The Putative Role Of MTOR/4E-BP1 Signal Pathway In The Carcinogenesis And Progression Of Gastric Cardiac Adenocarcinoma

Objective: Gastric cancer is one of the most common malignancies worldwide and ranks as the second leading cause of cancer-related death. During the past a few decades, the incidence of distal gastric carcinomas has decreased obviously, while adenocarcinoma of the oesophago-gastric junction, including adenocarcinomas of gastric cardia and distal oesophagus have shown a dramatic increase in Western countries. Simliar changing tendency in the incidence of gastric carcinoma at different subsites has also been found in some high risk areas of esophageal and gastric cancer in our country, but the reason is not clear enough. As we all know, that recent years have witnessed a variety of different studies on gastric cancer, yet studies only concentrating on the gastric cardiac adenocarcinoma have been relatively fewer. As previous papers showed that gastric cardiac adenocarcinoma may have some differences as compared with distal gastric carcinoma and esophagal carcinoma, the carcinogenesis and development of adenocarcinoma of gastric cardia and the possible difference from the adenocarcinoma of other subsites of stomach need to be further studied.One of the most clinically important molecular signaling networks quite frequently studied over the past decade is the mammalian target of rapamycin (mTOR) pathway. mTOR pathway is a critical nutritional and cellular energy checkpoint sensor and regulator of cell growth in mammalian cells. mTOR was originally identified as the target of the macrolide antibiotic rapamycin. It is a Ser/Thr protein kinase that mediates nutrient-dependent intracellular signalling related to cell growth, proliferation, and differentiation. Eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) is the first downstream substrate of mTOR, which is a small molecular protein. eIF4E (eukaryotic translation initiation factor 4E) is an oncogene encoding a cap binding protein, the most important translation initiator. eIF4E can specifically identify the mRNA 5'-end cap (m7 GPPPN, where N is any nucleotide and m is a methylgroup) structure, and initiate translation, so it is a very important regulative spot of translation in eukaryocyte. 4E-BP1 is a suppressor of eIF4E activity. It exerts the inhibitory activity by binding to eIF4E and preventing the assembly of the translation preinitiation complex. Thus, once 4E-BP1 is phosphorylated, it dissociates from eIF4E, hence permitting cap-dependent translation to take place. Under the stimulation of hormones, mitogens as well as other related factors, mTOR modulates the activity of eIF4E by regulating the phosphorylation of 4E-BP1. Activation of mTOR leads to the phosphorylation of 4E-BP1, resulting in the dissociating 4E-BP1 from the mRNA cap binding protein eIF4E and promotion of protein synthesis. In contrast, hypophosphorylated 4E-BP1 inhibits cap-dependent translation.As activation of mTOR growth pathway was seen in many malignant tumors, mTOR has been suggestted as an attractive molecular target for cancer therapy. Several studies showed that activation of mTOR signaling pathway and overexpression of mTOR was quite common in gastric cancers. Treatment strategies with the specific inhibitor of mTOR, Everolimus, has shown to be high efficacy and safety in the gastric cancer in a phaseⅡstudies, and the global phaseⅢstudy is under preparation. mTOR signaling pathway has become a new target for gastric cancer therapy. But the role of mTOR signaling pathway in different subsites of gastric cancer has not been reported.The aim of this study is to evaluate the activation of the mTOR/4E-BP1 signal transduction pathway in gastric cardiac adenocarcinoma, which is not commonly studied in the past, and to explore the putative role of mTOR/4E-BP1 signal transduction pathway in the carcinogenesis and progression of cardiac adenocarcinomas.Methods:1 CasesSpecimens of carcinoma tissue and corresponding normal gastric mucosa adjacent to carcinoma were taken from 33 patients underwent surgical resection for gastric cardiac adenocarcinoma. The specimens were freshly taken immediately after resection and kept in -80℃refrigerator. The pathlogical natures of all the specimens are confirmed by pathological examination with HE Staining. No radiotherapy or chemotherapy was used before surgery in all cases.2 Western Blot analysis of the expression and phosphorylation of mTOR, 4E-BP1 and eIF4E Protein2.1 Extraction and quantification of total protein of tissuesTotal protein was extracted from the tissues using total protein lysate. Ultraviolet spectrophotometer was employed for quantitation.2.2 Western BlotThe expression of mTOR, 4E-BP1, eIF4E and the level of p-mTOR, p-4E-BP1, p-eIF4E in gastric cardiac adenocarcinoma and normal gastric mucosa at protein level was detected by Western blotting.3 Reverse transcription PCR (RT-PCR) analysis of the expression of mTOR, 4E-BP1 and eIF4E at mRNA level3.1 Extraction and quantitation of the total RNATotal RNA was abstracted by single-step method with guanidinium isothiocyanate. The integrity of the total RNA was identified by 1% agarose electrophoresis. The quantitation is achieved by ultraviolet spectrophotometer.3.2 Reverse transcription PCR (RT-PCR)The expression of mTOR, 4E-BP1 and eIF4E in gastric cardiac adenocarcinoma and normal gastric mucosa at mRNA level was determined using RT-PCR method. The relative expression was calculated as the ratio between the density of target gene and that of GAPDH by BIO-LD densitometric image analyzer.4 StatisticsDatas from these studies were analyzed by Paired-samples t-test and independent samples t-test. The SPSS 13.0 was employed for all calculations. The p-values less than 0.05 were considered to be significant. Results:1 The relative expression of mTOR, 4E-BP1, eIF4E and the level of p-mTOR, p-4E-BP1, p-eIF4E in gastric cardiac adenocarcinoma and normal gastric mucosa at protein level1.1 The expression of mTOR in adenocarcinoma of gastric cardia and corresponding normal gastric moucosa at protein level1.1.1 The results of the expression of mTOR protein detected by Western blottingThe molecular weight of mTOR protein is 289KD. After SDS-PAGE electrophoresis and Western blotting, positive bands were found in lanes. Image analysis system was used for quantitative analysis of the hybrid bands withβ-actin (42KD) as internal reference. The optical density of mTOR protein was 0.296±0.08 in adenocarcinoma of gastric cardia and 1.3476±0.36 in corresponding normal gastric moucosa. Statistic results showed that the expression of mTOR protein in adenocarcinoma of gastric cardia was significantly lower than that in corresponding normal gastric moucosa (P<0.05).1.1.2 The clinical pathological significance of mTOR protein expression in adenocarcinoma of gastric cardiaThe expression of mTOR protein in adenocarcinom of gastric cardia was not related with age, sex, differentiation, tumor size, infiltrating depth and lymph node metastasis (P>0.05).1.2 The phosphoration of mTOR in adenocarcinoma of gastric cardia and corresponding normal tissues1.2.1 The level of phospho-mTOR protein detected by Western blottingThe molecular weight of phospho-mTOR protein is 289KD. SDS-PAGE electrophoresis and Western blotting showed that the optical density of phospho-mTOR protein was 0.348±0.09 in adenocarcinoma of gastric cardia and 0.475±0.11 in corresponding normal gastric moucosa respectively. Though no significant difference in the optical density of phospho-mTOR protein was found between cardiac adenocarcinoma and normal gastric mucosa (P>0.05), the p-mTOR to mTOR ratio in cardiac adenocarcinoma (1.152±0.32) was significantly higher than that in normal gastric mucosa (0.292±0.07, p<0.05), suggestting higher activation of mTOR in cardiac adenocarcinoma.1.2.2 The clinical pathological significance of the ratio of p-mTOR to mTOR in adenocarcinoma of gastric cardiaThe ratio of p-mTOR to mTOR in gastric cardiac adenocarcinom was not related with age, sex, differentiation, tumor size, infiltrating depth and lymph node metastasis (P>0.05).1.3 The expression of 4E-BP1 protein in gastric cardiac adenocarcinoma and corresponding normal mucosa at protein level1.3.1 The expression of 4E-BP1 protein detected by Western blottingThe molecular weight of 4E-BP1 protein is 15KD. SDS-PAGE electrophoresis and Western blotting results showed that the optical density of 4E-BP1 protein was 2.210±0.41 in cardiac adenocarcinoma, which was significantly decreased than that in the corresponding normal gastric mucosa (4.498±0.76, P<0.05).1.3.2 The clinical pathological significance of 4E-BP1 protein expression in gastric cardiac adenocarcinomaThe expression of 4E-BP1 protein in cardiac adenocarcinoma was not related with age, sex, differentiation, tumor size, infiltrating depth and lymph node metastasis (P>0.05).1.4 The phospho-4E-BP1 in gastric cardiac adenocarcinoma and corresponding normal mucosa at protein levelThe molecular weight of 4E-BP1 protein is 15KD. SDS-PAGE electrophoresis and Western blotting results showed that the optical density of 4E-BP1 protein was 2.210±0.41 in cardiac adenocarcinoma tissues, which was significantly decreased than that in the corresponding normal gastric mucosa tissues (4.498±0.76, P<0.05).1.4.1 The level of phospho-4E-BP1 protein detected by Western blottingThe molecular weight of phospho-4E-BP1 protein is 15KD. SDS-PAGE electrophoresis and Western blotting results showed that the optical density of phospho-4E-BP1 protein was 2.165±0.40 in cardiac adenocarcinoma, which was dramatically increased than that in corresponding normal gastric mucosa(1.184±0.23, P<0.05).1.4.2 The clinical pathological significance of the level of phospho-4E-BP1 in gastric cardiac adenocarcinomaThe level of phospho-4E-BP1 protein in cardiac adenocarcinom was not related with age, sex, differentiation, tumor size, infiltrating depth and lymph node metastasis (P>0.05).1.5 The expression of eIF4E in gastric cardiac adenocarcinoma and corresponding normal mucosa at protein level1.5.1 The expression of eIF4E protein detected by Western blottingThe molecular weight of eIF4E protein is 25KD. SDS-PAGE electrophoresis and Western blotting results showed that the optical density of eIF4E protein was 2.194±0.43 in cardiac adenocarcinoma, which had no significant difference in comparision with that in corresponding normal gastric mucosa (2.033±0.35, P>0.05).1.5.2 The clinical pathological significance of eIF4E protein expression in gastric cardiac adenocarcinoma The expression of eIF4E protein in cardiac adenocarcinoma was not related with age, sex, differentiation, tumor size, infiltrating depth and lymph node metastasis (P>0.05).1.6 The phospho-eIF4E in gastric cardiac adenocarcinoma and corresponding normal mucosa at protein level1.6.1 The level of phospho-eIF4E protein detected by Western blotting The molecular weight of phospho-eIF4E protein is 25KD. SDS-PAGE electrophoresis and Western blotting results showed that the optical density of phospho-eIF4E protein was 1.822±0.55 in cardiac adenocarcinoma, which was markedly increased than that in corresponding normal gastric mucosa(0.997±0.21, P<0.05).1.6.2 The clinical pathological significance of the level of phospho-eIF4E protein in gastric cardiac adenocarcinoma The level of phospho-eIF4E in cardiac adenocarcinoma was closely related with lymph node metastasis. The phosphorylation of eIF4E in lymph node metastasis group was dramatically higher compared with that in non-lymph node metastasis group (p<0.05). No relationship between the level of phospho-eIF4E in cardiac adenocarcinoma and the age, sex, differentiation, tumor size, infiltrating depth was found (P>0.05).2 The relative expression of mTOR, 4E-BP1, eIF4E in gastric cardiac adenocarcinoma and corresponding normal tissues at mRNA level2.1 The expression of mTOR mRNA detected by semi-quantitative RT-PCR in gastric cardiac adenocarcinoma and corresponding normal gastric mucosa RT-PCR products were detected by 1.5% agarose gelelectrophoresis and analyzed by BIO-LD. The optical density of mTOR mRNA was 6.41±1.27 in tumor tissues and 4.39±1.12 in normal tissues. Statistic results showed that the mTOR mRNA expression in tumor tissues was obviously higher than that in the normal tissues (P<0.05).2.2 The expression of 4E-BP1 mRNA detected by semi-quantitative RT-PCR in gastric cardiac adenocarcinoma and corresponding normal gastric mucosa RT-PCR products were detected by 1.5% agarose gelelectrophoresis and analyzed by BIO-LD. The optical density of 4E-BP1 mRNA was 4.39±1.70 in tumor tissues, which was significantly lower than that in the normal control group (7.12±2.66, P<0.05).2.3 The expression of eIF4E mRNA detected by semi-quantitative RT-PCR in gastric cardiac adenocarcinoma and corresponding normal gastric mucosa RT-PCR products were detected by 1.5% agarose gelelectrophoresis and analyzed by BIO-LD. The optical density of eIF4E mRNA was 7.56±1.60 in tumor tissues, which was obviously higher than that in the normal control group (1.60±0.78, P<0.05).Conclusions:1 The expression of mTOR in cardiac adenocarcinoma at protein level was significantly lower than that in the corresponding normal gastric mucosa, but the p-mTOR/mTOR ratio was significantly increased in cardiac adenocarcinoma, suggestting that the activation of mTOR may play important role in the carcinogenesis of cardiac adenocarcinoma and the activation may be mainly attribute to the increased mTOR phosphorylation rather than protein overexpression.2 In comparison with that in their corresponding normal gastric mucosa, the expressions of 4E-BP1 protein were significantly decreased while phospho-4E-BP1 and phospho-eIF4E levels were dramaticly increased in cardiac adenocarcinoma. The phosphoration level of eIF4E was closely related with lymph node metastasis, suggesting that activation of 4E-BP1 and eIF4E may play important roles in the carcinogenesis and progression of cardiac adenocarcinoma.3 mTOR/4E-BP1 signal pathway was activated in adenocarcinoma of gastric cardia and may become a potential therapy target for the treatment of cardiac adenocarcinoma.