HMBOX1 reverses autophagy mediated 5-fluorouracil resistance through promoting HACE1-induced ubiquitination and degradation of ATG5 in colorectal cancer

Chemotherapy remains the primary treatment for unresectable or advanced postoperative colorectal cancers. However, its effectiveness is compromised by chemoresistance, which adversely affects patient outcomes. Dysregulated macroautophagy/autophagy is a proposed mechanism behind this resistance, with ubiquitination playing a key regulatory role. In this study, we identify the transcription factor HMBOX1 (homeobox containing 1) as a critical regulator of chemoresistance in colorectal cancer. RNA sequencing revealed that <i>HMBOX1</i> is downregulated in drug-resistant colorectal cancer cells and tissues, with its low expression linked to poor prognosis. An integrated analysis of genes associated with autophagy and 5-fluorouracil (5-FU) resistance was conducted, verified in the colorectal cancer tissues of patients by single-cell RNA sequencing and immunostaining. Mass-spectrometry-based proteomics and RNA sequencing were used to elucidate the underlying molecular mechanisms. Functionally, upregulation of HMBOX1 enhances the sensitivity of colorectal cancer cells to the first-line treatment with 5-FU by inhibiting autophagy. Mechanistically, HMBOX1 promotes the transcription of the E3 ubiquitin ligase HACE1, which in turn enhances ATG5 K63-ubiquitination and subsequent proteasome-mediated degradation. This results in decreased ATG5 levels, inhibiting autophagy and thus reducing 5-FU resistance in colorectal cancer cells both <i>in vitro</i> and <i>in vivo</i>. Furthermore, we confirm that HMBOX1 expression positively correlates with HACE1 expression and inversely correlates with autophagy levels in clinical colorectal cancer tissues. Our findings suggest that HMBOX1 downregulation drives 5-FU resistance through autophagy enhancement in colorectal cancer, highlighting HMBOX1 as a potential target for improving chemosensitivity and patient prognosis.<b>Abbreviation</b>: 3-MA: 3-methyladenine; 5-FU: 5-fluorouracil; ATG: autophagy related; CASP3: caspase 3; C-CASP3: cleaved caspase 3; C-PARP: cleaved PARP; CCK8: cell counting kit-8; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; CNV: copy number variation; co-IP: co-immunoprecipitation; COAD: colorectal adenocarcinoma; CQ: chloroquine; CRC: colorectal cancer; CR: complete response; FHC: fetal human colon; GEO: Gene Expression Omnibus; HACE1: HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1; HMBOX1: homeobox containing 1; IHC: immunohistochemistry; LC-MS/MS: liquid chromatography-tandem mass spectrometry; mIHC: multiplexed immunohistochemistry; MUT: mutant; NC: negative control; OS: overall survival; PBS: phosphate-buffered saline; PD: progressive disease; PFA: paraformaldehyde; PFS: progression-free survival; PR: partial response; qPCR: quantitative polymerase chain reaction; RAPA: rapamycin; SD: stable disease; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; TF: translation factor; USP22: ubiquitin specific peptidase 22; WT: wild type.