Abstract 3602: Differential effects of mTOR and PI3K inhibition on the phosphorylation of polysome-associated proteins

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The PI3K (phosphoinositide-3 kinase) pathway is upregulated in many malignancies, including a significant proportion of human breast cancers. PI3K dysregulation activates downstream targets, such as the serine-threonine kinase AKT (also known as protein kinase B) and the mammalian target of rapamycin (mTOR) complexes, TORC1 and TORC2, serving to enhance cell survival, growth, invasiveness, and angiogenic potential as well as altering cellular metabolism. Many of these downstream cellular responses are induced by PI3K/AKT/mTOR control of mRNA translation initiation and elongation via phosphorylation of polysome-associated effectors, such as eukaryotic translation initiation factor 4 components, its inhibitory binding protein (eIF4BP), and the 40S ribosomal protein S6. Due to the importance of the PI3K pathway in tumorigenesis, several different PI3K/AKT/mTOR pathway inhibitors are under clinical development, including dual TORC1/C2 inhibitors (e.g. INK128; Intellikine) and pan-inhibitors of all four isoforms of class 1 PI3K (e.g. GDC0941; Genentech). Despite the specificity of such mTOR and PI3K inhibitors, it remains unclear which subsets of breast cancer will respond to these agents and which biomarkers are best used to monitor and distinguish their target-specific antitumor responses. Furthermore, known translation-regulating phosphoproteins downstream of mTORC1 (e.g. eIF4B, S6) can also be activated by alternative kinase pathways (e.g. PDK1) downstream of PI3K. Therefore, we sought to discover translation-associated biomarkers capable of distinguishing the responsiveness of breast cancer cells to INK128 and GDC0941, focusing on post-translationally modified proteins contained within the polysome-enriched cell compartment. We used an antibody specific for phospho-AKT substrates to probe 1-D and 2-D gel separated polysome-enriched fractions from the HER2-positive breast cancer cell line, SKBR3, following control or growth inhibiting doses of INK128 or GDC0941. The 5-day SKBR3 IC50 doses (inhibiting cell proliferation by 50%) for INK128 and GDC0941 were found to be 16 nM and 565 nM, respectively. Following 24 h exposure to identical doses of these two drugs, INK128 significantly reduced the phosphorylation of a small subset of polysome-associated proteins (including ∼30 kD and ∼50 kD species) while GDC0941 had no apparent effect on these proteins, whose identification is pending. These findings suggest that phosphorylation of specific polysome-associated proteins likely mediates translational control exerted by some downstream effectors of the PI3K/AKT/mTOR pathway. Furthermore these specific polysome-associated targets may serve as biomarkers differentiating cell responses to TORC1/C2 from pan-PI3K inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3602. doi:1538-7445.AM2012-3602