Study Of The Role Of NCOA5 And SIRT1 Protein In The Progression And Prognosis Of Esophageal Squamous Cell Carcinoma

Background and significanceCancer is always one of the primary public health concerns worldwide because of its extremely high mortality rate. With the development of modern medicine, the war on cancer has been going on for decades. Although we haven’t conquered it, a lot of beneficial findings have been acquired through the fight against it. People have proposed some hallmarks of malignant tumor, such as sustaining self-sufficient proliferative signaling, continuous replication without limit, evading apoptosis and the control of anti-growth signals, inducing angiogenesis and activating signals that can help tumor cells invade and metastasize to adjacent and distant tissues. From these perspectives, a variety of malignant tumors has been well studied in an organized and reasonable way. Esophageal squamous cell carcinoma (ESCC), which has an extremely high degree of malignance, is a predominant subtype of esophageal cancer. In our country, the incidence of this disease is especially high. ESCC is excessively vigorous in growth, and develops extremely fast. Most of the patients suffering this disease were diagnosed at an advanced stage. Besides, ESCC has an extremely aggressive biological behavior. It has great potential in invading adjacent tissues, and intends to spread to other organs through the lymphatic system eagerly. All these make the treatment of ESCC extremely intractable. So far, methods that can improve the prognosis of patients with ESCC are still early stage diagnosis and timely and targeted comprehensive treatment. Therefore, studies of molecular biological markers that may affect the progress and prognosis of ESCC may greatly help us develop noninvasive examination methods, classify patients at an early stage and those that may have unfavorable prognosis, and provide therapeutic targets.Nuclear receptor coactivator 5 (NCOA5) is a unique coactivator that can interact with both ERa and ERβ. By interacting with the ligand-binding domain (LBD) of the receptors, NCOA5 modulates the nuclear receptors to activate targeted genes that involve in reproduction, growth, development and other physiological processes. Until now, the research on NCOA5 is still limited, and mainly concentrated on its interaction with ERa. However, its crucial role in tumor progression has been shown in these researches. In livers of NCOA5 Haplo-insufficient mice, phosphorylation of insulin receptor β (IR-β), insulin receptor substrate 1 (IRS-1) and AKT was greatly reduced. Meanwhile, changes have been found in the expression of a variety of genes involving in the NF-κB, androgen and insulin pathways. By participating in the regulation of the expression of IL-6 and some other pathways, NCOA5 contributes to the occurrence of hepatocellular carcinoma. Therefore, further researches on NCOA5 in ESCC may help deepen the understanding of the mechanism of disease progression, and provide new targets for diagnosis and treatment of this disease.Sirtuin1 (SIRT1), which is one of the orthologues of yeast silent information regulator 2 (Sir2) in mammals, is an NAD+-dependent type Ⅲ histone deacetylase (HDAC) that is highly conserved during evolution. By performing its multilayered regulation ability, SIRT1 participates in multiple important biological processes such as cell metabolism, longevity, and stress response. In recent years, its role in tumor development is getting more and more attention. Although SIRT1 has been found to be overexpressed in some types of cancer and be able to inhibit apoptosis induced by proteins such as p53, forkhead box (FOXO) transcription factor family and a variety of other important tumor suppressors, its role in the development of tumor is still complicated and confusing. Activated by resveratrol, SIRT1 didn’t promote tumor growth, but restrained the growth of some kind of tumor cells. And at the same time, its expression is greatly decreased in some kind of tumors. Recent studies showed that SIRT1 can also participate in the regulation of the expression of vascular endothelial growth factor-C (VEGF-C) in tumor cells. This suggests that it may be involved in tumor lymphangiogenesis.As NCOA5 and SIRT1 protein play important roles in tumor progression and prognosis, and there is no researches about them in ESCC, we carried out the three-part researches as follows:(1) Detect NCOA5 expression in ESCC and adjacent normal tissues, investigate the relationship between NCOA5 expression, clinicopathological characteristics and prognosis of ESCC patients; (2) Overexpress NCPA5 in ESCC cells, investigate its influences on various malignant biological behaviors of ESCC cells; (3) Detect SIRT1 expression in pNO ESCC, investigate the relationship between SIRT1 expression and various clinicopathologic features, tumor lymphangiogenesis, lymphovascular invasion and prognosis.PartⅠ NCOA5 low expression correlates with survival in esophageal squamous cell carcinomaBackground:The aims of the present study were to investigate NCOA5 expression in esophageal squamous cell carcinoma and validate its possible influence on patients’ prognosis.Methods:NCOA5 expression was examined by immunohistochemical staining in 119 ESCC patients’tissues.10 paired tumor and adjacent normal specimens were examined by western blot analysis. Statistical analysis was performed to assess its relevance with various clinicopathologic features and its influence on patients’survival.Results:By immunohistochemistry analysis, NCOA5 expression was found to be significantly correlated with differentiation (P=0.039), T status (P=0.047) and stage (P=0.036). Furthermore, we found NCOA5 higher expression in normal tissues than in tumor tissues by western blot analysis. Univariate analysis showed that poor differentiation (P=0.035, P=0.027), lymph node metastasis (P<0.001, P<0.001), high T status (P=0.010, P=0.012), advanced stage (P<0.001, P<0.001), and NCOA5 low expression (P<0.001, P<0.001) were significantly correlated with poor prognosis of both disease-free survival (DFS) and overall survival (OS). Multivariate analysis showed that NCOA5 low expression (P=0.019, P=0.047), high T status (P=0.015, P=0.012), lymph node metastasis (P=0.040, P=0.021) and advanced stage (P=0.017, P=0.046) were independent prognostic factors of poor DFS and OS.Conclusions:Our findings suggest that NCOA5 low expression is associated with ESCC progression and is a potential biomarker in predicting poor prognosis. Further studies of NCOA5 may help develop new therapeutic strategies against ESCC.Part II Study of the influence of NCOA5 expression on biological behavior of esophageal squamous cell carcinomaBackground:Investigate NCOA5 expression in ESCC cells, then explore the effects of NCOA5 overexpression on proliferation, apoptosis, migration and invasion in vitro.Methods:Western blot was used to detect NCOA5 expression in ESCC cell lines. By transfecting pcDNA3.1-NCOA5, NCOA5 expression was evaluated. Flow cytometry was applied to test the influence of NCOA5 expression on ESCC cell apoptosis. Bcl-2 and Bax expression was detected by western blot analysis. The effect of NCOA5 on cell migration and invasion ability was examined by wound healing and transwell assay.Results:NCOA5 expression was extremely low in EC 109, TE-13, and TE-8 cell lines. 48 hours after pcDNA3.1-NCOA5 transfection, NCOA5 expression was significantly upregulated (P<0.05). MTT test found that overexpression of NCOA5 in EC 109 and TE-13 significantly decreased their proliferative ability (P<0.05). Flow cytometry detected that, with the upregulation of NCOA5 expression in EC 109 and TE-13 cells, cell apoptosis rate increased significantly (P<0.05). Bcl-2 expression decreased (P<0.05), while Bax expression increased at the same time (P<0.05). In ESCC cells with NCOA5 overexpression, the migration and invasion ability were greatly weakened (P<0.05), and E-cadherin expression was found to be increased (P<0.05).Conclusions:NCOA5 expression has a great effect on ESCC cell’s malignant biological behavior such as proliferation, apoptosis, migration and invasion. Insufficient expression of NCOA5 may take an important part in the progression of ESCC. Performing more researches on NCOA5 will help us better understand this disease, and provide more efficient treatment in the future.Part Ⅲ SIRT1 expression is associated with lymphangiogenesis, lymphovascular invasion and prognosis in pNO esophageal squamous cell carcinomaBackground:This research aims at investigating the prevalence of SIRT1 protein expression and its prognostic influence in pNO esophageal squamous cell carcinoma, and validating its possible role in lymphangiogenesis and lymphovascular invasion (LVI).Methods:206 patients were enrolled in this retrospective study. Immumohistochemical staining was used for SIRT1, VEGF-C protein detection. Peritumoral lymphatic microvessel density (LVD) and LVI were evaluated by immunostaining for D2-40. Statistical analysis was then preformed to investigate the relevance of SIRT1 expression and various clinicopathologic features, and to detect the effect of SIRT1 on tumor induced lymphangiogenesis, LVI and prognosis.Results:SIRT1 positive expression was recognized in 95 cases in the nucleus and was significantly correlated with T status (P<0.001), disease stage (P=0.001), VEGF-C positive expression (P=0.015), high LVD (P=0.013) and positive LVI (P=0.015). Patients with SIRT1 positive expression, high LVD and positive LVI had significantly unfavorable 5-year disease free survival (P<0.001, P=0.030, P<0.001) and overall survival (P<0.001, P=0.017, P<0.001), respectively. However, by multivariate Cox regression analysis, only SIRT1 positive expression and positive LVI remained as significant independent prognosticators for poor disease free survival (P=0.029 and 0.018) and overall survival (P=0.045 and 0.031), respectively.Conclusions:SIRT1 protein positive expression was significantly associated with tumor progression, lymphangiogenesis, LVI and poor survival in pNO ESCC patients. Our research shows a utilization of SIRT1 in prognosing poor survival and providing possibly target for ESCC patients through its lymphangiogenesis activity.