PO.CL08.01 · 临床研究
限制O-GlcNAc糖基化通过代谢和表观遗传重编程支持前列腺癌的放射敏感性
Limiting O-GlcNAcylation support prostate cancer radiation sensitivity through metabolic and epigenetic reprogramming
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：前列腺癌（PCa）常发生对放射治疗（RT）的抵抗，部分由DNA修复机制驱动。我们此前发现，谷氨酰胺（L-Gln）能够促进蛋白质O-GlcNAc糖基化这一与DNA修复和治疗抵抗相关的翻译后修饰。这与PCa转移部位（如骨和肝微环境）的照射高度相关，这些部位具有显著较高的L-Gln浓度，相应地对照射反应较差。在此，我们研究了使用苯丁酸钠（SPB）进行药理学谷氨酰胺耗竭是否会破坏该轴并增强放射敏感性。
方法：通过迭代慢性照射生成放射抵抗的22Rv1和ARCaP M细胞系。用溶剂、SPB、RT或SPB+RT处理皮下和肝脏异种移植瘤。通过bulk RNA-seq、蛋白质组学和免疫印迹分析肿瘤，揭示了NDRG1和PRDX1 O-糖基化的重要性。采用CRISPR/Cas9诱变生成O-GlcNAc缺陷型NDRG1和PRDX1变体。通过gammaH2AX染色、流式细胞术和Seahorse实验评估DNA损伤修复、细胞周期动态和线粒体功能。
结果：与单独RT或单独SPB相比，SPB+RT产生了最大的肿瘤缩小效果，并显著降低了循环和肿瘤内谷氨酰胺水平。转录组和蛋白质组分析显示氨基酸转运、脂肪酸代谢和组蛋白去甲基化酶活性下调，提示广泛的代谢和表观遗传重编程。质谱鉴定NDRG1和PRDX1为受SPB抑制的放疗诱导O-GlcNAc靶点。CRISPR工程改造的O-GlcNAc缺陷型NDRG1和PRDX1变体表现出更高的RT敏感性，其原因是持续的gammaH2AX病灶、延长的G2/M阻滞、核定位减少以及蛋白稳定性下降。这些变体的RNA-seq显示p53信号通路富集、内质网（ER）应激伴随通过c-Myc活性增加的代谢代偿以及氧化磷酸化。核苷补充并未逆转SPB介导的放射增敏，表明SPB的效应超出了核苷酸耗竭的范围。相反，数据提示照射PCa肿瘤中L-Gln成瘾的作用归因于NDRG1和PRDX1参与ER应激反应蛋白并激活未折叠蛋白反应。
结论：NDRG1和PRDX1的O-GlcNAc糖基化稳定了应激反应蛋白，以支持照射后的DNA修复和代谢适应性。SPB破坏了这一L-Gln驱动的O-GlcNAc糖基化轴，从而损害蛋白质翻译并支持显著的放射增敏。由于SPB已用于尿素循环障碍的长期管理，将其重新用于克服放射抵抗为PCa患者提供了近期可实现的治疗转化机会。
查看英文原文 English abstract
Background: Prostate cancer (PCa) frequently develops resistance to radiation therapy (RT), driven in part by DNA repair mechanisms. We previously found that glutamine (L-Gln) enables protein O-GlcNAcylation post-translational modifications linked to DNA repair and therapy resistance. This is highly relevant to irradiation of PCa metastatic sites like the bone and liver microenvironments that have markedly high concentrations of L-Gln and accordingly poor response to irradiation. Here, we investigated whether pharmacologic glutamine depletion using sodium phenylbutyrate (SPB) disrupts this axis and enhances radiosensitivity.
Methods: Radio-resistant 22Rv1 and ARCaP M lines were generated by iterative chronic irradiation. Subcutaneous and liver xenografts were treated with vehicle, SPB, RT, or SPB+RT. Tumors analyzed by bulk RNA-seq, proteomics, and immunoblotting revealed the importance of NDRG1 and PRDX1 o-glycosylation. CRISPR/Cas9 mutagenesis was used to generate O-GlcNAc-deficient NDRG1 and PRDX1 variants. DNA-damage repair, cell-cycle dynamics, and mitochondrial function were assessed by gammaH2AX staining, flow cytometry, and Seahorse assays.
Results: SPB+RT produced the greatest tumor reduction and significantly reduced circulating and intratumoral glutamine, compared to either RT or SBP alone. Transcriptomic and proteomic analyses showed downregulation of amino-acid transport, fatty-acid metabolism, and histone demethylase activity, indicating broad metabolic and epigenetic reprogramming. Mass spectrometry identified NDRG1 and PRDX1 as radiation-induced O-GlcNAc targets suppressed by SPB. CRISPR-engineered O-GlcNAc-deficient NDRG1 and PRDX1 variants exhibited greater RT sensitivity as a result of persistent gammaH2AX foci, prolonged G2/M arrest, reduced nuclear localization, and decreased protein stability. RNA-seq of these variants showed enrichment of p53 signaling, endoplasmic reticular (ER) stress with metabolic compensation through increased c-Myc activity, and oxidative phosphorylation. Nucleoside supplementation did not reverse SPB-mediated radio-sensitization, indicating that SPB's effects extended beyond nucleotide depletion. Instead, the data suggested the role of L-Gln addiction in irradiated PCa tumors was due to NDRG1 and PRDX1 in ER stress-response proteins and activating the unfolded protein response.
Conclusions: O-GlcNAcylation of NDRG1 and PRDX1 stabilizes stress-response proteins to support DNA-repair and metabolic fitness after irradiation. SPB disrupts this L-Gln-driven O-GlcNAcylation axis, to impair protein translation supporting significant radio-sensitization. As SPB is used for chronic management of urea cycle disorders, repurposing to overcome radiation resistance provides a near-term therapeutic translation opportunity for PCa patients.
利益披露 Disclosure
M. Thiruvalluvan, None..
S. Billet, None..
S. Kailasam Mani, None..
J. Watson, None..
N. A. Bhowmick, None.