Characterizing the Role of NOTCH2 in Neuroendocrine Tumors

项目总结 胰腺神经内分泌肿瘤(PNETs)是胰腺第二常见的恶性肿瘤，具有 总体生存时间为3.6年，手术成功是唯一有治愈潜力的治疗方法。然而， 大约40%-95%的PNETs在最初诊断时是转移的，局部复发在 以切除床为标准。此外，PNETs肝转移的患者通常有虚弱的 症状如无法控制的腹泻、潮红、皮疹和心力衰竭。很少有系统性的 那些已被证明在临床上有用的疗法，以及那些仍然具有很大差异的应答率的疗法 副作用也很差。这项提案的目标是确定Notch2在 Net的增殖、转移和激素分泌表型，并评估Notch2作为一种 患者预后的预测指标。我们实验室的初步数据表明，Notch2结果的过度表达 增加网状细胞的增殖率，同时减少其激素分泌。我们的数据也 提示与原发肿瘤相比，Notch2在转移性PNETs中表达上调。开创性数据来自一个 迄今为止关于PNETs的最大基因组研究也发现Notch2是pNETs的关键主调节因子 转移，代表了疾病进展所需的依赖关系和 转移性的确立。因此，我的假设是Notch2在肿瘤发生中起作用 Net并推动肿瘤进展，导致更具侵袭性的表型并预示着更糟糕的情况 患者预后。为了评估这一假设，我们将进行表型特征(增殖， 激素产生，迁移)在Notch2过表达的pNET细胞系上，这些细胞株已经短暂和 稳定的转染体。我们将通过siRNA类似地评估具有Notch2基因敲除的pNET细胞系，以及 用CRISPR-Cas9获得稳定的基因敲除pNET细胞系。然后我们将在体内进行类似的研究 通过注射Notch2过表达和Notch2 pNET细胞系建立小鼠肝转移模型 变成无性系小鼠。使用这个模型，我们将通过MicroCT来评估肿瘤的生长和转移，除了 16周的荷尔蒙分泌。然后我们将对肿瘤和小鼠进行终点分析 器官。最后，我们将使用pNET组织用免疫组织化学方法分析Notch2的表达 来自阿拉巴马大学接受手术切除的人类患者的微阵列 并评价Notch2、Notch2通路的表达之间的关系 组件，以及各种患者结果衡量标准。这项研究将提供有价值的信息，说明 Notch2在PNETs中的作用，有助于指导未来的靶向治疗努力，并有助于了解 PNET生物学。

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.