The Role And Mechanism Of SHMT2 Activating PI3K/AKT Signaling Pathway To Promote PTC Metastasis Via Enhancing PTEN Methylation

BackgroundPapillary thyroid carcinoma(PTC)is the most common subtype of thyroid cancer,approximately 80-90%of cases.And PTC is prone to lymph node metastasis in the early stage and even distant metastasis in some cases with poor prognosis.But the metastasis mechanism has not been elucidated.Uncovering the metastasis mechanism of PTC is helpful to provide a novel target for clinical treatment,which has important theoretical significance and potential application value.Metabolic reprogramming is a common feature of tumours.Serine metabolic reprogramming plays an important role in tumour development.Serine metabolic fluxes are increased in a variety of tumours,and inhibiting enzymes of serine metabolism can block tumour growth.Serine hydroxymethyltransferase 2(SHMT2)is a key enzyme in mitochondrial serine metabolism,catalyzing the catabolism of serine to glycine and methylenetetrahydrofolate,and is involved in reactive oxygen species homeostasis,nucleotide synthesis,and epigenetic modifications.It is essential for tumor survival,affecting tumorigenesis,survival,proliferation,poor prognosis and drug resistance through different mechanisms.The literature and our previous studies have shown that SHMT2 is highly expressed in PTC and correlates with prognosis,but the specific role and mechanism have not been reported.ObjectOur study aims to clarify the role and molecular mechanism of SHMT2 in the occurrence and development of PTC with experiments in vitro and in vivo and clinical studies,to provide novel targets and ideas for the prevention and treatment of PTC.Methods and resultsProteomics was performed on 27 PTC tumour and para-tumour clinical samples,and KEGG enrichment analysis revealed the most significant differences in metabolic pathways.Since mitochondria are cellular metabolic hubs,the study was localized to mitochondrial metabolic enzymes to help uncover metabolic regulatory information.Fifty-one differential proteins were obtained after screening,among which SHMT2 was significantly differential and over-expressed in PTC.The high expression of SHMT2 in thyroid cancer tissues was verified using Real time-quantitative PCR(RT-qPCR)and immunohistochemistry(IHC).Analysis of the TCGA database revealed that high SHMT2 expression decreased disease-free survival in PTC patients.Over-expressed or knock-down SHMT2 PTC cells was constructed and migration assays showed that SHMT2 promoted the migration of PTC cells.RT-qPCR and WB results showed that SHMT2 expression was positively correlated with the metastasis-promoting factor Ncadherin and Vimentin,and negatively correlated with the metastasis-inhibiting factor Ecadherin.Tumour lung metastasis model with mice demonstrated that enhanced ability to form metastases toumour for over-expressed SHMT2 PTC cells.In summary,SHMT2 promoted PTC metastasis in vivo and in vitro.To clarify the specific molecular mechanism of SHMT2 promoting PTC metastasis,proteomics data analyzed by GSEA revealed that PI3K/AKT signaling pathway was activated in PTC.Phosphorylated AKT levels were detected in tissue microarrays and clinical samples using IHC and WB,verified that SHMT2 expression was positively correlated with AKT phosphorylation levels.The detection of phosphorylated AKT levels in over-expressed or knock-down SHMT2 PTC cells verified that SHMT2 promoted the activation of PI3K/AKT signaling pathway.Meanwhile,migration assays and mice lung metastasis model classified that the AKT phosphorylation inhibitor MK-2206 significantly reversed the enhanced AKT activity and enhanced tumour metastasis caused by SHMT2.PTEN is a classic inhibitor of the PI3K/AKT signaling pathway,and PTEN expression was reported to be reduced in PTC in the literature.Using RT-qPCR and IHC assays,it was demonstrated that SHMT2 was negatively correlated with PTEN expression in PTC in clinical samples.Cellular assays demonstrated that SHMT2 negatively regulated PTEN expression.SHMT2 increased the level of SAM and genome-wide DNA methylation in cells,as confirmed by SAM content assay and DNA dot blotting.PTEN gene methylation sequencing and methylation-specific PCR experiments further verified that SHMT2 promoted the methylation of PTEN CpG islands to inhibit PTEN expression.We further treated overexpressed SHMT2 cells with the methylation enzyme inhibitor 5-azacytidine to derepress its inhibitory effect on PTEN expression.In addition,overexpression of PTEN reversed the enhanced AKT activity and enhanced metastasis of PTC cells caused by high SHMT2 expression,and knockdown of PTEN partially rescued the reduced AKT activation and inhibited metastasis of PTC cells caused by SHMT2 knockdown.Animal experiments further demonstrated that SHMT2-mediated PTEN silencing promoted tumour metastasis in vivo.The above results revealed that SHMT2 promotes PTEN CpG island methylation and activates PI3K/AKT signaling pathway to promote PTC metastasis by increasing SAM levels in PTC cells.Conclusions and implicationsThis study revealed the molecular mechanism.SHMT2 promoted PTC metastasis by increasing the level of SAM in PTC cells,promoting the methylation of PTEN CpG islands and leading to a decrease in their protein expression,which activated the PI3K/AKT signaling pathway and promoted PTC metastasis.The above findings revealed new targets and ideas for the prevention and treatment of PTC.