The role of PAK4 in extracellular matrix degradation

Abstract

Extracellular Matrix (ECM) Degradation is an essential process for cancer cell invasion from the tumor to blood stream. A membrane structure called invadopodia is thought to be responsible to this process.

p21-activated kinase 4 (PAK4), which is a serine/threonine protein kinase, has been found to be overexpressed in various cancers. PAK4 has been shown to participate in cell survival and cytoskeleton dynamics, so as to promote the proliferation and migration of cancer cells. Overexpression of PAK4 can also promote podosomes formation, a homolog of invadopodia in macrophages. This research aims to investigate the role of PAK4 in the ECM degrading properties of hepatocellular carcinoma (HCC).

Here, a panel of HCC cell lines was tested by ECM degradation assay. Among all the cell lines tested, only Hep3Band HLE cells possessed invadopodia to degrade the gelatin matrix. However, a new gelatin degradation pattern, characterized by large patches around cell peripheral, namely around-cell-peripheral degradation (ACPD), was observed in two HCC cell lines, SMMC-7721 (SMMC) and MHCC-97L. PAK4 overexpression in SMMC was shown to promote ACPD and it showed a faster recovery of the degradation after removal of MMPs inhibitor. Quantitative real-time PCR analysis showed PAK4 overexpression promoted transcription of MMP-2 and MMP-9, which are gelatin-degrading MMPs. In addition, the secretion of active dimeric form of MMP-9 was upregulated, as shown by gelatin zymography, suggesting that MMP activity is required for ACPD. Also, I found that PAK4 enhanced the induction of MMP-9 under the treatment of TNF-α, an NFκB pathway activator. Immunofluorescence staining experiments showed that overexpression of wild type and dominant active (DA, S445N)PAK4, but not its dominant negative form(DN, K350/351M),could significantly induce the nuclear translocation of NFκB, even without treatment of TNF-α. The ectopic expression of PAK4 DN in SMMC blocked the nuclear translocation of NFκB induced by TNF-α. Taken together, these results suggested that PAK4 can promote the translocation of NFκB in both TNF-α dependent and independent pathway. The effect of PAK4 on NFκB translocation was further confirmed by subcellular fractionation experiment. In summary, our data suggest PAK4 induces MMP-9 transcription by activation of NFκB pathway, to promote ACPD activity in SMMC cells.