Tyrosine Phosphorylation Proteomics Study On The Mechanism Of Liver Cancer Metastasis

Hepatocellular carcinoma（HCC）is one of the most common cause of cancer related death worldwide.Although many therapeutic methods,including early diagnosis,surgical resection,transplantation,and local ablation therapy,have been applied in the treatment of HCC,HCC is still ranked the second leading cause of cancer-related death globally.One of the major reasons for this frequent intrahepatic metastasis and distant metastasis.Thus,investigation of underlying mechanisms of HCC metastasis will provide novel insight for preventing HCC patients from poor prognosis.Many studies revealed that aberrant tyrosine phosphorylation were closely related with tumor metastasis.As one of post translational modification,tyrosine phosphorylation is regarded as key mediator of signaling pathway with profound physiological functions.Only hundreds of tyrosine phosphosites could be identified which were not sufficient to elucidate aberrant tyrosine mediated signaling pathway in previous tyrosine phosphoproteomics studies employing either immobilized meta affinity chromatography（IMAC）,meta oxide affinity chromatography（MOAC）or affinity tyrosine antibody.Yangyang Bian,et al.recently developed a new strategy for systematic identification of phosphotyrosine by affinity purification mass spectrometry（AP-MS）using a Src homology 2（SH2）-domain-derived pTyr superbinder as the affinity reagent and identified 10,030 reliable tyrosine phosphosites from 9tumor cell lines.We employed the superbinder and made some minor modification to establish a high throughput and specific enrichment strategy for tyrosine phosphopeptides which was applied in the investigation of underlying mechanisms of HCC metastasis.MHCC97L and MHCC97H,with different metastatic potential and same genetic background were considered as high metastasis cells.Meanwhile,Hep3B and HepG2 were regarded as low metastasis cells.1,140⁴,691 tyrosine phosphosites were identified in these four HCC cells with 2 mg starting materials after Ti⁴⁺-IMAC and SH2 superbinder enrichment.We found that more tyrosine phosphosites were identified in high metastasis cells than that in low metastasis cells.The uniquely identified and significantly upregulated high metastasis cells were mainly enriched in pathways of endocytosis,migration and invasion that were closely related with metastasis.More importantly,phosphorylation level ofβ-catenin Y489 were found to be significantly upregulated in high metastasis cells implying its potential role in HCC metastasis.After validating the higher level of phosphorylatedβ-catenin Y489 by western blot,mutantsβ-catenin Y489E orβ-catenin Y489F,which mimics the active or inactive Y489,were constructed to perform cell migration and transwell analysis.We found that HCC cell metastasis was promoted by phosphorylation ofβ-catenin Y489.