Characterizing the Role of NOTCH2 in Neuroendocrine Tumors

项目摘要 胰腺神经内分泌肿瘤（pNET）是胰腺的第二大常见恶性肿瘤， 总生存期为3.6年，成功的手术是唯一有治愈潜力的治疗方法。然而，在这方面， 大约40-95%的pNET在初次诊断时是转移性的，在肿瘤的局部复发中， 切除床作为规范。此外，患有来自pNET的肝转移的患者通常具有使人衰弱的 症状如无法控制的腹泻、潮红、皮疹和心力衰竭。很少有系统的 已被证明在临床上有用的疗法，以及那些仍然具有广泛可变反应率的疗法 并且具有不良的副作用特征。该提案的目标是确定Notch 2在 增殖、转移和分泌Notch 2的表型，并评估Notch 2作为 患者预后的预测指标。我们实验室的初步数据表明，Notch 2的过度表达导致 NET细胞增殖率增加，激素分泌减少。我们的数据还 表明与原发性肿瘤相比，Notch 2在转移性pNET中上调。精液数据来自一个 迄今为止，对pNET的最大规模的基因组研究也将Notch 2确定为pNET的关键主调节因子 转移，代表了疾病进展所需的依赖性的关键收敛， 建立转移。因此，我的假设是Notch 2在肿瘤中起致癌作用。 NET并驱动肿瘤进展，导致更具侵袭性的表型，并预示着更糟 患者预后为了评估这一假设，我们将进行表型表征（增殖， 激素产生、迁移）对Notch 2过表达pNET细胞系的作用， 稳定转染。我们将类似地评估通过siRNA敲低Notch 2的pNET细胞系，以及 使用CRISPR-Cas9产生的稳定敲除pNET细胞系。然后我们将在体内进行类似的研究 使用肝转移小鼠模型，其中注射Notch 2过表达和Notch 2 pNET细胞系 植入无胸腺小鼠体内。使用该模型，我们将通过microCT评估肿瘤生长和转移，此外， 荷尔蒙分泌的变化然后，我们将对肿瘤和小鼠进行终点分析， 机关最后，我们将使用pNET组织化学分析Notch 2的表达， 来自亚拉巴马大学接受手术切除的人类患者的微阵列 并评估Notch 2表达与Notch 2通路之间的关系 组件和各种患者结果测量。这项研究将提供有关下列因素影响的有价值的信息： pNET中的Notch 2可以帮助指导未来的靶向治疗工作，并了解 pNET biology.

PROJECT SUMMARY Pancreatic neuroendocrine tumors (pNETs) are the second most common malignancy of the pancreas, with an overall survival of 3.6 years and successful surgery the only treatment offering potential for cure. However, around 40-95% of pNETs are metastatic at the time of initial diagnosis, with local recurrence within the resection bed as the norm. Furthermore, patients with liver metastases from pNETs often have debilitating symptoms such as uncontrollable diarrhea, flushing, skin rashes, and heart failure. There are few systemic therapies that have proven to be clinically useful, and those that have still bear widely variable response rates and have poor side effect profiles. The goal of this proposal is to determine the role of Notch2 in the proliferation, metastasis, and hormone-secreting phenotype of NETs, and evaluate Notch2 as a predictor of patient outcomes. Preliminary data from our lab suggests that overexpression of Notch2 results in an increase of the proliferative rate of NET cells, while decreasing their hormone secretion. Our data also suggest that Notch2 is upregulated in metastatic pNETs compared to primary tumors. Seminal data from one of the largest genomic studies to date on pNETs has also identified Notch2 as a key master-regulator of pNET metastasis, representing a key convergence of dependencies required for disease progression and the establishment of metastasis. Therefore, it is my hypothesis that Notch2 functions in an oncogenic role in NETs and drives tumor progression, resulting in a more aggressive phenotype and portending worse patient prognosis. To evaluate this hypothesis, we will conduct phenotypic characterization (proliferation, hormone production, migration) on Notch2-overexpressing pNET cell lines that have been transiently and stably transfected. We will similarly evaluate pNET cell lines with Notch2 knockdown via siRNA, as well as stable knockout pNET cell lines generated using CRISPR-Cas9. We will then conduct similar studies in vivo using a liver metastasis mouse model whereby Notch2-overexpressing and Notch2 pNET cell lines are injected into athymic mice. Using this model, we will evaluate tumor growth and metastasis via microCT, in addition to hormone secretion over a 16-week period. We will then conduct endpoint analysis of tumors and mouse organs. Lastly, we will immunohistochemically analyze the expression of Notch2 using pNET tissue microarrays derived from human patients that have undergone surgical resection at the University of Alabama at Birmingham and evaluate the relationships between the expression of Notch2, Notch2 pathway components, and various patient outcome measures. This study will yield valuable information on the effects of Notch2 in pNETs that can help to guide future targeted therapeutic efforts and inform an understanding of pNET biology.