Xanthatin overcomes osimertinib resistance by disturbing cholesterol metabolism via targeting HMGCR protein degradation in NSCLC.

BACKGROUND: Osimertinib is a third-generation, irreversible EGFR tyrosine kinase inhibitor (TKI), nevertheless, the inevitable occurrence of osimertinib resistance after a long-term administration limits its clinical application in non-small cell lung cancer (NSCLC). Xanthatin, a naturally active component from Xanthium Strumarium L., has been shown to possess multiple bioactive effects. However, whether xanthatin can ameliorate osimertinib resistance remains unclear.

PURPOSE: The present study aimed to clarify the role and detailed mechanism of xanthatin in reversing osimertinib resistance in NSCLC.

METHODS: Combenefit analysis, LDH release assay, and EdU staining were employed to evaluate the effect of xanthatin on osimertinib cytotoxicity in osimertinib-resistant (OR) NSCLC cells. Quantitative proteomics combined with bioinformatics and network pharmacology analysis was used to uncover the specific target of xanthatin. Xanthatin-triggered HMGCR protein degradation was assessed by bio-layer interferometry, drug affinity responsive target stability assay, cycloheximide chase assay and ubiquitination determination. The function of xanthatin on cholesterol homeostasis was measured via the detection of HMGCR activity, total cholesterol, triglycerides and lipid droplets as well as MDR1 distribution in the lipid rafts. The function of MDR1 was examined via a rhodamine 123 efflux assay. The accumulation and efflux of osimertinib were monitored using FITC-labeled osimertinib. The effect of HMGCR on the cytotoxicity of xanthatin was evaluated through a series of in vitro and in vivo experiments in HMGCR-overexpressing NSCLC OR cells.

RESULTS: Xanthatin restored the sensitivity of NSCLC OR cells to osimertinib, accompanied by the induction of oxidative stress and mitochondrial dysfunction. Proteomic data indicate that oxidative stress and metabolic reprogramming contribute to xanthatin-induced apoptosis in NSCLC OR cells. Through further integration with network pharmacology, we identified HMGCR (a key enzyme of cholesterol metabolism) as a potential target of xanthatin. We confirmed xanthatin directly binds to HMGCR protein (KD=6.753 μM), subsequently facilitating AMFR-mediated K48-linked ubiquitination and degradation of HMGCR. Xanthatin diminished intracellular cholesterol biosynthesis and MDR1 levels in lipid rafts. HMGCR overexpression mitigated the cytotoxicity of xanthatin and sustained cholesterol homeostasis, enabling tolerance to the combined effects of xanthatin and osimertinib. Conversely, HMGCR-deficient NSCLC OR cells were more vulnerable to osimertinib.

CONCLUSION: Our study demonstrates for the first time that xanthatin directly binds to HMGCR and promotes AMFR-mediated K48-linked ubiquitination of HMGCR, which in turn accelerates HMGCR degradation through the ubiquitin-proteasome pathway, leading to reduced cholesterol synthesis, less distribution of MDR1 in lipid rafts, ROS accumulation, and mitochondrial dysfunction-associated apoptosis, ultimately restoring osimertinib sensitivity in NSCLC cells. Therefore, xanthatin may represent a preclinical natural product candidate for the reversal of osimertinib resistance in NSCLC cells, which requires further translational verification in future studies.