Molecular Mechanism Of Down-Regulation Of MMP9 Expression By KLF9 And Its Role In Breast Cancer Metastasis

In recent years,breast cancer is the most common malignancy in females worldwide.Breast cancer metastasis remains the main cause of failed therapy,poor prognosis and cancer-related death.It is shown that aberrant activity of NF-?B/MMP9(nuclear factor-?B,NF-?B/matrix metalloproteinase 9,MMP9)pathway is present in aggressive breast cancer types,which is associated with the degree of malignancy and metastasis.KLF9(Kriippel-like factor 9,KLF9)is a relatively unexplored transcription factor,and the function of KLF9 in cancer metastasis is still unknown.Recently,in a microarray-based gene expression study,the expression of KLF9 was significantly down-regulated in breast cancer and highly invasive breast cancer when compared to normal and non-invasive breast tissues,indicating a potential association of KLF9 with tumor metastasis.Owing to the negative correlation of KLF9 and MMP9 expressed in tumor tissues,we speculate that there might be potential mechanism involving the both factors to regulate cancer metastasis.However,the regulation of NF-?B/MMP9 by KLF9 still remains unknown.Therefore,we investigated the molecularmechanism that KLF9 regulate MMP9 expression to inhibit metastatic ability of breast cancer.The main works of this research are as follows:1)In the present study,we found that the expression of KLF9 was significantly down-regulated in highly invasive breast cancer cells when compared to non-invasive breast cancer cells by Western blot in different breast cancer cell lines.It was detected that KLF9 can suppress the migration and invasiveness of breast cancer cells,and was a cancer suppressive factor,as revealed by scratch wound-healing and Transwell assay.2)RT-PCR and Western blot assays indicated that KLF9 could inhibit the mRNA and protein expression of MMP9.Reporter-gene assay demonstrated that KLF9 down-regulated the activity of MMP9 transcription to inhibit MMP9 expression.KLF9 repressed human MMP9 gene promoter activity by binding to the-449 bp CACCC motif and interacting with NF-?B p50/p65,which interacted with the NF-?B response element of the MMP9 promoter,determined by truncated and mutant reporter gene assay and chromatin-immunoprecipitation(ChIP)assay.3)Co-immunoprecipitation(CoIP)showed that KLF9 can interact with p50/p65.ChIP assay suggested that the binding of KLF9 to NF-?B element on MMP9 promoter was dependent on p65.4)Reporter gene assay indicated that there was a synergistical suppression of KLF9 and HDAC1 to MMP9 promoter activity.CoIP confirmed that there was a positive interaction between KLF9 and HD AC1.CoIP and ChIP assays showed that KLF9 might increase the recruitment of HD AC 1 to p65,and also decrease the acetylation level of histone around the binding region of KLF9 with MMP9 promoter.Reporter gene and RT-PCR indicated that KLF9 could down-regulate the transcriptional activity of NF-?B target gene promoter,and also the mRNA levels of several targets.5)KLF9 can inhibit breast cancer metastasis through regulating the expression and activity of MMP9,revealed by scratch wound-healing,Transwell and the using of inhibitor of MMPs.Immunohistochemistry(IHC)analysis showed that the expression of KLF9 was negatively associated with the expression of MMP9,indicating a inverse relationship between the two proteins.6)In BALB/c mice,KLF9 could obviously inhibit lung metastasis of 4T1 cells when 4T1 cells were transfected with KLF9,indicating that KLF9 could suppress breast cancer metastasis in vivo.In summary,our data identified a new target gene of KLF9,and elucidated a novel mechanism that KLF9 transcriptionally regulated MMP9 expression.Mechanistically,KLF9 repressed human MMP9 promoter activity by binding to the CACCC motif and interacting with NF-?B p50/p65 at the NF-?B response element of the MMP9 promoter,accompanied with the recruitment of HDAC1,leading to decreased expression of MMP9.Furthermore,the expression of KLF9 was inversely correlated with the expression of MMP9 in breast cancer patients.This work emphasized an important role for KLF9 in suppressing human breast cancer metastasis and provided new insight into the underlying molecular mechanisms,which involved NF-?B/MMP9 pathway,a highly enhanced pathway in aggressive cancers.