XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas
Hamid Reza Rezvani, Arianna L. Kim, Rodrigue Rossignol, Nsrein Ali, Meaghan Daly, Walid Mahfouf, Nadège Bellance, Alain Taïeb, Hubert de Verneuil, Frédéric Mazurier, David R. Bickers
Hamid Reza Rezvani, Arianna L. Kim, Rodrigue Rossignol, Nsrein Ali, Meaghan Daly, Walid Mahfouf, Nadège Bellance, Alain Taïeb, Hubert de Verneuil, Frédéric Mazurier, David R. Bickers
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Research Article Oncology

XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas

Abstract

DNA damage is a well-known initiator of tumorigenesis. Studies have shown that most cancer cells rely on aerobic glycolysis for their bioenergetics. We sought to identify a molecular link between genomic mutations and metabolic alterations in neoplastic transformation. We took advantage of the intrinsic genomic instability arising in xeroderma pigmentosum C (XPC). The XPC protein plays a key role in recognizing DNA damage in nucleotide excision repair, and patients with XPC deficiency have increased incidence of skin cancer and other malignancies. In cultured human keratinocytes, we showed that lentivirus-mediated knockdown of XPC reduced mitochondrial oxidative phosphorylation and increased glycolysis, recapitulating cancer cell metabolism. Accumulation of unrepaired DNA following XPC silencing increased DNA-dependent protein kinase activity, which subsequently activated AKT1 and NADPH oxidase-1 (NOX1), resulting in ROS production and accumulation of specific deletions in mitochondrial DNA (mtDNA) over time. Subcutaneous injection of XPC-deficient keratinocytes into immunodeficient mice led to squamous cell carcinoma formation, demonstrating the tumorigenic potential of transduced cells. Conversely, simultaneous knockdown of either NOX1 or AKT1 blocked the neoplastic transformation induced by XPC silencing. Our results demonstrate that genomic instability resulting from XPC silencing results in activation of AKT1 and subsequently NOX1 to induce ROS generation, mtDNA deletions, and neoplastic transformation in human keratinocytes.

Authors

Hamid Reza Rezvani, Arianna L. Kim, Rodrigue Rossignol, Nsrein Ali, Meaghan Daly, Walid Mahfouf, Nadège Bellance, Alain Taïeb, Hubert de Verneuil, Frédéric Mazurier, David R. Bickers

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Figure 2

XPC silencing–induced ROS production leads to mtDNA deletions.

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XPC silencing–induced ROS production leads to mtDNA deletions. (A) ROS l...
(A) ROS levels in different cells were measured by flow cytometry using cytoplasmic- and mitochondrial-specific probes at the indicated days after transduction. The ROS level in the shCtrl-transduced cells was arbitrarily set to 1. Results are then assessed as shown at the top of the panels and expressed as the mean ± SD of 3 independent experiments. (B) Genomic and mtDNA oxidation were assessed by quantification of 8-oxodG levels in nuclear genome and mtDNA of different cells. Results are expressed as ng of 8-oxodG per μg DNA. (C) mtDNA was extracted and subjected to PCR, amplifying either reference fragments (Ref) representing total mitochondrial genome or 2 known deletions (3895 del and 4977 del). (D) NADPH oxidase activity was assessed as shown at the top of the panels and expressed as the mean ± SD of 3 independent experiments. (E) Glucose consumption and lactate production as well as the total endogenous ATP levels and ATP levels produced by mitochondria were measured in the different transduced cells. *P < 0.05 for different cells versus shCtrl-transduced cells at the indicated time points.