Hsa-miR-200 and hsa-miR34 miRNA family members target cellular and viral genes regulating HCMV infection in head and neck paraganglioma

Paragangliomas (PGLs) are rare hypervascular tumours of the paraganglia, organelles connected with the autonomic branches of the cranial (head and neck) and thoraco-lumbar nerves (1). PGLs have a strong genetic basis, being frequently (~ 40%) associated with predisposing mutations in one of at least 15 nuclear genes (1), most relevantly SDHA/B/C/D, that encode the four subunits of succinate dehydrogenase (SDH), a mitochondrial enzyme participating in both oxidative phosphorylation and the Krebs cycle, and SDHAF2, required for SDHA flavination. However, the penetrance of these mutations is incomplete and genetically-modeled mice mutated in Sdhb, homolog of the human SDHB gene, do not develop any cancer. In 1971 an isolated report described the retrieval of an infectious virus, then identified as human cytomegalovirus (CMV) in a retroperitoneal PGL (2). This report was not followed-up, and only in the last decade HCMV has emerged as an oncomodulatory agent in neural-derived cancers and as a target of therapy in gliomas (3). We show here evidence of a CMV-like herpesvirus, in high abundance, in head and neck PGLs. We collected over 100 HN-PGLs and examined them by electron microscopy, rapid in situ hybridation, IHC, IF and WB. All had mitochondrial changes, and all resulted positive for CMV-like viruses, regardless of SDH mutation status. We show here that the PGL-associated CMV-like viruses expresses viral proteins integrated in oncomodulatory pathways controlled by microRNAs of the miR200 and miR34 families. We previously reported that the miR-200a,b,c and the miR34b,c are constitutively downregulated in PGLs (4), and that this results in upregulated expression of NOTCH1 (4), PDGFRA and ZEB1 (5) in PGL cells. We enforced miR34s and miR200s overexpression by lentiviral reintroduction in PGL cells (PTJ64i) endogenously infected by the CMV-like PGL virus. This resulted in downregulation of IE2 (UL122), pp65 (UL83) and vMIA viral proteins, therefore affecting viral load. In the same cell system the IFN-inducible antiviral host protein viperin, encoded by the RSAD2 gene and highjacked by the CMV protein vMIA to deregulate mitochondrial metabolism by inhibition of fatty acids beta oxidation (6), was downregulated by reintroduction of miR34s, but not of miR200s. We found that viperin, the most upregulated host protein induced by HCMV infection, is highly expressed in PGLs and derived cells together with its viral partner protein vMIA. This was consistent with evidence of lipogenesis and glycolysis in PGLs. The 3’UTR of RSAD2 is predicted to contain binding seed regions for the miR34s and the miR200s. Li et al. recently showed that the 3’UTR of RSAD2 is a direct target of miR200a and miR200b (7). We are verifying whether the 3’UTR of RSAD2 gene is direct target of the miR34s. We also show that in PGL cells treatment with the antiviral drug ganciclovir reduces viral load and significantly upregulates the miR200s and the miR34s levels. These results suggest that epigenetic deregulation of the miR-200s and of the miR-34s supports HCMV infection in PGLs and that infection by the CMV-like PGL viruses contributes to the metabolic phenotype of PGLs. 1) Pacak K, Wimalawansa SJ. Pheochromocytoma and paraganglioma. Endocr Pract 2015 2) Heine U, Ultrastructural and biological properties of a cytomegalovirus rescued from a human paraganglioma. Cancer Res. 1971. 3) Söderberg-Nauclér C et al., Cytomegalovirus in human brain tumors: Role in pathogenesis and potential treatment options. World J Exp Med. 2015. 4) Cama et al., Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling. Acta Neuropatol. 2013 5) Verginelli F, et al. Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib. Acta Neuropathol. 2018 6) Seo JY and Cresswell P. Viperin regulates cellular lipid metabolism during human cytomegalovirus infection. PLoS Pathog. 2013. 7) Li Z, et al. miR-200 family promotes podocyte differentiation through repression of RSAD2. Sci Rep. 2016