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Tamoxifen Response Pathway Characterization by Reversible Gene Entrapment of Breast Cancer Cells
Antiestrogens such as tamoxifen (TAM) act by competing with estrogens for binding to the estrogen receptor (ER), resulting in inhibition of the estrogen-mediated cell growth signaling pathway. However, most cancers that initially respond to TAM develop resistance. Despite extensive experience with this drug, mechanisms that confer resistance remain unknown. In general, TAM resistance is not attributed to loss or alteration of the ER. Thus, it is our hypothesis that resistance is a result of acquired changes within the ER signaling pathway at some point downstream of the receptor. The goal of this study is to identify genes whose products are required for TAM sensitivity and test whether these genes are altered in cells from patients displaying TAM resistance. Gene entrapment is a powerful technique that has been successfully used to identify gene functions. Our laboratory has recently found that gene entrapment can be carried our in somatic cells with the aid of agents that induce loss of heterozygosity (LOH). Additionally, because of the unique design of the vector, gene trap-mediated disruption of genes can be “reversed” by excision of the vector from the genome. Therefore, the specific aims of this proposal are to: 1) use a reversible gene entrapment strategy coupled with induction of LOH to identify genes whose products are necessary for sensitivity to TAM in ER-positive breast cancer cells, and 2) analyze patient derived cells to determine if these genes have undergone genetic mutation or epigenetic repression as cells acquire TAM resistance. The ER-positive cell lines MCF7 and T47D will be infected with a retrovirus encoding the reversible gene entrapment vector. A library of clones with at least one retroviral insertion in every gene will be generated, treated transiently with an agent that induces LOH, and grown in the presence of TAM to select for clones with disruptions in genes required for TAM sensitivity. Surviving clones will be subjected to Cre recombinase-mediated excision of the retroviral DNA from the genome, and the identity of genes that were disrupted in clones whose TAM sensitivity can be reversed will be determined. Finally, the status of these genes will be assessed in patient derived cells that are TAM sensitive and resistant. It is expected that some of the identified genes will be involved in the ER signaling pathway, providing important insight into the mechanisms of estrogen-mediated cell growth. These studies also have the potential to identify new targets for therapeutic intervention for patients who are resistant to antiestrogens.
The steroid hormone estrogen influences breast cancer development and progression. The presence of cellular estrogen receptors (ER) plays a critical role in the clinical care of breast cancer patients both as a prognostic factor as well as a therapeutic target. Estrogen interacts with its cellular receptor to activate a protein signaling pathway within cells that leads to cell growth. Drugs that inhibit the interaction of estrogen with its receptor, or antiestrogens, have been used to both prevent and treat breast cancer. Tamoxifen is the most widely prescribed antiestrogen for treating ER-expressing breast cancers at all stages. However, eventually most breast cancer cells acquire genetic changes that allow them to activate the estrogen receptor signaling pathway even in the presence of tamoxifen. Unfortunately because the protein signaling pathway that is activated by estrogen and inhibited by tamoxifen has not yet been characterized, the genetic alterations leading to tamoxifen resistance are unknown.
Gene entrapment is a powerful technique that has been successfully used to characterize novel genes and analyze their importance in biological phenomena and disease states. This technique is based on the integration of a reporter gene into a random site in the genome, tagging the insertion site and generating a mutation. Theoretically, all of the >30,000 genes in the human genome could be tagged and functionally inactivated. Our laboratory has recently developed an approach to enhance the gene entrapment technology with new reversible gene entrapment vectors and methods to improve the mutagenesis of mammalian genes. Importantly, these technical advances now make it feasible to carry out gene entrapment studies in breast cancer cells. Thus, the goal of this proposal is to use this enhanced gene entrapment technology to identify genes required for resistance to the antiestrogen tamoxifen in model breast cancer cells. Genes identified through this process will be examined in cells from patients with breast cancers that are both sensitive and resistant to tamoxifen to determine if there are genetic mutations or other alterations in the genes from resistant cells. Information obtained from these studies will yield valuable insight into estrogen-mediated cell growth mechanisms and shed light onto the ways cells acquire resistance to anti-cancer agents. Furthermore, new therapeutic targets for the treatment of antiestrogen resistant cancers may be identified.