Folate Deficiency Facilitates Genomic Integration of Human Papillomavirus Type 16 DNA In Vivo in a Novel Mouse Model for Rapid Oncogenic Transformation of Human Keratinocytes

Abstract

Background Epidemiologic and in vitro studies suggest independent linkages between poor folate and/or vitamin B-12 nutrition, genomic human papillomavirus (HPV) type 16 viral integration, and cancer. However, there is no direct evidence in vivo to support the causative role of poor folate nutrition in HPV16 integration into the cellular genome. Objective We tested the hypothesis that folate deficiency enables the integration of HPV16 into the genome of HPV16-harboring keratinocytes, and could thereby influence earlier transformation of these cells to cancer in an animal model. Methods HPV16-harboring human keratinocytes [(HPV16)BC-1-Ep/SL] were differentiated into 3-dimensional HPV16-organotypic rafts under either folate-replete or folate-deficient conditions in vitro. These were then subcutaneously implanted in severely immunocompromised female Beige Nude XID (Hsd: NIHS-LystbgFoxn1nuBtkxid) mice (4–6 wk old, 16–18 g) fed either a folate-replete diet (1200 nmol folate/kg diet) or a progressively folate-deficient diet (600 or 400 nmol folate/kg diet) for 2 mo prior to raft-implantation surgery, and indefinitely thereafter. The tumors that subsequently developed were characterized for onset, pattern of growth, morphology, HPV16 oncogene expression, and HPV16-genomic integration. Results All HPV16-organotypic rafts developed in either folate-replete or physiologic low-folate media in vitro and subsequently implanted in folate-replete mice eventually transformed into aggressive malignancies within weeks. When compared to HPV16-high folate-organotypic raft-derived tumors from mice fed either a 1200 or 600 nmol folate/kg diet, those raft-derived cancers that developed in mice fed a 400 nmol folate/kg diet expressed significantly more HPV16 E6 (1.8-fold more) and E7 (2.8-fold more) oncogenic proteins (P = 0.001), and revealed significantly more HPV16-integration sites in genomic DNA (2-fold more), either directly into, or in the vicinity of, cellular genes (P < 0.05). Conclusions This unprecedented animal model for the consistent rapid transformation of differentiated (HPV16)BC-1-Ep/SL-derived organotypic raft-keratinocytes to cancer in Beige Nude XID mice confirms that dietary folate deficiency can profoundly influence and modulate events leading to HPV16-induced carcinogenesis, and facilitates genomic integration of HPV16 DNA in vivo.