Expression And Molecular Mechanism Of L1CAM In Esophageal Squamous Cell Carcinoma

Background and objective: Esophageal carcinoma accounts for a large proportion of gastrointestinal carcinoma in China, and Henan Province is recognized as a high-incidence area in the world. Due to the difficulties of early diagnosis, most patients are diagnosed with advanced stage disease. Also, these cancers progress rapidly and metastasize early. Esophageal squamous cell carcinoma(ESCC) is the more common pathological type of esophageal carcinoma in China. Although great success has been made in the treatment esophageal carcinoma, progress towards better prognosis for most advanced stage esophageal carcinoma remains frustratingly slow and many patients develop relapsed diseases or metastases. Therefore, there is an urgent need to develop novel and effective strategies for the treatment of this disease. With great advances in biological science and further studies of cancer-related genes, gene therapies and target therapies have been becoming the new promising treatment options in tumor therapy. The selection of specific target molecular is considered as the most important step for these strategies. L1CAM(L1 cell adhesion molecule) is a novel member of immunoglobulin superfamily cell adhesion molecules. Early research has demonstrated that L1 CAM played an important role in the process of development of the nervous system. Subsequently, L1 CAM was found to be abnormally expressed on tumors, including ovarian cancer, melanoma, breast cancer, prostate cancer and endometrial cancer, and it was considered to be associated with poor prognosis in late stage patients. These finding suggest that L1 CAM is a gene which promotes tumor progression. Thus, this novel molecule can be used as the target of treatment. Firstly, L1 CAM plays an important role in tumor progression and metastasis; Secondly, it is highly expressed on a wide variety of human tumors(i.e. ovarian cancer and pancreas cancer), and it can be used in the patients with failure of the traditional chemotherapy or poor prognosis. Finally, L1 CAM is defined as a tissue selective molecule, whose expression can be observed in the adult peripheral nervous system and tubular cells. Nevertheless, L1 CAM expression in ESCC is still unknown, and its molecular mechanisms underlying esophageal tumor development and progression remain poorly understood. In this study, we analyzed the correlation between L1 CAM expression and clinical parameters, and evaluated its prognostic values in ESCC patients. Then further studies were conducted to investigate the role of L1 CAM in the process of tumorigenesis. We inhibited the L1 CAM gene expression in order to explore its function in the proliferation, invasion and motility of esophageal cancer cells. The in-vivo xenograft esophageal tumor model would be used to confirm its functionality.Part 1 L1 CAM expression in Esophageal Squamous Cell Carcinoma Patients and its Relationship with the Clinicopathological ParametersMethods 1) Real time PCR was used to detect L1 CAM m RNA expression in esophageal cancer and adjacent normal esophageal mucosal tissues. 2) The relationship of L1 CAM m RNA expression and the clinicopathological parameters was analyzed. 3) Immunohistochemistry(IHC) was performed to detect L1 CAM protein expression in the esophageal cancer tissues. 4) The relationship of L1 CAM protein expression and clinicopathological parameters was assessed.5) The association of L1 CAM protein expression with prognosis was analyzed in patients with esophageal cancer.Results 1) The L1 CAM m RNA expression was significantly higher in 117 esophageal cancer tissues than the one in normal tissues, P<0.001. The level of L1 CAM expression in cancer(56.41%) was twice higher than the one in normal tissues. 2) The L1 CAM m RNA expression was significantly associated with the tumor differentiation, invasion and clinical stages, P<0.05. 3) The positive rate of L1 CAM protein was 78.26% in 69 esophageal cancer tumor tissues. 4) L1 CAM protein expression was significantly associated with the tumor differentiation, invasion, clinical stages and survivals, P<0.05. 5) The survival rate in patients with high L1 CAM protein expression was lower than the one in patients with low L1 CAM protein expression, P<0.05.Summary 1) The expression of L1 CAM m RNA was significantly higher in esophageal cancer tissues than the one in normal tissues. The expression of L1 CAM was related to the clinical parameters. The results show that L1 CAM might play an important role in the development and progression of esophageal cancer. 2) The L1 CAM protein expression was positively related to patients' survivals, demonstrating that L1 CAM might be a prognostic factor in esophageal carcinoma.Part 2 The Biological Significance of L1 CAM in Esophageal Cancer Cell Lines TE1 and EC1Methods 1) Real time PCR was performed to measure L1 CAM m RNA expression in human esophageal cancer cells and immortalized esophageal epithelial cells. 2) The lentiviral-mediated short hairpin RNA(sh RNA) interference was used to knockdown L1 CAM in cell lines TE1 and EC1. 3) CCK8 method was used to detect the effects of L1 CAM suppression on cell proliferation in cell lines TE1 and EC1. 4) The flow cytometric analysis was performed to investigate effects of its suppression on cell cycle and apoptosis in esophageal cancer cells. 5) The sphere forming assay was performed to observe the effect of its knockdown on the capability of esophageal cancer cells. 6) The Transwell migration assay was utilized to observe the effect of its knockdown on cell migration and invasion of esophageal cancer cells. 7) The in-vivo xenograft esophageal tumor model of nude mice was established to observe the effect of its knockdown on the tumor growth.Results 1) L1 CAM expression was relatively high in esophageal cancer cells EC1 and TE1, thus these two cell lines were chose to construct stable transduced sh RNA-mediated L1 CAM knockdown cells. 2) Real time PCR and Western blotting were used to verify the efficiency of L1 CAM knockdown. 3) L1 CAM knockdown could inhibit the proliferation of esophageal cancer cells. 4) L1 CAM knockdown could block the cycle at G0/G1 phase, and increase the apoptosis in esophageal cancer cells. 5) L1 CAM knockdown could inhibit the sphere-forming capability of esophageal cancer cells. 6) L1 CAM knockdown could reduce migration and invasion of esophageal cancer cells. 7) In the in-vivo xenograft esophageal tumor model, L1 CAM knockdown could weaken the ability of tumor formation, and decrease the tumor volume and weight.Summary 1) L1 CAM knockdown could reduce the cell proliferation and self-renewal, arrest cell cycle, reduce the migration and invasion of esophageal carcinoma cells, suggesting that L1 CAM plays an important role in esophageal cancer malignant behavior. 2) L1 CAM knockdown could influence the tumor formation and development in the in-vivo xenograft esophageal tumor model of nude mice.Part 3 Promoting effect of L1 CAM in the recruitment of Tregs via up-regulating NF-kBMethods 1) The cytometric bead array was used to analyze the changes of cytokines in the supernatant of esophageal cancer cells with L1 CAM knockdown. The cytokine with the biggest fold changes was selected and then confirmed by ELISA and real time PCR. 2) The in-vitro Transwell assay was used to detect the effect of L1 CAM knockdown on the recruitment of CD4+FOXP3+ Tregs from fresh peripheral blood of human healthy donors. 3) CD4+ T cells from fresh peripheral blood of human healthy donors were injected into the in-vivo xenograft esophageal tumor model of nude mice to verify the effect of L1 CAM suppression on the recruitment of Tregs. 4) Real time PCR and immunohistochemistry were employed to test the expression of L1 CAM, CCL22, FOXP3 and CCR4 in tumor xenografts of nude mice. 5) Western blot was applied to analyze the effect of L1 CAM knockdown on the NF-kB activity. The expression of CCL22 in TE1 cells after the treatment of NF-kB inhibitor was examined by ELISA and real time PCR. 6) Real time PCR and the flow cytometry assay were performed to investigate the correlation between L1 CAM m RNA expression and the number of Tregs in esophageal cancer samples. 7) The Kaplan–Meier survival curve was utilized to assess the association between the proportion of CD4+FOXP3+ Tregs and prognosis in patients with esophageal cancer.Results 1) With the decrease of L1 CAM expression, the levels of CCL22 secreted by TE1 cells were most obviously changed, and significantly decreased. 2) The decreased in-vitro production of CCL22 upon L1 CAM knockdown could reduce the number of Tregs recruitment to tumor sites significantly. 3) The flow cytometry assay showed the significantly reduced number of Tregs recruitment to tumor sites upon L1 CAM knockdown. 4) L1 CAM downregulation could reduce the in-vivo expression of CCL22, FOXP3 and CCR4 m RNA and protein at tumor sites. 5) L1 CAM downregulation could inhibit the activity of NF-kB pathway. The secretion level of CCL22 from tumor cells decreased upon the treatment of the NF-kB inhibitor. 6) The L1 CAM m RNA expression was positively correlated with the number of Tregs in esophageal cancer tissues, P<0.05. 7) The higher proportion of Tregs in esophageal cancer tissues might reflect a worse prognosis.Summary 1) The decreased production of CCL22 upon L1 CAM knockdown in esophageal cell line TE1 cells could reduce the number of Tregs recruitment to tumor sites. 2) The L1 CAM expression was positively correlated with the number of infiltrated Tregs in esophageal cancer tissues, and the number of Tregs was associated with the patients' prognosis. 3) L1 CAM could regulate CCL22 expression through NF-kB pathway.Conclusion:1) L1 CAM is highly expressed in esophageal cancer tissues, and correlates with clinicopathological characteristics and prognosis, suggesting that L1 CAM plays an important role in the development of esophageal cancer. 2) L1 CAM knockdown inhibits the cell proliferation, migration and invasion capability, induces cell cycle arrest and promotes apoptosis in esophageal cancer cells. 3) L1 CAM suppression downregulates CCL22 expression and decreases the number of Tregs recruited to tumor sites via inhibiting NF-kB activity.