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.

Figure 1. Effects of XPC downregulation on mitochondrial metabolism and glycolysis.

Glucose consumption and lactate production (A) as well as the total endogenous ATP levels and ATP levels produced by mitochondria (B) were measured at the indicated time points after transduction. The glucose uptake, lactate production, and the total endogenous and mitochondrial ATP levels by shCtrl-transduced cells were set to 100% at each time point. The results were then compared with the shCtrl and are expressed as the average percentage of shCtrl ± SD of 3 independent experiments. *P < 0.05 (black) for shXPC-transduced cells versus shCtrl-transduced cells; *P < 0.05 (purple) for XPC-KC versus control keratinocytes at the indicated time points. (C) The protein expression levels of XPC, ND1, COX3, GLUT1, HK-2, PFKPB-3, and G6PD were determined by Western blot at the indicated days after transduction. β-actin was used as a loading control. (D) The relative activity of complex IV of the mitochondrial respiratory chain was assessed. The mRNA levels of COX1 and COX3, ND1 and ND5 (E), HK-2 and PFKFB3, and GLUT1 and G6PD (F) were quantified by qRT-PCR. The results are shown as the average percentage of control ± SD of 3 independent experiments. (G) The mitochondrial network morphology in XPC-KC, shCtrl-, and shXPC-transduced keratinocytes was determined by microscopy using MitoTracker. Scale bars: 10 μM. (H) Length of mitochondrial tubules was measured in 50 cells (25 mitochondrial tubules per cell) per condition. Results are expressed as average percentage of mitochondrial tubule size distribution ± SD of 3 independent experiments. *P < 0.05 (black) for shXPC cells versus shCtrl-transduced cells. shCtrl, keratinocytes transduced with control shRNA; shXPC, keratinocytes transduced with shXPC; XPC-KC, keratinocytes isolated from XPC patients.

Figure 2. XPC silencing–induced ROS production leads to mtDNA deletions.

(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.

Figure 3. XPC^KD cells display increased proliferative capacity associated with an increased fraction of S phase cells and decreased length of S phase following an initial stalled phase.

(A) The proliferation capacities of shCtrl- or shXPC-transduced keratinocytes were measured by serial cell counts on different days after transduction. (B) The distribution of cells in the G₁, S, and G₂ phases was measured at the indicated time intervals after transduction using 7-AAD and BrdU staining. (C) Graphic representation of the distribution of cells in the G₁, S, and G₂ phases. The percentage of cells in each phase for shCtl-transduced keratinocytes was considered to be 100%. The results were then compared with the shCtrl and are expressed as the mean ± SD of 3 independent experiments. (D) DNA synthesis time was measured by a BrdU pulse assay. Results are shown as the average percentages of shCtrl ± SD of 3 independent experiments. *P < 0.05 for shXPC-transduced cells versus shCtrl-transduced cells at the indicated time points. (E) Total protein extracts were assessed for cell-cycle regulators by Western blot analysis. β-actin was used as a loading control.

Figure 4. Knockdown of XPC results in epithelial hyperplasia.

Epidermis reconstructed with XPC-KC, shCtrl-, or shXPC-transduced keratinocytes on days 5 and 15 after transduction. (A) Architectures of epidermis were evaluated with H&E staining. Proliferation and differentiation status of epidermis were assessed using immunofluorescence staining of K10, K14, integrin α6, integrin β1, and Ki67. The nuclei were marked in blue with DAPI. (B) High proliferation detected with Ki67 staining in a large extension of rete pegs in epidermis reconstructed with XPC^KD cells and XPC-KC. Scale bars: 200 μm.

Figure 5. XPC silencing drives neoplastic transformation of human keratinocytes via NOX1.

Keratinocytes were transduced with indicated lentivirus. On the 15th day after transduction, cells were injected subcutaneously into NOD/SCID mice. (A) The in vivo tumor growth rate of XPC^KD cells injected in mice is enhanced by overexpression of NOX1. Results are presented as mean ± SD. (B) H&E staining (upper panels) shows that tumors are well-differentiated SCCs with keratin pearl formation. Expression of GFP in tumors was confirmed using immunostaining with an anti-GFP antibody (lower panels). Scale bar: 100 μm.

Figure 6. AKT activation in XPC^KD cells triggers NADPH oxidase activation and metabolic alteration.

(A) To assess activation of different cancer pathways, luciferase-reporter lentivirus was used to transduce the keratinocytes in the indicated conditions at days 5 and 15. For each reporter, the mean ± SD luciferase activity is presented as the relative value to the activity in shCtrl-transduced cells (n = 3). (B and C) Glucose consumption and lactate production (B) as well as the total ATP levels and ATP levels produced by mitochondria (C) were measured in the different cells. The results were then compared with the shCtrl and are expressed as the average percentage of shCtrl ± SD of 3 independent experiments. (D) NADPH oxidase activity and cytoplasmic and mitochondrial ROS levels were measured in different transduced cells. (E) The effect of AKT on XPC silencing–induced genomic and mtDNA oxidation were assessed by quantification of 8-oxodG levels in nuclear and mtDNA. (F) To assess the effect of NOX1 on activation of AKT, WNT, and P53 pathways following XPC downregulation, luciferase reporter lentivirus was used as described in A. *P < 0.05 for indicated cells versus shCtrl; ^†P < 0.05 for indicated cells versus shXPC-transduced cells. Results are presented as mean ± SD.

Figure 7. NHEJ activation in XPC^KD cells results in overcoming stalled cell-cycle progression and AKT activation.

(A) The percentage of apoptotic keratinocytes was evaluated by flow cytometry. Results are expressed as the average percentage of apoptotic and necrotic cells ± SD of 3 independent experiments. *P < 0.05 for different cells versus shCtrl-transduced cells at the indicated time points. (B) The effects of XPC and/or AKT downregulation on the protein expression level of pro- and antiapoptotic proteins and the major components of NHEJ repair were determined by Western blot. (C) The mRNA levels of DNA-PKcs and KU70 were quantified by qRT-PCR. (D) DNA-PK kinase activity was measured in nuclear extracts of various cells. The results were then compared with the shCtrl and are expressed as the mean ± SD of 3 independent experiments. (E) Nuclear extract of cells transduced with shCtrl or shXPC were used in in vitro end-joining assays. The pUC19 plasmid digested with EcoRI was used as monomer DNA substrate. The amount of ligated pUC19 following incubation with various nuclear extracts shows repair efficiency (left side of photomicrograph). Fidelity of repair was assessed by redigestion of end-jointed products with the EcoRI (right side of photomicrograph). (F) Graphic representation of the end-joining experiment in E. Data are expressed as mean ± SD of the 3 independent experiments.

Figure 8. A model outlining cellular responses to XPC downregulation.

Lentivirus-mediated XPC silencing in normal human keratinocytes impairs DNA repair efficiency. To evade damaged base–induced cell-death apoptosis, some cells use bypass repair systems (such as NHEJ repair). Activation of the DNA-PK pathway results in upregulation of the AKT pathway, which in turn activates NADPH oxidase. Cytoplasmic ROS generation will consequently increase, leading to enhanced oxidation of nuclear and mitochondrial DNA, followed by the induction of mtDNA deletions, mitochondrial ROS, and alterations in mitochondrial bioenergetics. Meanwhile, it is likely that increased cytoplasmic ROS enhances the mutation of oncogenes and/or tumor suppressor genes, leading to aberrant cell-cycle progression, enhanced cell proliferation, and tumorigenesis.