Abstract 3431: PI3Kbeta regulates macropinocytosis in PTEN-null tumor cells

Abstract

Abstract Class I phosphoinositide 3-kinases (PI3Ks) have been previously implicated in the regulation of vesicular trafficking, including receptor-mediated endocytosis and macropinocytosis. Using NIH3T3 cells in which the genes for the PI3Kbeta or PI3Kalpha catalytic subunits were deleted by CRISPR/Cas9, we were unable to detect any defects in receptor-mediated endocytosis of [125I]-PDGF or in pinocytosis of Lucifer yellow. However, we observed a marked inhibition of macropinocytosis in PI3Kbeta knockout cells; deletion of the other major PI3K isoform in these cells, PI3Kalpha, had no effect. These studies were confirmed using isoform-selective inhibitors: only inhibition of PI3Kbeta blocked macropinocytosis. Similarly, in MDA-MB-231 breast cancer cells, HGF-stimulated macropinocytosis was markedly reduced by inhibition of PI3Kbeta, but not the other PI3K isoforms. Notably, expression of a mutant PI3Kbeta that cannot be activated by Gbeta-gamma, or treatment of WT cells with pertussis toxin, also inhibited macropinocytosis in MDA-MB-231 cells. A requirement for Gbeta-gamma binding to PI3Kbeta was also seen in primary macrophages from mice expressing WT or Gbeta-gamma-uncoupled PI3Kbeta, as both CSF-1 and C5a-stimulated macropinocytosis was inhibited by mutant PI3Kbeta. To define the mechanism by which PI3Kbeta regulates macropinocytosis, we measured circular dorsal ruffle (CDR) formation. Genetic ablation or inhibition of PI3Kbeta completely suppressed CDR formation in PDGF-stimulated NIH3T3 cells, whereas inhibition or ablation of other PI3K isoforms had no effect. While previous studies have suggested that Class I PI3Ks act late in macropinosome formation, just prior to macropinosome sealing, our data suggest that PI3Kbeta functions early in macropinocytosis, as it mediates CDR formation in a Gbeta-gamma-dependent manner. Given previous work showing a positive feedback loop involving PI3Kbeta and Rac1, we tested whether macropinosome formation was rescued by expression of CA-Rac1. CA-Rac1 expression potently stimulated macropinocytosis in MDA-MB-231 cells. Strikingly, this was completely blocked by expression of the Gbeta-gamma-uncoupled PI3Kbeta, and partially blocked by expression of a mutant PI3Kbeta that cannot bind Rac1. Finally, given the well established role of PI3Kbeta in the growth of tumor cells lacking the PTEN tumor suppressor, as well as recent studies showing that high rates of basal macropinocytosis can serve as a nutrient a uptake mechanism in PTEN null cells, we measured macropinocytosis in two PTEN-deficient tumor lines, BT549 (breast) and PC3 (prostate). Both lines showed elevated basal levels of macropinocytosis, which were specifically blocked by inhibition of PI3Kbeta. Our data support a highly specific role for PI3Kbeta in the regulation of macropinocytosis, which may contribute the role of PI3Kbeta in the growth of PTEN-null tumor cells. Citation Format: Gilbert Salloum, Charles Jakubik, Anne R. Bresnick, Jonathan M. Backer. PI3Kbeta regulates macropinocytosis in PTEN-null tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3431.