Department of Radiation Oncology, The First Hospital of Lanzhou University, Lanzhou University, Lanzhou, Gansu 730000, PR China; The First School of Clinical Medicine, Lanzhou University, Lanzhou, Gansu 730000, PR China.
The First School of Clinical Medicine, Lanzhou University, Lanzhou, Gansu 730000, PR China.
J Adv Res. 2024 Jul;61:239-251. doi: 10.1016/j.jare.2023.09.011. Epub 2023 Sep 19.
Despite radiotherapy being one of the major treatments for triple-negative breast cancer (TNBC), new molecular targets for its treatment are still required due to radioresistance. CDK2 plays a critical role in TNBC. However, the mechanism by which CDK2 promotes TNBC radioresistance remains to be clearly elucidated.
We aimed to elucidate the relationship between CDK2 and TRIM32 and the regulation mechanism in TNBC.
We performed immunohistochemical staining to detect nuclear TRIM32, CDK2 and STAT3 on TNBC tissues. Western blot assays and PCR were used to detect the protein and mRNA level changes. CRISPR/Cas9 used to knock out CDK2. shRNA-knockdown and transfection assays also used to knock out target genes. GST pull-down analysis, immunoprecipitation (IP) assay and in vitro isomerization analysis also used. Tumorigenesis studies also used to verify the results in vitro.
Herein, tripartite motif-containing protein 32 (TRIM32) is revealed as a substrate of CDK2. Radiotherapy promotes the binding of CDK2 and TRIM32, thus leading to increased CDK2-dependent phosphorylation of TRIM32 at serines 328 and 339. This causes the recruitment of PIN1, involved in cis-trans isomerization of TRIM32, resulting in importin α3 binding to TRIM32 and contributing to its nuclear translocation. Nuclear TRIM32 inhibits TC45-dephosphorylated STAT3, Leading to increased transcription of STAT3 and radioresistance in TNBC. These results were validated by clinical prognosis confirmed by the correlative expressions of the critical components of the CDK2/TRIM32/STAT3 signaling pathway.
Our findings demonstrate that regulating the CDK2/TRIM32/STAT3 pathway is a promising strategy for reducing radioresistance in TNBC.
尽管放射疗法是三阴性乳腺癌 (TNBC) 的主要治疗方法之一，但由于放射抗性，仍需要新的分子靶标进行治疗。CDK2 在 TNBC 中发挥着关键作用。然而，CDK2 促进 TNBC 放射抗性的机制仍有待阐明。
我们旨在阐明 CDK2 与 TRIM32 之间的关系及其在 TNBC 中的调节机制。
我们通过免疫组织化学染色检测 TNBC 组织中的核 TRIM32、CDK2 和 STAT3。使用 Western blot 分析和 PCR 检测蛋白和 mRNA 水平的变化。使用 CRISPR/Cas9 敲除 CDK2。还使用 shRNA 敲低和转染实验敲除靶基因。还使用 GST 下拉分析、免疫沉淀 (IP) 分析和体外异构化分析。肿瘤发生研究也用于验证体外结果。
在此，发现三基序蛋白 32 (TRIM32) 是 CDK2 的底物。放射治疗促进 CDK2 和 TRIM32 的结合，从而导致 CDK2 依赖性磷酸化 TRIM32 的丝氨酸 328 和 339 增加。这导致 PIN1 的募集，PIN1 参与 TRIM32 的顺反异构化，导致 importin α3 与 TRIM32 结合并促进其核转位。核 TRIM32 抑制 TC45 去磷酸化的 STAT3，导致 STAT3 转录增加和 TNBC 放射抗性增加。这些结果通过临床预后得到验证，即 CDK2/TRIM32/STAT3 信号通路的关键成分的相关性表达得到证实。
我们的研究结果表明，调节 CDK2/TRIM32/STAT3 通路是减少 TNBC 放射抗性的一种有前途的策略。
