RESUMEN
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.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Cirrosis Hepática/genética , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Calnexina/genética , Calnexina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dipeptidasas/genética , Dipeptidasas/metabolismo , Chaperón BiP del Retículo Endoplásmico , Femenino , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Persona de Mediana Edad , Anotación de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Proteómica/métodos , Transducción de Señal , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteína que Contiene ValosinaRESUMEN
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.