Quantitative analysis of castration resistant prostate cancer progression through phosphoproteome signaling

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Figure 1

An overview of the measured signaling pathways and responses to treatment. A) A diagrammatic overview illustrating the cell signaling between the phosphoproteins measured (blue lettering), the treatments used (green lettering), and the inhibitors which were used on the LNCaP cells (red lines). B) Heatmap with hierarchical clustering illustrating the mean centered and variance scaled (z-score) changes in phosphoprotein values in response to varying treatments (see Methods), as well as survival values of LNCaP, PC3, or MDA-PCa-2b cells as measured via a MTT assay.

Figure 2

Partial least squares regression results with three principal components. A) A scatter plot illustrating the measured versus predicted values for each different treatment group across all three cell lines (LNCaP, PC3, MDA-PCa-2b). B) The regression coefficients for all 8 phosphoproteins across the 3 time points. C) The mean absolute value of the regression coefficients, indicating the contribution of that phosphoprotein to the overall model. D) The R-squared value as additional phosphoproteins are added to a 3 principal component partial least squares regression model. The R-squared value of Erk is for a model built on Erk data alone. The R-squared value for RPS6 is for a model built on Erk and RPS6 data. The R-squared value for p38 is for the complete model for all data and is 0.577.

Figure 3

Modeling the effect of androgen treatment of cell signaling. A) The percent change in phosphoprotein levels due to DHT treatment of LNCaP cells in androgen depleted media as compared to the control condition. A HSP27 phosphorylation value of 3200% was observed at the 30 minute time point. B) The relative survival of LNCaP cells under various treatment conditions in response to androgen treatment (DHT), a PI3K inhibitor (LY294002), or a combination of DHT and LY294002 in androgen depleted media as compared to the control condition of androgen depleted media. DHT is significantly greater than all other groups (** equals P-value < 0.01). Error bars are std. dev. from mean. Values are normalized to the untreated control condition’s mean. C)A diagrammatic overview of the proposed signaling interactions between the androgen receptor, PI3K signaling pathway, RPS6, and cell cycle targets.

Figure 4

The mean phosphorylation value for each phosphoprotein across the three cell lines. A) These phosphoprotein levels were averaged from the EGF, IGF, IL6, TNFα, DHT, and docetaxel treatment in androgen depleted media for each cell line. B) JNK phosphorylation values at 30 minutes for all three cell lines. C) JNK phosphorylation values at 4 hours for all three cell lines. D) JNK phosphorylation values at 24 hours for all three cell lines. In B, C, and D all groups are significantly different from each other (* equals P-value < 0.05, ** equals P-value < 0.01, and *** equals P-value < 0.001).

Figure 5

The relative activation of each phosphoprotein induced by each ligand treatment. Line thickness is proportional to percent increase over untreated control of cells in androgen depleted media. Red lines indicate a reduction in phosphoprotein levels. A) The activation of phosphoproteins in PC3 cells in response to ligand treatment. Red lines indicate a reduction in phosphoprotein levels. B) The activation of phosphoproteins in MDA-PCa-2b cells in response to ligand treatment. C) The activation of phosphoproteins in LNCaP cells in response to ligand treatment.

Figure 6

Response of LNCaP cells to targeted inhibitors. A) The relative cell survival of LNCaPs treated with targeted inhibitors in androgen depleted media after 72 hours. Relative survival was normalized to untreated control LNCaPs in androgen depleted media (100%). Error bars are std. dev. from mean. B) The degree to which each targeted inhibitor reduced phosphorylation of the phosphoprotein as compare to control conditions. A thicker line indicates a stronger reduction in phosphorylation. Edges in average effect over 30 minutes, 4 hours, and 24 hours. C) The degree to which each targeted inhibitor activated phosphorylation of the phosphoprotein as compare to control conditions. A thicker line indicates an increase phosphorylation. Edges in average effect over 30 minutes, 4 hours, and 24 hours. D) The effect of LY294002 treatment on p-Akt, p-GSK3, and p-RPS6. PI3K and mTor were not measured. A stronger color red indicates increased inhibition. E) The effect of Temsirolimus treatment on p-Akt, p-GSK3, and p-RPS6. PI3K and mTor were not measured. A stronger color red indicates increased inhibition.

Figure 7

The correlation values between phosphoproteins across the three different cell lines. Black lines indicate a positive correlation while red lines indicate a negative correlation. Solid lines indicate statistical significance when adjusted for multiple hypothesis testing while dotted lines indicate a P-value of less than 0.05 before multiple hypothesis correction.