Abstract
Background and Objectives: Docetaxel remains the standard of care and one of the two chemotherapy drugs proven to prolong survival in men with castration-resistant prostate cancer. However, 40% of patients' progress by 18 weeks and some patients suffer all the toxicities of docetaxel with limited benefit. We are prospectively validating an expression-based docetaxel response signature in castration resistant prostate cancer (CRPC) and using pathway analysis to identify mechanisms of resistance.
Brief Description of Methodologies: Signature- and pathway-focused expression analysis of microarray data from clinical samples and cell lines is performed to determine predictors of docetaxel response and mechanisms of resistance. Western blot analysis is used to assess protein and phospho-protein expression. Immunofluorescent staining of cells using microtubule targeting antibodies is used to identify mitotic figures. Spinning disk confocal time-lapse microscopy is used to analyze microtubule dynamics.
Results to Date: We have accrued 18 patients onto our prospective validation protocol that has expanded to Oregon Health & Sciences University, the Fred Hutchinson Cancer Research Center, and the University of California San Francisco. Further accrual is required before we will determine the accuracy of the docetaxel response signature. Analysis of docetaxel-resistant cell lines have implicated increased p38 MAPK activity with docetaxel resistance. Importantly, in pair wise comparisons between parental cell lines (i.e. prostate cancer cell lines prior to selection for resistance) and the derivative, docetaxel-resistant cell lines, we noted a consistent increase in expression of the genes within the p38 MAPK pathway using Gene Set Enrichment Analysis. We have validated the increase in p38 MAPK activity by demonstrating corresponding changes in p38 MAPK phosphorylation status and phosphorylation of p38 targets (e.g. STAT1). Importantly, the combination of SB203580, a p38 MAPK inhibitor, and docetaxel is synergistic across resistant cell lines using the methods of Chao and Talalay with combination indices below 0.50. Moreover, combination of SB203580 and docetaxel has shown a significant growth inhibition in xenografts compared with tumors treated with any drug alone. We have become specifically interested in the interaction between specific p38 MAPK family members (alpha, beta, gamma, and delta) and their interactions with microtubule interacting proteins. Resistant cell lines have a lower proportion of abnormal mitotic figures (median 0.081, n=459 total nuclei counted) when exposed to similar concentrations of docetaxel compared to sensitive cell lines (median 0.31, n=733)(Mann Whitney p<0.001).
Conclusions: p38 MAPK activity is associated with docetaxel resistance and inhibition of p38 MAPK is synergistic with docetaxel in vitro and in vivo. The mechanism remains unclear but may involve destabilization of microtubules.
Impact Statement: Our project aims to improve the quality and duration of life for men with castration resistant prostate cancer by identifying men who will respond well to single-agent docetaxel and by identifying pathway targets for molecularly-rational combination therapy for men likely to be resistant to docetaxel. A second drug recently approved as a second line therapy following docetaxel for mCRPC patients is cabazitaxel, which also belongs to the Taxane family. While cabazitaxel has been shown to provide a survival advantage, the effectiveness is limited (only 2-3 months) and similar mechanisms of resistance may be involved in both docetaxel and cabazitaxel resistance. The future direction of this project will include testing our hypothesis in cabazitaxel resistant prostate cancers and trying to identify the common resistant mechanisms. Prostate cancer is generally considered a chemoresistant cancer, therefore combining targeted therapy with chemotherapy reagents is an important approach to improve survival.
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