Proteomics Analysis Of Lung Cancer Cells Reveals A Critical Role Of BCAT1 In Cancer Metastasis

The invasiveness and metastasis of lung cancer result in poor prognosis and low survival rate of the patients.Metastasis is a multiple-step biological process involved with numerous regulatory networks,systematic exploring of the key mechanisms and regulated targets could provide new therapeutics for lung cancer.However,there is still lacking of proteomic studies on lung cancer patients with comparison between primary tumor and metastasis tissues,as majority large-scaled comprehensive proteogenomic studies focused on tumorigenesis of primary tumor.Cell line models of cancer malignancy were generated from spinal metastasis subclones A549L2 and L6 that underwent in vivo selection of nude mice,as well as the pre-malignancy cells A549(designated L0).In this study,we applied quantitative mass spectrometry analysis based on stable isotopic labeling of amino acids in cell culture(SILAC),revealing consistent regulation of amino acid metabolism.Involved enzymes like CPS1,BCAT1 were significantly upregulated in metastasis cells and tissues derived from lymph node of lung cancer patients.Comprehensive survival analysis of transcriptome and clinical information of LUAD and LUSC patients,suggesting that patients expressed high level of BCAT1 had a shorter median survival time,as well as aberrant CPS1 was correlated with poor prognosis in LUAD patients.Overall,amplification of metabolic enzyme BCAT1,CPS1 promote lung cancer metastasis.Metabolism reprogram is a marker of malignancy,as a result of comprehensive regulation of micro-environment induction and inner driven gene mutations.Amino acid metabolism supports protein and nucleotides synthesis of tumor cells as a nitrogen and energy donation,and participates in ammonia elimination and sustain redox state,plays an essential role in cancer progression.Searching for the key metabolism pathways modulated in metastasis could navigate to new strategy for intervention.The investigation of mechanism in lung cancer metastasis related with amino acid metabolism,the regulatory mechanism of BCAT1 has been further studied.In terms of function,the depletion of BCAT1 was found to inhibit the migration and distal metastasis of in vitro and in vivo.In terms of mechanism,we found that reducing of BCAT1 inhibited the expression of SOX2 in transcription and protein levels,resulting in decreased proportion of CD133~+cancer stem cells.Further studies showed that amplification of BCAT1 maintained SOX2 expression by depleting its co-substrate?-KG,and the addition of cell permeable DM-?-KG inhibited SOX2 protein level.Since?-KG is a cofactor of DNA demethylase TET2,an?-KG/Fe2+dependent dioxygenases,reducing?-KG concentration may lead to hypermethylation of target genes.While mi R200c,which is regulated by TET2,acts as a negative regulator by targeting and degrading SOX2 m RNA.We found the level of mi R200c was significantly decreased in A549L2 and L6 cells with high expression of BCAT1,and recovered after BCAT1reduction.Meanwhile,the expression of E-cadherin was up-regulated when BCAT1knocked down.These results suggested that high expression of BCAT1 could promote tumor progression and metastasis by maintaining the expression of SOX2 trough reducing mi R200c.In conclusion,we have found a new mechanistic understanding of BCAT1,an enzyme involved in amino acid metabolism,promoting cancer metastasis through stemness factor SOX2,which provides potential therapeutic strategy to blocking cancer progression.