Human papillomavirus insertions identify the PIM family of serine/threonine kinases as targetable driver genes in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) insertions in cancer genomes have been linked to various forms of focal genomic instability and altered expression of neighboring genes. Here we tested the hypothesis that investigation of HPV insertions in a head and neck cancer squamous cell carcinoma (HNSCC) cell line would identify targetable driver genes contributing to oncogenesis of other HNSCC. In the cell line UPCI:SCC090 HPV16 integration amplified the PIM1 serine/threonine kinase gene ~16-fold, thereby increasing transcript and protein levels. We used genetic and pharmacological approaches to inhibit PIM kinases in this and other HNSCC cell lines. Knockdown of PIM1 transcripts by transfected short hairpin RNAs reduced UPCI:SCC090 viability. CRISPR/Cas9-mediated mutagenesis of PIM1 caused cell cycle arrest and apoptosis. Pharmacological inhibition of PIM family kinases decreased growth of UPCI:SCC090 and additional HNSCC cell lines in vitro and a xenograft UPCI:SCC090 model in vivo. Based on established interactions between intracellular signaling pathways and relatively high levels of gene expression in almost all HNSCC, we also evaluated combinations of PIM kinase and epidermal growth factor receptor (EGFR) inhibitors. Dual inhibition of these pathways resulted in supra-additive cell death. These data support clinical testing of PIM inhibitors alone or in combination in HNSCC.

Genome-wide analysis of HPV integration in human cancers reveals recurrent, focal genomic instability.

Genomic instability is a hallmark of human cancers, including the 5% caused by human papillomavirus (HPV). Here we report a striking association between HPV integration and adjacent host genomic structural variation in human cancer cell lines and primary tumors. Whole-genome sequencing revealed HPV integrants flanking and bridging extensive host genomic amplifications and rearrangements, including deletions, inversions, and chromosomal translocations. We present a model of "looping" by which HPV integrant-mediated DNA replication and recombination may result in viral-host DNA concatemers, frequently disrupting genes involved in oncogenesis and amplifying HPV oncogenes E6 and E7. Our high-resolution results shed new light on a catastrophic process, distinct from chromothripsis and other mutational processes, by which HPV directly promotes genomic instability.

The prevalence and incidence of oral human papillomavirus infection among young men and women, aged 18-30 years.

BACKGROUND: Oral human papillomavirus (HPV) infection is a cause of oropharyngeal squamous cell carcinoma, yet little is known about the epidemiology and natural history of infection. METHODS: At a baseline and 3-month follow-up visit, 1000 young adults aged 18 to 30 years provided an oral rinse sample and completed a survey assessing demographic and behavioral risk factors. The oral rinse sample was analyzed for 37 types of HPV by use of a multiplex polymerase chain reaction assay. Factors associated with oral HPV detection were analyzed using univariate and bivariate logistic regression. RESULTS: The prevalence of oral HPV infection was 2.4% (95% CI: 1.4-3.4). Ever having consumed alcohol (OR, 0.2; 95% CI: 0.1-0.8), 5 or more lifetime open-mouth kissing (OR, 4.0; 95% CI: 1.1-14.8) or lifetime oral sex (OR, 4.0; 95% CI: 1.3-11.9) partners were associated with infection, controlling for lifetime vaginal sex partners. The incidence rate for oral HPV infection was 5.67 (95% CI: 3.12-8.16) per 1000 person-months. Incident infection was associated in univariate analysis with black race (OR, 4.7; 95% CI: 1.7-13.5) and having open-mouth kissed a new partner in the previous 3 months (OR, 2.6; 95% CI: 1.0-6.4). CONCLUSIONS: This study provides further evidence that oral sexual contact in the form of both oral-oral and oral-genital contact could play a role in the transmission of oral HPV.

Automated high throughput DNA isolation for detection of human papillomavirus in oral rinse samples.

BACKGROUND: Oral HPV infection elevates risk of oropharyngeal cancer, but its natural history is unknown. Natural history studies necessitate validation of an automated, high-throughput method for HPV genomic DNA detection in oral rinse samples (ORS). OBJECTIVES: To compare agreement of oral HPV detection in ORS processed by a magnetic-bead based automated platform to a previous gold-standard, manual protein-precipitation method. Agreement was compared to that of repeat sampling and repeat HPV testing. STUDY DESIGN: HIV-infected individuals (n=100) provided two ORS collected 15 min apart. DNA was isolated from equal aliquots by either a protein-precipitation based (Puregene, Qiagen) or magnetic bead-based (QIAsymphony™ SP, Qiagen) method. HPV DNA was detected and type-specified by consensus primer PCR and reverse line blot hybridization. The kappa statistic was used to assess overall agreement (OA) and agreement on a positive test (Ps+). RESULTS: The DNA purification methods had very high agreement for categorizing an individual as HPV infected (OA=0.95; Ps+=0.94) as well as for detection of HPV type-specific infection (OA=0.99; Ps+=0.88) in ORS. Agreement for detection of HPV type-specific infection was greater than that observed with repeat oral rinse sampling (OA=0.99, Ps+=0.76) but comparable to inter-assay agreement (OA=1.00, Ps+=0.90). CONCLUSIONS: HPV detection in ORS processed with a magnetic-bead based automated platform will facilitate large natural history studies of oral HPV infection necessary to evaluate the potential use of oral HPV detection in oral cancer screening.

Binding and neutralization characteristics of a panel of monoclonal antibodies to human papillomavirus 58.

Human papillomavirus (HPV) 58 is a high-risk HPV type associated with progression to invasive genital carcinomas. We developed six monoclonal antibodies (mAbs) against HPV58 L1 virus-like particles that bind conformational epitopes on HPV58. The hybridoma cell lines were adapted to serum- and animal component-free conditions and the mAb supernatants were affinity-purified. The six mAbs neutralized HPV58 pseudoviruses (PsVs) and 'quasivirions' with different capacities. The mAbs differed in their ability to prevent PsV58 attachment to HaCaT cells, to the extracellular matrix (ECM) deposited by HaCaT cells, to heparin and to purified human laminin 5, a protein in the ECM. These mAbs provide a unique set of tools to study the binding properties of a previously untested, high-risk HPV type and the opportunity to compare these characteristics with the binding of other HPV types.

Differential binding patterns to host cells associated with particles of several human alphapapillomavirus types.

The focus of this research was to compare the binding profiles of human papillomavirus (HPV) 11, 16, 18 and 45 virus-like particles (VLPs) to HaCaT cells and to the extracellular matrix (ECM) secreted by these cells. All four HPV types tested bind to a component(s) of the ECM. HPV11 VLP binding is blocked when the ECM is pretreated with an anti-laminin 5 (LN5) polyclonal antibody. A series of treatments utilizing heparins and heparinase revealed that HPV18 VLPs are dependent on heparan sulfates (HS) for binding to cells and ECM. HPV16 and HPV45 VLPs are dependent on HS for binding to HaCaT cells and dependent on both HS and LN5 for binding to ECM. These studies emphasize the need to study the binding characteristics of different HPV types before applying universal binding principles to all papillomaviruses.