Abstract PR01: Inhibitory role of phosphatidylinositol-3,4-bisphosphate in triple-negative breast cancers

Abstract

Abstract Triple-negative breast cancer (lacking expression of estrogen receptor, progesterone receptor and amplification of HER2/Neu) remains one of the most aggressive subtypes, affects the youngest patients and yet still lacks an effective targeted therapy. Novel insights into the molecular mechanisms that drive these cancers are imperative to guide development and application of such targeted therapies. Data from The Cancer Genome Atlas and other sources have suggested that phospho-Akt (pAkt) levels are significantly higher in triple-negative tumors compared to either hormone receptor positive tumors (express estrogen and/or progesterone receptor) or Her2/Neu amplified tumors. This has led to the hypothesis that these tumors are dependent on phosphatidylinositide-3-kinase (PI3K) and that PI3K pathway inhibitors may be effective therapeutically in triple-negative tumors. Both PI3K-α and β are ubiquitously expressed and therefore may be important for the oncogenesis of solid tumors, including those of the breast. INPP4B catalyzes the removal of the 4′ phosphate of phosphatidylinositol-(3,4)-bisphosphate creating phosphatidylinositol-(3)-phosphate. There has been debate concerning whether phosphatidylinositol-(3,4)-bisphosphate can contribute to activation of Akt and other downstream effectors, in addition to phosphatidylinositol-(3,4,5)-trisphosphate, the canonical signaling second messenger effector downstream of PI3K. If this were true, INPP4B would have a negative effect on signaling. Utilizing a panel of cell lines derived from triple-negative breast tumors that are null for PTEN, we used both siRNA-mediated silencing and stable transfectants expressing either Flag-INPP4B or a mutant of Flag-INPP4B(Cys842Ala) lacking phosphatase activity to manipulate levels of phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(3,4)-bisphosphate. We then employed a panel of PI3K pan- and isoform-selective inhibitors to investigate how changing the levels of phosphatidylinositol phosphate species effected inhibitor sensitivity. We have found that silencing INPP4B in several triple-negative breast cancer cell lines sensitizes cells to PI3Kα-isoform selective inhibitors and, to an even greater degree, PI3Kβ-isoform selective inhibitors. This is evidenced by both a decrease in pAkt levels, as detected by Western blot, as well as a decrease in GI50, as detected by proliferation assays following siRNA treatment. Conversely, introduction of an extra copy of INPP4B into the same cell lines results in a decrease in sensitization to both PI3Kα- and PI3Kβ-isoform selective inhibitors, in this case to a greater degree with the PI3Kα-isoform selective inhibitor than the PI3Kβ-isoform selective inhibitor. This is especially evident by a marked dampening of the decrease in pAkt levels that is seen in response to the inhibitors in both the parental cell line and stable cell lines expressing a catalytically dead mutant of INPP4B. Thus, this is an effect requiring the phosphatase activity of INPP4B. Unexpectedly, our data support a model in which phosphatidylinositol-(3,4)-bisphosphate is in fact inhibitory toward PI3K, at least under conditions of strong signaling, as in triple negative breast cancers. When levels of this inhibitor accumulate following siRNA-mediated knockdown of INPP4B, cells become sensitized to PI3K pathway inhibitors. Conversely, when levels are depleted following overexpression of INPP4B, cells become resistant to PI3K pathway inhibitors. This may indicate a novel feedback inhibition by a downstream substrate within the PI3K pathway under conditions of excessive signaling. This work has important implications for the future of targeted therapy by PI3K pathway inhibitors. Furthermore, it may reveal a unique indication for PI3Kβ-isoform selective inhibitors in triple-negative breast cancers that have lost both PTEN and INPP4B. This abstract is also being presented as Poster A21. Citation Format: Darien E. Reed, Kevan M. Shokat. Inhibitory role of phosphatidylinositol-3,4-bisphosphate in triple-negative breast cancers. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR01.