The Mechanisms Of Signal Transducer And Activator Of Transcription3and Its Target Gene On Biological Behaviour Of Human Esophageal Sqamous Cell Carcinoma

BackgroundThe development and progression of esophageal sqamous cell carcinoma(ESCC) is a multifactor and multistage process involving quite some genes abnormality.Many genes and proteins were involved in the carcinogenesis and progression of esophagus.However, the precise mechanisms that underlie the development and progression of ESCC are far from clear. Up to date, no biomarkers of ESCC have been proposed.Recent studies have revealed that several genes and molecules are involved in the origin and progression of esophageal cancer. Signal transducer and activator of transcription3(STAT3) is one of the transcription factors reported to play an important role in tumor cell growth, proliferation, apoptosis, and has been classified as an oncogene.STAT3signaling pathway is activated after receiving extra cellular signals such as epidermal growth factor, cytokines. Activated STAT3(phosphorylated-STAT3)enters cellular nucleus and binds to specific DNA promoter to regulate target genes transcription which control fundamental physiologic processes such as cell proliferation, aptosis and angiogenesis. In normal condition, activation of STAT3signaling pathway is temporary and is controlled accurately. But with various kinds of tumor genesis signal stimulation, STAT3presents in the cell nucleus permanently and regulates target genes transcription continuously being involved in malignant transformation of tumor cells, called constitutively activation of STAT3signaling pathway. STAT3signaling pathway could convert various kinds of oncogenic tyrosine kinases signals and promote tumor cell generation, invasion and metastasis by inducing transcriptional activation of downstream target genes like VEGF, cyclinDl, Bcl-xL and MUC1.Constitutive activation of STAT3has been documented in some kinds of tumors.Resent studies show blocking activation of STAT3signaling pathway could inhibit cell growth and induce apoptosis in many different kinds of tumors. Therefore only targeting one protein-STAT3is enough to oppose many different oncogenic or anti-oncogene in the upstream or downstream of STAT3in tumor cells. These findings place STAT3in a pivotal position in oncogenic tyrosine-kinase signaling, providing a compelling rationale for its suitability as molecular target for cancer therapy.So far, there are seldom reports about whether STAT3signaling pathway is constitutively activated in ESCC and whether activated STAT3signaling pathway panicipates in malignant transformation and progression of STAT3. To investigate the molecular mechanism of STAT3signaling pathway in ESCC, the correlation between STAT3signaling pathway and its target gene in patients with ESCC, and the prognostic significance of STAT3signaling pathway and its target gene in patients with ESCC, STAT3and its target gene VEGF, cyclinDl, Bcl-xL and MUC1were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC).We also evaluate the levels of STAT3and p-STAT3expression in ESCC specimens and paracancerous normal tissues by1HC and western Blotting. This finding will provide theory bases for the opinion that STAT3signaling pathway could be considered potential therapeutic target for ESCC.Part I:Clinicopathological Significance of STAT3and p-STAT3Expression in Patients with Esophageal Sqamous Cell CarcinomaObjective:This study was to investigate the clinicopathological significance of STAT3and p-STAT3expression in patients with esophageal sqamous cell carcinoma (ESCC).Methods:The subjects were71patients with ESCC who underwent resection from August2008to January2009.71ESCC specimens and20paracancerous normal tissue specimens were obtained from these patients. All the specimens were examined by RT-PCR, immunohistochemistry and western blot assays to detect STAT3expression. We also evaluate the levels of p-STAT3expression in the specimens by immunohistochemistry and western blot.According to the clinicopathologic factors, the enumeration data was calculated me positive rate, the comparison of positive rates uses the Chi-square; measurement data was represented with average±standard deviation and t-rest or F-test coupled with SNK test (q-test)were used. Differences were considered significant when the P value was less than0.05.The statistical data were obtained using an SPSS software package (SPSS13.0Inc., Chicago, IL, USA).Results:The expression of STAT3mRNA in the cancerous tissue group was significantly higher than that in the paracancerous normal tissues group (80.3%:55%, P<0.05). STAT3mRNA expression was correlated with pT (P<0.05) and pTNM (P <0.05). The positive expressions of STAT3protein were located both in cytoplasm and in cell nucleus. And the positive expressions of p-STAT3protein were located in cell nucleus. The expression of STAT3protein and p-STAT3protein in the cancerous tissue group was significantly higher than that in the paracancerous normal tissues group respectively (P<0.01, P<0.01). STAT3protein expression was correlated with pT (P <0.01) and pTNM (P<0.01). p-STAT3protein expression was correlated with pT (P <0.05), pN (P<0.01) and pTNM (P<0.05).Conclusions:STAT3mRNA expression was correlated with pT and pTNM. p-STAT3expression was correlated with pT, pN and pTNM.Overexpression of STAT3and p-STAT3might plays an important role in carcinogenesis, invasion and metastasis of ESCC. Part II:Correlation between STAT3Signaling Pathway and Its Target Gene VEGF, cyclinDl, Bcl-xL and MUC1in Patients with Esophageal Squamous Cell CarcinomaObjective:The purpose of the present study was to investigate the correlation between STAT3signaling pathway and its target gene VEGF, cyclinDl, Bcl-xL and MUC1in patients with esophageal squamous cell carcinoma (ESCC).Methods:The subjects were71patients with ESCC who underwent resection from August2008to January2009.71ESCC specimens and20paracancerous normal tissues were obtained from these patients. Expression levels of STAT3,VEGF, cyclinDl, Bcl-xL and MUC1in ESCC specimens and paracancerous normal tissues were studied using immunohistochemistry and RT-PCR. We also evaluate the levels of p-STAT3expression in ESCC specimens and paracancerous normal tissues by immunohistochemistry. The correlation between STAT3expression, p-STAT3expression, VEGF expression, cyclinDl expression, Bcl-xL expression and MUC1expression and clinicopathological factors was analyzed using Fisher's exact probability test or x2test. Spearman's rank correlation analysis was used to determine the relationships among the above variables. Differences were considered significant when the P value was less than0.05.The statistical data were obtained using an SPSS software package (SPSS13.0Inc., Chicago,1L, USA).Results:The expression of STAT3mRNA, VEGF mRNA, cyclinDl mRNA, Bcl-xL mRNA and MUC1mRNA in the cancerous tissue group was significantly higher than that in the paracancerous normal tissues group respectively (P<0.05, P<0.01, P<.01, P<0.01, P<0.01). STAT3mRNA expression was correlated with pT (P<0.05) and pTNM (P <0.05).VEGF mRNA expression was correlated with pT (P<0.05), pN (P<0.01) and pTNM (P<0.05). CyclinD1mRNA expression was correlated with pN (P<0.05). Bcl-xL mRNA expression was correlated with pT (P<0.05). And MUC1mRNA expression was correlated with pT (P<0.05) and pN (P<0.05). The positive expressions of STAT3protein were located both in cytoplasm and in cell nucleus. The positive expressions of p-STAT3protein were located in cell nucleus. The positive expressions of VEGF protein were located in cytoplasm. The positive expressions of cyclinDl protein were located in cell nucleus.The positive expressions of Bcl-xL protein were located in cytoplasm. And the positive expressions of MUC1protein were located in cytoplasm. The expression of STAT3protein, VEGF protein, cyclinD1protein, Bcl-xL protein and MUC1protein in the cancerous tissue group was significantly higher than that in the paracancerous normal tissues group respectively (P<0.05, P<0.01, P<0.01, P<0.01, P<0.01, P<0.01). STAT3protein expression was correlated with pT (P<0.05) and pTNM (P<0.05). p-STAT3protein expression was correlated with pT (P<0.05), pN (P<0.01) and pTNM (P<0.05). VEGF protein expression was correlated with pT (P<0.05), pN (P<0.01) and pTNM (P <0.05). CyclinDl protein expression was correlated with pN (P<0.05). Bcl-xL protein expression was correlated with pT (P<0.05). And MUC1protein expression was correlated with pT (P<0.05) and pN (P<0.05). There was a positive correlation between STAT3mRNA expression and VEGF mRNA expression, between STAT3protein and VEGF protein expression in the cancerous tissue group (r=0.285, P<0.05; r=0.255, P <0.05). There was a positive correlation between STAT3protein expression and p-STAT3protein expression in the cancerous tissue group (r=0.421, P<0.01).And there was a positive correlation between p-STAT3protein expression and VEGF protein expression, between p-STAT3protein expression and cyclinDl protein expression, and between p-STAT3protein expression and MUC1protein expression in the cancerous tissue group (r=0.352, P<0.01; r=0.305, P<0.05; r=0.273, P<0.05).Conclusions:VEGF, cyclinD1, Bcl-xL and MUC1were significantly increased in ESCC tissue compared with paracancerous normal tissues, respectively. VEGF expression was correlated with pT, pN and pTNM. CyclinDl expression was correlated with pN. Bcl-xL expression was correlated with pT. And MUC1expression was correlated with pT and pN. There was a positive correlation between STAT3expression and VEGF expression in the cancerous tissue group.And there was a positive correlation between p-STAT3expression and VEGF expression, between p-STAT3expression and cyclinDl expression, and between p-STAT3expression and MUC1expression in the cancerous tissue group.The mechanism of STAT3signaling pathway contributing to carcinogenenisis might be due to promoting transcriptional activation of VEGF, cyclinD1and MUC1in ESCC Part Ⅲ Prognostic Significance of STAT3, p-STAT3, VEGF, CyclinD1, Bcl-xL and MUC1Expression in Patients with Esophageal Sqamous Cell CarcinomaObjective:The purpose of the present study was to investigate the prognostic significance of STAT3, p-STAT3, VEGF, cyclinD1, Bcl-xL and MUC1in patients with esophageal squamous cell carcinoma (ESCC).Methods:The subjects were71patients with ESCC who underwent resection from August2008to January2009.71ESCC specimens were obtained from these patients. Expression levels of STAT3, VEGF, cyclinD1, Bcl-xL and MUC1in ESCC specimens were studied using immunohistochemistry and RT-PCR. We also evaluate the levels of p-STAT3expression in ESCC specimens by immunohistochemistry.Follow-up was complete for all the71patients. Univariate analysis was performed by modeling Kaplan-Meier survival curves. The log-rank test was used to calculate the survival rate. Multivariate analysis was carried out by the use of the Cox proportional hazard model. Differences were considered significant when the P value was less than0.05.The statistical data were obtained using an SPSS software package (SPSS13.0Inc., Chicago, IL, USA).Results:The Kaplan-Meier method indicated that the3-year survival rates of the71patients were52.1%. In univariate analysis by the log-rank test, the3-year survival rate in patients after operation was significantly associated with pT (P<0.01), pN (P<0.01), pTNM stage (P<0.01). Moreover, the3-year survival rate of the patients without VEGF mRNA expression and without VEGF protein expression in ESCC tissues was significantly lower than that of the patients without VEGF mRNA expression (35.6%:80.8%, P<0.01) and VEGF protein expression (35.7%:75.9%, P<0.01). The3-year survival rate of the patients with cyclinD1mRNA expression and protein expression in ESCC tissues was significantly lower than that of the patients without cyclinDl mRNA expression (41.9%:67.9%, P<0.05) and protein expression (40.0%:67.7%, P<0.05) The3-year survival rate of the patients with MUC1mRNA expression and protein expression in ESCC tissues was significantly lower than that of the patients without MUC1mRNA expression (41.7%:73.9%, P<0.05) and protein expression (41.3%:72.0 %, P<0.05). And the3-year survival rate of the patients with p-STAT3protein expression in ESCC tissues was significantly lower than that of the patients without P-STAT3protein expression (34.1%:81.5%,<0.01).However, no statistically significant correlations with gender, age, tumor length, tumor location, histological differentiation, STAT3expression and Bcl-xL expression were demonstrated for the3-year survival rate.The results of Cox regression multivariate analysis confirmed that pT and pN were independent relevant factors.Conclusions:The3-year survival rate in patients after operation was significantly associated with pT, pN, pTNM stage, p-STAT3expression, VEGF expression, cyclinDl expression and MUC1expression. However, pT and pN were independent relevant factors.