Phosphatidylinositol 4,5-bisphosphate and PIP5-kinase Ialpha 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) delta1-pleckstrin homology (PH) domain, a specific probe for PI(4,5)P(2), also blocked invadopodia formation, while a mutated PLCdelta1-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 PIP5KIalpha, Ibeta, and Igamma. Real-time quantitative PCR analysis showed that PIP5KIalpha is a dominant isoform expressed in MDA-MB-231 cells. Knockdown of PIP5KIalpha by small-interfering RNA (siRNA) inhibited invadopodia formation and gelatin degradation. Immunofluorescence analysis revealed that endogenous PIP5KIalpha protein localizes at invadopodia, which is corroborated by the observation that exogenously expressed green fluorescent protein (GFP)-fused PIP5KIalpha protein also accumulates at gelatin degradation sites. These results indicate that localized production of PI(4,5)P(2) by PIP5KIalpha is required for invadopodia formation and ECM degradation by human breast cancer cells.
Yamaguchi
Hideki
H
Laboratory of Genome and Biosignal, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
Yoshida
Shuhei
S
Muroi
Emi
E
Kawamura
Masahiro
M
Kouchi
Zen
Z
Nakamura
Yoshikazu
Y
Sakai
Ryuichi
R
Fukami
Kiyoko
K
D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D002460:Cell Line; D017931:DNA Primers; D005260:Female; D006801:Humans; D007700:Kinetics; D009361:Neoplasm Invasiveness; D010641:Phenotype; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D019269:Phosphatidylinositol 4,5-Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase