Nitrosative stress due to GSNOR deficiency induces agingrelated mitochondrial impairment and enhances vulnerability to mitochondrial complex II-targeting drugs: a potential therapeutic approach for HCC

The denitrosylating enzyme GSNOR profoundly impact s on cellular signal transduction by controlling cellular concentrations of protein-SNOs. Excessive S-nitrosylation is a well-documented phenomenon known as nitrosative stress, which increases during age. In this phD thesis we provide evidence that GSNOR physiologically may undergo epigenetic silencing during aging probably due to DNA methylation of its promoter, carried out by TET proteins. It is plausible to speculate, therefore, that nitrosative stress occurring upon GSNOR deficiency, could compromise mitochondrial function, resembling, in such a way, aging-like conditions. By promoting mitochondrial defects, indeed, nitrosative stress has been associated with several features related to aging and to pathological states typical of advanced age, such as cancer. Coherently, GSNOR depletion has been recently demonstrated to contribute to development of hepatocellular carcinoma (HCC) and , being downregulated in almost the half of HCC cases, GSNOR deficiency characterizes a large subclass of this tumor. In this work we demostrate that hepatocarcinoma HepG2 cells stably downregulating GSNOR (shGSNOR HepG2) exhibit defective and fragmented mitochondria and that this condition is associated with the upregulation of succinate dehydrogenase (SDH) in order to sustain ATP production. Therefore, GSNOR deficiency, compromising mitochondrial function, prompts mitochondria to a higher vulnerability and this molecular adaptation enhances cell sensitivity of shGSNOR HepG2 cells to even low doses of different SDH-directed drugs (mitocans). In particular, we demonstrate that shGSNOR HepG2 cells undergo necroptosis via PARP1/RIP1 pathway. As a matter of fact, pharmacological inhibition of both proteins, as well as reverse genetics experiments completely rescue cell viability. We show that non-apoptotic cell death depends on caspase 3 Snitrosylation, which leads to its inactivation, and on the capability of SDHtargeting mitocans to produce ROS as side-effect of their mechanism of action. Indeed, pre-treatment with antioxidants fully abolishes cell demise. In addition, we revealed that SDH upregulation is associated with the cytosolic degradation of the mitochondrial chaperone TNF-associate protein 1 (TRAP1) via the ubiquitin-proteasome pathway. In particular, we provide the first evidence that TRAP1 is S-nitrosylated and that this modification is mandatory for its degradation. TRAP1 silencing by siRNA, indeed, recapitulates the molecular and cellular phenotype of shGSNOR HepG2 cells, and increases cell sensitivity to SDH-targeting mitocans. Overall, our results indicate that GSNOR is implicated in aging process, being epigenetically regulated during age and impacting on mitochondrial homeostasis, the first determinant of cell senescence/aging. Furthermore, excessive S-nitrosylation due to GSNOR downregulation elicits TRAP1 Snitrosylation and degradation, which in turn leads to increased SDH levels and enhanced sensitivity to SDH -targeting mitocans. Being GSNOR depletion distinctive of the 50% of HCC cases, our results argue for this class of molecules as new promising drugs to selectively eradicate HCC.