Mutation of the Cell Cycle Regulator p27kip1 Drives Pseudohypoxic Pheochromocytoma Development

SIMPLE SUMMARY: Pheochromocytomas and paragangliomas (PPGLs) can be subdivided into at least three different subgroups associated with different clinical manifestations and depending on the risk to metastasize. A shortage in human tumor material, the lack of a functional human cell line and very limited animal models were major drawbacks for PPGL research and consequently for the development of patient-tailored targeted therapies. We have previously reported that the MENX rat model develops pheochromocytoma with a full penetrance at the age of 8–10 months, however, it was unclear which human group the rat tumors modeled best. In order to characterize the rat pheochromocytomas, we analyzed gene expression, the catecholamine profile, TCA-cycle metabolism, methylation, angiogenesis, histology and mitochondrial ultrastructure. In all aspects, rat MENX pheochromocytomas resemble the features of the human pseudohypoxia group, the most aggressive one and in need of effective therapeutic approaches. ABSTRACT: Background: Pseudohypoxic tumors activate pro-oncogenic pathways typically associated with severe hypoxia even when sufficient oxygen is present, leading to highly aggressive tumors. Prime examples are pseudohypoxic pheochromocytomas and paragangliomas (p-PPGLs), neuroendendocrine tumors currently lacking effective therapy. Previous attempts to generate mouse models for p-PPGLs all failed. Here, we describe that the rat MENX line, carrying a Cdkn1b (p27) frameshift-mutation, spontaneously develops pseudohypoxic pheochromocytoma (p-PCC). Methods: We compared rat p-PCCs with their cognate human tumors at different levels: histology, immunohistochemistry, catecholamine profiling, electron microscopy, transcriptome and metabolome. The vessel architecture and angiogenic potential of pheochromocytomas (PCCs) was analyzed by light-sheet fluorescence microscopy ex vivo and multi-spectral optoacoustic tomography (MSOT) in vivo. Results: The analysis of tissues at various stages, from hyperplasia to advanced grades, allowed us to correlate tumor characteristics with progression. Pathological changes affecting the mitochrondrial ultrastructure where present already in hyperplasias. Rat PCCs secreted high levels of norepinephrine and dopamine. Transcriptomic and metabolomic analysis revealed changes in oxidative phosphorylation that aggravated over time, leading to an accumulation of the oncometabolite 2-hydroxyglutarate, and to hypermethylation, evident by the loss of the epigenetic mark 5-hmC. While rat PCC xenografts showed high oxygenation, induced by massive neoangiogenesis, rat primary PCC transcriptomes possessed a pseudohypoxic signature of high Hif2a, Vegfa, and low Pnmt expression, thereby clustering with human p-PPGL. Conclusion: Endogenous rat PCCs recapitulate key phenotypic features of human p-PPGLs. Thus, MENX rats emerge as the best available animal model of these aggressive tumors. Our study provides evidence of a link between cell cycle dysregulation and pseudohypoxia.