Analysis of a hit-and-run tumor model by HPV in oropharyngeal cancers

Abstract

Several viruses are known to be associated with the development of certain cancers, including human papilloma virus (HPV), an established causative agent for a range of anogenital and head and neck cancers. However, the causality has been based on the presence of the virus, or its genetic material, in the sampled tumors. We have long wondered if viruses cause cancer via a "hit and run" mechanism such that they are no longer present in the resulting tumors. Here, we hypothesize that the absence of viral genes from the tumor does not necessarily exclude the viral aetiology. To test this, we used an HPV-driven oropharyngeal cancer (OPC) tumor model and CRISPR to delete the viral oncogene, E7. Indeed, the genetic removal of HPV E7 oncogene eliminates tumors in vivo. Remarkably, E7 deleted tumors recurred over time and develop new mutations not previously seen in HPV⁺ OPC tumors. Importantly, a number of these new mutations are found to be already present in HPV^- OPC tumors.

Keywords: E7; human papilloma virus; oropharyngeal cancer.

Figure 1

Genetic deletion of E7 results in effective amelioration of OPC tumor growth before recurring over time. (A) Schematic representation of the experimental plan. (B) SCC2_Cas9 (−), SCC2_Cas9_16E7 (E7) and SCC2_Cas9_18E7 (Off‐target) cells were either treated with sterile water (Vehicle) or with doxycycline (10 μg/ml) (DOX) before performing an MTT assay at indicated timepoints. Data representative of one out of three independent experiments. Bars denote mean percentage viability to SCC2_Cas9 control cells within its respective treatment groups. Error bars denotes standard error of mean (SEM) of technical quadruplicate treatments. The Student t test, *p = 0.0442; **p = 0.0015; ****p < 0.0001. (C) SCC2_Cas9_16E7 cells were either treated with sterile water (Vehicle) or with doxycycline (10 μg/ml) (DOX) for 72 h before extracting genomic DNA for Sanger sequencing. Percentage indel was determined by TIDE analysis (n = 3 independent cell treatments ± SEM). Proteins were also extracted from cells and immunoblotted for HPV 16 E7 and Rb. S6 protein was used as a loading control. Immunoblotting data is representative of one out of three independent experiments. (D) SCC2_Cas9 (−), SCC2_Cas9_16E7 (E7) and SCC2_Cas9_18E7 (Off‐target) cells were either treated with sterile water (Vehicle) or with doxycycline (10 μg/ml) (DOX) over 7 days before performing a colony forming assay. Data representative of one out of three independent experiments. (E) Nude mice were xenografted with either SCC2_Cas9 (−), SCC2_Cas9_16E7 (E7) or SCC2_Cas9_18E7 (Off‐target) cells and tumors allowed to grow to 100 mm³ before feeding mice with either normal (Vehicle) or DOX fodder (DOX). Each point represents mean of n = 5‐6 mice/group. Mean tumor volumes are shown with error bars representing SEM. (F) Tumors excised at indicated days (SCC2_Cas9 (−)—Day 37, SCC2_Cas9_16E7 (E7)—Day 89 and SCC2_Cas9_18E7 (Off‐target)—Day 37) were processed for genomic DNA extraction before performing Sanger sequencing. Percentage indel was determined by TIDE analysis (n = 6 tumors ± SEM). (G) Mouse survivorship (percentage survival) were evaluated and plotted throughout the experiment for DOX‐treated groups. (H) Proteins were extracted from tumors at indicated days (SCC2_Cas9 (−)—Day 37, SCC2_Cas9_16E7 (E7)—Day 89 and SCC2_Cas9_18E7 (Off‐target)—Day 37) and immunoblotted for HPV 16 E7. S6 protein was used as a loading control. Data representative of one out of three independent experiments, using tumors from different mice within its respective treatment groups. gRNA, guide RNA; OPC, oropharyngeal cancer

Figure 2

Recurred OPC tumors have gained novel mutations not previously present in HPV⁺ OPC tumors. (A) Heatmap of significantly mutated genes, corresponding to genes mutated only in HPV‐ samples or E7‐edited mice (DOX‐treated SCC2_Cas9 [control] and SCC2_Cas9_E7 [treatment]) and/or genes associated with cellular proliferation. For comparison, the mutation percentage of 302 HPV⁻ and 58 HPV⁺ head and neck squamous cell carcinoma (HNSCC) patients were compiled from the TCGA. The scale color bar represents the number of mutations encountered in each gene or the percentage of patients exhibiting mutation. Genes mutated in the SCC2 parental cell line were bioinformatically filtered and excluded. (B) Circular heatmap of type of mutations in the gene set. Combined number of SNV (gain or loss of stop codon), frameshift insertion or deletion for each examined gene. (C) The top 30 relevant enriched pathways for carcinogenesis. Plot showing −log‐transformed p value of enriched KEGG pathways for the 24 selected genes. KEGG, Kyoto Encyclopedia of Genes and Genomes; OPC, oropharyngeal cancer; SNV, single nucleotide variation; TCGA, The Cancer Genome Atlas