Roles And Mechanisms Of Osterix In Bone Metastasis Of Breast Cancer

In recent years,breast cancer is among the most prevalent cancers worldwide.A majority of breast cancer-related deaths are due to metastatic tumor growth in distant organs,with bone being the most common site of metastasis.Despite the treatment of breast cancer improved continually,approximately 70% of breast cancer patients suffer from bone metastases,which are often leads to bone fractures,severe and persistent bone pain,spinal cord compression,anemia,hypercalcemia and a series of complications,seriously affects life quality of patients.Osterix(Osx/Sp7),a member of the Sp family of zinc finger-containing transcription factors,is a master regulator of osteogenesis and bone formation during embryonic development.It has been reported that during bone development Osx upregulated the expression of VEGF,MMP13 and MMP9.Moreover,VEGF and MMP13 have been proven to be the direct target genes of Osx.But the mechanism of Osx raised MMP9 was not reported.Through bioinformatics analysis,we found that there exist three potential Osx binding sites in the MMP9 promoter.So we constructed pGL3 luciferase reporter plasmid containing 1,2 and 3 binding sites or 3 binding sites were mutated.By luciferase assays we demonstrated that Osx was able to increase the promoter activity of MMP9.Electrophoretic mobility shift assay(EMSA)provided the evidence that Osx enhanced MMP9 promoter activity by binding to CCAAT sequence located in MMP9 promoter.In addition,we found that Osx presented a higher expression in highly metastatic breast cancer cells,while did not expressed in low metastatic breast cancer cells.So we speculated that Osx may promote the invasion of breast cancer by raising the expression of MMP9.We constructed the MDA-MB 231 stable cell lines with Osx overexpression or knockdown,respectively.By Real-time PCR and zymography assays,we demonstrated that the mRNA expression and enzyme activity of MMP9 were upregulated by Osx.Moreover,we demonstrated that Osx promoted the invasion of breast cancer cells by upregulating MMP9.At the same time,we found that angiogenesis factors VEGF and CD34 as well as the osteoclast activation factors IL-8 and PTHrP were also upregulated by Osx in breast cancer cells.By establishing the orthotopic and tibia model with nude mice,we proved that the enzyme activity of MMP9 and the expression of MMP13,VEGF,CD34,IL-8 and PTHrP were upregulated by Osx in vivo further.Finally,we examined the expression of Osx in breast cancer tissues using the clinical tissue microarray.Results showed that Osx expression was significantly correlated with the patient's survival and lymph node metastasis.Taken together,we demonstrated for the first time that Osx promoted the bone metastasis of breast cancer in in vitro and in vivo,suggesting that Osx may be a novel target for the prevention and treatment of breast cancer bone metastases.