Targeting DNA damage response to overcome taxane resistance.

Abstract

312 Background: Overcoming taxane resistance in metastatic castrate-resistant prostate cancer (mCRPC) is an unmet need. We and others have observed that a significant proportion of patients with docetaxel (Dox)-refractory disease exhibit sensitivity to DNA-damaging agents. Conversely, loss of DNA repair proteins has been shown to confer resistance to anti-microtubule agents. We hypothesize that Dox exposure alters expression of DNA damage response (DDR) genes, promoting Dox-resistance; and targeting these changes would restore Dox sensitivity. Methods: Dox-resistant prostate cancer cells were developed by exposing DU145 cells to stepwise increasing Dox concentration. Resistance was confirmed by comparing viability (MTT assay) of DU-R (Dox-resistant) and DU-P (age-matched parent) cells after 72 h exposure to Dox (0.1 – 100 nM). Changes in gene expression between DU-DR and DU-P were measured using a DDR-specific 84 gene PCR array. Genes with > 2-fold change in expression were confirmed by RT-PCR and immunoblotting. Sensitivity to Dox of DU-DR was measured in presence of candidate gene inhibitors. Effects of Dox + inhibitor on cell cycle and apoptosis were analyzed by flow cytometry. Results: DU-DR was significantly more resistant to Dox than DU-P (IC50 DU-DR vs DU-P: > 100nM vs 0.86 nM), cabazitaxel (Cab) (9.48 nM vs 0.32 nM), and etoposide (Eto) (4.67 μM vs 1.32 μM) but not 4-hydroperoxy cyclophosphamide (4-HC) (4.50 μM vs 3.16 μM). DDR PCR array revealed three genes were significantly upregulated ( TP73, PRKDC, CDK7) and two downregulated ( BBC3, MSH3); and confirmed by RT-PCR and immunoblotting. DNA-PKc inhibitor, NU7441, treatment significantly increased sensitivity of DU-DR cells to Dox (IC50 = 30.7 nM). Similarly, NU7441 also re-sensitized DU-DR cells to Cab and Eto but not 4-HC. Dox did not induce apoptosis in DU-DR cells (sub-G1 = 2.54 ± 0.15 %; Annexin V+/PI- = 3.60 ± 0.58%) unlike in DU-P (sub-G1 = 39.4 ± 0.28 %; Annexin V+/PI- 28.6 ± 1.1%). NU7441 increased sub-G1 (15.8 ± 0.99 %) and Annexin V+/PI- (14.5 ± 1.4 %) population in Dox-treated DU-DR cells. Conclusions: Targeting prostatic DNA-PKc restores sensitivity to taxanes in vitro. Co-targeting these genes may lead to novel mCRPC therapies.