Contributions of retinoblastoma and p53 to breast carcinogenesis

The lifetime risk for a woman to develop breast cancer is 1 in 9 and yet relatively little is known about the early events and conditions that allows for this disease to develop. Tumor suppressor genes are fundamental to the regulation of cellular proliferation and are frequent targets of mutations in tumors. Abrogation of these tumor suppressors is thus a critical event during carcinogenesis. Understanding how these perturbations promote cancer development in imperative. Two such genes, retinoblastoma (RB) and p53, have well-defined roles in modulating cell growth and have been found to be mutated in a high proportion of breast tumors. To study the contributions of RB and p53 mutations to breast carcinogenesis, human mammary epithelial cells (HMEC) deficient for these two proteins were constructed. Oncogenic viral proteins, including human papillomavirus type 16 (HPV16) E6 and E7 proteins, bind to and inactivate p53 and RB protein (pRB), respectively, through sequestration and induction of proteolysis. Primary outgrowth cells from normal human breast organoids were infected with retroviruses expressing the HPV16 E6, E6, or E6 and E7 (E6/E7) genes. Stably infected cells were selected by culturing in antibiotic-containing medium. HMEC=E6 and HMEC+E7 were shown to be deficient for p53 and pRB, respectively, by western immunoblot analysis. The extracellular matrix (ECM) has a key role in mediating cellular signaling that regulates proliferation, Differentiation, apoptosis, and morphogenesis. These processes can be recapitulated for breast epithelial cells in vitro by culturing cells within a reconstituted basement membrane (rBM). This rBM culture system allows for normal HMEC to differentiate into polarized acinar structures. In rBM, HMEC+E7 formed acini-like structures that were not fully differentiated. Culturing HMEC+E6 in this ECM, induced apoptosis as determined by presence of fragmented DNA and caspase activity, both hallmarks of apoptosis. Up-regulation of the Fas receptor (FasR) was observed in these HMEC-E6 cultures and other necessary components of the Fas signaling pathway were expressed. Finally, co expression of HPV16 E6 and E7 circumvented apoptosis and instead permitted acinar structure formation in ECM. These data suggest new functions for pRB and p53 in breast epithelial cells in vivo and further define the cooperative relationship between RB and p53.