Functional analysis of Manic Fringe in the claudin-low breast cancer

描述(申请人提供)：雌激素受体、孕激素受体和Her2(此后称为TNBC)三重阴性的乳腺癌占乳腺癌的15%-20%，并以侵袭性著称，但目前还没有针对这些恶性肿瘤的靶向治疗。基底样型(BLBC)和克拉丁低型(CLBC)是TNBC的两种主要亚型。CLBC具有上皮向间充质转化(EMT)的特征，被认为起源于乳腺干细胞(MASC)。Noch是干细胞的强大调节器，也是包括乳腺在内的许多组织中的EMT。最近我们发现，Lfng是哺乳动物的一种条纹，可以修饰Notch受体并调节Notch的激活，在MASC和腔前体细胞中发挥肿瘤抑制功能。有趣的是，在人类乳腺癌的各个亚型中，Lfng在BLBC中的表达最低，而另一种哺乳动物的边缘躁狂(Mfng)在CLBC中的表达最高。因此，条纹可能不同地调节不同类型乳腺上皮细胞中Notch的激活，并且条纹的遗传改变可能导致不同起源细胞中Notch信号的失调，最终导致不同的乳腺癌亚型。虽然Lfng抑制BLBC，但Mfng可能促进CLBC的启动和/或进展。事实上，我们对CLBC细胞系的初步分析表明，Mfng增强了Notch4信号，这已被证明可以调节乳腺癌干细胞的活动。在这项提案中，我们计划研究Mfng在CLBC中的作用，具体目的如下：1)利用乳腺癌细胞系确定Mfng的功能。我们将在CLBC细胞系中敲除Mfng，并在非CLBC细胞系中过表达Mfng，以测试对Notch激活、EMT、肿瘤干细胞浓缩及其肿瘤起始和转移能力的影响。2)识别CLBC中Mfng的潜在下游目标。我们打算在Mfng基因敲除或过表达的稳定细胞系中进行微阵列基因表达分析(来自Aim 1)，并检测候选基因在CLBC组织中的表达。3)验证Mfng在CLBC小鼠模型中的作用。我们将把Mfng缺失的小鼠培育成CLBC小鼠模型，以确定Mfng的缺失是否抑制了CLBC肿瘤的形成。我们希望获得数据来证明Mfng促进CLBC的启动和/或进展。作为一种“可药物”的酶，Mfng可能是一个理想的CLBC靶点，因为它影响特定的细胞类型，而不是全面抑制Notch信号的广泛影响。此外，了解CLBC的独特生物学特性以及Mfng调控的Notch激活在CLBC中的具体作用，将有助于深入了解癌症干细胞，这被认为是启动复发和转移的因素。

DESCRIPTION (provided by applicant): Breast cancers that are triple negative for estrogen receptor, progesterone receptor, and Her2 (thereafter referred as TNBC) account for 15-20% of breast carcinomas and are notoriously aggressive, yet there is no targeted therapy against these malignancies. Basal-like (BLBC) and claudin-low (CLBC) are the two major subtypes of TNBC. CLBCs show characteristic of epithelial-to-mesenchymal transition (EMT) and are thought to originate from mammary stem cells (MaSC). Notch is a powerful regulator for stem cells as well as EMT in many tissues including the mammary gland. Most recently we have revealed that Lunatic Fringe (Lfng), one of the mammalian Fringes that modify Notch receptors and modulate Notch activation, exerts tumor suppressor function in MaSC and luminal progenitor cells. Interestingly, while Lfng expression is lowest in BLBC among various subtypes of human breast cancer, another mammalian Fringe, Manic Fringe (Mfng), shows highest expression in CLBC. Thus Fringes may differentially regulate Notch activation in different cell types of mammary epithelial hierarchy, and genetic alterations of Fringes may cause dysregulation of Notch signaling in different cells of origin, ultimately leading to different subtypes of breast cancer. While Lfng suppresses BLBC, Mfng may facilitate CLBC initiation and/or progression. Indeed, our preliminary analysis in a CLBC cell line suggests that Mfng enhances Notch4 signaling, which has been shown to regulate breast cancer stem cell activity. In this proposal, we plan to investigate Mfng roles in CLBC with the following Specific Aims: 1) Define Mfng functions using breast cancer cell lines. We will knockdown Mfng in CLBC cell lines and overexpress Mfng in non-CLBC cell lines to test for effects on Notch activation as well as EMT, cancer stem cell enrichment and its tumor initiating and metastatic capacity. 2) Identify potential downstream targets of Mfng in CLBC. We intend to perform microarray gene expression analysis in stable cell lines with Mfng knockdown or overexpression (from Aim 1), and to examine the expression of candidate genes in CLBC tissues. 3) Validate Mfng roles in CLBC mouse models. We will breed the Mfng null mice into CLBC mouse models to determine whether loss of Mfng suppresses CLBC tumor formation. We hope to obtain data to demonstrate that Mfng promotes CLBC initiation and/or progression. As a "druggable" enzyme, Mfng could be an ideal CLBC target since it affects specific cell types rather than broad impact from overall inhibition of Notch signaling. Furthermore, understanding the unique biology of CLBC and specific roles of Mfng-modulated Notch activation in CLBC will shed insight on cancer stem cell, which is thought to initiate recurrence and metastasis.