Endocrine Abstracts

Context: Nicotinamide nucleotide transhydrogenase (NNT) is a NADPH-generating mitochondrial proton pump with a central role in mitochondrial antioxidant pathways. Recent studies revealed inactivating NNT mutations in patients with familial glucocorticoid deficiency, indicating a selective susceptibility of the adrenal cortex to NNT deficiency and oxidative stress. Here we explored the potential value of NNT as a therapeutic target in adrenocortical cancer.

Methods: We delineated the distinct effects of NNT loss on cellular proliferation and steroidogenesis employing two in vitro knockdown models: transient, siRNA-mediated and stable, shRNA-mediated NNT silencing, both utilising the adrenocortical carcinoma cell line NCI-H295R.

Results: Transient NNT knockdown impaired cellular redox balance, as indicated by a decline in the ratio of reduced to oxidised glutathione, and impeded cellular proliferation (30±8% decrease in proliferation compared to scrambled siRNA-transfected cells, P<0.01). Importantly, NNT-deficient cells also became exceedingly sensitive to chemically induced oxidative stress (55±10% loss of cellular viability when co-treated with 20 μM Paraquat, P<0.01). After long-term culture, H295R cells with stable NNT knockdown appeared to develop compensatory mechanisms, with evidence of improved redox balance and proliferative potential. This adaptation was associated with alterations in glycolytic and oxygen consumption rates, as demonstrated by extracellular flux analysis using Seahorse XF technology. Steroid profiling by liquid chromatography-tandem mass spectrometry revealed a distinct profile induced by transient NNT knockdown, surprisingly characterised by enhanced cortisol and androstenedione synthesis. This was underpinned by an increase in the activity of several key steroidogenic enzymes including CYP11B1, CYP17A1 and CYP21A2.

Conclusion: Our study suggests a potential role of NNT inhibition as a novel therapeutic approach in advanced adrenocortical carcinoma. Steroid profiling reveals a surprising increase in glucocorticoid and androgen synthesis in response to NNT loss, challenging our current understanding of the complex association between redox balance and adrenal function.