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Nicotinamide Nucleotide Transhydrogenase as a Novel Treatment Target in Adrenocortical Carcinoma.

ABSTRACT: Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. In this study, we evaluated a potential new treatment target for ACC, focusing on the mitochondrial reduced form of NAD phosphate (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 that 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 nontargeted metabolome analysis, revealing major alterations in nucleotide synthesis, protein folding, and polyamine metabolism. This study provides preclinical evidence of the therapeutic merit of antioxidant targeting in ACC as well as illuminating the long-term adaptive response of cells to oxidative stress.

SUBMITTER: Chortis V

PROVIDER: S-EPMC6093335 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Nicotinamide Nucleotide Transhydrogenase as a Novel Treatment Target in Adrenocortical Carcinoma.

Chortis Vasileios V Taylor Angela E AE Doig Craig L CL Walsh Mark D MD Meimaridou Eirini E Jenkinson Carl C Rodriguez-Blanco Giovanny G Ronchi Cristina L CL Jafri Alisha A Metherell Louise A LA Hebenstreit Daniel D Dunn Warwick B WB Arlt Wiebke W Foster Paul A PA

Endocrinology 20180801 8

Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. In this study, we evaluated a potential new treatment target for ACC, focusing on the mitochondrial reduced form of NAD phosphate (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 that NNT s ...[more]

PMID: 29850793

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Project description:Endothelial dysfunction is a critical, initiating step in the development of hypertension (HTN) and mitochondrial reactive oxygen species (ROS) are important contributors to endothelial dysfunction. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in the nicotinamide nucleotide transhydrogenase (Nnt) gene that are associated with endothelial dysfunction and increased risk for HTN. NNT is emerging as an important enzyme that regulates mitochondrial NADPH levels and mitochondrial redox balance by supporting the thiol dependent peroxidase systems in the mitochondria. We have previously shown that the absence of NNT in C57Bl/6J animals promotes a more severe hypertensive phenotype through reductions in •NO and endothelial dependent vessel dilation. However, the impact of NNT on human endothelial cell function remains unclear. We utilized NNT directed shRNA in human aortic endothelial cells to test the hypothesis that NNT critically regulates mitochondrial redox balance and endothelial function in response to angiotensin II (Ang II). We demonstrate that NNT expression and activity are elevated in response to the mitochondrial dysfunction and oxidative stress associated with Ang II treatment. Knockdown of NNT led to a significant elevation of mitochondrial ROS production and impaired glutathione peroxidase and glutathione reductase activities associated with a reduction in the NADPH/NADP+ ratio. Loss of NNT also promoted mitochondrial dysfunction, disruption of the mitochondrial membrane potential, and impaired ATP production in response to Ang II. Finally, we observed that, while the loss of NNT augmented eNOS phosphorylation at Ser1177, neither eNOS activity nor nitric oxide production were similarly increased. The results from these studies clearly demonstrate that NNT is critical for the maintenance of mitochondrial redox balance and mitochondrial function. Loss of NNT and disruption of redox balance leads to oxidative stress that compromises eNOS activity that could have a profound effect on the endothelium dependent regulation of vascular tone.

Dataset Information

Nicotinamide Nucleotide Transhydrogenase as a Novel Treatment Target in Adrenocortical Carcinoma.

Publications

Nicotinamide Nucleotide Transhydrogenase as a Novel Treatment Target in Adrenocortical Carcinoma.

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