The Molecular Mechanism And Significance Of RUNX2 In EMT And Metastasis Of Breast Cancer Cells

Breast cancer is one of the most common malignant tumors for females,and about 1.2 million women each year suffer it worldwide,accounting for 7-10%of all malignant tumors.Metastasis is the main cause of death for breast cancer patients,epithelial-mesenchymal transition(EMT)is the key stage for early metastasis.To study the molecular mechanisms of breast cancer metastasis and EMT would help us to estimate the prognosis andguide us for better treatment of breast cancer patients.Methods:To investigate the key role of RUNX2 in the process of EMT in breast cancer and its regulatory mechanism,we first studied the relationship between expression of Runx2 and clinical pathological parameters and also the prognosis of breast cancer patients through immunohistochemical technique in 493 cases.Then,we constructed TGF-betal-induce EMT model of breast cancer.In order to research the key role of RUNX2 in EMT,we applied the siRNA to silence the expression of RUNX2.We further studied the molecular mechanism of RUNX2 regulation.Besides the Smad pathway,TGF-beta 1 pathway can also activate other signal pathways at the same time,for example,PI3K/AKT and MAPK pathways,which also played an important role in the regulation of RUNX2.Our previous studies suggested that the influence of RUNX2 on the prognosis of patients with breast cancer was associated with the expression of ER alpha.So there may be a crosstalk between the ER alpha and RUNX2 in the regulation of EMT.Since we have confirmed that Slug is the common downstream transcription factor of ER alpha and RUNX2,we further studied the role of Slug in the process of EMT.Results:The results showed that there were significant correlation between RUNX2 and ER(p=0.014),HER2(p=0.02),histological grade(p=0.046),tumor staging(p= 0.0004),and distant metastasis(p<0.0001).We further analyzed the correlation between expression of RUNX2 and prognosis in the 463 cases without distant metastasis.The inducing experiment showed that shape of EMT cells changed into long spindle,which was accompanied by the change of a series of EMT-related proteins,such as increased E-cadherin and decreased Vimentin,Fibronectin and so on.However,the expression of RUNX2 did not change significantly,Then we discovered that lack of RUNX2 could inhibit the EMT-inducing effect of TGF beta 1 in breast cancer MCF-7 cell lines.The outcome of Western Blot and RT-PCT also support this conclusion.In addition,the invasion of the cells was also significantly reduced in the absent of RUNX2,accompanied by the decline of mRNA transcription of MMP9,MMP13 and VEGF,which is involved in the cell invasion.It was discovered that Smad2 and RUNX2 combined to form a complex in the stimulation of TGF-beta 1,which was accompanied with phosphorylation of Smad2 and RUNX2.In our previous work,we found that the TGF-beta 1 could not increase the expressoin of RUNX2.Studies found that in the presence of specific inhibitors of PI3K/AKT and MAPK pathways,the phosphorylation level of RUNX2 decreased,accompanied by the up-regulation of E-cadherin,and down-regulation of Vimentin,Fibronectin,Snail,Slug and so on,which were all important EMT-related proteins.We further detected the effects of PI3K/AKT and MAPK pathways on distribution of RUNX2 in breast cancer cells by immunofluorescence method.Results showed that the MAPK pathway plays a main role in the entrance of RUNX2 into nucleus,and RUNX2 would not be unable to enter the nucleus of the activity of MAPK pathway was inhibited.However,PI3K/AKT pathway did not have obvious regulation effects in this process.Studies have found that ER alpha signaling pathway inhibits the expression of RUNX2 and downstream transcription factors Slug,which was the common downstream target of ER alpha and RUNX2.Slug could receive inhibiting or stimulating signals from the ER alpha or RUNX2 respectively,thereby accurately regulate EMT process of breast cancer cells.In addition,ER signal could also inhibit the expression of RUNX2 directly.Results show that the Slug plays a crucial role in EMT,and in the absence of Slug expression,TGF-beta 1-induced EMT process is suppressed.Therefore,ER signal by suppressing the expression of RUNX2 and Slug,negatively regulates the process of EMT.Conclusion:There were significant negative correlation between the expression of RUNX2 and disease-free survival(p=0.01)and overall survival(p=0.003),which may be associated with ER status.RUNX2 plays an important role in the process of EMT and cell invasion in breast cancer and it may be changing its phosphorylation status that regulated RUNX2 in the EM process.These findings suggest that PI3K/AKT and MAPK pathways play important roles in the regulation of RUNX2 and also in the process of EMT,the MAPK pathway,however,plays a leading role in regulating the process of RUNX2 entrance into nuclear.ER signal pathway and RUNX2,mainly through the regulation of the downstream Slug,formed the common regulation of EMT,which is a novel mechanism of EMT and will provide a new method in the treatment of breast cancer metastasis.