Abstract PD1-9: P-REX1 employs a positive feedback loop to activate growth factor receptor/PI3K signaling

Abstract A mass spectrometry-based proteomic screen in ER+ breast cancer cells revealed that levels of P-REX1 are decreased upon PTEN loss, and increased upon PI3K inhibition. P-REX1 integrates signaling inputs from receptor tyrosine kinases (RTKs)/PI3K (via the PI3K phospholipid product PIP3) and G protein-coupled receptors (via Gbg subunits) to drive guanine exchange factor (GEF) activity on Rac1. RNAi knockdown of PREX1 and overexpression of exogenous PREX1 in ER+ breast cancer cells respectively decreased and increased activation of insulin-like growth factor receptor-1 (IGF-1R)/insulin receptor (InsR), PI3K/AKT/SGK3, and MEK/Erk under steady-state and growth factor (IGF-1, Heregulin)-stimulated conditions. While the P-REX1 homologue P-REX2a was previously shown to inhibit PTEN phosphatase activity to activate the PI3K/AKT pathway, we did not detect an effect of P-REX1 on PTEN activity. PREX1 knockdown suppressed PI3K/AKT signaling in PTEN-null breast cancer cells; therefore, P-REX1 and P-REX2a may not be functionally redundant. Knockdown and overexpression of PREX1 respectively increased and decreased doxorubicin-induced apoptosis in ER+ breast cancer cells, linking PREX1 with a pro-survival phenotype. Inhibition of signaling nodes downstream of PI3K (AKT, mTOR) derepresses feedback to activate RTKs and PI3K; knockdown of PREX1 abrogated the PI3K activation induced by inhibition of mTORC1/mTORC2. Structural analysis of P-REX1 revealed that the DH domain (which binds Gβγ and is required for GEF activity) is dispensable for P-REX1 effects on PI3K signaling, while the PH domain [which binds PIP3 and PI(3,4)P2] is required. These data place P-REX1 in a positive feedback loop, whereby PI3K generates PIP3 and PI(3,4)P2, and P-REX1 binds these phospholipids at the plasma membrane. In turn, P-REX1 promotes RTK activation, and RTKs activate PI3K/AKT and MEK/Erk signaling. Gene expression profiling of diverse types of solid tumors (n = 2,009) and cancer cell lines (n = 807) revealed that PREX1 mRNA is most abundant in ER+ breast tumors compared to other subtypes. Reverse-phase protein array (RPPA) analysis of lysates from 712 breast tumors showed that P-REX1 levels are inversely correlated with markers of PI3K/AKT/mTOR pathway activation. Further, P-REX1 levels are higher in ER+ tumors than ER- tumors. In another series of 1,293 carcinomas, PREX1 was genetically amplified or mutated in 6.2% of cases, and in 5% of breast cancers. We then tested whether PREX1 lesions co-exist with other PI3K pathway-activating lesions. Among genes encoding proteins implicated in RTK/PI3K signaling and phosphatidylinositol metabolism, we found a significant enrichment for PREX1 mutation/amplification in 54/79 (68%) genes across 1,523 carcinomas. We tested the effects of 7 PREX1 mutants found in breast tumors on PI3K signaling in vitro. A G344R mutation in the PREX1 PH domain conferred increased affinity for PIP3 and PI(3,4)P2, and increased the levels of phospho-AKT. These findings suggest that P-REX1 is an ER+ breast tumor-specific oncogene, and PREX1 mutations increase its oncogenic effects in breast cancer. We propose that neutralizing P-REX1 function is a novel therapeutic approach to selectively abrogate oncogenic signaling in ER+ breast cancers while sparing normal tissues. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-9.