Targeting radioresistance and replication fork stability in prostate cancer
Xiangyi Li, GuemHee Baek, Suzanne Carreira, Wei Yuan, Shihong Ma, Mia Hofstad, Sora Lee, Yunpeng Gao, Claudia Bertan, Maria de los Dolores Fenor de la Maza, Prasanna G. Alluri, Sandeep Burma, Benjamin P.C. Chen, Ganesh V. Raj, Johann de Bono, Yves Pommier, Ram S. Mani
Xiangyi Li, GuemHee Baek, Suzanne Carreira, Wei Yuan, Shihong Ma, Mia Hofstad, Sora Lee, Yunpeng Gao, Claudia Bertan, Maria de los Dolores Fenor de la Maza, Prasanna G. Alluri, Sandeep Burma, Benjamin P.C. Chen, Ganesh V. Raj, Johann de Bono, Yves Pommier, Ram S. Mani
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Research Article Oncology

Targeting radioresistance and replication fork stability in prostate cancer

Abstract

The bromodomain and extraterminal (BET) family of chromatin reader proteins bind to acetylated histones and regulate gene expression. The development of BET inhibitors (BETi) has expanded our knowledge of BET protein function beyond transcriptional regulation and has ushered several prostate cancer (PCa) clinical trials. However, BETi as a single agent is not associated with antitumor activity in patients with castration-resistant prostate cancer (CRPC). We hypothesized novel combinatorial strategies are likely to enhance the efficacy of BETi. By using PCa patient-derived explants and xenograft models, we show that BETi treatment enhanced the efficacy of radiation therapy (RT) and overcame radioresistance. Mechanistically, BETi potentiated the activity of RT by blocking DNA repair. We also report a synergistic relationship between BETi and topoisomerase I (TOP1) inhibitors (TOP1i). We show that the BETi OTX015 synergized with the new class of synthetic noncamptothecin TOP1i, LMP400 (indotecan), to block tumor growth in aggressive CRPC xenograft models. Mechanistically, BETi potentiated the antitumor activity of TOP1i by disrupting replication fork stability. Longitudinal analysis of patient tumors indicated that TOP1 transcript abundance increased as patients progressed from hormone-sensitive prostate cancer to CRPC. TOP1 was highly expressed in metastatic CRPC, and its expression correlated with the expression of BET family genes. These studies open new avenues for the rational combinatorial treatment of aggressive PCa.

Authors

Xiangyi Li, GuemHee Baek, Suzanne Carreira, Wei Yuan, Shihong Ma, Mia Hofstad, Sora Lee, Yunpeng Gao, Claudia Bertan, Maria de los Dolores Fenor de la Maza, Prasanna G. Alluri, Sandeep Burma, Benjamin P.C. Chen, Ganesh V. Raj, Johann de Bono, Yves Pommier, Ram S. Mani

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Figure 4

Overexpression of RAD51 partially rescues the effect of BETi-TOP1i combination treatment on replication fork stability.

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Overexpression of RAD51 partially rescues the effect of BETi-TOP1i combi...
(A) Labeling scheme. Cells were transfected with control or RAD51 overexpression plasmid (72 hours), then were pulse labeled first with IdU (100 μM, 30 minutes) and then with CldU (100 μM, 30 minutes), followed by drug treatment for 5 hours. (B and C) The length of labeled DNA tracks, IdU (red) and CldU (green), was scored (n ≥ 110) and displayed in scatter dot plots (left panel). CldU/IdU ratio of each track was calculated and displayed in box-and-whisker plots (right panel). Boxes: 25th–75th percentile; whiskers: 10th–90th percentile. Two-tailed Mann-Whitney U test was applied; the P values were adjusted by Benjamini-Hochberg procedure for multiple comparisons (**P < 0.01; *P < 0.05). (D) Representative images of DNA tracks (fork direction: red to green). Scale bar: 10 μm. (E) Immunoblot verification of RAD51 overexpression upon plasmid transfection in LNCaP cells (72 hours). (F) RAD51 foci were analyzed in U2OS cells upon RAD51 overexpression with or without irradiation (IR 6 Gy). pcDNA3.1 was used as a mock control. Cells were analyzed 4 hours after IR treatment. Cells with ≥5 foci were counted. **P < 0.01, Mann-Whitney U test. (G) The effect of treatment with JQ1 and/or CPT on the proliferation of LNCaP, VCaP, 22Rv1, and DU145 PCa cells. Two-tailed Student’s t test was applied; the P values were adjusted by Benjamini-Hochberg procedure for multiple comparisons; error bars, SEM of 3 technical triplicates. The P values are listed in Supplemental Table 4. (H) Immunoprecipitation (IP) was performed using BRD4 or TOP1 antibodies in VCaP, 22Rv1, and DU145 cells and analyzed by immunoblot with the indicated antibodies.