Dataset Information

Cytosolic phospholipase A2? modulates cell-matrix adhesion via the FAK/paxillin pathway in hepatocellular carcinoma.

ABSTRACT: Objective:To explore the effect of cytosolic phospholipase A2? (cPLA2?) on hepatocellular carcinoma (HCC) cell adhesion and the underlying mechanisms. Methods:Cell adhesion, detachment, and hanging-drop assays were utilized to examine the effect of cPLA2? on the cell-matrix and cell-cell adhesion. Downstream substrates and effectors of cPLA2? were screened via a phospho-antibody microarray. Associated signaling pathways were identified by the functional annotation tool DAVID. Candidate proteins were verified using Western blot and colocalization was investigated via immunofluorescence. Western blot and immunohistochemistry were used to detect protein expression in HCC tissues. Prognosis evaluation was conducted using Kaplan-Meier and Cox-proportional hazards regression analyses. Results:Our findings showed that cPLA2? knockdown decreases cell-matrix adhesion but increases cell-cell adhesion in HepG2 cells. Microarray analysis revealed that phosphorylation of multiple proteins at specific sites were regulated by cPLA2?. These phosphorylated proteins were involved in various biological processes. In addition, our results indicated that the focal adhesion pathway was highly enriched in the cPLA2?-relevant signaling pathway. Furthermore, cPLA2? was found to elevate phosphorylation levels of FAK and paxillin, two crucial components of focal adhesion. Moreover, localization of p-FAK to focal adhesions in the plasma membrane was significantly reduced with the downregulation of cPLA2?. Clinically, cPLA2? expression was positively correlated with p-FAK levels. Additionally, high expression of both cPLA2? and p-FAK predicted the worst prognoses for HCC patients. Conclusions:Our study indicated that cPLA2? may promote cell-matrix adhesion via the FAK/paxillin pathway, which partly explains the malignant cPLA2? phenotype seen in HCC.

SUBMITTER: Guo P

PROVIDER: S-EPMC6713643 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Cytosolic phospholipase A2α modulates cell-matrix adhesion <i>via</i> the FAK/paxillin pathway in hepatocellular carcinoma.

Guo Piao P He Yuchao Y Chen Lu L Qi Lisha L Liu Dongming D Chen Ziye Z Xiao Manyu M Chen Liwei L Luo Yi Y Zhang Ning N Guo Hua H

Cancer biology & medicine 20190501 2

<h4>Objective</h4>To explore the effect of cytosolic phospholipase A2α (cPLA2α) on hepatocellular carcinoma (HCC) cell adhesion and the underlying mechanisms.<h4>Methods</h4>Cell adhesion, detachment, and hanging-drop assays were utilized to examine the effect of cPLA2α on the cell-matrix and cell-cell adhesion. Downstream substrates and effectors of cPLA2α were screened <i>via</i> a phospho-antibody microarray. Associated signaling pathways were identified by the functional annotation tool DAVI ...[more]

PMID: 31516757

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Project description:Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase A2α (cPLA2α) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLA2α activity, which produces lysophospholipids (LPLs) by cleaving at the sn-2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i) viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLA2α inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA2α activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development.IMPORTANCE Examples of highly conserved RNA virus proteins that qualify as drug targets for broad-spectrum antivirals remain scarce, resulting in increased efforts to identify and specifically inhibit cellular functions that are essential for the replication of RNA viruses belonging to different genera and families. The present study supports and extends previous conclusions that enzymes involved in cellular lipid metabolism may be tractable targets for broad-spectrum antivirals. We obtained evidence to show that a cellular phospholipase, cPLA2α, which releases fatty acid from the sn-2 position of membrane-associated glycerophospholipids, is critically involved in coronavirus replication, most likely by producing lysophospholipids that are required to form the specialized membrane compartments in which viral RNA synthesis takes place. The importance of this enzyme in coronavirus replication and DMV formation is supported by several lines of evidence, including confocal and electron microscopy, viral replication, and lipidomics studies of coronavirus-infected cells treated with a highly specific cPLA2α inhibitor.

Dataset Information

Cytosolic phospholipase A2? modulates cell-matrix adhesion via the FAK/paxillin pathway in hepatocellular carcinoma.

Publications

Cytosolic phospholipase A2α modulates cell-matrix adhesion <i>via</i> the FAK/paxillin pathway in hepatocellular carcinoma.

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