Efficacy of Topically Administered Dihydroartemisinin in Treating Papillomavirus-Induced Anogenital Dysplasia in Preclinical Mouse Models.

The artemisinin family of compounds is cytopathic in certain cancer cell lines that are positive for human papillomaviruses (HPV) and can potentially drive the regression of dysplastic lesions. We evaluated the efficacy of topical dihydroartemisinin (DHA) on cervical dysplasia and anal dysplasia in two papillomavirus mouse models: K14E6/E7 transgenic mice, which express HPV16 oncogenes; and immunodeficient NOD/SCID gamma (NSG) mice infected with Mus musculus papillomavirus (MmuPV1). Mice started treatment with DHA at 25 weeks of age (K14E6/E7) or 20 weeks post infection (MmuPV1-infected), when the majority of mice are known to have papillomavirus-induced low- to high-grade dysplasia. Mice were treated with or without topical DHA at the cervix or anus and with or without topical treatment with the chemical carcinogen 7,12 dimethylbenz(a)anthracene (DMBA) at the anus of in transgenic mice to induce neoplastic progression. Mice were monitored for overt tumor growth, and tissue was harvested after 20 weeks of treatment and scored for severity of histological disease. For MmuPV1-infected mice, anogenital lavages were taken to monitor for viral clearance. Tissues were also evaluated for viral gene expression at the RNA and/or protein levels. Treatment with topical DHA did not reduce dysplasia in the anogenital tract in either papillomavirus-induced mouse model and did not prevent progression to anal cancer in the DMBA-treated K14E6/E7 mice.

A Novel Model for Papillomavirus-Mediated Anal Disease and Cancer Using the Mouse Papillomavirus.

Up to 95% of all anal cancers are associated with infection by human papillomavirus (HPV); however, no established preclinical model exists for high-grade anal disease and cancer mediated by a natural papillomavirus infection. To establish an infection-mediated model, we infected both immunocompromised NSG and immunocompetent FVB/NJ mice with the recently discovered murine papillomavirus MmuPV1, with and without the additional cofactors of UV B radiation (UVB) and/or the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Infections were tracked via lavages and swabs for MmuPV1 DNA, and pathology was assessed at the endpoint. Tissues were analyzed for biomarkers of viral infection and papillomavirus-mediated disease, and the localization of viral infection was investigated using biomarkers to characterize the anal microanatomical zones. IMPORTANCE We show, for the first time, that MmuPV1 infection is sufficient to efficiently mediate high-grade squamous intraepithelial lesions in the anal tract of mice using the NSG immunocompromised strain and that MmuPV1, in combination with the chemical carcinogen DMBA, has carcinogenic potential. We further show that MmuPV1 is able to persist for up to 6 months in the anal tract of FVB/NJ mice irradiated with UVB and contributes to high-grade disease and cancer in an immunocompetent strain. We demonstrate that MmuPV1 preferentially localizes to the anal transition zone and that this localization is not an artifact of infection methodology. This study presents a valuable new preclinical model for studying papillomavirus-mediated anal disease driven by a natural infection.

Activating Mutations in Pik3ca Contribute to Anal Carcinogenesis in the Presence or Absence of HPV-16 Oncogenes.

PURPOSE: Over 95% of human anal cancers are etiologically associated with high-risk HPVs, with HPV type 16 (HPV16) the genotype most commonly found. Activating mutations in the catalytic subunit of Phosphatidylinositol (3,4,5)-trisphosphate kinase (PI3K), encoded by the Pik3ca gene, are detected in approximately 20% of human anal cancers.Experimental Design: We asked if common activating mutations in Pik3ca contribute to anal carcinogenesis using an established mouse model for anal carcinogenesis in which mice are topically treated with the chemical carcinogen 7,12-Dimethylbenz(a)anthracene (DMBA). Mice expressing in their anal epithelium one of two activating mutations in Pik3ca genes, Pik3caH1047R or Pik3caE545K , were monitored for anal carcinogenesis in the presence or absence of transgenes expressing the HPV16 E6 and E7 oncogenes. RESULTS: Both mutant forms of Pik3ca increased susceptibility to anal carcinogenesis in the absence of HPV16 oncogenes, and cooperated with HPV16 oncogenes to induce the highest level and earliest onset of anal cancers. The combination of HPV16 oncogenes and Pik3ca mutations led to anal cancers even in the absence of treatment with DMBA. We further observed that the investigational mTOR1/2 dual inhibitor, TAK-228, significantly reduced the size of anal cancer-derived tumor spheroids in vitro and reduced the growth rates of anal cancer-derived tumor grafts in vivo. CONCLUSIONS: These data demonstrate that activating mutations in Pik3ca drive anal carcinogenesis together with HPV16 oncogenes, and that the PI3K/mTOR pathway is a relevant target for therapeutic intervention.

High incidence of HPV-associated head and neck cancers in FA deficient mice is associated with E7's induction of DNA damage through its inactivation of pocket proteins.

Fanconi anemia (FA) patients are highly susceptible to solid tumors at multiple anatomical sites including head and neck region. A subset of head and neck cancers (HNCs) is associated with 'high-risk' HPVs, particularly HPV16. However, the correlation between HPV oncogenes and cancers in FA patients is still unclear. We previously learned that FA deficiency in mice predisposes HPV16 E7 transgenic mice to HNCs. To address HPV16 E6's oncogenic potential under FA deficiency in HNCs, we utilized HPV16 E6-transgenic mice (K14E6) and HPV16 E6/E7-bi-transgenic mice (K14E6E7) on genetic backgrounds sufficient or deficient for one of the fanc genes, fancD2 and monitored their susceptibility to HNCs. K14E6 mice failed to develop tumor. However, E6 and fancD2-deficiency accelerated E7-driven tumor development in K14E6E7 mice. The increased tumor incidence was more correlated with E7-driven DNA damage than proliferation. We also found that deficiency of pocket proteins, pRb, p107, and p130 that are well-established targets of E7, could recapitulate E7's induction of DNA damage. Our findings support the hypothesis that E7 induces HPV-associated HNCs by promoting DNA damage through the inactivation of pocket proteins, which explains why a deficiency in DNA damage repair would increase susceptibility to E7-driven cancer. Our results further demonstrate the unexpected finding that FA deficiency does not predispose E6 transgenic mice to HNCs, indicating a specificity in the synergy between FA deficiency and HPV oncogenes in causing HNCs.

Requirement for stromal estrogen receptor alpha in cervical neoplasia.

The major etiological factor for cervical cancer is the high-risk human papillomavirus (HPV), which encodes E6 and E7 oncogenes. However, HPV is not sufficient, and estrogen has been proposed as an etiological cofactor for the disease. Its requirement has been demonstrated in mouse models for HPV-associated cervical cancer (e.g., K14E7 transgenic mice). Although germline knockout of estrogen receptor alpha (ERα) renders mice resistant to cervical cancer, the cell-type-specific requirement for ERα is not known. In this study, we demonstrate that temporal deletion of stromal ERα induced complete regression of cervical dysplasia in K14E7 mice. Our results strongly support the hypothesis that stromal ERα is necessary for HPV-induced cervical carcinogenesis and implicate paracrine mechanisms involving ERα signaling in the development of estrogen-dependent cervical cancers. This is the first evidence to support the importance of stromal ERα in estrogen-dependent neoplastic disease of the female reproductive tract.

Inactivating all three rb family pocket proteins is insufficient to initiate cervical cancer.

Human papillomavirus-16 (HPV-16) is associated etiologically with many human cervical cancers. It encodes 3 oncogenes E5, E6, and E7. Of these oncogenes, E7 has been found to be the dominant driver of cervical cancer in mice. More than 100 cellular proteins have been reported to associate with HPV-16 E7, which is thought to dysregulate the cell cycle in part by binding and inducing the degradation of pRb and its related pocket protein family members, p107 and p130. The ability of E7 to inactivate the pRb family correlates with its ability to induce head and neck cancers in mice. We previously showed that the inactivation of pRb is itself not sufficient to recapitulate the oncogenic properties of E7 in cervical carcinogenesis. In this study, we evaluated mice that were deficient in multiple pocket proteins, including mice that lacked pRb, p107, and p130. Strikingly, combined loss of two or all 3 pocket proteins resulted in development of high-grade cervical intraepithelial neoplasia, but not frank cervical carcinoma. These findings strongly argue that the oncogenic properties of HPV-16 E7 in human cervical carcinogenesis may involve disruption of E7 binding proteins beyond simply the pRb family members.

Pocket proteins suppress head and neck cancer.

Head and neck squamous cell carcinomas (HNSCC) is a common cancer in humans long known to be caused by tobacco and alcohol use, but now an increasing percentage of HNSCC is recognized to be caused by the same human papillomaviruses (HPV) that cause cervical and other anogenital cancers. HPV-positive HNSCCs differ remarkably from HPV-negative HNSCCs in their clinical response and molecular properties. From studies in mice, we know that E7 is the dominant HPV oncoprotein in head and neck cancer. E7 is best known for its ability to inactivate pRb, the product of the retinoblastoma tumor susceptibility gene. However, loss of pRb function does not fully account for potency of E7 in causing head and neck cancer. In this study, we characterized the cancer susceptibility of mice deficient in the expression of pRb and either of two related "pocket" proteins, p107 and p130, that are also inactivated by E7. pRb/p107-deficient mice developed head and neck cancer as frequently as do HPV-16 E7 transgenic mice. The head and neck epithelia of the pRb/p107-deficient mice also displayed the same acute phenotypes and biomarker readouts as observed in the epithelia of E7 transgenic mice. Mice deficient for pRb and p130 in their head and neck epithelia showed intermediate acute and tumor phenotypes. We conclude that pRb and p107 act together to efficiently suppress head and neck cancer and are, therefore, highly relevant targets of HPV-16 E7 in its contribution to HPV-positive HNSCC.

Insertion and deletion mutagenesis by overlap extension PCR.

Mutagenesis by the overlap extension PCR has become a standard method of creating mutations including substitutions, insertions, and deletions. Nonetheless, the established overlap PCR mutagenesis is limited in many respects. In particular, it has been difficult to make an insertion larger than 30 nt, since all sequence alterations must be embedded within the primer. Here, we describe a rapid and efficient method for creating insertions or deletions of any length at any position in a DNA molecule. This method is generally applicable, and therefore represents a significant improvement to the now widely used overlap extension PCR method.

Human papillomavirus type 16 E6 and E7 oncoproteins act synergistically to cause head and neck cancer in mice.

High-risk human papillomaviruses (HPVs) contribute to cervical and other anogenital cancers, and they are also linked etiologically to a subset of head and neck squamous cell carcinomas (HNSCC). We previously established a model for HPV-associated HNSCC in which we treated transgenic mice expressing the papillomaviral oncoproteins with the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO). We found that the HPV-16 E7 oncoprotein was highly potent in causing HNSCC, and its dominance masked any potential oncogenic contribution of E6, a second papillomaviral oncoprotein commonly expressed in human cancers. In the current study, we shortened the duration of treatment with 4-NQO to reduce the incidence of cancers and discovered a striking synergy between E6 and E7 in causing HNSCC. Comparing the oncogenic properties of wild-type versus mutant E6 genes in this model for HNSCC uncovered a role for some but not other cellular targets of E6 previously shown to contribute to cervical cancer.

Human papillomavirus E7 oncoprotein overrides the tumor suppressor activity of p21Cip1 in cervical carcinogenesis.

The E7 oncoprotein of the high-risk human papillomaviruses (HPV) is thought to contribute to cervical carcinogenesis at least in part by abrogating cell cycle regulation. E7 can dysregulate the cell cycle through its interaction with several cellular proteins including the retinoblastoma suppressor protein pRb, as well as the cyclin-dependent kinase inhibitor p21(Cip1). Inactivation of pRb in cervical epithelia is not sufficient to explain the ability of E7 to cause cervical cancers in transgenic mice. In the current study, we focused on the role of p21(Cip1) in cervical cancer. Cervical disease was significantly increased in p21(-/-) mice compared with p21(+/+) mice, showing that p21(Cip1) can function as a tumor suppressor in this tissue. Importantly, the ability of E7 to induce cervical cancers was not significantly enhanced on the p21-null background, consistent with the hypothesis that the ability of E7 to inhibit p21(Cip1) contributes to its carcinogenic properties. Further supportive of this hypothesis, cervical carcinogenesis in mice expressing a mutant form of HPV-16 E7, E7(CVQ), which fails to inactivate p21(Cip1), was significantly reduced compared with that in K14E7(WT) mice expressing wild-type HPV-16 E7. However, K14E7(CVQ) mice still displayed heightened levels of cervical carcinogenesis compared with that in nontransgenic mice, indicating that activities of E7 besides its capacity to inactivate p21(Cip1) also contribute to cervical carcinogenesis. Taken together, we conclude that p21(Cip1) functions as a tumor suppressor in cervical carcinogenesis and that p21(Cip1) inactivation by HPV-16 E7 partially contributes to the contribution of E7 to cervical carcinogenesis.

Polyadenylation is dispensable for encapsidation and reverse transcription of hepatitis B viral pregenomic RNA.

A hepadnaviruses replicates its DNA genome via reverse transcription of an RNA template (pregenomic RNA or pgRNA), which has a cap structure at the 5' end and a poly(A) tail at the 3' end. We have previously shown that the 5' cap is indispensable for encapsidation of the pgRNA. A speculative extension of the above finding is that the cap contributes to encapsidation via its interaction with the poly(A) tail, possibly involving eIF4E-eIF4G-PABP interaction. To test this hypothesis, poly(A)-less pgRNAs were generated via cleavage by a cis-acting hepatitis delta virus ribozyme sequence. We found that accumulation of the poly(A)-less pgRNA was markedly diminished, mostly likely due to its reduced stability. Importantly, however, the remaining poly(A)-less pgRNAs were nonetheless encapsidated and reverse transcribed normally when the reduced stability was taken account. Our finding clearly contradicts the notion that the poly(A) tail has any function in encapsidation and viral reverse transcription.

Circularization of an RNA template via long-range base pairing is critical for hepadnaviral reverse transcription.

Although an overall genetic strategy for hepadnaviral reverse transcription has been established, the mechanism that underlies the minus-strand transfer is still poorly defined. We and others independently identified a novel cis-acting element, termed beta or varphi, respectively, that is critical for the minus-strand DNA synthesis of hepatitis B virus. A 5'-3', long-range interaction of the RNA template was proposed that involves the 5' epsilon sequence (encapsidation signal) and the 3' beta/varphi sequence. We subjected the hypothesized base pairing to genetic analysis. The data indicated that mutations abrogating the hypothesized base pairing markedly impaired minus-strand DNA synthesis, while compensatory mutations that restored the base pairing rescued the minus-strand DNA synthesis. These results demonstrated the critical role of the 5'-3', long-range interaction in minus-strand DNA synthesis. We speculate that such a long-range interaction may precisely juxtapose a donor to an acceptor during minus-strand transfer.

Three novel cis-acting elements required for efficient plus-strand DNA synthesis of the hepatitis B virus genome.

Synthesis of the relaxed-circular (RC) DNA genomes of hepadnaviruses by reverse transcriptase involves two template switches during plus-strand DNA synthesis. These template switches require repeat sequences (so-called donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. To determine cis-acting elements apart from the donor and acceptor sites that are required for plus-strand RC DNA synthesis by hepatitis B virus (HBV), a series of mutants bearing a small deletion were made and analyzed for their impact on the viral genome synthesis. We found three novel cis-acting elements in the HBV genome: one element, located in the middle of the minus strand, is indispensable, whereas the other two elements, located near either end of the minus strand, contribute modestly to the plus-strand RC DNA synthesis. The data indicated that the first element facilitates plus-strand RNA primer translocation or subsequent elongation during plus-strand RC DNA synthesis, while the last two elements, although distantly located on the minus strand, act at multiple steps to promote plus-strand RC DNA synthesis. The necessity of multiple cis-acting elements on the minus-strand template reflects the complex nature of hepadnavirus reverse transcription.

A novel cis-acting element facilitates minus-strand DNA synthesis during reverse transcription of the hepatitis B virus genome.

Hepadnaviruses replicate through reverse transcription of an RNA pregenome, resulting in a relaxed circular DNA genome. The first 3 or 4 nucleotides (nt) of minus-strand DNA are synthesized by the use of a bulge in a stem-loop structure near the 5' end of the pregenome as a template. This primer is then transferred to a complementary UUCA motif, termed an acceptor, within DR1* near the 3' end of the viral pregenome via 4-nt homology, and it resumes minus-strand DNA synthesis: this process is termed minus-strand transfer or primer translocation. Aside from the sequence identity of the donor and acceptor, little is known about the sequence elements contributing to minus-strand transfer. Here we report a novel cis-acting element, termed the beta5 region (28 nt in length), located 20 nt upstream of DR1*, that facilitates minus-strand DNA synthesis. The deletion or inversion of the sequence including the beta5 region diminished minus-strand DNA synthesis initiated at DR1*. Furthermore, the insertion of the beta5 region into its own position in a mutant in which the sequences including the beta5 region were replaced restored minus-strand DNA synthesis at DR1*. We speculate that the beta5 region facilitates minus-strand transfer, possibly by bringing the acceptor site in proximity to the donor site via base pairing or by interacting with protein factors involved in this process.