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Cervical cancer is caused almost exclusively by the human papillomavirus (HPV) and has a high mortality burden, especially in developing countries. HPV has a 7.9 kb genome and replicates in the nucleus as a circular extrachromosomal element, called an episome, by hijacking the host cell’s machinery. It encodes two oncogenes, E6 and E7, which inhibit the host tumor suppressor genes p53 and RB1, respectively. Integration into the genome frequently leads to suppression of viral replication proteins E1 and E2 and contributes to chromosomal instability.
The HPV genome is integrated into 70% of cervical cancers. Integration events frequently occur near oncogenes, which is associated with their increased expression. These events can also be quite complex, making them difficult to resolve with short-read sequencing technologies. At London Calling 2022, Vanessa Porter and Michael Dean showed how long nanopore readouts could be used to examine the impact of integration on the human genome and HPV*.
The complex landscape of HPV integration events
Using PromethIONMT, Vanessa Porter (Michael Smith Genome Sciences Centre, Canada) and her team generated whole genome sequencing data for 66 cervical cancer clinical research samples from Ugandan and US cohorts. Vanessa developed her own custom pipeline to call HPV integration breakpoints as translocations. Vanessa pointed out how the benefit of long reads is that “you can identify integration events that may span multiple chromosomes, or that are linked together over large distances by structural rearrangements.” Through the samples, Vanessa observed integration events that could be classified into one of six categories. While the actual content of the inserted HPV sequence was often variable, the E6 and E7 sequences remained constant, suggesting that they are a prerequisite for the formation of cervical cancer.
One of the six integration events involved two breakpoints existing on separate chromosomes and resulted in complete translocations of the chromosomal arm, shown by chr12 and chr4 sandwiching a piece of HPV DNA. Vanessa noted how ‘this is exactly the type of event that nanopore sequencing allows us to identify, since the reads are able to skip over the HPV, sequencing itself into the next chromosome.Vanessa also looked at how the landscape of integration events varies between HPV types. Critical multipoint integration events were most frequently observed in HPV16.
Leveraging a ‘added bonus of nanopore sequencing’, through PCR-free direct detection of DNA, Vanessa sought to analyze the impact of HPV methylation patterns on the human genome surrounding the integration event. With long nanopore readouts, the data could be easily transformed into haplotypes. Vanessa observed that the human genome had a demethylation scan for large regions upstream of the breakpoint (up to 400 kb), compared to the unintegrated haplotype. Additionally, Vanessa explored how HPV methylation patterns were affected by the integration event. The upstream regulatory regions of HPV remained unmethylated, while the gene parts were significantly more methylated compared to unintegrated episomal HPV, although variability was observed between HPV types.
HPV – pulling the epigenetic strings
Michael Dean (National Cancer Institute, USA) uses nanopore sequencing to unravel the role of HPV in driving cancer phenotypes. He, too, used long nanopore readouts to delineate complex integration events. Michael is now focusing on the impact of HPV infection on genes regulating the epigenome, thereby altering gene expression and driving carcinogenesis. Michael drew comparisons to embryology and the differentiation of cells into specialized cell lineages: each stem cell contains the same DNA, but it is their unlocking of certain epigenetic programs that governs their differentiated fate. Parallels can be drawn in cancer cells; for example, if a cancer cell needs more oxygen, it can alter the epigenome to activate angiogenesis pathways. The question Michael asked was: how are these genes that modify the epigenome impacted by HPV?
To start answering this question, Michael used the MinIONMT device – or as he called it, ‘this little beast’ – which is the same size as a Swiss army knife he had growing up. He started by performing full cDNA sequencing on 20 HPV+ cancer cell lines and acquired “unprecedented detail” on HPV expression. In the CaSki cell line, in which hundreds of copies of HPV are integrated into the human genome, only one copy of HPV was expressed; the others were silent. Moreover, of the twenty or so isoforms that HPV can produce, the most predominant transcript was an isoform of the oncogene E7. Similar observations were made in the Snu1000 cell line, which contains many copies of episomal HPV. Michael described how the virus is so’regulate its epigenome in order to express the oncogene it will need later in life.’ HPV integration was found to up-regulate genes near the integration site, some of which were oncogenes which Michael hypothesized’probably played a role in the process of transformation‘. Michael also turned to direct RNA nanopore sequencing, in which native RNA molecules are sequenced, avoiding reverse transcription bias and allowing for RNA edit calling. He applied this method to HeLa cell lines, in which HPV integrated near the MYC oncogenic, when significant MYC overexpression was observed. Michael shared how his PhD focused on studying MYC expression; he pointed out how he ‘never imagined that…[he] would directly see RNA transcripts.
Like Vanessa, Michael explored how methylation was affected by HPV integration. He compared his results to short-read bisulfite sequencing and found that “Nanopore sequencing gave very precise methylation calls”indicating that the ‘Beauty of [nanopore sequencing] is that you can see every CpG site across the HPV genome on individual viral genomes.” In one of the cell lines, FOXE1 – a potential driver of cancer transformation – was up-regulated near the site of integration. Michael observed marked demethylation in and around FOXE1. To take this a step further, Michael used Pore-C, an Oxford Nanopore workflow that combines chromatin conformation capture with long nanopore readouts. His results revealed that most interactions between HPV and human DNA occur in the activator regions. This phenomenon is consistent with the notion that HPV provides an activator and interacts with other nearby genes. To better understand cancer phenotypes, Michael explored the entire mitochondrial genome to begin to decipher the mechanisms behind cancer energetics.
Michael is currently focusing on the most common aberrant human gene in cervical cancer, PIK3 – an important kinase. The drug Piqray acts as a PIK3CA inhibitor and is currently an FDA-approved drug to treat breast cancer, but its therapeutic potential for the treatment of cervical cancer is unknown. To investigate this, Michael took a clinical research sample of cervical cancer cells with activation in this pathway, and treated with micromolar concentrations of the drug, which reduced expression of the checkpoint protein. immune, PD-L1, reduced expression of HPV transcripts and cessation of proliferation. This would make the cancer more treatable with immunotherapies.
Both Vanessa and Michael have used nanopore sequencing to further our understanding of how HPV drives cervical cancer transformation. From delineation of complex integration events to methylation analysis and chromatin conformation studies, nanopore sequencing opens new avenues to explore cervical cancer in unprecedented detail.
* London Calling 2022 hybrid conference, organized by Oxford Nanopore Technologies; May 18-20, 2022
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