ABSTRACT: Background: Microtubule-actin cross-linking factor 1 (MACF1) is a cytoskeletal cross-linker implicated in cancer pathogenesis, yet its role in colorectal cancer (CRC) progression remains unclear. This study investigated MACF1 expression, function, and its mechanistic link to the ATF4-ASNS regulatory axis in CRC. Methods: MACF1 expression was assessed in CRC tissues and cell lines (HCT116, HT29, SW620, Caco2, LoVo) via immunohistochemistry and qPCR. Functional assays—including proliferation, colony formation, wound healing, Transwell migration, and cell-cycle analysis—were performed following MACF1 knockdown and overexpression. In vivo tumor growth was evaluated using a subcutaneous xenograft model. RNA-seq screening identified ASNS as a downstream effector, and the MACF1–ATF4–ASNS relationship was explored through ChIP, immunofluorescence, nuclear fractionation, and Co-IP. Results: MACF1 was significantly elevated in CRC tissues and cells versus controls (p < 0.05). MACF1 knockdown suppressed proliferation, colony formation, migration, and xenograft tumor growth, while overexpression promoted aggressive phenotypes (p < 0.05). Mechanistically, ASNS was identified as a critical MACF1 target, with ATF4 serving as a transcriptional mediator linking MACF1 to ASNS regulation. Conclusion: Our findings demonstrate that MACF1 promotes CRC cell growth and migration, at least in part, via ATF4-associated ASNS regulation and asparagine metabolism. This work establishes a novel functional bridge between cytoskeletal remodeling and amino acid metabolic reprogramming in CRC, offering potential insights for future metabolism-oriented therapeutic strategies.
