HPV16 E6 and E7 proteins induce a chronic oxidative stress response via NOX2 that causes genomic instability and increased susceptibility to DNA damage in head and neck cancer cells

Abstract

Human papillomavirus (HPV) is the causative agent of a subgroup of head and neck cancer characterized by an intrinsic radiosensitivity. HPV initiates cellular transformation through the activity of E6 and E7 proteins. E6 and E7 expression is necessary but not sufficient to transform the host cell, as genomic instability is required to acquire the malignant phenotype in HPV-initiated cells. This study reveals a key role played by oxidative stress in promoting genomic instability and radiosensitivity in HPV-positive head and neck cancer. By employing an isogenic human cell model, we observed that expression of E6 and E7 is sufficient to induce reactive oxygen species (ROS) generation in head and neck cancer cells. E6/E7-induced oxidative stress is mediated by nicotinamide adenine dinucleotide phosphate oxidases (NOXs) and causes DNA damage and chromosomal aberrations. This mechanism for genomic instability distinguishes HPV-positive from HPV-negative tumors, as we observed NOX-induced oxidative stress in HPV-positive but not HPV-negative head and neck cancer cells. We identified NOX2 as the source of HPV-induced oxidative stress as NOX2 silencing significantly reduced ROS generation, DNA damage and chromosomal aberrations in HPV-positive cells. Due to their state of chronic oxidative stress, HPV-positive cells are more susceptible to DNA damage induced by ROS and ionizing radiation (IR). Furthermore, exposure to IR results in the formation of complex lesions in HPV-positive cells as indicated by the higher amount of chromosomal breakage observed in this group of cells. These results reveal a novel mechanism for sustaining genomic instability in HPV-positive head and neck tumors and elucidate its contribution to their intrinsic radiosensitivity.

Figure 1.

HPV-positive head and neck cancer cells are characterized by a status of chronic oxidative stress. (A) Intracellular ROS levels and (B) superoxide level in a panel of head and neck cancer cells divided in groups based on HPV status. The line in the middle of each box is plotted at the median, and whiskers represent minimum and maximum value for each group. A two-tailed Mann–Whitney test was used to compare medians between the two groups. (C) Endogenous DNA damage level in HPV-positive and HPV-negative head and neck cancer cell lines following treatment with NAC (1mM). DNA damage level in treated versus non-treated cells for each cell line was analysed by two-tailed t-test. *P < 0.05, **P < 0.005.

Figure 2.

Expression of E6 ad E7 promotes NOX-dependent oxidative stress in head and neck cancer cells. (A) Representative flow cytometry curve of total intracellular ROS levels in PCI-15A cells (HPV negative) transfected with an empty plasmid (CTRL) or a plasmid expressing E6 and E7 (E6/E7). (B) Quantitative representation of previous experiment. ROS level in treated and non-treated cells for each genotype was compared by two-way analysis of variance (ANOVA; treatment × genotype interaction P = 0.0053; Bonferroni post-test for multiple comparison: **P < 0.005). (C) Representative flow cytometry curve of mitochondrial ROS levels in CTRL and E6/E7 cells. (D–E) Representative flow cytometry curves of ROS levels in (D) CTRL and (E) E6/E7 cells following treatment with DPI (1 µM). (F) DNA damage levels in E6/E7 transfected and isogenic CTRL cells following treatment with NAC (1mM). DNA damage level in treated and non-treated cells for each genotype was compared by two-way ANOVA (treatment × genotype interaction P < 0.005; Bonferroni post-test for multiple comparison: *P < 0.05, **P < 0.005; ***P < 0.0005). (G) Expression of full-length (E6) and truncated (E6* and E6**) mRNA transcripts in HPV-positive head and neck cancer cell lines. Genomic DNA for each cell line was used as control.

Figure 3.

NOX2 is the source of HV16-induced chronic oxidative stress in head and neck cancer cells. (A) Intracellular ROS level in HPV-positive and HPV-negative head and neck cancer cell lines following treatment with DPI (1 µM) or NAC (1mM). ROS level in treated and non-treated cells for each cell line was analysed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.005, ***P < 0.0005. (B) NOX2 mRNA expression levels and (C) intracellular ROS levels in SCC47 cells (HPV positive) transfected with plasmids carrying non-targeted shRNA (pLKo1 cells) or NOX2-targeted shRNA (SH88 and SH90). (D) Amount of DNA damage in pLKo1, SH88 or SH90 cells. (E) Representative comets in each cell line. NOX2 mRNA expression, intracellular ROS levels and DNA damage amount in pLKo1, SH88 and SH90 cells were analysed by one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.005. (F) Representative western blot analysis of p47phox expression in the membrane enriched and the soluble cytosolic fraction of HPV-negative (A: PCI-15A; B: PCI-13) and HPV-positive (C: SCC47; D: 93VU147) cells lysate. Na, K-ATPase and β-actin were used as loading control for membrane-enriched fraction and soluble cytosolic fraction, respectively. Average of p47phox expression for each group (A + B versus C + D) was compared and numbers represent normalized fold changes.

Figure 4.

Chronic oxidative stress contributes to chromosomal instability in HPV-positive head and neck cancer cells. (A) Micronuclei frequency in SCC47 cells (HPV positive) transfected with non-targeted shRNA (pLKo1 cells) or NOX2-targeted shRNA (SH88 and SH90 cells). Micronuclei frequency was compared by one-way analysis of variance (ANOVA; P = 0.006; Dunnett’s post-test for multiple comparison: *P < 0.05, **P < 0.005. (B) Micronuclei frequency in PCI-15A cells (HPV negative) transfected with empty plasmid (CTRL) or plasmid expressing HPV16 E6 and E7 (E6/E7). Micronuclei frequency was compared by two-way ANOVA (Bonferroni post-test for multiple comparison: *P < 0.05, **P < 0.005). (C) Micronuclei frequency in HPV-positive and HPV-negative head and neck cancer cells following treatment with NAC (1mM). Micronuclei amount in treated and non-treated cells for each cell line was analysed by two-tailed t-test. *P < 0.05, **P < 0.005.

Figure 5.

HPV-positive head and neck cancer cells are more susceptible to ROS-induced DNA damage. (A) DNA damage level following exposure to a dose range of H₂O₂ in HPV-positive and HPV-negative head and neck cancer cell lines. Column represents mean for each group, whereas black dots represent individual values for each cell line. DNA damage amount in treated versus non-treated cells for each group was analysed by two-way analysis of variance (ANOVA; treatment × HPV status interaction P < 0.005; Bonferroni post-test for multiple comparison: **P < 0.005). (B) DNA damage levels and (C) micronuclei frequency following exposure to IR (2 or 6 Gy) in HPV-positive and HPV-negative head and neck cancer cell lines. DNA damage and micronuclei amount in treated versus non-treated cells for each group were analysed by two-way ANOVA followed by Bonferroni’s multiple comparison test. **P < 0.005.

Figure 6.

Proposed model for establishment of NOX-induced chronic oxidative stress and genomic instability in HPV-positive head and neck cancer cells. Expression of HPV16 E6/E7 generates a ROS response in host cell via NOX2 oxidase activation. Activation of NOX2 oxidase occurs via increased formation of NOX2 active complex at membrane. NOX2-generated ROS causes DNA damage, such as bases oxidation, single DNA strand breaks and double DNA strand breaks, leading to chromosomal aberration and genomic instability. This state of chronic oxidative stress contributes to the increased sensitivity to IR of HPV-positive head and neck cancer cells. Additional mechanisms for HPV16-induced genomic instability comprise replication stress and centrosome amplification.