Co-targeting MADD and Wnt/β-catenin signaling in Anaplastic Thyroid Cancer

项目简介：在美国，甲状腺癌的发病率正在迅速上升。退伍军人扯平了 他们一生中罹患甲状腺癌的风险更高，这是因为他们暴露在 武装部队。此外，患有甲状腺癌的退伍军人自我报告的患病率更高 橙剂暴露的可能性，增加了这种恶性肿瘤与退伍军人管理局人群的相关性。尽管使用了 在一系列治疗组合中，ATC的预后很差，中位生存期为6个月。 ATC肿瘤比所有其他形式的甲状腺癌具有更大的突变负担，这些类型的甲状腺癌会带来显著的 因此，确定在“结节”起作用的蛋白质是很重要的。 与ATC有关的不同信号通路的“点”，因此可以代表ATC的“阿喀琉斯跟”。这个 CTNNB1(β-catenin)是Wnt信号通路的效应分子，在上皮-间充质形成过程中起重要作用 转移所需的过渡(EMT)。PI3K/Akt/GSK3β信号在ATC中由于突变而过度活跃。这 能增强β-连环蛋白活性，还能磷酸化MAPK激活死亡结构域含蛋白 (MADD)，并有助于其有利于生存的功能。PMADD使ATC细胞抵抗凋亡。因此，在 ATC、pMADD和β-连环蛋白可以作为两个潜在的“结点”。 最重要的是，我们最近的新发现表明，MADD基因敲除可以显著抑制 肿瘤坏死因子α通过阻止PERK激活和随后的pGSK3β而介导PERK-连环蛋白信号的激活 激活。ERK缺乏Gsk3β的磷酸化，促进β-连环蛋白的泛素化导致ITS 降解和由此产生的对EMT激活的阻断。此外，肿瘤内给药MADD siRNA 显著降低治疗小鼠原位ATC肿瘤生长和肺转移。因此，MADD是一种 ATC的潜在治疗靶点可以单独使用，也可以与Wnt/β-Catenin抑制剂联合使用。 基于一个非常强大的科学前提，我们假设MADD下调制可以是有效的 抑制生长和克服针对高活性MAPK、PI3K/Akt和Wnt/β-catenin的药物耐药性 信号，这是ATC的标志。为了解决这个问题，在AIM-1中，我们将从功能上描述 CRISPR/Cas9介导的ATC细胞MADD基因敲除对Wnt信号转导的影响 将确定下调MADD表达的影响，以及MAPK和PI3K/Akt信号通路， 在AIM-3中，我们将测试与MADD联合治疗的体内疗效 原位和自发性ATC小鼠模型的基因敲除。 ATC不成比例地占了甲状腺癌相关死亡的大部分。癌细胞特异性 MADD的表达，它在激活几个关键信号通路中的作用，以及它作为促进生存的能力 ATC中的因子和促进转移使其成为治疗开发的理想靶点。重要的是，MADD 缺失对正常细胞无明显影响。因此，拟议的新颖研究与退伍军人高度相关。

Project Description: Thyroid cancer incidence is rapidly increasing in the United States. Veterans are at even higher risk of developing thyroid cancer in their lifetime due to the increased potential of radiation exposure in the armed services. Furthermore, veterans who suffered from thyroid cancer self-reported a higher prevalence of Agent Orange exposure, increasing the relevance of this malignancy to the VA population. Despite the use of a range of combinations of treatments, ATC exhibits a dismal prognosis with a median survival of < six months. ATC tumors possess a greater mutation burden than all other forms of thyroid cancer which impart significant growth benefit and high metastatic potential Therefore, it is important to identify proteins that function at “nodal points” of different signaling pathways implicated in ATC, and thus could represent ATC “Achilles Heel.” The CTNNB1 (β-catenin) is an effector molecule of Wnt signaling which is critical for epithelial-mesenchymal transition (EMT) required for metastasis. PI3K/Akt/GSK3β signaling is hyperactive in ATC due to mutations. This can enhance β-catenin activity and also phosphorylate Map kinase Activating Death Domain-containing protein (MADD) and contribute to its pro-survival function. pMADD renders ATC cells resistant to apoptosis. Thus in ATC, pMADD and β-catenin can serve as two potential “nodal points.” Most importantly, our recent novel findings have shown that MADD knockdown can significantly inhibit TNFα mediated activation of β-catenin signaling by preventing pERK activation and consequent pGSK3β activation. Lack of GSK3β phosphorylation by ERK, facilitated ubiquitination of β-catenin leading to its degradation and resultant blockage of EMT activation. Furthermore, intra-tumoral administration of MADD siRNA significantly reduced orthotpic ATC tumor growth and lung metastasis in treated mice. Therefore, MADD is a potential therapeutic target in ATC either alone or in combination with Wnt/β-catenin inhibitors. Based on a very strong scientific premise, we hypothesize that MADD down modulation can be effective in inhibiting growth and overcoming resistance to drugs targeting hyperactive MAPK, PI3K/Akt and Wnt/β-catenin signaling, which are hallmarks of ATC. To address this, in aim-1, we will functionally characterize the impact of CRISPR/CAS9 mediated MADD gene knock-out in ATC cells on Wnt signaling in vitro and ex vivo; in aim-2, we will determine the impact of down-modulating MADD expression, and MAPK and PI3K/Akt signaling pathways, on Wnt signaling; and in aim-3, we will test the in vivo therapeutic efficacy of combination treatment with MADD knockdown in orthotopic and spontaneous mouse models of ATC. ATC disproportionately accounts for the majority of thyroid cancer-related deaths. Cancer cell-specific expression of MADD, its role in activating several key signaling pathways, and its ability to act as a pro-survival factor and promote metastasis in ATC makes it an ideal target for therapeutic development. Importantly, MADD deletion had no apparent effect on normal cells. Thus the proposed novel studies are highly relevant to veterans.