Involvement of Hypoxia-Inducing Factor-1 Alpha-Dependent Plasminogen Activator Inhibitor-1 up-Regulation in Cyr61/Ccn1-Induced Gastric Cancer Cell Invasion

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1 alpha (HIF-1 alpha) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF -1 alpha protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1 alpha protein by recombinant Cyr 61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl2 treatment, both well known for induction of HIF-1 alpha. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1 alpha up- regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1 alpha protein accumulation. Blockage of HIF -1 alpha activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1 alpha strongly inhibited their invasion ability, suggesting that elevation in HIF-1 alpha protein is vital for Cyr 61-mediated gastric cancer cell invasion. In addition, several HIF-1 alpha -regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI -1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61- overexpressing cells. Transfection with dominant negative- HIF-1 alpha to block HIF-1 alpha activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1 alpha-dependent up-regulation of PAI-1.