Elevated expression of ISY1, APOA-1, SYNE1, MTG1, and MMP10 at HCC initiation: HCC specific protein network involving interactions of key regulators of lipid metabolism, EGFR signaling, MAPK, and splicing pathways.

Identification of molecular regulators of hepatocellular carcinoma (HCC) initiation and progression is not well understood. We chemically induced HCC in male Wistar rats by administration of diethyl nitrosamine (DEN) and 2-acetylaminofluorene (2-AFF). Using 2D-electrophoresis and MALDI-TOF-MS/MS analyses, we characterized differentially expressed proteins in liver tissues at early stage of HCC progression. Using RT-PCR analysis, we quantified the mRNA expression of the characterized proteins and validated the transcript expression with tumor tissues of clinically confirmed HCC patients. Using bioinformatic tools, we analyzed a network among the introduced proteins that identified their interacting partners and analyzed the molecular mechanisms associated with signaling pathways during HCC progression. We characterized a protein, namely, pre-mRNA splicing factor 1 homolog (ISY1), which is upregulated at both transcriptome and proteome levels at HCC initiation, progression, and tumor stages. We analyzed the interacting partners of ISY1, namely, APOA-1, SYNE1, MMP10, and MTG1. Real-time PCR analysis confirmed elevated expression of APOA-1 mRNA at HCC initiation, progression, and tumor stages in animals undergoing tumorigenesis. The mRNA expression of the interacting partners was validated with tumor tissues of clinically confirmed liver cancer patients; the analysis revealed significant elevation in expression of transcripts. The transcriptome and proteome analyses complement each other and dysregulation in mRNA and protein expression of these regulators may play critical role in HCC initiation and progression.

Elevated expression of sepiapterin reductase, regulator of G protein signaling 1, hypothetical protein CXorf58 homolog, and zinc finger and BTB domain-containing protein 21 isoform X2 is associated with progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers associated with high mortality rate. Understanding of events leading to HCC pathophysiology is essential for its better management. We earlier reported development of a novel rodent model by administrating chemical carcinogens, DEN, and 2-AAF for study of HCC at very early stage. 2D-Electrophoresis analysis of total serum proteins identified several differentially expressed proteins in animals undergoing tumorigenesis. MALDI-TOF-MS/MS analyses were performed to characterize the differentially expressed proteins. Further real-time PCR analyses were taken place to quantify the transcript expression for the identified proteins at HCC initiation and tumor stages. Considering protein-protein interactions among the experimentally identified proteins and their interacting neighbors, a protein network has been analyzed that provided further insight into molecular events taking place during HCC development. Histological changes confirmed HCC initiation and hepatotumorigenesis at 1 and 4 months post carcinogen treatment, respectively. Four differentially expressed proteins were identified which were further characterized as regulator of G protein signaling 1 (RGS1), sepiapterin reductase (SPR), similar to zinc finger and BTB domain-containing protein 21 isoform X2 (ZNF295), and a hypothetical protein CXorf58 homolog. Quantification of transcripts for these proteins revealed elevation in their expression both at initiation and tumorigenesis stages. The study deciphers the regulatory role of these proteins during HCC progression.

Elevated expression of cellular SYNE1, MMP10, and GTPase1 and their regulatory role in hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy resulting in high mortality. HCC progression is associated with abnormal signal transduction that changes cell signaling pathways and ultimately leads to dysregulation of cell functions and uncontrolled cell proliferation. Present study was undertaken with the objective to identify differentially expressed proteins and quantify their transcript expression in the liver of HCC-bearing rats vis-à-vis controls and to decipher the network involving interaction of genes coding for the characterized proteins to an insight into mechanism of HCC tumorigenesis. 2D-Electrophoresis and MALDI-TOF-MS/MS were used to characterize differentially expressed proteins in DEN (diethylnitrosamine)-induced HCC tissue using the protocol reported by us earlier. Real-time PCR was performed to quantify the expression of transcripts for the identified proteins. GENEMANIA, an interacting network of genes coding for selected proteins, was deciphered that provided the functional role of these proteins in HCC progression. Upregulation of proteins SYNE1, MMP10, and MTG1 was observed. The mRNA quantification revealed elevated expression of their transcripts at HCC initiation, progression, and tumor stages. Network analysis showed the involvement of the genes coding for these proteins in dysregulation of signaling pathways during HCC development. The elevated expression of SYNE1, MMP10, and MTG1 suggests the role of these proteins as potential players in HCC progression and tumorigenesis.

Elevated Expression of Cytosolic Phospholipase A2 Delta Is Associated with Lipid Metabolism Dysregulation during Hepatocellular Carcinoma Progression.

OBJECTIVE: Liver cancer is the third rank amongst the common malignancies, causing maximum death in the patients diagnosed with cancers. Currently available biomarkers are not enough sensitive for early diagnosis of hepatocellular carcinoma (HCC). This makes difficult management of HCC. With the aim of finding new generation of proteomic-based biomarkers, the represented study was designed to characterize the differentially expressed proteins at different stages of HCC initiation and at progression. This could lead to find potential biomarkers for early detection of HCC. MATERIALS AND METHODS: In this experimental study, we report induction of HCC by administrating chemical carcinogens in male Wistar rats. Disease progression was monitored by histological evaluation. Serum proteomic analyses such as 2 dimensional (2D)-electrophoresis, MALDI-TOF-MS/MS and Western blot have been used to analyze and characterize the differentially expressed proteins during HCC development. RESULTS: HCC initiation and tumorigenesis were observed at one and four months post carcinogen treatment, respectively. One of the differentially-expressed proteins, namely, cytosolic phospholipase A2 delta was significantly up-regulated at very early stage of HCC development. Its expression continued to increase during cancer progression and hepatotumorigenesis stages. Its elevated expression has been confirmed by Western blot analysis. Consistent to this, analyses of the sera in the clinically confirmed liver cancer patients showed elevated expression of this protein, further validating our experimental results. CONCLUSION: This study suggests that elevation in the expression of cytosolic phospholipase A2 delta is associated with progression of HCC.

Elevated Expression of A-Raf and FA2H in Hepatocellular Carcinoma is Associated with Lipid Metabolism Dysregulation and Cancer Progression.

BACKGROUND: Identification of events leading to hepatocellular carcinoma (HCC) progression is essential for understanding its pathophysiology. The aims of this study are to identify and characterize differentially expressed proteins in serum of HCC-bearing rats and the corresponding controls during cancer initiation, progression and tumorigenesis. METHODS: Chemical carcinogens, N-Nitrosodiethylamine and 2-aminoacetylfluorine are administered to induce HCC to male Wistar rats. The 2D-Electrophoresis and PD-Quest analyses are performed to identify several differentially expressed proteins in serum of HCC-bearing animals. These proteins are further characterized by MALDI-TOF-MS/MS analyses. Using pathwaylinker a HCC-specific network is analyzed among the MALDITOF- MS/MS characterized proteins and their interactors. RESULTS: Carcinogen administration caused inflammation leading to liver injury and HCC development. Liver inflammation was confirmed by increase in the levels of TNF-α and IL-6 in carcinogen treated rats. We report significant increase in expression of two differentially expressed proteins, namely, A-Raf and Fatty Acid 2- Hydroxylase (FA2H), at early stage of HCC initiation, during its progression and at tumor stage. Real-time PCR analysis of mRNA for these proteins confirmed up-regulation of their transcripts. Further, we validated our experimental data with sera of clinically confirmed liver cancer patients. CONCLUSION: The study suggests that FA2H and A-Raf play a major role in the progression of HCC.

HCC Specific Protein Network Involving Interactions of EGFR with A-Raf and Transthyretin: Experimental Analysis and Computational Biology Correlates.

BACKGROUND: The network interactions link human disease proteins to regulatory cellular pathways leading to better understanding of protein functions and cellular processes. Revealing the network of signaling pathways in cancer through protein-protein interactions at molecular level enhances our understanding of Hepatocellular Carcinoma (HCC). OBJECTIVE: A rodent model for study of HCC was developed to identify differentially expressed proteins at very early stage of cancer initiation and throughout its progression. METHODOLOGY: HCC was induced by administrating N-Nitrosodiethylamine (DEN) and 2-aminoacetylfluorine (2-AAF) to male Wistar rats. Proteomic approaches such as 2D-Electrophoresis, PD-Quest, MALDI-TOF-MS and Western blot analyses have been used to identify, characterize and validate the differentially expressed proteins in HCC-bearing animals vis-a-vis controls. RESULTS: The step-wise analysis of morphological and histological parameters revealed HCC induction and tumorigenesis at 1 and 4 months after carcinogen treatment, respectively. We report a novel protein network of 735 different proteins out of which eight proteins are characterized by MALDI-TOF-MS analysis soon after HCC was chemically induced in rats. We have analyzed four different novel routes representing the association of experimentally identified proteins with HCC progression. CONCLUSION: The study suggests that A-Raf, transthyretin and epidermal growth factor receptor play major role in HCC progression by regulating MAPK signaling pathway and lipid metabolism leading to continuous proliferation, neoplastic transformation and tumorigenesis.

Novel mutations in transthyretin gene associated with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major health problems with increasing incidence worldwide. We report the elevation in transthyretin (TTR) expression following HCC induction using diethylnitrosamine (DEN) and 2-aminoacetylfluorine (2-AAF) in male Wistar rats. The increase in its expression took place at very early stage and remained elevated throughout HCC progression. The analysis of TTR gene in HCC bearing rats revealed four novel mutations that alter three amino acids at positions 61, 100, and 115. While these mutations do not directly affect the binding of TTR to thyroxine (T4 ), the mutation in amino acid 115 interferes with TTR tetramer formation that leads to its accumulation. Further, the mutated TTR is unable to cleave C-terminal of apolipoprotein A1 (APOA1) that results in abnormal lipid metabolism. This has correlation with development of liver cirrhosis and HCC. Furthermore, the mutated TTR seems to have potential as biomarker for early detection of HCC.