Phosphatidylinositol 4,5-bisphosphate and PIP5-kinase Iα are required for invadopodia formation in human breast cancer cells

Invadopodia are ventral cell protrusions formed in invasive cancer cells. Because invadopodia have extracellular matrix (ECM) degradation activity, they are thought to function in cancer invasion. In this study, we examined the roles of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and PI(4,5)P(2)-producing enzymes in invadopodia formation in MDA-MB-231 human breast cancer cells. Immunofluorescence analysis showed that PI(4,5)P(2) accumulates at invadopodia on the ventral cell surface. Injection of an anti-PI(4,5)P(2) antibody inhibited invadopodia formation along with gelatin degradation activity. Sequestering of PI(4,5)P(2) by overexpression of the phospholipase C (PLC) delta 1-pleckstrin homology (PH) domain, a specific probe for PI(4,5)P(2), also blocked invadopodia formation, while a mutated PLC delta 1-PH domain that lacks PI(4,5)P(2)-binding activity had no effect. Cellular PI(4,5)P(2) production is mainly mediated by type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) family proteins, which include PIP5KI alpha, I beta, and I gamma. Real-time quantitative PCR analysis showed that PIP5KI alpha is a dominant isoform expressed in MDA-MB-231 cells. Knockdown of PIP5KI alpha by small-interfering RNA (siRNA) inhibited invadopodia formation and gelatin degradation. Immunofluorescence analysis revealed that endogenous PIP5KI alpha protein localizes at invadopodia, which is corroborated by the observation that exogenously expressed green fluorescent protein (GFP)-fused PIP5KI alpha protein also accumulates at gelatin degradation sites. These results indicate that localized production of PI(4,5)P(2) by PIP5KI alpha is required for invadopodia formation and ECM degradation by human breast cancer cells. (Cancer Sci 2010).