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

Abstract

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) d1-pleckstrin homology (PH) domain, a specific probe for PI(4,5)P 2 , also blocked invadopodia formation, while a mutated PLCd1-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 PIP5KIa, Ib, and Ic. Real-time quantitative PCR analysis showed that PIP5KIa is a dominant isoform expressed in MDA-MB-231 cells. Knockdown of PIP5KIa by small-interfering RNA (siRNA) inhibited invadopodia formation and gelatin degradation. Immunofluorescence analysis revealed that endogenous PIP5KIa protein localizes at invadopodia, which is corroborated by the observation that exogenously expressed green fluorescent protein (GFP)-fused PIP5KIa protein also accumulates at gelatin degradation sites. These results indicate that localized production of PI(4,5)P 2 by PIP5KIa is required for invadopodia formation and ECM degradation by human breast cancer cells. (Cancer Sci 2010; 101: 1632-1638 I nvadopodia are extracellular matrix (ECM)-degrading protrusions formed at the ventral surface of invasive cancer cells.(1,2) In the case of breast cancer cell lines, the ability to form invadopodia is closely related to their invasive and metastatic properties.(3,4) Additionally, during intravasation, invadopodia-like protrusions in tumor cells have been observed in vivo by intravital imaging.(5,6) Therefore, invadopodia are proposed to function in local ECM degradation during cancer invasion and metastasis, although the in vivo relevance of invadopodia has yet to be determined. To date, many components of invadopodia have been reported, including proteins involved in the regulation of the actin cytoskeleton, cell signaling, cell-ECM adhesion, ECM degradation, and membrane remodeling.(7-9) We and other researchers previously proposed that invadopodia formation occurs in several steps. Invadopodia precursors are assembled by an actin polymerization machinery containing neural Wiskott-Aldrich syndrome protein (N-WASP), the Arp2 ⁄ 3 complex, and cortactin in response to extracellular stimuli. (3,10) The invadopodia precursors are then stabilized by further actin polymerization induced by cofilin and Mena, and finally these structures gather matrix metalloproteinases to mature into functional invadopodia. (3,11,12) However, it i...

Membrane lipids in invadopodia and podosomes: Key structures for cancer invasion and metastasis

“…We recently reported that PI(4,5)P 2 is enriched at invadopodia and blockage of the PI(4,5)P 2 function suppresses invadopodia formation and ECM degradation by invasive human breast cancer cells [39]. We also found that a kinase generating PI(4,5)P 2 , phosphatidylinositol-4-phosphate 5-kinase type Iα (PIPK Iα), accumulates at the invadopodia and that a knockdown of PIPK Iα inhibits invadopodia formation.…”

“…Nck is required for N-WASP-dependent actin polymerization induced by PI(4,5)P 2 and Nck also stimulates PI(4,5)P 2 production via recruitment of PIPK Iα. Considering that Nck and PI(4,5)P 2 are essential for invadopodia formation [9,39], these components may interdependently activate N-WASP to assemble invadopodia structures. Other invadopodia components, such as cofilin and dynamin-2, are also regulated by PI(4,5)P 2 [31].…”

Signaling inputs to invadopodia and podosomes

“…PtdIns(4,5)P 2 has been shown to surround the invadopodium protrusion and might be generated locally by phosphatidylinositol 4-phosphate 5-kinase type Ia (PIP5KIa) (Yamaguchi et al, 2010). Both PtdIns(4,5)P 2 and PIP5KIa are required for ECM degradation by breast cancer cell invadopodia, but a requirement for invadopodia formation itself was not assessed in that study (Yamaguchi et al, 2010).…”

“…PtdIns(4,5)P 2 has been shown to surround the invadopodium protrusion and might be generated locally by phosphatidylinositol 4-phosphate 5-kinase type Ia (PIP5KIa) (Yamaguchi et al, 2010). Both PtdIns(4,5)P 2 and PIP5KIa are required for ECM degradation by breast cancer cell invadopodia, but a requirement for invadopodia formation itself was not assessed in that study (Yamaguchi et al, 2010). Interestingly, the Ras family GTPase ARF6, which activates PIP5KIa downstream of EGF (Honda et al, 1999), localizes to invadopodia and is required for their activity (Hashimoto et al, 2004;Tague et al, 2004).…”

The guanine nucleotide exchange factor Arhgef5 plays crucial roles in Src-induced podosome formation

“…For the fluorescent gelatin degradation assay, Alexa-Fluor-594-gelatin-coated dishes were prepared as described previously (Yamaguchi et al, 2010). Confocal images were obtained using a confocal laser scanning microscopy (Olympus, FV-1000).…”