Proteomics Discovery Of Tumor Markers For Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is unquestionably the most serious and dreaded complication of chronic liver disease. Its mortality accounted for the second of cancer in China. The average life expectancy after diagnosis of clinically apparent HCC is less than 12 months. HCC develops typically in patients with chronic liver disease and cirrhosis. In these targeted populations serum markers are most urgently needed. Metastases are the cause of 80%~90% of human cancer deaths. However, there is no marker at present to diagnose or predict the metastasis for HCC patients. Therefore, detection of HCC at an early, and hence potentially resectable stage, offers the best hope for cure. Unfortunately, tumor markers that are currently utilized for the detection of HCC in clinical practice lack the sensitivity and specificity needed to detect potential curable lesions.Proteomics as a powerful comprehensive analysis tool in protein expression research has been widely applied in diseases, particularly in cancer research. Quantitative protein expression profiling is a crucial part of proteomics and requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. ClinProt, as one novel protein-chip technology, uses different chemical chromatographic surfaces on an outer layer of magnetic beads to selectively purify certain subsets of proteins, allowing unbound impurities to be removed by washing with buffers. Proteins bound to the magnetic beads are then eluted, diluted, and directly analyzed by MALDI-TOF MS. This approach is sensitive and fast, which features are essential for clinical use. Moreover, the cost is low, and further protein identification can be easily performed from the eluted material without complex purification. SILAC (Stable isotope-labelled animo acids in cell culture) has recently emerged as a valuable proteomic technique. Using SILAC, cells representing two biological conditions can be cultured in amino acid-deficient growth media supplemented with 12C- or 13C-labeled amino acids. The proteins in these two cell populations effectively become isotopically labeled as"light"or"heavy."The technology is a breakthrough for analysis of serum-free secreted proteins and discovery of tumor markers for the early diagnosis of cancer metastasis and recurrence.Studies assessingα-fetaprotein (AFP) value as a screening tool varied widely in their design and in the characteristics of the patients (type of viral infection, type and severity of liver disease, and so forth). Especially the sera AFP of approximately one-third of the HCC patients were always negative in their proceeding of disease. There is significant heterogeneity of cancer cells in HCC tissues, and studies showed that AFP negative HCC cells and AFP positive HCC cells are two different subgroups of liver cancer cells. Therefore, we discovered biomarkers with the sera of AFP negative HCC patients which will reduce the biological interference from research materials and improve the diagnostic specificity for the AFP negative HCC, and then may provide a complementary biomarker for HCC. To obtain the best experimental results, a series of preliminary experiments were done to evaluate the reproducibility of this method and to select the optimum experimental materials, e.g. sample type, kits and target. Then we used ClinProt to assess protein expression profiles and to identify potential serum tumor markers in AFP negative HCC. Totally, 218 cases of sera were tested. In learning and evaluation stage, sera of patients with cirrhosis which have been infected with HBV and their sera AFP level are all normal (≤20ng/ml), either with (n=28) or without (n=31) HCC, were tested to establish an AFP negative diagnosis model. The sensitivities of this model was 92.86 %, and the specificities was 93.94 %. In the validation sample set, patients included AFP negative HCC (n=31), AFP positive HCC (serum AFP≥100ng/ml) (n=21), liver cirrhosis (n=30), chronic liver disease (n=35), all patients displayed chronic liver disease related to HBV infection, and 30 healthy blood donors were also included in the validation groups. The sensitivities of the model was 91.67%, and the specificities was 90.91%. The diagnosis model can also recognize all the AFP positive HCC into cancer group correctly. Of the five diagnosis peaks, 2467.83, 7757.73, 9277.89Da peaks are significantly up-regulated in HCC than healthy donors and cirrhosis groups. The model is not only specific, but also more sensitive than other HCC markers in AFP negative HCC and may improve the clinical diagnosis of HCC in future. Three up-regulated proteins in HCC serum were identified to be cleavages of C-terminal parts of PRG4, vitronectin and SERPINC1. Among them, vitronectin was identified in HCV-related HCC by SELDI previously. PRG4 was one novel marker of AFP negative HCC and was further validated by western blotting and immunohistochemistry.Metastases are the main cause of treatment failure and death for cancer patients. Cancer metastasis is a complex multistep process that involves alterations in dissemination, invasion, survival, and growth of new cancer cell colonies and the development of cancer-associated vasculature. Many molecular events and signal transduction pathways including cell-cell interactions and adhesion molecules, extracellular matrix proteolysis, growth factors, chemotactic factor, angiogenesis and lymphangiogenesis are associated with metastasis procedure. Secreted proteins control or participate in the growth of cell division, differentiation or apoptosis and many other important biological processes. They are also closely related with body's overall level of the immune defense, blood agglutination and tumor formation and development. Therefore, the proteins secreted from tumor cells are good targets for cancer treatment or diagnosis and prognosis markers. In this study, we used stable isotope labeling with amino acids in cell culture (SILAC) method to compare the'secretomes'of three human HCC cell lines, MHCC97-L, MHCC97-H and HCCLM6, their spontaneous metastasis potential are stepwise increasing. We identified totally 1,251 proteins from the secreted supernatant of the three cell lines, including 993 accurate quantitative proteins and 549 differentially expressed proteins (≥1.5-fold change). Of them, 158 proteins were found to be regularly alterred in the'secretome'of MHCC97-H and HCCLM6 cells versus MHCC97-L cells with fold changes consistent with the cell lines'metastasis capacity; 42 were also identified from Human Plasma Proteome core database, of which several were previously reported as either up-regulated or down-regulated proteins in HCC, confirming the validity of our approach. According to the classification of biological functions, some differential proteins participate in the adjustment of tumor-host microenvironment and hydrolysis of the basement membrane, cells migration and movement; and also include many proteins such as heparan sulfate-polysaccharides (HSPG2), matrix metalloproteinases (TIMP2) and gastrointestinal cancer metastasis-related proteins. Meanwhile, many differential proteins are firstly identified in metastatic sample and their biological function are un-definited in cancer metastasis. Because some proteins are significantly up- or down- regulated in HCCLM6 or MHCC97-H agaist MHCC97-L and their expression are obviously related to the cell lines'metastasis phenotype, many of these proteins could be candidates of diagnosis biomarkers or treatment targets. According to the scores of the identified proteins, biological function and the relevance with tumor metastasis, three up-regulated proteins (clusterin, lumican, HSPG2) and three down-regulated proteins (gelsolin, C3, IGFBP2) were validated with secreted supernatant by western blotting. Results of four proteins (clusterin, gelsolin, C3 and IGFBP2) were consistent with the quantitative results of SILAC. Clusterin, gelsolin and C3 were also validated positively with human sera from metastatic HCC patients. Thus they might be the candidate markers for diagnosis of HCC metastasis.Three features of the study increased our yield of potential biomarkers: firstly, we directly compared a well established in vitro model of a''stepwise metastatic HCC cell model system''for in-depth study of the mechanisms of HCC metastasis; secondly, by restricting our analyses to the secreted proteins alone, we maximize the possibility of identifying candidates that have the greatest potential for serum-based biomarkers of HCC metastasis detection; and at last, we validated our best biomarker candidates from the proteomics study by western blotting in patients'sera.