Abstract
The synthetic rexinoid bexarotene (Targretin, LGD1069) inhibits the formation of both estrogen receptor-negative and estrogen receptor-positive breast cancer in preclinical models and controls the expression of growth-regulatory biomarkers, such as IGFBP-6 (insulin-like growth factor-binding protein 6), RARbeta, or cyclin D1. In this study, we identified a classical retinoic acid-responsive element in the first intron in the IGFBP-6 gene adjacent to a consensus AP-1 binding site, both elements essential for rexinoid-induced expression of IGFBP-6. In chromatin binding experiments, bexarotene increased the occupancy of the identified enhancer element by RXRalpha, RARbeta, cJun, cFos, and p300. In normal mammary epithelial cells and T47D breast cancer cells, small interfering RNA-mediated knockdown of all RXR isoforms or RARbeta, but not RARalpha or RARgamma alone, blocked the induction of IGFBP-6 by bexarotene. Simultaneous knockdown of RARalpha and RARgamma abrogated both the induction of RARbeta and the up-regulation and secretion of IGFBP-6. The suppression of either RARbeta or cJun by small interfering RNA blocked the recruitment of RXRalpha and cJun to the enhancer. These results demonstrate a novel cooperative interaction between retinoid receptors and AP-1 orchestrated by RARbeta and highlight a novel mechanism by which RARbeta can mediate the cancer-preventive effects of rexinoids.
Highlights
We and others have shown that the panagonist 9-cis-retinoic acid suppresses the development of breast cancer in mice and rats [6, 7]
In a previous study of gene expression profiles of rexinoidinduced biomarkers, we discovered that bexarotene modulated the expression of IGFBP-6, along with other growth-regulatory genes, such as COX-2, cyclin D1, and RAR [11, 12]
IGF-II is a mitogen for many different cancer cells, and overexpression of IGF-II gives rise to mammary tumors [14, 15]
Summary
Ligands—The synthetic RXR-ligand bexarotene (Targretin, LGD1069) was a kind gift of Dr William Lamph (Ligand Pharmaceuticals) and was used at a 1 M final concentration, unless otherwise indicated. siRNA SmartPools and Dharmafect 1 transfection reagent were purchased from Dharmacon (Lafayette, CO). Selective Gene Knockdown Experiments—To assess the effect of gene silencing on the consequent changes in transcript levels, 104 cells were seeded on 24-well tissue culture plates 24 h prior to transfection. Reporter Assays—Reporter assays of luciferase expression vectors were carried out to assess the retinoid responsiveness of various regulatory regions of the IGFBP-6 gene promoter and introns. Forty-eight hours later transfected cells were treated with bexarotene or vehicle Another 24 h later, cell supernatants were harvested, and proteins from whole cell lysates were extracted using methods previously described [11]. Binding proteins 1– 6 in HMECs after a 24-h exposure to the drug using real time quantitative reverse transcription-PCR. These studies showed that IGFBP-2 mRNA was most abundant in HMECs, whereas.
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