Identification of Critical Transducers of Rexiniod-Mediated Growth Suppression in Normal Breast Cells.
Authors: J. Rodenberg, I. Uray, R. Bissonnette, P. Brown
Journal: Cancer Research (2009)

Abstract Background: Targeted agents against the estrogen receptor are effective in treating and preventing many breast cancers, but these agents are not effective for the treatment or prevention of estrogen receptor-negative (ER-neg) breast cancer. Our laboratory has demonstrated that rexinoids are capable of preventing ER-neg breast cancer in transgenic mice; however, this treatment is not 100% effective. Therefore, it is imperative to delineate the mechanism by which rexiniods prevent breast cancer to develop more effective prevention strategies. 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 transduce 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 (1μg/mL) and EdU (10mM) was used to analyze growth. If a receptor's corresponding siRNA reversed the growth inhibitory effects of rexinoids 75% or greater, it was considered for further investigation. Receptors that did not reverse the effect of rexiniods or enhanced growth suppression when knocked-down were identified, but were not studied further in this analysis. Co-immunoprecipitation (coIP) and GST pull-down assays are being conducted to investigate whether the identified nuclear hormone receptors form a complex with, or bind to, 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 (including COUP TF1, EAR1 and ERRβ) affect growth of HMECs but do not reverse the effects of rexinoid treatment. Loss of ERRα and AR induced growth, but rexiniod treatment suppressed growth to a greater extent than controls. Receptors that appeared to reverse the growth suppressive effect of rexiniods include GCNF1, THRα, PNR and PPARγ. Loss of PPARγ alone stimulated growth, but reversed effects of rexiniod-induced growth suppression. Currently, we are further investigating these receptors in HMECs and other cell types.Conclusions: These experiments have identified nuclear hormone receptors that are critical for the growth suppressive effects of rexiniods in normal human breast cells. Identification of such receptors provides possible targets for preventative therapy used alone, or in combination with rexinoids, to increase the efficacy of rexinoids for more effective prevention of estrogen receptor-negative breast cancer. Support: NCI R01 CA78480 (PHB, IPU) and NIH/NCI T32 CA90221 (JMR) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3134.