Targeting the ARL4C/RAP1/PI3K-Akt-mTOR signaling loop promotes ARL4C ubiquitination and reverses oxaliplatin resistance in colorectal cancer

Background: Oxaliplatin resistance poses a significant therapeutic challenge in colorectal cancer (CRC), contributing to disease progression and poor clinical outcomes. There is an urgent need to identify novel molecular targets to overcome chemoresistance and inhibit metastatic dissemination. Methods: We conducted integrative multi-omics analyses to identify genes associated with oxaliplatin resistance in CRC and detected ARL4C, a small GTPase, as a candidate driver. Functional experiments, including gene knockdown/overexpression, mutant construction, cell viability, apoptosis, migration, and invasion assays, as well as in vivo mouse models, were used to evaluate the role of ARL4C. Signaling pathways were examined using proteomics and molecular biology techniques. We employed network pharmacology and molecular docking to identify ARL4C-targeting compounds and selected β-Lapachone for further validation. Results: ARL4C was significantly overexpressed in oxaliplatin-resistant CRC tissues and correlated with poor prognosis and increased metastatic potential. Mechanistic studies revealed that ARL4C activates RAP1/PI3K-Akt-mTOR and RAC1/Arp2/3 signaling axes, promoting cell survival, epithelial-mesenchymal transition, and invasion. ARL4C also inhibited its own ubiquitination by regulating USP38, forming a positive feedback loop that enhanced protein stability following chemotherapy. β-Lapachone was identified as a direct ARL4C inhibitor that binds competitively at the LYS128 residue, disrupting USP38 interactions and promoting ARL4C degradation. Combination therapy with β-Lapachone and oxaliplatin significantly suppressed tumor growth, reduced metastasis, reversed drug resistance, and mitigated oxaliplatin-induced renal toxicity in preclinical models. Conclusions: Our study identifies ARL4C as a critical mediator of chemoresistance and metastasis in CRC. Targeting ARL4C with β-Lapachone restores oxaliplatin sensitivity and enhances therapeutic efficacy, offering a promising combinatorial strategy with strong potential for clinical translation in drug-resistant CRC.