DNA repair hijacking by HPV

From the Galloway lab, Human Biology Division

It is now common knowledge that some cancers are caused by viral infection. The explanation for this direct causal relationship differs between viruses. In the case of the human papilloma virus (HPV), the expression of two oncoproteins called E6 and E7 is sufficient to cause dysregulation of cell cycle and DNA repair pathways, resulting in a dramatic increase in genomic instability. Persistent infection allows the aberrant accumulation of DNA anomalies predisposing to cancer. HPV causes about 5% of all solid-organ cancers worldwide. While virtually all cervical cancers are HPV-positive, other anogenital or oropharyngeal cancers are also highly associated with HPV.

In previous work, the Galloway lab (Human Biology Division) demonstrated that HPV oncogenes E6 an E7 dysregulate the DNA damage repair (DDR) processes by impairing the homologous recombination pathway, a mechanism using a homologous unaffected DNA strand to “overwrite” the mutation with the correct sequence. In a recent publication in the journal PLoS Pathogens, Drs. Sujita Khanal and Denise Galloway investigated the effect of HPV oncogenes on another DDR process named Fanconi anemia-BRCA (FA-BRCA) pathway.

The FA-BRCA pathway oversees the repair of intra- or inter-strand crosslinks (ICLs) within DNA. ICLs prevent DNA replication and transcription. Under physiological conditions, unrepaired crosslinks lead to cell death. When a crosslink is detected, ATR/CHK1 signaling pathway is activated, leading to the phosphorylation of FA proteins. FancD2 is one such FA protein and when monoubiquitinated, re- locates to an ICL-stalled replication fork and recruits other DNA repair proteins. After repair, FancD2 is deubiquitinated by the ubiquitin-specific protease USP1 in coordination with UAF1 (USP1-associated factor), which ends the cell cycle arrest and prevents cell death. While some studies anticipated the interaction between HPV oncogenes and the FA-BRCA pathway, little was known on the molecular mechanism underlying this link. There is more than a fundamental understanding at stake here since cisplatin, a crosslinking chemotherapeutic agent, is the most commonly used treatment against cervical cancer.

The authors started their investigations with primary human foreskin keratinocytes (HFKs) in which they demonstrated that overexpression of high-risk HPV-E6, HPV- E7 or both, sensitized cells to cisplatin-induced cell death. As they wanted to test whether this was related to the FA-BRCA pathway, they knocked down FancD2 in these cells. Whereas FancD2 depletion sensitized control cells to cisplatin, it did not have an effect on E6/E7 expressing cells, suggesting that the FA pathway is already downregulated downstream of E6 and E7 HPV oncogenes. The researchers reasoned that the dysregulation of the FA pathway should result in the inability to repair cisplatin-induced ICLs. To test this hypothesis, they treated the cells with cisplatin for a few hours and performed a comet assay at different timepoints after removing cisplatin. In the comet assay, when subjected to gel electrophoresis, DNA migrates at different velocities depending on the level of DNA damage. In the case of ICLs, DNA migrates slower, resulting on a shorter “tail moment”. Although all cells treated with cisplatin displayed a shorter tail moment before the removal of the chemotherapy independently of the expression of E6/E7, the authors observed that only cells overexpressing the HPV oncogenes displayed a shorter tail moment 72h after cisplatin removal, demonstrating the inability of these cells to repair ICLs.

Comet assay showing the inability of E6/E7 expressing cells to repair ICLs (shorter tail moment) after removal of cisplatin treatment.
Comet assay showing the inability of E6/E7 expressing cells to repair ICLs (shorter tail moment) after removal of cisplatin treatment. Illustration from publication.

In order to dissect the molecular mechanisms underlying this striking observation, Khanal and colleagues assessed the FA pathway functionality by immunoblots and immunofluorescence in the presence or in absence of HPV oncogenes, with or without cisplatin. Surprisingly, E6 and E7 oncogenes both triggered the FA pathway by increasing the monoubiquitination of FancD2. The binding of monoubiquitinated FancD2 to chromatin was not impaired. However, FancD2 did not colocalize with the sites of DNA damage when HPV oncogenes were expressed, and E6/E7 expressing cells were unable to recruit the different component of the repair pathway downstream of FancD2 monoubiquitination. In addition, E6-mediated P53 degradation prevented FancD2 deubiquitination. Thus, the cells can trigger the FA pathway when treated with cisplatin, but the pathway is not functional.

Dr. Khanal comments: “We provided an in-depth mechanistic framework for how HPV oncogenes disrupt the FA DNA repair pathway. The work described in the paper advances our knowledge of how dysregulated FA pathway contributes to the development of cancers in patients with persistent viral infection. Further, our work has important therapeutic implications. Disrupted FA pathway explains why HPV+ oropharyngeal cancers benefit more with cisplatin treatment than HPV negative head and neck cancer patients. As HPV associated cancers are more sensitive to cisplatin therapy, attempts should be made to de-escalate the doses to reduce toxicities in these patients”.

This work was supported by the National Institute of Health.

Fred Hutch/UW Cancer Consortium member Dr. Denise Galloway contributed to this research.

Khanal S, Galloway DA. 2019.  High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and deubiquitination of FancD2. PLoS Pathog 15(2): e1007442. https://doi.org/10.1371/journal.ppat.1007442