P21-activated Kinase Research Articles

Combined blockade of mTOR and p21-activated kinases pathways prevents tumour growth in KRAS-mutated colorectal cancer.

The identification of novel therapeutic strategies for metastatic colorectal cancer (mCRC) patients harbouring KRAS mutations represents an unmet clinical need. In this study, we aimed to clarify the role of p21-activated kinases (Paks) as therapeutic target forKRAS-mutated CRC. Paks expression and activation levels were evaluated in a cohort of KRAS-WT or -mutated CRC patients by immunohistochemistry. The effects of Paks inhibition on tumour cell proliferation and signal transduction were assayed by RNAi and by the use of three pan-Paks inhibitors (PF-3758309, FRAX1036, GNE-2861), evaluating CRC cells, spheroids and tumour xenografts' growth. Paks activation positively correlated withKRASmutational status in both patients and cell lines. Moreover, genetic modulation or pharmacological inhibition of Paks led to a robust impairment of KRAS-mut CRC cell proliferation. However, Paks prolonged blockade induced a rapid tumour adaptation through the hyper-activation of the mTOR/p70S6K pathway. The addition of everolimus (mTOR inhibitor) prevented the growth ofKRAS-mut CRC tumoursin vitro and in vivo,reverting the adaptive tumour resistance to Paks targeting. In conclusion, our results suggest the simultaneous blockade of mTOR and Pak pathways as a promising alternative therapeutic strategy for patients affected byKRAS-mut colorectal cancer.

Abstract P1155: P62 Overexpression Rescues Defective Autophagy Flux In Cardiomyocytes Deficient In P21 Activated Kinase 1

P21-Activated Kinase 1 (PAK1) is a serine-threonine kinase that plays a critical role in cardiomyocyte survival under numerous stressful conditions. However, the underlying mechanisms for PAK1’s cardioprotective effects remain unclear. Autophagy is a lysosome-dependent degradation pathway for eliminating and recycling long-lived proteins and unwanted cellular components, which is essential for cellular homeostasis. In this study, we investigated whether and how PAK1 regulates autophagy in cardiomyocytes. PAK1 was either silenced by siRNA knockdown or overexpressed by adenovirus-mediated gene transfer in H9c2 cardiac myoblast cells. Autophagy flux was determined by measuring LC3-II protein levels or by a fluorescent autophagy reporter with and without the lysosomal protease inhibitors pepstatin A (pepA) and Aloxistatin (E64d). PAK1 downregulation reduced LC3-II levels in the total cell lysates, which were not affected by pepA and E64d, suggesting that knockdown of PAK1 inhibited autophagy flux. Inversely, PAK1 overexpression increased LC3-II levels, which were further elevated by pepA and E64d, suggesting that PAK1 was sufficient to accelerate autophagy flux. Interestingly, the expression of p62, a ubiquitin-binding autophagic adaptor, was concurrently upregulated by PAK1 overexpression or downregulated by PAK1 knockdown, suggesting that p62 may mediates PAK1-dependent autophagy. Indeed, overexpression of p62 restored autophagy flux in the absence of PAK1 (Figure 1). Together, these results suggest that PAK1 is both sufficient and necessary for maintaining normal autophagy function in cardiomyocytes, which is likely mediated by p62.

Circular RNA circ_0000119 promotes cervical cancer cell growth and migration via miR-433-3p/PAK2 axis.

The purpose of this study was to investigate the role of circ_0000119 on CC progression and its molecular mechanism. The expression levels of circ_0000119, miR-433-3p, and p21-activated kinase 2 (PAK2) in CC tissues and cell lines were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay. Cell cycle and apoptosis were assessed by flow cytometry. Cell migration and invasive ability were examined by Transwell assays. Downstream binding targets of circ_0000119 were predicted by online bioinformatics tools and confirmed by dual luciferase reporter gene assay, RNA immunoprecipitation (RIP) assay, and RNA pull-down assay. The role of circ_0000119/miR-433-3p/PAK2 axis in regulating the CC process was explored by rescue experiments. A xenograft model was constructed to further determine the effect of circ_0000119 on CC tumor growth in vivo. Immunohistochemistry (IHC) assay was conducted for Ki67 expression. Circ_0000119 was aberrantly upregulated in CC tissues and cell lines. Knockdown of circ_0000119 inhibited CC cell proliferation, cell cycle progress, migration, invasion, and promoted apoptosis of CC cells. MiR-433-3p was a binding target of circ_0000119, and PAK2 was a downstream gene of miR-433-3p. MiR-433-3p inhibition reversed the inhibitory effect of silencing circ_0000119 on CC progression. In addition, PAK2 overexpression reversed the effect of miR-433-3p on CC progression. PAK2 expression was regulated by circ_0000119 and miR-433-3p. Moreover, circ_0000119 knockdown reduced tumor growth of CC in vivo. Circ_0000119 was upregulated in CC, and circ_0000119 knockdown suppressed CC malignant development through the miR-433-3p/PAK2 axis.

ATRA sensitized the response of hepatocellular carcinoma to Sorafenib by downregulation of p21-activated kinase 1

BackgroundSorafenib resistance greatly reduces the efficacy of treatments in advanced hepatocellular carcinoma (HCC) patients, but the underlying mechanisms are not thoroughly understood. All-trans retinoic acid (ATRA), an anti-leukaemia agent, has attracted considerable attention due to its role in sensitizing cells to other anticancer treatments. We aimed to investigate the combined effect of ATRA and Sorafenib on HCC and the underlying mechanisms.MethodsCCK-8, cell sphere formation, trans-well migration, and wound-healing assays were used to analyse the biological behaviours of HCC cells in vitro. Western blotting and qRT-PCR analysis were conducted to measure the expression of p21 activated kinase 1 (PAK1) and phospho-p21 activated kinase 1 (pPAK1). Xenograft models were established to confirm the synergistic effects of ATRA and Sorafenib in vivo. TUNEL assays and immunohistochemistry were utilized to determine apoptosis, proliferation, PAK1 and pPAK1 levels in tumour tissues.ResultsWe observed that PAK1 was overexpressed in HCC, and its expression was negatively correlated with the survival of patients. PAK1 promoted the proliferation, self-renewal and epithelial-mesenchymal transition of HCC cells. Correlation analysis indicated that the IC50 of Sorafenib was positively correlated with the level of pPAK1 in HCC cell lines. ATRA inhibited the progression of HCC and sensitized HCC response to Sorafenib by downregulation of PAK1, as shown by the calculated coefficient of drug interaction and the data obtained from xenograft models.ConclusionsOur findings indicated that instead of treatment with Sorafenib alone, the combination of ATRA and Sorafenib provides a more effective treatment for HCC patients.2_mPhFeBDQdvWNSurq11yxVideo

Proteomic analysis identifies subgroups of patients with active systemic lupus erythematosus

ObjectiveSystemic lupus erythematosus (SLE) is a clinically and biologically heterogenous autoimmune disease. We aimed to investigate the plasma proteome of patients with active SLE to identify novel subgroups, or endotypes, of patients.MethodPlasma was collected from patients with active SLE who were enrolled in the British Isles Lupus Assessment Group Biologics Registry (BILAG-BR). The plasma proteome was analysed using a data-independent acquisition method, Sequential Window Acquisition of All theoretical mass spectra mass spectrometry (SWATH-MS). Unsupervised, data-driven clustering algorithms were used to delineate groups of patients with a shared proteomic profile.ResultsIn 223 patients, six clusters were identified based on quantification of 581 proteins. Between the clusters, there were significant differences in age (p = 0.012) and ethnicity (p = 0.003). There was increased musculoskeletal disease activity in cluster 1 (C1), 19/27 (70.4%) (p = 0.002) and renal activity in cluster 6 (C6) 15/24 (62.5%) (p = 0.051). Anti-SSa/Ro was the only autoantibody that significantly differed between clusters (p = 0.017). C1 was associated with p21-activated kinases (PAK) and Phospholipase C (PLC) signalling. Within C1 there were two sub-clusters (C1A and C1B) defined by 49 proteins related to cytoskeletal protein binding. C2 and C6 demonstrated opposite Rho family GTPase and Rho GDI signalling. Three proteins (MZB1, SND1 and AGL) identified in C6 increased the classification of active renal disease although this did not reach statistical significance (p = 0.0617).ConclusionsUnsupervised proteomic analysis identifies clusters of patients with active SLE, that are associated with clinical and serological features, which may facilitate biomarker discovery. The observed proteomic heterogeneity further supports the need for a personalised approach to treatment in SLE.

Nitric oxide-induced ribosome collision activates ribosomal surveillance mechanisms

Impairment of protein translation can cause stalling and collision of ribosomes and is a signal for the activation of ribosomal surveillance and rescue pathways. Despite clear evidence that ribosome collision occurs stochastically at a cellular and organismal level, physiologically relevant sources of such aberrations are poorly understood. Here we show that a burst of the cellular signaling molecule nitric oxide (NO) reduces translational activity and causes ribosome collision in human cell lines. This is accompanied by activation of the ribotoxic stress response, resulting in ZAKα-mediated activation of p38 and JNK kinases. In addition, NO production is associated with ZNF598-mediated ubiquitination of the ribosomal protein RPS10 and GCN2-mediated activation of the integrated stress response, which are well-described responses to the collision of ribosomes. In sum, our work implicates a novel role of NO as an inducer of ribosome collision and activation of ribosomal surveillance mechanisms in human cells.

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.

Molecular docking analysis of natural unusual triterpenoids towards PAK1 protein

Triterpenoids are secondary metabolites produced by different natural sources and present in natural sources in the form of free acid or aglycones for triterpenoid saponins in nature. Stellettin B, rhabdaprovidine B, and neviotine A are unusual triterpenoids with their unique skeletal backbone. The serine/threonine p21-activating PAK kinases which play an important pivotal role in intracellular signaling pathways as mediator and join the biochemical process in a cell, such as cytoskeleton dynamics, in cell adhesion, polarity, motility, migration, proliferation, apoptosis, cell division, and in vesicle-mediated transport processes. In this study, molecular docking technique was used for detecting free binding of energy and inhibition constant values of selected triterpenoids towards PAK1 protein. Results showed that that ligand 1 (stellettin B) significantly inhibited the PAK1 enzyme at the value of -8.69 kcal/mol.

Targeting P21-activated kinase suppresses proliferation and enhances chemosensitivity in T-cell lymphoblastic lymphoma.

T-cell lymphoblastic lymphoma (T-LBL) is a highly aggressive non-Hodgkin lymphoma with a poor prognosis. P21-activated kinase (PAK) is a component of the gene expression-based classifier that can predict the prognosis of T-LBL. However, the role of PAK in T-LBL progression and survival remains poorly understood. Herein, we found that the expression of PAK1 was significantly higher in T-LBL cell lines (Jurkat, SUP-T1, and CCRF-CEM) compared to the human T-lymphoid cell line. Moreover, PAK2 mRNA level of 32 relapsed T-LBL patients was significantly higher than that of 37 cases without relapse (P = .012). T-LBL patients with high PAK1 and PAK2 expression had significantly shorter median RFS than those with low PAK1 and PAK2 expression (PAK1, P = .028; PAK2, P = .027; PAK1/2, P = .032). PAK inhibitors, PF3758309 (PF) and FRAX597, could suppress the proliferation of T-LBL cells by blocking the G1/S cell cycle phase transition. Besides, PF could enhance the chemosensitivity to doxorubicin in vitro and in vivo. Mechanistically, through western blotting and RNA sequencing, we identified that PF could inhibit the phosphorylation of PAK1/2 and downregulate the expression of cyclin D1, NF-κB and cell adhesion signaling pathways in T-LBL cell lines. These findings suggest that PAK might be associated with T-LBL recurrence and further found that PAK inhibitors could suppress proliferation and enhance chemosensitivity of T-LBL cells treated with doxorubicin. Collectively, our present study underscores the potential therapeutic effect of inhibiting PAK in T-LBL therapy.

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.

PAK1 contributes to cerebral ischemia/reperfusion injury by regulating the blood-brain barrier integrity

Globally, stroke is one of the leading causes of death and significant contributors to disability. Gaining a thorough comprehension of the underlying pathogenic processes is essential for stroke treatment and prevention. In this study, we investigated the role of p21-activated kinase 1 (PAK1) in stroke by using oxygen-glucose deprivation (OGD) and transient middle cerebral artery occlusion and reperfusion (tMCAO/R) models. We reported that focal ischemia and reperfusion affect the PAK1 expression and activity levels. We further demonstrated that PAK1 is responsible for the endothelial hyperpermeability that occurs in the early stages of ischemia and reperfusion. Additionally, inhibition of PAK1 was discovered to alleviate blood-brain barrier disruption and protect against brain injury induced by tMCAO/R. Mechanistically, we provide the evidence that PAK1 regulates the formation of stress fibers and expression of surface junctional proteins. Together, our findings reveal a pathogenic function of PAK1 in stroke.

PAK5 potentiates slug transactivation of N-cadherin to facilitate metastasis of renal cell carcinoma

Renal cell carcinoma (RCC) is an aggravating cancer with a poor prognosis and a high rate of metastasis. PAK5, a p21-activated kinases, has shown to be overexpressed in a variety of cancers, including RCC. In previous studies, we discovered that PAK5 regulates cell migration and invasion in RCC cell lines. However, the underlying mechanisms remain obscure. In this study, we consolidated that PAK5 confers a pro-metastatic phenotype RCC cells in vitro and exacerbates metastasis in vivo. High PAK5 expression was associated with an advanced TNM stage and a lower overall survival. Furthermore, PAK5 increases the expression level of N-cadherin. In terms of mechanism, PAK5 bound to Slug and phosphorylated it at serine 87. As a result, phosphorylated Slug transactivated N-cadherin, accelerating the epithelial-mesenchymal transition. Collectively, Slug is a novel PAK5 substrate, and PAK5-mediated phosphorylation of Slug-S87 increases N-cadherin and the pro-metastatic phenotype of RCC, implying that phosphorylated Slug-S87 could be a therapeutic target in progressive RCC.

USP24-dependent stabilization of Runx2 recruits a p300/NCOA3 complex to transactivate ADAMTS genes and promote degeneration of intervertebral disc in chronic inflammation mice

BackgroundIntervertebral disc degeneration (IDD) naturally occurs during the aging process. Its occurrence is closely related to chronic inflammation; however, the causal relationship between them is controversial. This study aimed to investigate if inflammation would promote IDD incidence and explore the underlying mechanism.MethodsA chronic inflammation mouse model was established by intraperitoneal injection of lipopolysaccharide (LPS). Enzyme-linked immunosorbent assay was performed to determine proinflammatory cytokines in serum. Histological staining was used to evaluate the degeneration of IVDs. Immunoblots and RT-qPCR analyses were performed to measure protein and mRNA expression levels. Immunoprecipitation, mass spectrometry, and co-immunoprecipitation assays were used to determine the assembly of protein complex.ResultsWe found that an inflammatory microenvironment activated p38 kinase, which phosphorylated the Runx2 transcription factor at the Ser28 site. The phosphorylated Runx2 (pRunx2) then recruited a deubiquitinase, ubiquitin-specific peptidase 24 (USP24), which stabilized pRunx2 and protected it from ubiquitin-dependent proteasomal degradation. The stabilized pRunx2 recruited histone acetyltransferase p300 and nuclear receptor coactivator 3 (NCOA3) to assemble a complex. This NCOA3-p300-pRunx2 complex then transactivated the expression of 13 ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) genes, thereby promoting the degradation of extracellular matrix (ECM) in intervertebral discs (IVDs) and causing IDD. Administration of either a p38 inhibitor (doramapimod), a NCOA3 inhibitor (bufalin), or a p300 inhibitor (EML425) significantly decreased the expression of the 13 ADAMTS genes and slowed the degeneration of IVDs.ConclusionIn summary, our results demonstrate that USP24 protects pRunx2 from proteasomal degradation under chronic inflammation conditions, enabling pRunx2 to transactivate ADAMTS genes and degrade ECM. Our findings provide direct evidence that chronic inflammation triggers IDD and offer a therapeutic strategy for retarding IDD in patients with chronic inflammation.

<i>Alpinia zerumbet</i>: A Review of the Chemistry, Quantity, and Pharmacological Properties of Selected Kavalactones

Alpinia zerumbet or shell ginger is a ginger plant with diverse chemical constituents and medicinal and non-medicinal uses. Dihydro-5,6-dehydrokawain (DDK) and Dehydrokawain (DK) are two kavalactones (also known as kava pyrones or styrylpyrones) from A. zerumbet. Both DDK and DK have a carbonyl group at C2, a methoxy group at C4, and a double bond at C5 and C6. DK has a double bond at C7 and C8 that is absent in DDK. Quantity of DDK in A. zerumbet can be ranked as rhizome > leaf > flower > stem > seed. The pericarp and seed placenta of the fruit has higher quantity of DDK than the leaf. In most plant parts, the contents of DDK are higher than those of DK. Hispidin (HP) is synthesized from DK by hydrolysis. These three kavalactones from A. zerumbet have the most promising pharmacological properties that include insecticidal, fungicidal, antioxidant, inhibition of enzymes, inhibition of Advanced Glycation End-products (AGEs), inhibition of p21-activated kinase 1 (PAK1), inhibition of LIM domain kinase 1 (LIMK1), promotion of hair growth, anti-cancer, inhibition of melanogenesis, anti-inflammatory, anti-obesity, HIV-1 integrase inhibition, neuraminidase inhibition, osteogenic, anti-platelet aggregation, cytoprotective, anti-ulcerative, and singlet oxygen quenching activities. Some fields for further research are suggested. Sources of information in this review were from Google, Google Scholar, Science Direct, PubMed, J-Stage, China National Knowledge Infrastructure (CNKI), and PubChem.

Long-Term Impact of Daily E-cigarette Exposure on the Lungs of Asthmatic Mice.

Although the greater popularity of electronic cigarettes (EC) among asthmatics is alarming, there is limited knowledge of the long-term consequences of EC exposure in asthmatics. Mild asthmatic C57/BL6J adult male and female mice were established by intranasal insufflation with three combined allergens. The asthmatic and age and sex-matched' naïve mice were exposed to air, nicotine-free (propylene glycol [PG]/vegetable glycerin [VG]-only), or PG/VG+Nicotine, 4 hours daily for 3 months. The effects of EC exposure were accessed by measuring cytokines in bronchoalveolar lavage, periodic acid-schiff (PAS) staining, mitochondrial DNA copy numbers (mtCN), and the transcriptome in the lung. Significance was false discovery rate <0.2 for transcriptome and 0.05 for the others. In asthmatic mice, PG/VG+Nicotine increased PAS-positive cells and IL-13 compared to mice exposed to air and PG/VG-only. In naïve mice exposed to PG/VG+Nicotine and PG/VG-only, higher INF-γ was observed compared to mice exposed only to air. PG/VG-only and PG/VG+Nicotine had significantly higher mtCN compared to air exposure in asthmatic mice, while the opposite pattern was observed in non-asthmatic naïve mice. Different gene expression patterns were profoundly found for asthmatic mice exposed to PG/VG+Nicotine compared to PG/VG-only, including genes involved in mitochondrial dysfunction, oxidative phosphorylation, and p21-activated kinase (PAK) signaling. This study provides experimental evidence of the potential impact of nicotine enhancement on the long-term effects of EC in asthmatics compared to non-asthmatics. The findings from this study indicate the potential impact of EC in asthmatics by addressing multiple biological markers. The long-term health outcomes of EC in the susceptible group can be instrumental in supporting policymaking and educational campaigns and informing the public, healthcare providers, and EC users about the underlying risks of EC use.

Neuronal Histone Methyltransferase EZH2 Regulates Neuronal Morphogenesis, Synaptic Plasticity, and Cognitive Behavior in Mice.

The histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)-mediated trimethylation of histone H3 lysine 27 (H3K27me3) regulates neural stem cell proliferation and fate specificity through silencing different gene sets in the central nervous system. Here, we explored the function of EZH2 in early post-mitotic neurons by generating a neuron-specific Ezh2 conditional knockout mouse line. The results showed that a lack of neuronal EZH2 led to delayed neuronal migration, more complex dendritic arborization, and increased dendritic spine density. Transcriptome analysis revealed that neuronal EZH2-regulated genes are related to neuronal morphogenesis. In particular, the gene encoding p21-activated kinase 3 (Pak3) was identified as a target gene suppressed by EZH2 and H3K27me3, and expression of the dominant negative Pak3 reversed Ezh2 knockout-induced higher dendritic spine density. Finally, the lack of neuronal EZH2 resulted in impaired memory behaviors in adult mice. Our results demonstrated that neuronal EZH2 acts to control multiple steps of neuronal morphogenesis during development, and has long-lasting effects on cognitive function in adult mice.

LncRNA PCBP1-AS1 induces osteoporosis by sponging miR-126-5p/PAK2 axis.

Long non-coding RNAs (lncRNAs) act as crucial regulators in osteoporosis (OP). Nonetheless, the effects and potential molecular mechanism of lncRNA PCBP1 Antisense RNA 1 (PCBP1-AS1) on OP remain largely unclear. The aim of this study was to explore the role of lncRNA PCBP1-AS1 in the pathogenesis of OP. Using quantitative real-time polymerase chain reaction (qRT-PCR), osteogenesis-related genes (alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2)), PCBP1-AS1, microRNA (miR)-126-5p, group I Pak family member p21-activated kinase 2 (PAK2), and their relative expression levels were determined. Western blotting was used to examine the expression of PAK2 protein. Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. To examine the osteogenic differentiation, Alizarin red along with ALP staining was used. RNA immunoprecipitation assay and bioinformatics analysis, as well as a dual-luciferase reporter, were used to study the association between PCBP1-AS1, PAK2, and miR-126-5p. The expression of PCBP1-AS1 was pre-eminent in OP tissues and decreased throughout the development of human bone marrow-derived mesenchymal stem cells (hBMSCs) into osteoblasts. PCBP1-AS1 knockdown and overexpression respectively promoted and suppressed hBMSC proliferation and osteogenic differentiation capacity. Mechanistically, PCBP1-AS1 sponged miR-126-5p and consequently targeted PAK2. Inhibiting miR-126-5p significantly counteracted the beneficial effects of PCBP1-AS1 or PAK2 knockdown on hBMSCs' ability to differentiate into osteoblasts. PCBP1-AS1 is responsible for the development of OP and promotes its progression by inducing PAK2 expression via competitively binding to miR-126-5p. PCBP1-AS1 may therefore be a new therapeutic target for OP patients.

A genome-wide genetic screen identifies CYRI-B as a negative regulator of CEACAM3-mediated phagocytosis.

Opsonin-independent phagocytosis mediated by human carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) has evolved to control a subset of human-restricted bacterial pathogens. CEACAM3 engagement triggers rapid GTP-loading of the small GTPase Rac as a master regulator of cytoskeletal rearrangements and lamellipodia-driven internalization. To identify components of the CEACAM3-initiated signaling cascade, we performed a genome-wide CRISPR/Cas9-based screen in human myeloid cells. Following infection with fluorescently labeled bacteria, cells exhibiting elevated phagocytosis (gain-of-function) as well as cells showing reduced phagocytosis (loss-of-function) were sorted and enrichment of individual single-guide RNAs (sgRNAs) was determined by next generation sequencing. Concentrating on genes whose targeting by three distinct sgRNAs consistently resulted in a gain-of-function phenotype, we identified the Rac-GTP-sequestering protein CYRI-B as a negative regulator of CEACAM3-mediated phagocytosis. Clonal HL-60 cell lines with CYRI-B knockout showed enhanced CEACAM3-downstream signaling, such as Rac GTP loading and phosphorylation of PAK kinases, leading to increased phagocytosis of bacteria. Complementation of the CYRI-B knockout cells reverted the knockout phenotype. Our results unravel components of CEACAM3-initiated opsonin-independent phagocytosis on a genome-wide level and highlight CYRI-B as a negative regulator of CEACAM3-initiated signaling in myeloid cells.

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.

Correction to: 'Cardiac deficiency of P21-activated kinase 1 promotes atrial arrhythmogenesis in mice following adrenergic challenge'.

Correction to: 'Cardiac deficiency of P21-activated kinase 1 promotes atrial arrhythmogenesis in mice following adrenergic challenge'.