Retinoids are promising chemopreventive agents in animals and in humans. However, because currently available retinoids are relatively toxic, they are not generally used for cancer prevention. Therefore, receptor-selective retinoids are being tested to develop agents which might prevent cancer with reduced toxicity. The long-term goal of our studies is to identify genes which are regulated by these receptor-selective retinoids and to determine whether the protein products of these genes can serve as surrogate-endpoint biomarkers for clinical chemoprevention trials. Our preliminary studies demonstrated that RCR-selective retinoids including LGD1069 can inhibit the proliferation of normal and malignant breast cells and suppress mammary tumor development in a transgenic mouse model, perhaps with less toxicity than naturally occurring broad- spectrum retinoids. We now propose to identify a set of genes which are modulated by RXR- selective retinoids in breast cells and which regulate growth and invasive properties of these cells, and to determine whether changes in the expression of these genes are associated with successful chemoprevention by retinoids. First, we will determine the set of genes which are up- or down-regulated in human breast and ovarian cells upon treatment by the RCR-selective ligand LDG1069. We will use cDNA expression arrays to determine the genes which are up- or down-regulated panels of normal, premalignant, and malignant normal breast and ovarian cells treated with LGD1069. Second, we will determine whether these specific RXR- regulated genes are involved in controlling the growth and invasion of breast and ovarian cells. Genes identified in Specific Aim 1 which are involved in signal transduction pathways, or are related to proteins which regulate growth or invasion, will have expression and antisense constructs of these genes, and transfect cells to determine how expression or suppression prepared. Transfected cells will show how expression or suppression of these genes affects growth or invasiveness, or affects the actions of LGD1069 on the cells. Third, we will determine whether LGD1069 induces changes in the expression of retinoid-regulated genes in vivo in mammary glands. Using stored tissues representing stages of mammary tumor formation from an SV40 Tag transgenic mouse model treated with LGD1069, we will investigate whether LGD1069 modulated the in vivo expression of markers identified above in normal, and dysplastic, and in malignant tissue. markers tested will also include the proliferation marker Histone H3 (the phosphorylated form), as well as ER, ErbB2, and the retinoid receptors (RARalpha, beta, gamma). Through these studies we will identify promising markers regulated by RXR-selective retinoids which could serve as surrogate endpoint biomarkers in chemoprevention trials. Potential biomarkers altered by LGD1060 will then be tested directly in Projects 1 and 2 using human breast and ovarian tissues.
