An Association Of Fra-1 With Oncogenesis, Invasiveness And Metastasis Of Breast

A proto-oncogene, Fra-1 is a Fos family member and an AP-1 transcription factor component. Emerging evidence suggests an important role for Fra-1 in cell motility, invasion, and progression of the transformed state in several cell types. The recent data indicated that overexpressed Fra-1 in fibroblasts causes anchorage-independent growth and oncogenic transformation. Elevated Fra-1 mRNA and protein have been detected in multiple tumors as well as transformed cell lines, including breast, colon and lung.In the present work, The expression of Fra-1 was investigated by immunohistochemistry in neoplastic breast diseases ranging from benign fibroadenoma to very aggressive undifferentiated carcinoma. All neoplastic breast tissues, either benign or malignant breast tissues, were nuclear immunoreactive for Fra-1-recognizing antibody. The pattern of Fra-1 expression by benign neoplastic cells was predominantly nuclear. However, the nuclear/cytoplasmic concomitant immunoreactivity was observed in all types of breast carcinomas. No correlations were found between Fra-1 expression and breast cancer prognostic markers: ER, PR and HER2. A clear shift in Fra-1 immunoreactivity, from an exclusively nuclear to a simultaneous nuclear and cytoplasmic locmatalization was noticed in～90% of breast carcinomas. By RT-PCR and Western blot, Fra-1 were found to be consistently overexpressed in the more highly aggressive breast cancer cells.To illustrate the relationship between Fra-1 with motility and invasiveness of breast cancer cell, in this study, Fra-1 was overexpressed in MCF-7 cell. By colony formation assay, Matrigel adhesion assay and wound healing assay, the enhanced growth, adhesion and motility of MCF-7 cells stably overexpressing Fra-1 were detected.To investigate how Fra-1 influences the growth, adhesion and motility of MCF-7 cell, MMP-9 promoter were cloned and ligated into the luciferase reporter constructs. The transcriptional activity of the MMP-9 was analyzed promoter through the measurement of luciferase activities in the MCF-7 cells transiently transfected with Fra-1.The positive regulation of Fra-1 on MMP-9 promoter was not detectable. By the analysis of MMP-9 promoter, a potential Stat3 binding site, just juxtaposed AP-1 consensus sequence, was noticed. The reporter assay showed that MMP-9 promoter was activated remarkably by cotransfection with Fra-1 and Stat3C. DNA affinity precipitation assay confirmed the binding of Stat3 and Fra-1 to the elements of MMP-9 promoter and also revealed c-Jun recruited to Stat3-Fra-1 complex. ChIP-PCR further confirmed the binding of Stat3 and Fra-1 to the elements of MMP-9 promoter in vivo. By immunoprecipitation assay, the Stat3/Fra-1, Stat3/c-Jun and Fra-1/c-Jun complexes were identified in vivo. Our study demonstrated that the activation of MMP-9 promoter is dependent upon interactions of Fra-1/c-Jun with Stat3. A juxtaposed Stat3/AP-1 element plays a crucial role in the manner of enhancersome in the activation of MMP-9 gene. The functional cooperation of the Stat3 and AP-1 transcription factors is required for the transcription of MMP-9 gene.Taken together, our findings suggest that overexpression of Fra-1, leading to a persistent high cytoplasmic accumulation, may play a role in the process of breast carcinogenesis. Overexpression of Fra-1 increased the growth, adhesion and motility of MCF-7 cells. Fra-1 and Stat3 synergistically regulate activation of human MMP-9 gene.