Quantitative analysis of castration resistant prostate cancer progression through phosphoproteome signaling

Although recent progress has been made in treating castration resistant prostate cancer, the interplay of signaling pathways which enable castration resistant growth is incompletely understood. A data driven, multivariate approach, was used in this study to predict prostate cancer cell survival based on the phosphorylation levels of key proteins in PC3, LNCaP, and MDA-PCa-2b cell lines in response to EGF, IGF1, IL6, TNFα, dihydrotestosterone, and docetaxel treatment.

The prostate cancer cell lines were treated with ligands or inhibitors, cell lyates were collected, and the amount of phosphoprotein quantified using 384 well ELISA assays. In separate experiments, relative cell viability was determined using an MTT assay. Normalized data was imported into Matlab where regression analysis was performed.

Based on a linear model developed using partial least squares regression, p-Erk1/2 was found to correlate with castration resistant survival along with p-RPS6, and this model was determined to have a leave-one-out cross validated R2 value of 0.61. The effect of androgen on the phosphoproteome was examined, and increases in PI3K related phosphoproteins (p-Akt, p-RPS6, and p-GSK3) were observed which accounted for the majority of the significant increase in androgen-mediated cell survival. Simultaneous inhibition of the PI3K pathway and treatment with androgen resulted in a non-significant increase in survival. Given the strong effect of PI3K related signaling in enabling castration resistant survival, the specific effect of mTor versus complete inhibition was examined using targeted inhibitors. It was determine that mTor inhibition accounts for 52% of the effect of complete PI3K inhibition on cell survival. The differences in signaling between the cell lines were explored it was observed that MDA-PCa-2b exhibited far less activation of p-Erk in response to varying treatments, explaining one of the reasons for the lack of castration resistance.

In this work, regression analysis to the phosphoproteome was used to illustrate the sources of castration resistance between the cell lines including reduced p-Erk signaling in MDA-PCa-2b and variations in p-JNK across the cell lines, as well as studying the signaling pathways which androgen acts through, and determining the response to treatment with targeted inhibitors.