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

描述（由申请人提供）：目前我们缺乏生物标志物来预测雌激素受体阴性（ER(-)）浸润性乳腺癌的发展。由于缺乏对ER（-）乳腺癌起始/进展的生物学理解，这一领域的进展受到阻碍。本应用研究了一种新的信号网络对ER（-）乳腺癌侵袭的调控，该信号网络涉及一种被ER沉默的转录抑制因子Snail-1。在Aim 1中，我们将研究一个涉及两个转录抑制因子（Snail-1和ZEB1）的新型信号网络对ER（-）乳腺肿瘤细胞侵袭的调节。Snail-1和ZEB1通过抑制上皮基因的转录促进侵袭性肿瘤行为。Snail-1和ZEB1在乳腺癌细胞系中经常共表达，但尚不清楚它们如何合作促进侵袭行为。MicroRNA200 （miRNA200）家族成员抑制乳腺肿瘤细胞侵袭，其表达水平在浸润性乳腺肿瘤细胞中相对于非浸润性乳腺肿瘤细胞降低。然而，在乳腺肿瘤细胞中最初抑制miRNA200从而驱动ZEB1表达和侵袭行为的因素尚不清楚。我们将探讨Snail-1通过调节miRNA200/ZEB1轴促进ER（-）乳腺肿瘤细胞侵袭的假说。为了验证我们的体外研究结果，我们将研究核Snail-1/miRNA200/核ZEB1网络在原发性人ER（-）乳腺癌中的表达，并回顾性确定该网络的表达是否预测患者预后。Snail-1的mRNA水平不能反映Snail-1的活性，因为：1)Snail-1的活性依赖于其核定位，2)Snail-1的定位受Snail-1翻译后修饰的调控。先前对人类癌症中Snail-1蛋白表达的研究使用了特征较差的抗体，这些抗体在某些情况下会与Snail-1家族的其他成员发生反应。我们利用高特异性的Snail-1抗体优化了一种检测核Snail-1蛋白的免疫组化方案。我们的初步研究表明，核蜗牛-1在ER（-）中频繁表达，而在ER（+）导管乳腺癌中不表达。在Aim 2中，我们将研究该网络在恶性乳腺病变前的表达，这些病变来自一组独特的乳腺癌高风险女性。我们将测试该网络在这些癌前病变中的表达是否能预测浸润性ER（-）乳腺癌的后续发展。这项为期两年的研究结果将使我们能够进行必要的功率计算，以进行未来的前瞻性研究，研究核Snail-1/miRNA200/核ZEB1网络作为预测浸润性ER（-）乳腺癌发展的癌前病变生物标志物的价值。总的来说，这项为期两年的研究将确定ER（-）乳腺肿瘤细胞侵袭行为的分子决定因素，作为鉴定：1)早期发现浸润性乳腺癌的生物标志物，以及2)预防和治疗浸润性ER（-）乳腺疾病的靶点的一种手段。

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.