Pro-tumoural effects of vitamin C supplementation in a zebrafish model for SDHB-associated PPGL

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine, chromaffin cell-derived tumours of the adrenal medulla or paraganglia. Germline pathogenic variants in succinate dehydrogenase subunit B (SDHB) are most prevalent and associated with malignancy and poor prognosis. Treatment options are limited, and therapy development is hindered by knowledge gaps concerning the pathomechanism and lack of suitable models. Previously, homozygous sdhb mutant zebrafish larvae showed disease characteristics, and adult heterozygous mutants represented human heterozygous SDHB carriers, portraying low-grade systemic succinate accumulation. Since spontaneous PPGL formation remains absent, we applied vitamin C supplementation as potential trigger to induce tumourigenesis in adult heterozygous sdhb mutant zebrafish. In addition, sdhb mutant larvae were exposed to vitamin C to investigate dose-dependent effects in an sdhb-deficient context. Twelve percent of vitamin C-supplemented adult sdhb mutants showed local proliferation of chromaffin cells with tissue-specific sdhb deficiency. Furthermore, metabolite profiling showed an increase in succinate/fumarate ratios upon vitamin C in heterozygous sdhb mutants. Gene expression analysis showed activation of the hypoxia-inducible factor pathway in larval and adult zebrafish. In addition, high dose vitamin C increased nrf2a expression, representing oxidative stress, in homozygous and heterozygous sdhb mutants. Ultimately, we have successfully employed vitamin C supplementation to initiate chromaffin cell proliferation in adult heterozygous sdhb mutant zebrafish, resembling early-stage PPGL tumourigenesis. Furthermore, we gained insights into underlying mechanisms, including HIF stabilisation, demethylation, oxidative stress, iron metabolism and glucose transport. Our model provides a unique platform to investigate possible triggers for PPGL development in the human mutation carrier state and investigate early stages of tumourigenesis.