Sun Yu-Jie, Zhang Qiang, Cao Shi-Jie, Sun Xiao-Hu, Zhang Ji-Chao, Zhang Bing-Yang, Shang Ze-Bin, Zhao Chong-Yan, Cao Zhi-Yong, Zhang Qiu-Ju, Gao Xiu-Mei, Qiu Feng, Kang Ning
School of Medical Technology, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, PR China; State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, PR China.
School of Medical Technology, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, PR China; State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, PR China.
J Adv Res. 2024 Nov 5. doi: 10.1016/j.jare.2024.11.004.
Triple-negative breast cancer (TNBC) has a high mortality rate and limited treatment options. Tetrahydrocurcumin (THC), a major metabolite of curcumin, has potential antitumor activities. However, the antitumor effects and mechanism of THC in TNBC remain elusive.
To investigate the mechanism of THC in combating TNBC by targeting TRIP13 to disrupt the interaction of the TRIP13/USP7/c-FLIP complex and mediate c-FLIP ubiquitination both in vitro and in vivo.
Apoptosis was measured by TUNEL and flow cytometry. Click chemistry-based target fishing, CETSA, DARTS, and SPR were used to identify direct target of THC. Protein interactions was examined using co-immunoprecipitation. The role of USP7 in THC-mediated c-FLIP ubiquitination was evaluated by in vitro deubiquitination assay. Human breast cancer clinical samples were employed to assess the expression of c-FLIP, TRIP13, and USP7. The impact of THC on USP7/TRIP13/c-FLIP was analyzed using co-immunoprecipitation, confocal microscopy, molecular docking and dynamics simulations.
THC effectively inhibits TNBC cell proliferation and tumor growth in vitro and in vivo without significant toxicity. Mechanistically, THC induces extrinsic apoptosis in TNBC primarily by promoting degradation of c-FLIP, a key negative regulator in the apoptotic pathway. Furthermore, utilizing click chemistry-based target fishing, we identified TRIP13, a component of the highly conserved AAA ATPase family, as a direct target of THC in combating TNBC. Interestingly, contrary to previous drug-target studies, the knockdown of TRIP13 further amplified the antitumor effects of THC. After in-depth investigation, it was revealed that TRIP13 forms a trimeric complex with USP7 and c-FLIP in TNBC cells. THC specifically targets TRIP13 to disrupt the interaction of TRIP13/USP7/c-FLIP, leading to the ubiquitination of c-FLIP, ultimately inducing extrinsic apoptosis.
These findings offer new insights into the novel molecular mechanisms of anti-TNBC effects of THC and present a promising targeted therapeutic strategy for TNBC.
三阴性乳腺癌（TNBC）死亡率高且治疗选择有限。姜黄素的主要代谢产物四氢姜黄素（THC）具有潜在的抗肿瘤活性。然而，THC在TNBC中的抗肿瘤作用及机制仍不清楚。
通过在体外和体内靶向TRIP13破坏TRIP13/USP7/c-FLIP复合物的相互作用并介导c-FLIP泛素化，研究THC对抗TNBC的机制。
通过TUNEL和流式细胞术检测细胞凋亡。使用基于点击化学的靶点筛选、CETSA、DARTS和SPR来鉴定THC的直接靶点。采用免疫共沉淀检测蛋白质相互作用。通过体外去泛素化试验评估USP7在THC介导的c-FLIP泛素化中的作用。用人乳腺癌临床样本评估c-FLIP、TRIP13和USP7的表达。使用免疫共沉淀、共聚焦显微镜、分子对接和动力学模拟分析THC对USP7/TRIP13/c-FLIP的影响。
THC在体外和体内均能有效抑制TNBC细胞增殖和肿瘤生长，且无明显毒性。机制上，THC主要通过促进凋亡途径中的关键负调节因子c-FLIP的降解，诱导TNBC细胞发生外源性凋亡。此外，利用基于点击化学的靶点筛选，我们鉴定出高度保守的AAA ATP酶家族成员TRIP13是THC对抗TNBC的直接靶点。有趣的是，与以往的药物靶点研究相反，敲低TRIP13进一步增强了THC的抗肿瘤作用。深入研究发现，TRIP13在TNBC细胞中与USP7和c-FLIP形成三聚体复合物。THC特异性靶向TRIP13破坏TRIP13/USP7/c-FLIP的相互作用，导致c-FLIP泛素化，最终诱导外源性凋亡。
这些发现为THC抗TNBC作用的新分子机制提供了新见解，并为TNBC提供了一种有前景的靶向治疗策略。
