Thymidylate synthase O-GlcNAcylation: a molecular mechanism of 5-FU sensitization in colorectal cancer

Abstract

Alteration of O-GlcNAcylation, a dynamic post-translational modification, is associated with tumorigenesis and tumor progression. Its role in chemotherapy response is poorly investigated. Standard treatment for colorectal cancer (CRC), 5-fluorouracil (5-FU), mainly targets Thymidylate Synthase (TS). TS O-GlcNAcylation was reported but not investigated yet. We hypothesize that O-GlcNAcylation interferes with 5-FU CRC sensitivity by regulating TS. In vivo, we observed that combined 5-FU with Thiamet-G (O-GlcNAcase (OGA) inhibitor) treatment had a synergistic inhibitory effect on grade and tumor progression. 5-FU decreased O-GlcNAcylation and, reciprocally, elevation of O-GlcNAcylation was associated with TS increase. In vitro in non-cancerous and cancerous colon cells, we showed that 5-FU impacts O-GlcNAcylation by decreasing O-GlcNAc Transferase (OGT) expression both at mRNA and protein levels. Reciprocally, OGT knockdown decreased 5-FU-induced cancer cell apoptosis by reducing TS protein level and activity. Mass spectrometry, mutagenesis and structural studies mapped O-GlcNAcylated sites on T251 and T306 residues and deciphered their role in TS proteasomal degradation. We reveal a crosstalk between O-GlcNAcylation and 5-FU metabolism in vitro and in vivo that converges to 5-FU CRC sensitization by stabilizing TS. Overall, our data propose that combining 5-FU-based chemotherapy with Thiamet-G could be a new way to enhance CRC response to 5-FU.

Targeting O-GlcNAcylation to overcome resistance to anti-cancer therapies

“…(2021) demonstrated that cisplatin enhances UDP-GlcNAc production and global O -GlcNAcylation levels in vitro and in vivo by OGT, GFAT1 activation, and OGA inhibition ( 33 ). Conversely, we recently demonstrated that 5-FU decreases intracellular O -GlcNAcylation in vitro by reducing OGT at both protein and transcriptional levels but also in vivo most likely by reducing OGT activity ( 34 ). Owing to the fact that OGT enzymatic activity is inhibited by elevated UDP, UTP, and UDP-GlcNAc ( 35 ), we suggest that 5-FU metabolites may inhibit OGT by producing fluorinated derivates or uridine compounds ( 36 ).…”

“…OGT was also identified in a cluster of co-expressed genes associated with 5-FU resistance in colorectal cancer (CRC) ( 79 ). Moreover, the 5-FU pathway actors SLC29A1 ( 80 ), TP ( 10 ), TK ( 81 , 82 ), and TS ( 34 ) are direct targets for O -GlcNAcylation ( Figure 2 ). Nevertheless, modified sites and roles of this PTM on these proteins as well as their impacts on 5-FU sensitivity are largely not elucidated yet.…”

“…Very recently, we demonstrated that TS is stabilized by O -GlcNAcylation at Thr 251 and Thr 306 residues. OGT knock-down decreases 5-FU-induced apoptosis by enhancing TS proteasomal degradation and, by reducing TS level and enzyme activity in HT-29 cell line but not in 5-FU resistant HT-29 counterpart ( 34 ) ( Figure 2 ). The latter exhibits TS gene amplification and TS overexpression which is a current biomarker of 5-FU resistance in CRC ( 83 ).…”

“…We have shown that the regulation of 5-FU response by O -GlcNAcylation is finely tuned and depends on a proper quantity of TS proteins. In a CRC mouse model, the combination of 5-FU with the OGA inhibitor Thiamet-G had a synergistic inhibitory effect on tumor grade and progression ( 34 ). As the O -GlcNAcylation homeostasis-TS axis mediates the response to 5-FU, we propose to combine an OGA inhibitor with 5-FU-based therapies to enhance CRC patient response to 5-FU.…”

Genome-wide CRISPR Screening Reveals Pyrimidine Metabolic Reprogramming in 5-FU Chronochemotherapy of Colorectal Cancer

“…The drug 5-fluorouracil (5-FU) is a synthetic fluorinated pyrimidine analog that is widely used in the treatment of cancers including colorectal cancer (CRC), breast cancer, and respiratory and digestive tract cancers ( 6 ). This drug is mainly converted into active metabolites such as fluorodeoxyuridine monophosphate (FdUMP), which inhibits the action of thymidylate synthase (TYMS), a rate-limiting enzyme of DNA synthesis, ultimately leading to cell damage ( 7 ). However, acquired drug resistance is the main issue limiting its clinical application ( 8 ).…”