Compounds Blocking Assembly of LRH-1 in Breast Cancer

Institution: University of California, San Francisco
Investigator(s): Cindy Benod, Ph.D. -
Award Cycle: 2009 (Cycle 15) Grant #: 15FB-0103 Award: $90,000
Award Type: Postdoctoral Fellowship
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2009)

The focus of this project involves a nuclear receptor, called LRH-1 (liver receptor homolog-1) recently found to be present at high levels in breast tumor cells and surrounding adipose tissue. LRH-1 plays a critical role in the regulation of development, cholesterol transport, bile acid homeostasis and steroid formation. The link to breast cancer was demonstrated by studies showing that LRH-1 inactivation would block estrogen-dependent proliferation of breast cancer cells. In addition, LRH-1 controls the expression of genes coding for two cell cycle regulatory cyclins that are thought to be important in tumor maintenance. Finally, LRH-1 powerfully enhances expression of aromatase, the only enzyme that converts androgen to estrogen, stimulating tumor progression. Thus, inhibitors of LRH-1 would potentially be the key to shutting off multiple pathways associated with breast cancer progression.

The goal of this project is to identify small molecules that inhibit the LRH-1 transcriptional activity in breast cancer cells. Our approach is to employ an innovative screen for inhibitors of the LRH-1 transcriptional assembly. First, we will screen chemical libraries for compounds targeting the regulatory binding sites and interaction surfaces of LRH-1 that participate in binding to other essential co-regulators. The technologies used will be Surface Plasmon Resonance (SPR) followed by X-ray crystallography to “map” protein-compounds interaction sites. Finally, we will evaluate the effects of these new LRH-1 antagonists by testing them in breast cancer cells, such as MCF-7, MDAMB-453 and ZR-75-1 cell lines.

The discovery of LRH-1 specific inhibitors would provide a new route to block aromatase. The critical role of LRH-1 in regulating aromatase transcription in malignant breast, but not in normal breast, bone or brain suggests that these inhibitors should be more selective and could not have the side effects observed for current generation aromatase inhibitors. Furthermore, because the role of LRH-1 in breast cancer merits further study, these compounds could represent powerful tools to validate key molecular mechanisms of LRH-1 in breast cancer development and tumor progression.




Final Report (2010)

Information regarding tumor size can be obtained from contrast-enhanced magnetic resonance imaging. This size-based information can be valuable in monitoring tumor response to chemotherapy: a significantly decreased tumor size is interpreted as a good response, which can correlate with better long-term patient outcomes; however, this correlation is imperfect. In-vivo, three-dimensional information regarding tumor cellularity, measured as the apparent diffusion coefficient (ADC), can be obtained from diffusion-weighted magnetic resonance imaging (DW-MRI) and we wanted to investigate if tumor ADC could improve the ability to predict tumor response to chemotherapy.

The project funded by this grant has allowed us to investigate the ability of tumor ADC to predict treatment response in patients enrolled in neoadjuvant studies at our institution. Our results from limited patient numbers suggest that tumor ADC may be a predictor of recurrence-free survival and time to recurrence. A major barrier to analysis in current studies is image quality. The echo planar DW-MRI sequences used on scanners today are prone to distortion and other artifacts, impairing the ability to register contrast-enhanced MRI and DW-MRI information and making it sometimes difficult to obtain an accurate ADC measurement. This problem was addressed by collaborating with industry and academia and scanning with a new DW-MRI pulse sequence. The sequence improved in-plane spatial resolution and allowed for artifacts from air-tissue interfaces to be reduced. This sequence could improve DW-MRI acquisition in the breast, allowing for ADC to be more accurately monitored throughout treatment.

Funding from this grant also allowed for improved automation of ADC measurements to be explored. ADC is not the same throughout the tumor and this mixture of different ADCs is not adequately captured by a mean ADC. Methods were developed to calculate parameters related to variation in tumor ADC, and to quantify and map variations in longitudinal ADC change throughout the tumor. In order for ADC information to be analyzed, a region of interest (ROI) must first be defined in the breast and this task can be cumbersome. Tumor ROIs obtained from this manual method were compared to ROIs obtained using an automated method that utilized information regarding tumor volume. A faster method to segment breast tissue was also developed and this method could improve the ability to measure ADC in normal fibroglandular tissue, allowing for changes in normal tissue ADC to be measured throughout treatment.

The results of this study suggest that ADC measurement can be improved and made more time-effective, but these technical advances must be compared to standard methods. The results of this project also suggest that ADC may be valuable in predicting treatment response but validation in large, prospective studies is needed.




Symposium Abstract (2010)

Cindy Benod, PhD (PI), Robert Fletterick, PhD (mentor)

The nuclear receptor LRH-1 (liver receptor homolog-1) is expressed at high levels in breast tumor cells and surrounding adipose tissue. Increased expression and its newly appreciated role in early development suggest that LRH-1 may be a worthy target in breast cancer research. Its roles in carcinogenesis are unknown, but in breast cancer cells uniquely, recent studies showed that LRH-1 powerfully enhances expression of aromatase, the enzyme that converts androgen to estrogen, stimulating tumor progression. Among the hundreds of genes under LRH-1 control are two cyclins regulating the G to S transition in cell cycles. Enhanced expression of these may be important in tumor maintenance. I hypothesize that inhibitors of LRH-1 transcriptional activity would slow or inhibit multiple pathways associated with breast cancer progression. Thus, the goal of this project is to identify small molecules that inhibit LRH-1 transcriptional activity in breast cancer cells.

In collaboration with Dr. Shoichet’s lab, sophisticated computational chemistry calculations were carried out to examine the fit of test compounds into the LRH-1 hormone binding site. The experiments were run to favor discovery of compounds which fit the hormone binding site but extended outwards to deny formation of the surface binding site on the receptor for attachment of essential coregulators. We screened 8 million commercially available compounds from the Zinc database. At the end of this computational and careful chemical evaluation, we purchased a small number of potential LRH-1 inhibitors. To assess the binding of these compounds to LRH-1, we employed two innovative biochemical assays: Differential Scanning Fluorimetry and Surface Plasmon Resonance technology. The first assay was coarse, and the second quantitative. Together, these methods identified three compounds that bound to LRH-1 and predicted that the LRH-1 molecule would be destabilized when the compounds bind. The biological activity of these candidates is being investigated using cell-based assays of LRH-1 function. LRH-1 transcriptional activity in the presence of these compounds is being measured for several breast cancer cell lines. Effects on breast cancer cells proliferation will be quantified.  For compounds that have desired effects, crystallographic studies will be performed to reveal the mode of binding and conformational changes in the receptor.

The major impact of this study is to provide breast cancer researchers with compounds that could be powerful tools to study the importance of LRH-1 in tumor progression. Furthermore, the discovery of LRH-1 specific inhibitors would provide a new route to block aromatase and possibly lead to pharmaceuticals with new and unexplored mechanisms of action to treat breast cancer. The critical role of LRH-1 in regulating aromatase transcription in malignant breast, but not in normal breast, bone or brain, suggests that these inhibitors may be more selective and might not have the side effects observed for current aromatase inhibitors.



Structure-based Discovery of Antagonists of Nuclear Receptor LRH-1.
Periodical:Journal of Biochemistry and Cell Biology
Index Medicus: J Biochem Cell Biol
Authors: Carlsson J, Uthayaruban R, Hwang P, Irwin JJ, Doak AK, Shoichet BK, Sablin EP...C. Benod
Yr: 2013 Vol: 2013 Nbr: 288 Abs: Pg:19830-44