IDENTIFICATION OF NEW RETINOID REGULATED BIOMARKERS IN BREAST AND OVARIAN CELLS

类维生素A在动物和人类中是有前途的化学预防剂。然而，由于目前可用的类维生素A相对有毒，它们通常不用于癌症预防。因此，正在测试受体选择性类维生素A，以开发可能以降低毒性预防癌症的药物。我们研究的长期目标是确定受这些受体选择性维甲酸调控的基因，并确定这些基因的蛋白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，RCR选择性类维生素A（包括LGD 1069）可抑制转基因小鼠模型中正常和恶性乳腺细胞的增殖，并抑制乳腺肿瘤的发展，其毒性可能低于天然广谱类维生素A。我们现在提出，以确定一组基因的调节RXR-选择性类维生素A在乳腺细胞中，并调节这些细胞的生长和侵袭性，并确定这些基因的表达的变化是否与成功的化学预防类维生素A。首先，我们将确定在人乳腺和卵巢细胞中通过RCR选择性配体LDG 1069处理后上调或下调的基因组。我们将使用cDNA表达阵列来确定LGD 1069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调节基因是否参与控制乳腺和卵巢细胞的生长和侵袭。在特异性目的1中鉴定的参与信号转导途径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构建体，并转染细胞以确定如何制备表达或抑制。转染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LGD 1069对细胞的作用。第三，我们将确定LGD 1069是否诱导体内乳腺中类维生素A调节基因表达的变化。使用代表用LGD 1069处理的SV 40 Tag转基因小鼠模型的乳腺肿瘤形成阶段的储存组织，我们将研究LGD 1069是否调节上述在正常、发育异常和恶性组织中鉴定的标志物的体内表达。测试的标记物还包括增殖标记物组蛋白H3（磷酸化形式），以及ER、ErbB 2和类维生素A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调节的有希望的标志物，这些标志物可以作为化学预防试验中的替代终点生物标志物。LGD 1060改变的潜在生物标志物将直接在项目1和2中使用人类乳腺和卵巢组织进行检测。

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.