Proteome Analysis Of Cell Membrane And Serum From Hepatocellular Carcinoma Model System With Multiple Metastatic Potentials

Hepatocellular carcinoma (HCC), accounting for an estimated 600,000 deaths annually, is the third leading cause of cancer-related mortality worldwide. Most cases occur in Asia and sub-Saharan Africa, however, the incidence is also expected to double over the next 10 to 20 years in the West, possibly due to the increased HCV infection. While curative therapies are possible if the lesion remains early and localized, almost 70% of resected cases recurred within 5 years. Impressive progression has been made in providing an increasingly comprehensive portrayal of HCC. However, biomarkers that indicate the risk of invasion and metastatic potential of HCC and can be widely used in clinical settings are not currently available.For a better insight into the characteristic of HCC metastasis, the stepwise metastatic human HCC cells MHCC97L and HCCLM9, with low and high metastatic potentials, were established via repeated in vivo selection and characterized by a similar genetic background but with significant differences in spontaneous metastasis behavior, providing appropriate model systems for comparative study on the molecular events correlated with HCC metastasis.Plasma membrane, the structure surrounding all living cells and acting as the primary interface between the cellular contents and the extracellular environment, plays crucial roles in cell functions. Membrane proteins and other components maintain cell structure, motility and recognition involved in receptor-binding and further transport of bound components into the cell, cell-cell and cell-matrix interactions, and the organization of the cytoskeleton. The composition and characteristics of membrane proteins of tumor cells are modified during malignant transformation and make them likely candidates for cancer biomarkers.Serum and tissue are optimal sources for discovery and analysis of cancer biomarker. Metastasis is the result of cancer cell adaptation to a tissue microenvironment at a distance from the primary tumor. Proteins derived from diseased tissue compartments may leak into lymph and blood, eventually becoming detectable in serum and tissues and representing potential biomarkers of disease.The current work was firstly to identify potential proteins from cell membrane and serum related to HCC invasive progression, using human HCC cells with different metastasis potentials, by proteomics analysis, experimental animal studies and clinical validation.Part One:Proteome analysis of cell membrane from hepatocellular carcinoma model system with multiple metastatic potentialsObjective To search for hepatocellular carcinoma (HCC) invasion and metastasis related biomarkers using the cell membrane proteomics approaches, and to validate the markers using experimental and clinical specimens. Methods HCCLM9 and MHCC97L cell, with a similar genetic background and remarkably different metastasis behaviors, were used for comparative membrane proteome profiling using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrospray ionization mass spectrometry (ESI-MS) technologies. Candidate protein makers were further validated by Western blot on cells, immunohistochemistry (IHC) on animal tumor tissues, and tissue micro-array on clinical specimens. Results Membrane proteins from MHCC97L and HCCLM9 cells were compared by SDS-PAGE analyses. A total of 14 proteins were identified by ESI-MS/MS among the differential bands. Coronin-1C was overexpressed in HCCLM9 (7.31±0.73) versus MHCC97L (2.84±0.99), and validated by western blot and IHC from both nude mice tumor tissues and clinical specimens. Elevated coronin-1C expression was observed in liver cancer tissues of HCCLM9 nude mice. IHC study in 115 human HCC specimens demonstrated that patients with higher coronin-1C expession showed greater invasiveness and more advanced stage. Conclusion The study suggests coronin-1C could be a candidate biomarker to predict HCC invasive behavior.Part One:Proteome analysis of serum from hepatocellular carcinoma model system with multiple metastatic potentialsObjective Serum proteomics approaches were applied to search for and validate hepatocellular carcinoma (HCC) invasion and metastasis related biomarkers. Methods 16 male athymic BALB/c nu/nu mice were randomly divided into two groups. HCCLM9-and MHCC97L-nude mice models of human HCC, with a similar genetic background and remarkably different pulmonary metastasis potential, were established.2D gel electrophoresis (2-DE) and MALDI-TOF/TOF technologies were used to construct a comparative proteome profile of nude mice sera from HCCLM9-and MHCC97L-nude mice. Candidate protein was confirmed by western blot and measured in sera from tumor development of spontaneous pulmonary metastasis in HCCLM9-nude mice. This result was further was analyzed by immunohistochemistry (IHC) on animal tumor tissues, and tissue micro-array on clinical specimens including 170 male cases and 38 female cases. Results Serum proteins from HCCLM9-and MHCC97L-nude mice were compared by 2-DE. A total of 11 proteins were identified by MALDI-TOF/TOF. Complement factor H (CFH) was overexpressed in HCCLM9-(213.83±55.17) versus MHCC97L-nude mice serum (122.48±48.91) by western blot, and validated by IHC from both nude mice tumor tissues and clinical specimens. Serum CFH level showed a remarkable upsurge when pulmonary metastasis occurred. Elevated CFH expression was observed in liver cancer tissues of HCCLM9 nude mice. IHC study in 208 human HCC specimens including demonstrated that patients with higher CFH expession showed greater invasiveness and more advanced stage. Conclusion The study suggests CFH could be a candidate biomarker to predict HCC invasive behavior.