We have recently identified that homocysteinylation of heterogeneous nuclear ribonucleoprotein-E1 (hnRNP-E1) during physiological folate deficiency, activates a nutrition-sensitive posttranscriptional RNA operon that also includes an important interaction with two loci in Human Papillomavirus type-16 (HPV16) RNA. This HPV16 RNA-protein interaction with homocysteinylated-hnRNP-E1 led to a profound perturbation in the generation of both HPV16 major (L1) and minor (L2) viral capsid proteins in vitro; in HPV16-harboring keratinocytes that were propagated as monolayers; as well as when these HPV16-keratinocytes were developed into organotypic rafts in physiologic low-folate medium. Despite a similar HPV16 DNA viral load in HPV16-high folate- and low folate-organotypic rafts, the latter contained a high-level of integration of HPV16 DNA into genomic DNA. Subcutaneous implantation of 18-day HPV16-low folate-organotypic rafts in Beige Nude XID immunodeficient mice led to an aggressive HPV16-induced cancer within 12 weeks. Thus, we have developed a new model of HPV16-induced carcinogenesis within a time frame of less than 4 months. Because folate deficiency can induce single-strand nicks in genomic DNA and also double-strand DNA fragmentation, our overarching hypothesis is that the unstable genomic DNA in HPV16-low folate-organotypic rafts is primarily responsible for the high-level integration of HPV16 DNA into genomic DNA and transformation of this benign tissue to cancer. So we will test various aspects of this hypothesis using three specific aims:
In Specific Aim #1, we will characterize the time-course relationship, extent, and consequence of integration of HPV16 DNA into the genomic DNA of HPV16-organotypic rafts in vitro and in vivo, and assess the frequency of transformation of benign HPV16-organotypic rafts into cancer in immunodeficient mice.
In Specific Aim #2 we will assess the potential of amplified 'capsid-less' HPV16 DNA to integrate into unperturbed and intact [stable] genomic DNA after transduction and expression of novel genes encoding various strengths of molecular mimics of homocysteinylated-hnRNP-E1 into HPV16-harboring keratinocytes that are subsequently developed into organotypic rafts under high-folate conditions.
In Specific Aim #3, we will assess the permissiveness of unperturbed and transiently perturbed genomic DNA to integrate HPV16 DNA in AAV2-transduced [high folate] HPV16-organotypic rafts that either do or do not contain an abundance of 'capsid-less' HPV16 DNA, and then evaluate the potential for such genomic HPV16 DNA integration to induce carcinogenesis within implants of rafts in Beige Nude XID mice. Such investigations will ultimately provide a better understanding of the mechanism of transformation of HPV16-infected tissues to cancer and benefit HPV-infected elderly Veterans with poor nutrition; Veterans with HPV16 and human immunodeficiency virus (HIV); and those in developing countries where the combination of poor nutrition, and co-infection with HIV and HPV16 places individuals at high risk for HPV-induced cancers
to Veterans Health: The potential significance of these new studies to Veteran's Health is that they will set the stage for a better understanding of the mechanism of how HPV16 transforms normal cells to cancer. Specifically, these studies will clarify whereby folate or vitamin-B12 -deficiency acts as a co-factor in transforming HPV16-infected cells to cancer and thereby provide new leads to prevent these cancers in patients at risk. Because up to 25,000 cases of HPV-induced cancers occur in the USA, this grant proposal is highly relevant to Veterans Health Care.