Integrated quantative and spatial proteomic analysis of PI3kinase signaling in high grade prostate cancer (PCa) treated with Temsirolimus with or without androgen ablation

Abstract

5071 Background: Temsirolimus modulates the PI3kinase (PI3K) pathway in PCa models. An integrated proteomic strategy of reverse phase protein arrays (RPPA) and tissue microarrays (TMA) was applied to assess PI3K pathway, proliferation and apoptosis following Temsirolimus (T) in high risk PCa. Methods: Thirty-Five patients (pts) received oral T (20pts) or T+androgen ablation (T+AA) (15pts) for a median of 4 weeks followed by radical prostatectomy(RP). We constructed TMAs from RP specimens of: a) (T±AA) 33 pts treated with T (19pts) or T+AA (14pts) b)(Control) 42 untreated matched pts. 2320 cores were obtained [median cores: 18 (range 12–90), median blocks: 5 (range 3–9) per RPS]. Immunohistochemical expression of PI3K pathway, Ki67 and caspase 3 were assessed. Mixed model was used for fitting the correlated data. RPPAs were performed on protein lysates from tumor of 15 T±AA pts and 17 matched controls to compare expression of 25 proteins (PI3K, MAPK and cell cycle control signaling pathways). Cancer duplicates were harvested by UV/laser capture microdissection from a median of 4 sections per case. Unsupervised hierarchical clustering approaches were used for analysis. Separate controls were used for RPPA and TMA. Results: RPPAs segregate the samples in two clusters. Treated tumors had a distinct profile that was observed by both methodologies and included: a)Reduced levels of phospho-S6 in the treated group versus the control (p value <0.005) b) Increased phospho-mTOR [by both methodologies (p<0.005)] and phospho-AKT [by TMA (0.019)]. These were more evident in the T+AA arm (p: 0.0007 and 0.007 respectively). TMA data analysis indicate: a) extent of pS6 expression is lower in the T arm compared to T+AA b) T+AA had a higher rate of apoptosis compared to both T alone and control (p values 0.004 and 0.024 respectively). We noted a trend for lower Ki67 (p 0.07 by TMA ) and increased Caveolin and phospho-p38 in treated compared to controls (p<0.005 by RPPA). Conclusions: RPPA and TMA are complementary methodologies strengthening the validity of conclusions. Temsirolimus effectively blocks mTOR signaling in high grade PCa. Increase in upstream pathway components most noted with T+AA warrants study. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Wyeth