Protein Signatures for Classification and Prognosis of Gastric Cancer

Abstract

Current methods have limited accuracy in predicting survival and stratifying patients with gastric cancer for appropriate treatment. We sought to identify protein signatures of gastric cancer for classification and prognostication. The Protein Pathway Array (initial study) and Western blot (confirmation) were used to assess the protein expression in a total of 199 fresh frozen gastric samples. There were 56 paired samples divided into a training set (n ‫؍‬ 37) and a validation set (n ‫؍‬ 19) for the identification of differentially expressed proteins between tumor and normal tissues. There were 56 tumor samples used to identify proteins correlating with tumor and nodal staging. All 93 tumor samples were used to identify candidate proteins for predicting survival. We confirmed the survival prediction of the candidate proteins by using an additional cohort of gastric cancer samples (n ‫؍‬ 50). There were 22 proteins differentially expressed between normal and tumor tissues. Nine proteins were selected to build the predictor to classify normal and tumor samples. Ten proteins were differentially expressed among different T stages and four of these were associated with invasive behavior. An additional four proteins were associated with lymph node metastasis. Two proteins were identified as independent risk factors for overall survival. This study indicated that some dysregulated signaling proteins could be selected Gastric cancer is the fourth most common malignancy and ranked as the second leading cause of cancer death worldwide.1 The geographic distribution of incidence and mortality of gastric cancer varies remarkably worldwide. Areas with high incidence include Japan, Korea, China, Eastern Europe, and parts of Latin America. The mortality of gastric cancer has declined in past decades, mainly due to early detection by gastric endoscopy. 2 However, unlike that of other common cancers, the prognosis for most gastric cancer is poor and has improved little for the past several decades. Despite recent advances in chemotherapy and surgical techniques, the overall 5-year survival rate is lower than 40%.3 Perplexingly, the prognosis varies widely in patients with stage II or III disease for undetermined biological reasons.

Protein predictive signatures for lymph node metastasis of gastric cancer

“…10 Briefly, 1 ml of 1Â lysis buffer (Cell Signaling Technology, Danvers, MA) with 1Â protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and 1Â phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN) was added to each tissue sample and the lysate was sonicated twice for 15 sec each time on ice, and then centrifuged at 14,000 rpm for 30 min at 4 C. The protein concentration was determined using BCA Protein Assay kit (PIERCE, Rockford, IL). Three hundred lg of protein lysate was loaded in one well across the entire width of a 10% SDS polyacrylamide gel and separated by electrophoresis as described previously.…”

“…The backgroundsubtracted intensity was normalized by ''global median subtraction'' method to reduce variation among different experiments, that is, the intensity of each protein from each sample divided by total intensities of all proteins from the same sample and then multiplied by average intensities of all proteins in all samples. 10 …”

“…Three hundred lg of protein lysate was loaded in one well across the entire width of a 10% SDS polyacrylamide gel and separated by electrophoresis as described previously. 10,13 After electrophoresis, the proteins were transferred electrophoretically to a nitrocellulose membrane (Bio-Rad, Hercules, CA), which was then blocked for 1 hr with blocking buffer consisting of 3% BSA in 1Â TBST containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.1% Tween-20. Next, the membrane was clamped on a Western blotting manifold (Mini-PROTEAN II Multiscreen apparatus, Bio-Rad, Hercules, CA) which isolates 20 channels across the membrane.…”

“…[9][10][11][12][13] PPA is a recently developed highthroughput protein assay, which combines multiplex immunoblot with computational analysis. We were able to identify several differentially expressed proteins which are associated with vascular/lymphatic invasion and lymph node metastasis.…”

Regulation of a Novel Androgen Receptor Target Gene, the Cyclin B1 Gene, through Androgen-Dependent E2F Family Member Switching

“…Total cellular proteins were extracted from PShTert or PShTertAR cells grown in androgen-free or androgen-supplemented (10 nM R1881) medium, using a lysis buffer containing 20 mmol/liter Tris-HCl (pH 7.5), 20 mmol/liter sodium pyrophosphate, 40 mmol/liter ␤-glycerophosphate, 30 mmol/liter sodium fluoride, 2 mmol/liter EGTA, 10 mmol/liter NaCl, and 0.5% NP-40. PPAA was performed as described previously (35,36,39,41). Chemiluminescence signals were captured using the ChemiDoc XRS system.…”