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    INK4/ARF in Parity-Induced Protection of Breast Cancer

    Scientific Abstract:
    Title: Role of p16INK4/p19ARF in parity-induced protection of breast cancer Background. The rationale to study the roles of p16INK4a and p19ARF in mammary gland protection against tumorigenesis in parity is based on preliminary data demonstrating that significant increases (p<0.001) and (p<0.003) in p16INK4a and p53 (Lys373, Lys382) protein levels, respectively, were observed in the parous as compared to adult virgin mammary gland. During mammary gland development, p16INK4 protein level increased significantly (p<0.001) in lactation and remained high at 15-day of involution. The p19ARF null mammary gland demonstrated a similar pattern of alteration of p16INK4 level during development, but was ~60% lower than WT at all stages. Furthermore, in the WT, p53 (Lys373, Lys382) protein levels increased significantly (p<0.003) by 15-day of involution, but not in the p19ARF null mammary glands. These data suggest that p16INK4 and p19ARF may be involved in protective pathways induced via parity. Our recent study demonstrated that one consequence of the lack of p19ARF is that mammary epithelial cells achieve an indefinite division potential or immortalization in vivo. The mammary gland goes through a rapid ductal proliferation at puberty, then a proliferative cycle of pregnancy that includes ductal side branching and lobuloalveolar development. The lobuloalveolar structures persist until the end of lactation. The cells undergo growth arrest in early lactation followed by rapid tissue regression culminating in a return to normal cellularity 10 days after weaning. Women who have undergone a full-term pregnancy before the age of 20 have one-half the risk of developing breast cancer compared with women who have never gone through full-term pregnancy. The alternative reading frame product, p19ARF, is derived from the INK4a/ARF tumor suppressor locus. Together, the p16INK4a/p19ARF and p53 represent the two most frequently inactivated genes in human cancer, including breast cancer. Our recent study demonstrated that p19ARF is upregulated in pregnancy and involution during normal mammary gland development suggesting that p19ARF may function to protect cells from exceeding the normal rate of cell proliferation and apoptosis during development. Lack of p19ARF caused a significant increase (p< 0.05) in cell proliferation during pregnancy and a decrease in apoptosis (p< 0.02) associated with a delay in involution as compared to WT. Thus, the focus of this study is to determine whether p16INK4a or/and p19ARF provide protective measures against mammary carcinoma via parity. Objective/hypothesis: To determine whether p16INK4a or p19ARF provide protection against mammary carcinoma after treatment with estrogen (E) and progesterone (P). Furthermore, to determine the effects of p16INK4a and p19ARF on expression of genes related to E+P-induced mammary gland protection. Our previous study demonstrated that p19ARF increased in mammary glands that were stimulated with progesterone. Other studies suggested that pregnancy may provide protection to mammary epithelial cells against cancer, and that progesterone plays a critical role in the protection against mammary tumorigenesis afforded by early parity. Published results demonstrated that mammary epithelial cells of parous mice exhibit a block in proliferation that is induced by chemical carcinogens. I postulate that p16INK4a/p19ARF function have a potential role in this proliferation block and provide one pathway for protection against mammary carcinoma in parous mice. Specific Aim 1. Determine whether p19ARF provides protection against mammary carcinoma after short-term hormone treatment utilizing Balb/c p19ARF null mouse model. Aim 2. Determine whether p16INK4a provides protection against mammary carcinoma after short-term hormone treatment utilizing Balb/c p16INK4a null mouse model. Aim 3. Determine alterations in gene expression in mammary glands related to p16INK4a and p19ARF pathways upon hormone treatment of WT, p16INK4a and p19ARF null mice. Study Design: To measure mammary tumor incidence, cohort of Balb/c virgin WT, p16INK4a null and p19ARF mice will be treated with E+P to mimic pregnancy, then the hormones removed to allow mammary glands to completely involute for 15 days, followed by 4 doses of 1mg/week DMBA to induce mammary tumorigenesis. In parallel, similar groups of mice will receive DMBA without prior treatment with hormones. Mammary tumor incidence and latencies will be calculated and evaluated in correlation to the presence or absence of p16INK4a or p19ARF in the E+P treated and age-matched untreated mice. We will also determine alteration in the expression of genes in mammary glands of WT, p16INK4a null and p19ARF null mice after E+P treatment using DNA oligonucleotide microarrays to identify pathways that involve p16INK4a and p19ARF in mammary gland protection. Relevance: Our previous study demonstrated that p19ARF prevents normal mammary epithelial cells from exceeding the normal rate of cell proliferation and apoptosis during development. Loss of p19ARF alone in mammary epithelial cells may not lead to breast cancer, but my recent study confirmed that lacking p19ARF gene causes immortalization of mammary epithelial cells in vivo. Together with p16INK4a, p19ARF appear to provide an appropriate protection to the normal mammary epithelial cells that have gone through full-term pregnancy. Thus, this study is to understand the mechanisms of cancer prevention provided by p16INK4a/p19ARF through treatment with E+P.

    Lay Abstract:
    Title: Role of p16INK4/p19ARF in parity-induced protection of breast cancer Women who have undergone a full-term pregnancy before the age of 20 have one-half the risk of developing breast cancer compared with women who have never gone through full-term pregnancy. This prospective effect is observed universally among women of all ethnic groups. Parity in rats and mice also protect them against chemically induced mammary carcinogenesis. p16INK4a and p19ARF are two proteins that are produced from one gene, but each is a product of different region of that gene. When normal breast cells are stimulated by hormones, growth factors, or DNA damage cased by UV light, radiation or chemicals, to divide, signals pass through the breast cell to the nucleus to induce ARF synthesis to prevent abnormal increase in cell division. P19ARF functions as a cell guard, to stop cell division or induce cell death through p53-dependent or independent pathways. p53 is another tumor suppressor gene that p19ARF protects from being degraded. On the other hand, p16INK4a is increased when cells receive signals to senesce, so p16INK4a helps to inhibit cell division and remains high as long as the cells are not dividing. These types of functions are very important for protection against breast cancer. Together, the INK4a/ARF and p53 represent the two most frequently inactivated genes in human cancer, including breast cancer. My recent study demonstrated that p19ARF potential function is to determine the balance between normal cell division rate and cell death during mammary gland development. The study also demonstrated that p19ARF increased during stages of pregnancy and involution (after weaning), and that progesterone hormone upregulated p19ARF protein level in the mammary glands. Other studies suggested that pregnancy may provide protection to mammary epithelial cells against cancer, and that progesterone plays a critical role in the protection against mammary tumorigenesis afforded by early parity. We reported that lack of p19ARF allowed mammary epithelial cells to divide indefinitely in the mammary gland. Our preliminary data revealed that p16INK4a, a potential protein to arrest mammary cell growth, increased significantly in normal mammary cells that have gone through full-term pregnancy as compared to virgin cells. This increase was not observed in cells lacking p19ARF. We also found that the tumor suppressor gene, p53, which is protected by p19ARF, was activated in mammary glands of parous, but not in virgin. I hypothesize that p16INK4a/p19ARF gene products have a pivotal role in providing protection against beast cancer after estrogen and progesterone treatment. The outcome from the specific aims of this study will answer the following questions: 1) how frequent and how much time for mammary tumors to develop in mammary glands of mice treated or untreated with estrogen and progesterone prior to exposure to carcinogen in the presence or absence of p16INK4a or p19ARF. 2) What are the genes that are increased or decreased after short-term treatment with estrogen and progesterone in mammary glands lacking p16INK4a or p19ARF compared to normal mammary glands? This proposal will provide at the end of the three years new and valuable information on the role of p16INK4a or/and p19ARF proteins in breast cancer prevention through short-term treatment with E+P to mimic pregnancy. If this research is completed in 3 years, future research will be focused to study pathways related to p16INKa or p19ARF that are induced to provide protection to the mammary gland through short-term hormone stimulation. The projected time to achieve better preventive measures to the consumers is too early to predict as this area of research is at its early stages in breast cancer prevention.