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Functional cooperativity by direct interaction between PAK4 and MMP-2 in the regulation of anoikis resistance, migration and invasion in glioma.

ABSTRACT: Gliomas display anoikis resistance, enhanced invasion in to the adjacent brain parenchyma and eventually recur despite using the standard therapies. Our studies on increased anoikis sensitization in matrix metalloproteinase-2 (MMP-2)-knockdown 4910 and 5310 human glioma xenograft cells were interestingly correlated with p21-activated kinase 4 (PAK4) inhibition, prompting us to further investigate the role of PAK4 in glioma. Here, we report the PAK4 upregulation in positive correlation with increasing glioma pathological grades. The siRNA-mediated PAK4 knockdown elevated anoikis, and inhibited invasion and migration by downregulating MMP-2, ?v?3-integrin and phospho-epidermal growth factor receptor (phospho-EGFR). The cDNA-PCR arrays revealed a transcriptional suppression of essential proteins involved in cell proliferation and adhesion in PAK4-knockdown cells. Most importantly, glutathione S-transferase pull-down assays demonstrated the MMP-2 as a new PAK4-interacting protein which binds to PAK4 kinase domain. Individual EGFR/ErbB2 inhibitor and ?v?3 antibody treatments in PAK4si-treated cells indicated the regulation of ?v?3/EGFR survival signaling by PAK4. Overexpression of PAK4 significantly reversed the MMP2si-induced cell death in both cell lines. Codepletion of PAK4 and MMP-2 resulted in robust anoikis-mediated cell death, and severely inhibited invasive and migratory properties in these cells. PAK4si inhibited in vivo tumor growth in nude mice by inhibiting MMP-2, ?3-integrin and phospho-EGFR levels in tumors. Our findings indicate a physical association between PAK4 and MMP-2, and suggest the future therapeutic potential of PAK4/MMP-2 dual targeting in glioma treatment.

SUBMITTER: Kesanakurti D

PROVIDER: S-EPMC3542618 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Functional cooperativity by direct interaction between PAK4 and MMP-2 in the regulation of anoikis resistance, migration and invasion in glioma.

Kesanakurti D D Chetty C C Rajasekhar Maddirela D D Gujrati M M Rao J S JS

Cell death & disease 20121220

Gliomas display anoikis resistance, enhanced invasion in to the adjacent brain parenchyma and eventually recur despite using the standard therapies. Our studies on increased anoikis sensitization in matrix metalloproteinase-2 (MMP-2)-knockdown 4910 and 5310 human glioma xenograft cells were interestingly correlated with p21-activated kinase 4 (PAK4) inhibition, prompting us to further investigate the role of PAK4 in glioma. Here, we report the PAK4 upregulation in positive correlation with incre ...[more]

PMID: 23254288

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Project description:Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-?, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with ?5 and ?1 integrins and MMP-2 downregulation inhibited ?5?1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/?5?1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of ?5?1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking ?5?1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/?5?1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/?5?1 interaction in the regulation of ?5?1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/?5?1 interaction in glioma treatment.

Dataset Information

Functional cooperativity by direct interaction between PAK4 and MMP-2 in the regulation of anoikis resistance, migration and invasion in glioma.

Publications

Functional cooperativity by direct interaction between PAK4 and MMP-2 in the regulation of anoikis resistance, migration and invasion in glioma.

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