Human papillomavirus 16 promotes microhomology-mediated end-joining

Abstract

Squamous cell carcinomas (SCCs) arising from aerodigestive or anogenital epithelium that are associated with the human papillomavirus (HPV) are far more readily cured with radiation therapy than HPV-negative SCCs. The mechanism behind this increased radiosensitivity has been proposed to be secondary to defects in DNA repair, although the specific repair pathways that are disrupted have not been elucidated. To gain insight into this important biomarker of radiosensitivity, we first examined genomic patterns reflective of defects in DNA double-strand break repair, comparing HPV-associated and HPV-negative head and neck cancers (HNSCC). Compared to HPV-negative HNSCC genomes, HPV+ cases demonstrated a marked increase in the proportion of deletions with flanking microhomology, a signature associated with a backup, error-prone double-strand break repair pathway known as microhomology-mediated end-joining (MMEJ). Then, using 3 different methodologies to comprehensively profile double-strand break repair pathways in isogenic paired cell lines, we demonstrate that the HPV16 E7 oncoprotein suppresses canonical nonhomologous end-joining (NHEJ) and promotes error-prone MMEJ, providing a mechanistic rationale for the clinical radiosensitivity of these cancers.

Keywords: E7; HPV; alternative end-joining; microhomology-mediated end-joining; radiation.

Fig. 1.

HPV-associated head and neck cancers are associated with increased utilization of microhomology at deletion breakpoints without signatures of homologous recombination deficiency. (A–E) The proportion of deletions with microhomology, LST scores, and contribution of genomic Stratton Signature 3 were measured in breast and ovarian cancers (n = 1,487) with or without biallelic BRCA1 or BRCA2 mutation, and in HPV+ and HPV− head and neck cancers (n = 510). HPV+ head and neck cancers demonstrated an increase in microhomology usage (D) without an associated increase in LST score of Signature 3 contribution (A and B). (C) Schematic of MMEJ. (E) BRCA mutations and HPV positivity were associated with an increase in microhomology length in these tumors. (F) Whole exome sequencing data from all cancer types available in TCGA were analyzed for the presence of microhomology at mapped deletion sites, only deletions of 3+ bp in length were considered. The proportion of deletions with 3+ bp of microhomology across all cancer types is shown. (G) Disease-free survival (DFS) of TCGA HPV+ HNSCC cases classified by <50% of deletions containing MH and ≥50% deletions containing MH. Assessable cases defined as those having a least 1 characterized deletion with MH. *P < 0.05, ***P < 0.001.

Fig. 2.

HPV E7 protein expression down-regulates canonical nonhomologous end-joining (cNHEJ) and up-regulates MMEJ. (A–C) U2OS cells with integrated DSB repair reporter systems were transiently cotransfected with Isce1 restriction enzyme and plasmids expressing individual HPV16 oncoproteins (E1, E2, E5, E6, E7). The percentage of GFP-positive cells was measured 72 h following transfection by flow cytometry (n = 3+ repeats per condition). Cells expressing exogenous E7 oncoprotein showed an increase in HR and MMEJ but decreased cNHEJ. (D) Stable transfection of U2OS cells with the HPV16 E7 expression plasmid conferred significant reduction in pRB protein levels. (Lower) Survival curves based on clonogenic growth of U2OS cells with integrated EJ5 reporter system transfected with empty vector (red) and HPV16-E7 (blue). Curves represent the mean of 4 experiments. (E) Decrease in NHEJ activity measured by the percentage of GFP-positive cells in U2OS-EJ5 reporter cells with HPV16 E7 overexpression. (F) HR activity measured in DR-GFP cells showed increase in HR activity with E7 expression but not in the cells expressing mutant E7 C24G protein defective in pRb binding. (G) cNHEJ activity measured in U2OS EJ5-GFP cells showed down-regulation of cNHEJ activity with E7 expression but no difference in cells expressing E7 C24G. (H) MMEJ activity measured in U2OS EJ2-GFP cells showed up-regulation of MMEJ activity with E7 expression but no significant difference in cells expressing E7 C24G. At least 3 independent experiments were performed for each group. (I) Relationship between E7 mRNA expression in TCGA HPV+ HNSCC cases and percentage of deletions with MH. Cases partitioned into quartiles of increasing E7 expression levels *P < 0.05; **P < 0.01; ***P < 0.001; N.S., not significant.

Fig. 3.

Effect of E7 on cNHEJ in the setting of IR-mediated DNA damage. (A) Representative immunofluorescent images of phospho-DNA-PKcs foci at 30 and 60 min after radiation in U2OS cells stably transfected with an HPV16 E7-expressing vector or empty vector control. Green, phospho-DNA-PKcs foci; blue, DAPI staining. (B) The number of pDNA-pk foci per cell detected was significantly decreased in the E7-expressing cell line compared to the EV-expressing cell line. (C) Data from 3 independent experiments represented as fold change in foci per cell shows significant fold change in cells expressing E7 after 60 and 240 min after IR. *P < 0.05, ***P < 0.001.

Fig. 4.

E7 expression shifts DNA DSB repair pathway choice toward MMEJ. (A) Schematic of DSBR-Seq Assay. The sequencing reads are classified into NHEJ (<5 bp deletion) in B, MMEJ (>5 bp deletion with at least 2 bp MH) in C, and HR (substitutions from HR donor) in D. The DNApk inhibitor Nu7741 was used at 1 μM concentration. (E) cNHEJ measured as a fraction of mutant reads with deletion sizes less than 5 bp. (F) E7 expression favors mutant reads with deletion sizes larger than 5 bp representing DSBs repaired by MMEJ. (G) E7 expression results in lower deletion frequency of small deletions near the DSB. Results represent the mean of 3 independent experiments ± the SEM. *P < 0.05.