Abstract A08: PI3Kbeta regulates beta-1 integrin signaling in invadopodia through formation of PI(3,4)P2

The PI3Kbeta isoform of PI 3-kinase, which is activated downstream from both RTKs and GPCRs, is an important regulator of invadopodia formation. We have previously reported that in MDA-MB-231 breast cancer cells, replacement of endogenous PI3Kbeta with kinase dead (KD) or GPCR-uncoupled (KK-DD) mutants leads to defects in invadopodia-mediated gelatin degradation. We have now studied the mechanism by which loss of PI3Kbeta signaling affects invadopodia. While the number of invadopodia precursors is unaffected by mutation of PI3Kbeta, formation of mature invadopodia is reduced in cells expressing KK-DD and KD PI3Kbeta mutants. Previous studies have demonstrated a role for beta-1 integrins in invadopodia maturation, and PI3Kbeta has been implicated in integrin signaling in platelets. We therefore tested whether mutation of PI3Kbeta would affect integrin signaling in breast cancer cells. In both haptotaxis and cell spreading assays on collagen I, cells expressing KD or KK-DD PI3Kbeta showed a significant impairment. Haptotaxis and cell spreading was also inhibited by TGX221 (PI3Kbeta inhibitor) and pertussis toxin (PTX), but not by specific inhibitors of other Class I PI3Ks. To distinguish whether PI3Kbeta 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 gelatin, but this was blocked by TGX221, suggesting that PI3Kbeta acts downstream of beta-1 integrins. Importantly, haptotaxis and cell spreading were not affected by an Akt inhibitor, but were blocked by an inhibitor of SHIP2, which hydrolyzes PIP3 to produce PI[3,4]P2. These data suggest that integrin signaling requires the coupling of Gbeta/gamma-mediated activation of PI3Kbeta to the production of PI[3,4]P2. Finally, to determine whether PI3Kbeta was involved in beta-1 integrin signaling in invadopodia, we plated starved cells on high-density fibrillar collagen (HDFC), which drives invadopodia formation through beta-1 integrin activation. Collagen degradation on HDFC was blocked by inhibition of PI3Kbeta or SHIP2, but not PI3Kalpha. Moreover, recruitment of the PI(3,4)P2 binding protein lamellipodin to invadopodia was reduced by inhibition of PI3Kbeta. In summary, beta-1 integrin signaling in the context of invadopodia maturation requires signaling through PI3Kbeta. We propose that heterotrimeric G-protein activation of PI3Kbeta is necessary for formation of PI(3,4)P2 in invadopodia, which is required for invadopodial maturation. Citation Format: Zahra Erami, Anne R. Bresnick, Jonathan M. Backer. PI3Kbeta regulates beta-1 integrin signaling in invadopodia through formation of PI(3,4)P2 [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A08.