Tetrahydrocurcumin targets TRIP13 inhibiting the interaction of TRIP13/USP7/c-FLIP to mediate c-FLIP ubiquitination in triple-negative breast cancer.

Introduction: 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.

Objectives: 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.

Methods: 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.

Results: 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.

Conclusions: 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.