Transforming growth factor-beta1 regulation of ATF-3 and identification of ATF-3 target genes in breast cancer cells.

Transforming growth factor-beta1 (TGF-beta1) is a crucial molecule for stimulation of breast cancer invasion and formation of bone metastases. The molecular mechanisms of how TGF-beta1 mediates these effects have yet to be completely determined. We have found that activating transcription factor-3 (ATF-3) is strongly stimulated and its level is sustained by TGF-beta1 in highly invasive and metastatic human breast cancer (MDA-MB231) and in mouse mammary pad tumor cells (r3T). ATF-3 is also overexpressed in human primary breast cancer tissue. Overexpression of ATF-3 increased normal human mammary epithelial cell number and DNA synthesis suggesting a role for ATF-3 in cell proliferation. The functional role of ATF-3 in breast cancer progression was determined by the RNA interference technique. Knockdown of ATF-3 by ATF-3 shRNA in MDA-MB231 cells decreased expression of cell cycle gene, cyclin A1 in MDA-MB231 cells. ATF-3 shRNA also decreased expression of an invasive and metastatic gene, matrix metalloproteinase-13 (MMP-13; collagenase-3) in these cells. Chromatin immunoprecipitation experiments identified the direct physical interaction of ATF-3 protein on the human MMP-13 promoter. Thus, the dysregulation of ATF-3 by TGF-beta1 is likely to activate cyclin A1 and MMP-13 genes in breast cancer cells and that would be key to the subsequent cancer cell invasion and metastasis.

Kwok S ，Rittling SR ，Partridge NC ，Benson CS ，Thiyagaraj M ，Srinivasan N ，Selvamurugan N ... -  《-》

Silencing of transforming growth factor-beta1 in situ by RNA interference for breast cancer: implications for proliferation and migration in vitro and metastasis in vivo.

Moore LD ，Isayeva T ，Siegal GP ，Ponnazhagan S ... -  《CLINICAL CANCER RESEARCH》

Transforming growth factor-β1 regulation of ATF-3, c-Jun and JunB proteins for activation of matrix metalloproteinase-13 gene in human breast cancer cells.

Transforming growth factor-beta1 (TGF-β1) plays a significant role in breast cancer mediated bone metastasis, and it stimulated expression of matrix metalloproteinase-13 (MMP-13; an invasive and metastasis gene) via activating transcription factor-3 (ATF-3) in human breast cancer cells (MDA-MB231). We further dissected the role of ATF-3 and its interacting proteins (activator protein-1; AP-1) for TGF-β1-stimulation of MMP-13 expression in these cells. Chromatin immunoprecipitation (ChIP) experiment identified the TGF-β1-stimulation of ATF-3 interaction at the AP-1 site of the MMP-13 promoter in a sustained and prolonged manner in MDA-MB231 cells. In silico protein-protein interaction, co-immunoprecipitation and western blot experiments identified the ATF-3 interaction and regulation of c-Jun and JunB proteins in these cells. The sequential ChIP assay confirmed the presence of c-Jun/ATF-3 complex at the AP-1 site of the MMP-13 promoter in MDA-MB231 cells upon TGF-β1-treatment. Hence, our results suggested that TGF-β1-treatment stimulated a sustained and prolonged expression of ATF-3, and its interaction and regulation of c-Jun protein and their assembly as a protein complex at the AP-1 site of the MMP-13 promoter could be responsible for MMP-13 gene activation in MDA-MB231 cells.

Gokulnath M ，Swetha R ，Thejaswini G ，Shilpa P ，Selvamurugan N ... -  《-》

Runx2, a target gene for activating transcription factor-3 in human breast cancer cells.

Gokulnath M ，Partridge NC ，Selvamurugan N 《-》

MiR-4638-3p regulates transforming growth factor-β1-induced activating transcription factor-3 and cell proliferation, invasion, and apoptosis in human breast cancer cells.

TGF-β1 (transforming growth factor-beta1), a secreted polypeptide cytokine, stimulates ATF-3 (activating transcription factor-3) expression in a sustained and prolonged manner in human breast cancer cells (MDA-MB231), but not in normal human mammary epithelial cells (MCF-10A). Cyclin A (cell proliferation gene), Runx2 (metastasis gene), and MMP-13 (matrix metalloproteinase-13; invasive gene) were identified as ATF-3 target genes in these cells. Because ATF-3 has very few druggable sites, its direct targeting is difficult. Recent evidence has indicated that microRNAs (miRNAs) are key players in the post-transcriptional modulation of gene expression under several conditions. Bioinformatic analysis suggested a list of putative miRNAs that target ATF-3. Therefore, we hypothesized that TGF-β1 downregulates the miRNAs that target ATF-3, resulting in the activation of genes that participate in breast cancer progression and skeletal metastasis. Our findings indicate that TGF-β1 downregulated the expression of miR-4638-3p in MDA-MB231 cells. At the molecular level, forced expression of miR-4638-3p reduced the expression of ATF-3 and its downstream targets, Runx2 and MMP-13, in these cells. At the cellular level, overexpression of miR-4638-3p reduced proliferation, invasion, and migration, and induced G0/G1 cell cycle arrest and apoptosis in MDA-MB231 cells. Overall, this study highlights the possibility of utilizing miR-4638-3p as a therapeutic molecule to curb skeletal metastasis of breast cancer cells.

Akshaya RL ，Rohini M ，He Z ，Partridge NC ，Selvamurugan N ... -  《-》