The Function Of SARI In Modulating Epithelial-Mesenchymal Transition And Lung Adenocarcinoma Metastasis

Lung cancer is the leading cancer all over the world, and lung adenocarcinoma is the most common kind of lung cancer. In the absence of metastasis, lung adenocarcinoma is largely a treatable disease. Thus, early diagnosis of patients who develop lung adenocarcinoma metastasis could reduce the mortality and morbidity associated with this disease. The development of metastasis depends on the migration and invasion of cancer cells from the primary tumor into the surrounding tissues. To acquire such invasive abilities, carcinoma cells may acquire unique phenotypic changes such as epithelial-mesenchymal transition (EMT). EMT is a highly conserved cellular process that allows polarized, generally immotile epithelial cells to convert to motile mesenchymal-appearing cells. This process was initially recognized during several critical stages of embryonic development and has more recently been implicated in promoting carcinoma invasion and metastasis. During EMT,3major changes occur:(i) morphological changes from a cobblestone-like monolayer of epithelial cells to dispersed, spindle-shaped mesenchymal cells with migratory protrusions;(ii) changes in the differentiation markers from cell-cell junction proteins and cytokeratin intermediate filaments to vimentin filaments and fibronectin; and (iii) acquisition of invasiveness through the extracellular matrix. Decreased E-cadherin expression or gain of vimentin expression is closely correlated with various indices of lung adenocarcinoma progression, including the grade, local invasiveness, dissemination into blood, and tumor relapse after radiotherapy.SARI, also known as suppressor of AP-1, is regulated by IFN and has been implicated in cell-growth inhibition and apoptosis. SARI is down-regulated in various types of human cancers and plays an important role in tumor development.Thus, it is very likely that SARI functions as a tumor suppressor in cancer development; however, its role and mechanism in lung adenocarcinoma metastasis is largely unknown. In the current study, we have shown that loss of SARI facilitates EMT, leading to lung adenocarcinoma metastasis:The first part explores the SARI and EMT markers expression in lung adenocarcinoma patients. In accordance with the preoperative PET-CT results, we will have lymph node metastasis and non-lymph node metastasis groups. According to postoperative lung cancer TNM staging system,6cases of stage I patients with adenocarcinoma, and7cases of stage III patients were screened using the conventional H&E staining of lymph node metastasis and lymph node metastasis of lung adenocarcinoma specimens and metastatic lymph node lesions. We found that the primary lesions and metastases of the cancer are the same cells type. Compared with the non-metastasis group, lymph node metastasis of lung adenocarcinoma patients were showing that SARI was high expression but E-cadherin missing.We also find positive for vimentin, p-GSK-3β and (3-catenin, increased expression. In all test specimens, a significant correlation (r=0.8390) between SARI and E-cadherin, and SARI and vimentin has a significant negative correlation (r=0.7255).The second part to further explore the effect and mechanism of regulation of EMT by SARI in vitro.SARI expression is detected in a series of lung adenocarcinoma cell line, found a high-expression in the NCI-1650, NCI-of H1299and CRL5908SARI; in NCI-H1975, Calu-3and A549, are the relatively low expression; GLC-82, PG49and HTB-55, are almost no expression. When transfected SARI to GLC-82and PG49cells, cell morphology changed from long hammer-shaped fibroblast-like mesenchymal type (control group) into a pebble-like epithelial phenotype (experimental group), the connection between adjacent cells become closer, and also enhances cell polarity. GLC-82and PG49cells, effect of SARI expression is to enhance the E-cadherin and reduce expression of vimentin. Conversely, when endogenous SARI gene knockout, lung adenocarcinoma cell lines NCI-H1650and NCI-of H1299, for example, both can be detected the process of EMT, changed cell morphology and biomarkers.For understanding the possible mechanisms SARI adjustment EMT, we examined the effect of SARI in GSK-3β-β-catenin signaling pathway. SARI gene downregulated by siRNA, and we observed in the cytoplasm of (3-catenin accumulation and translocation to the nucleus, which attached to the membrane of (3-catenin,which expression was also decreased when transfected SARI to GLC-82. Based on immunoprecipitation experiments, GSK-3β and SARI are in close contaction. The mechanism may be due to the SARI is not a phosphorylase, so SARI activation of GSK-3β is mediated by an independent phosphorylase that is in close contact with the compound. And based on Ser9(S9, a negative regulator of the site) phosphorylation of GSK-3β level was significantly increased but β-catenin/TCF transcriptional activity (TOP/FOP) decreased. Similarly, by transient transfection SARI-siRNA to NCI-H1650cells, SARI expression can increase GSK-3β phosphorylation (S9) and β-catenin/TCF transcriptional activity by Wnt medium processing, the effect is much more pronounced. Therefore, SARI is used to activate the by S9reduction phosphate into GSK-3β, thereby regulating GSK-3β-β-catenin signal. Although Wnt was only a slight to the EMT in the NCI-H1650cells, but SARI-siRNA knockout of EMT markers increased significantly. Conversely, the restoration of the SARI expression in GLC-82cells was to prevent Wnt-induced EMT. This indicates that the SARI is the antagonist of the Wnt-mediated EMT. SARI can activate GSK-3β activity and thus degradation of cytoplasmic P-catenin and reduce the nucleus of P-catenin transcriptional activity, but overexpression of P-catenin can reverse SARI inhibition is unknown, in this regard to do the testing. In cells transfected with SARI, a gradual increasing of β-catenin cDNA dose increase beta-catenin transcriptional activity, restore the EMT markers and changes in cell morphology. Similar in the knockout the SARI NCI-H1650cells, overexpression of P-catenin and active beta-catenin transcription can be induced by the EMT, showing a dose-dependent, and cell morphology changed.The third part in situ animal models, we compare the knockout the SARI genes in the NCI-H1650cells and the control group transfer potential in situ animal models,. Before the inoculated cells, we must make sure that the fluorescence activity in each subgroup was consistent. Using bioluminescence imaging (BLI), we monitor tumor growth and metastasis initiating. A week later, the BLI detected in different parts of the mice inoculated with NCI-H1650cells-the KD obvious tumor metastasis damage. On the contrary, the control mice only can see the original tumor site and no transfer of signs after5weeks. The immunohistochemical staining revealed that most of the tumor cells strongly positive for vimentin, but weak expression of E-cadherin and cytokeratin. The above experimental data provide strong evidence that SARI in vivo inhibition of lung adenocarcinoma metastasis. Conclusion:This study delineates the functional role of SARI in EMT, which also explains how loss of SARI in lung adenocarcinoma underlines the onset of aggressive metastatic lung adenocarcinoma. We believe that the assessment of SARI expression in lung adenocarcinoma specimens can be a valuable prognostic biomarker for the risk of lung adenocarcinoma metastasis, and that the delineation of SARI function could provide a potential intervention strategy for lung adenocarcinoma metastasis.