Nicotinamide Nucleotide Transhydrogenase as a novel treatment target in adrenocortical carcinoma

Abstract

Adrenocortical Carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. Here we evaluated a potential new treatment target for ACC, focusing on the mitochondrial NADPH generator Nicotinamide Nucleotide Transhydrogenase (NNT). NNT has a central role within mitochondrial antioxidant pathways, protecting cells from oxidative stress. Inactivating human NNT mutations result in congenital adrenal insufficiency. We hypothesized NNT silencing in ACC cells will induce toxic levels of oxidative stress. To explore this, we transiently knocked down NNT in NCI-H295R ACC cells. As predicted, this manipulation increased intracellular levels of oxidative stress; this resulted in a pronounced suppression of cell proliferation and higher apoptotic rates, as well as sensitization of cells to chemically-induced oxidative stress. Steroidogenesis was paradoxically stimulated by NNT loss, as demonstrated by mass spectrometry-based steroid profiling. Next, we generated a stable NNT knockdown model in the same cell line to investigate the longer-lasting effects of NNT silencing. After long-term culture, cells adapted metabolically to chronic NNT knockdown, restoring their redox balance and resilience to oxidative stress, although their proliferation remained suppressed. This was associated with higher rates of oxygen consumption. The molecular pathways underpinning these responses were explored in detail by RNA sequencing and non-targeted metabolome analysis, revealing major alterations in nucleotide synthesis, protein folding and polyamine metabolism. Our study provides the first pre-clinical evidence of the therapeutic merit of antioxidant targeting in ACC as well as illuminating the long-term adaptive response of cells to oxidative stress.

Nicotinamide nucleotide transhydrogenase mutation analysis in Chinese patients with thyroid dysgenesis

“…In addition, knock down of NNT resulted in a pronounced suppression of cell proliferation and higher apoptotic rate. (Chortis et al, 2018). As both the thyroid gland and cancer cells are highly exposed to ROS (Poncin et al, 2008), the effect of NNT deficiency might be similar, NNT dysfunction might cause the growth suppression in thyroid progenitors.…”

“…NNT catalyzes the reaction: NADH + NADP + + H + (0ut) ↔ NAD + + NADPH + H + (In) , maintaining the balance between NADH and NADPH in mitochondria (Murphy, 2015). Based on the capacity to detoxify ROS, NNT is a key antioxidative enzyme, protecting cells from the deleterious effects of oxidative stress (Chortis et al, 2018; Nesci et al, 2020). In this study, the ability to generate NADPH of NNT A271S , NNT R693H , and NNT V861M was decreased when compared with NNT.…”

“…The results of the two‐way experiences demonstrated that all the three mutations have a negative effect on enzyme activity of NNT. Both in human and animal, NNT dysfunction usually leads to oxidative stress (Chortis et al, 2018; Meimaridou et al, 2018).…”

The C-terminal tail of polycystin-1 suppresses cystic disease in a mitochondrial enzyme-dependent fashion

“…NNT knockdown, which leads to decreased proliferation and increased apoptosis, is being explored as a therapeutic approach in models of various neoplasms, including gastric cancer (Li et al, 2018), adrenocortical carcinoma (Chortis et al, 2018) and hepatic adenocarcinoma (Ho et al, 2017).…”