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p16 Mediated supression of hTERT in Breast Cancer
Telomerase activity is commonly thought to be required for the unlimited proliferation of most breast cancer cells. We hypothesize that telomerase is downregulated by a p16Ink4a (p16) mediated epigenetic program as part of the normal maturation of breast stem and progenitor cells. Our preliminary data indicate that, independent of its effects on growth, transient p16 exposure leads to stable telomerase repression in primary HMEC cultures as well as a breast tumor-derived cell line. Although under some cell culture conditions, HMEC spontaneously arise in which p16 has been silenced through promoter methylation, the hTERT gene encoding the catalytic subunit of telomerase remains stably repressed in these cells and cannot be reactivated in response to introduction of individual oncogenes (e.g., c-myc) or inactivation of individual tumor suppressor genes (e.g., p53). We hypothesize that p16 induces stable chromatin modifications at the hTERT locus and other loci, which serve as stringent tumor suppressor mechanisms in cells that have left the stem/progenitor cell niche. The overall goals of our current studies are to determine how p16 normally causes stable repression of telomerase and other oncodevelopmental genes, and how this program can be activated pharmacologically in breast tumor cells that have lost p16 expression. There are three specific aims: 1. Investigate the mechanism of stable hTERT suppression by transient expression of p16. 2. Identify targets of the p16/pRb pathway in MCF7 cells and HMEC. 3. Investigate the therapeutic potential of transient Rb pathway activation using a highly specific small molecule CDK inhibitor. These experiments will significantly advance the understanding of telomerase inactivation and cellular differentiation as potent cancer defense mechanisms, and provide rationale for targeted therapeutic strategies.
Cancer cells are similar to stem cells in that both have the ability to divide indefinitely. Stem cells, however, lose the capacity to divide indefinitely when they mature or "differentiate" into cells with characteristics of a particular organ. Most conventional cancer therapies target DNA replication and cell cycle progression indiscriminately. These therapies are not always effective, and they are also highly toxic to normal cells. An alternative approach which may be less toxic and more effective is to induce the differentiation of malignant cells - make them less like stem cells and more like mature cells - so that they lose the ability to divide indefinitely. While the idea for such an approach was proposed decades ago, it has not been employed mainly because how malignant cells maintain their stem-ness and how they can lose it have not been understood. An important trait that contributes to the ability of both malignant cells and stem cells to divide indefinitely is the expression of telomerase, an enzyme that maintains the chromosome ends and ensures that genetic material is faithfully replicated when cells divide. In cultured breast cells from non-malignant tissues, we have observed that the levels of telomerase decrease rapidly while the levels of a tumor suppressor protein, p16Ink4a, increase. Interestingly, p16 has also been recently implicated in the differentiation of stem cells. When we manipulated p16 in a breast cancer cell line, we found to our surprise that transient p16 exposure leads to stable repression of telomerase. As such stable suppression may constitute an important mechanism for preventing unlimited growth of tumor cells, we propose to study the mechanism responsible for this surprising phenomenon and to explore the possibility of using it to develop novel less toxic anti-cancer treatments. We hypothesize that p16 causes stable repression of hTERT, a rate limiting catalytic subunit of telomerase by the triggering changes in the accessibility of the gene that encodes it. We will investigate this hypothesis by studying the binding of accessibility determining proteins to the hTERT gene. We will also determine whether transient p16 exposure causes similar changes in other genes associated with breast cancer. Finally, we will investigate whether a new drug already in clinical trials, PD 0332991, can inhibit human breast tumor cell growth by a similar mechanism. We hypothesize that short term exposure to this drug will eliminate stem cell properties in tumor cells, leading to cessation of tumor growth. We hope that our research will provide a rationale for targeting tumors with certain characteristics with PD 0332991 or similar drugs.