Research Projects

Transforming growth factor-beta1 (TGF-β1), a multipotent cytokine, plays a vital role in normal and pre-malignant cells by maintaining homeostasis and suppressing tumor progression. Nevertheless, breast cancer advances when this TGF-β1 anti-proliferative response is lost. The effect of TGF-β1 on epithelial cell homeostasis and the significance of its disturbance in cancer have been attributed to a small collection of immediate TGF-β1 target genes that have been identified through independent studies over time. The development and metastasis of breast cancer can be changed by transcriptional factors. In contrast to normal TGF-β1 repression of ATF-3 in normal human mammary epithelial cells (MCF-10A), we found that TGF-β1 strongly stimulates and maintains the level of activating transcription factor-3 (ATF-3) in highly invasive and metastatic human breast cancer cells (MDA-MB-231) and mouse mammary pad tumor cells (r3T). In human primary tumor tumors, ATF-3 was also highly expressed. Cyclin A1 (cell proliferation gene), MMP-13 (matrix metalloproteinase-13; invasive gene), and Runx2 (bone metastasis gene) were found to be ATF-3 target genes, suggesting a role for ATF-3 in breast cancer progression and bone metastasis. We recently found that TGF-β1 stimulates ATF-3 to undergo posttranslational modification, such as sumoylation in human breast cancer cells. Hence, we hypothesized that the extended and sustained expression of ATF-3 by TGF-β1 could be due to its sumoylation, and this effect could protect ATF-3 from proteasomal degradation. Hence, in this proposal, we will identify and functionally characterize the TGF-β1-stimulated ATF-3 sumoylation and its sites using a cell culture in vitro system and a mouse model of breast cancer bone metastasis in vivo system. Consequently, the objectives of this proposal are to: 1. Determine the TGF-β1-stimulation of ATF-3 sumoylation in human normal mammary epithelial cells and human breast cancer cells by immunoprecipitation and western blot analyses. 2. Determine the role of TGF-β1-stimulated ATF-3 sumoylation for the expression of its target genes with sumoylation inhibitors and siRNAs in human breast cancer cells. 3. Identify the TGF-β1-stimulated ATF-3 sumoylation sites in human breast cancer cells using a strategy based on mass spectrometry. 4. Determine the functional role of the TGF-β1-stimulated ATF-3 sumoylation sites for the expression of its target genes with the wild or mutant ATF-3 sumoylation site(s) constructs in human breast cancer cells. 5. Determine the functional role of the TGF-β1-stimulated ATF-3 sumoylation sites using a mouse model of breast cancer bone metastasis in vivo with the wild or mutant ATF-3 sumoylation site(s) constructs. Identifying the importance of ATF-3 stability by sumoylation would further lead to translational research for developing small molecules against ATF-3 sumoylation in controlling breast cancer progression and bone metastasis.