RESUMO
Our recent studies have identified p21-activated kinase 4 (PAK4) as a key regulator of lipid catabolism in the liver and adipose tissue, but its role in glucose homeostasis in skeletal muscle remains to be explored. In this study, we find that PAK4 levels are highly upregulated in the skeletal muscles of diabetic humans and mice. Skeletal muscle-specific Pak4 ablation or administering the PAK4 inhibitor in diet-induced obese mice retains insulin sensitivity, accompanied by AMPK activation and GLUT4 upregulation. We demonstrate that PAK4 promotes insulin resistance by phosphorylating AMPKα2 at Ser491, thereby inhibiting AMPK activity. We additionally show that skeletal muscle-specific expression of a phospho-mimetic mutant AMPKα2S491D impairs glucose tolerance, while the phospho-inactive mutant AMPKα2S491A improves it. In summary, our findings suggest that targeting skeletal muscle PAK4 may offer a therapeutic avenue for type 2 diabetes.
Assuntos
Proteínas Quinases Ativadas por AMP , Diabetes Mellitus Tipo 2 , Glucose , Resistência à Insulina , Músculo Esquelético , Quinases Ativadas por p21 , Animais , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Glucose/metabolismo , Fosforilação , Músculo Esquelético/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Humanos , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Masculino , Transportador de Glucose Tipo 4/metabolismo , Transportador de Glucose Tipo 4/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismoRESUMO
Background: Lenvatinib is the most common multitarget receptor tyrosine kinase inhibitor for the treatment of advanced hepatocellular carcinoma (HCC). Acquired resistance to lenvatinib is one of the major factors leading to the failure of HCC treatment, but the underlying mechanism has not been fully characterized. Methods: We established lenvatinib-resistant cell lines, cell-derived xenografts (CDXs) and patient-derived xenografts (PDXs) and obtained lenvatinib-resistant HCC tumor tissues for further study. Results: We found that ubiquitin-specific protease 14 (USP14) was significantly increased in lenvatinib-resistant HCC cells and tumors. Silencing USP14 significantly attenuated lenvatinib resistance in vitro and in vivo. Mechanistically, USP14 directly interacts with and stabilizes calcium- and integrin-binding protein 1 (CIB1) by reversing K48-linked proteolytic ubiquitination at K24, thus facilitating the P21-activated kinase 1 (PAK1)-ERK1/2 signaling axis. Moreover, in vivo adeno-associated virus 9 mediated transduction of CIB1 promoted lenvatinib resistance in PDXs, whereas CIB1 knockdown resensitized the response of PDXs to lenvatinib. Conclusions: These findings provide new insights into the role of CIB1/PAK1-ERK1/2 signaling in lenvatinib resistance in HCC. Targeting CIB1 and its pathways may be a novel pharmaceutical intervention for the treatment of lenvatinib-resistant HCC.
Assuntos
Carcinoma Hepatocelular , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas , Compostos de Fenilureia , Quinolinas , Ubiquitina Tiolesterase , Quinases Ativadas por p21 , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Humanos , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Animais , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Camundongos , Linhagem Celular Tumoral , Sistema de Sinalização das MAP Quinases , Camundongos Nus , UbiquitinaçãoRESUMO
Psychiatric disorders are highly inheritable, and most psychiatric disorders exhibit genetic overlap. Recent studies associated the 3q29 recurrent deletion with schizophrenia (SCZ) and autism spectrum disorder (ASD). In this study, we investigated the association of genes in the 3q29 region with SCZ and ASD. TM4SF19 and PAK2 were chosen as candidate genes for this study based on evidence from previous research. We sequenced TM4SF19 and PAK2 in 437 SCZ cases, 187 ASD cases and 524 controls in the Japanese population. Through targeted sequencing, we identified 6 missense variants among the cases (ASD & SCZ), 3 missense variants among controls, and 1 variant common to both cases and controls; however, no loss-of-function variants were identified. Fisher's exact test showed a significant association of variants in TM4SF19 among cases (p=0.0160). These results suggest TM4SF19 variants affect the etiology of SCZ and ASD in the Japanese population. Further research examining 3q29 region genes and their association with SCZ and ASD is thus needed.
Assuntos
Povo Asiático , Transtorno do Espectro Autista , Predisposição Genética para Doença , Esquizofrenia , Humanos , Transtorno do Espectro Autista/genética , Esquizofrenia/genética , Feminino , Masculino , Japão , Povo Asiático/genética , Predisposição Genética para Doença/genética , Quinases Ativadas por p21/genética , Cromossomos Humanos Par 3/genética , Adulto , Mutação de Sentido Incorreto/genética , Estudos de Casos e Controles , Estudos de Associação Genética , População do Leste AsiáticoRESUMO
The GTPase Cdc42 regulates polarized growth in most eukaryotes. In the bipolar yeast Schizosaccharomyces pombe, Cdc42 activation cycles periodically at sites of polarized growth. These periodic cycles are caused by alternating positive feedback and time-delayed negative feedback loops. At each polarized end, negative feedback is established when active Cdc42 recruits the Pak1 kinase to prevent further Cdc42 activation. It is unclear how Cdc42 activation returns to each end after Pak1-dependent negative feedback. We find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. Using experimental and mathematical approaches, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Polaridade Celular , Endocitose , Retroalimentação Fisiológica , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteína cdc42 de Ligação ao GTP , Quinases Ativadas por p21 , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Actinas/metabolismoRESUMO
BACKGROUND: An increasing number of studies have demonstrated the association of circular RNAs (circRNAs) with the pathological processes of various diseases and their involvement in the onset and progression of multiple cancers. Nevertheless, the functional roles and underlying mechanisms of circRNAs in the autophagy regulation of gastric cancer (GC) have not been fully elucidated. METHODS: We used transmission electron microscopy and the mRFP-GFP-LC3 dual fluorescent autophagy indicator to investigate autophagy regulation. The cell counting kit-8 assay, colony formation assay, 5-ethynyl-2'-deoxyuridine incorporation assay, Transwell assay, and Western blot assay were conducted to confirm circPTPN22's influence on GC progression. Dual luciferase reporter assays validated the binding between circPTPN22 and miR-6788-5p, as well as miR-6788-5p and p21-activated kinase-1 (PAK1). Functional rescue experiments assessed whether circPTPN22 modulates PAK1 expression by competitively binding miR-6788-5p, affecting autophagy and other biological processes in GC cells. We investigated the impact of circPTPN22 on in vivo GC tumors using a nude mouse xenograft model. Bioinformatics tools predicted upstream regulatory transcription factors and binding proteins of circPTPN22, while chromatin immunoprecipitation and ribonucleoprotein immunoprecipitation assays confirmed the binding status. RESULTS: Upregulation of circPTPN22 in GC has been shown to inhibit autophagy and promote cell proliferation, migration, and invasion. Mechanistically, circPTPN22 directly binds to miR-6788-5p, subsequently regulating the expression of PAK1, which activates protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation. This modulation ultimately affects autophagy levels in GC cells. Additionally, runt-related transcription factor 1 (RUNX1) negatively regulates circPTPN22 expression, while RNA-binding proteins such as FUS (fused in sarcoma) and ELAVL1 (recombinant ELAV-like protein 1) positively regulate its expression. Inhibition of the autophagy pathway can increase FUS expression, further upregulating circPTPN22 in GC cells, thereby exacerbating the progression of GC. CONCLUSION: Under the regulation of the transcription factor RUNX1 and RNA-binding proteins FUS and ELAVL1, circPTPN22 activates the phosphorylation of Akt and Erk through the miR-6788-5p/PAK1 axis, thereby modulating autophagy in GC cells. Inhibition of autophagy increases FUS, which in turn upregulates circPTPN22, forming a positive feedback loop that ultimately accelerates the progression of GC.
Assuntos
Autofagia , Movimento Celular , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core , Proteína Semelhante a ELAV 1 , MicroRNAs , RNA Circular , Proteína FUS de Ligação a RNA , Neoplasias Gástricas , Quinases Ativadas por p21 , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Autofagia/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Proliferação de Células/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Movimento Celular/genética , Linhagem Celular Tumoral , Animais , Proteína Semelhante a ELAV 1/metabolismo , Proteína Semelhante a ELAV 1/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Camundongos , Invasividade Neoplásica , Camundongos Endogâmicos BALB CRESUMO
Neurofibromatosis type 2 is a genetic disorder that results in the formation and progressive growth of schwannomas, ependymomas, and/or meningiomas. The NF2 gene encodes the Merlin protein, which links cell cortical elements to the actin cytoskeleton and regulates a number of key enzymes including Group I p21-activated kinases (PAKs), the Hippo-pathway kinase LATS, and mTORC. While PAK1 and PAK2 directly bind Merlin and transmit proliferation and survival signals when Merlin is mutated or absent, inhibition of Group 1 PAKs alone has not proven sufficient to completely stop the growth of NF2-deficient meningiomas or schwannomas in vivo, suggesting the need for a second pathway inhibitor. As the Hippo pathway is also activated in NF2-deficient cells, several inhibitors of the Hippo pathway have recently been developed in the form of YAP-TEAD binding inhibitors. These inhibitors prevent activation of pro-proliferation and anti-apoptotic Hippo pathway effectors. In this study, we show that PAK inhibition slows cell proliferation while TEAD inhibition promotes apoptotic cell death. Finally, we demonstrate the efficacy of PAK and TEAD inhibitor combinations in several NF2-deficient Schwannoma cell lines.
Assuntos
Proliferação de Células , Via de Sinalização Hippo , Neurilemoma , Neurofibromina 2 , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Fatores de Transcrição , Quinases Ativadas por p21 , Humanos , Neurilemoma/metabolismo , Neurilemoma/genética , Neurilemoma/patologia , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genética , Proliferação de Células/efeitos dos fármacos , Neurofibromina 2/genética , Neurofibromina 2/deficiência , Neurofibromina 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Apoptose/efeitos dos fármacos , Sinergismo Farmacológico , Neurofibromatose 2/metabolismo , Neurofibromatose 2/genética , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
BACKGROUND: The intermediate filament protein vimentin is widely recognized as a molecular marker of epithelial-to-mesenchymal transition. Although vimentin expression is strongly associated with cancer metastatic potential, the exact role of vimentin in cancer metastasis and the underlying mechanism of its pro-metastatic functions remain unclear. RESULTS: This study revealed that vimentin can enhance integrin ß1 surface expression and induce integrin-dependent clustering of cells, shielding them against anoikis cell death. The increased integrin ß1 surface expression in suspended cells was caused by vimentin-mediated protection of the internal integrin ß1 pool against lysosomal degradation. Additionally, cell detachment was found to induce vimentin Ser38 phosphorylation, allowing the translocation of internal integrin ß1 to the plasma membrane. Furthermore, the use of an inhibitor of p21-activated kinase PAK1, one of the kinases responsible for vimentin Ser38 phosphorylation, significantly reduced cancer metastasis in animal models. CONCLUSIONS: These findings suggest that vimentin can act as an integrin buffer, storing internalized integrin ß1 and releasing it when needed. Overall, this study provides insights regarding the strong correlation between vimentin expression and cancer metastasis and a basis for blocking metastasis using this novel therapeutic mechanism.
Assuntos
Anoikis , Integrina beta1 , Vimentina , Vimentina/metabolismo , Vimentina/genética , Integrina beta1/metabolismo , Integrina beta1/genética , Humanos , Animais , Sobrevivência Celular , Camundongos , Linhagem Celular Tumoral , Fosforilação , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genéticaAssuntos
Carcinoma de Células Renais , Neoplasias Renais , Terapia de Alvo Molecular , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/antagonistas & inibidores , Imunoterapia/métodos , Quimera de Direcionamento de ProteóliseRESUMO
Acral lentiginous melanoma (ALM) is the most common subtype of acral melanoma. Even though recent genetic studies are reported in acral melanomas, the genetic differences between in-situ and invasive ALM remain unclear. We aimed to analyze specific genetic changes in ALM and compare genetic differences between in-situ and invasive lesions to identify genetic changes associated with the pathogenesis and progression of ALM. We performed whole genome sequencing of 71 tissue samples from 29 patients with ALM. Comparative analyses were performed, pairing in-situ ALMs with normal tissues and, furthermore, invasive ALMs with normal and in-situ tissues. Among 21 patients with in-situ ALMs, 3 patients (14.3%) had SMIM14 , SLC9B1 , FRG1 , FAM205A , ESRRA , and ESPN mutations, and copy number (CN) gains were identified in only 2 patients (9.5%). Comparing 13 invasive ALMs with in-situ tissues, CN gains were identified in GAB2 in 8 patients (61.5%), PAK1 in 6 patients (46.2%), and UCP2 and CCND1 in 5 patients (38.5%). Structural variants were frequent in in-situ and invasive ALM lesions. Both in-situ and invasive ALMs had very low frequencies of common driver mutations. Structural variants were common in both in-situ and invasive ALMs. Invasive ALMs had markedly increased CN gains, such as GAB2 , PAK1 , UCP2 , and CCND1 , compared with in-situ lesions. These results suggest that they are associated with melanoma invasion.
Assuntos
Biomarcadores Tumorais , Ciclina D1 , Variações do Número de Cópias de DNA , Melanoma , Invasividade Neoplásica , Neoplasias Cutâneas , Sequenciamento Completo do Genoma , Quinases Ativadas por p21 , Humanos , Melanoma/genética , Melanoma/patologia , Masculino , Feminino , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Quinases Ativadas por p21/genética , Pessoa de Meia-Idade , Idoso , Ciclina D1/genética , Biomarcadores Tumorais/genética , Adulto , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso de 80 Anos ou mais , Predisposição Genética para Doença , Mutação , Fenótipo , Proteína Desacopladora 2RESUMO
This study aimed to explore the expression, function, and mechanisms of TBC1D10B in colon cancer, as well as its potential applications in the diagnosis and treatment of the disease.The expression levels of TBC1D10B in colon cancer were assessed by analyzing the TCGA and CCLE databases. Immunohistochemistry analysis was conducted using tumor and adjacent non-tumor tissues from 68 colon cancer patients. Lentiviral infection techniques were employed to silence and overexpress TBC1D10B in colon cancer cells. The effects on cell proliferation, migration, and invasion were evaluated using CCK-8, EDU, wound healing, and Transwell invasion assays. Additionally, GSEA enrichment analysis was used to explore the association of TBC1D10B with biological pathways related to colon cancer. TBC1D10B was significantly upregulated in colon cancer and closely associated with patient prognosis. Silencing of TBC1D10B notably inhibited proliferation, migration, and invasion of colon cancer cells and promoted apoptosis. Conversely, overexpression of TBC1D10B enhanced these cellular functions. GSEA analysis revealed that TBC1D10B is enriched in the AKT/PI3K/mTOR signaling pathway and highly correlated with PAK4. The high expression of TBC1D10B in colon cancer is associated with poor prognosis. It influences cancer progression by regulating the proliferation, migration, and invasion capabilities of colon cancer cells, potentially acting through the AKT/PI3K/mTOR signaling pathway. These findings provide new targets and therapeutic strategies for the treatment of colon cancer.
Assuntos
Apoptose , Movimento Celular , Proliferação de Células , Neoplasias do Colo , Proteínas Ativadoras de GTPase , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Quinases Ativadas por p21 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genéticaRESUMO
By taking advantage of forward genetic analysis in mice, we have demonstrated that Pak1 plays a crucial role during DMBA/TPA skin carcinogenesis. Although Pak1 has been considered to promote cancer development, its overall function remains poorly understood. To clarify the functional significance of Pak1 in detail, we sought to evaluate the possible effect of an allosteric inhibitor against PAK1 (NVS-PAK1-1) on a syngeneic mouse model. To this end, we established two cell lines, 9AS1 and 19AS1, derived from DMBA/TPA-induced squamous cell carcinoma (SCC) that engrafted in FVB mice. Based on our present results, NVS-PAK1-1 treatment significantly inhibited the growth of tumors derived from 9AS1 and 19AS1 cells in vitro and in vivo. RNA-sequencing analysis on the engrafted tumors indicates that NVS-PAK1-1 markedly potentiates the epidermal cell differentiation and enhances the immune response in the engrafted tumors. Consistent with these observations, we found an expansion of Pan-keratin-positive regions and potentially elevated infiltration of CD8-positive immune cells in NVS-PAK1-1-treated tumors as examined by immunohistochemical analyses. Together, our present findings strongly suggest that PAK1 is tightly linked to the development of SCC, and that its inhibition is a promising therapeutic strategy against SCC.
Assuntos
Carcinoma de Células Escamosas , Modelos Animais de Doenças , Neoplasias Cutâneas , Quinases Ativadas por p21 , Animais , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Camundongos , Linhagem Celular Tumoral , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Feminino , Diferenciação Celular/efeitos dos fármacos , Acetato de Tetradecanoilforbol , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
BACKGROUND: Erectile dysfunction (ED) is a common male sexual dysfunction, with an increasing incidence, and the current treatment is often ineffective. METHODS: Vascular endothelial growth factor (VEGFA) was used to treat bone marrow-derived mesenchymal stem cells (BM-MSCs), and their cell migration rates were determined by Transwell assays. The expression of the von Willebrand Factor (vWF)VE-cadherin, and endothelial nitric oxide synthase(eNOS) endothelial markers was determined by qRTâPCR and Western blot analyses. The MALAT1-induced differentiation of BM-MCs to ECs via the CDC42/PAK1/paxillin pathway was explored by transfecting VEGFA-induced BM-MSC with si-MALAT1 and overexpressing CDC42 and PAK1. The binding capacity between CDC42, PAK1, and paxillin in VEGFA-treated and non-VEGFA-treated BM-MSCs was examined by protein immunoprecipitation. MiR-206 was overexpressed in VEGFA-induced BM-MSC, and the binding sites of MALAT1, miR-206, and CDC42 were identified using a luciferase assay. Sixty male SpragueâDawley rats were divided into six groups (n = 10/group). DMED modelling was demonstrated by APO experiments and was assessed by measuring blood glucose levels. Erectile function was assessed by measuring the intracavernosa pressure (ICP) and mean arterial pressure (MAP). Penile erectile tissue was analysed by qRTâPCR, Western blot analysis, and immunohistochemical staining. RESULTS: MALAT1 under VEGFA treatment conditions regulates the differentiation of BM-MSCs into ECs by modulating the CDC42/PAK1/paxillin axis. In vitro experiments demonstrated that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs. CDC42 binds to PAK1, and PAK1 binds to paxillin. In addition, CDC42 in the VEGFA group had a greater ability to bind to PAK1, whereas PAK1 in the VEGFA group had a greater ability to bind to paxillin. Overexpression of miR-206 in VEGFA-induced BM-MSCs demonstrated that MALAT1 competes with the CDC42 3'-UTR for binding to miR-206, which in turn is involved in the differentiation of BM-MSCs to ECs. Compared to the DMED model group, the ICP/MAP ratio was significantly greater in the three BM-MSCs treatment groups. CONCLUSIONS: MALAT1 facilitates BM-MSC differentiation into ECs by regulating the miR-206/CDC42/PAK1/paxillin axis to improve ED. The present findings revealed the vital role of MALAT1 in the repair of BM-MSCs for erectile function and provided new mechanistic insights into the BM-MSC-mediated repair of DMED.
Assuntos
Diferenciação Celular , Disfunção Erétil , Células-Tronco Mesenquimais , MicroRNAs , Paxilina , RNA Longo não Codificante , Ratos Sprague-Dawley , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP , Quinases Ativadas por p21 , Masculino , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Diferenciação Celular/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Ratos , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Células-Tronco Mesenquimais/metabolismo , Disfunção Erétil/terapia , Disfunção Erétil/genética , Disfunção Erétil/metabolismo , Paxilina/metabolismo , Paxilina/genética , Células Endoteliais/metabolismo , Células Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Hypoxia-inducible factors (HIF) 1 and 2 regulate similar but distinct sets of target genes. Although HIFs are best known for their roles in mediating the hypoxia response accumulating evidence suggests that under certain conditions HIFs, particularly HIF2, may function also under normoxic conditions. Here we report that HIF2α functions under normoxic conditions in kidney epithelial cells to regulate formation of adherens junctions. HIF2α expression was required to induce Dock4/Rac1/Pak1-signaling mediating stability and compaction of E-cadherin at nascent adherens junctions. Impaired adherens junction formation in HIF2α- or Dock4-deficient cells led to aberrant cyst morphogenesis in 3D kidney epithelial cell cultures. Taken together, we show that HIF2α functions in normoxia to regulate epithelial morphogenesis.
Assuntos
Junções Aderentes , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Polaridade Celular , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP , Junções Aderentes/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Caderinas/metabolismo , Caderinas/genética , Camundongos , Humanos , Células Epiteliais/metabolismo , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Linhagem CelularRESUMO
Pancreatic ductal adenocarcinoma (PDA) is a common malignancy with a 5-year survival <10 %. Immunosuppressive tumor microenvironment (TME) plays a critical role in the progression of PDA. In recent years, programmed death-ligand 1 (PD-L1)/programmed cell death protein-1 (PD-1) blockade has emerged as a potent anti-tumor immunotherapy, while is yet to achieve significant clinical benefits for PDA patients. P21-Activated kinase 1 (PAK1) is highly upregulated in PDA and has been reported to be involved in the regulation of anti-tumor immunity. This study aims to investigate the combined effect of PAK1 inhibition and anti-PD-1 therapy on PDA and the underlying mechanisms. We have shown that PAK1 expression positively correlated with PD-L1 in PDA patients, and that inhibition of PAK1 downregulated PD-L1 expression of PDA cells. More importantly, we have demonstrated that PAK1 competed with PD-L1 in binding to tripartite motif-containing protein 21 (TRIM21), a ubiquitin E3 ligase, resulting in less ubiquitination and degradation of PD-L1. Moreover, PAK1 inhibition promoted CD8+ T cells activation and infiltration. In a murine PDA model, the combination of PAK1 inhibition and anti-PD-1 therapy showed significant anti-tumor effects compared with the control or monotherapy. Our results indicated that the combination of PAK1 inhibition and anti-PD-1 therapy would be a more effective treatment for PDA patients.
Assuntos
Antígeno B7-H1 , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Quinases Ativadas por p21 , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genética , Humanos , Animais , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/imunologia , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Camundongos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/imunologia , Linhagem Celular Tumoral , Feminino , Masculino , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Proteólise/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Ovarian cancer is the most common cause of gynecological cancer death. Pak4 has been proved to be tumorigenic in many types of cancers, but its role in ovarian cancer is still not clarified. METHODS: In this study, we used immunohistochemistry to investigate into Pak4 expression in different histological types of ovarian cancer. TIMER, TISCH2, GEPIA, ualcan, KM plotter, GSCA and GeneMANIA were used to identify the prognostic roles and gene regulation networks of Pak4 in ovarian cancer. Immune infiltration levels were investigated using TIMER database. RESULTS: Pak4 was highly expressed in ovarian cancers, regardless of different FIGO stages and histological grades. Single cell sequencing database proved that Pak4 was highly expressed in malignant ovarian cancer cells. Pak4 level was significantly correlated with different histological types of ovarian cancer. Pak4 expression was negatively connected with OS and PFS of ovarian cancer patients. Functions of Pak4 and its interacted genes were mainly involved in protein serine/threonine kinase activity, regulation of actin filament-based process and regulation of cytoskeleton organization. Pak4 level was negatively correlated with immune biomarkers of B cell infiltration (p = 2.39e-05), CD8 + T cell infiltration (p = 1.51e-04), neutrophil (p = 1.74e-06) and dendritic cell (p = 4.41e-08). Close correlation was found between Pak4 expression and T cell exhaustion (p < 0.05). CONCLUSIONS: Our results demonstrated the expression level, gene interaction networks and immune infiltration levels of Pak4 in ovarian cancer. And the results revealed role of Pak4 in tumorigenesis and the possibility to be a potential immunotherapeutic target.
Assuntos
Neoplasias Ovarianas , Quinases Ativadas por p21 , Humanos , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Feminino , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/imunologia , Regulação Neoplásica da Expressão Gênica , Prognóstico , Carcinogênese/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Redes Reguladoras de GenesRESUMO
OBJECTIVE: This study aimed to investigate the expression and functional role of NISCH in skin cutaneous melanoma (SKCM), exploring its association with clinical characteristics and its potential impact on human skin melanoma cell behavior. METHODS: The research assessed differential NISCH expression in SKCM tissues using the GEPIA (Gene Expression Profiling Interactive Analysis) database and validated these findings through immunohistochemical staining of 45 clinical samples. To affirm NISCH expression at the cellular level, three human skin melanoma cell lines (RPMI-7951, A375, MEL-5), and the human normal skin cell line HEMa underwent quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. Transwell experiments evaluated the migration and invasion capabilities of RPMI-7951 and A375 cells post-transduction with NISCH or PAK1 lentiviral activation particles. Additionally, qRT-PCR analysis of epithelial-mesenchymal transition (EMT)-related gene expression (Vimentin, E-cadherin, N-cadherin) was conducted in A375 and RPMI-7951 cells. RESULTS: SKCM tissues exhibited significantly reduced NISCH expression compared to normal tissues. Immunohistochemical analysis revealed predominant nuclear localization of NISCH in melanoma cells, with reduced expression significantly correlating with sex, advanced stage, and lymph node metastasis. Melanoma cell lines displayed lower NISCH expression levels compared to normal skin cells. Functional experiments showcased that NISCH overexpression suppressed p-PAK1/PAK1, while PAK1 upregulation notably increased melanoma cell migration, invasion, and induced EMT. Remarkably, NISCH overexpression counteracted PAK1-induced effects on EMT, migration, and invasion in melanoma cells. CONCLUSION: NISCH may significantly influence the aggressive behavior of SKCM cells via the PAK1 pathway, making it a potential therapeutic target for managing melanoma metastasis.
Assuntos
Movimento Celular , Regulação para Baixo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Melanoma , Invasividade Neoplásica , Neoplasias Cutâneas , Quinases Ativadas por p21 , Humanos , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Melanoma/patologia , Melanoma/metabolismo , Melanoma/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/genética , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Linhagem Celular Tumoral , Masculino , Feminino , Pessoa de Meia-Idade , Regulação para Baixo/genética , Melanoma Maligno Cutâneo , Idoso , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Relevância ClínicaRESUMO
Successful regeneration of missing tissues requires seamless integration of positional information along the body axes. Planarians, which regenerate from almost any injury, use conserved, developmentally important signaling pathways to pattern the body axes. However, the molecular mechanisms which facilitate cross talk between these signaling pathways to integrate positional information remain poorly understood. Here, we report a p21-activated kinase (smed-pak1) which functionally integrates the anterior-posterior (AP) and the medio-lateral (ML) axes. pak1 inhibits WNT/ß-catenin signaling along the AP axis and, functions synergistically with the ß-catenin-independent WNT signaling of the ML axis. Furthermore, this functional integration is dependent on warts and merlin-the components of the Hippo/Yorkie (YKI) pathway. Hippo/YKI pathway is a critical regulator of body size in flies and mice, but our data suggest the pathway regulates body axes patterning in planarians. Our study provides a signaling network integrating positional information which can mediate coordinated growth and patterning during planarian regeneration.
Assuntos
Planárias , Via de Sinalização Wnt , Quinases Ativadas por p21 , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Planárias/fisiologia , Planárias/genética , Planárias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Regeneração , Transativadores/metabolismo , Transativadores/genéticaRESUMO
Cell stiffness is regulated by dynamic interaction between ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1) proteins, besides other biochemical and molecular regulators. In this study, we investigated how the Placental Growth Factor (PlGF) changes endometrial mechanics by modifying the actin cytoskeleton at the maternal interface. We explored the global effects of PlGF in endometrial stromal cells (EnSCs) using the concerted approach of proteomics, atomic force microscopy (AFM), and electrical impedance spectroscopy (EIS). Proteomic analysis shows PlGF upregulated RhoGTPases activating proteins and extracellular matrix organization-associated proteins in EnSCs. Rac1 and PAK1 transcript levels, activity, and actin polymerization were significantly increased with PlGF treatment. AFM further revealed an increase in cell stiffness with PlGF treatment. The additive effect of PlGF on actin polymerization was suppressed with siRNA-mediated inhibition of Rac1, PAK1, and WAVE2. Interestingly, the increase in cell stiffness by PlGF treatment was pharmacologically reversed with pravastatin, resulting in improved trophoblast cell invasion. Taken together, aberrant PlGF levels in the endometrium can contribute to an altered pre-pregnancy maternal microenvironment and offer a unifying explanation for the pathological changes observed in conditions such as pre-eclampsia (PE).
Assuntos
Endométrio , Fator de Crescimento Placentário , Pré-Eclâmpsia , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP , Feminino , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Humanos , Pré-Eclâmpsia/metabolismo , Gravidez , Endométrio/metabolismo , Endométrio/patologia , Fator de Crescimento Placentário/metabolismo , Fator de Crescimento Placentário/genética , Células Estromais/metabolismo , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Microscopia de Força AtômicaRESUMO
The intricate orchestration of osteoporosis (OP) pathogenesis remains elusive. Mounting evidence suggests that angiogenesis-driven osteogenesis serves as a crucial foundation for maintaining bone homeostasis. This study aimed to explore the potential of the endothelial platelet-derived growth factor receptor-ß (PDGFR-ß) in mitigating bone loss through its facilitation of H-type vessel formation. Our findings demonstrate that the expression level of endothelial PDGFR-ß is reduced in samples obtained from individuals suffering from OP, as well as in ovariectomy mice. Depletion of PDGFR-ß in endothelial cells ameliorates angiogenesis-mediated bone formation in mice. The regulatory influence of endothelial PDGFR-ß on H-type vessels is mediated through the PDGFRß-P21-activated kinase 1-Notch1 intracellular domain signaling cascade. In particular, the endothelium-specific enhancement of PDGFR-ß facilitates H-type vessels and their associated bone formation in OP. Hence, the strategic targeting of endothelial PDGFR-ß emerges as a promising therapeutic approach for the management of OP in the near future.
Assuntos
Neovascularização Fisiológica , Osteogênese , Osteoporose , Receptor Notch1 , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Transdução de Sinais , Quinases Ativadas por p21 , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Humanos , Feminino , Camundongos , Receptor Notch1/metabolismo , Receptor Notch1/genética , Osteoporose/metabolismo , Osteoporose/patologia , Células Endoteliais/metabolismo , Camundongos Endogâmicos C57BL , Células Endoteliais da Veia Umbilical Humana/metabolismo , AngiogêneseRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal types of cancer, and KRAS oncogene occurs in over 90% of cases. P21-activated kinases (PAK), containing six members (PAK1 to 6), function downstream of KRAS. PAK1 and PAK4 play important roles in carcinogenesis, but their combinational effect remains unknown. In this study, we have determined the effect of dual inhibition of PAK1 and PAK4 in PDA progression using knockout (KO) cancer cell lines. METHODS: Murine wild-type (WT) and PAK1KO pancreatic cancer cell lines were isolated from PAK1+/+ and PAK1-/- KPC (LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx-1-Cre) mice. KPC PAK4KO and KPC PAK1&4 KO cell lines were generated from KPC WT and KPC PAK1KO cell lines respectively using the CRISPR-CAS9 gene knockout technique. PAK WT and KO cell lines were used in mouse models of pancreatic tumours. Cells and tumour tissue were also used in flow cytometry and proteomic studies. A human PDA tissue microarray was stained by immunohistochemistry. RESULTS: Double knock out of PAK1 and PAK4 caused complete regression of tumour in a syngeneic mouse model. PAK4KO inhibited tumour growth by stimulating a rapid increase of cytotoxic CD8+ T cell infiltration. PAK1KO synergistically with PAK4KO increased cytotoxic CD8+ T cell infiltration and stimulated a sustained infiltration of CD8+ T cells at a later phase to overcome the immune evasion in the PAK4KO tumour. The human PDA tissue microarray study showed the important role of PAK1 and PAK4 in intra-tumoral T-cell function. CONCLUSION: Our results demonstrated that dual inhibition of PAK1 and PAK4 synergistically suppressed PDA progression by stimulating cytotoxic CD8 + T cell response.