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Roles of p38? and p38? mitogen?activated protein kinase isoforms in human malignant melanoma A375 cells.

ABSTRACT: Skin cancer is one of the most common cancers worldwide. Melanoma accounts for ~5% of skin cancers but causes the large majority of skin cancer?related deaths. Recent discoveries have shown that the mitogen?activated protein kinase (MAPK) signaling pathway is critical for melanoma development and progression. Many oncogenic pathways that cause melanoma tumorigenesis have been identified, most of which are due to RAF/MEK/ERK (MAPK) pathway activation. However, the precise role of p38 remains unclear. Using specific short hairpin (sh) RNA to silence p38? and p38?, the present findings demonstrated that p38? was a crucial factor in regulating cell migration in the A375 melanoma cell line. Silencing p38? downregulated the expression of epithelial?mesenchymal transition markers, such as matrix metallopeptidase (MMP) 2, MMP9, twist family bHLH transcription factor 1, snail family transcriptional repressor 1 and vimentin, while mesenchymal?epithelial transition markers, such as E?cadherin, were upregulated. Of note, the results also demonstrated that p38? silencing impaired vascular endothelial growth factor expression, which regulates tumor angiogenesis. Furthermore, p38? knockdown inhibited cell proliferation in melanoma cells. In addition, silencing p38? induced senescence?like features, but not cell cycle arrest. Expression of the senescence markers p16, p21, p53 and ??galactosidase was upregulated, and an increase in the number of senescence?associated ??galactosidase?positive cells was observed in a p38? knockdown stable clone. However, no significant difference was found between control and p38? stable knockdown cells. Taken together, the present results suggested that p38? knockdown impaired migration and proliferation, and increased senescence, in A375 melanoma cells. However, p38? may not be involved in melanoma tumorigenesis. Therefore, targeting p38? may be a valuable approach towards inhibiting tumor growth and metastasis in patients with melanoma.

SUBMITTER: Wen SY

PROVIDER: S-EPMC6844598 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Roles of p38α and p38β mitogen‑activated protein kinase isoforms in human malignant melanoma A375 cells.

Wen Su-Ying SY Cheng Shi-Yann SY Ng Shang-Chuan SC Aneja Ritu R Chen Chih-Jung CJ Huang Chih-Yang CY Kuo Wei-Wen WW

International journal of molecular medicine 20191024 6

Skin cancer is one of the most common cancers worldwide. Melanoma accounts for ~5% of skin cancers but causes the large majority of skin cancer‑related deaths. Recent discoveries have shown that the mitogen‑activated protein kinase (MAPK) signaling pathway is critical for melanoma development and progression. Many oncogenic pathways that cause melanoma tumorigenesis have been identified, most of which are due to RAF/MEK/ERK (MAPK) pathway activation. However, the precise role of p38 remains uncl ...[more]

PMID: 31661126

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Project description:Background and aimsNeutrophil infiltration is a hallmark of nonalcoholic steatohepatitis (NASH), but how this occurs during the progression from steatosis to NASH remains obscure. Human NASH features hepatic neutrophil infiltration and up-regulation of major neutrophil-recruiting chemokines (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1] and interleukin [IL]-8). However, mice fed a high-fat diet (HFD) only develop fatty liver without significant neutrophil infiltration or elevation of chemokines. The aim of this study was to determine why mice are resistant to NASH development and the involvement of p38 mitogen-activated protein kinase (p38) activated by neutrophil-derived oxidative stress in the pathogenesis of NASH.Approach and resultsInflamed human hepatocytes attracted neutrophils more effectively than inflamed mouse hepatocytes because of the greater induction of CXCL1 and IL-8 in human hepatocytes. Hepatic overexpression of Cxcl1 and/or IL-8 promoted steatosis-to-NASH progression in HFD-fed mice by inducing liver inflammation, injury, and p38 activation. Pharmacological inhibition of p38α/β or hepatocyte-specific deletion of p38a (a predominant form in the liver) attenuated liver injury and fibrosis in the HFD+Cxcl1 -induced NASH model that is associated with strong hepatic p38α activation. In contrast, hepatocyte-specific deletion of p38a in HFD-induced fatty liver where p38α activation is relatively weak exacerbated steatosis and liver injury. Mechanistically, weak p38α activation in fatty liver up-regulated the genes involved in fatty acid β-oxidation through peroxisome proliferator-activated receptor alpha phosphorylation, thereby reducing steatosis. Conversely, strong p38α activation in NASH promoted caspase-3 cleavage, CCAAT-enhancer-binding proteins homologous protein expression, and B cell lymphoma 2 phosphorylation, thereby exacerbating hepatocyte death.ConclusionsGenetic ablation of hepatic p38a increases simple steatosis but ameliorates oxidative stress-driven NASH, indicating that p38α plays distinct roles depending on the disease stages, which may set the stage for investigating p38α as a therapeutic target for the treatment of NASH.

Project description:The p38 members of the mitogen-activated protein kinases (MAPKs) family mediate various cellular responses to stress conditions, inflammatory signals, and differentiation factors. They are constitutively active in chronic inflammatory diseases and some cancers. The differences between their transient effects in response to signals and the chronic effect in diseases are not known. The family is composed of four isoforms, of which p38α seems to be abnormally activated in diseases. p38α and p38β are almost identical in sequence, structure, and biochemical and pharmacological properties, and the specific unique effects of each of them, if any, have not yet been revealed. This study aimed to reveal the specific effects induced by p38α and p38β, both when transiently activated in response to stress and when chronically active. This was achieved via large-scale proteomics and phosphoproteomics analyses using stable isotope labeling of two experimental systems: one, mouse embryonic fibroblasts (MEFs) deficient in each of these p38 kinases and harboring either an empty vector or vectors expressing p38αWT, p38βWT, or intrinsically active variants of these MAPKs; second, induction of transient stress by exposure of MEFs, p38α-/-, and p38β-/- MEFs to anisomycin. Significant differences in the repertoire of the proteome and phosphoproteome between cells expressing active p38α and p38β suggest distinct roles for each kinase. Interestingly, in both cases, the constitutive activation induced adaptations of the cells to the chronic activity so that known substrates of p38 were downregulated. Within the dramatic effect of p38s on the proteome and phosphoproteome, some interesting affected phosphorylation sites were those found in cancer-associated p53 and Hspb1 (HSP27) proteins and in cytoskeleton-associated proteins. Among these, was the stronger direct phosphorylation by p38α of p53-Ser309, which was validated on the Ser315 in human p53. In summary, this study sheds new light on the differences between chronic and transient p38α and p38β signaling and on the specific targets of these two kinases.

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Project description:BackgroundPatients with malignant melanoma often relapse after treatment with BRAF and/or mitogen-activated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance.ObjectivesTo establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy.MethodsImmunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours. Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations. Flow cytometry and immunofluorescence microscopy was used to evaluate trametinib-induced cell death and CD271 expression. MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinib-resistant melanoma cell survival and invasion, respectively.ResultsCD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi. In vitro studies demonstrate MEKi of BRAF-mutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271-expressing subpopulations. Trametinib-induced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance. Treatment of CD271-expressing melanoma subpopulations with RNA interference and small-molecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents - including Δ9-tetrahydrocannabinol and Vps34 - reduced survival of MEKi-resistant melanoma cells. Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKi-resistant cells in an in vivo zebrafish xenograft.ConclusionsThese results highlight a novel mechanism of MEKi-induced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drug-resistant melanoma cells to the cytotoxic effects of MEKi.

Dataset Information

Roles of p38? and p38? mitogen?activated protein kinase isoforms in human malignant melanoma A375 cells.

Publications

Roles of p38α and p38β mitogen‑activated protein kinase isoforms in human malignant melanoma A375 cells.

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