Abstract Introduction Lymph node metastasis in colorectal cancer (CRC) correlates with poor prognosis, yet its molecular drivers remain unknown. This study aimed to identify novel targets associated with lymph node metastasis. Methods Colon cancers were classified into "expanding" and "metastatic" phenotypes using TCGA data. Transcriptomic profiles were analyzed by RNA-seq and GSEA. C6ORF15 knockout (KO) and overexpression (OE) in HCT116 and SW480 cells were achieved via CRISPR-Cas9 and lentiviral transduction, validated by Sanger sequencing and Western blot. Functional effects were assessed through proliferation, migration, and invasion assays, and Western blot for EMT markers. Immunoprecipitation-mass spectrometry (IP-MS) was performed in C6ORF15-OE HCT116 cells to identify potential interactors. Results TCGA analysis identified a median CRC tumor size of 5.375 cm, enabling classification into ‘expanding’ (5 cm, no lymph node involvement) and ‘metastatic’ (≤5 cm, early lymph node involvement) phenotypes. Kaplan-Meier analysis showed significantly poorer survival in the ‘metastatic’ group (p = 0.0145). GSEA identified epithelial-mesenchymal transition (EMT) as the top enriched Hallmark in ‘metastatic’ tumors (NES = 1.81, FDR q = 0), with C6ORF15 highly upregulated (LogFC = 2.37, p = 2.06E-07), confirmed by immunohistochemistry in patient-derived tissue specimens. In isogenic cell lines, C6ORF15 KO downregulated the EMT gene set (NES = -1.44, FDR q = 0.1), reducing SNAI1/2 while preserving CDH1/2, shifting cells toward a non-metastatic epithelial phenotype with reduced proliferation, migration, and invasion. Conversely, OE cells exhibited aggravated partial EMT characteristics, marked by downregulation of CDH1 and upregulation of selected mesenchymal markers (CDH2, SLUG, and SNAI1) in a cell line-dependent manner, shifting cells toward a more aggressive metastatic phenotype with enhanced proliferation, migration, and invasion. IP-MS identified FAT1 as one of two potential C6ORF15 interactors. FAT1 OE is known to upregulate EMT and is associated with CRC. In isogenic cell lines, C6ORF15 OE rescued EMT activity in FAT1 KO cells, while C6ORF15 KO downregulated EMT in FAT1 OE cells, suggesting that C6ORF15 effects on EMT may persist independent of FAT1 expression. Conclusion C6ORF15 may promote EMT and early CRC metastasis involving lymph nodes, showing potential as a target for future therapies. Citation Format: Dedrick Kok Hong Chan. Characterisation of C6ORF15 as a marker for epithelial-mesenchymal transition and lymph node metastasis in colorectal cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4847.
Dedrick Kok Hong Chan (Fri,) studied this question.