The low efficiency of current HCC therapy urges the identification of tumor-specific markers as potential therapeutic targets, and for the early detection of the disease. As consequence of the strong association between HCV infection, chronic liver disease, and progression to HCC, this high risk group population can be monitored on a regular basisto detect early cancerous lesions. Detection and diagnosis of HCC an early stage may significantly improve the survival of patients. Currently used methods for detection of liver tumors, however, rely largely on radiographic imaging techniques that are not practical for population-based screening. Thus, considerable effort has been expended toward identification of practical approaches for noninvasive detection of HCC. The ideal biomarker for this type of applicationis one that can be detected with good sensitivity in a biological sample from the patient in a noninvasive manner (e.g., blood, urine). For HCC, blood representsthe best source for detection of cancer-related biomarkers. However, despite an increasing number of noninvasive tests and imaging techniques, detection of liver cirrhosis and hepatocellular cancer is often difficult in chronic hepatitis C infected patients (6
Recent technological advances in transcriptomics and proteomics make it possible to examine expression profiles at the mRNA and protein level. Such approaches are expected to establish the molecular definition of the non-tumor and cancer states of each patient and contribute to the discovery of diagnostic markers and therapeutic targets.
The present study therefore aimed at the identification of plasma protein patterns and single protein markers to differentiate HCV-liver cirrhosis and HCV-hepatocellular carcinoma for monitoring high risk HCV-cirrhotic patients awaiting liver transplantation.
Studying the protein profiles in plasma samples of HCV-cirrhotic patients with and without HCC (HCV-HCC samples included different TNM stages), we were able to identify a panel of differentially expressed proteins. Specifically, when plasma samples from HCV-cirrhotic patients were compared to HCV-HCC samples, seven proteins were significantly differentially expressed between groups (q-Value<0.05).
Precursors of Apolipoprotein D, Apolipoprotein C-III, Apolipoprotein C-IV, Apolipoprotein C-I, and Apolipoprotein C-II were identified under expressed in HCV-HCC samples when compared with HCV-cirrhosis (FDR<10%). Most plasma apolipoproteins, endogenous lipids and lipoproteins are synthesized in the liver (21
), which depends on the integrity of cellular functions of liver (22
). Under normal physiological conditions, liver ensures homeostasis of lipid and lipoprotein metabolism (24
). Hepatic cellular damage and HCC impairs these processes, leading to alterations in plasma lipid and lipoprotein patterns.
Apolipoprotein C-I has been identified as a marker to differentiate between liver fibrosis and cirrhosis. A role for apolipoproteins in liver fibrosis has been previously published (25
). Moreover, apolipoproteins have not only been identified as a serum discriminator of fibrosis but also as a marker in different types of cancer (26
). Specifically, apolipoprotein C-I down-regulation was detected to reliably distinguish colorectal cancer patients from healthy controls (27
). Thus, besides the function of apolipoprotein C-I in lipid metabolism (28
) an additional pathogenic role in liver fibrosis and cancerogenesis appears possible (29
It was of great interest to evaluate differences in protein expression between HCV-cirrhotic and early HCV-HCC samples. Interestingly, 18 proteins were statistically differentially expressed (FDR<5%) between these groups. Galectin-4 and Interleukin-27 were some of the proteins over expressed in the HCV-cirrhotic samples. Galectins are a family of β-galactoside-binding lectins with related amino acid sequences. They are soluble proteins and are generally localized in the cytosol. However, they can accumulate on the cell surface under certain conditions to play an important role in cell-cell and cell-matrix interactions. Galectin-1 mRNA expression is elevated in fibrosarcoma cells (32
) and squamous cell carcinoma (33
), whereas Galectin-3 is overexpressed in thyroid tumors (34
), colon cancer (35
), and squamous cell carcinoma (33
). Kondoh et al. (36
) reported the association between Galectin-4 and HCC. In contrast to their results in liver, Galectin-4 expression was reported to be down-regulated in colon carcinoma (37
), suggesting that the tissue-specific background may be important in determining the functional significanceof this molecule for tumor development.
IL-27 is a heterodimeric cytokine that consists of an EBV-transformed gene 3 (EBI3), an IL-12p40-related protein, and p28, a newly discovered IL-12p35-relatedpolypeptide. The former subunit was originally described as a factor secreted by EBI3, while the latter was identified through its homology to the IL-6/IL-12 cytokine family. IL-27 is produced early by activated APCs and induces a rapid clonal expansion of naive but not memory CD4+
T cells and synergizes with IL-12to trigger IFN-γ production in naive CD4+
T cells (38
). IL-27Rconsists of WSX-1 (also known as T cell cytokine receptor) and gp130 subunits (39
). Researchers have recently demonstrated that IL-27 has a potent ability to induce tumor-specific anti-tumor and protective immunity using colon carcinoma colon 26 (C26) (40
) and TBJ neuroblastoma (42
). Proteins of the complement system (C1r, C7) were also over expressed in cirrhotic HCV samples were compared to HCV-HCC while apolipoptotein precursors were down expressed.
Transferrin (Protein ID: 2815575) was also statistically significant down expressed in HCV-HCC plasma samples. The function of this encoded protein is to transport iron from the intestine, reticuloendothelial system, and liver parenchymal cells to all proliferating cells in the body. Perturbations in iron metabolism have been described in HCC (30
). However, the molecular mechanisms underlying the iron perturbations seen in hepatic malignant cells remain poorly understood. Interesting, transferrin was one of the protein identified when a forward variable selection strategy was applied to assess the diagnostic utility of the identified proteins. Moreover, using a second independent method, we were able to correlate the values of transferrin with our proteomic results.
To date, both proteomic and genomic studies have highlighted the heterogeneity of HCC. This heterogeneity emphasizes the complex nature of HCC carcinogenesis and disease progression in which multiple pathogenesis mechanisms seem to be involved. Diagnostic and prognostic molecular markers are being identified by transcriptomic and proteomic analysis of HCC today. However, many of these analyses have been performed on HCC in general, and the studied tissues were HCV infected, HBV infected, infected with both or neither, or the infection status may be unknown (43
). Because the HCC etiology might have an important effect in the proteomic profiles, our study was limited to samples from HCV-HCC patients.
Our findings provide additional confirmation thata proteomic approach can accurately identify HCC inpatients with cirrhosis. We also showed a different pattern of proteins between HCV-cirrhosis and early HCV-HCC. This might have important implications for its utility in screeningfor HCC in high risk patients. Evaluations using HCC cells lines might allow functional studies to determine the effects of modulating the expression or activity of the differentially expressed proteins identified in the present study. Based on the identification of differential proteins in HCC cells, these investigations might provide valuable information to recognize changes in cellular pathways that might participate in the development and maintenance of the transformed phenotype (46
). However, HCC cell lines are relatively homogeneous systems when compared with liver cancer tissue which is composed of multiple cell subpopulations. Further validation studies of large cohorts of patients will be the ultimate step toward clinical use.