ABSTRACT
Due to its increasing incidence and relatively poor prognosis, esophageal adenocarcinoma (EAC) is becoming a significant health problem. Elucidating the mechanisms underlying EAC development is of great importance to improve upon current conventional treatment strategies. Insight into phosphorylation has proven to be useful for the development of diagnostic and molecular treatment strategies in cancer. A pathway largely dependent on phosphorylation and frequently deregulated in cancer is the cell cycle regulating p16-retinoblastoma (Rb) pathway. We investigated kinase activity, specifically phosphorylation within the p16-Rb pathway, in EAC. A high-throughput peptide tyrosine kinase array containing short peptides representing 100 proteins with known phosphorylation sites, was used to assess phosphorylation activity in EAC. Also, specific phosphorylation changes of the cell cycle protein Rb and its upstream regulator P16 were validated through immunoblotting in EAC and normal esophageal cells and tissues. Phosphorylation activity was higher in EAC tissues as compared to normal squamous esophageal tissues. A majority of the proteins significantly higher phosphorylated in EAC were found to be involved in cell structure maintenance and immunity. Validation of Rb phosphorylation in EAC biopsy specimens and cell lines showed hyper phosphorylation of Rb associated with aberrant P16 expression in the cancer tissues. The specific Rb (S795) residue was significantly higher phosphorylated in EAC compared to normal esophageal tissue (Wilcoxon paired rank test, p=0.004). Investigation of Rb (S795) phosphorylation may indicate targets for intervention and give more molecular insight in EAC.
Subject(s)
Adenocarcinoma/metabolism , Esophageal Neoplasms/metabolism , Neoplasm Proteins/metabolism , Protein Array Analysis/methods , Retinoblastoma Protein/metabolism , Serine/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Aged , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p16 , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Neoplasm Proteins/genetics , Phosphorylation , Retinoblastoma Protein/geneticsABSTRACT
Barrett's esophagus (BE) goes through a sequence of low grade dysplasia (LGD) and high grade dysplasia (HGD) to esophageal adenocarcinoma (EAC). The current gold standard for BE outcome prediction, histopathological staging, can be unreliable. TP53 abnormalities may serve as prognostic biomarkers. TP53 protein accumulation detected by immunohistochemistry (IHC) indirectly assesses TP53 mutations. DNA fluorescent in situ hybridization (FISH) on brush cytology specimens directly evaluates gene locus loss. We evaluated if IHC and FISH are complementary tools to assess TP53 abnormalities and tested their prognostic value in a long-term prospective follow-up of a BE cohort. TP53 IHC on tissue sections and FISH on brush cytology specimens were evaluated for 116 BE patients with respect to the different histological stages. The TP53 abnormalities were further studied in a panel of cell lines representative of the Barrett's carcinogenic sequence. For 91patients, the predictive value of TP53 abnormalities with respect to progression to HGD/EAC was tested after long term follow-up. The frequency of IHC and FISH TP53 abnormalities increased significantly with increasing histological stage (P < 0.001, Chi(2) -test). Combining the techniques detected TP53 abnormalities in 100% of patients with LGD, HGD, and EAC. Multivariate analysis showed that IHC (hazard ratio: 17, 95% CI: 3.2-96, P = 0.001) and FISH (hazard ratio: 7.3, 95% CI: 1.3-41, P = 0.02) were both independent significant predictors of progression. Combining FISH and IHC in assessing TP53 abnormalities leads to an increased detection rate of TP53 aberrations and improved accuracy for predicting BE progression.
