Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Department of Radiation Oncology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Prostate. 2024 Dec;84(16):1456-1467. doi: 10.1002/pros.24786. Epub 2024 Sep 1.
Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, cell growth, and survival. PCNA also enhances androgen receptor (AR) signaling in prostate cancer (PC) cells. We identified a PCNA interaction protein (PIP) box at the N-terminal domain of AR and developed a small peptide PCNA inhibitor R9-AR-PIP containing AR PIP-box. We also identified a series of small molecule PCNA inhibitors (PCNA-Is) that bind directly to PCNA and interrupt PCNA functions. The present study investigated the effects of the PCNA inhibitors on the sensitivity of PC cells to X-ray radiation.
The effects of targeting PCNA on radio sensitivity of PC cells were investigated in four lines of castration-resistant PC (CRPC) cells with different AR expression statuses. The cells were treated with the PCNA inhibitors and X-ray radiation alone or in combination. The effects of the treatment on expression of AR target genes, DNA damage response, DNA damage, homologous recombination repair (HRR), and cytotoxicity were evaluated.
We found that the androgen response element (ARE) occupancy of the DNA damage response gene PARP1 by AR is significantly attenuated by PCNA-I1S or R9-AR-PIP combined with X-ray radiation, while X-ray radiation alone does not enhance the ARE occupancy. PCNA-I1S or R9-AR-PIP alone significantly inhibits occupancy of the AR-occupied regions (AROR) in PRKDC and XRCC2 genes. R9-AR-PIP and PCNA-I1S inhibit expression of AR-Vs target gene cyclin A2 and show the additive effects with radiation in AR-positive CRPC cells. Targeting PCNA by PCNA-I1S and R9-AR-PIP downregulates expression of DNA damage response genes EXO1, Rad54L, Rad51, and/or PARP1 and shows the additive effects with radiation as compared with their respective controls in AR-positive CRPC LNCaP-AI, 22Rv1, and R1-D567 cells, but not in AR-negative PC-3 cells. R9-AR-PIP and PCNA-I1S elevate the levels of phospho-DNA-PKcs(S) and γH2AX, indicating DNA damage in response to radiation in AR-positive cells. The HRR is significantly attenuated by PCNA inhibitors PCNA-I1S, R9-AR-PIP, and T2AA in all four CRPC cells examined, and inhibited by Enzalutamide (Enz) only in 22RV1 cells. The cytotoxicity induced by X-ray radiation in androgen-dependent LNCaP cells is enhanced by Enz and a lower concentration of R9-AR-PIP in the colony formation assay. R9-AR-PIP at higher concentration reduces the colony formation and has an additive effect with X-ray radiation in all AR expressing cells, regardless of AR-FL and AR-Vs, but does not significantly alter the colony formation in AR-negative PC-3 cells. PCNA-I1S attenuates colony formation and has an additive effect with ionizing radiation in all four CRPC cells, regardless of AR expression status.
These data provide a strong rationale for the therapy studies using PCNA-I1S or R9-AR-PIP in combination with X-ray radiation against CRPC tumors in preclinical models.
增殖细胞核抗原（PCNA）对于 DNA 复制和修复、细胞生长和存活至关重要。PCNA 还增强了前列腺癌细胞（PC）中的雄激素受体（AR）信号。我们在 AR 的 N 端结构域中发现了一个 PCNA 相互作用蛋白（PIP）盒，并开发了一种含有 AR PIP 盒的小肽 PCNA 抑制剂 R9-AR-PIP。我们还鉴定了一系列直接与 PCNA 结合并中断 PCNA 功能的小分子 PCNA 抑制剂（PCNA-Is）。本研究探讨了 PCNA 抑制剂对 PC 细胞对 X 射线辐射敏感性的影响。
在具有不同 AR 表达状态的 4 株去势抵抗性 PC（CRPC）细胞中，研究了靶向 PCNA 对 PC 细胞放射敏感性的影响。用 PCNA 抑制剂和 X 射线单独或联合处理细胞。评估处理对 AR 靶基因表达、DNA 损伤反应、DNA 损伤、同源重组修复（HRR）和细胞毒性的影响。
我们发现，PCNA-I1S 或 R9-AR-PIP 与 X 射线联合使用时，雄激素反应元件（ARE）在 DNA 损伤反应基因 PARP1 中的 AR 占有率显著降低，而 X 射线单独照射不会增强 ARE 占有率。PCNA-I1S 或 R9-AR-PIP 单独显著抑制 PRKDC 和 XRCC2 基因中 AR 占据区域（AROR）的占有率。R9-AR-PIP 和 PCNA-I1S 抑制 AR-Vs 靶基因 cyclin A2 的表达，并在 AR 阳性 CRPC 细胞中与辐射表现出相加效应。用 PCNA-I1S 和 R9-AR-PIP 靶向 PCNA 可下调 EXO1、Rad54L、Rad51 和/或 PARP1 等 DNA 损伤反应基因的表达，并与 AR 阳性 CRPC LNCaP-AI、22Rv1 和 R1-D567 细胞中的各自对照相比，与辐射具有相加效应，但在 AR 阴性 PC-3 细胞中没有。R9-AR-PIP 和 PCNA-I1S 提高了磷酸化 DNA-PKcs(S) 和 γH2AX 的水平，表明 AR 阳性细胞对辐射的 DNA 损伤。在所有 4 株 CRPC 细胞中，PCNA 抑制剂 PCNA-I1S、R9-AR-PIP 和 T2AA 均显著减弱 HRR，而在 22RV1 细胞中仅恩杂鲁胺（Enz）减弱 HRR。X 射线辐射在雄激素依赖性 LNCaP 细胞中诱导的细胞毒性，通过在集落形成试验中用 Enz 和较低浓度的 R9-AR-PIP 增强。R9-AR-PIP 浓度较高时会降低集落形成率，并与所有表达 AR 的细胞（无论 AR-FL 和 AR-Vs 如何）中的 X 射线辐射具有相加效应，但不会显著改变 AR 阴性 PC-3 细胞中的集落形成率。PCNA-I1S 减弱集落形成并与所有 4 株 CRPC 细胞中的电离辐射具有相加效应，无论 AR 表达状态如何。
这些数据为使用 PCNA-I1S 或 R9-AR-PIP 联合 X 射线辐射治疗 AR 阳性和阴性 CRPC 肿瘤的临床前模型提供了强有力的依据。
