To obtain more precise, objective expression measures, we used a newly developed method of automated, quantitative analysis of tissue microarrays. EPO906 Epothilone B As redundant activators of the PI3K AKT signaling pathway and negative feedback loops limit the efficacy of single agent therapies, our next purpose was to study the effects of targeting the PI3K AKT signaling pathway at multiple levels in NSCLC cell lines. We found that higher expression of p85 correlated with poor survival and advanced stage. Expression of p110a correlated with that of mTOR. Concurrent inhibition of PI3K and mTOR resulted in synergistic growth suppression. Adding EGFR inhibition further enhanced the growth inhibitory effects of a dual PI3K mTOR inhibitor. Materials and Methods Tissue Microarray Construction A NSCLC cohort was obtained from the H.
Lee Moffitt Cancer Center. The Moffitt Cancer Center cohort contains cores from primary NSCLC tumors of patients diagnosed between 1991 and 2001. Follow up time ranged between 0.8 months and 146.4 months, mean follow up time of 52.3 months. Age at diagnosis ranged from 40.8 to 84.4. The cohort included 54.5 males and 45.5 females. The Yale University cohort was GSK256066 constructed from paraffin embedded, formalin fixed tissue blocks obtained from the Yale University Department of Pathology Archives. The specimens were resected between 1995 and 2003, with a follow up range between 0.1 months and 182.25 months, and a mean followup time of 41 months. Age at diagnosis ranged from 21 to 90. The cohort included 51 males and 49 females. TMAs were constructed as previously described.
Two 0.6 mm cores were obtained from different, representative areas of each primary NSCLC specimen and spaced 0.8 mm apart on glass slides. Cell line pellets consisting of SW480, HT29, A431, MB435, MCF7, BT474, and SKBR3 were used as controls and were embedded in the array, as previously described. The cohorts for MTMA and YTMA were collected with approval of the institutional review boards and have been used in prior publications. Immunofluorescent staining TMA slides were stained for each of the two target markers, PI3K p85 and p110a subunits. Staining was performed for AQUA as described previously. Slides were incubated with the primary antibody diluted in Tris buffered saline containing 0.3 bovine serum albumin at 4uC.
Primary antibodies used for the respective incubations were mouse monoclonal anti human PI3K p85, clone 4 PI3 Kinase or rabbit anti human PI3K p110a clone C73F8, at 1:200 or 1:50 dilutions, respectively. Either goat anti mouse or goat anti rabbit horseradish peroxidase decorated polymer backbone was utilized to visualize the target protein. To create a tumor mask, slides were simultaneously incubated with either mouse or rabbit anti cytokeratin at 1:100. For visualization of cytokeratin staining a goat anti mouse or antirabbit IgG conjugated to Alexa 546 at 1:200 was utilized. The target marker was visualized with Cy5 tyramide. Coverslips wer