Abstract

Background Cetuximab is an approved therapy for metastatic colorectal cancer (CRC) with wild-type RAS and BRAF; however, additional resistance mechanisms beyond genetic mutations remain poorly understood. Butyrate, a key metabolite produced by the gut microbiome and present in the circulatory system, has been reported to supply cellular energy and modulate the epidermal growth factor receptor (EGFR) downstream signaling pathway. However, whether butyrate affects the resistance to cetuximab is still unknown.Methods In this work, Cell Counting Kit-8 (CCK-8) and colony formation assays were used to evaluate the efficacy of cetuximab. Glycolysis/oxidative phosphorylation (OXPHOS) Assay Kit was applied to assess metabolic activity. Human Phospho-Kinase Array and RNA sequencing were employed to screen targets of butyrate. Overexpression plasmids and short hairpin RNAs (shRNAs) targeting these molecules were transfected into cells for further validation. Subcutaneous tumor and pulmonary metastasis models were used for in vivo studies.Results The findings showed that physiological concentrations of butyrate increased cetuximab resistance in KRAS wild-type cells only. Further investigation found that butyrate upregulated EGFR signaling through facilitating the binding reaction between epidermal growth factor (EGF) and EGFR. In parallel, butyrate activated AMP-activated protein kinase (AMPK)–wild-type p53-induced phosphatase 1 (Wip1) signaling, leading to suppression of p53 and p38 mitogen-activated protein kinase (p38 MAPK)-mediated pro-apoptotic signaling. These two mechanisms are the reason that butyrate attenuates the efficacy of cetuximab. Results of subcutaneous tumor and pulmonary metastasis models exhibited a similar conclusion to in vitro experiments.Conclusion Butyrate reduces cetuximab efficacy in KRAS wild-type colorectal cancer through EGFR and AMPK–Wip1 signaling, and may represent a candidate predictive biomarker for treatment response.