GPCR Signaling to PI3Kbeta: Regulation and Role in Breast Cancer Metastasis
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p110beta, the catalytic subunit of P13Kbeta, is an isoform of phosphoinositide 3-kinase (PI3K), a family of lipid kinases that control critical cellular functions, including proliferation, growth, metabolism, and migration. The PI3K pathway is one of the most commonly deregulated signaling pathways in human cancers. p110beta is unique among the Class IA isoforms as it is the only isoform that is directly activated downstream of receptor tyrosine kinases (RTKs) as well as G protein-coupled receptors (GPCRs). GPCR activation of p110beta occurs by its direct binding to the Gbetagamma subunit of trimeric G proteins. Additionally, p110beta has unique binding partners, including the small GTPases Rac, Cdc42, and Rab5.;While most studies on the role of PI3K in breast cancer focus on the p110alpha isoform due to its frequent mutation, a few reports have suggested a role for p110beta in breast cancer progression and metastasis. Given the crucial role of GPCR signaling within the tumor microenvironment in breast cancer metastasis, we decided to investigate the importance of GPCR-mediated activation of p110beta in breast cancer metastasis. Our lab previously identified a mutation that specifically abolishes p110beta binding to Gbetagamma (p110beta526KK-DD). My data show that expression of this mutant in p110beta knockdown breast cancer cells inhibits lysophosphatidic acid (LPA)-stimulated phosphorylation of AKT, a major downstream effector in the PI3K pathway, and migration. Cells expressing p110beta526 KK-DD do not invade in response to other GPCR ligands, including stromal cell-derived factor 1 (SDF-1) and interleukin 8 (IL8). This is correlated with inhibition of multiple steps of the metastatic cascade in vitro and in vivo, including macrophage-stimulated invasion, in vitro extravasation, as well as experimental metastasis following tail vein injection into SCID mice. These results can be explained, in part, by the fact that p110beta526KK-DD cells show an intrinsic defect in invadopodia-mediated extracellular matrix degradation. However, we also demonstrate that GPCR signaling to p11013 is required for paracrine interactions with macrophages, since cells expressing p110beta526 KK-DD show defects in a macrophage-stimulated migration assay that does not require invasion or invadopodia function. Our data demonstrate a previously unidentified role for GPCR signaling to p110beta in metastasis and suggest that disruption of Gbetagamma-p110beta binding might constitute a novel pharmacologic target for the prevention of metastatic disease.;In addition to directly binding and activating p110beta, Gbetagamma has also been shown to indirectly activate p110beta via the binding and activation of Dock180/EImo1, a Rac1 guanine nucleotide exchange factor. Dock180/EImol activates Rac1, which in turn binds to and activates p110beta. Given the dual pathways by which GPCRs activate P13Kbeta, we examined the integration of these inputs in intact cells. We find that GRgamma and Rac1 additively activate P13Kbeta in breast cancer cells. Surprisingly, Rac1-mediated activation of P13Kbeta is greatly attenuated in cells expressing p110beta 526KK-DD. Similarly, Rac1- -mediated activation of P13Kbeta is reduced in cells treated with pertussis toxin or expressing a fragment of GRK2 that sequesters Gbetagamma subunits. The GRgamma binding-site mutation has no effect on in vitro binding or activation of P13Kbeta by Rac1. However, Rac1-mediated membrane translocation of P13Kbeta is reduced in cells expressing mutant P13Kbeta. Our data suggest that in intact cells, Gbetagamma and Rac1 activate P13Kbeta in a hierarchical fashion, in which binding to Gbetagamma subunits is a prerequisite for P13Kbeta interactions with activated Rac1 in cell membranes.;Taken together, my work shows that GPCR signaling to p110beta is required for breast cancer metastasis, and that inhibition of the p110beta-Gbetagamma interaction will block both Gbetagamma- and Rac1-mediated activation of p110beta. The data suggests the disruption of p110beta-Gbetagamma binding as a potential therapeutic target for the treatment of metastasis.