Abstract 3165: PI 3-kinase-beta regulates invadopodia maturation and beta-1 integrin signaling

Abstract

Abstract The invasion of tumors cells during metastasis is mediated by invadopodia, actin rich protrusive organelles that secrete matrix metalloproteases and mediate matrix degradation. The PI3K-beta isoform of PI 3-kinase, which is regulated by both receptor tyrosine kinases and GPCRs, is an important regulator of invadopodia formation. We have previously reported that in MDA-MB-231 breast cancer cells, replacement of endogenous PI3K-beta with kinase dead (p110-betaK799R; KD) or GPCR-uncoupled (p110-beta526KK-DD; KK-DD) mutants leads to defects in gelatin degradation. We have now studied the mechanism by which loss of PI3K-beta signaling affects invadopodia formation and matrix degradation. While the number of invadopodia precursors (cortactin-Tks5 positive, degradation negative) is unaffected by mutation of PI3K-beta, formation of mature invadopodia (cortactin-Tks5 positive, degradation positive) is reduced in cells expressing KK-DD and KD PI3K-beta. Previous studies have demonstrated a role for beta-1 integrins in invadopodia maturation, and PI3K-beta has been implicated in integrin signaling in platelets. We therefore tested whether mutation of PI3K-beta would affect integrin signaling in breast cancer cells. In a haptotaxis assay, MDA-MB-231 cells expressing KD or KK-DD PI3K-beta showed a significant decrease in migration. Consistent with these data, cell spreading on collagen I was significantly impaired in cells expressing KD or KK-DD PI3K-beta, and was inhibited by TGX221 (PI3K-beta inhibitor) and LY294002 (pan-PI3K inhibitor), but not by specific inhibitors of other Class I PI3Ks (alpha, delta and gamma). Spreading was also impaired in cells treated with pertussis toxin, which blocks GPCR activation of PI3K-beta. To distinguish whether PI3K-beta was acting on integrins by inside-out versus outside-in mechanisms, we treated cells with the activating beta-1 integrin antibody TS2/16, which bypasses inside-out regulation of integrin binding. TS2/16 increased cell spreading on collagen I, but TS2/16-stimulated spreading was blocked by treatment of cells with TGX221, suggesting that PI3K-beta acts downstream of beta-1 integrins. Finally, to determine whether PI3K-beta was involved in beta-1 integrin signaling in invadopodia, we plated cells on high density fibrillar collagen (HDFC), which enhances invadopodia formation through beta-1 integrin activation. Invadopodia formation on HDFC was blocked by treatment of cells with TGX221. In summary, beta-1 integrin signaling in the context of invadopodia requires GPCR signaling to PI3K-beta. We propose that GPCR activation of PI3K-beta is a critical downstream component of beta-1 integrin signaling in invadopodia. Citation Format: Zahra Erami, Anne R. Bresnick, Jonathan M. Backer. PI 3-kinase-beta regulates invadopodia maturation and beta-1 integrin signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3165.