Expressions Of RSK4 And P63 In Esophageal Squamous Cell Carcinoma And Their Correlation With Clinicopathologic Features

【Background】 Esophageal carcinoma(EC) is a malignant tumor of the esophagus epithelium, with men having a two or fourfold higher risk than women at the age over 40. The global incidence of esophageal reaches about 500,000 cases per year, accounting for 2% of all kinds of malignancies and the number of cases in death ranks sixth among all cancer deaths worldwide with an approximate 400,000 annual death cases. Incidence of esophageal cancer in China accounts for 50% of that in the world, with a 5-year survival rate of less than 10%. In areas with a high risk of esophageal cancer, squamous cell carcinoma as the main histological type, ranks seventh in the cancer death. Patients with Esophageal Squamous Cell Carcinoma(ESCC) suffer from poor prognosis, bear relatively higher risks of operations, and have no specific diagnostic and prognostic evaluation to follow. Additionally, the pathogenesis of ESCC still remains unclear.p90 ribosomal protein S6 kinase 4(RSK4) is a member of ribosomal S6 kinase(RSK) gene family. RSK4 was originally discovered in 1999 by Yntema et al. It locates in the human Xq21, which functions in the Ras-MAPKs downstream signaling pathway, and was reported as the most important downstream regulating factor in the MAPK /ERK signaling pathway and a vital signaling molecule in the Ras signaling pathway. RSK4, a member of serine/threonine protein kinases, participates in cell cycle regulation, influencing cell proliferation, differentiation and apoptosis. According to recent studies, the abnormal expression of RSK4 in colon cancer, renal carcinoma, breast cancer, endometrial cancer and other tumors may link to tumorigenesis and the development of tumors. However, there has been no report about the association between RSK4 and esophageal cancer. p63 was first discovered by Yang et al. in 1998 and shows a high homology with p53 and p73 in the gene sequence and functions. p63 is mainly expressed in basal cells of normal squamous epithelium and catheter myoepithelial cells. In invasive tumors, the expression of p63 often turned to be negative due to the absence of either basal epithelial cells or muscle cells. According to a latest study, the down regulation of RSK4 was found in squamous cells of the skin of p63 knockout mice, which suggests some correlation between RSK4 and p63 in squamous epithelium. Our previous studies found that the expression of RSK4 was higher in renal cell carcinoma and pancreatic cancer cells than that in normal tissues, indicating a possible oncogenes function of RSK4. However, the expressions of RSK and p63 in ESCC, as well as the progress of ESCC, have not yet been made manifest, thus our research aims at revealing their expressions and relations.【Objectives】 This study is designed to identify the relationship between the expressions of RSK4 and p63, their clinic pathological features, and prognosis of patients with esophageal squamous cell carcinoma, by using histopathological and molecular biological methods. Experiments were performed to investigate the possibility of their function as molecular markers of malignant progression and prognosis factor, which will assist researchers in exploring the mechanism of ESCC and providing theoretical support for the early diagnosis, treatment and prognosis of ESCC.【Methods】 1. In the tissues of 72 cases of esophageal squamous cell carcinoma, the expressions and correlation of RSK4 and p63 were analyzed by referring to immunohistochemical Envision two-step method. 2. q RT-PCR was used to detect the expression levels of p63 and RSK4 with m RNA extracted from esophageal squamous cells and normal esophageal cells. 3. Western blot: Protein was extracted from esophageal squamous cells and normal esophageal cells. When the quality was settled by SDS-PAGE electrophoresis, the first and secondary antibody were incubated after trarsmembrane. Finally ECL chemiluminescence detection was employed to detect the protein levels of RSK 4 and P63. 4. SPSS19.0 software applications, Kaplan–Meier method and Cox hazard regression model were employed for correlation analysis and survival analysis, in an attempt to articulate the relationship between RSK4 and p64 expression and their clinical pathological features in esophageal squamous cell carcinoma patients.【Results】 1. The expression and distribution of RSK4 and p63 of esophageal squamous cell carcinoma and adjacent tissues Immunohistochemical staining suggested that RSK4 and p63 expressions were higher than in the surrounding tumor tissues. 2. Expressions and correlation analysis of RSK4 and p63 in esophageal squamous cell carcinoma The positive rate of RSK4 expression reached 62.5%(45/72) in 72 cases of esophageal squamous cell carcinoma while positive rate of p63 was 83.3%(60/72). Among these cases, 42 cases were proved both RSK4 and p63 positive and 9 cases were proved RSK4 and p63 negative. According to the Pearson correlation analysis, RSK4 and p63 expressions have a positive correlation.3. Expression of RSK4 and p63 in esophageal squamous cancer cell line The observed m RNA and protein levels of RSK4 and p63 were higher in esophageal squamous cancer cell line than in normal cells, and the expression levels of RSK4 and p63 varied among different esophageal squamous cancer cell lines. 4. The correlation between RSK4 expression and clinic pathological features of esophageal squamous cell carcinoma Cases with RSK4 positive, in a total of 72 cases of esophageal squamous cell carcinoma, were observed.(45 / 72, 62.5%). χ2 test was used to analyze the relation between RSK4 expression and clinicopathological features of 66 cases of esophageal squamous cell carcinoma. The result showed a significant difference in the positive rate of RSK4 expression of patients at different stages of esophageal squamous cell carcinoma. The positive rate of p T1 ~ p T2 reached 48.3%(14/29) while of p T3 ~ p T4 positive rate marked a significant increase to 73.0%(27/37), as was illustrated by statistical analysis. What’s more, the expression of RSK4 did not only have a positive relation with the high p T stage of tumor but was also relevant to tumor differentiation and invasion(lymph node metastasis) and so on. However, RSK4 expression and any pathological features such as gender, age, tumor size and gross type, showed no correlation. 5. The relationship between p63 expression and clinicopathological features Among a total of 72 cases, 60 cases of esophageal squamous cell carcinoma(60 / 72, 83.3%) were positive p63. The correlation between the expression of p63 and clinical pathological features of 66 cases with surgery was analyzed and the results are as follows: p T1 ~p T2 positive 75.9%(22/29), and p T3 ~ p T4 positive rate of 89.2%(33/37), but the statistical analysis demonstrated no statistically significant difference in the expression of p63.No correlation was found between the expression of p63 and other clinicopathological characteristics of patients such as age, tumor size and the presence of distant metastases. 6. The survival and prognosis relation between RSK4 and p63 and the patients with ESCC Univariate survival analysis suggested that the positive expression of RSK4 has correlation with the poor prognosis of esophageal squamous cancer patients, and the staging, classification degree, the presence of lymphatic metastasis, along with other factors may also affect the life-span of esophageal squamous cancer patients whereas smoking and tissue type have no correlation with prognosis. Statistical results shows that the positive expression of p63 and clinicopathologic features has no relationship with the prognosis of esophageal squamous cancer.【Conclusions】 This study showed a considerable incresase of the expressions of RSK4 and p63 in esophageal squamous carcinoma tissues and cell lines, and it also identified a significant relation between RSK4 and the p T staging, the degree of staging, lymphatic metastasis, and survival prognosis among patients with esophageal cancer, which suggests that RSK4 might serve as a molecular marker for the possible malignant progress of ESCC and function as a prognosis indicator. These findings will lay foundation for the further investigation of the relation between RSK4 and p63, as well as their their mechanism at molecular level in ESCC.