ABSTRACT
Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen(®), and AMMP(®)) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253.
Subject(s)
Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Liver Cirrhosis/genetics , Liver Neoplasms/genetics , Neoplasm Proteins/genetics , Phosphoproteins/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Aged , Aged, 80 and over , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Calnexin/genetics , Calnexin/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Dipeptidases/genetics , Dipeptidases/metabolism , Endoplasmic Reticulum Chaperone BiP , Female , Gene Expression Profiling , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Liver/metabolism , Liver/pathology , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Middle Aged , Molecular Sequence Annotation , Neoplasm Proteins/metabolism , Phosphoproteins/metabolism , Phosphorylation , Procollagen-Proline Dioxygenase/genetics , Procollagen-Proline Dioxygenase/metabolism , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , Proteomics/methods , Signal Transduction , Thioredoxins/genetics , Thioredoxins/metabolism , Valosin Containing ProteinABSTRACT
The acoustic membrane micro particle (AMMP) technology has been used to quantify single analytes out of multiple sample types. In this study the technology is used to reveal molecular interactions of components of kinase pathways. Specifically, the downstream kinase activity of the EGFR receptor in the presence or absence of EGFR inhibitors is investigated. These experiments substantiate that EGFR stimulation predominantly activates the MEK/ERK pathway. The EGFR inhibitors tested had varying effectiveness at preventing phosphorylation at the EGFR or downstream kinase activity. These experiments reveal the use of the AMMP technology for observing multiple signaling pathways in a single experiment.
