Estrogen Promotes Tumor Metastasis Though Estrogen Receptor ?(ER?) By Regulating Matrix-metalloproteinase-2(MMP-2) In Non-small Cell Lung Cancer

Objectve: Mortality rates in non–small-cell lung cancer(NSCLC) patients remain overly high among malignant tumors worldwide, of which deaths spread metastatic accounts for >70%. Several Studies including our previous work also evidently proved that Estrogen Receptor(ER?) is the dominant ER in the development of human NSCLC. The present study revealed that estrogen induces cell proliferation of NSCLC cells in vitro, developing in human tumor xenografts and in urethane-induced NSCLC animal models, raising the question of whether estrogen-induced ER? would promote lung cancer metastasis progression and what's its mechanisms.Based on this, we focus on the expression of ER? and MMPs and the relationships between them in the tumor tissue from NSCLC patients. We also demostrate the overexpression of ER?,MMPs and the activation of p38 MAPK and AKT singal pathway in lung adenocarcinoma cell line that is induced by E2, the ER?-selective agonist PPT,the ER?-selective agonist DPN and the ER antagonist fulvestrant.Additionally,we developed of a novel mouse models of NSCLC lung metastasis and evaluated it in vivo. Our research attempt to clarify the role and mechanism ER? acted in NSCLC.Methods: Our study were divided into three parts:(A) Firstly we evaluated the expressions of ER?, MMP1,MMP2 and MMP9 by immunohistochemistry in tumor and corresponding normal lung tissues and analysised the correlation between their expressions and clinical pathological features.Then to clarified the relationship ofthe ER? expression between primary tumors and metastatic lymph node from the same NSCLC patient,we evaluated 30 samples in the paired primary tumors and metastatic lymph node also by Immunohistochemical analysis.(B)We stimulated NSCLC cell line A549 and H1793 by E2, the ER?-selective agonist PPT,the ER?-selective agonist DPN and the ER antagonist fulvestrant to find the their change in ablitiies of invasion, migration and protein expression.Additonlly,we used RNAi technich to regulated the expression of ER? in NSCLC cell line to further verify our results.(C)Finally,to investigate whether Estrogen promote the metastatic ability of A549 cells in lung metastatic mouse model,we injected A549 cells into 4-week-old female BALB/c nude mice via the tail vein. Then we intraperitoneally injected with E2,PPT,DPN,and E2+Ful mentioned above in each group twice weekly.In the end lungs of murine were removed and weighed, the number of metastatic lesions was determined macroscopically.Results:(A)Our study data clearly revealed that the overexpression of ER? was significantly correlated with poorer tumor differentation and distant metastasis. Tumor samples showed increased levels of MMPs compared with normal lung tissues,however it was only significant correlation between ER? and MMP2.Negative results was observed in MMP1 and MMP9. Then the expression of ER? was significantly higher in metastasis lymph node tissue than in its primary NSCLC tumor tissue,which further suggest ER? activation may enhance aggressiveness of lung cancer cells metastasis.(B)In vivo,we first found that Estrogen could activate ER? and MMP-2 in a dose-dependent manner and also time-dependent manner in NSCLC cell A549. Estrogen and specific-ER? agonist DPN increased ER? and MMP-2 expression of NSCLC cells and also incresed the aggresiveness of invasion and migration. In vitro, overexpressing ER? increased MMP-2 expression,promoted cell migration andinvasion of lung cancer cells, while silencing ER? had the opposite effects.The results also demonstrated that ER? and MMP-2 activation significantly associated with p38 MAPK and AKT phosphorylation without alternation in total protein expression of p38 MAPK and AKT.(C) In vivo experiment,the tumor nodes formation rate of A549 cells induced by E2 and DPN was larger and more numerous compared with the control group.Unsurprisingly, Treatment with ER-antagonist Fulvestrant results in a significantly decreased tumor formation in lung metastasis.Considering the alternation in protein expression in murine lung cancer tissue,we found ER?,MMP-2, phosphorylated p38 MAPK and phosphorylated AKT overexpressed in E2 and DPN group. Opposite effects were observed in Ful group.Conclusion: In conclusion, this study reveal evidently that overexpression of ER? related with patients who had distant metastasis and poor tumor differentation grade and the expression of ER? was higher in metastasis lymph node tissue than in its primary NSCLC tumor tissue. Furthermore, the activation of ER? induced by estrogen promotes non-small lung cancer metastasis by upregrulating MMP-2 and is involved in the p38 MAPK and AKT pathway.ER? targeting treatment could serve as an appealing strategy for advanced lung cancer patient therapy,particulally for whom suffering metastasis.