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1.
BMC Med Genomics ; 17(1): 148, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38807162

ABSTRACT

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.


Subject(s)
Ovarian Neoplasms , p21-Activated Kinases , Humans , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , Female , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/immunology , Gene Expression Regulation, Neoplastic , Prognosis , Carcinogenesis/genetics , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/genetics , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Gene Regulatory Networks
2.
Cell Commun Signal ; 22(1): 287, 2024 May 27.
Article in English | MEDLINE | ID: mdl-38797819

ABSTRACT

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.


Subject(s)
Pancreatic Neoplasms , p21-Activated Kinases , p21-Activated Kinases/metabolism , p21-Activated Kinases/genetics , p21-Activated Kinases/antagonists & inhibitors , Animals , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/genetics , Mice , Cell Line, Tumor , Humans , Cell Proliferation , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/genetics , Mice, Knockout
3.
Commun Biol ; 7(1): 530, 2024 May 04.
Article in English | MEDLINE | ID: mdl-38704457

ABSTRACT

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).


Subject(s)
Endometrium , Placenta Growth Factor , Pre-Eclampsia , Signal Transduction , rac1 GTP-Binding Protein , Female , rac1 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/genetics , Humans , Pre-Eclampsia/metabolism , Pregnancy , Endometrium/metabolism , Endometrium/pathology , Placenta Growth Factor/metabolism , Placenta Growth Factor/genetics , Stromal Cells/metabolism , p21-Activated Kinases/metabolism , p21-Activated Kinases/genetics , Microscopy, Atomic Force
4.
Nature ; 630(8015): 198-205, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38720074

ABSTRACT

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.


Subject(s)
Class Ib Phosphatidylinositol 3-Kinase , Signal Transduction , p21-Activated Kinases , Humans , Class Ib Phosphatidylinositol 3-Kinase/metabolism , Class Ib Phosphatidylinositol 3-Kinase/genetics , p21-Activated Kinases/metabolism , p21-Activated Kinases/antagonists & inhibitors , p21-Activated Kinases/genetics , Mice , Animals , Female , Leukemia/genetics , Leukemia/enzymology , Leukemia/pathology , Leukemia/metabolism , Phosphorylation , Cell Line, Tumor , Mitochondria/metabolism , Male
5.
Sci Rep ; 14(1): 12153, 2024 05 27.
Article in English | MEDLINE | ID: mdl-38802496

ABSTRACT

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.


Subject(s)
Adherens Junctions , Basic Helix-Loop-Helix Transcription Factors , Cell Polarity , Signal Transduction , rac1 GTP-Binding Protein , Adherens Junctions/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , rac1 GTP-Binding Protein/metabolism , GTPase-Activating Proteins/metabolism , GTPase-Activating Proteins/genetics , Cadherins/metabolism , Cadherins/genetics , Mice , Humans , Epithelial Cells/metabolism , p21-Activated Kinases/metabolism , p21-Activated Kinases/genetics , Cell Line
6.
Proc Natl Acad Sci U S A ; 121(20): e2321919121, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38713625

ABSTRACT

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.


Subject(s)
Planarians , Wnt Signaling Pathway , p21-Activated Kinases , Animals , Body Patterning/genetics , Body Patterning/physiology , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , p21-Activated Kinases/metabolism , p21-Activated Kinases/genetics , Planarians/physiology , Planarians/genetics , Planarians/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Regeneration , Trans-Activators/metabolism , Trans-Activators/genetics
7.
J Parkinsons Dis ; 14(3): 495-506, 2024.
Article in English | MEDLINE | ID: mdl-38640169

ABSTRACT

Background: Parkinson's disease is a progressive neurodegenerative disorder mainly distinguished by sporadic etiology, although a genetic component is also well established. Variants in the LRRK2 gene are associated with both familiar and sporadic disease. We have previously shown that PAK6 and 14-3-3γ protein interact with and regulate the activity of LRRK2. Objective: The aim of this study is to quantify PAK6 and 14-3-3γ in plasma as reliable biomarkers for the diagnosis of both sporadic and LRRK2-linked Parkinson's disease. Methods: After an initial quantification of PAK6 and 14-3-3γ expression by means of Western blot in post-mortem human brains, we verified the presence of the two proteins in plasma by using quantitative ELISA tests. We analyzed samples obtained from 39 healthy subjects, 40 patients with sporadic Parkinson's disease, 50 LRRK2-G2019S non-manifesting carriers and 31 patients with LRRK2-G2019S Parkinson's disease. Results: The amount of PAK6 and 14-3-3γ is significantly different in patients with Parkinson's disease compared to healthy subjects. Moreover, the amount of PAK6 also varies with the presence of the G2019S mutation in the LRRK2 gene. Although the generalized linear models show a low association between the presence of Parkinson's disease and PAK6, the kinase could be added in a broader panel of biomarkers for the diagnosis of Parkinson's disease. Conclusions: Changes of PAK6 and 14-3-3γ amount in plasma represent a shared readout for patients affected by sporadic and LRRK2-linked Parkinson's disease. Overall, they can contribute to the establishment of an extended panel of biomarkers for the diagnosis of Parkinson's disease.


Subject(s)
14-3-3 Proteins , Biomarkers , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Parkinson Disease , p21-Activated Kinases , Humans , Parkinson Disease/blood , Parkinson Disease/diagnosis , Parkinson Disease/genetics , 14-3-3 Proteins/blood , Male , p21-Activated Kinases/blood , p21-Activated Kinases/metabolism , p21-Activated Kinases/genetics , Female , Aged , Biomarkers/blood , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Middle Aged , Aged, 80 and over , Prospective Studies , Adult , Mutation
8.
Elife ; 132024 Apr 25.
Article in English | MEDLINE | ID: mdl-38661167

ABSTRACT

Osteoblast adherence to bone surfaces is important for remodeling bone tissue. This study demonstrates that deficiency of TG-interacting factor 1 (Tgif1) in osteoblasts results in altered cell morphology, reduced adherence to collagen type I-coated surfaces, and impaired migration capacity. Tgif1 is essential for osteoblasts to adapt a regular cell morphology and to efficiently adhere and migrate on collagen type I-rich matrices in vitro. Furthermore, Tgif1 acts as a transcriptional repressor of p21-activated kinase 3 (Pak3), an important regulator of focal adhesion formation and osteoblast spreading. Absence of Tgif1 leads to increased Pak3 expression, which impairs osteoblast spreading. Additionally, Tgif1 is implicated in osteoblast recruitment and activation of bone surfaces in the context of bone regeneration and in response to parathyroid hormone 1-34 (PTH 1-34) treatment in vivo in mice. These findings provide important novel insights in the regulation of the cytoskeletal architecture of osteoblasts.


Subject(s)
Cytoskeleton , Homeodomain Proteins , Osteoblasts , Repressor Proteins , Signal Transduction , p21-Activated Kinases , Animals , Mice , Cell Adhesion , Cell Movement , Cytoskeleton/metabolism , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Osteoblasts/metabolism , p21-Activated Kinases/metabolism , p21-Activated Kinases/genetics , p21-Activated Kinases/deficiency , Repressor Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/deficiency
9.
Drug Discov Ther ; 18(2): 134-139, 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38569833

ABSTRACT

Both PAK1 (RAC/CDC42-activating kinase 1) and TOR (Target of Rapamycin) are among the major oncogenic/ageing kinases. However, they play the opposite role in our immune system, namely immune system is suppressed by PAK1, while it requires TOR. Thus, PAK1-blockers, would be more effective for therapy of cancers, than TOR-blockers. Since 2015 when we discovered genetically that PDGF-induced melanogenesis depends on "PAK1", we are able to screening a series of PAK1-blockers as melanogenesis-inhibitors which could eventually promote longevity. Interestingly, rapamycin, the first TOR-inhibitor, promotes melanogenesis, clearly indicating that TOR suppresses melanogenesis. However, a new TOR-inhibitor called TORin-1 no longer suppresses immune system, and blocks melanogenesis in cell culture. These observations strongly indicate that TORin-1 acts as PAK1-blockers, instead of TOR-blockers, in vivo. Thus, it is most likely that melanogenesis in cell culture could enable us to discriminate PAK1-blockers from TORblockers.


Subject(s)
Imatinib Mesylate , Pyrimidines , Sirolimus , TOR Serine-Threonine Kinases , p21-Activated Kinases , p21-Activated Kinases/metabolism , p21-Activated Kinases/genetics , Humans , TOR Serine-Threonine Kinases/metabolism , Pyrimidines/pharmacology , Sirolimus/pharmacology , Sirolimus/therapeutic use , Imatinib Mesylate/pharmacology , Imatinib Mesylate/therapeutic use , Animals , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Melanins/biosynthesis , Melanins/metabolism , MTOR Inhibitors/pharmacology , MTOR Inhibitors/therapeutic use , Naphthyridines
10.
Biochem Pharmacol ; 224: 116206, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38615921

ABSTRACT

Long-chain fatty acyl-Coa ligase 4 (ACSL4) is an important enzyme that converts fatty acids to fatty acyl-Coa esters, there is increasing evidence for its role in carcinogenesis. However, the precise role of ACLS4 in hepatocellular carcinoma (HCC) is not clearly understood. In the present study, we provide evidence that ACSL4 expression was specifically elevated in HCC and is associated with poor clinical outcomes. ACSL4 significantly promotes the growth and metastasis of HCC both in vitro and in vivo. RNA sequencing and functional experiments showed that the effect of ACSL4 on HCC development was heavily dependent on PAK2. ACSL4 expression is well correlated with PAK2 in HCC, and ACSL4 even transcriptionally increased PAK2 gene expression mediated by Sp1. In addition, emodin, a naturally occurring anthraquinone derivative, inhibited HCC cell growth and tumor progression by targeting ACSL4. In summary, ACSL4 plays a novel oncogene in HCC development by regulating PAK2 transcription. Targeting ACSL4 could be useful in drug development and therapy for HCC.


Subject(s)
Carcinoma, Hepatocellular , Coenzyme A Ligases , Disease Progression , Liver Neoplasms , Mice, Nude , p21-Activated Kinases , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Humans , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/genetics , p21-Activated Kinases/metabolism , p21-Activated Kinases/genetics , Coenzyme A Ligases/metabolism , Coenzyme A Ligases/genetics , Animals , Mice , Male , Cell Line, Tumor , Mice, Inbred BALB C , Transcription, Genetic/drug effects , Gene Expression Regulation, Neoplastic , Emodin/pharmacology , Female
11.
ACS Infect Dis ; 10(4): 1370-1378, 2024 Apr 12.
Article in English | MEDLINE | ID: mdl-38503263

ABSTRACT

Sepsis is a severe syndrome characterized by organ dysfunction, resulting from a systemic imbalance in response to infection. PAK1 plays a critical role in various diseases. The present study aimed to explore and delineate the mechanism of PAK1 in inflammation induced by sepsis. Bioinformatics analysis was performed to assess PAK1, snail, and CXCL2 expression in the whole blood of septic patients and the pathways enriched with PAK1. To simulate the sepsis model, THP-1 cells were stimulated with lipopolysaccharide. Gene expression was evaluated using qRT-PCR, while cell viability was assessed using CCK-8 assay. Cell apoptosis was tested with flow cytometry. Expression of inflammatory factors in cells following different treatments was analyzed using the enzyme linked immunosorbent assay (ELISA). Dual-luciferase and chromatin immunoprecipitation assays were conducted to verify the binding relationship between PAK1 and the snail. Mouse models of cecal ligation and puncture were established, and hematoxylin and eosin staining and ELISA were employed to detect the infiltration levels of inflammatory cells and the expression of related protective factors in lung, liver, and kidney tissues. The results demonstrated upregulation of PAK1, snail, and CXCL2 in the whole blood of septic patients, with PAK1 being enriched in the chemokine-related pathway. Knockdown of PAK1 significantly promoted the apoptosis of LPS-stimulated THP-1 cells and inhibited the expression of inflammatory factors. PAK1 upregulated the expression of the snail, which in turn promoted the expression of CXCL2. Thus, PAK1 mediated the sepsis-induced inflammatory response through the snail/CXCL2 pathway. In conclusion, PAK1 played a role in promoting inflammation induced by sepsis through the snail/CXCL2 axis, thereby providing a potential therapeutic target for the management of sepsis.


Subject(s)
Sepsis , Signal Transduction , Mice , Animals , Humans , Inflammation , Apoptosis , Liver/metabolism , Chemokine CXCL2/genetics , Chemokine CXCL2/metabolism , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism
12.
Int Heart J ; 65(2): 339-348, 2024.
Article in English | MEDLINE | ID: mdl-38556341

ABSTRACT

Myocarditis, a severe inflammatory disease, is becoming a worldwide public health concern. This study aims to elucidate the effect of Chemokine (C C motif) receptor-like 2 (CCRL2) in experimental autoimmune myocarditis (EAM) occurrence and its potential regulatory mechanisms.EAM was simulated in a mouse model injected with α-myosin-heavy chain. The changes on EAM were assessed through histological staining of heart tissues, including measuring cardiac troponin I (cTnI), proinflammatory cytokines, transferase-mediated dUTP nick end labeling (TUNEL) assay, and cardiac function. Then, the heart tissues from the EAM mouse model and control groups were analyzed through transcriptome sequencing to identify the differential expressed genes (DEGs) and hub genes related to pyroptosis. Downregulation of CCRL2 further verified the function of CCRL2 on EAM and p21-activated kinase 1/NOD-like receptor protein 3 (PAK/NLRP3) signaling pathways in vivo.The EAM model was constructed successfully, with the heart weight/body weight ratio, serum level of cTnI, and concentrations of proinflammatory cytokines elevation. Moreover, cell apoptosis was also significantly increased. Transcriptome sequencing revealed 696 and 120 upregulated and downregulated DEGs, respectively. After functional enrichment, CCRL2 was selected as a potential target. Then, we verified that CCRL2 knockdown improved cardiac function, alleviated EAM occurrence, and reduced PAK/NLRP3 protein expression.CCRL2 may act as a novel potential treatment target in EAM by regulating the PAK1/NLRP3 pathway.


Subject(s)
Autoimmune Diseases , Myocarditis , Animals , Mice , Autoimmune Diseases/pathology , Cytokines , Disease Models, Animal , Myocarditis/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , NLR Proteins , p21-Activated Kinases/genetics
13.
Front Immunol ; 15: 1347683, 2024.
Article in English | MEDLINE | ID: mdl-38343537

ABSTRACT

Background: Pancreatic cancer remains an extremely malignant digestive tract tumor, posing a significant global public health burden. Patients with pancreatic cancer, once metastasis occurs, lose all hope of cure, and prognosis is extremely poor. It is important to investigate liver metastasis of Pancreatic cancer in depth, not just because it is the most common form of metastasis in pancreatic cancer, but also because it is crucial for treatment planning and prognosis assessment. This study aims to delve into the mechanisms of pancreatic cancer liver metastasis, with the goal of providing crucial scientific groundwork for the development of future treatment methods and drugs. Methods: We explored the mechanisms of pancreatic cancer liver metastasis using single-cell sequencing data (GSE155698 and GSE154778) and bulk data (GSE71729, GSE19279, TCGA-PAAD). Initially, Seurat package was employed for single-cell data processing to obtain expression matrices for primary pancreatic cancer lesions and liver metastatic lesions. Subsequently, high-dimensional weighted gene co-expression network analysis (hdWGCNA) was used to identify genes associated with liver metastasis. Machine learning algorithms and COX regression models were employed to further screen genes related to patient prognosis. Informed by both biological understanding and the outcomes of algorithms, we meticulously identified the ultimate set of liver metastasis-related gene (LRG). In the study of LRG genes, various databases were utilized to validate their association with pancreatic cancer liver metastasis. In order to analyze the effects of these agents on tumor microenvironment, we conducted an in-depth analysis, including changes in signaling pathways (GSVA), cell differentiation (pseudo-temporal analysis), cell communication networks (cell communication analysis), and downstream transcription factors (transcription factor activity prediction). Additionally, drug sensitivity analysis and metabolic analysis were performed to reveal the effects of LRG on gemcitabine resistance and metabolic pathways. Finally, functional experiments were conducted by silencing the expression of LRG in PANC-1 and Bx-PC-3 cells to validate its influence to proliferation and invasiveness on PANC-1 and Bx-PC-3 cells. Results: Through a series of algorithmic filters, we identified PAK2 as a key gene promoting pancreatic cancer liver metastasis. GSVA analysis elucidated the activation of the TGF-beta signaling pathway by PAK2 to promote the occurrence of liver metastasis. Pseudo-temporal analysis revealed a significant correlation between PAK2 expression and the lower differentiation status of pancreatic cancer cells. Cell communication analysis revealed that overexpression of PAK2 promotes communication between cancer cells and the tumor microenvironment. Transcription factor activity prediction displayed the transcription factor network regulated by PAK2. Drug sensitivity analysis and metabolic analysis revealed the impact of PAK2 on gemcitabine resistance and metabolic pathways. CCK8 experiments showed that silencing PAK2 led to a decrease in the proliferative capacity of pancreatic cancer cells and scratch experiments demonstrated that low expression of PAK2 decreased invasion capability in pancreatic cancer cells. Flow cytometry reveals that PAK2 significantly inhibited apoptosis in pancreatic cancer cell lines. Molecules related to the TGF-beta pathway decreased with the inhibition of PAK2, and there were corresponding significant changes in molecules associated with EMT. Conclusion: PAK2 facilitated the angiogenic potential of cancer cells and promotes the epithelial-mesenchymal transition process by activating the TGF-beta signaling pathway. Simultaneously, it decreased the differentiation level of cancer cells, consequently enhancing their malignancy. Additionally, PAK2 fostered communication between cancer cells and the tumor microenvironment, augments cancer cell chemoresistance, and modulates energy metabolism pathways. In summary, PAK2 emerged as a pivotal gene orchestrating pancreatic cancer liver metastasis. Intervening in the expression of PAK2 may offer a promising therapeutic strategy for preventing liver metastasis of pancreatic cancer and improving its prognosis.


Subject(s)
Liver Neoplasms , Pancreatic Neoplasms , Humans , Gemcitabine , Cell Proliferation , Pancreatic Neoplasms/pathology , Transcription Factors , Transforming Growth Factor beta/pharmacology , Liver Neoplasms/genetics , Tumor Microenvironment , p21-Activated Kinases/genetics
14.
Cancer Lett ; 587: 216725, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38364963

ABSTRACT

Next-generation androgen receptor signaling inhibitors (ARSIs), such as enzalutamide (Enza) and darolutamide (Daro), are initially effective for the treatment of advanced prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). However, patients often relapse and develop cross-resistance, which consequently makes drug resistance an inevitable cause of CRPC-related mortality. By conducting a comprehensive analysis of GEO datasets, CRISPR genome-wide screening results, ATAC-seq data, and RNA-seq data, we systemically identified PAK1 as a significant contributor to ARSI cross-resistance due to the activation of the PAK1/RELA/hnRNPA1/AR-V7 axis. Inhibition of PAK1 followed by suppression of NF-κB pathways and AR-V7 expression effectively overcomes ARSI cross-resistance. Our findings indicate that PAK1 represents a promising therapeutic target gene for the treatment of ARSI cross-resistant PCa patients in the clinic. STATEMENT OF SIGNIFICANCE: PAK1 drives ARSI cross-resistance in prostate cancer progression.


Subject(s)
Prostatic Neoplasms, Castration-Resistant , Male , Humans , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Receptors, Androgen/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Early Detection of Cancer , Neoplasm Recurrence, Local/genetics , Nitriles/pharmacology , Drug Resistance, Neoplasm/genetics , Cell Line, Tumor , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism
15.
Oncogene ; 43(13): 921-930, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38336988

ABSTRACT

Neurofibromatosis Type 2 (NF2)-related schwannomatosis is a genetic disorder that causes development of multiple types of nervous system tumors. The primary and diagnostic tumor type is bilateral vestibular schwannoma. There is no cure or drug therapy for NF2. Recommended treatments include surgical resection and radiation, both of which can leave patients with severe neurological deficits or increase the risk of future malignant tumors. Results of our previous pilot high-throughput drug screen identified phosphoinositide 3-kinase (PI3K) inhibitors as strong candidates based on loss of viability of mouse merlin-deficient Schwann cells (MD-SCs). Here we used novel human schwannoma model cells to conduct combination drug screens. We identified a class I PI3K inhibitor, pictilisib and p21 activated kinase (PAK) inhibitor, PF-3758309 as the top combination due to high synergy in cell viability assays. Both single and combination therapies significantly reduced growth of mouse MD-SCs in an orthotopic allograft mouse model. The inhibitor combination promoted cell cycle arrest and apoptosis in mouse merlin-deficient Schwann (MD-SCs) cells and cell cycle arrest in human MD-SCs. This study identifies the PI3K and PAK pathways as potential targets for combination drug treatment of NF2-related schwannomatosis.


Subject(s)
Indazoles , Neurilemmoma , Neurofibromatoses , Neurofibromatosis 2 , Skin Neoplasms , Sulfonamides , Humans , Animals , Mice , Neurofibromatosis 2/drug therapy , Neurofibromatosis 2/genetics , Neurofibromatosis 2/metabolism , Neurofibromin 2/genetics , Neurofibromin 2/metabolism , Phosphatidylinositol 3-Kinases , p21-Activated Kinases/genetics , Phosphatidylinositol 3-Kinase/therapeutic use , Neurilemmoma/drug therapy , Neurilemmoma/genetics
16.
Thyroid ; 34(5): 583-597, 2024 May.
Article in English | MEDLINE | ID: mdl-38411500

ABSTRACT

Background: Familial non-medullary thyroid carcinoma (FNMTC) is a genetically predisposed disease with unclear genetic mechanisms. This makes research on susceptibility genes important for the diagnosis and treatment options. Methods: This study included a five-member family affected by papillary thyroid carcinoma. The candidate genes were identified through whole-exome sequencing and Sanger sequencing in family members, other FNMTC patients, and sporadic non-medullary thyroid carcinoma patients. The pathogenicity of the mutation was predicted using in silico tools. Cell phenotype experiments in vitro and models of lung distant metastasis in vivo were conducted to confirm the characteristics of the mutation. Transcriptome sequencing and mechanistic validation were employed to compare the disparities between PAK4 wild-type (WT) and PAK4 mutant (MUT) cell lines. Results: This mutation alters the protein structure, potentially increasing instability by affecting hydrophobicity, intra-molecular hydrogen bonding, and phosphorylation sites. It specifically promotes phosphorylated PAK4 nuclear translocation and expression in thyroid tissue and cell lines. Compared with the WT cells line, PAK4 I417T demonstrates enhanced proliferation, invasiveness, accelerated cell division, and inhibition of cell apoptosis in vitro. In addition, it exhibits a significant propensity for metastasis in vivo. It activates tumor necrosis factor signaling through increased phosphorylation of PAK4, JNK, NFκB, and c-Jun, unlike the WT that activates it via the PAK4-NFκ-MMP9 axis. In addition, PAK4 MUT protein interacts with matrix metalloproteinase (MMP)3 and regulates MMP3 promoter activity, which is not observed in the WT. Conclusions: Our study identified PAK4: c.T1250C: p.I417T as a potential susceptibility gene for FNMTC. The study concludes that the mutant form of PAK4 exhibits oncogenic function, suggesting its potential as a novel diagnostic molecular marker for FNMTC.


Subject(s)
Genetic Predisposition to Disease , Mutation , Thyroid Cancer, Papillary , Thyroid Neoplasms , p21-Activated Kinases , Humans , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology , Thyroid Cancer, Papillary/genetics , Thyroid Cancer, Papillary/pathology , Thyroid Cancer, Papillary/metabolism , Female , Male , Cell Line, Tumor , Animals , Pedigree , Cell Proliferation , Adult , Apoptosis , Exome Sequencing , Middle Aged , Mice
17.
Anticancer Res ; 44(2): 593-604, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38307587

ABSTRACT

BACKGROUND/AIM: Protein arginine methyltransferase 5 (PRMT5), a member of the arginine methyltransferases, is an enzyme catalyzing the methylation of arginine residuals of histones and non-histone proteins to serve as one of many critical posttranslational modifications (PTMs). Phosphorylated P21-activated kinase 1 (p-PAK1), a serine/threonine protein kinase family member, is a cytoskeletal protein that plays a critical role in metastasis. We examined the expression of PRMT5 and PAK1 in esophageal squamous cell carcinoma (ESCC) and evaluated the correlation between PRMT5/p-PAK1 and both clinicopathological parameters and prognosis of ESCC patients. MATERIALS AND METHODS: 106 tumor tissues collected from ESCC patients were assessed for PRMT5 and PAK1 expression using immunohistochemistry. Pearson's correlation and Kaplan-Meier analysis were used to estimate the correlation with the clinicopathological parameters and effect on patient survival. Western blot analysis was used to determine the PRMT5/p-PAK1 protein expression. The wound healing assay was performed to assess the effect of PRMT5 on the migration of ESCC cells. RESULTS: PRMT5 is upregulated in ESCC and the level of PRMT5 is correlated with metastasis and can serve as an independent prognostic factor for overall survival (OS). PRMT5 knockdown remarkably inhibited ESCC cell migration with concomitantly reduced levels of phosphorylated PAK1 (p-PAK1) but not total PAK1. Kaplan-Meier analysis showed that the OS of the subgroup of patients with PRMT5high/p-PAK1high is remarkably shorter than those of other subgroups (i.e., PRMT5high/p-PAK1low, PRMT5low/p-PAK1low and PRMT5low/p-PAK1high). CONCLUSION: PRMT5-PAK1 signaling participates in ESCC metastasis and can predict patients' outcomes.


Subject(s)
Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Humans , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/pathology , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , Biomarkers, Tumor/metabolism , Prognosis , Histones , Arginine , Kaplan-Meier Estimate , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism
18.
Nat Metab ; 6(1): 94-112, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38216738

ABSTRACT

Adipose tissue lipolysis is mediated by cAMP-protein kinase A (PKA)-dependent intracellular signalling. Here, we show that PKA targets p21-activated kinase 4 (PAK4), leading to its protein degradation. Adipose tissue-specific overexpression of PAK4 in mice attenuates lipolysis and exacerbates diet-induced obesity. Conversely, adipose tissue-specific knockout of Pak4 or the administration of a PAK4 inhibitor in mice ameliorates diet-induced obesity and insulin resistance while enhancing lipolysis. Pak4 knockout also increases energy expenditure and adipose tissue browning activity. Mechanistically, PAK4 directly phosphorylates fatty acid-binding protein 4 (FABP4) at T126 and hormone-sensitive lipase (HSL) at S565, impairing their interaction and thereby inhibiting lipolysis. Levels of PAK4 and the phosphorylation of FABP4-T126 and HSL-S565 are enhanced in the visceral fat of individuals with obesity compared to their lean counterparts. In summary, we have uncovered an important role for FABP4 phosphorylation in regulating adipose tissue lipolysis, and PAK4 inhibition may offer a therapeutic strategy for the treatment of obesity.


Subject(s)
Lipolysis , Sterol Esterase , Animals , Mice , Fatty Acid-Binding Proteins/genetics , Fatty Acid-Binding Proteins/metabolism , Lipolysis/physiology , Obesity/metabolism , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , Sterol Esterase/genetics , Sterol Esterase/metabolism
19.
Am J Hypertens ; 37(1): 46-52, 2024 Jan 01.
Article in English | MEDLINE | ID: mdl-36634025

ABSTRACT

BACKGROUND: P21-activated kinase 1 (Pak1) has an effect on cell apoptosis and has recently been reported to play an important role in various cardiovascular diseases, in which vascular smooth muscle cell (VSMC) apoptosis is a key process. Thus, we hypothesized that Pak1 may be a novel target to regulate VSMC behaviors. METHODS AND RESULTS: In the present study, we found that the expression of Pak1 was dramatically upregulated in vascular smooth muscle cells (VSMCs) on H2O2 administration and was dependent on stimulation time. Through a loss-of-function approach, Pak1 knockdown increased apoptosis of VSMCs, as tested by TUNEL (TdT-mediated dUTP Nick-End Labeling) immunofluorescence staining, whereas it inhibited the proliferation of VSMCs examined by EdU staining. Moreover, we also noticed that Pak1 silencing promoted the mRNA and protein levels of pro-apoptosis genes but decreased anti-apoptosis marker expression. Importantly, we showed that Pak1 knockdown reduced the phosphorylation of Bad. Moreover, increased Pak1 expression was also noticed in carotid arteries on the wire jury. CONCLUSIONS: Our study identified that Pak1 acted as a novel regulator of apoptosis of VSMCs partially through phosphorylation of Bad.


Subject(s)
Muscle, Smooth, Vascular , p21-Activated Kinases , Phosphorylation , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , p21-Activated Kinases/pharmacology , Muscle, Smooth, Vascular/metabolism , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Apoptosis , Myocytes, Smooth Muscle/metabolism , Cell Proliferation , Cells, Cultured
20.
Cancer Chemother Pharmacol ; 93(1): 41-54, 2024 01.
Article in English | MEDLINE | ID: mdl-37741955

ABSTRACT

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.


Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Animals , Mice , Humans , Ivermectin/pharmacology , Mice, Nude , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/pathology , Lung Neoplasms/pathology , Autophagy , Cell Proliferation , Apoptosis , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , p21-Activated Kinases/pharmacology
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