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The Role of p21 in Tamoxifen Agonism
Background: Tamoxifen is a first line therapy in the treatment of Estrogen Receptor alpha (ERa) positive breast cancers. However, resistance to Tamoxifen is a common, but poorly understood process. We have identified a case report demonstrating a particular form of Tamoxifen resistance, and have shown that this is mediated by the loss of expression of the cyclin dependent kinase inhibitor, p21. We have recently developed a model system using isogenic ERa positive MCF-10A p21 -/- cell lines that recapitulates the same Tamoxifen resistance phenotype seen in our case report. Moreover, using a high throughput drug screen, we have identified small molecules that can overcome this form of Tamoxifen resistance. Objective/Hypothesis: We propose that p21 loss of function is a pathway leading to Tamoxifen resistance. We hypothesize that loss of p21 results in aberrant ERa phosphorylation leading to the recruitment of coactivators with subsequent gene expression of growth promoting genes. In this study, we will address the molecular mechanisms of Tamoxifen resistance and our small molecule inhibitors using our model system. Specific Aims: 1) Elucidation of the mechanism of Tamoxifen growth stimulation/resistance as mediated by cyclin/cdk complexes. 2) Gene expression and mutational analysis of ERa regulated genes in p21 null cells 3) Molecular characterization of compounds that overcome p21-/- mediated Tamoxifen resistance. Study Design: Our results suggest that the lack of p21?s CDK inhibitory activity is critical for Tamoxifen-mediated growth. We will use a combination of ChIP and RNAi studies to elucidate the molecular interactions between ERa and cyclin/cdk complexes. In addition, we have found that aberrant phosphorylation of ERa occurs following growth-stimulation with Tamoxifen in p21-/- cells. We will mutate critical serine residues of ERa and analyze the effects of these mutations on downstream ERa target genes. Finally, we will perform the above studies in parallel with p21-/- cells exposed to our newly isolated drugs to determine their mechanisms of action. Potential Outcomes/Benefits of the Research: Tamoxifen is one of the most effective therapies for ERa positive breast cancers. Revelation of mechanisms leading to drug resistance will direct the development of compounds that can overcome hormonal drug resistance. We have isolated drugs that kill Tamoxifen resistant cells in a targeted fashion. A more complete understanding of the pathways involved with Tamoxifen resistance will enable us to move these studies forward toward clinical development.
Increased exposure to estrogen over a woman?s lifetime may increase her risk for developing breast cancer. Drugs used to treat this disease either compete with targets of estrogen (Selective Estrogen Receptor Modulators, SERMS) or prevent the production of estrogen (aromatase inhibitors). The drug Tamoxifen (a SERM) is a first line of treatment for primary breast cancers that express estrogen receptor. Currently, the standard of care for pre-menopausal women with estrogen receptor positive breast cancers is to take Tamoxifen for five years as adjuvant therapy. However, many breast cancers develop Tamoxifen resistance, and in some cases this resistance is associated with Tamoxifen stimulated growth of the breast cancer. We have identified a patient demonstrating this phenomenon. Furthermore, we have strong evidence to suggest that this Tamoxifen-resistant disease was due to the silencing of a gene called p21. Based on this data, we have developed a model system that recapitulates this situation. Our data and the data of others suggest that the loss of the p21 gene or loss of p21 function leads to Tamoxifen-resistant breast cancer. In addition, we have used this model system in a high throughput drug screen to identify drugs that specifically target this phenomenon. In this study, we will elucidate the molecular mechanisms of this particular form of Tamoxifen resistance. Furthermore, we will molecularly characterize the effects of the lead compounds we have identified that specifically target Tamoxifen resistant/dependent cells. The work proposed here may lead to a better understanding of how our new drugs target Tamoxifen-resistant cancers and will allow for the development of more advanced drug therapies with fewer toxic side effects for the treatment of breast cancers.