PO.ET03.04 · 实验与分子治疗
CDK4/6 抑制通过抑制 TYMS 驱动的 R-loop 相关 DNA 损伤克服结直肠癌中的 5-FU 耐药
CDK4/6 inhibition overcomes 5-FU resistance by suppressing TYMS-driven R-loop-associated DNA damage in colorectal cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：对 5-氟尿嘧啶（5-FU）的耐药仍是结直肠癌（CRC）治疗中的重大障碍。作为一种抗代谢药物，5-FU 通过抑制胸苷酸合成酶（TYMS）诱导细胞毒性，从而损害 DNA 合成与修复。然而，CRC 细胞常通过上调 TYMS 来适应，进而增强其 DNA 修复能力并降低药物疗效。R-loop 是由 RNA 与其 DNA 模板杂交形成的三链核酸结构，是复制应激和 DNA 损伤的关键促成因素。由于 TYMS 调控核苷酸合成，它可能影响 R-loop 相关的 DNA 损伤。周期蛋白依赖性激酶 4 和 6（CDK4/6）通过 E2F 激活控制 G1-S 细胞周期进程，同时也调控 TYMS 转录。因此，我们假设 CDK4/6 抑制可能下调 TYMS 并增强 R-loop 相关的 DNA 损伤，从而恢复 5-FU 敏感性。
方法：我们建立了 5-FU 耐药的 CRC 细胞系（HCT116-5FUR 和 SW480-5FUR）。采用 MTT 法评估细胞活力，分别通过 Western blot 和 RT-PCR 定量蛋白和 mRNA 水平。采用流式细胞术进行细胞周期分析。通过 gammaH2AX 免疫荧光评估 DNA 损伤，通过 S9.6 和抗核仁蛋白免疫染色检测 R-loop 形成。采用荧光素酶报告基因实验监测双链断裂（DSB）修复效率。同时评估了 5-FU 与 CDK4/6 抑制剂 abemaciclib 的协同效应。
结果：与亲本细胞系相比，TYMS 表达在 HCT116 5FUR（p<0.001）和 SW480 5FUR（p<0.01）中显著上调。与亲本细胞系相比，CDK4 表达在 HCT116 5FUR 和 SW480 5FUR 中上调（p<0.01）。CDK6 表达在 HCT116 5FUR 和 SW480 5FUR 中相较其亲本细胞系也上调（p<0.01）。与未处理的 5-FU 耐药 CRC 细胞系相比，abemaciclib 下调了 HCT116 5FUR（p = 0.011）和 SW480 5FUR（p = 0.0002）中的 TYMS 表达。TYMS 下调促进了 R-loop 相关的 DNA 损伤，表明核苷酸代谢与基因组不稳定性之间存在机制关联。5-FU 与 abemaciclib 联合处理通过诱导 G1 期阻滞和增强 DNA 损伤，协同降低了耐药细胞的活力。单用 abemaciclib 即可诱导 R-loop 形成和 DNA 损伤信号，与 TYMS 抑制及复制应激放大相一致。
结论：TYMS 调节 R-loop 相关的 DNA 损伤，参与 CRC 中的 5-FU 耐药。CDK4/6 抑制通过抑制 TYMS 并增强 R-loop 驱动的基因组应激恢复化疗敏感性。将 CDK4/6 抑制剂与 5-FU 联用是克服结直肠癌耐药的一种有前景的治疗策略。
查看英文原文 English abstract
Background: Resistance to 5-fluorouracil (5-FU) remains a significant obstacle in colorectal cancer (CRC) therapy. As an antimetabolite, 5-FU induces cytotoxicity by inhibiting thymidylate synthase (TYMS), thereby impairing DNA synthesis and repair. However, CRC cells frequently adapt through TYMS upregulation, thereby strengthening their DNA repair capacity and reducing the efficacy of drugs. R-loops, three-stranded nucleic acid structures formed by hybridization of RNA to its DNA template, are key contributors to replication stress and DNA damage. Since TYMS regulates nucleotide synthesis, it may influence R-loop-associated DNA damage. Cyclin-dependent kinases 4 and 6 (CDK4/6), which control G1-S cell cycle progression via E2F activation, also regulate TYMS transcription. Therefore, we hypothesized that CDK4/6 inhibition may downregulate TYMS and enhance R-loop-related DNA damage, restoring 5-FU sensitivity.
Methods: We established 5-FU-resistant CRC cell lines (HCT116-5FUR and SW480-5FUR). Cell viability was assessed using the MTT assay, and protein and mRNA levels were quantified by Western blot and RT-PCR, respectively. Flow cytometry was used for cell cycle profiling. DNA damage was assessed by gammaH2AX immunofluorescence, and R-loop formation was detected via S9.6 and anti-nucleolin immunostaining. A luciferase reporter assay was used to monitor the efficiency of double-strand break (DSB) repair. Synergistic effects of 5-FU and the CDK4/6 inhibitor abemaciclib were also evaluated.
Results: TYMS expression was markedly upregulated in HCT116 5FUR (p<0.001) and SW480 5FUR (p<0.01) compared to parental lines. CDK4 expression was upregulated in HCT116 5FUR and SW480 5FUR (p<0.01) compared to parental lines. CDK6 expression was also upregulated in HCT116 5FUR and SW480 5FUR (p<0.01) compared to their parental cell lines. Abemaciclib downregulated TYMS expression in HCT116 5 FUR (p = 0.011) and SW480 5FUR (p = 0.0002) compared to untreated 5-FU-resistant CRC cell lines. TYMS downregulation promoted R-loop-associated DNA damage, indicating a mechanistic link between nucleotide metabolism and genomic instability. Combination treatment with 5-FU and abemaciclib synergistically reduced the viability of resistant cells by inducing G1 arrest and enhancing DNA damage. Abemaciclib alone induced R-loop formation and DNA damage signaling, consistent with TYMS suppression and the amplification of replication stress.
Conclusions: TYMS modulates R-loop-associated DNA damage, contributing to 5-FU resistance in CRC. CDK4/6 inhibition restores chemosensitivity by repressing TYMS and enhancing R-loop-driven genomic stress. Combining CDK4/6 inhibitors with 5-FU represents a promising therapeutic strategy to overcome drug resistance in colorectal cancer.
利益披露 Disclosure
L. Xu, None..
J. Sun, None..
M. Li, None.