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. IntroductionEarly studies of the metabolic changes that accompany the development of cancer led Otto Warburg to propose that a respiratory deficiency might drive neoplastic transformation (1), prompting many investigators to analyze the metabolism of tumor cells. These analyses revealed that a large number of cancer cell lines have a higher rate of glycolysis, an increased rate of glucose transport, increased pentose phosphate pathway (PPP) activity, decreased numbers of mitochondria, and a reduction in mitochondrial oxidative phosphorylation (OXPHOS) proteins and activities (2-4). These alterations in cancer cell energy metabolism could be related to somatic mutations in mitochondrial DNA (mtDNA); oxidative stress as a result of increased ROS level; adaptation to tissue hypoxia (4-7); the activation of oncogenes and/or inactivation of tumor suppressors (TP53,; as well as deregulation of PI3K/AKT, which influences the glycolytic flux through regulation of different factors (8).However, the etiologic relationship among genomic mutations, the Warburg effect, and increased ROS levels in tumor induction remains unclear (3,5,9). Furthermore, there is no clear mechanism(s) linking genomic mutations and modified cellular bioenergetics. To understand the relationships between these factors, we speculated that cells with heightened predisposition to malignant transformation, or cells with the capacity to accumu-

Cancer Cell Survival Following DNA Damage-mediated Premature Senescence Is Regulated by Mammalian Target of Rapamycin (mTOR)-dependent Inhibition of Sirtuin 1

“…of three independent experiments. Intracellular Reactive Oxygen Species (ROS)-Intracellular ROS were measured as described previously using a cell-permeable fluorogenic dye, 2Ј,7Ј-dichlorofluorescein diacetate, which detects hydrogen peroxide (15). The results were obtained as arbitrary absorbance units/mg of protein.…”

Loss of epidermal hypoxia-inducible factor-1α accelerates epidermal aging and affects re-epithelialization in human and mouse

“…Cell cycle analysis was performed using APC-linked anti-BrdU and 7-AAD according to the manufacturer's protocols (BD Biosciences), as already described (Rezvani et al, 2011b). For apoptosis analysis, cells were incubated with 2.5 mg/ml propidium iodide (PI; Sigma) and immediately analyzed by flow cytometry.…”

Lexeroderma pigmentosum

“…Comme l'activation de NOX a été constatée dans plusieurs tumeurs [9,13], l'inhibition de cette activation pourrait être une piste originale pour la prévention et la thérapie des cancers liés à une augmentation des ERO. ‡ Xeroderma pigmentosum: a useful model to study the relation between genomic mutations and cell transformation ERO, les délétions de l'ADN mitochondrial, les modifications métaboliques ainsi que la transformation tumorale des kératino-cytes XPC KD (Figure 2) [9,10]. Compte tenu de l'importance de l'activation de NOX1 dans la transformation tumorale des kératinocytes XPC KD , nous nous sommes intéressés au mécanisme de cette activation.…”

“…Notre approche [9,10] a été d'analyser le comportement métabolique et la transformation tumorale des kératino-cytes normaux lorsqu'on invalide l'expression de XPC en utilisant des vecteurs lentiviraux qui expriment des petits ARN en « épingle à cheveux » (shRNA) (Figure 2). …”