The Expression And Biological Significance Of SATB2 In Esophageal Squamous Cell Carcinoma

Background and research purposeEsophageal squamous cell carcinoma is the most common histological type of esophageal cancer. However, its early symptoms are not obvious, leading to the low early detection rate. Most patients are in advanced stages of esophageal cancer when first diagnosed, and have the poor treatment effect and prognosis. This forces the clinicians and researchers to identify new genes regulating the development, invasion and metastasis processes of esophageal cancer and further clarify the underlying molecular mechanisms, which may be applied to overcome the occurrence and metastasis of esophageal carcinoma. Special AT-rich sequence binding protein 2（SATB2） is a newly discovered transcription factor that binds to the matrix attachment region. SATB2 resides on human 2 chromosome 2q33, encoding a 733-amino acids protein containing a DNA binding domain（residues 614-677）, a Pfam-B₁0016 or SATB domain required for dimerization（57-231）, and two CUT domains（352-437 and 482-560）. SATB2 is involved in the generation and development of tumors and the development of central nervous system and skeletal system via regulating gene expression and modifying chromosomes. SATB2 expression is found to be positively correlated with the pathological grade and worse prognosis in breast cancer, lymphoma, and head and neck cancers. However, whether SATB2 also participates in the regulation of esophageal carcinoma is still unknown. This study is aimed to investigate the role and mechanisms of SATB2 in the occurrence, proliferation, prognosis, migration and invasion of esophageal carcinoma, which may provide experimental basis for genetic intervention for esophageal cancer therapy. Methods 1. The expression of SATB2 was detected in esophageal carcinoma tissues and paired adjacent non-carcinoma tissues by quantitative PCR and Western blot, and in 203 cases of esophageal tumor paraffin embedded samples by immunohistochemical staining. Then the correlation between SATB2 expression and clinicopathologic characteristics of esophageal cancer patients, including gender, pathological type, age, clinical TNM stage, lymph node status and prognosis, were analyzed. 2. Esophageal carcinoma EC109 cells were transfected with pcDNA3.1 empty vector and pcDNA-SATB2 eukaryotic expression vector, and then treated with G418 to establish SATB2 stably-expressed EC109 cell line and the paired control cell line, which were used in the following experiments. Effects of SATB2 on the proliferation, migration, invasion and cell cycle progression of EC109 cells were analyzed by using MTT assay, wound healing assay, matrigel invasion assay and flow cytometry, respectively. 3. Western blot were used to compare the expression levels of MMP2, MMP9, and proteins involved in the AKT/mTOR, ERK, p38 and STAT3 pathways in three EC109 cell lines, including untreated control cells, pCDNA3.1 vector stably-expressed cells, and pcDNA-SATB2 stably-expressed cells, and the molecular mechanisms of SATB2 on the invasion and migration of EC109 cells was then analyzed. Results 1. The expression of SATB2 was lower in esophageal tumor tissue compared to the adjacent non-tumor tissue in 103 cases out of 203（50.7%）. Statistical analysis showed that the expression level of SATB2 was negatively correlated with clinical stage（p<0.05） and tissue differentiation（p<0.05）, and positively correlated with the total survival rate and survival time of the patients（p<0.05）. Multivariate analysis indicated that the expression level of SATB2 could be used as an independent prognostic factor for esophageal cancer. 2. qPCR and Western blot were used to confirm the overexpression of SATB2 in the SATB2 stably-expressed EC109 cell line. Results from MTT assay showed that the proliferation rate of SATB2 stably-expressed cells was significantly lower than the untreated group and empty vector pcDNA3.1 group（p<0.05）,suggesting that SATB2 could inhibit the proliferation of esophageal cancer cells. Results from wound healing assay and matrigel invasion assay showed that SATB2 could inhibit the migration and invasion ability of esophageal cancer EC109 cells. 3. Western blot assay showed that the expression level of MMP2 and MMP9, and the phosphorylation of Akt and mTOR were decreased in cells overexpressing SATB2, implying that SATB2 may regulate cell proliferation, invasion and metastasis via Akt/mTOR pathway. Conclusions 1. Low SATB2 expression was detected in a great part of esophageal cancer tissues, and its expression was negatively correlated with the degree of malignancy of esophageal cancer, indicating that SATB2 can be used as a potential molecular marker for the prognosis of esophageal cancer patients. 2. SATB2 stably-expressed EC109 cell line was successfully constructed. Results from MTT assay, wound healing assay and matrigel invasion assay indicated that SATB2 could inhibit the proliferation, migration and invasion of esophageal cancer EC109 cells. 3. Overexpression of SATB2 in esophageal cancer EC109 cells can reduce the expression of MMP2 and MMP9 proteins and the phosphorylation levels of Akt and mTOR. The results suggest that SATB2 may modulate the biological behavior of esophageal cancer cells through regulating the Akt/mTOR pathway. In this study, the expression of SATB2 in esophageal cancer tissues, the role of SATB2 in the proliferation, migration and invasion of esophageal cancer EC109 cells and the relationship between SATB2 and Akt/mTOR signaling pathway were studied. It is proved that SATB2 expression is closely correlated with the clinical stage of esophageal cancer, and SATB2 can inhibit the proliferation, invasion and migration of esophageal cancer cells via regulating Akt/mTOR pathway. This study provides a new strategy for the prevention and treatment of esophageal cancer.