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Elucidating the Cause for the Insrease in Telomerase in Human Breast Cancer
Tumor Cell Biology V
Although activation of the gene for the catalytic subunit of telomerase, hTERT, results in the elevation of telomerase in 90% of breast cancers, the causes for this process remain unresolved. Instead of studying breast cancer "after the fact", we seek to understand the causes for telomerase elevation during transformation of normal breast cells. Using defined genetic elements, we are developing a model system in which the endogenous hTERT promoter becomes activated early in tumorigenesis and the DNA methyltransferases also become elevated in these cells. In addition, we have shown that the hTERT promoter is epigenetically controlled. Therefore, this system provides an excellent opportunity to resolve the causes for hTERT gene activation during early breast tumorigenesis. Objective/Hypothesis: The objective is to determine the causes for activation of the hTERT gene as normal human mammary epithelial cells (HMECs) are induced to undergo tumorigenesis. The hypothesis is that tumorigenesis alters the epigenetics of breast cells leading to hTERT gene activation and unlimited cellular proliferation. Specific Aims: We propose aims (1) to measure changes in DNA methylation and histone acetylation of the hTERT promoter in HMECs induced to undergo tumorigenesis, (2) to detect the binding of the E2F-1 methylation-sensitive repressor and other factors to the hTERT promoter during early breast tumorigenesis, and (3) to assess the epigenetic mechanisms for endogenous hTERT activation in breast cancer using an in vivo model. Study Design: We will transform HMECs using SV40 T antigen, hTERT , and activated H- ras to assess changes in the methylation and histone acetylation of the endogenous hTERT promoter. Use of the hypomethylating agent, 5-azacytidine, and the histone deacetylase inhibitor, trichostatin A, will allow us to assess whether the observed changes are required for hTERT deregulation or transformation. Additional studies will be performed in breast cells that have alternative lengthening of telomeres and are not dependent upon exogenous hTERT. We will perform mobility shifts of the hTERT promoter to assess the effects of epigenetic changes on binding of the methylation-sensitive hTERT repressor, E2F-1, in these cells. Lastly, we will assess the in vivo epigenetic causes for hTERT activation using breast xenograft implants. Potential Outcome and Benefits: We have solid data showing that the endogenous hTERT gene is activated during early induced tumorigenesis and that this gene is epigenetically regulated. We feel that the probability that we will be able to elucidate the causes for telomerase elevation in our developing in vitro and in vivo "endogenous telomerase activation" breast cancer model is excellent. If so, efforts to find specific inhibitors of the factors involved in this process will greatly facilitate cancer prevention and lead to novel approaches for breast cancer therapy.
New model systems are needed to assess the basis for the genetic causes of breast cancer. It is now clear that certain genes can become activated in normal breast cells and lead to cancer. What is much less clear is how and why these genes become activated. One gene, telomerase, is activated in about 90% of breast cancers but is barely detectable in most normal cells. We know that the resultant elevation of the telomerase enzyme allows cancer cells to proliferate indefinitely by maintaining the ends of their chromosomes, but the early causes for the increases of this enzyme in breast cancer are unknown. Although most studies of breast cancer focus on cells after they are fully cancerous, we have begun development of a new model system in which the telomerase gene can be induced to activate as the cancer is in very early stages . In other words, we can add specific genes to normal cells causing these cells to become cancerous and explore the reasons for telomerase elevation in these cells. We seek to now apply this system to resolve the underlying causes for telomerase gene activation in breast cancer. More specifically, we will study the role of epigenetic changes of the telomerase gene in this system. These epigenetic alterations involve changes in DNA or its associated proteins but do not involve mutations. Examples of epigenetic processes are the addition of methyl groups to certain bases of DNA or the loss of acetyl groups from DNA-associated proteins such as histones. The telomerase gene is greatly influenced by these epigenetic processes but no one has yet analyzed the early causes for this in a model system whereby normal breast cells are converted to cancer cells. That is the goal of this investigation. Ultimately, these studies will lead to facilitation of more effective uses of inhibitors of the factors that lead to increases in telomerase in breast cancer. Such studies could have an impact on up to 90% of the existing breast cancers that are known to express telomerase. This investigation will also contribute to the development of novel therapeutic approaches to breast cancer with very little risk of harm to normal cells.