The ETS transcription factor family is highly redundant, with many members serving to promote tumorigenesis. Several ETS factors have been implicated in breast cancer, but ESE-1 is the most relevant. ESE-1 mRNA and protein are over-expressed in human breast cancer and its co-expression with HER2 correlates with a highly malignant phenotype. Indeed, a positive-feedback loop exists between ESE-1 and HER2. Despite these advances, the ability of any ETS factor to transform human mammary epithelial cells (MECs) remained unknown. We discovered that ESE-1 initiates transformation of benign, ESE-1-negative MCF-12A and MCF-10A human mammary epithelial cell lines via a novel cytoplasmic mechanism in which its unique 40-amino acid, serine and aspartic rich domain is necessary and sufficient for transformation. As our studies progressed with tumorigenic breast cancer cell lines, we discovered an additional, classical nuclear transcriptional role for ESE-1 in maintaining mammary tumorigenesis. Using highly-specific monoclonal anti-ESE-1 antibodies that we generated (mAb405 and mAb1534), we found ESE-1 to be nuclear in established luminal and HER2+ breast cancer cell lines, while preliminary IHC studies of breast cancer tissue microarrays revealed ESE-1 protein to be expressed both in the nucleus and cytoplasm. Furthermore, T47D cells stably expressing HA-ESE-1 injected into nude mice resulted in orthotopic tumors that were 2-fold larger than vector controls. Also, we've shown that interfering with ESE-1 function or expression, via dominant-negative, antisense or shRNA methods, is sufficient to reverse the transformed phenotype in MCF-7, T47D and HER2+ ZR-75-1 breast cancer cells, revealing a crucial role for ESE-1 in maintaining mammary tumorigenesis, via a classic nuclear transcriptional mechanism. Our goal is to define the ESE-1 cistrome that drives transformation in HER2+ breast cancer cells, thus elucidating ESE-1 mechanism and identifying novel therapeutic targets. The hypothesis of this proposal is that the ETS transcription factor, ESE-1, maintains the transformed phenotype in HER2+ breast cancer cells by controlling the transcription of ESE-1-specific, growth-promoting target genes (ESE-1 cistrome). To test this hypothesis, we propose four specific aims: (1) to determine the HER2-dependence and mechanistic role of ESE-1 in maintaining the transformed phenotype in breast cancer cell lines;(2) to apply unbiased chromatin immunoprecipitation (ChIP)- and DNAse I-deep DNA sequencing, coupled with genome-wide RNA expression studies, to identify the ESE-1 cistrome;(3) to identify key downstream ESE-1 targets by ectopically expressing candidates that meet rigorous selection criteria and testing their ability to rescue the transformation-inhibitory effect of shESE-1;and (4) to use highly-specific anti-ESE-1 mAbs to probe breast cancer specimens, to determine whether nuclear and/or cytoplasmic ESE-1 expression correlates with tumor cell type, grade or outcome. We have gathered expert collaborators and generated new data supporting the project feasibility. Insights gained will provide new drug targets and markers to use in our battle against this deadly disease.
Breast cancer remains the most common and one of the deadliest malignancies in women, with the HER2+ subtype displaying a poor overall prognosis and frequent eventual resistance to HER2-specific therapies. To date, we still do not understand all of the biological mechanisms causing HER2+ breast tumors, and how they acquire resistance to HER2+-targeted therapies. In this proposal, we plan to use highly-specific anti-ESE-1 monoclonal antibodies that we generated and HER2+ cell lines that are sensitive and resistant to trastuzumab (Herceptin) treatment, to define the role of ESE-1 in maintaining the transformed state, the HER2/ESE-1 interdependence, the key pro-tumorigenic ESE-1-regulated genes, and to probe for ESE-1 expression in human tumor samples, in order to correlate with tumor subtype and clinical behavior.
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|Kar, Adwitiya; Gutierrez-Hartmann, Arthur (2013) Molecular mechanisms of ETS transcription factor-mediated tumorigenesis. Crit Rev Biochem Mol Biol 48:522-43|