Characterizing the Role of NOTCH2 in Neuroendocrine Tumors

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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.