Clinical Study Of The Correlation Between Expression Of STAT3and Lymph Node Micrometastasis And Prognosis In Non-smali Cell Lung Cancer(NSCLC)

BackgroundLung cancer is the leading cause of tumor-specific death in many countries of the world nowadays, and its incidence rate is arising every year. The main pathological type of lung cancer, which covers more than80%is non-small cell lung cancer (NSCLC). It appears that lymph node metastasis of lung cancer may occur early, and latter lung cancer can metastasize to other distant organs. Metastatic spread constitutes the primary source of morbidity and mortality, and influences the effect of the patients' treatment and prognosis. In recent years, researchers have done much work in the study of the etiology and disease mechanism of lung cancer in order to improve the patients' diagnosis and prognosis. Many genes and factors have been proved to be related with the carcinogenesis and development of lung cancer. STAT3(signal transducer and activator of transcription3) is one of them. And it has been recognized as an oncogene. Although the regional development of NSCLC is not as aggressive as small-cell lung cancer (SCLC), micrometastasis may occur in NSCLC and it will seriously influence the patients' prognosis. So the research and detection of lymph node micrometastasis in lung cancer is becoming more and more important.Signal transducers and activators of transcription (STATs) family is a cytoplasmic protein family. It is identified by Fu etc in1992when they were studying how interferon (IFN) induces genetic transcription. It is coupled with tyrosine phosphorylation signaling pathway, and related with transduction regulation in many biological effects. In mammalian cells, the family members of STATs are STAT1(a/(3), STAT2, STAT3(α/β/γ), STAT4, STAT5(a/b) and STAT6. The STATs proteins with similar molecular weight are composed by750～900amino acids and coded by different genes. They have some conservative construction and function regions, including NH2-terminal, DNA binding domain (DBD), coiled-coil domain (CCD), linker domain and Src-homology-2domain (SH2domain). The diverse transcriptional activation domain (TAD)of the carboxyl-terminal determines the specificity of STATs proteins (Fig1).signal transducer and activator of transcription3(STAT3) protein was isolated as an acute-phase response factor (APRF) of interleukin-6(IL-6) by Zhong etc in1994. Naturally, there are three isomers of STAT3:STAT3α, STAT3β and STAT3γ. STAT3α usually correlates with cell proliferation and transformation; STAT3β imposes greater impact on granulocyte colony-stimulating factor-mediated cell differentiation than the other two isomers; STAT3γ (dominant negative isomer of STAT3α)is the restrictive protein hydrolysate of STAT3α. And it does not have the transcription activation domain (TAD)of STAT3α. The active form of STAT3γ mainly exists in differentiated neutrophils [6]. STAT3is widely present in different types of tissues and cells, especially in the brain, heart, liver, testes and the thymus. It is also can be detected in the spleen, lung and the esophagus. In recent years, many studies have confirmed that STAT3correlates with the cell proliferation and differentiation, immune evasion, angiogenesis, invasion and metastasis of a variety of different tumors. It has become a research focus of oncomolecularbiology. Phosphorylated-STAT3(pSTAT3) is the active and functional form of STAT3protein.Mucins are a calss of high molecular weight (>200kD) glycoprotein with15species. MUC1is type Ⅰ transmembrane protein and usually expressed in a variety of tissues, epithelial cells near the lumen or glandular cavity surface and several types of cells in the hematopoietic system (T, B and dendritic cells etc). Many studies have proved that MUC1usually abnomally expressed in a variety of tumors, mainly as follows:(1)expression increases up to even100times higher than normal;(2) distribution changes in cell surface-loss of polarity and expression in the entire cell surface;(3) construction changes of molecules. The abnomal expression of MUC1in tumors correlates with tumor proliferation, transformation and progression, so it has also become the research focus of oncomolecularbiology. Many studies have confirmed that detecting MUC1levels in bone marrow, blood or lymph nodes dissected in surgery using reverse transcription polymerase chain reaction (RT-PCR) method can detect whether there is tumor micrometastasis and facilitate assessing the risk of disease recurrence. This study was to test the expression of MUC1mRNA in the dissected lymph nodes to determine micrometastasis status, then test the expression of STAT3in the tumor tissues using RT-PCR, western blot assays (WB) and immunohistochemistry (IHC)methods to investigate the relationship between overexpression of STAT3and lymph node micrometastasis in early-stage NSCLC and explore the mechanism of lymph node micrometastasis of early-stage NSCLC. This study was also aimed to investigate the correlation between pSTAT3expression and the prognosis of NSCLC through detection of pSTAT3in tumor tissues and clinical follow-up. We hope we can make some progress in the study of NSCLC micrometastasis, targeted gene therapy and factors influencing prognosis of NSCLC.Part I Correlation between expression of STAT3and lymph node micrometastasis in early-stage NSCLCObjective:The incidence of lymph node micrometastasis of lung cancer was an important cause of tumor recurrence, and it may influence the prognosis of NSCLC. This study was to investigate the relationship between STAT3overexpression and lymph node micrometastasis in early-stage NSCLC and make some progress in improving the patients'prognosis.Methods:The samples of cancer tissues, lymph nodes from50patients with early-stage NSCLC (20stage IA samples;20stage IB samples;10stage II samples without lymph node metastasis)and20normal lung tissue samples,40normal lymph node samples from20patients with benign lung tumors, who underwent complete resection in Provincial Hospital Affiliated to Shandong University from Jun2008to Jan2010, were collected. All cancer samples and normal lung tissues were examined by RT-PCR to detect STAT3mRNA expression and the200lymph node samples from NSCLC patients and40lymph node samples from patients with benign lung tumors were examined by RT-PCR to detect MUC1mRNA expression. Western blot assays and immunohistochemistry methods were performed to detect STAT3protein and pSTAT3protein expression in lung cancer samples and normal lung tissue samples. The SPSS11.5software package was used for statistical analysis. Measurement data was represented with average±standard deviation and t-rest or F-test coupled with SNK test (q-test)were used. Chi-squared (χ2)test was used in enumeration data. Logistic regression analysis was performed to determine the independent risk factors influencing lymph node micrometastasis of early-stage NSCLC.Results:RT-PCR was performed to detect the expression of MUC1mRNA in all lymph node samples, and19patients with NSCLC were confirmed the existence of lymph node micrometastasis. No normal lymph node samples were found expression of MUC1mRNA. STAT3mRNA was detected in50NSCLC tissue samples and20normal lung tissue samples using RT-PCR method, and the expression values were0.8019±0.1462and0.3303±0.0236, respectively (t'=22.105, P=0.000). The expression of STAT3mRNA in lung cancer samples was significantly correlated with lymph node micrometastasis (t=9.308, P=0.000). The expression value of STAT3protein and pSTAT3protein in lung cancer samples were0.9459±0.1375and0.6503±0.1510, which were much higher than that expressed in normal lung tissue samples (0.3887±0.0269and0.2192±0.0215) using western blot method. The expression of STAT3protein and pSTAT3protein in lung cancer samples were both correlated with lymph node micrometastasis (P<0.05). The expression rate of STAT3protein and pSTAT3protein in lung cancer samples with lymph node micrometastasis were94.74%and73.68%using immunohistochemistry method, which were also higher than that in lung cancer samples without lymph node micrometastasis (both were38.71%). No STAT3protein and pSTAT3protein were detected in normal lung tissue samples using immunohistochemistry method. Logistic regression analysis revealed that STAT3protein expression in tumors (OR=13.379, P=0.033) and the differentiation degree of tumors (OR=0.131,P=0.016) were independent risk factors for lymph node micrometastasis of early-stage NSCLC.Conclusions:STAT3mRNA, STAT3protein and pSTAT3protein were all highly expressed in early-stage NSCLC with lymph node micrometastasis, and significantly correlated with it. Overexpression of STAT3might promote lymphatic micrometastasis of early-stage NSCLC and might be a clinical predictor of lymph node micrometastasis in early-stage NSCLC. Part Ⅱ Relationship between phosphorylated-STAT3(pSTAT3) expression and prognosis of NSCLCObjective:With the incidence rate of NSCLC becoming higher all over the world, people pay more attention in the research of factors influencing the prognosis of NSCLC. pSTAT3is the active form of STAT3and the relationship between pSTAT3and biological behavior of NSCLC is always the research focus. This study was to investigate the relationship between pSTAT3expression and prognosis of NSCLC.Methods:The NSCLC tissue samples from82patients, who underwent complete resection and lymphadenectomy in Provincial Hospital Affiliated to Shandong University from Mar2005to Jul2006, were collected. All samples were examined by immunohistochemistry to detect pSTAT3protein expression. The differences between pSTAT3protein expression and the clinicalpathological factors of patients were compared by Chi-squared test. Estimation of survival was calculated using the Kaplan-Meier method, and the statistical differences were analyzed using the Log-rank test. Cox regression analysis was performed to identify prognostic risk factors of NSCLC patients.Results:Of the82lung cancer tissue samples, pSTAT3protein was observed in49(59.76%) lung cancer samples by immunohistochemistry. The expression of pSTAT3protein correlated with the pN stage, pTNM stage and the differentiation degree of tumors (P<0.05), but not correlated with the patients'gender, age, tumor histological type and pT stage of tumors. The1-year,3-year and5-year cumulative survival rates of the82patients were89.02%,51.22%and31.71%respectively. Cox regression analysis showed that patients'gender, age, tumor histological type, tumor differentiation degree and pT stage of tumors did not significantly affect survival of NSCLC patients (P>0.05). But pN stage, pTNM stage and pSTAT3protein overexpression in lung cancer correlated with the survival of NSCLC patients (P<0.05). Conclusions:pSTAT3protein expressed highly in lung cancer tissues. Patients with high expression of pSTAT3protein always have poor prognosis, which indicates that pSTAT3protein overexpression may be related to prognosis of NSCLC patients.