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Piperine Targets the FANCL/UBE2T Complex to Inhibit the FA Pathway and Sensitize Bladder Cancer to Cisplatin
Chen Li1, Guanglin Lv1, Ying Yue1
1Department of Urology, Wuxi No.2 People's Hospital, Wuxi, China.
Objectives:
Platinum-based chemotherapy remains a mainstay for bladder cancer treatment, yet resistance often arises through activation of the Fanconi anemia (FA) DNA repair pathway. The monoubiquitination of the FANCI-FANCD2 (ID2) complex by FANCL and UBE2T is a critical step in repairing cisplatin-induced interstrand crosslinks (ICLs). Identifying small molecules that block this process may improve the therapeutic efficacy of cisplatin.
Methods:
We investigated the effects of piperine, a natural alkaloid from black pepper, on FA pathway activation in bladder cancer cells. A combination of immunoblotting, immunofluorescence, co-immunoprecipitation, qPCR-blocking assays, dot blot analyses, in vitro ubiquitination/discharge assays, biolayer interferometry (BLI), and differential scanning fluorimetry (DSF) were employed to characterize the molecular mechanism. Xenograft models were used to evaluate in vivo efficacy.
Results:
Piperine pretreatment markedly suppressed cisplatin-induced monoubiquitination of FANCI and FANCD2 and reduced FANCD2 foci formation in T24, 5637, and RT4 cells. Co-immunoprecipitation confirmed diminished recruitment of downstream nucleases and repair factors (FANCP, FANCQ, PCNA). qPCR-blocking assays showed delayed ICL repair, while dot blot analyses revealed that intrastrand cisplatin adduct removal was unaffected, indicating selective inhibition of ICL repair. Piperine did not alter mRNA or protein expression of FANCL, UBE2T, USP1, or UAF1, nor did it enhance deubiquitinase activity. Instead, in vitro assays demonstrated that piperine blocked FANCL-mediated ubiquitin transfer from UBE2T∼Ub to the ID2 complex, without impairing E2 charging or FANCL-UBE2T binding. BLI confirmed unaltered binding affinity, whereas DSF revealed a significant ΔTm shift for UBE2T, consistent with allosteric modulation. In xenografts, combined cisplatin and piperine treatment significantly reduced tumor growth and attenuated FANCI/FANCD2 monoubiquitination.
Conclusion:
Our findings uncover piperine as a natural compound that allosterically inhibits UBE2T activity within the FA pathway, thereby impairing ID2 monoubiquitination and enhancing cisplatin sensitivity in bladder cancer. This study highlights the therapeutic potential of piperine and provides a rationale for targeting the FA repair axis to overcome platinum resistance.
Insights
Piperine, a natural compound, inhibits the Fanconi anemia (FA) DNA repair pathway by blocking UBE2T activity. This enhances cisplatin sensitivity in bladder cancer, offering a new strategy against platinum resistance.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Platinum-based chemotherapy is a primary bladder cancer treatment.
- Resistance to chemotherapy often involves the Fanconi anemia (FA) DNA repair pathway.
- Targeting the FA pathway, specifically FANCI-FANCD2 (ID2) complex monoubiquitination, may overcome resistance.
Purpose of the Study:
- To investigate piperine's effect on the FA pathway in bladder cancer.
- To elucidate the molecular mechanism by which piperine impacts FA pathway activation.
- To evaluate the therapeutic potential of combining piperine with cisplatin.
Main Methods:
- Utilized immunoblotting, immunofluorescence, co-immunoprecipitation, qPCR-blocking assays, dot blot analyses, in vitro ubiquitination/discharge assays, biolayer interferometry (BLI), and differential scanning fluorimetry (DSF).
- Investigated piperine's impact on FANCI-FANCD2 monoubiquitination and ID2 complex recruitment.
- Assessed ICL repair kinetics and in vivo efficacy using xenograft models.
Main Results:
- Piperine suppressed cisplatin-induced FANCI/FANCD2 monoubiquitination and reduced FANCD2 foci formation.
- Piperine selectively inhibited interstrand crosslink (ICL) repair without affecting intrastrand adduct removal.
- In vitro studies showed piperine allosterically inhibits UBE2T, blocking ubiquitin transfer to the ID2 complex.
- Combined cisplatin and piperine treatment significantly reduced tumor growth in vivo.
Conclusions:
- Piperine acts as a natural allosteric inhibitor of UBE2T in the FA pathway.
- Piperine impairs ID2 monoubiquitination, enhancing cisplatin sensitivity in bladder cancer.
- Piperine presents a promising therapeutic strategy for overcoming platinum resistance by targeting the FA repair axis.
