The molecules that control Retinoblastoma (Rb) phosphorylation called "the Rb pathway" consisting of D-type cyclins, cyclin dependent kinases (cdks) and cdk inhibitors (cdkis) as well as Rb itself are regularly disrupted in most cancer types including breast cancer leading to excessive phosphorylation (hyperphosphorylation) of the Rb protein. Hyperphosphorylation of Rb is known to promote proliferation and block apoptosis. Treatments that inhibit cdk activity have begun to exhibit promise in the clinic in several cancer types. Although Rb can be phosphorylated on 15 amino acid residues, very little is known about the contribution any individual phosphorylated amino acid has on the function of Rb in proliferation and apoptosis. In this project we propose to undertake the first investigation of Rb phosphorylation in proliferation and apoptosis utilizing a model of breast epithelial cells using three-dimensional cultures. This model system is employed to recapitulate the physiological context in which breast epithelial cells regulate cellular processes. We propose to target Rb hyper- phosphorylation by using a novel method of activating Rb-specific phosphatase activity in breast epithelial cells grown in 3D cultures. In this way we can determine the effect of dephosphorylation of Rb on proliferation and apoptosis in these cells. In addition, we will perform site directed mutagenesis of each Rb phosphorylation site to either alanine or glutamic acid to block phosphorylation or mimic phosphorylation at each site and evaluate the effects on proliferation and apoptosis in breast epithelial cells grown in the 3D culture model. Finally, we will clarify the functional significance of complex formation between Rb and the pro-apoptotic protein Bak that is regulated by Rb dephosphorylation in apoptosis. Thus, the proposed studies will elucidate the role of specific Rb phosphorylation sites involved in the regulation of proliferation and apoptosis in breast cancer and will yield useful information that could inform the development of future therapies that target Rb phosphorylation in cancer.
In cancer, cells divide and proliferate in an uncontrolled manner. One protein, Retinoblastoma (Rb) is dysfunctional in breast cancer. This project is designed to study the function and regulation of this protein in a physiologically relevant model of breast cancer tumors.