The negative effect of G1958A polymorphism on MTHFD1 protein stability and HCC growth

Purpose: Methylenetetrahydrofolate dehydrogenase (MTHFD1), a key enzyme on the folate pathway, has been implicated in the tumor development of distinct types of cancers. The single nucleotide polymorphism (SNP) of 1958G > A mutation in the coding region of MTHFD1 (arginine 653 is mutated into glutamine) has been detected in a significant proportion of clinical samples of hepatocellular carcinoma (HCC). METHODS : Hepatoma cell lines, 97H and Hep3B were used. The expression of MTHFD1 and SNP mutation protein was determined by immunoblotting analysis. The protein ubiquitination of MTHFD1 was detected by immunoprecipitation analysis. The post-translational modification sites and interacting proteins of MTHFD1 in the presence of G1958A SNP were identified by mass spectrometry. Metabolic flux analysis was used to detect the synthesis of relevant metabolites sourced from serine isotope.

Results: The present study showed G1958A SNP of MTHFD1, encoding MTHFD1 R653Q, was associated with the attenuated protein stability caused by ubiquitination-mediated protein degradation. Mechanistically, MTHFD1 R653Q displayed an enhanced binding to the E3 ligase TRIM21, which was responsible for the augmented ubiquitination, and MTHFD1 K504 was identified to be the primary ubiquitination site. The subsequent metabolite analysis revealed MTHFD1 R653Q resulted in the repressed flux of serine-derived methyl group into metabolite precursors for purine synthesis, and the compromised purine synthesis was demonstrated to be responsible for the impeded growth capability in MTHFD1 R653Q-expressing cells. Moreover, the suppressive effect of MTHFD1 R653Q expression in tumorigenesis was verified by xenograft analysis, and the relationship between MTHFD1 G1958A SNP and its protein levels was revealed in clinical human liver cancer specimens.

Conclusion: Our results uncovered an unidentified mechanism underlying of the impact of G1958A SNP on MTHFD1 protein stability and tumor metabolism in HCC. which provides a molecular basis for the according clinical management when considering MTHFD1 as a therapeutic target.