RUNX2 TRANSCRIPTIONAL ACTIVATION MECHANISMS IN ANGIOGENESIS

Jasjit Saini¹, Michele Vitolo^2,4, and Antonino Passaniti^2,3,4

¹Breast Cancer Undergraduate Research Experience Program, University of Maryland, Baltimore County, ²Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore (UMB), ³Department of Pathology, UMB, ⁴The Greenbaum Cancer Center Program in Oncology, UMB

Angiogenesis, or the growth of new blood vessels from existing vessels, occurs during embryonic development, wound healing, and the menstrual cycle. This process is rare in adults and, therefore, tightly regulated. However, dysregulation of angiogenesis may worsen many diseases. Insufficient angiogenesis may exacerbate coronary artery disease and stroke, and excessive angiogenesis can intensify ailments such as macular degeneration, rheumatoid arthritis, psoriasis, and breast cancer. Angiogenesis is a multi-step process that depends on the release of growth factors from injured or diseased tissue, leading to endothelial cell (EC) activation, migration, and invasion into the surrounding tissue to form new vessels. The transcription factor RUNX2, with its cofactor, CBFß, is expressed in endothelial cells, and has been implicated as a possible regulator of EC migration and/or invasion. Determining the mechanism of activation of downstream genes by RUNX2 can, therefore, provide insight into these biological processes.

The RUNX2 binding site was cloned into an expression vector containing a luciferase reporter gene, and the expression of luciferase activity in human bone marrow EC (HBME) and human embryonic kidney 293T cells was determined. RUNX2 promoter-luciferase activity was found to be 2-3 fold higher than control vector and this induction could be inhibited by the introduction of a dominant negative (DN) form of the gene, known as Runt, which consists only of the DNA and CBFß binding domain of the full-length Runx protein. Mutated sequences of the DN Runt, with altered DNA and CBFß binding sites, have been sub-cloned into expression vectors, and will be employed in the luciferase assay to determine which domains of Runt are responsible for the inhibition. Investigating RUNX2 activation and inhibition will aid in clarification of the basic mechanisms of angiogenesis, which is an important target in developing treatments for breast cancer and other diseases.