Identification of Biomarkers Modulated by the Rexinoid LGD1069 (Bexarotene) in Human Breast Cells Using Oligonucleotide Arrays

Abstract

Retinoids have been found to be promising chemopreventive agents that play an important role in regulating cell growth, differentiation, and apoptosis. The action of retinoids is mediated by retinoid receptors (retinoic acid receptors and retinoid X receptors), which are nuclear transcription factors that, when bound to retinoids, regulate gene expression.LGD1069 is a highly selective RXR agonist that has reduced toxicity compared with retinoids. Our previous studies have shown that RXR-selective ligands (or ''rexinoids''), including LGD1069, can inhibit the growth of normal and malignant breast cells and can suppress the development of breast cancer in transgenic mice. For the current study, we attempted to identify biomarkers of the chemopreventive effect of the RXR-selective retinoid LGD1069. In these experiments, we used Affymetrix microarrays to identify target genes that were modulated by LGD1069 in normal human breast cells. Affymetrix and dChip analysis identified more than 100 genes that were up-regulated or down-regulated by LGD1069 treatment. We then tested 16 of these genes in validation experiments using quantitative reverse transcription-PCR and Western blotting of independently prepared samples, and found that 15 of 16 genes were modulated in a similar manner in these validation experiments as in the microarray experiments. Genes found to be regulated include known retinoidregulated genes, growth regulatory genes, transcription factors, and differentiation markers. We then showed that the expression of several of these rexinoid-regulated biomarkers is modulated in vivo in mammary glands from mice treated with LGD1069. These critical growth-regulating proteins will be promising targets of future agents for the prevention and treatment of breast cancer. (Cancer Res 2006; 66(24): 12009-18)

A New Rexinoid, NRX194204, Prevents Carcinogenesis in Both the Lung and Mammary Gland

“…2), and tumor-associated macrophages and other stromal cells in the tumor microenvironment are emerging as important targets for chemoprevention (32,41). Although additional studies such as the identification of biomarkers or oligonucleotide arrays, which have been previously described for bexarotene (42), are clearly needed to help define the molecular pathways targeted in the tumor stroma, our data support testing 4204 in upcoming clinical trials. NOTE: When tumors in female MMTV-neu mice grew to at least 32 mm 3 in volume, the mice were fed a control diet or 4204 for up to 4 wk.…”

Bexarotene Induces Cellular Senescence in MMTV-Neu Mouse Model of Mammary Carcinogenesis

“…Transfection of RARb2 in tumor cells lacking the receptor was associated with cell growth inhibition and induction of apoptosis (44). Our previous data obtained from the promoter luciferase activity assay (45) and those of others obtained by gene analysis of human breast epithelial cells (26) revealed that bexarotene, in addition to RXRs, can also induce RARb transcription and thus promotes cellular senescence. Here, we showed that atRA is a more powerful inducer of RARb than bexarotene, which correlates well with differences in their binding affinity to RARs (38,46).…”

“…Bexarotene also decreased RXRa expression in MDA-MB-231 and BT474 cells, suggesting receptor protein degradation, as has been reported for RARa in MCF-7 cells treated with atRA (47). The role of RARb and RXRa in mediating antitumor potential of retinoids and rexinoids seems to be overestimated, as reported by gene profiling as well (25,26). Previous studies (15,22,25) and our data in this study showed that retinoids and rexinoids can, in addition to modulating RARs and RXRs activity, also affect expression of p21, cyclins D-A, cdk2/4, pRb, E2F, and other genes/ proteins and thus facilitate retinoid receptor-independent cell and tumor growth inhibition as well as development of cellular senescence.…”

“…Recently, we found that 9cRA suppressed prostate carcinogenesis in p27-deficient mice, and this was associated with inhibition of cell proliferation and induction of cellular senescence (24). Gene analysis studies on MCF-7 cells treated with atRA (25) and on normal human breast epithelial cells treated with bexarotene (26) revealed up-and downregulation of hundred of genes, most of them associated with cell-cycle regulation, intercellular matrix proteins, cell differentiation, retinoid metabolism, various transcription factors, and secretion of cytokines that may affect surrounding and distant cells and tissues. However, the affected genes in both studies, except for RARb transcription, were not directly associated with cellular senescence (26).…”

“…Gene analysis studies on MCF-7 cells treated with atRA (25) and on normal human breast epithelial cells treated with bexarotene (26) revealed up-and downregulation of hundred of genes, most of them associated with cell-cycle regulation, intercellular matrix proteins, cell differentiation, retinoid metabolism, various transcription factors, and secretion of cytokines that may affect surrounding and distant cells and tissues. However, the affected genes in both studies, except for RARb transcription, were not directly associated with cellular senescence (26). Here, we used the MMTV-Neu mammary carcinogenesis model in mice and breast cancer cell lines to determine potential target genes associated with the bexaroteneinduced cellular senescence.…”