| Protein is the main undertaker of life activities and the target of most drugs,so protein science is one of the most important research fields.Proteomics is applied to studying the composition and variation of all the proteins in cells,tissues or organisms using the advanced mass spectrometry technology.Mass spectrometry-based proteomics enables in-depth qualitative and quantitative analysis of proteins in biological samples,providing a powerful tool to life science and disease-related research.Primary liver cancer ranked the fourth in incidence rate and the second most common malignancy in China,and it is mainly divided into hepatocellular carcinoma(HCC)and intrahepatic cholangiocarcinoma(i CCA).Early stage of primary liver cancer patients has no obvious clinical symptoms.Although some early detected patients are treated by surgery,the prognosis is still poor with high recurrence rate and poor life quality.Most patients diagnosed at advanced stages are with high degree of malignancy and mortality,because the available therapies are of limited effectiveness.Studies of primary liver cancer for systematical understanding of the pathogenesis and discovery of potential targets are urgently needed.Therefore,on the basis of our previous proteogenomic analysis of HCC,the proteogenomic research of i CCA was performed following the mass spectrometry-based proteomics procedures.We provide a theoretical basis for clinical development of new diagnostic and therapeutic methods by revealing the panoramic molecular mapping,determining the disease classification and characteristics analysis,and identifying potential biomarkers and drug targets.In addition,proteomic study of HCC NAT and healthy liver tissues provides the promising strategy for the early diagnosis of HCC and the development of prevention and treatment methods for relapse.And these omics data can be helpful to dissect the heterogeneity and molecular characteristics,to investigate the new biological insights and to discover the biomarkers.Firstly,genomic,transcriptomic,proteomic,phosphoproteomic and microbiome studies were performed for the clinical tissue samples from Chinese i CCA patients.The genetic characteristics of i CCA were revealed by analyzing the genetic information.It was found that the samples contained aflatoxin-induced gene mutation "fingerprint",and the mutation "fingerprint" was associated with tumor genesis,proliferation and immunosuppression.Correlation analysis of somatic chromatin copy number alterations showed that cis/trans effects of CNA caused changes in m RNA,protein and phosphorylation.Pathway enrichment analysis showed that cis/trans effects were significantly correlated with changes in some protein and related pathways.In addition,the related signal of genetic mutations showed that TP53,KRAS,BAP1 and IDH1/2gene mutations could affect downstream related protein and phosphorylation abundance,thus affecting the occurrence and development of disease.It was found that TP53/KRAS dual mutations may promote i CCA metastasis through the integrin-FAK/SRC pathway,resulting in poorer prognosis.In conjunction with the drug target database,it was found that higher expression of the target protein in gene mutationrelated patient group could be used for personalized treatment,which was validated by patient-derived primary cancer cell lines.The i CCA patients were divided into four subgroups by proteomic analysis,and these four subgroups have specific characteristics.Four subgroup-specific protein biomarkers from each proteomic subgroup were identified by omics data dimension reduction analysis.And these four biomarkers were further validated in this and another independent patient cohort by immunofluorescence staining.Two potential protein biomarkers of i CCA,SLC16A3 protein and HKDC1 protein,were identified by clinical risk model analysis of proteomic data.Further analysis and verification showed that these two proteins were associated with cellular metabolic reprogramming of i CCA.Phosphoproteomic analysis revealed that FGFR2 gene fusion can activate Rho GTPase pathway,contributing to the development of i CCA.Immunopeptidomic analysis was employed for successfully identifying immunopeptides in FGFR2-BICC1 fusion protein-overexpressed Hu CCT1 cells.Secondly,DIA-based quantitative proteomic technology was applied for the deep proteomic analysis of 106 samples including healthy liver tissues,NAT and tumor tissues.The NATs proteins from the previously published TMT labeling-based proteomic data from the 159 patients were differentially expressed in the three subgroups from the tumor tissues suggesting that the paired NATs can reflect the intrinsic characteristics of tumor subgroups.In order to reveal the heterogeneity of NATs,the significantly changed 759 proteins were used for unsupervised hierarchical cluster analysis,resulting in two subtypes with significant difference of the prognosis and recurrence.The two subtypes have different molecular,clinical and immune characteristics.We performed DIA-based proteomic experiment to compare the proteomic differences between NATs from each NAT subtype and the healthy liver tissues.It was revealed that NAT Subtype 1 was close to healthy liver.Proteins that may be involved in early events of HCC were identified by analysis of differentially expressed proteins in healthy liver tissues and Subtype 1 NATs,including 13 upregulated secreted proteins.By comparing the protein expression of Subtype 2 NATs and normal liver tissues,it was found that down-regulated proteins were mainly enriched in metabolity-related pathways.In addition,by mfuzz clustering the proteins in the order of healthy,NAT Subtype 1,NAT Subtype 2 and tumor,we identified 762 proteins with a decreasing expression trend.These proteins were mainly enriched in metabolic biological processes,such as fatty acid metabolism,cellular amino acid metabolism,lipid catabolism,organic anion transport and nucleoside diphosphate metabolism.We also identified 365 proteins in an increasing expression trend,suggesting the potential function in promoting cancer development.Comparing with multiple databases,we found nine metabolism-related proteins with unfavorable prognosis.7 proteins among them have been reported to be associated with the development of HCC.And the cellular function experiments of the remaining two proteins(GPD1L and SRM)was performed,showing that these two proteins may be involved in the occurrence and development of HCC.In summary,the systematical studies for the iCCA and the HCC NATs by MSbased proteomics were performed to reveal the intrinsic biological mechanism of primary liver cancer and provide more possibilities for its clinical diagnosis and treatment.These omics data also provide useful resources to support the basic and clinical research in liver cancer. |
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