FOXP3-microRNA146-NFkB Axis in Tumor Suppression

摘要 背景：X连锁基因FOXP3是叉头盒/翼状螺旋转录因子的成员 家族成员并对X连锁的IPEX负责(免疫失调、多内分泌病和肠病，X连锁) 小鼠和人类的综合症。在发育过程中，Foxp3似乎扮演着主要调节者的角色 和调节性T细胞的功能。除了固有的T细胞功能外，我们还证明了FOXP3是 首个针对乳腺癌的X连锁肿瘤抑制因子。Foxp3直接靶向关键癌基因HER2/ErbB2 以及Skp2和肿瘤抑制基因p21。最近，我们报道了FOXP3抑制前列腺 通过抑制c-myc的转录而致癌。FOXP3在非编码调控中的潜在作用 RNA还没有被研究过。使用microRNA(MiRNA)阵列分析，我们筛选了所有已知的miRNA 人类以FOXP3为目标。值得注意的是，我们观察到FOXP3显著诱导了 人乳腺癌细胞中的miRNA(MiR)-146a/b(miR-146a是7倍，miR-146b是14倍)。这 在人类乳腺和前列腺癌细胞系中，TaqMan miRNA检测证实了这一观察结果。更多 重要的是，小鼠前列腺组织中FoxP3的组织特异性缺失导致miR- 146，人miR-146a/b的小鼠同源物。从其他人的积累数据表明，miR- 146a/b抑制癌细胞增殖、侵袭和转移，包括乳腺癌、前列腺癌、 胰腺癌和神经胶质瘤。此外，其他研究也表明miR-146a/b负向调节核因子B。 通过抑制IRAK1和TRAF6的表达而发挥活性。然而，除了乳腺癌转移外， 抑制因子1，miR-146a/b的反式激活因子在很大程度上尚未确定。因为我们的初步数据显示 FOXP3在体内外对癌细胞miR-146a/b的强烈诱导作用，对其进行鉴定具有重要意义 FOXP3-miR146-NF&B轴对肿瘤抑制的影响 假设和目标：这个项目的中心假设是FOXP3-miR146-NF-B轴扮演着一个 FOXP3在肿瘤抑制功能中的关键作用。本项目的总体目标是确立以下原则 肿瘤抑制因子在FOXP3和非编码RNA之间的传递。这一假设将在三个具体的方面进行检验 目标。1)明确miR-146a/b在FOXP3介导的肿瘤抑制中的作用。2)澄清 FOXP3诱导miR-146a/b转录的机制。3)确定功能是否为 MIR-146a/b是通过抑制NF-954；B介导的。 方法：首先，我们将测试miR-146a/b对乳腺癌细胞增殖和存活的影响， 使用miR-146a/b抑制剂。这些抑制剂还将用于确定miR-146a/b是否需要 FOXP3对生长的抑制作用。其次，我们将调查FOXP3诱导如何导致快速和渐进的 MiR-146a/b转录本的诱导。利用生物信息学工具，我们已经确定了候选叉头绑扎 MiR-146a(5个位点)/b(3个位点)潜在启动子区域内的基序(RYMAAYA)。我们将使用 染色质免疫沉淀、凝胶迁移率改变分析、荧光素酶报告分析以及 定点突变以鉴定功能叉头基序。第三，使用miR-146a/b抑制剂，我们 将确定FOXP3诱导的miR-146a/b是否抑制癌细胞中的核因子-B活性。一旦我们进入 体外研究为FOXP3-miR146-NF&B轴提供了证据，我们将利用遗传模型 以确定该轴在体内的功能作用。 创新和意义：我们提出的研究建立在一种新的观察基础上。如果得到验证，我们将拥有 提供了X连锁肿瘤抑制基因和非编码RNA之间缺失的链接和一种新的 FOXP3调控miR-146a/b抑瘤机制的研究由于特定血统的消融 小鼠前列腺上皮细胞中的Foxp3导致前列腺增生和前列腺上皮内瘤变， 这一机制应该会对我们对早期癌症的理解产生重大影响，并对 对改进癌症早期检测、干预和预防的潜在意义。此外，有缺陷的 Foxp3功能可能为肿瘤细胞中的结构性核因子B活性提供了一种新的机制，目前仍未见报道 很大程度上无法解释。将FOXP3的肿瘤抑制功能与核因子B激活联系起来可能提供一种新的 FOXP3缺陷癌症的治疗途径。因为已经有治疗癌症的核因子B抑制剂 在人类的治疗中，我们的假设，如果被拟议的工作证实，将表明癌症与 可识别的FOXP3缺陷可能对抑制剂有反应。

Abstract Background: The X-linked gene FOXP3 is a member of the forkhead-box/winged-helix transcription factor family and responsible for X-linked IPEX (Immunodysregulation, Polyendocrinopathy, and Enteropathy, Xlinked) syndrome in mice and humans. FOXP3 appears to function as the master regulator in the development and function of regulatory T cells. Apart from the intrinsic T-cell function, we have demonstrated that FOXP3 is the first X-linked tumor suppressor for breast cancer. FOXP3 directly targets critical oncogenes HER2/ErbB2 and SKP2 and a tumor suppressor gene p21. More recently, we reported that FOXP3 suppresses prostate cancer by repressing transcription of c-MYC. The potential role in FOXP3-mediated regulation on noncoding RNA has not been studied. Using a microRNA (miRNA) array analysis, we screened all known miRNAs in humans for FOXP3 targets. Remarkably, we observed that FOXP3 drastically induced the expressions of miRNA (miR)-146a/b (7-fold for miR-146a and 14-fold for miR-146b) in human breast cancer cells. This observation was validated by a TaqMan miRNA assay in human breast and prostate cancer cell lines. More importantly, tissue-specific deletion of FoxP3 in mouse prostate tissue caused a significant reduction of miR- 146, the mouse homologue of human miR-146a/b. Accumulating data from others demonstrate that miR- 146a/b inhibit cancer cell proliferation, invasion, and metastasis in human cancers, including breast, prostate, pancreatic cancers and glioma. Moreover, others also showed that miR-146a/b negatively regulate NF-κB activity by inhibiting expression of IRAK1 and TRAF6. However, apart from breast cancer metastasis suppressor 1, transactivators of the miR-146a/b are largely unidentified. Since our preliminary data reveal a strong induction of miR-146a/b by FOXP3 in the cancer cells in vitro and in vivo, it is of great interest to identify the impact of FOXP3-miR146-NFκB axis on tumor suppression. Hypothesis and Goals: The central hypothesis of this project is that the FOXP3-miR146-NF-κB axis plays a critical role in tumor suppressor function of FOXP3. The overall goal of this project is to establish the principle of tumor suppressor relay between FOXP3 and noncoding RNA. This hypothesis will be tested in three specific aims. 1) To identify the functional role of miR-146a/b in FOXP3-mediated tumor suppression. 2) To elucidate the mechanism by which FOXP3 induces miR-146a/b transcriptions. 3) To determine whether the function of miR-146a/b is mediated by repression of NF-κB. Approach: First, we will test the effects of the miR-146a/b on proliferation and survival of breast cancer cells, using miR-146a/b inhibitors. The inhibitors will also used to determine whether the miR-146a/b are required for growth inhibition by FOXP3. Second, we will investigate how FOXP3 induction causes a rapid and progressive induction of miR-146a/b transcripts. Using bioinformatic tools, we have identified the candidate forkheadbinding motifs (RYMAAYA) within potential promoter region of miR-146a (5 sites)/b (3 sites). We will use chromatin immunoprecipitation, electrophoretic mobility shift assay and luciferase reporter assays as well as site-directed mutagenesis to identify the functional forkhead motif. Third, using the miR-146a/b inhibitors, we will determine whether FOXP3-induced miR-146a/b represses the NF-κB activity in cancer cells. Once our in vitro studies provide evidence for the FOXP3-miR146-NFκB axis, we will take advantage of the genetic model to determine the functional role of this axis in vivo. Innovation and Significance: Our proposed studies build on a novel observation. If validated, we would have provided a missing link between the X-linked tumor suppressor gene and noncoding RNA and a novel mechanism for regulating miR-146a/b by FOXP3 in tumor suppression. Since lineage-specific ablation of FoxP3 in the mouse prostate epithelial cells leads to prostate hyperplasia and prostatic intraepithelial neoplasia, this mechanism should have a substantial impact on our understanding of early stage cancers and have a potential significance in improving early cancer detection, intervention, and prevention. Furthermore, defective FOXP3 function may provide a novel mechanism for constitutive NF-κB activity in tumor cells, which is still largely unexplained. Linking tumor suppressor function of FOXP3 to NF-κB activation may provide a new therapeutic approach for cancers with FOXP3 defects. Since an NF-κB inhibitor is already available for cancer therapy in human, our hypothesis, if validated by the proposed work, would suggest that cancer with identifiable FOXP3 defects may be responsive to the inhibitor.