Nuclear Snail-1 in the progression of Estrogen Receptor(-) invasive breast cancer

描述（由申请人提供）：目前我们缺乏预测雌激素受体阴性[ER（-）]浸润性乳腺癌发展的生物标志物。 由于我们对ER（-）乳腺癌发生/进展的生物学缺乏了解，这一领域的进展受到阻碍。 本申请研究了ER（-）乳腺癌侵袭的调节，通过一种涉及Snail-1的新型信号网络，Snail-1是一种被ER沉默的转录阻遏物。 在目的1中，我们将研究一个新的信号网络涉及两个转录抑制因子（蜗牛-1和ZEB 1）的ER（-）乳腺肿瘤细胞侵袭的调节。 Snail-1和ZEB 1通过抑制上皮基因的转录促进侵袭性肿瘤行为。 Snail-1和ZEB 1经常在乳腺癌细胞系中共表达，但尚不清楚它们如何合作促进侵袭行为。 MicroRNA 200（miRNA 200）家族成员抑制乳腺肿瘤细胞的侵袭，并且相对于非侵袭性乳腺肿瘤细胞，它们的表达水平在侵袭性中降低。 然而，最初抑制乳腺肿瘤细胞中miRNA 200的因素，从而驱动ZEB 1表达和侵袭行为，是未知的。 我们将研究Snail-1通过调节miRNA 200/ZEB 1轴促进ER（-）乳腺肿瘤细胞侵袭的假设。 为了验证我们的体外研究结果，我们将研究核Snail-1/miRNA 200/核ZEB 1网络在原发性人ER（-）乳腺癌中的表达，并回顾性地确定该网络的表达是否预测患者预后。 Snail-1 mRNA水平不反映Snail-1活性，因为：1）Snail-1活性依赖于其核定位，以及2）Snail-1定位受Snail-1翻译后修饰调节。 以前对人类癌症中Snail-1蛋白表达的研究使用了特征不佳的抗体，在某些情况下，这些抗体与其他Snail-1家族成员发生反应。 我们已经优化了免疫组化方案检测核蜗牛-1蛋白使用高度特异性的蜗牛-1抗体。 我们的初步研究表明，核Snail-1在ER（-）而不是ER（+）导管乳腺癌中频繁表达。 在目标2中，我们将研究该网络在癌前乳腺病变中的表达，这些病变来自一个独特的乳腺癌高危女性队列。 我们将检测这种网络在这些癌前病变中的表达是否可以回顾性地预测浸润性ER（-）乳腺癌的后续发展。 这项为期两年的研究的结果将使我们能够进行未来前瞻性研究所需的功效计算，研究核Snail-1/miRNA 200/核ZEB 1网络作为癌前病变生物标志物的价值，预测侵袭性ER（-）乳腺癌的发展。 总的来说，这项为期两年的提案中的研究将定义ER（-）乳腺肿瘤细胞侵袭行为的分子决定因素，作为识别的一种手段：1）早期检测侵袭性乳腺癌的生物标志物，2）预防和治疗侵袭性ER（-）乳腺疾病的靶点。 公共卫生相关性：只有一小部分诊断为癌前乳腺病变的女性会发展为浸润性乳腺肿瘤。 目前，我们缺乏预测侵袭性肿瘤生长高风险的生物标志物。 这种生物标志物的鉴定和验证将需要：1）侵袭的关键调节因子的分子研究，2）确定这些侵袭标志物在癌前病变和恶性病变中的表达频率的研究，和3）确定这些标志物在癌前病变中的表达是否预测随后侵袭性癌症的发展的前瞻性研究。 本两年期提案中的试验将：1）研究通过涉及转录抑制因子Snail-1的新型信号网络对雌激素受体阴性[ER（-）]乳腺癌侵袭的调节，2）检查该Snail-1网络的决定子在恶性和癌前乳腺病变中的表达频率，和3）回顾性地检查该Snail-1网络在癌前乳腺病变中的表达是否预测侵袭性ER（-）乳腺癌的发展。 使用所获得的结果，我们将进行规划未来前瞻性研究所需的功效计算，该研究测试该信号网络是否是预测侵袭性ER（-）乳腺癌发展的有价值的生物标志物。 总的来说，这些研究将确定ER（-）乳腺肿瘤细胞侵袭行为的分子决定因素，作为识别以下因素的一种手段：1）早期检测侵袭性ER（-）乳腺癌的生物标志物，2）预防和治疗侵袭性ER（-）乳腺疾病的靶点。

DESCRIPTION (provided by applicant): Currently we lack biomarkers to predict development of Estrogen Receptor-negative [ER(-)] invasive breast cancer. Progress in this area is hindered by our lack of understanding of the biology of ER(-) breast cancer initiation/progression. This application studies the regulation of ER(-) breast cancer invasion by a novel signaling network involving Snail-1, a transcriptional repressor silenced by ER. In Aim 1, we will investigate the regulation of ER(-) breast tumor cell invasion by a novel signaling network involving two transcriptional repressors (Snail-1 and ZEB1). Snail-1 and ZEB1 promote invasive tumor behavior by suppressing transcription of epithelial genes. Snail-1 and ZEB1 are frequently co-expressed in breast cancer cell lines, but it is unknown how they cooperate to promote invasive behavior. MicroRNA200 (miRNA200) family members suppress breast tumor cell invasion, and their expression levels are reduced in invasive relative to non-invasive breast tumor cells. However, the factors that initially suppress miRNA200 in breast tumor cells, thus driving ZEB1 expression and invasive behavior, are unknown. We will investigate the hypothesis that Snail-1 promotes ER(-) breast tumor cell invasion by regulating the miRNA200/ZEB1 axis. To validate our in vitro findings, we will investigate expression of the nuclear Snail-1/miRNA200/nuclear ZEB1 network in primary human ER(-) breast cancers, and retrospectively determine if expression of this network predicts patient prognosis. Snail-1 mRNA levels do not reflect Snail-1 activity because: 1) Snail-1 activity is dependent on its nuclear localization, and 2) Snail-1 localization is regulated by Snail-1 post-translational modifications. Previous studies of Snail-1 protein expression in human cancers used poorly-characterized antibodies that in some cases react with other Snail-1 family members. We have optimized an immunohistochemistry protocol for detecting nuclear Snail-1 protein using a highly-specific Snail-1 antibody. Our preliminary studies indicate that nuclear Snail-1 is expressed frequently in ER(-) but not in ER(+) ductal breast cancers. In Aim 2, we will study expression of this network in pre-malignant breast lesions obtained from a unique cohort of women at high risk for developing breast cancer. We will test if expression of this network in these pre-malignant lesions retrospectively predicts subsequent development of invasive ER(-) breast cancer. The results from this two-year study will allow us to perform power calculations necessary for a future prospective study investigating the value of a nuclear Snail-1/miRNA200/nuclear ZEB1 network as a biomarker in pre-malignant lesions that predicts development of invasive ER(-) breast cancer. Collectively, the studies in this two-year proposal will define molecular determinants of ER(-) breast tumor cell invasive behavior as a means of identifying: 1) biomarkers for early detection of invasive breast cancer, and 2) targets for prevention and treatment of invasive ER(-) breast disease. PUBLIC HEALTH RELEVANCE: Only a fraction of women diagnosed with pre-malignant breast lesions develop invasive breast tumors. Currently we lack biomarkers that predict a high risk for invasive tumor growth. Identification and validation of such a biomarker will require: 1) molecular studies of key regulators of invasion, 2) studies determining the frequency of expression of these invasion markers in pre-malignant and malignant lesions, and 3) prospective studies determining if expression of these markers in pre-malignant lesions predicts subsequent development of invasive cancer. The experiments in this two year proposal will: 1) investigate the regulation of Estrogen Receptor-negative [ER(-)] breast cancer invasion by a novel signaling network involving the transcriptional repressor Snail-1, 2) examine the frequency of expression of determinants of this Snail-1 network in malignant and pre-malignant breast lesions, and 3) retrospectively examine if expression of this Snail-1 network in pre-malignant breast lesions predicts development of invasive ER(-) breast cancer. Using the results obtained, we will perform a power calculation necessary for planning a future prospective study that tests if this signaling network is a valuable biomarker for predicting development of invasive ER(-) breast cancer. Collectively, these studies will define molecular determinants of ER(-) breast tumor cell invasive behavior as a means of identifying: 1) biomarkers for early detection of invasive ER(-) breast cancer, and 2) targets for prevention and treatment of invasive ER(-) breast disease.