The Molecular Mechanisms Of Estrogen Induced The Transcription Factor ZEB-1on Bone Metastasis Of NSCLC

The morbidity and mortality rates caused by NSCLC are among the highestin malignant tumor. Bone metastasis of NSCLC is a common complication whichcan cause severe pain, spinal cord compression, hypercalcemia, pathologicfractures, which affecting NSCLC patients’ quality of life. Our laboratory havestudied some mechnisims of bone metastasis in lung cancer, and discoveredseveral molecules, such as S100B, calcineurin which are closely related to bonemetastasis of lung cancer. However, the molecular mechanisms of bonemetastasis of NSCLC are far under elucidation.Recent study showed that estrogen could promote NSCLC bone progression,we found estrogen receptor was highly expressed in bone metastasis of NSCLCtissue compared to that without bone metastasis by immunohistochemistry, andthe expression of ER beta is more important than ER alpha.thus, further study toilluminate the exact mechanism of estrogen in the NSCLC bone metastasis maybe of some interest for developing the new therapy targeting in estrogen. PurposeThe purposes of present works are to investigate the effects of estrogen onbone metastasis in NSCLCcell and the role of EMT involved in it. Further studyis to characterize the role of ZEB-1contributing to estrogen-induced EMT andsubesquently promoting bone metastasis in NSCLC.Methods1Effects of estrogen on bone metastasis are studied by immunohistochemistry, invitro and in vivo invasion assay, ELISA detection of bone related moleculeexpression in NSCLC;2Effects of estrogen and its receptor antagonist ICI on EMT are detected byWestern blot, RT-PCR and cell morphology.3Important transcription factors which promote NSCLC cell EMT are screenedby high throughput RT-PCR. GFP labeled ZEB-1-shRNA was built, and packed,then infected into NSCLC A549. Effects of estrogen induced EMT and bonemetastasis of NSCLCafter ZEB-1silencing were detected by Western, blot,ELISA and in vivo experiments.Results:1Estrogen receptor was highly expressed in bone metastasis of NSCLC tissuecompared to that without bone metastasis, and the expression of ER beta.is moreimportant than ER alpha Cell invasion assay confirmed that estrogen can promoteNSCLC cell invasion, and estrogen receptor antagonist ICI can reduce estrogeninduced NSCLC cell invasion. In vivo experiments confirmed that estrogen canpromote NOD-SCID mice bone metastasis number, while ICI can reduce the theincidence of bone metastases.2Estrogen treated A549cells presented round to spindle-shaped transformationcompared to untreated A549cells. Western Blot confirmed it was through NSCLC cells EMT, which mesenchymal marker was promoted, and the epithelialMarker was inhibited (P <0.05). Estrogen receptor antagonist ICI can reverseestrogen induced EMT.3Key transcription factor ZEB-1in estrogens promoted NSCLC cell EMT wasscreened by high-throughput RT-PCR. Estrogen can promote the expression ofZEB-1, and ICI can inhibit expression of ZEB-1, which were comfirmed byWestern Blot. Inhibition of ZEB-1by ZEB-1shRNA exhibits cell morphologicalrecovery, through inhibition of fibronectin, promotion of E-cadherin.4Bone related molecule PTHrP expression was decreased after ZEB-1kockdown,and can decrease the NOD-SCID mice bone metastases numbers.Conclusion1Estrogen can promote NSCLC cell EMT, then promoting the NSCLC bonemetastases.2Estrogen receptor antagonists ICI could inhibit the estrogen induced EMT andNSCLC bone metastases.3ZEB-1may play a important role in the process of estrogen promoted EMT andNSCLC bone metastasis.4Interference of ZEB-1expression can effectively reverse estrogen induced EMT,and inhibit estrogen induced NSCLC bone metastases.