Liquidambaric acid inhibits cholangiocarcinoma progression by disrupting the STAMBPL1/NRF2 positive feedback loop

Background

Abnormal antioxidant capacity in cancer cells is intimately linked to tumor aggressiveness. Modulating oxidative stress status and inhibiting ferroptosis represents a novel anticancer therapeutic strategy. STAM Binding Protein Like 1 (STAMBPL1), a deubiquitinase, is implicated in various malignancies, yet its function in inhibiting ferroptosis and therapeutic potential for cholangiocarcinoma (CCA) remains unexplored.

Purpose

This study elucidates STAMBPL1's function in ferroptosis and evaluates liquidambaric acid (LDA) as its inhibitor for therapeutic applications.

Methods

Using bioinformatics, WB, IHC, the expression and prognostic value of STAMBPL1 in CCA tissue was detected. The carcinogenic capacity of STAMBPL1 and LDA were assessed through CCK-8, EdU, cloning, transwell, scratch, apoptosis, and cell cycle assays. Flow cytometry and fluorescence microscopy, as well as transmission electron microscopy (TEM), examines the effects of STAMBPL1 and LDA on intracellular reactive oxygen species (ROS) and changes in mitochondrial membrane potential. The tumorigenic ability of STAMBPL1 and LDA in vivo was evaluated through subcutaneous tumor model and lung metastasis model. The underlying mechanism of STAMBPL1 was explored using immunoprecipitation coupled with Mass spectrometry (IP/MS), Co-immunoprecipitation (Co-IP), GST pull-down, DNA pull-down, and Dual-luciferase reporter assays. Molecular docking simulations, SPR, DARTS and CETSA predict the putative binding site of LDA on STAMBPL1 protein. Rescue experiments further confirmed the above conclusions.

Results

This study unveils the upregulation and oncogenic role of STAMBPL1 in CCA. Functionally, STAMBPL1 notably enhances CCA cell proliferation and metastasis while impeding ferroptosis. STAMBPL1 stabilizes NRF2, a pivotal regulator of antioxidant enzymes, through K63 deubiquitination. Elevated NRF2, stabilized by STAMBPL1 overexpression, triggers GPX4 activation and reactive oxygen species (ROS) elimination. Particularly, sites 251–436 of STAMBPL1 interact with sites 228–605 of NRF2, facilitating DUB activity and eliminating ubiquitin molecules attached to NRF2, thus protecting it from proteasome-mediated degradation. Moreover, NRF2, acting as a transcription factor, binds to the promoter region of STAMBPL1 and activates its transcription, thus forming STAMBPL1/NRF2 positive feedback loop and regulating redox homeostasis. Molecular docking and in vitro/in vivo experiments identified that LDA binds to and inhibits STAMBPL1, thereby disrupting the STAMBPL1/NRF2 positive feedback loop, consequently suppressing CCA progression.

Conclusion

This study firstly reveals that STAMBPL1 promotes cholangiocarcinoma progression by upregulating NRF2, indicating that targeting the STAMBPL1/NRF2 axis is a novel therapeutic strategy. Additionally, our findings firstly suggest that LDA can bind to STAMBPL1, inhibiting NRF2 deubiquitination and offering significant therapeutic potential.