Hx Npm1cAmice (and to a lesser extent, from Npm1cA mice),but not Dnmt3aR878H mice or WT mice ( Figure 1A), suggesting that the mutations in Npm1 and Dnmt3a synergize to account for this phenotype. Strikingly, overexpression of GAB2 in Dnmt3aR878Hx Npm1cAbone marrow led to development of AML in 5 of 5 engrafted mice in less than 5 months (p<10 -6 compared to untransduced marrow; Figure 1B). 0 of 3 engrafted mice with “Empty Vector” (GFP only) in Dnmt3aR878Hx Npm1cAbone marrow and 0 of 3 engrafted mice with overexpression of PAK1 in Dnmt3aR878Hx Npm1cAbone marrow developed leukemia during this 5 month period. Taken together, these data suggest that AML-associated mutations in DNMT3A and NPM1 allow for the selective expansion of cells overexpressing GAB2 as an important step towards leukemic transformation. In human myeloid neoplasms, amplification of chromosome 11q (containing human GAB2) is a recurrent cytogenetic event, observed in 1.5% of cases (Papaemmanuil, NEJM, 2016), and expression of GAB2 across AML samples containing NPM1 mutations is increased ~50% compared to healthy CD34+ cells or promyelocytes. GAB2 is known to bind to GRB2 and SHP2/PTPN11 to facilitate signaling from receptor tyrosine kinases (including FLT3) to downstream pathways (including RAS/RAF/MEK/ERK, PI3K and AKT, in some contexts). These data nominate GAB2 overexpression as a factor that may be relevant for the progression of founding clones with DNMT3A and NPM1 mutations to overt AML. Additional studies to define its protein interactome in preleukemic cells, its mechanism of action, and how leukemia-causing mutations in DNMT3A and NPM1 shape the fitness landscape to select for GAB2 overexpression are underway.

Ivermectin induces nonprotective autophagy by downregulating PAK1 and apoptosis in lung adenocarcinoma cells.

LUAD (Lung adenocarcinoma), the most common subtype of lung carcinoma and one of the highest incidences and mortality cancers in the world remains still a substantial treatment challenge. Ivermectin, an avermectin derivative, has been traditionally used as an antiparasitic agent in human and veterinary medicine practice during the last few decades. Though ivermectin has been shown to be effective against a variety of cancers, however, there is few available data reporting the antitumor effects of ivermectin in LUAD. The effect of ivermectin on cell viability and proliferative ability of LUAD cells was evaluated using CCK-8 and colony formation assay. Apoptosis rate and autophagy flux were detected using flow cytometry based on PI/Annexin V staining and confocal laser scanning microscope based on LC3-GFP/RFP puncta, respectively. Western blotting experiment was conducted to verify the results of changes in apoptosis and autophagy. LUAD-TCGA and GEO databases were used to analyse the expression and predictive value of PAK1 in LUAD patients. Xenograft model and immumohistochemical staining were used for verification of the inhibitor effect of ivermectin in vivo. Ivermectin treatment strikingly impeded the colony formation, and the viability of the cell, along with cell proliferation, and caused the apoptosis and enhanced autophagy flux in LUAD cells. In addition, ivermectin-induced nonprotective autophagy was confirmed by treating LUAD cells with 3-MA, an autophagy inhibitor. Mechanistically, we found that ivermectin inhibited PAK1 protein expression in LUAD cells and we confirmed that overexpression of PAK1 substantially inhibited ivermectin-induced autophagy in LUAD cells. Based on TCGA and GEO databases, PAK1 was highly expressed in LUAD tissues as compared with normal tissues. Furthermore, LUAD patients with high PAK1 level have poor overall survival. Finally, in vivo experiments revealed that ivermectin efficiently suppressed the cellular growth of LUAD among nude mice. This study not only revealed the mechanism of ivermectin inhibited the growth of LUAD but also supported an important theoretical basis for the development of ivermectin during the therapy for LUAD.

Identification of Balanol As a Potential Inhibitor of PAK1 That Induces Apoptosis and Cytoprotective Autophagy in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common malignancy of the gastrointestinal tract, often accompanied by poor prognosis and high incidence and mortality. p21 activated kinases (PAKs) have been used as therapeutic targets because of their central role in many oncogenic signaling networks. By exploring tumor databases, we found that PAK1 overexpression is associated with poor prognosis in colorectal cancer, and therefore, PAK1-targeted inhibition is a new potential therapeutic strategy for colorectal cancer. We identified that Balanol (compound 6, DB04098) can effectively target PAK1 by high-throughput virtual screening. In vitro, compound 6 exhibited favorable PAK1 inhibition with potent anti-proliferative and anti-migration activity in SW480 cells. Additionally, we also found that compound 6 induced apoptosis and cytoprotective autophagy in SW480 cells. Together, these results indicate that compound 6 is a potential novel PAK1 inhibitor, which would be utilized as a candidate compound for future CRC treatment.

Pak1 pathway hyper-activation mediates resistance to endocrine therapy and CDK4/6 inhibitors in ER+ breast cancer

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) have been approved in combination with endocrine therapy (ET) to treat estrogen receptor-positive (ER+) metastatic breast cancer (BC). However, drug resistance represents the leading cause of breast cancer patients mortality. This study aimed to identify novel resistance mechanisms to ER antagonists in combination with CDK4/6 inhibitors. We generated two ER+ BC cell lines, T47D and MCF7, resistant to the combination of the ER antagonist fulvestrant and CDK4/6i abemaciclib, named T47D-FAR and MCF7-FAR. Transcriptomic analysis revealed common up-regulation of genes involved in MAPK and epithelial to mesenchymal transition (EMT) pathways in FAR cells, sustaining their hyper-invasive phenotype and increased anchorage-independent growth, compared to sensitive cells. FAR cells showed higher p21-activated kinase 1 (Pak1) expression and phosphorylation levels than parental cells. PAK1 knockdown by siRNAs hampered cell proliferation, reduced anchorage-independent growth and invasive properties of T47D-FAR and MCF7-FAR, re-sensitizing them to fulvestrant and abemaciclib. Conversely, over-expression of PAK1 in MCF7 and T47D cells increased tumor spheroids’ growth and invasion and reduced sensitivity to fulvestrant and abemaciclib, confirming its role in inducing drug resistance. Finally, treatment with Pak1 inhibitors, PF-3758309 (PF309) and NVS-PAK1-1, restored cell sensitivity to fulvestrant and abemaciclib of MCF7-FAR and T47D-FAR cells, both in vitro and in vivo. In conclusion, our data suggested a pivotal role for Pak1 in resistance to ET and CDK4/6i in ER+ breast cancers. These data might promote the rationale for the development of novel Pak1 inhibitors for treatment of patients with ER+ BC progressing on ET plus CDK4/6i.

Mechanism by Which PF-3758309, a Pan Isoform Inhibitor of p21-Activated Kinases, Blocks Reactivation of HIV-1 Latency.

The "block and lock" strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The "block" refers to a compound's ability to inhibit latent HIV-1 proviral transcription, while the "lock" refers to its capacity to induce permanent proviral silencing. We previously identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal. The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ naïve and central memory T cells express high levels of PAK2 and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant, decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-κB signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency.

PAK1 overexpression promotes myxofibrosarcoma angiogenesis through STAT5B-mediated CSF2 transactivation: clinical and therapeutic relevance of amplification and nuclear entry.

Myxofibrosarcoma is genetically complex without established nonsurgical therapies. In public datasets, PAK1 was recurrently gained with mRNA upregulation. Using myxofibrosarcoma cells, we explored the oncogenic underpinning of PAK1 with genetic manipulation and a pan-PAK inhibitor (PF3758309). Myxofibrosarcoma specimens were analyzed for the levels of PAK1, phospho-PAKT423, CSF2 and microvascular density (MVD) and those of PAK1 gene and mRNA. PAK1-expressing xenografts were assessed for the effects of PF3758309 and CSF2 silencing. Besides pro-proliferative and pro-migrator/pro-invasive attributes, PAK1 strongly enhanced angiogenesis in vitro, which, not phenocopied by PAK2-4, was identified as CSF2-mediated using antibody arrays. PAK1 underwent phosphorylation at tyrosines153,201,285 and threonine423 to facilitate nuclear entry, whereby nuclear PAK1 bound STAT5B to co-transactivate the CSF2 promoter, increasing CSF2 secretion needed for angiogenesis. Angiogenesis driven by PAK1-upregulated CSF2 was negated by CSF2 silencing, anti-CSF2, and PF3758309. Clinically, overexpressed whole-cell phospho-PAKT423, related to PAK1 amplification, was associated with increased grades, stages, and PAK1 mRNA, higher MVD, and CSF2 overexpression. Overexpressed whole-cell phospho-PAKT423 and CSF2 independently portended shorter metastasis-free survival and disease-specific survival, respectively. In vivo, both CSF2 silencing and PF3758309 suppressed PAK1-driven tumor proliferation and angiogenesis. Conclusively, the nuclear entry of overexpressed/activated PAK1 endows myxofibrosarcomas with pro-angiogenic function, highlighting the vulnerable PAK1/STAT5B/CSF2 regulatory axis.

Development and Utility of a PAK1-Selective Degrader.

Overexpression of PAK1, a druggable kinase, is common in several malignancies, and inhibition of PAK1 by small molecules has been shown to impede the growth and survival of such cells. Potent inhibitors of PAKs 1-3 have been described, but clinical development has been hindered by recent findings that PAK2 function is required for normal cardiovascular function in adult mice. A unique allosteric PAK1-selective inhibitor, NVS-PAK1-1, provides a potential path forward, but has modest potency. Here, we report the development of BJG-05-039, a PAK1-selective degrader consisting of NVS-PAK1-1 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon. BJG-05-039 induced selective degradation of PAK1 and displayed enhanced anti-proliferative effects relative to its parent compound in PAK1-dependent, but not PAK2-dependent, cell lines. Our findings suggest that selective PAK1 degradation may confer more potent pharmacological effects compared with catalytic inhibition and highlight the potential advantages of PAK1-targeted degradation.

High tumour mutational burden and EGFR/MAPK pathway activation are therapeutic targets in metastatic porocarcinoma.

Eccrine porocarcinoma (EPC) is a rare skin cancer arising from the eccrine sweat glands. Due to the lack of effective therapies, metastasis is associated with a high mortality rate. To investigate the drivers of EPC progression. We carried out genomic and transcriptomic profiling of metastatic EPC (mEPC), validation of the observed alterations in an EPC patient-derived cell line, confirmation of relevant observations in a large patient cohort of 30 tumour tissues, and successful treatment of a patient with mEPC under the identified treatment regimens. mEPC was characterized by a high tumour mutational burden (TMB) with an ultraviolet signature, widespread copy number alterations and gene expression changes that affected cancer-relevant cellular processes such as cell cycle regulation and proliferation, including a pathogenic TP53 (tumour protein 53) mutation, a copy number deletion in the CDKN2A (cyclin dependent kinase inhibitor 2A) region and a CTNND1/PAK1 [catenin delta 1/p21 (RAC1) activated kinase 1] gene fusion. The overexpression of EGFR (epidermal growth factor receptor), PAK1 and MAP2K1 (mitogen-activated protein kinase kinase 1; also known as MEK1) genes translated into strong protein expression and respective pathway activation in the tumour tissue. Furthermore, a patient-derived cell line was sensitive to EGFR and MEK inhibition, confirming the functional relevance of the pathway activation. Immunohistochemistry analyses in a large patient cohort showed the relevance of the observed changes to the pathogenesis of EPC. Our results indicate that mEPC should respond to immune or kinase inhibitor therapy. Indeed, the advanced disease of our index patient was controlled by EGFR-directed therapy and immune checkpoint inhibition for more than 2years. Molecular profiling demonstrated high TMB and EGFR/MAPK pathway activation to be novel therapeutic targets in mEPC.

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.

P21-Activated kinase 3 promotes cancer stem cell phenotypes through activating the Akt-GSK3β-β-catenin signaling pathway in pancreatic cancer cells.

Relative to several other p21-activated kinase (PAK) family members, the role of PAK3 in regulating cancer cell functions remains unclear. Our study obtained evidence that PAK3 regulates the Akt-GSK3β-β-catenin signaling by acting as Ser473-Akt kinase in several pancreatic cancer cell lines. Specifically, knockdown of PAK3 or overexpression of dominant-negative PAK3 inhibited the phosphorylation of Ser473-Akt and GSK3β, resulting in the proteasomal degradation of β-catenin. Conversely, overexpression of PAK3 led to activation of Akt signaling and increased β-catenin expression. These changes, however, were not noted with the silencing and/or overexpression of PAK1, PAK2, or PAK4, which underlies the impetus of PAK3 as a key effector in governing malignant phenotypes in these pancreatic cancer cells, including cancer stem cell (CSC) expansion. Accordingly, PAK3 depletion effectively suppresses tumorsphere formation, ALDH activity, and the expression of CSC surface markers. Moreover, we used a stable knockdown clone of AsPC-1 cells to demonstrate the in vivo efficacy of PAK3 inhibition in suppressing tumorigenesis and xenograft tumor growth. Together, these findings suggest the potential role of PAK3 as a target for pancreatic cancer therapy, which warrants further investigations.

Group I Paks support muscle regeneration and counteract cancer-associated muscle atrophy.

BackgroundSkeletal muscle is characterized by an efficient regeneration potential that is often impaired during myopathies. Understanding the molecular players involved in muscle homeostasis and regeneration could help to find new therapies against muscle degenerative disorders. Previous studies revealed that the Ser/Thr kinase p21 protein‐activated kinase 1 (Pak1) was specifically down‐regulated in the atrophying gastrocnemius of Yoshida hepatoma‐bearing rats. In this study, we evaluated the role of group I Paks during cancer‐related atrophy and muscle regeneration.MethodsWe examined Pak1 expression levels in the mouse Tibialis Anterior muscles during cancer cachexia induced by grafting colon adenocarcinoma C26 cells and in vitro by dexamethasone treatment. We investigated whether the overexpression of Pak1 counteracts muscle wasting in C26‐bearing mice and in vitro also during interleukin‐6 (IL6)‐induced or dexamethasone‐induced C2C12 atrophy. Moreover, we analysed the involvement of group I Paks on myogenic differentiation in vivo and in vitro using the group I chemical inhibitor IPA‐3.ResultsWe found that Pak1 expression levels are reduced during cancer‐induced cachexia in the Tibialis Anterior muscles of colon adenocarcinoma C26‐bearing mice and in vitro during dexamethasone‐induced myotube atrophy. Electroporation of muscles of C26‐bearing mice with plasmids directing the synthesis of PAK1 preserves fiber size in cachectic muscles by restraining the expression of atrogin‐1 and MuRF1 and possibly by inducing myogenin expression. Consistently, the overexpression of PAK1 reduces the dexamethasone‐induced expression of MuRF1 in myotubes and increases the phospho‐FOXO3/FOXO3 ratio. Interestingly, the ectopic expression of PAK1 counteracts atrophy in vitro by restraining the IL6‐Stat3 signalling pathway measured in luciferase‐based assays and by reducing rates of protein degradation in atrophying myotubes exposed to IL6. On the other hand, we observed that the inhibition of group I Paks has no effect on myotube atrophy in vitro and is associated with impaired muscle regeneration in vivo and in vitro. In fact, we found that mice treated with the group I inhibitor IPA‐3 display a delayed recovery from cardiotoxin‐induced muscle injury. This is consistent with in vitro experiments showing that IPA‐3 impairs myogenin expression and myotube formation in vessel‐associated myogenic progenitors, C2C12 myoblasts, and satellite cells. Finally, we observed that IPA‐3 reduces p38α/β phosphorylation that is required to proceed through various stages of satellite cells differentiation: activation, asymmetric division, and ultimately myotube formation.ConclusionsOur data provide novel evidence that is consistent with group I Paks playing a central role in the regulation of muscle homeostasis, atrophy and myogenesis.

Abstract TP263: Activation of Endothelial p21-Activated Kinase 1 Provides Neuroprotection After Stroke

Stroke is a major cause of death and long-term disability. Pak1 is implicated in many neurological disorders as it has been suggested to promote neuronal survival. However, its function and mechanisms have not been investigated in stroke. We assessed the hypothesis that Pak1 provides neuroprotection in stroke by stimulating the secretion of brain-derived neurotrophic factor (BDNF) in endothelial cells. Transient focal ischemia was induced by middle cerebral artery occlusion (MCAO, 90 mins) in young WT male mice. Two doses of IPA3, a Pak1 specific inhibitor, were injected (i.p.) at 30 minutes prior to stroke and 1.5 hours after stroke. For mechanism studies, lentivirus (LV) encoding Pak1 or IPA3 was used to overexpress or pharmacologically inhibit Pak1 in mouse primary brain microvascular endothelial culture. Then the cells were subjected to OGD (16h) with 48 h re-oxygenation and conditioned media (Endo-CM) were collected subsequently. Mouse primary cortical neuron culture was treated with Endo-CM after OGD (90 mins) and neuronal death was assessed 24 hours later. Pak1 activation (phosphorylated Pak1) was observed in the ischemic hemispheres 6 and 72 hours after MCAO (p-Pak1: sham 94.5± 4.7% versus 6-hour stroke 192.5 ± 10.4% ; sham 94.5± 4.7% versus 72-hour stroke 254.4 ± 32.3%, n = 4 p/g, p&lt;0.05 for both). Administration of IPA3 increased brain infarct size assessed 24 hours after MCAO stroke. In endothelial cultures, overexpression of Pak1 increased BDNF secretion in Endo-CM after OGD (LV-GFP 22.7 ± 2.8 versus LV-Pak1 48.6± 5.5, n = 4 p/g, p&lt;0.05). Accordingly, inhibition of Pak1 attenuated levels of BDNF in Endo-CM (Vehicle 24.5 ± 1.5 versus IPA3 9.4± 0.7, n = 3 p/g, p&lt;0.05). Finally, treatment with Endo-CM collected from Pak1 overexpressing culture reduced neuronal death after OGD (LV-GFP 36 ± 2% versus LV-Pak1 10± 1%, n = 3 p/g, p&lt;0.05), while Endo-CM from cells treated with Pak1 inhibitor increased neuronal mortality (Vehicle 24± 7% versus IPA3 43± 2%, n = 3 p/g, p&lt;0.05). Taken together, we demonstrated that endothelial Pak1 provides neuroprotection after ischemia. Mechanically, our data revealed that activation of Pak1 stimulates endothelial production of a well-known potent neuroprotectant, BDNF.

Transcriptional regulation of ataxia-telangiectasia and Rad3-related protein by activated p21-activated kinase-1 protects keratinocytes in UV-B-induced premalignant skin lesions.

Sun-induced skin lesions, in particular actinic keratosis, are generally considered as premalignant skin lesions that can progress into squamous cell carcinoma (SCC) and invasive SCC if left untreated. Therefore, understanding the molecular mechanisms by which the ultraviolet-B (UV-B)-exposed cells are being protected and the signaling pathways that promote the progression of certain premalignant skin lesions to malignant lesions will permit us to prevent or cure skin cancers. In the current study, we found that phospho-p21-activated kinase-1 (Pak1) and Pak1 expression was high in clinical samples of sunlight-induced premalignant skin lesions assessed by immunohistochemistry. Further, we observed that phospho-Pak1 and Pak1 levels are high in UV-B-exposed hairless SKH mouse model skin samples as compared with unexposed skin tissue. Our results from cell line and animal models showed that Pak1 is activated in response to UV-B radiation, and this activated Pak1 translocates from the cytoplasm to the nucleus. Inside the nucleus, Pak1 via C-Fos binds to a specific promoter region of DNA repair kinase ATR (ataxia-telangiectasia and Rad3-related protein) and acts as a transcriptional regulator of ATR. Results from our analysis showed that Pak1 overexpression, knockdown and Pak1 knockout cell line models showed that Pak1 confers protection to keratinocytes from UV-B-induced apoptosis and DNA damage via ATR. To our knowledge, this is the first study that evaluates the functional and clinical significance of a signaling molecule, Pak1, in sun-induced premalignant skin lesions and indicates that increased Pak1 activation and expression could serve as an early warning sign of progression toward non-melanoma skin cancer, if ignored.

Hsa-miR-485-5p reverses epithelial to mesenchymal transition and promotes cisplatin-induced cell death by targeting PAK1 in oral tongue squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is currently a highly prevalent disease worldwide. Cisplatin (CDDP) is widely used for the chemotherapy of OSCC. Yet, the molecular mechanisms responsible for cisplatin resistance have not been fully elucidated. In this study, we showed that overexpression of p21 (RAC1) activated kinase 1 (PAK1) induced epithelial to mesenchymal transition (EMT) and significantly promoted the invasion and migration of oral squamous cell carcinoma SCC25 cells. Emerging evidence indicates a strong link between resistance to therapy and the induction of EMT in cancer. We showed that overexpression of PAK1 induced cisplatin resistance in SCC25 cells. ERCC1 and YAP can promote cisplatin resistance in human OSCC. We showed that ERCC1 and YAP protein were upregulated by PAK1 in SCC25 cells. We found that miR-485-5p inhibited PAK1 protein expression in the SCC25 cells. Contrary to PAK1, we demonstrated that overexpression of miR-485-5p reversed EMT and significantly inhibited invasion and migration. Moreover, its overexpression sensitized SCC25-CR cells (cisplatin-resistant cells) to cisplatin. Thus, we conclude that miR-485-5p reverses EMT and promotes cisplatin-induced cell death by targeting PAK1 in oral tongue squamous cell carcinoma. This study suggests that PAK1 plays an essential role in the progression of OSCC and it is a potential therapeutic target for OSCC.

P21-activated protein kinase 1 induces the invasion of gastric cancer cells through c-Jun NH2-terminal kinase-mediated activation of matrix metalloproteinase-2.

Gastric cancer(GC) is one of the most common malignancies worldwide. The prognosis of GC is poor, mostly due to widespread metastasis. p21-activated kinase1 (Pak1), the best characterized member of an evolutionarily conserved family of serine/threonine kinases, plays an important role in the regulation of cell morphogenesis, motility, mitosis and angiogenesis. By qRT-PCR and Gelatin zymograph assay, we demonstrated in the present study that stable overexpression of Pak1 induced matrix metalloproteinase (MMP)-2 mRNA expression and activity in the human MKN45 GC cell line. Conversely, knockdown of endogenous Pak1 expression by small interfering RNA (siRNA) decreased MMP-2 mRNA expression and activity in the MKN45 GC cells. Activation of c-Jun N-terminal kinase (JNK) was required for Pak1-induced upregulation of MMP-2 mRNA level and activity. Moreover, upregulation of MMP-2 by Pak1 via the JNK pathway notably promoted the invasion of MKN45 GC cells. Overexpression of MMP-2 mRNA was once again confirmed to be associated with GC metastasis. In conclusion, our results demonstrated for the first time that Pak1 stimulated MMP-2 mRNA expression and activity in MKN45 GC cells. The JNK signaling pathway was involved in Pak1 modulation of MMP-2, which was important for MKN45 GC cell invasiveness.

Overexpression of PAK1 Correlates with Aberrant Expression of EMT Markers and Poor Prognosis in Non-Small Cell Lung Cancer.

Objective: p21-activated kinases (PAKs) are serine/threonine protein kinases. PAK1 and epithelial-mesenchymal transition (EMT) are key therapeutic targets in cancer. The clinical significance of PAK1 and its potential association with EMT phenotype in non-small cell lung cancer (NSCLC) was investigated.Methods: Immunohistochemistry was used to detect the expression of PAK1, and mesenchymal and epithelial markers (vimentin, N-cadherin, and E-cadherin) in 186 cases of NSCLC tissues and 50 cases of tumor-adjacent normal tissues. The correlation of PAK1 with the clinicopathological characteristics, prognosis, and mesenchymal and epithelial markers in NSCLC were analyzed.Results: Compared with the non-tumor tissues, PAK1, vimentin, and N-cadherin levels were markedly elevated in NSCLC tissues, whereas the E-cadherin levels were significantly decreased (P<0.05). The aberrant expression of PAK1 was significantly associated with TNM stage and metastasis (P<0.001). Patients who displayed high expression of PAK1 may achieve a poorer progression-free survival (PFS) and overall survival (OS), compared to those with low expression of PAK1 (P=0.001 and P<0.001). Univariate and multivariate analysis showed that high expression of PAK1 was an independent predictor of poor prognosis [hazard ratio (HR) =2.121, P<0.001, HR=1.928, P=0.001, respectively]. In addition, significant correlations were observed between the EMT markers and OS or PFS (P<0.01). Interestingly, PAK1 expression was positively correlated with vimentin and N-cadherin levels (r=0.473, P<0.001; r=0.526, P<0.001, respectively) and negatively correlated with E-cadherin levels (r=-0.463, P<0.001) in NSCLC tissues.Conclusion: PAK1 may promote NSCLC progression and metastasis through EMT, thereby exhibiting the potential of an efficient prognostic predictor in NSCLC patients.

P21-activated kinase 1 (Pak1) signaling influences therapeutic outcome in pancreatic cancer

Therapeutic resistance to gemcitabine in pancreatic ductal adenocarcinoma (PDAC) is attributed to various cellular mechanisms and signaling molecules that influence as a single factor or in combination. In this study, utilizing in vitro p21-activated kinase 1 (Pak1) overexpression and knockdown cell line models along with in vivo athymic mouse tumor xenograft models and clinical samples, we demonstrate that Pak1 is a crucial signaling kinase in gemcitabine resistance. Pak1 kindles resistance via modulation of epithelial-mesenchymal transition and activation of pancreatic stellate cells. Our results from gemcitabine-resistant and -sensitive cell line models showed that elevated Pak1 kinase activity is required to confer gemcitabine resistance. This was substantiated by elevated levels of phosphorylated Pak1 and ribonucleotide reductase M1 levels in the majority of human PDAC tumors when compared with normal. Delineation of the signaling pathway revealed that Pak1 confers resistance to gemcitabine by preventing DNA damage, inhibiting apoptosis and regulating survival signals via NF-κB. Furthermore, we found that Pak1 is an upstream interacting substrate of transforming growth factor β-activated kinase 1-a molecule implicated in gemcitabine resistance. Molecular mechanistic studies revealed that gemcitabine docks with the active site of Pak1; furthermore, gemcitabine treatment induces Pak1 kinase activity both in vivo and in cell-free system. Finally, results from athymic mouse tumor models illustrated that Pak1 inhibition by IPA-3 enhances the cytotoxicity of gemcitabine and brings about pancreatic tumor regression. To our knowledge, this is the first study illustrating the mechanistic role of Pak1 in causing gemcitabine resistance via multiple signaling crosstalks, and hence Pak1-specific inhibitors will prove to be a better adjuvant with existing chemotherapy modality for PDAC.

The p21-activated kinase (PAK1) is involved in diet-induced beta cell mass expansion and survival in mice and human islets.

Human islets from type 2 diabetic donors are reportedly 80% deficient in the p21 (Cdc42/Rac)-activated kinase, PAK1. PAK1 is implicated in beta cell function and maintenance of beta cell mass. We questioned the mechanism(s) by which PAK1 deficiency potentially contributes to increased susceptibility to type 2 diabetes. Non-diabetic human islets and INS 832/13 beta cells cultured under diabetogenic conditions (i.e. with specific cytokines or under glucolipotoxic [GLT] conditions) were evaluated for changes to PAK1 signalling. Combined effects of PAK1 deficiency with GLT stress were assessed using classic knockout (Pak1 (-/-) ) mice fed a 45% energy from fat/palmitate-based, 'western' diet (WD). INS 832/13 cells overexpressing or depleted of PAK1 were also assessed for apoptosis and signalling changes. Exposure of non-diabetic human islets to diabetic stressors attenuated PAK1 protein levels, concurrent with increased caspase 3 cleavage. WD-fed Pak1 knockout mice exhibited fasting hyperglycaemia and severe glucose intolerance. These mice also failed to mount an insulin secretory response following acute glucose challenge, coinciding with a 43% loss of beta cell mass when compared with WD-fed wild-type mice. Pak1 knockout mice had fewer total beta cells per islet, coincident with decreased beta cell proliferation. In INS 832/13 beta cells, PAK1 deficiency combined with GLT exposure heightened beta cell death relative to either condition alone; PAK1 deficiency resulted in decreased extracellular signal-related kinase (ERK) and B cell lymphoma 2 (Bcl2) phosphorylation levels. Conversely, PAK1 overexpression prevented GLT-induced cell death. These findings suggest that PAK1 deficiency may underlie an increased diabetic susceptibility. Discovery of ways to remediate glycaemic dysregulation via altering PAK1 or its downstream effectors offers promising opportunities for disease intervention.

Abstract PD3-04: PAK-1 amplified breast cancer cell lines are preferentially sensitive to PAK inhibition with G-5555

Abstract The small GTP-binding proteins Rac1 and Cdc42 stimulate activity of the serine/threonine kinase p21-activated kinase-1 (PAK-1) to drive growth factor signaling networks and Ras-driven tumorigenesis. Genomic amplification and over-expression of PAK1 are prevalent in luminal breast cancer and correlate with poor clinical outcome. Here we use a novel and selective small molecule inhibitor, G-5555, of the group I PAKs (PAK1, 2, and 3) to evaluate the importance of PAK1 in promoting growth of PAK1 amplified breast cancer cells. Cell lines with amplification of PAK1 were found to be more sensitive to PAK1 inhibition than non-amplified cell lines. Additionally, reverse phase protein array (RPPA) was used to assess the effects of PAK1 inhibition on a wide range of signaling pathways in both amplified and non-amplified cell lines. Reduced levels of phosphorylation of MEK S298 was observed in all cell lines exposed to G-5555 irrespective of amplification status, consistent with PAK1 inhibition in these cell lines. However, modulation of this downstream PAK1 substrate did not correlate with inhibition of cell proliferation or induction of cell death. Cell lines that showed inhibition of proliferation in response to G-5555 also showed enhanced levels of cell death along with apoptosis. Moreover, G-5555 reduced tumor growth in the PAK1 amplified MDA-MB-175 xenograft tumor model. Finally, we compared the in vitro activity of G-5555 with palbociclib, a recently approved inhibitor of the cyclin-dependent kinases CDK4 and CDK6, in PAK1 amplified luminal breast cancer cell lines. Our data supports PAK1 as an attractive target in PAK1 amplified cells and tumors and suggests that inhibiting PAK1 rather than CDK4/6 in this context may be a more attractive therapeutic strategy. Citation Format: Ong CC, Blackwood E, Jakubiak D, Daemen A, Ramaswamy S, Heise C, Schmidt M, Sanders L, Wilson TR, Huw L, Ndubaku C, Rudolph J, Hoeflich KP, Friedman L, O'Brien T. PAK-1 amplified breast cancer cell lines are preferentially sensitive to PAK inhibition with G-5555. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-04.

The potential role of the NEK6, AURKA, AURKB, and PAK1 genes in adenomatous colorectal polyps and colorectal adenocarcinoma.

Colorectal adenomatous polyp (CRAP) is a major risk factor for the development of sporadic colorectal cancer (CRC). Histone modifications are one of the epigenetic mechanisms that may have key roles in the carcinogenesis of CRC. The objective of the present study is to investigate the alternations in the defined histone modification gene expression profiles in patients with CRAP and CRC. Histone modification enzyme key gene expressions of the CRC, CRAP, and control groups were evaluated and compared using the reverse transcription PCR (RT-PCR) array method. Gene expression analysis was performed in the CRAP group after dividing the patients into subgroups according to the polyp diameter, pathological results, and morphological parameters which are risk factors for developing CRC in patients with CRAP. PAK1, NEK6, AURKA, AURKB, HDAC1, and HDAC7 were significantly more overexpressed in CRC subjects compared to the controls (p < 0.05). PAK1, NEK6, AURKA, AURKB, and HDAC1 were significantly more overexpressed in the CRAP group compared to the controls (p < 0.005). There were no significant differences between the CRAP and CRC groups with regards to PAK1, NEK6, AURKA, or AURKB gene overexpression. PAK1, NEK6, AURKA, and AURKB were significantly in correlation with the polyp diameter as they were more overexpressed in polyps with larger diameters. In conclusion, overexpressions of NEK6, AURKA, AURKB, and PAK1 genes can be used as predictive markers to decide the colonoscopic surveillance intervals after the polypectomy procedure especially in polyps with larger diameters.