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

项目描述：甲状腺癌的发病率在美国迅速上升。老兵们甚至 由于受辐射照射的可能性增加， 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较高 橙子剂暴露，增加了这种恶性肿瘤与VA人群的相关性。尽管使用 在一系列的治疗组合中，ATC表现出令人沮丧的预后，中位生存期<6个月。 ATC肿瘤具有比所有其他形式的甲状腺癌更大的突变负荷，这赋予显著的 因此，重要的是鉴定在“结节”中起作用的蛋白质， 点”的不同信号通路参与ATC，因此可以代表ATC的“阿喀琉斯之踵”。的 CTNNB 1（β-catenin）是Wnt信号的效应分子，在上皮-间充质细胞间充质细胞的增殖和分化中起重要作用。 转移所需的EMT。由于突变，PI 3 K/Akt/GSK 3 β信号在ATC中过度活跃。这 可增强β-连环蛋白活性，也可使含有MAP激酶激活死亡结构域的蛋白磷酸化 （MADD），并有助于其促生存功能。pMADD使ATC细胞对凋亡具有抗性。因此在 ATC、pMADD和β-catenin可以作为两个潜在的“节点”。 最重要的是，我们最近的新发现表明，MADD敲低可以显著抑制 TNFα通过阻止pERK激活和随后的pGSK 3 β介导β-catenin信号转导的激活 activation. ERK缺乏GSK 3 β磷酸化，促进β-catenin的泛素化，导致其 降解和由此导致的EMT激活的阻断。此外，肿瘤内施用MADD siRNA 显著降低治疗小鼠的原位ATC肿瘤生长和肺转移。因此，MADD是一个 单独或与Wnt/β-catenin抑制剂组合的ATC中的潜在治疗靶点。 基于一个非常强的科学前提，我们假设MADD下调制可以是有效的 在抑制生长和克服对靶向过度活跃的MAPK、PI 3 K/Akt和Wnt/β-catenin的药物的耐药性方面， 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MADD基因敲除在ATC细胞中对体外和离体Wnt信号传导的影响;在aim-2中，我们研究了MADD基因敲除对ATC细胞中Wnt信号传导的影响。 将确定下调MADD表达以及MAPK和PI 3 K/Akt信号通路的影响， 在aim-3中，我们将测试与MADD联合治疗的体内治疗功效。 在ATC的原位和自发小鼠模型中的敲低。 ATC不成比例地占甲状腺癌相关死亡的大多数。癌细胞特异 MADD的表达，它在激活几个关键信号通路中的作用，以及它作为促生存因子的能力， 因子和促进ATC的转移使其成为治疗发展的理想靶点。重要的是，MADD 缺失对正常细胞没有明显影响。因此，拟议的新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.