Quantitative Proteome Analysis Of HCC Cell Lines With Different Metastatic Potentials

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with an annual death population of 650,000. Metastasis is the main cause for treatment failure and high fatality of HCC. To date, the mechanism of HCC metastasis is still unclear, and there is no available biomarker or biochemical test to predict HCC recurrence. It is urgent to deeply investigate the metastasis mechanism and to discover reliable prediction markers and therapeutic targets for the clinical diagnosis and treatment of HCC.HCC metastasis is closely related with multifactors, such as cancer cell adheresion and motility, extracellular matrix degradation, body immunity, tumor angiogenesis, etc. High-throughput proteomics would contribute to the integral study on tumor related proteins. Quantitative proteome analysis can profile the dynamic protein changes, and have great potentials for pathogenic mechanism clarification and biomarker discovery. Stable-isotope labeling by amino acids in cell culture (SILAC) is a metabolic labeling strategy that uses stable isotope labeled amino acids in culture medium to encode cellular proteomes for quantitative analysis. It is compatible for hydrophobic proteins and alkaline proteins separation, and the labeling efficiency is not affected by detergent. So it suits for not only whole-cell protein analysis, but also membrane protein identification and quantification.In order to make further exploration into the mechanism of HCC metastasis and to search for the candidates of diagnostic marker and therapeutic target, SILAC technique was employed to conduct differential proteome analysis on HCC cell lines with low and high metastatic potentials (MHCC97L and HCCLM6). In total, 2,335 reliable proteins were identified using LTQ-FT mass spectrometer, among which 91 proteins were up-regulated and 61 proteins were down-regulated in high metastatic cell line. Differential proteins were mainly localized in plasma membrane, mitochondrion and nucleus, suggesting the necessarity of subcellular proteome analysis. Most of the up-regulated proteins were involved in adherence, morphogenesis and lipid synthesis, while lots of the down-regulated proteins were involved in electron transport. These changes might be crucial for HCC metastasis. Besides the proteins reported to be associated with HCC metastasis, a series of important proteins were found to be dysregualted in our model. Syntenin-1, anterior gradient protein 2 homolog, estradiol 17-beta-dehydrogenase 12, protein disulfide-isomerase A4, solute carrier family 12 member 2, etc. were up-regulated, and cellular retinoic acid-binding protein 2, heme oxygenase 1, similar to SMT3 suppressor of mif two 3 homolog 2, estradiol 17-beta-dehydrogenase 2, etc. were down-regulated in high metastatic HCC cell. Six dysregulated proteins were validated by Western blotting in the cell lines. Interestingly, both solute carrier family 12 member 2 and protein disulfide-isomerase A4 were also elevated in high metastatic HCC patients'sera, which is well consistent with the SILAC results.Plasma membrane plays critical roles in cell adherence, morphogenesis, signal transduction and material transport. Plasma membrane proteins are associated with multiple steps of metastasis process, such as breakaway from primary site, adhesion to extracellular matrix, infiltration into blood and lymphatic vessels, cell migriation, and lodgment to target organs. Because of the accessibility, plasma membrane proteins constitute the major targets for protein-based drug targets. Some of them, such as carcino-embryonic antigen (CEA), can shed into body fluids and be used as biomarkers. Despite their importance in cancer research, plasma membrane proteins are usually challenging to study because they are difficult to isolate or enrich. The colloidal silica pellicle tehchnique is based upon the ionic interaction of the cell surface with a positively-charged solid support. After homogenation, the coated plasma membrane can be enriched by density gradient centrifugation due to the high density. We systematically evaluated the purity and efficiency by transmission electron microscopy, Western Blotting, and protein yield. Contamination from other cellular components was less and the enrichment of plasma membrane was significant.In this study, we coupled colloidal silica pellicle tehchnique and SILAC to quantitatively analyze the plasma membrane proteins of HCC cell lines with low and high metastatic potentials (MHCC97L and MHCC97H). Among the 1,268 identified proteins, 257 proteins have plasma membrane localization according to GO, IPA and Uniprot annotations, while some other proteins have also been reported with plasma membrane localization, such as heat shock proteins, protein disulfide-isomerases and histons. There were 42 plasma membrane dysregulated proteins. Most of them were involved in material transport and signal transduction, and lots of adhesion molecules and cytoskeleton associated proteins were up-regulated. Besides the known HCC metastasis related molecules, the alterations of an additional series of important signaling molecules were detected in this study. For example, multidrug resistance-associated protein 1 and 3, contributing to the low drug sensitivity, were up-regulated in high metastatic cell line. Sodium bicarbonate cotransporter 3 and solute carrier family 12 member 2, participating in the reabsorption of Na+, were both up-regulated and might be related to tumor cell proliferation, migration, adhesion, secretion, and invasion. CD109, negative modulating factor of TGF-β1 signaling, was upregulated and might facilitate cell proliferation. Four dysregulated proteins without HCC metastasis-related reports, including solute carrier family 12 member 2, CD109, myristoylated alanine-rich C-kinase substrate and syntaxin-4, were validated and confirmed by Western Blotting in cell lines and patients'sera.The quantitative data of whole-cell proteins, plasma membrane proteins and secretory proteins were compared. The results indicated that HCC metastasis might be accosicated with cell adherence and morphogenesis, mitochondria deterioration, p53 inhibition, Na+ reabsorption, lipid synthesis, cell proliferation, matrix metalloproteinase synthesis and transport and chemical resistance.In conclusion, SILAC technique was employed to quantative proteome analyses (whole-cell proteins and plasma membrane proteins) of high and low metastatic HCC cell lines. Many reliable differential proteins were identified. Our study provided not only the valuable insights into the HCC metastasis mechanisms, but also two potential candidate biomarkers for prediction of HCC metastasis with clinical prospects.