Genomic studies on nuclear receptor-mediated transcriptional networks in breast cancer cells

Nuclear receptors (NRs) govern a wide range of physiological and pathological processes through directly modulating gene expression in response to hormones. Previous work has established critical roles of NRs, most notably estrogen receptor-alpha (ERalpha), in breast carcinogenesis. However, the current picture of NR-regulated networks is fragmentary and knowledge of the functional interactions between different NRs is scarce. In order to achieve comprehensive understanding of NR function in breast cancer, we developed an integrated genomic approach to uncover NR-mediated transcriptional networks in the breast cancer cell line MCF-7.;We first focused on estrogen signaling in breast cancer. Using chromatin immunoprecipitation (ChIP) and genomic tiling arrays, whole genome mapping of binding sites for ERalpha and MYC, two essential transducers of the estrogen signaling cascade, was combined with gene expression profiling to identify target genes involved in the proliferative response to estrogen. Using RNA interference, selected ERalpha and MYC target genes were knocked down to identify mediators of estrogen-stimulated cell proliferation. Tissue microarray screening revealed that high expression of an epigenetic factor, the estrogen-inducible histone variant H2A.Z, was significantly associated with lymph node metastasis and poor breast cancer survival.;We developed a technology that enables ChIP experiments in cultured human cells without the need for protein-specific antibodies by physiologically expressing epitope-tagged fusion protein using bacterial artificial chromosome transgenesis. This approach enabled a systems-level study of essentially all NRs expressed in MCF-7 cells. Comparison of genomic binding sites of these NRs revealed a marked co-localization between ERalpha and retinoic acid receptors (RARs), mediators of retinoic acid signaling that triggers growth-suppressive effects in breast cancer cells. Further analysis revealed RAR-dependent regulation of many breast cancer-relevent genes and a widespread antagonistic crosstalk between retinoic acid and estrogen signaling. ERalpha and RAR binding sites appear to be co-evolved on a large scale throughout the genome, allowing for competitive binding between these transcription factors via nearby or overlapping cis-regulatory elements and often resulting in opposite effects on target gene transcription. Together these data indicate the existence of a highly coordinated intersection between these two critical NR signaling pathways that provides a mechanism for balancing gene expression output.