The Mechanisms Of Estrogen Receptor β Promoting The Progression Of Lung Adenocarcinoma

Lung cancer, especially non small cell lung cancer (NSCLC), remains the leading cause of cancer-related mortality for both men and women worldwide. The disease is largely preventable as most cases are due to tobacco smoking. However, global statistics estimate that 15% of lung cancers in men and 53% in women are not attributable to smoking. Growing evidence suggests that for women, the risk of all major histological types of lung cancer, especially lung adenocarcinoma, is approximately three times higher than that for men, independent of the number of cigarettes smoked per day. Furthermore, there is also evidence for a higher risk of lung cancer among females undergoing estrogen replacement therapy. It suggests that endocrine factors, namely estrogens, may play an important role in the pathogenesis of lung cancer.The effects of estrogens are mediated via two estrogen receptor (ER) isoforms, that is, ER alpha (ERa) and ER beta (ERP). ERαin NSCLC have been evaluated mostly by immunohistochemistry. However, the result of most research showed that there was no expression of ERa in NSCLC or ERa was not predictive of survival. Our previous study showed that ERβwas highly expressed in Chinese NSCLC. But the roles of ERβin Chinese NSCLC have not been clarified as yet. In addition, both estrogen and midkine (MK) can induce NSCLC epithelial-mesenchymal transition (EMT). MK expression is enhanced in many human solid tumors and it regulates the angiogenesis process of cancer. EMT plays a specific role in the migration of tumor cells. Although many reports indicate the correlation between estradiol (E2) and MK, the precise mechanism on their interreaction is still unknown. So in the present study, two Chinese lung adenocarcinoma cell lines, SPC-A1 and LTEP-a2, were used and the role of ERβin lung tumorigenesis was focused to be investigated by in vitro and in vivo experiments. In addition, we further to analyze the interreaction of E2, MK and EMT and then reveal the regulatory mechanisms of E2 on MK expression.So we checked and analyzed the level of estradiol and MK in serum from 21 lung adenocarcinoma. In addition, we also tested the expression of ERβand MK in lung adenocarcinoma tissues. The results showed that there was a significant association between E2 and MK or ERP and MK expression. These results suggested that estrogen may play a role in lung cancer and regulate the MK expression.In order to study the roles of ERβin NSCLC progression, we construct the ERP expression plasmid and designed the ERP specific siRNAs. Cell proliferation, apoptosis, clone formation and tumorigenesis were checked after transfection of plasmid and siRNAs into SPC-A1 and LTEP-a2. The results showed that over-expressed ERP can promote cell proliferation, clone formation and migration ability, while ERP-siRNA can inhibit the cell proliferation and promote the apoptosis of NSCLC cells. To further study the effects of ERβ-siRNA on promotion of NSCLC cell apoptosis, we checked the mitochondrial membrane potential, release of cytochrome c and the activation of caspase3/9. The results showed that siRNA targeting ERP gene can induce NSCLC cells apoptosis via mitochondrial depolarization and caspase-3 activation. These results indicated that ERβplays an important role in development of Chinese NSCLC. This suggests that ERP deactivation or down-regulation may possess potential therapeutic utility for the treatment of lung cancer.MK is a heparin-binding growth factor. It has various biological activities such as carcinogenesis and migration. To clarify the mechanisms of E2 in the regulation of MK expression. Estradiol, estrogen antagonist, ER selective agonist, inhibitor of MAPK and PI3K were used for treatment of LTEP-a2 and A549 cells, and the change of MK expression was analyzed. We found that E2 increased MK mRNA expression in LTEP-a2 and A549 lung adenocarcinoma cells in a time-dependent manner. E2-induced MK expression was inhibited by the estrogen receptor (ER) antagonist ICI 182,780 and Tamxifen but not by phosphoinositide-3 kinase and MAPK inhibitors, indicating a genomic mechanism of E2 on the regulation of MK transcription. Moreover, an estrogen response element (ERE) in the MK promoter bound ERs in vitro in lung adenocarcinoma cells. After construction of plasmid with the promoter of MK, E2 can induce the luciferase activity. Chromatin immunoprecipitation(CHIP) assays exhibited that E2 induced ERa and ERβrecruitment to the ERE in these cells. Furthermore, the results of confocal analysis of location of ER showed that ERa and ERβmainly located in nucleus of LTEP-a2 and A549. In further study, after transfection of ERa or ERβspecific siRNA, we found that knockdown of ERβhas more effect on MK expression than ERa. These data together indicate that both ERs, especially ERP mediate the E2-induced transcription of MK in LTEP-a2 cells.Although it is reported that MK can enhance proliferation and migration of tumor cells and EMT is an important step in migration of tumor cells, it is not clear what the roles of MK in the process of E2 induced EMT. So we firstly identified that estradiol can enhance the expression of MK protein. Then phage-contrast and qPCR technology were used to identify that E2 and MK can indeed induce the NSCLC cell EMT. Furthemore, we checked the status of NSCLC cell EMT after transfection of MK-siRNA. The results showed that knockdown of MK could block EMT under E2 stimulation. It suggests that MK plays a pivotal role in progression of E2-regulated EMT.In conclusion, in this study we found that both estrogen and ERβhave significant association with MK in NSCLC patients and ERβcan promote the progression of Chinese NSCLC. In addition, we also found that estrogen induced the expression of MK by recruiting the ERs to ERE of MK promoter and ERβmay play a predominant role in this regulation process. Furthermore, MK is found to be an important molecule in process of E2 induced EMT.