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Fanconi anemia deficiency stimulates HPV-associated hyperplastic growth in organotypic epithelial raft culture

Oncogene volume 28, pages 674–685 (2009)Cite this article

Abstract

Fanconi anemia (FA) is a recessive genome instability syndrome characterized by heightened cellular sensitivity to DNA damage, aplastic anemia and cancer susceptibility. Leukemias and squamous cell carcinomas (SCCs) are the most predominant FA-associated cancers, with the latter exhibiting markedly early disease onset and aggressiveness. Although studies of hematopoietic cells derived from FA patients have provided much insight into bone marrow deficiencies and leukemogenesis, molecular transforming events in FA-deficient keratinocytes, which are the cell type of origin for SCC, are poorly understood. We describe here the growth and molecular properties of FANCA-deficient versus FANCA-corrected HPV E6/E7 immortalized keratinocytes in monolayer and organotypic epithelial raft culture. In response to DNA damage, FANCA-deficient patient-derived keratinocyte cultures displayed a G2/M phase arrest, senescence and apoptosis. Organotypic raft cultures exhibited DNA repair-associated defects with more 53BP1 foci and TdT-mediated dNTP nick end labeling-positive cells over their corrected counterparts. Interestingly, together with reduced rates of DNA damage, FA correction resulted in a marked decrease in epithelial thickness and the presence of fewer cell layers. The observed FANCA-mediated suppression of hyperplasia correlated with the detection of fewer cells transiting through the cell cycle in the absence of gross differentiation abnormalities or apoptotic differences. Importantly, the knockdown of either FANCA or FANCD2 in HPV-positive keratinocytes was sufficient for increasing epithelial hyperplasia. Our findings support a new role for FA pathways in the maintenance of differentiation-dependent cell cycle exit, with the implication that FA deficiencies may contribute to the high risk of FA patients for developing HPV-associated SCC.

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Acknowledgements

We thank Paul Andreassen, Ruhikanta Meetei, Qishen Pang, James Mulloy, Harmut Geiger, Yi Zheng and Stella Davies for helpful discussions and critical comments on the manuscript. We thank members of the Fanconi Anemia Comprehensive Care Center (FACCC), particularly Lars Wagner, Richard Harris and Robin Mueller for human tissue samples, as well as Susan Radtke for regulatory support. We thank the patients of the FACCC for samples. We are grateful to Denis Lee and Paul Lambert for expert advice on the organotypic raft system, as well as to Maureen Hoatlin for the FANCA and to James Lessard for the monoclonal actin antibody. We thank Christopher Baum for the retroviral SF91 expression vector, and Neeraj Singh for generation of the SF91-18E6/E7 retroviral construct. We thank the flow cytometry core facility and Dan Marmer for assistance with flow sorting. Vector supernatant was provided by the CCHMC Viral Vector Core and Translational Trials Development and Support Lab. This research was supported by Public Health Service Grant CA102357, a grant from the Translational Research Initiative at CCHMC and a grant from the Fanconi Anemia Research Fund to SIW. SD and NS are supported by NIH/NCI R01 CA112598. DAW was supported by Public Health Service Grant HL081499. KAW-B was supported by HL079193.

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Authors and Affiliations

  1. Division of Hematology/Oncology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    E E Hoskins, T A Morris, J M Higginbotham & S I Wells

  2. Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    N Spardy, E Cha & S Duensing

  3. Wyeth Pharmaceuticals, Cambridge, MA, USA

    P Kelly

  4. Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA

    D A Williams

  5. Division of Pathology and Laboratory Medicine and Pulmonary Biology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    K A Wikenheiser-Brokamp

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  1. E E Hoskins
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Correspondence to S I Wells.

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Hoskins, E., Morris, T., Higginbotham, J. et al. Fanconi anemia deficiency stimulates HPV-associated hyperplastic growth in organotypic epithelial raft culture. Oncogene 28, 674–685 (2009). https://doi.org/10.1038/onc.2008.416

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