Background: Drugs targeting estrogen signaling or production have successfully prevented or treated estrogen receptor‐positive breast cancers, but not estrogen receptor‐negative (ER‐negative) breast cancers. Our laboratory has demonstrated that rexinoids prevent ER‐negative breast cancer in transgenic mice by 90% at high‐doses. Since the prevention is not 100% effective, it is imperative to develop more efficacious prevention strategies by determining the mechanism of rexinoids. We hypothesized that rexinoids are specifically able to prevent breast cancer by suppressing growth though interactions with key nuclear hormone receptors. To test this hypothesis we conducted high‐throughput screens to determine which nuclear hormone receptors are critical to the growth suppressive effects of rexinoids.
Methods: We used quantitative RT‐PCR to determine the quantity of mRNA transcripts for all 48 nuclear hormone receptors in human mammary epithelial cells (HMECs). To identify nuclear hormone receptors that when knocked‐down reverse the effects of rexiniod treatment, a final concentration of 30nM of pools of three independent siRNAs specific to each nuclear hormone receptor was used. High‐throughput microscopy of the siRNA‐treated HMECs stained with DAPI (1ug/mL) and EdU (10mM) was used to analyze cell number and percent S‐phase. Those nuclear receptors whose corresponding siRNA reversed the growth inhibitory effects of the rexinoid 75% or greater, were considered for further investigation. Co‐immunoprecipitation (coIP) and GST pull‐down assays are being conducted to investigate whether the identified nuclear hormone receptors form a complex with RXR proteins.
Results: qRT‐PCR analysis confirmed that many of known RXR‐binding nuclear hormone receptors are expressed in HMECs. High‐throughput screens indicate that knockdown of most receptors alone affect growth of HMECs, but do not reverse the effects of rexinoid treatment. Receptors that did reverse the growth suppressive effect of rexiniods include RXRα, VDR, PNR and PPARγ. Currently, we are further investigating the function of these receptors in HMECs and other normal, premalignant, and malignant breast cells.
Conclusions: These experiments identified nuclear hormone receptors that are critical for the growth suppressive effects of rexinoids in normal human breast cells. Such studies identify possible targets for preventative therapy, alone or in combination with rexinoids to increase the efficacy of drugs for more effective prevention of estrogen receptor‐negative breast cancer. Support: NCI R01 CA78480 (PHB, IPU) and NIH/NCI T32 CA90221 (JMR)
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A91.