Breast cancer is the second leading cause of death in women following lung cancer. The incidence of breast cancer mortality has decreased in recent years as a result of more efficient preventative medicine and improved detection technologies. However, women who suffer from breast cancer face physical and mental distress associated with treatment. Importantly, there is lack of a subjective and reliable evaluation system for clinical prognosis of breast cancer patients. This makes the process of choosing optimal therapeutic strategies and strategies for aftercare more difficult for both patients and clinicians. As we know, uncontrolled proliferation and invasion of transformed cells is the distinct character of poorly differentiated malignant tumors and poses the greatest health risk to the patient. On the contrary, the tumors with higher degree of differentiation usually present a less invasive phenotype and better clinical outcome. It is well known that all-trans retinoic acid (RA) induces differentiation of a wide variety of malignancies and may prevent breast cancer or improve clinical outcome.

The epithelial cells of mammary gland of a reproductive woman undergo continuous and well regulated regenerative cycles initiated by a small population of stem cells. These cells retain their ability to grow and resist differentiation and programmed cell death in a process known as self-renewal. These cells have a prolonged life-span and may shield harmful genetic mutations from the protective effects of programmed cell death. This suggests that stem cells may be a sensitive target of causative lesions of breast cancer and promising site for chemo-prevention.

The ability of mammary stem cells to preserve the process of self-renewal has been linked to the activity of a gene called Delta-N-p63 (DN-p63). Our preliminary data have shown RA contributes to differentiation in a stem cell model called immortalized mammary epithelial cell (IMEC). Treatment of IMECs with RA results in growth arrest and the loss of DN-p63 expression, which we believe is critical for initiation of differentiation. DN-p63 is a member of the p53 family of transcriptional regulators and opposes the activities of the tumor suppressor, p53. It is believed that the ability of DN-p63 to oppose the activity of p53 accounts for its ability to preserve self-renewal. Robust expression of DN-p63 is a distinct quality of IMEC and is essential for proliferation. Therefore, we hypothesize that differentiation induced by RA treatment of IMECs must depend on downregulation of DN-p63. This loss of DN-p63 will cause changes in the expression pattern of p53/DN-p63-target genes and these genes are predicted to be mediators of differentiation. Furthermore, we predict that expression of these genes will be correlated to a positive effect on clinical prognosis.

We propose to identify genes that represent early mediators of cellular differentiation and to determine whether these genes have a positive correlation with improved clinical outcome. We hypothesize that RA causes the suppression of DN-p63 and this results in increased expression of p53 target genes associated with differentiation. Specific Aim 1 proposes to use micro-array technology to discover gene expression profile differences between differentiation and non-differentiation status. Our goal will be to identify genes that are positively regulated by RA in a manner that is sensitive to DN-p63. In Specific Aim 2 we propose to further analyze this list of candidate genes for RA-inducible expression in the mouse mammary gland. This will allow for confirmation and localization of such pro-differentiation genes in mice. Finally, in Specific Aim 3, we plan to determine the pattern of expression of validated target genes in clinical breast cancer samples. Our goal will be to determine whether expression of pro-differentiation target genes in breast cancers correlates with reduced tumor aggression and improved clinical outcome.

Our research project will highlight the role of DN-63 in differentiation of stem cells and uses RA as an effective tool to induce differentiation in IMEC cells. Successful fulfillment of these studies will definitively identify a set of pro-differentiation genes in breast cancer and will be very useful to establish a subjective evaluation system for diagnosis, staging and prediction of therapeutic efficacy.