Abstract
Simple SummaryHigh levels of global genome methylation in HPV-associated head and neck cancer prompted us to explore demethylation as a potential treatment by determining mechanisms of its toxicity in HPV-positive head and neck cancer cells. Previously, we reported that demethylating drug 5-azaC stabilizes p53 and reduces the expression of HPV genes and matrix metalloproteinases in HPV+ head and neck cancer cells and tumors from patients enrolled in a 5-azaC window clinical trial. Here, we extended our understanding of toxicity caused by global demethylation in HPV-associated head and neck cancer cells by finding that 5-azaC treatment results in formation of DNA double strand breaks that depend on transcription and replication.High levels of DNA methylation at CpG loci are associated with transcriptional repression of tumor suppressor genes and dysregulation of DNA repair genes. Human papilloma virus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) have high levels of DNA methylation and methylation has been associated with dampening of an innate immune response in virally infected cells. We have been exploring demethylation as a potential treatment in HPV+ HNSCC and recently reported results of a window clinical trial showing that HNSCCs are particularly sensitive to demethylating agent 5-azacytidine (5-aza). Mechanistically, sensitivity is partially due to downregulation of HPV genes expression and restoration of tumor suppressors p53 and Rb. Here, for the first time, we show that 5-azaC treatment of HPV+ HNSCC induces replication and transcription-associated DNA double strand breaks (DSBs) that occur preferentially at demethylated genomic DNA. Blocking replication or transcription prevented formation of DNA DSBs and reduced sensitivity of HPV-positive head and neck cancer cells to 5-azaC, demonstrating that both replication and active transcription are required for formation of DSBs associated with 5-azaC.
Highlights
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world and is associated with poor prognosis in advanced cases [1,2]
DNA from DSBs and intact DNA contained reads that mapped randomly to the genome with more reads mapping to longer chromosomes. These results demonstrate that 5-azaC-induced DSBs in Human papilloma virus (HPV)+ head and neck squamous cell carcinomas (HNSCC) were entions mapping to intron, exons, or intergenic regions in both, intact DNA and double strand breaks, samples; no strand bias was found for the genes in either intact or DSB DNA
Using BRCA2 deficient cells, we found that HR defects sensitize cells to demethylation (Figure 4A) consistent with reports that 5-azaC-induced DNA double strand breaks require homologous recombination [20]
Summary
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world and is associated with poor prognosis in advanced cases [1,2]. Advanced HNSCC are treated with combinations of primary surgical resection, cervical lymphadenectomy, radiation, or radiation given with platin drugs, depending on institutional preference and tumor characteristics. Extended survival analysis of trials comparing radiation and chemotherapy regimens suggests that side effects of concomitant chemotherapy and radiation may decrease overall survival in the absence of recurrent tumor [6]. Despite aggressive and morbid therapy, up to 25% of patients with HPVassociated (HPV+) head and neck tumors suffer recurrent or metastatic disease, for which treatment options are limited. Available HPV vaccines hold tremendous promise for prevention of HPV+ head and neck cancer; given the latency between infection and the development of HPV+ HNSCC, estimates suggest that the HPV vaccine will not decrease HNSCC prevalence until 2060 [7]. Insight into vulnerabilities and development of less morbid, yet effective treatments for primary and recurrent HPV+
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