Interplay between O-GlcNAcylation and 5-FU response in colorectal cancer: Novel insights from an examination of primary and metastatic cell lines

5-fluorouracil (5-FU) is the cornerstone of colorectal cancer (CRC) chemotherapy. O-linked β-N-Acetyl-glucosaminylation (O-GlcNAcylation) plays an essential role in cancer biology, including CRC, but its impact on chemotherapy response remains underexplored. Our previous study revealed that O-GlcNAcylation enhances 5-FU sensitization in vivo and in vitro in HT-29 colon primary cancer cells by reducing Thymidylate Synthase (TS) degradation, the main 5-FU target. The present study analyzed the impact of O-GlcNAcylation on HT-29 and LoVo metastatic colon cancer cells response to 5-FU and further explored its effect on Thymidine Phosphorylase (TP) and Thymidine Kinase (TK), key-actors of 5-FU conversion into active compounds.

Using Human Protein Atlas (HPA) and GEPIA2 databases, combined with comparative analysis of non-cancerous colon CCD841CoN, HT-29 and LoVo cells, we showed elevated TK1 and TYMS expressions in CRC tissues and cell lines, alongside increased O-GlcNAcylation, OGA, OGT, TK and TS levels. Notably, TP overexpression was specific to LoVo. 5-FU treatment induced a time-dependent decrease of OGT and O-GlcNAcylation in LoVo, as previously observed in HT-29; but this was not correlated with OGT mRNA drop. Functionally, OGT knockdown reduced 5-FU sensitivity in HT-29 via decreased TK and TS levels, while enhancing LoVo sensitivity, correlated with increased TP levels. Importantly, we identified TP, TK and TS as O-GlcNAcylated in both cell lines.

These findings reveal distinct regulatory effects of O-GlcNAcylation on 5-FU metabolic enzymes and on chemotherapy response between primary and metastatic colon cancer cells. This underscores the potential for tailored therapeutic strategies considering the unique enzymatic profile of different tumor stages.