Abstract C39: Rad51 mediates prostate cancer cells' resistance to topoisomerase I inhibitor under hypoxia.

Abstract

Abstract Background: Metastatic castration resistant prostate cancer (mCRPC) remains an incurable disease and intra-tumoral hypoxia has been proposed to create a “mutator” phenotype with increased genomic instability and drug resistance. Rad51 is an essential protein in homologous recombination repair of DNA double strand breaks. Down-regulation of Rad51 expression has been reported in prostate, lung, breast, colon, and cervical cancer cell lines grew under hypoxic conditions. Such down regulation was shown to lead to diminished DNA repair and the genomic instability. We therefore study whether the diminished DNA repair under hypoxia would sensitize mCRPC cells to DNA damaging drugs like topoisomerase I inhibitor. Method: mCRPC cell lines, PC3 and Du145 were grown under normoxia (21% oxygen) and hypoxia (0.2% oxygen). These cells were then treated with SN38, the active metabolite of topoisomerase I inhibitor, CPT-11. DNA damages, DNA repairs and cell deaths were evaluated with western blot, immunoprecipitation, immunofluorescence and flowcytometry. Changes in DNA repair proteins like Rad51 were compared with quantitative RT-PCR and western blot. Dual luciferase reporter assay was used to study the Rad51 promoter activities. Results: Both PC3 and DU145 cells continue to proliferate under hypoxia. Compared to PC3 and Du145 cells treated with SN38 under normoxia, there were much less DNA damages and cell deaths observed in these cells treated under hypoxia. Although Rad51 was initially down regulated under hypoxia, increasing Rad51 RNA and proteins were observed soon after treatment with SN38 under hypoxia. Such increase in Rad51 is associated with increasing Rad51 promoter activity, Rad51 nuclear foci formation, co-localization of Rad51 and r-H2AX, and the formation of the Rad51-BRCA1 complex. No changes in the proteins involved in non-homologous end joining pathway were observed. Furthermore, inhibiting Rad51 expression with siRNA under hypoxia reversed the mCRPC cells' resistance to SN38 induced DNA damage and cell death. Ongoing studies focuses on the mechanisms of transcriptional regulation of Rad51 in response to hypoxia and DNA damage. Of note, no changes in rad51 were observed when the PC3 and Du145 cells were treated with non-DNA damaging docetaxel and they remain sensitive to docetaxel under both normoxia and hypoxia. Conclusions: mCRPC cells can quickly adapt to hypoxia and drug induced DNA damage by modulating the levels of key DNA repair proteins like Rad51. Altering the regulation of Rad51 expression in response to DNA damage can be exploitable as a new therapeutic approach for mCRPC. Such approach could directly target the “mutator” phenotype created by tumor micro environmental factors like hypoxia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C39.