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, αvβ3-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 αvβ3 antibody treatments in PAK4si-treated cells indicated the regulation of αvβ3/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, β3-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.
Highlights
The role of matrix metalloproteases (MMPs) is significant in the degradation of extracellular matrix (ECM), thereby facilitating tumor cell invasion.[1]
Matrix metalloproteinase-2 (MMP-2)-knockdown resulted in B42.3% death in adhered cells and more sensitized to anoikis-mediated cell death (B62.4%) in suspended cells compared with the corresponding controls (Figure 1a)
In adhered MMP2sitreated cells, we observed a substantial phospho-p21-activated kinase 4 (PAK4) and total-PAK4 downregulation accompanied by decreased phospho-epidermal growth factor receptor (EGFR), phospho-c-Src and phospho-focal adhesion kinase (FAK) levels, which were further drastically decreased in suspended cultures compared with respective controls (Figure 1h)
Summary
The role of matrix metalloproteases (MMPs) is significant in the degradation of ECM, thereby facilitating tumor cell invasion.[1]. EGFR signaling has a key role in maintaining GBM hallmark characteristics, including rapid cell proliferation, diffuse invasion and metastases.[16] A possible cross-talk between PAK4 and EGFR was suggested to enhance malignancy in ovarian cancer.[13]. To our knowledge, this is the first comprehensive study demonstrating the PAK4 upregulation in positive correlation with increasing glioma pathological grades. Cosuppression of PAK4 and MMP-2 conferred anoikis-mediated cell death in cells and inhibited in vivo tumor growth, thereby suggests the therapeutic potential of PAK4/MMP-2 dual targeting in glioma treatment
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