RETINOID INHIBITION OF BREAST CANCER GROWTH

维甲酸类化合物被用作抗肿瘤或化学预防药物 包括乳腺癌妇女在内的临床试验。我们发现 维甲酸仅抑制雌激素受体(ER)阳性的生长 细胞。我们现在发现，乳腺癌细胞拥有一种独特的 维甲酸核受体-α(RAR-α)的亚型 雌激素必须提高水平，才能使维甲酸抑制 成长。具体目标1：确定人类乳房中是否存在ER阳性 癌组织活检标本中RAR和RXR基因表达水平升高。 ER阴性的乳腺癌细胞株难以生长 RA抑制RAR-α的表达，而ER-α则呈下降趋势。 阳性细胞株表达高水平的RAR-α信使核糖核酸特异性目的2： 证明RA通过RAR-α对基因的调节起着至关重要的作用 在RA中抑制乳腺癌的生长。2a)确定 RA耐药ER阴性乳腺癌细胞的横切术 RAR-α表达载体及随后RAR-α表达的增强 阿尔法导致RA抑制生长。2b)证明抑制作用 利用选择性RAR-α拮抗剂结果的RAR-α功能 在抗RA-抑制生长2C)中选择性抑制野生型 利用RAR-α显性负突变体的类型RAR-α功能 结果导致细胞对维甲酸介导的生长抑制无效。 具体目标3：我们发现人乳腺癌细胞株表达一种独特的 受雌二醇转录调控的RAR-α亚型 在ER阳性细胞中。我们将识别和表征RAR-Alpha 异构体(S)在人乳腺癌细胞中的表达 并在功能上表征了5‘-上游调控区域( RAR-α基因的启动子区域)，并鉴定 雌激素介导的RAR-α基因调控元件 表情。具体目标4：阐明发病机制(S) 雌激素对人乳腺RAR-α基因转录的调节 癌细胞。确定利用分离的启动子是否 雌二醇通过与启动子结合来增强基因转录 雌激素反应元件(ERE)或刺激或抑制 其他反过来调节基因活性的因素。具体目标5： 乳腺组织中特异性视黄酸受体-α亚型(S)基因水平的研究 石蜡包埋的癌细胞及其周围间质 从患者身上获得的浸润性导管癌标本，以及 将标本的ER状态与RAR-αmRNA水平进行相关性分析。 特定目标6：确定RA是否也抑制体内生长 和/或ER转基因的MDA-MB-231细胞的转移潜能。

Retinoids are employed as anti-neoplastic or chemopreventative agents in clinical trials including women with breast carcinoma. We found retinoids only inhibit the growth of estrogen receptor (ER)-positive cells. We have now found that breast carcinoma cells possess a unique isoform of the retinoic acid nuclear receptor-alpha (RAR-alpha) whose levels must be enhanced by estrogens to allow retinoids to inhibit growth. Specific Aim 1: Determine whether ER positivity in human breast carcinoma biopsy specimens results in increased RAR and RXR mRNA levels. ER negative breast carcinoma cell lines are refractory to growth inhibitory by RA, and possess decreased RAR-alpha mRNA levels, while ER- positive cell lines express high RAR-alpha mRNA level Specific Aim 2: Demonstrate that RA modulation of genes via RAR-alpha plays a vital role in RA inhibition of breast carcinoma growth. 2A) Determine if transection of RA-refractory ER-negative breast carcinoma cells with a RAR-alpha expression vector and subsequent increased expression of RAR- alpha results in RA-inhibition of growth. 2B) Demonstrate that inhibition of RAR-alpha function utilizing a selective RAR-alpha antagonist results in resistance to RA-inhibition of growth 2C) Selective inhibition of wild type RAR-alpha function utilizing a RAR-alpha dominant negative mutant results in cells refractory to RA-mediated inhibition of growth. Specific Aim 3: We found human breast cancer cell lines express a unique isoform of RAR-alpha which is transcriptionally regulated by estradiol in ER-positive cells. We will identify and characterize the RAR-alpha isoform(s) expressed in human breast carcinoma cells; isolate, sequence and functionally characterize the 5'-upstream regulatory region (the promoter region) of the RAR-alpha gene and identify the regulatory elements responsible for estrogen-mediated modulation of RAR-alpha gene expression. Specific Aim 4: Elucidate the mechanism(s) involved in estrogen regulation of RAR-alpha gene transcription in human breast carcinoma cells. Ascertain utilizing the isolated promoter whether estradiol enhances genes transcription by binding to the promoter via an estrogen response element (ERE) or stimulates or represses the levels of other factors which in turn modulate gene activity. Specific Aim 5: Study the mRNA levels of the specific RAR-alpha isoform(s) in breast carcinoma cells and the surrounding stroma in paraffin embedded infiltrating ductal carcinoma specimens obtained from patients, and correlate the ER-status of the specimen and the level of RAR-alpha mRNA. Specific Aim 6: Determine whether RA also inhibits the in vivo growth and/or metastatic potential of ER-transfected MDA-MB-231 cells.