The Role of Estrogen Receptor in Endocrine Resistance

Institution: Beckman Research Institute of the City of Hope
Investigator(s): Hei Chan, B.S. -
Award Cycle: 2009 (Cycle 15) Grant #: 15GB-0027 Award: $76,000
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2009)

The estrogen receptor (ER) is a key regulator of proliferation and survival in breast cancer cells. Anti-estrogens that block ER activity can effectively inhibit tumor growth. Aromatase inhibitors are also effective inhibitors of tumor growth, but they function differently by blocking the synthesis of estrogen. Both anti-estrogens and aromatase inhibitors show significant survival benefits for breast cancer patients, however, most patients would eventually develop resistance to these drugs. Results from our lab shows that ER still plays a critical role in the resistant cells, but the mechanism is not yet completely understood. Characterization of the changes in ER signaling will confer a better understanding of how resistance arises.

The genome-wide profiling of ER binding sites in hormone responsive cells has been published, but this approach has not been used to study endocrine resistant cells. We hypothesize that the ER binding site profile in resistant cells is different from that in the hormone responsive cells. We will identify these differences in ER binding which will reveal the changes in ER signaling that are important for the resistant phenotype.

To compare genome-wide profiles of ER binding sites in the hormone-responsive and resistant lines, we will characterize the ER binding profiles in breast cancer cell lines show resistance to: (1) estrogen deprivation, or (2) tamoxifen, letrozole, anastrozole, or exemestane treatment. Next, we will identify and characterize the functional significance of ER signaling changes in the hormone-responsive and resistant cell lines. The binding site profiles in combination with gene expression array data previously generated in our lab will provide further insights into the role of ER in endocrine resistance. Our methods include chromatin immunoprecipitation (ChIP) followed by massively parallel Illumina instrument-based DNA sequencing (ChIP-seq). Bioinformatics analyses of the binding site locations and organizations will unravel differences in ER binding properties and co-regulator involvement among different types of resistant lines. Correlation of the ChIP-seq data with gene expression array data will allow us to explain the functional significance of the binding events.

The detailed elucidation of ER functions in the drug-resistant cells is a key step to understanding the mechanism of endocrine resistance, which is crucial for the design of novel strategies to overcome this widespread problem.




Final Report (2011)

Endocrine resistance is a major hindrance to the successful use of aromatase inhibitors (AIs) and anti-estrogens for the treatment of estrogen receptor (ER) positive breast cancer. To understand the mechanisms of acquired endocrine resistance, we use a cell culture model called Long Term Estrogen Deprived (LTEDaro), which was derived by culturing the MCF7aro cell line (i.e., cells stably expressing aromatase) in the absence of estrogen for a long term. To study the transcriptional (gene regulatory) role of ER in endocrine resistance, we have performed chromatin immunoprecipitation followed by Solexa “deep sequencing” (ChIP-seq) for genome-wide detection of ER binding sites. Comparison of the ER binding profiles showed interesting differences between the LTEDaro and the parental MCF7aro cell lines.

We further studied an ER variant found in LTED cells using the HC20 and 1D5 antibodies. From the ChIP-seq analysis we found several interesting candidate genes and binding sites that we are trying to validate. We also investigated the functional role of the ER variant gene by cloning and expressing it in 293T cells, which are ER negative, as well as MCF7aro and LTEDaro cells. Overall, our results show that the ER variant has partial transcriptional activity when compared to the wild-type ER activity, and variant ER's activity may dominate over the wild-type ER, and may act as a competitor.

To summarize the overall findings of the project, we started with ChIP-seq study on the LTEDaro and MCF7aro cells, and found that the LTEDaro cells has significantly more ER binding. In order to understand the mechanism for this ER behavior, we investigated the ER activity and protein expression levels in LTEDaro cells compared to MCF7aro. This leads to the identification of an ER variant that we believe plays an important role in endocrine resistance. Current studies are ongoing to validate the role of this ER variant in DNA binding and transcriptional regulation.




Symposium Abstract (2010)

Hei Jason Chan,1,2 Haiqing Li,3 Hanlin Gao,4 Yate-Ching Yuan,3 and Shiuan Chen2
1Irell & Manella Graduate School of Biological Sciences, 2Division of Tumor Cell Biology, 3Bioinformatics Core, 4DNA Sequencing/Solexa Core
Beckman Research Institute of City of Hope, Duarte, CA

Aromatase inhibitors (AIs) and anti-estrogens are two main therapeutic strategies currently in use for the treatment of estrogen receptor (ER) positive breast cancer. Both strategies are very successful in the treatment of this type of breast cancer, but unfortunately, many patients eventually develop resistance to these agents, and treatment options are limited for these patients who has acquired resistance. To overcome this problem, it is necessary to understand the mechanism underlying the development of acquired resistance to these agents using clearly defined model systems. The ER is a steroid nuclear receptor that plays a key role not only in breast cancer cell proliferation, but also in resistance to endocrine therapy. One of the main actions of ER is the transcriptional activation or repression of genes in response to E2 stimulation. This occurs through the “classical” mechanism of direct binding to Estrogen Response Element (ERE), and through “non-classical” interactions with other DNA-binding complexes. All of these physical interactions of ER with chromatin will be captured by chromatin immunoprecipitation followed by Solexa deep sequencing (ChIP-seq) for detection across the entire genome. Our lab has generated an MCF7 cell line that stably expresses the aromatase gene (MCF7aro) as a model for hormone dependent breast cancer. In order to study endocrine resistance, we derived another cell line called the Long Term Estrogen Deprived (LTEDaro) cell line, which was derived from the MCF7aro cell line by culturing MCF7aro cells without any hormones over a long period. By comparing the genome-wide profiles of ER binding sites in these hormone-responsive and resistant lines, we were able to detect differences in ER binding. Our ChIP-seq results show that LTEDaro cells had enrichment of ER binding sites near transcription start sites (TSS) under both DMSO and E2 treatments, whereas in the MCF7aro cells this is observed only in the E2 treated cells. De novo motif discovery confirmed the presence of the estrogen response element (ERE), and also the consensus sequence for Fox transcription factor binding. Mapping of known motifs in LTEDaro ER binding sites showed an increase in Sp1 and AP1 occurrence. Overlap analysis shows a high degree of similar binding sites between E2 and DMSO treatment for both cell lines. Interestingly, comparison of LTEDaro DMSO with MCF7aro E2 also shows a high number of overlapping sites. Taken together, the distance to nearest TSS and overlap analyses shows clear difference of ER behavior in LTEDaro cells. Coactivators such as Sp1 and AP1, and other proteins such as the Fox family of transcription factors may play an important role in the resistance phenotype. Overall, our data supports the hypothesis that ER in LTEDaro cells actively binds DNA and drives cell proliferation independently of E2.