Detection of HPV E7 Transcription at Single-Cell Resolution in Epidermis.

Persistent human papillomavirus (HPV) infection is responsible for at least 5% of human malignancies. Most HPV-associated cancers are initiated by the HPV16 genotype, as confirmed by detection of integrated HPV DNA in cells of oral and anogenital epithelial cancers. However, single-cell RNA sequencing may enable prediction of HPV involvement in carcinogenesis at other sites. We conducted single-cell RNA sequencing on keratinocytes from a mouse transgenic for the E7 gene of HPV16 and showed sensitive and specific detection of HPV16-E7 mRNA, predominantly in basal keratinocytes. We showed that increased E7 mRNA copy number per cell was associated with increased expression of E7 induced genes. This technique enhances detection of active viral transcription in solid tissue and may clarify possible linkage of HPV infection to development of squamous cell carcinoma.

[1]  P. Lemey,et al.  Single Cell Analysis of Lymph Node Tissue from HIV-1 Infected Patients Reveals that the Majority of CD4+ T-cells Contain One HIV-1 DNA Molecule , 2013, PLoS pathogens.

[2]  Grace X. Y. Zheng,et al.  Massively parallel digital transcriptional profiling of single cells , 2016, Nature Communications.

[3]  H. Soyer,et al.  Clinical features of actinic keratoses and early squamous cell carcinoma. , 2015, Current problems in dermatology.

[4]  A. Gewirtzman,et al.  Epidermodysplasia verruciformis and human papilloma virus , 2008, Current opinion in infectious diseases.

[5]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[6]  M. Feltkamp,et al.  The presence of betapapillomavirus antibodies around transplantation predicts the development of keratinocyte carcinoma in organ transplant recipients: a cohort study. , 2015, The Journal of investigative dermatology.

[7]  B. Smoller,et al.  Immunohistochemical Comparison of P16 Expression in Actinic Keratoses and Squamous Cell Carcinomas of the Skin , 2002, Modern Pathology.

[8]  M. Michal,et al.  Human papillomavirus infection and p16 expression in the immunocompetent patients with extragenital/extraungual Bowen’s disease , 2016, Diagnostic Pathology.

[9]  S. Feldman,et al.  Progression of actinic keratosis to squamous cell carcinoma revisited: clinical and treatment implications. , 2011, Cutis.

[10]  M. Poljak,et al.  Anogenital hairs are an important reservoir of alpha-papillomaviruses in patients with genital warts. , 2009, The Journal of infectious diseases.

[11]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer , 2011, Nature Biotechnology.

[12]  K. Münger,et al.  Dissection of human papillomavirus E6 and E7 function in transgenic mouse models of cervical carcinogenesis. , 2003, Cancer research.

[13]  C. la Vecchia,et al.  Site distribution of different types of skin cancer: New aetiological clues , 1996, International journal of cancer.

[14]  I. Frazer,et al.  A Mouse Model of Hyperproliferative Human Epithelium Validated by Keratin Profiling Shows an Aberrant Cytoskeletal Response to Injury , 2016, EBioMedicine.

[15]  M. Jackson,et al.  Epidermal cancer associated with expression of human papillomavirus type 16 E6 and E7 oncogenes in the skin of transgenic mice. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Hogewoning,et al.  Detection of Human Papillomavirus Types 6 and 11 in Pubic and Perianal Hair from Patients with Genital Warts , 1999, Journal of Clinical Microbiology.

[17]  Maria Kasper,et al.  Single-Cell Transcriptomics Reveals that Differentiation and Spatial Signatures Shape Epidermal and Hair Follicle Heterogeneity , 2016, Cell systems.

[18]  S. Arron,et al.  Transcriptome Sequencing Demonstrates that Human Papillomavirus is not Active in Cutaneous Squamous Cell Carcinoma , 2011, The Journal of investigative dermatology.

[19]  A. Jemal,et al.  Global cancer statistics, 2012 , 2015, CA: a cancer journal for clinicians.

[20]  D. O’Connell,et al.  Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction , 2016, Cancer.

[21]  Alix Warburton,et al.  The role of integration in oncogenic progression of HPV-associated cancers , 2017, PLoS pathogens.

[22]  W. Sterry,et al.  Identification of differentially expressed genes in cutaneous squamous cell carcinoma by microarray expression profiling , 2006, Molecular Cancer.

[23]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[24]  Rob Patro,et al.  Salmon provides fast and bias-aware quantification of transcript expression , 2017, Nature Methods.

[25]  D. Whiteman,et al.  Multiplicity of skin cancers in Queensland and their cost burden to government and patients , 2018, Australian and New Zealand journal of public health.

[26]  Harald Kittler,et al.  Dermatoscopy of facial actinic keratosis, intraepidermal carcinoma, and invasive squamous cell carcinoma: a progression model. , 2012, Journal of the American Academy of Dermatology.

[27]  A. Toland,et al.  Epidemiology of keratinocyte carcinomas after organ transplantation , 2017, The British journal of dermatology.

[28]  J. McLauchlan,et al.  Determining the Cellular Diversity of Hepatitis C Virus Quasispecies by Single-Cell Viral Sequencing , 2013, Journal of Virology.

[29]  Ludo Waltman,et al.  A smart local moving algorithm for large-scale modularity-based community detection , 2013, The European Physical Journal B.

[30]  P. Boukamp Non-melanoma skin cancer: what drives tumor development and progression? , 2005, Carcinogenesis.

[31]  H. Pfister Chapter 8: Human papillomavirus and skin cancer. , 2003, Journal of the National Cancer Institute. Monographs.

[32]  I. Frazer,et al.  Expression of a Single, Viral Oncoprotein in Skin Epithelium Is Sufficient to Recruit Lymphocytes , 2013, PloS one.

[33]  V. Sondak,et al.  Case–control study of genus‐beta human papillomaviruses in plucked eyebrow hairs and cutaneous squamous cell carcinoma , 2014, International journal of cancer.

[34]  Martin R. Johnson,et al.  Quantitation of human papillomavirus 16 E6 and E7 DNA and RNA in residual material from ThinPrep Papanicolaou tests using real‐time polymerase chain reaction analysis , 2002, Cancer.

[35]  L. Naldi,et al.  Human Papillomavirus Load in Eyebrow Hair Follicles and Risk of Cutaneous Squamous Cell Carcinoma , 2013, Cancer Epidemiology, Biomarkers & Prevention.

[36]  Monika S. Kowalczyk,et al.  Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells , 2015, Genome research.

[37]  M. Karagas,et al.  Cutaneous alpha, beta and gamma human papillomaviruses in relation to squamous cell carcinoma of the skin: A population‐based study , 2013, International journal of cancer.

[38]  H. Pitot,et al.  Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene , 1996, Journal of virology.

[39]  I. Frazer,et al.  Invariant NKT Cells in Hyperplastic Skin Induce a Local Immune Suppressive Environment by IFN-γ Production , 2009, The Journal of Immunology.

[40]  I. Frazer,et al.  Murine HPV16 E7-expressing transgenic skin effectively emulates the cellular and molecular features of human high-grade squamous intraepithelial lesions , 2017, Papillomavirus research.

[41]  Qiang Sun,et al.  The Papillomavirus Episteme: a major update to the papillomavirus sequence database , 2016, Nucleic Acids Res..

[42]  R. Moll,et al.  The human keratins: biology and pathology , 2008, Histochemistry and Cell Biology.

[43]  J. Mellors,et al.  Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART , 2017, Proceedings of the National Academy of Sciences.

[44]  Huanming Yang,et al.  Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells , 2015, GigaScience.