Jasmijn Miltenburg1, Marnix Gorissen2, Niek Strijkers2, Femke ten Seldam2, Henri Timmers3, Margo Dona4
(1) Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands. (2) Department of Plant and Animal Biology, Radboud Institute for Biological and Ecological Sciences, Radboud University, Nijmegen, The Netherlands. (3) Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands. (4) Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.
Background:
Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours arising from chromaffin cells. Germline mutations in succinate dehydrogenase subunit B (SDHB) are associated with malignancy and poor prognosis when acquiring a genetic second hit, but the pathophysiology of tumorigenesis remains largely unknown. To gain knowledge of PPGL pathogenesis and improve treatment possibilities, a functional model is essential. However, current disease models lack natural tumour development following an SDHB mutation. Earlier, sdhb mutant zebrafish showed PPGL tumour characteristics in larval stages, while tumour development remained absent in adult zebrafish. Here, we aim to accelerate tumorigenesis in an adult zebrafish model for SDHB-associated PPGLs through vitamin C supplementation.
Methods:
Adult heterozygous sdhb mutant and wild-type zebrafish were fed a normal or vitamin C-supplemented diet for 6 months. Fish were monitored for abnormalities and analysed morphologically via immunohistochemistry of chromaffin cell marker tyrosine hydroxylase. Metabolically, we profiled the hypoxia-inducible factor (hif) pathway and downstream targets via qPCR, and Kreb’s cycle metabolites using LC-MS.
Results:
In heterozygous sdhb mutants, vitamin C supplementation increased levels of oncometabolite succinate. In addition, vitamin C induced prominent effects on the hifpathway, increasing expression of key genes hif1aa, hif1ab and hif2aa. Moreover, 10% of vitamin C supplemented mutant zebrafish showed an increase and disorganisation of chromaffin cells. Importantly, these chromaffin cells showed a tissue-specific sdhb protein deficiency, indicative for a second hit.
Conclusion:
In conclusion, we successfully employed vitamin C as trigger to accelerate tumorigenesis in adult heterozygous sdhb mutant zebrafish. By investigating triggers for natural tumour development following a germline sdhb mutation, we provide insight into PPGL disease pathogenesis and establish a unique platform to investigate tumour development, aiming to identify novel therapeutic targets.