Generation and characterization of CRISPR/Cas9-mediated MEN1 knockout BON1 cells: a human pancreatic neuroendocrine cell line

• Published in: Scientific reports Vol. 10; no. 1; p. 14572
• Main Authors: Monazzam, Azita, Li, Su-Chen, Wargelius, Hanna, Razmara, Masoud, Bajic, Duska, Mi, Jia, Bergquist, Jonas, Crona, Joakim, Skogseid, Britt
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 03.09.2020; Nature Publishing Group
• Subjects: 631/67; 631/80; Apoptosis; Biomarkers, Tumor - metabolism; CRISPR-Cas Systems; CYP1A2 protein; Cytochrome; Cytochrome P450; DNA adducts; DNA microarrays; DNA repair; Gene Editing; Gene expression; Gene Expression Regulation, Neoplastic; Humanities and Social Sciences; Humans; Hyperoxia; Inactivation; Lungs; multidisciplinary; Mutation; Neuroendocrine tumors; Neuroendocrine Tumors - metabolism; Neuroendocrine Tumors - pathology; Pancreatic cancer; Pancreatic Neoplasms - metabolism; Pancreatic Neoplasms - pathology; Proteome - analysis; Proto-Oncogene Proteins - antagonists & inhibitors; Proto-Oncogene Proteins - genetics; Respiratory distress syndrome; Respiratory therapy; Science; Science (multidisciplinary); Tumor Cells, Cultured
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-71516-7

Among patients with the rare diagnosis of pancreatic neuroendocrine tumor (P-NET), a substantial proportion suffer from the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1), which is caused by germline mutations of the MEN1 suppressor gene. Somatic mutations and loss of the MEN1 protein (menin) are frequently also found in sporadic P-NETs. Thus, a human neuroendocrine pancreatic cell line with biallelic inactivation of MEN1 might be of value for studying tumorigenesis. We used the polyclonal human P-NET cell line BON1, which expresses menin, serotonin, chromogranin A and neurotensin, to generate a monoclonal stable MEN1 knockout BON1 cell line (MEN1-KO-BON1) by CRISPR/Cas9 editing. Changes in morphology, hormone secretion, and proliferation were analyzed, and proteomics were assessed using nanoLC-MS/MS and Ingenuity Pathway Analysis (IPA). The menin-lacking MEN1-KO-BON1 cells had increased chromogranin A production and were smaller, more homogenous, rounder and grew faster than their control counterparts. Proteomic analysis revealed 457 significantly altered proteins, and IPA identified biological functions related to cancer, e.g. , posttranslational modification and cell death/survival. Among 39 proteins with at least a two-fold difference in expression, twelve are relevant in glucose homeostasis and insulin resistance. The stable monoclonal MEN1-KO-BON1 cell line was found to have preserved neuroendocrine differentiation, increased proliferation, and an altered protein profile.