Effect of KRT3 on colorectal cancer growth through regulation glycolysis via the cebpd-LRRC8A-AKT signaling axis.

e15587 Background: Colorectal cancer (CRC) is one of the most common malignancies worldwide with high morbidity and mortality. Its development and progression are closely associated with metabolic reprogramming. Glycolysis, a critical pathway for energy metabolism in cancer cells, plays a pivotal role in CRC progression. KRT3 (Keratin 3) is aberrantly expressed in various cancers, but its function and mechanisms in CRC remain poorly understood. This study aims to investigate whether KRT3 promotes CRC growth by regulating glycolysis and to explore its underlying molecular mechanisms. Methods: The expression of KRT3 in CRC and its correlation with patient prognosis were analyzed using the TCGA database and CRC tissue microarrays. KRT3-knockdown CRC cell lines were established using lentiviral shRNA technology, and RNA sequencing was performed to identify downstream targets of KRT3. The regulatory effects of KRT3 on CEBPD, LRRC8A, and the AKT signaling pathway were validated using qPCR, Western blot, immunofluorescence, Co-IP, and dual-luciferase reporter assays. The impact of KRT3 on glycolysis was assessed through metabolomic analysis and Seahorse metabolic flux analysis. In vivo experiments were conducted using a nude mouse xenograft model to evaluate the role of KRT3 in CRC growth. Results: KRT3 was significantly overexpressed in CRC tissues and associated with poor patient prognosis. Knockdown of KRT3 markedly inhibited CRC cell proliferation, invasion, migration, and glycolytic capacity. Mechanistic studies revealed that KRT3 upregulates LRRC8A expression through the transcription factor CEBPD, thereby activating the AKT signaling pathway and promoting the expression and activity of key glycolytic enzymes. Metabolomic analysis confirmed that KRT3 knockdown significantly reduced glycolytic metabolite levels. In vivo experiments demonstrated that KRT3 knockdown significantly suppressed tumor growth and glycolytic activity in nude mouse xenografts. Conclusions: This study unveils a novel mechanism by which KRT3 promotes CRC growth through the CEBPD-LRRC8A-AKT signaling axis to regulate glycolysis. KRT3 may serve as a potential therapeutic target for CRC, providing a new theoretical foundation for metabolic-targeted therapy in colorectal cancer.