IDENTIFICATION OF NEW RETINOID REGULATED BIOMARKERS IN BREAST AND OVARIAN CELLS

类维生素a是很有前途的化学预防剂，在动物和人类。然而，由于目前可用的类维生素a是相对有毒的，它们通常不用于预防癌症。因此，受体选择性类维生素a正在进行测试，以开发可能在降低毒性的情况下预防癌症的药物。我们研究的长期目标是确定受这些受体选择性类维生素a调节的基因，并确定这些基因的蛋白质产物是否可以作为临床化学预防试验的替代终点生物标志物。我们的初步研究表明，在转基因小鼠模型中，包括LGD1069在内的rcr选择性类维甲酸可以抑制正常和恶性乳腺细胞的增殖，并抑制乳腺肿瘤的发展，可能比天然存在的广谱类维甲酸的毒性更小。我们现在建议在乳腺细胞中确定一组由RXR选择性类维生素a调节的基因，这些基因调节这些细胞的生长和侵袭特性，并确定这些基因表达的变化是否与类维生素a成功的化学预防有关。首先，我们将确定在rcr选择性配体LDG1069处理后，人类乳腺和卵巢细胞中上调或下调的一组基因。我们将使用cDNA表达阵列来确定LGD1069处理的正常、癌前和恶性正常乳腺和卵巢细胞中上调或下调的基因。其次，我们将确定这些特定的RXR调控基因是否参与控制乳腺和卵巢细胞的生长和侵袭。在Specific Aim 1中发现的参与信号转导途径的基因，或与调节生长或侵袭的蛋白质相关的基因，将具有这些基因的表达和反义结构，并转染细胞以确定如何准备表达或抑制。转染后的细胞将显示这些基因的表达或抑制如何影响生长或侵袭性，或影响LGD1069对细胞的作用。第三，我们将确定LGD1069是否会诱导乳腺体内类维甲酸调节基因的表达变化。我们将使用LGD1069处理SV40 Tag转基因小鼠模型中保存的乳腺肿瘤形成阶段的组织，研究LGD1069是否会调节正常组织、发育不良组织和恶性组织中上述标记物的体内表达。测试的标记还包括增殖标记组蛋白H3（磷酸化形式），以及ER， ErbB2和类视黄醛受体（rar α, β, γ）。通过这些研究，我们将确定由rxr选择性类维生素a调节的有希望的标记物，这些标记物可以作为化学预防试验的替代终点生物标记物。LGD1060改变的潜在生物标志物将直接在项目1和项目2中使用人类乳腺和卵巢组织进行测试。

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.