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1.  Genetic variation in the TLR and NF-κB genetic pathways and cervical and vulvar cancer risk: a population-based case-control study 
Genital infection with the oncogenic human papillomavirus (HPV) is the necessary cause of cervical cancer and of a large fraction of vulvar cancers. The toll-like receptor (TLR) and the nuclear factor κB (NF-κB) signaling pathways have been implicated in inflammation, autoimmune disease and cancer, but whether common nucleotide variation in these pathways is associated with the risk of cervical and vulvar cancers has received little study. Using data from a population-based case-control study of cervical and vulvar cancers, we genotyped 205 single nucleotide polymorphisms (SNPs) in and around 32 candidate gene regions within these pathways. Gene-based analyses were employed to estimate the associations between individual gene regions and the risk of cervical and vulvar cancers. Odds ratio (OR) and 95% confidence intervals (CI) were calculated to assess the risk of cervical and vulvar cancers for each SNP. P-values were adjusted for multiple testing. A total of 876 cervical cancer cases, 517 vulvar cancer cases and 1,100 controls were included in the analysis. The TNF region was significantly associated with the risks of cervical cancer (gene-based P-value: 2.0×10−4) and vulvar cancer (gene-based P-value: 1.0×10−4). The rare allele (A) of SNP rs2239704 in the 5′ UTR of the LTA gene was significantly associated with increased risks of cervical cancer (OR=1.31, 95% CI: 1.15–1.50; adjusted P-value: 0.013) and vulvar cancer (OR=1.51, 95% CI: 1.30–1.75; adjusted P-value: 1.9×10−5). These findings add to the evidence of the importance of the immune system in the etiology of cervical and vulvar cancers.
PMCID: PMC3883910  PMID: 23824834
cervical cancer; vulvar cancer; toll-like receptor; nuclear factor-κB; tumor necrosis factor
2.  Cohort Profile: The Skin Cancer After Organ Transplant Study 
The Skin Cancer after Organ Transplant (SCOT) study was designed to investigate the link between genus beta human papillomavirus (HPV) and squamous cell skin cancer (SCSC). We focused on a population receiving immunosuppressive therapy for extended periods, transplant patients, as they are at extremely high risk for developing SCSC. Two complementary projects were conducted in the Seattle area: (i) a retrospective cohort with interview data from 2004 recipients of renal or cardiac transplants between 1995 and 2010 and (ii) a prospective cohort with interview data from 328 people on the transplant waiting lists between 2009 and 2011. Within the retrospective cohort, we developed a nested case–control study (172 cases and 337 control subjects) to assess risk of SCSC associated with markers of HPV in SCSC tumour tissue and eyebrow hair bulb DNA (HPV genotypes) and blood (HPV antibodies). In the prospective cohort, 135 participants had a 1-year post-transplant visit and 71 completed a 2-year post-transplant visit. In both arms of the cohort, we collected samples to assess markers of HPV infection such as acquisition of new types, proportion positive for each type, persistence of types at consecutive visits and number of HPV types detected. In the prospective cohort, we will also examine these HPV markers in relation to levels of cell-mediated immunity. The goal of the SCOT study is to use the data we collected to gain a more complete understanding of the role of immune suppression in HPV kinetics and of genus beta HPV types in SCSC. For more information, please contact the principal investigator through the study website:
PMCID: PMC3887562  PMID: 23171871
3.  Beta Human Papillomavirus E6 Expression Inhibits Stabilization of p53 and Increases Tolerance of Genomic Instability 
Journal of Virology  2014;88(11):6112-6127.
Infections with the beta genus of human papillomaviruses (β-HPVs) may contribute to the development of nonmelanoma skin cancers. However, β-HPV genomes are found at too low a copy number in tumors for the virus to be necessary for tumor maintenance. Instead, they are hypothesized to destabilize the host genome by allowing the persistence of mutations that can drive tumorigenesis independently of the viral genome. Supporting this premise is our previous finding that the expression of some β-HPV E6 proteins can attenuate p53 signaling in response to DNA damage. We show that β-HPV E6 proteins can prevent the stabilization of p53 in response to two types of genome-destabilizing events, aberrant mitosis and dysregulated centrosome duplication. The inability to stabilize p53 in response to these stimuli allows cells expressing HPV5, HPV8, or HPV38 E6 to remain proliferatively active, leading to further genome deterioration in a proportion of these cells. These phenotypes are lost by the introduction of a mutation into the p300 binding domain of HPV8 E6 or by the transfection of mutated p300 that is resistant to the degradation mediated by HPV5 or HPV8 E6. These findings expand the understanding of the role played by p300 in promoting the faithful resolution of mitotic figures as well as proper centrosome duplication. Finally, we describe a phenomenon by which binucleated cells are resolved via cytokinesis into two cells, each with one nucleus. These data support the hypothesis that β-HPV infections may promote tumorigenesis via genome destabilization.
IMPORTANCE The work described in this report provides support for the hypothesis that β-HPV infections may contribute to nonmelanoma skin cancer by increasing the likelihood that tumorigenic mutations are introduced into the host cell's genome. We demonstrate that expression of the E6 proteins from some of these viruses increases the tolerance of two genome-destabilizing events, aberrant cell division and dysregulated centrosome duplication. Typically, these mutagenic occurrences elicit the stabilization of the tumor suppressor p53, which prevents further propagation of cells containing these errors. We show that the expression of β-HPV E6 restricts this stabilization of p53, leading not only to continued cellular proliferation but also to further accumulation of similar mutagenic events. Finally, in addition to supporting a role for β-HPV infections in certain skin cancers, we present studies with a mutated β-HPV E6 protein suggesting that the histone acetyltransferase p300 plays a role in promoting genome stability during replication.
PMCID: PMC4093859  PMID: 24648447
4.  An unbiased in vivo screen reveals multiple transcription factors that control HPV E6-regulated hTERT in keratinocytes 
Virology  2013;446(0):17-24.
Activation of telomerase by human papillomavirus 16 (HPV16) E6 is a critical step for cell immortalization and transformation in human foreskin keratinocytes (HFKs). Multiple transcription factors have been identified as being involved in E6-induced hTERT expression. Here, we adapted an unbiased in vivo screen using a LacO-LacI system in human cells to discover hTERT promoter-interacting regulators. This approach allowed us to identify a novel hTERT repressor, Maz, which bound the hTERT promoter. E6 expression reduced Maz binding and correspondingly increased Sp1 binding at the hTERT promoter. Knockdown of Maz further increased histone acetylation, as well as hTERT expression in the presence of E6. Overall, these data indicate the utility of a novel screen for promoter-interacting and transcription-regulating proteins. These data also highlight multiple factors that normally regulate hTERT repression in HFKs, and therefore are targeted by E6 for hTERT expression.
PMCID: PMC3787310  PMID: 24074563
HPV E6; hTERT; Keratinocytes; Maz; Sp1
5.  Characteristics of Memory B Cells Elicited by a Highly Efficacious HPV Vaccine in Subjects with No Pre-existing Immunity 
PLoS Pathogens  2014;10(10):e1004461.
Licensed human papillomavirus (HPV) vaccines provide near complete protection against the types of HPV that most commonly cause anogenital and oropharyngeal cancers (HPV 16 and 18) when administered to individuals naive to these types. These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies. However, almost nothing is known about the immunological memory that forms following HPV vaccination, which is required for long-term immunity. Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells. Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation. These findings suggest that the quadrivalent HPV vaccine provides an excellent model for studying the development of B cell memory; and, in the context of what is known about memory B cells elicited by influenza vaccination/infection, HIV-1 infection, or tetanus toxoid vaccination, indicates that extensive somatic hypermutation is not required to achieve potent vaccine-specific neutralizing antibody responses.
Author Summary
There is an urgent need to better understand how to reliably generate effective vaccines, particularly subunit vaccines, as certain pathogens are considered to pose too great of a safety risk to be developed as live, attenuated or killed vaccines (e.g., HIV-1). The human papillomavirus (HPV) vaccines are two of the most effective subunit vaccines ever developed and have continued to show protection against HPV associated disease up to and beyond five years post-vaccination. Moreover, the target population for these vaccines have essentially no pre-existing immunity to the HPV types covered by the vaccine; therefore, these vaccines provide an excellent model for studying the immunity elicited by a highly effective subunit vaccine. As the HPV vaccines, like most vaccines, protect by generating antibodies, we are interested in characterizing the memory B cells elicited by the HPV vaccine. Memory B cells help to sustain antibody levels over time by rapidly differentiating into antibody secreting cells upon pathogen re-exposure. Although previous studies have provided evidence that the HPV vaccines elicit memory B cells, they did not characterize these cells. Here, we have isolated HPV-specific memory B cells from adolescent females and women who received the quadrivalent HPV vaccine and have cloned antibodies from these cells. Importantly, we find that these antibodies potently inhibit HPV and that the memory B cells from which they derive exhibit hallmarks of long-lived memory B cells.
PMCID: PMC4199765  PMID: 25330199
6.  Risk of squamous cell skin cancer after organ transplant associated with antibodies to cutaneous papillomaviruses, polyomaviruses, and TMC6/8 (EVER1/2) variants 
Cancer Medicine  2014;3(5):1440-1447.
Squamous cell skin cancer (SCSC) disproportionately affects organ transplant recipients, and may be related to increased viral replication in the setting of immune suppression. We conducted a nested case–control study among transplant recipients to determine whether SCSC is associated with antibodies to cutaneous human papillomaviruses (HPV), to genes associated with a rare genetic susceptibility to HPV (TMC6/TMC8), or to human polyomaviruses (HPyV). Cases (n = 149) had histologically confirmed SCSC, and controls (n = 290) were individually matched to cases on time since transplant, type of transplant, gender, and race. All subjects had serum drawn immediately prior to transplant surgery. Antibodies to 25 cutaneous HPVs and six HPyVs were assayed by detection of binding to virus-like particles, and 11 TMC6/8 variants were genotyped. After correction for multiple comparisons, only antibodies to HPV37 were associated with SCSC (OR 2.0, 95% CI 1.2–3.4). Common genetic variants of TMC6/8 were not associated with SCSC, but three variants in TMC8 (rs12452890, rs412611, and rs7208422) were associated with greater seropositivity for species 2 betapapillomaviruses among controls. This study suggests that some betaHPVs, but not polyomaviruses, may play a role in the excess risk of SCSC among transplant recipients.
PMCID: PMC4302694  PMID: 24913986
Cutaneous human papillomavirus; epidemiology; organ transplant; polyomavirus; squamous cell skin cancer
7.  HPV 5 and 8 E6 Expression Reduces ATM Protein Levels and Attenuates LINE-1 Retrotransposition 
Virology  2013;443(1):10.1016/j.virol.2013.04.022.
The expression of the E6 protein from certain members of the HPV genus β (β HPV 5 and 8 E6) can disrupt p53 signaling by diminishing the steady state levels of two p53 modifying enzymes, ATR and p300. Here, we show that β-HPV 5 and 8 E6 are also capable of reducing the steady state levels of another p53 modifying enzyme, ATM, and as a result restrict LINE-1 retrotransposition. Furthermore, we show that the reduction of both ATM and LINE-1 retrotransposition is dependent upon the ability of β-HPV 8 E6 to bind and degrade p300. We use inhibitors and dominant negative mutants to confirm that ATM is needed for efficient LINE-1 retrotransposition. Furthermore, neither sensitivity to LINE-1 expression nor LINE-1 induced DSB formation is altered in an ATM deficient background. Together, these data illustrate the broad impact some β-HPVs have on DNA damage signaling by promoting p300 degradation.
PMCID: PMC3873879  PMID: 23706308
Retrotransposition; ATM; LINE-1; beta HPV E6; DNA Damage Repair
8.  Redetection of Cervical Human Papillomavirus Type 16 (HPV16) in Women With a History of HPV16 
The Journal of Infectious Diseases  2013;208(3):403-412.
Background. The purpose of this study was to examine the rate of and risks for cervical human papillomavirus type 16 (HPV16) redetection in women with documented or suspected HPV16 infection.
Methods. A convenience sample of women aged 13–21 years were seen at 4-month intervals for HPV DNA testing and cytology. Serum samples were obtained at baseline and annually.
Results. A total of 1543 women entered the study. Of the 295 women with detection of HPV16 DNA and subsequent clearance, 18.1% had HPV16 redetected by 8.5 years (88% cleared this second detection by 3 years). Of the 247 women who had antibodies to HPV16 and were HPV16 DNA negative at baseline, 15.3% had HPV16 redetected by year 5. Risks for redetection included douching, current use of medroxyprogesterone, reporting >1 sex partner or having a new sex partner, and having a sexually transmitted infection. Development of cervical intraepithelial neoplasia 2/3 was rare in women with redetection, except for those with prevalent HPV16 infection.
Conclusions. Reappearance of HPV16 DNA was observed in 18% of women. Most are associated with sexual exposure and appear benign. Interpretation of the studies is more complex in women with prevalent infections as it appears that this small subset reflects women with persistence already present at entry.
PMCID: PMC3699002  PMID: 23599313
redetection of HPV16; reactivation; risk for acquisition; risk for clearance
9.  Nucleotide variation in IL10 and IL12 and their receptors and cervical and vulvar cancer risk: a hybrid case-parent triad and case-control study 
Given the important role of cell mediated immunity in viral clearance and control of premalignant lesions, we hypothesize that variation in the IL12/IL10 cytokine and cytokine receptor genes may influence cervical and vulvar cancer risk. We evaluated 76 tagSNPs from 7 candidate genes (IL10, IL12A, IL12B, IL10RA, IL10RB, IL12RB1, and IL12RB2) in case-parent sets (n=43 cervical squamous cell carcinoma (SCC), n=96 cervical adenocarcinoma, n=53 vulvar SCC), additional cases (n=356 cervical SCC, n=406 cervical adenocarcinoma, and n=473 vulvar SCC) and population based controls (1,111). We calculated log-additive odds ratios (ORs) and 95% confidence intervals (CIs) for the association between tagSNP and cancer risk using a pseudo-likelihood based method which combined genotype information on cases, parents, and population controls. After correction for multiple comparisons, we identified several statistically significant SNP associations. Cervical SCC risk was associated with the minor alleles of the IL10RA rs9610 3′ UTR SNP (OR=1.76, 95% CI= 1.15–2.68) and two synonymous IL12RB2 SNPs (rs4297265, OR=0.46, 95% CI=0.26–0.82; rs2229546, OR=0.43, 95% CI=0.21–0.87). Cervical adenocarcinoma risk was associated with the minor alleles of the IL10RA rs4252314 intronic SNP (OR=2.23, 95% CI = 1.26–3.96) and IL12RB1 rs11575934 non-synonymous SNP (OR=1.51, 95% CI=1.12–2.05). Finally, the minor allele of the IL12B rs3181224 3′ UTR SNP was associated with a reduced risk of vulvar SCC (OR=0.30, 95% CI=0.12–0.74). These results raise the possibility that a shift in the balance of the immune response due to genetic variants in key cytokine genes could influence the development of cervical and vulvar cancer.
PMCID: PMC3833442  PMID: 23280621
Cervical cancer; vulvar cancer; case-parent; interleukin 12; interleukin 10
10.  Risk of Cervical Cancer Associated with Chlamydia trachomatis Antibodies by Histology, HPV Type, and HPV Cofactors 
Human papillomavirus (HPV) is the central etiologic factor for cervical cancer, and prior studies suggested C. trachomatis may act as an HPV cofactor. We examined the C. trachomatis—cervical cancer association by serotype, histology, HPV type in the tumor, and other HPV cofactors. We conducted a population-based study in the Seattle-Puget Sound area of 302 women with invasive squamous cell carcinomas (SCC), 185 women with adenocarcinomas of the cervix (AC), and 318 HPV seropositive control women. The risk of SCC associated with antibodies to C. trachomatis was increased (OR 1.6, 95% CI 1.1–2.2) but not for AC (OR 1.0, 95% CI 0.6–1.5). This association was independent of HPV type in the SCC tumor tissue. There was an association between specific serotypes of C. trachomatis and SCC for 6 of the 10 serotypes: B (OR 3.6, 95% CI 1.5–8.4), D (OR 2.1, 95% CI 1.2–3.5), E (OR 2.4, 95% CI 1.4–3.9), G (OR 3.0, 95% CI 1.1–7.9), I (OR 4.2, 95% CI 1.5–11.7), and J (OR 2.3, 95% CI 1.0–5.1), but not for the 4 types (C, F, H, and K) that were present at very low prevalence in this population. There was an increased risk of SCC, but not AC, associated with antibodies to C. trachomatis that was not serotype specific.
PMCID: PMC4049152  PMID: 17096345
Chlamydia trachomatis; HPV; cervical cancer; histologic type; microimmunofluorescence
Journal of medical virology  2009;81(4):713-721.
Human papillomavirus (HPV) RNA levels may be a more sensitive early indicator of predisposition to carcinogenesis than DNA levels. We evaluated whether levels of HPV-16 and HPV-18 DNA and messenger RNA (mRNA) in newly detected infections are associated with cervical lesion development. Female university students were recruited from 1990-2004. Cervical samples for HPV DNA, HPV mRNA, and Papanicolaou testing were collected tri-annually, and women were referred for colposcopically-directed biopsy when indicated. Quantitative real-time polymerase chain reaction of L1 and E7 DNA and E7 mRNA was performed on samples from women with HPV-16 and HPV-18 infections that were incidently detected by consensus PCR. Adjusting for other HPV types, increasing E7 cervical HPV-16 mRNA levels at the time of incident HPV-16 DNA detection were associated with an increased risk of cervical intraepithelial neoplasia grade 2 to 3 (HR per 1 log10 increase in mRNA=6.36,95%CI=2.00-20.23). Increasing HPV-16 mRNA levels were also associated with an increased risk of cervical squamous intraepithelial lesions; the risk was highest at the incident positive visit and decreased over time. Neither HPV-16 E7 DNA levels nor HPV-18 E7 DNA nor mRNA levels were significantly associated with cervical lesion development. Report of >1 new partner in the past 8 months (relative to no new partners) was associated with increased HPV mRNA (viral level ratio [VLR]=10.05,95%CI=1.09-92.56) and increased HPV DNA (VLR=16.80,95%CI=1.46-193.01). In newly detected HPV-16 infections, increasing levels of E7 mRNA appear to be associated with an increased risk of developing cervical pre-cancer.
PMCID: PMC3984467  PMID: 19235870
HPV; viral load; mRNA; cervical pre-cancer
12.  A history of allergies is associated with reduced risk of oral squamous cell carcinoma 
Cancer causes & control : CCC  2012;23(12):1911-1919.
A history of allergies is associated with a decreased risk of several types of cancers. Potential mechanisms include enhanced immune surveillance against tumor cells early in disease development and/or carcinogenic infectious agents. We tested whether allergies are inversely associated with oral squamous cell carcinoma (OSCC), accounting for factors that may modify the association, such as tumor site, stage, and HPV infection.
We estimated odds ratios (OR) and 95% confidence intervals (CI) for the association between allergy history (including different types of allergies) and OSCC, adjusted for potential confounders, among 400 cases and 613 controls. Analyses were also stratified by site, stage, and measures of HPV infection.
We observed a weak inverse association between history of any allergy and OSCC (OR=0.81, 95% CI, 0.61–1.08). This association was present only for allergies to airborne allergens (dust/pollen/mold); OR=0.67; 95% CI, 0.48–0.93. The inverse associations with airborne allergies were slightly stronger for oropharyngeal SCC (OR=0.56; 95% CI, 0.35–0.90) than for oral cavity SCC (OR=0.71; 95% CI, 0.49–1.05), and present only for later stage cancers (OR=0.42; 95% CI, 0.26–0.66) as opposed to earlier stage cancers (OR=0.98; 95% CI, 0.66–1.46). Inverse associations were not particularly present or stronger among HPV-16 seropositive individuals or for HPV DNA positive OSCC.
There is an inverse association between history of allergies to dust, pollen or mold and OSCC. Whether the inverse association involves heightened immune surveillance, increased immune response to HPV or other antigen, or other carcinogenic mechanism, remains to be determined in more definitive studies.
PMCID: PMC3499679  PMID: 23011537
allergies; oral squamous cell carcinoma; HPV; HSV
13.  Merkel cell polyomavirus-specific CD8+ and CD4+ T-cell responses identified in Merkel cell carcinomas and blood 
Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCCs) and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy.
Intracellular cytokine cytometry, IFN-γ-ELISPOT, and a novel HLA-A*2402-restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects.
We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6), but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402-restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor-infiltrating lymphocytes as compared to blood, implying intact T-cell trafficking into the tumor. While tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide.
Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients.
PMCID: PMC3207011  PMID: 21908576
14.  No evidence for human papillomavirus in the etiology of colorectal polyps 
While some studies have reported detection of oncogenic human papillomavirus (HPV) in colorectal tumors, others have not.
We examined the association between oncogenic HPV infection and colorectal polyps in a case-control study of individuals with colorectal adenomas (n=167), hyperplastic polyps (n=87), and polyp-free controls (n=250). We performed real-time PCR for HPV-16 /18 DNA, and SPF PCR covering 43 HPV types, on lesional and normal colorectal tissue samples. Plasma antibodies for oncogenic HPV types were assessed via a bead-based multiplex Luminex assay.
HPV DNA was not found in any of the 609 successfully assayed colorectal tissue samples from adenomas, hyperplastic polyps, normal biopsies adjacent to polyps, or normal biopsies of the rectum of disease-free controls. Also, there was no association between HPV seropositivity for all oncogenic HPV types combined, for either polyp type, and for men or women. When analyses were restricted to participants without a previous history of polyps, among men [adenomas (n=31), hyperplastic polyps (n=28), and controls (n=68)], there was an association between seropositivity and hyperplastic polyps when all oncogenic HPV types were combined (odds ratio=3.0; 95% confidence interval: 1.1–7.9).
Overall, our findings do not support an etiologic relationship between HPV and colorectal adenomas or hyperplastic polyps; however, our finding suggesting an association between HPV seropositivity and hyperplastic polyps in men may warrant further investigations.
After stringent controls for contamination and three methods to assess HPV infection, we report no evidence for HPV in the etiology of colorectal neoplasia for either men or women.
PMCID: PMC3236024  PMID: 21817125
HPV; colorectal adenomas; hyperplastic polyps; DNA; antibodies
15.  Gene expression signature of c-MYC-immortalized human fibroblasts reveals loss of growth inhibitory response to TGFβ 
Cell Cycle  2011;10(15):2540-2548.
Cancer cells exhibit the ability to proliferate indefinitely, but paradoxically, overexpression of cellular oncogenes in primary cells can result in a rapid and irreversible cell cycle arrest known as oncogene-induced senescence (OIS). However, we have shown that constitutive overexpression of the oncogene c-MYC in primary human foreskin fibroblasts results in a population of cells with unlimited lifespan; these immortalized cells are henceforth referred to as iMYC. Here, in order to further elucidate the mechanisms underlying the immortalization process, a gene expression signature of three independently established iMYC cell lines compared with matched early passage c-MYC overexpressing cells was derived. Network analysis of this “iMYC signature” indicated that a large fraction of the downregulated genes were functionally connected and major nodes centered around the TGFβ, IL-6 and IGF-1 signaling pathways. Here, we focused on the functional validation of the alteration of TGFβ response during c-MYC-mediated immortalization. The results demonstrate loss of sensitivity of iMYC cells to activation of TGFβ signaling upon ligand addition. Furthermore, we show that aberrant regulation of the p27 tumor suppressor protein in iMYC cells is a key event that contributes to loss of response to TGFβ. These findings highlight the potential to reveal key pathways contributing to the self-renewal of cancer cells through functional mining of the unique gene expression signature of cells immortalized by c-MYC.
PMCID: PMC3180192  PMID: 21720214
c-MYC; gene expression signature; immortalization; TGFβ; p27
16.  Serum Antibody Response Following Genital α9 Human Papillomavirus Infection in Young Men 
The Journal of Infectious Diseases  2011;204(2):209-216.
Background. Although the prevalence of human papillomavirus (HPV) genital infection is similarly high in males and females, seroprevalence is lower in males. This study assessed rates and determinants of seroconversion after detection of genital HPV infection in young men.
Methods. We investigated HPV type-specific seroconversion in a cohort of heterosexual male university students who had an α9 HPV type (HPV-16, -31, -33, -35, -52, -58, or -67) detected in the genital tract (n = 156). HPV DNA and antibodies were detected and typed using liquid bead-based multiplex assays. We calculated seroconversion using Kaplan–Meier survival analysis. Cox proportional hazards models with generalized estimating equations were used to examine associations with seroconversion.
Results. Within 24 months of detecting genital HPV infection, type-specific seroconversion ranged from 4% for HPV–52 to 36% for HPV-31. HPV-16 seroconversion at 24 months was 13% (95% confidence interval [CI], 7%–25%). Among incident HPV infections, ever cigarette smoking and infection site(s) (shaft/scrotum and glans/urine vs shaft/scrotum or glans/urine only) were positively associated with type-specific seroconversion.
Conclusions. For each of the α9 HPV types, type-specific seroconversion within 24 months was observed in 36% or less of infected men. Seroconversion might be related to cigarette smoking and genital site(s) infected.
PMCID: PMC3114468  PMID: 21673030
17.  HPV 5 and 8 E6 Abrogate ATR Activity Resulting in Increased Persistence of UVB Induced DNA Damage 
PLoS Pathogens  2012;8(7):e1002807.
The role of the E6 oncoprotein from high-risk members of the α human papillomavirus genus in anogenital cancer has been well established. However, far less is known about the E6 protein from the β human papillomavirus genus (β-HPVs). Some β-HPVs potentially play a role in non-melanoma skin cancer development, although they are not required for tumor maintenance. Instead, they may act as a co-factor that enhances the carcinogenic potential of UV damage. Indeed, the E6 protein from certain β-HPVs (HPV 5 and 8) promotes the degradation of p300, a histone acetyl transferase involved in UV damage repair. Here, we show that the expression of HPV 5 and 8 E6 increases thymine dimer persistence as well as the likelihood of a UVB induced double strand break (DSB). Importantly, we provide a mechanism for the increased DNA damage by showing that both extended thymine dimer persistence as well as elevated DSB levels are dependent on the ability of HPV 8 E6 to promote p300 degradation. We further demonstrate that HPV 5 and 8 E6 expression reduces the mRNA and protein levels of ATR, a PI3 kinase family member that plays a key role in UV damage signaling, but that these levels remain unperturbed in cells expressing a mutated HPV 8 E6 incapable of promoting p300 degradation. We confirm that the degradation of p300 leads to a reduction in ATR protein levels, by showing that ATR levels rebound when a p300 mutant resistant to HPV 8 mediated degradation and HPV 8 E6 are co-transfected. Conversely, we show that ATR protein levels are reduced when p300 is targeted for degradation by siRNA. Moreover, we show the reduced ATR levels in HPV 5 and 8 E6 expressing cells results in delayed ATR activation and an attenuated ability of cells to phosphorylate, and as a result accumulate, p53 in response to UVB exposure, leading to significantly reduced cell cycle arrest. In conclusion, these data demonstrate that β-HPV E6 expression can enhance the carcinogenic potential of UVB exposure by promoting p300 degradation, resulting in a reduction in ATR levels, which leads to increased thymine dimer persistence and increased UVB induced DSBs.
Author Summary
Human papillomaviruses are a large family of viruses that can cause ailments ranging from benign warts to anogenital cancer. Recently, interest has increased for a subgroup of these viruses, the β-HPVs, because of their potential involvement in squamous cell skin cancer. In this work, we show that the E6 protein from two of these viruses (HPV 5 and 8) is able to increase the damage that UV exposure causes to the host cell's DNA. The E6 protein from these viruses promotes the degradation of p300, a cellular protein involved in DNA damage repair. This in turn reduces amounts of another cellular protein, ATR, which facilitates the process of signaling the cell to repair its damaged DNA. The decrease in ATR levels delays the cell's recognition of the damaged DNA, allowing thymine dimers to remain unrepaired longer and more often leading to a double strand break in the DNA. Together, our data show that the β HPVs can cause UV exposures to be more deleterious to host cell DNA, potentially increasing the likelihood that these cells become cancerous.
PMCID: PMC3395675  PMID: 22807682
18.  Antibody responses in oral fluid following administration of prophylactic human papillomavirus vaccines 
The Journal of Infectious Diseases  2009;200(9):1452-1455.
We sought to determine whether oral fluid can be used to assess serum human papillomavirus (HPV) antibody status by enrolling women who had received a prophylactic HPV-16 vaccine in a new follow-up study. After the prophylactic HPV-6/11/16/18 vaccine was licensed in the United States, we administered it to consenting participants. The sensitivity of oral fluid, treating serology as the gold standard, before and after administration of the quadrivalent vaccine was 49.6% (95% confidence interval [CI]: 42.0%–57.3%) and 100% (95% CI: 92.0%–100%), respectively. Oral fluid may have the potential to be used for monitoring of prophylactic HPV vaccines in the future.
PMCID: PMC3392559  PMID: 19698077
Human papillomavirus; Prophylactic vaccines; Antibodies; Oral fluid
19.  Human papilloma viruses 
Retrovirology  2012;9(Suppl 1):I23.
PMCID: PMC3360389
20.  Viral Load in the Natural History of Human Papillomavirus Type 16 Infection: A Nested Case–control Study 
The Journal of Infectious Diseases  2011;203(10):1425-1433.
Background. Viral load may influence the course of human papillomavirus type 16 (HPV-16) infection.
Methods. This case-control study was nested within the 2-year Atypical Squamous Cells of Undetermined Significance and Low-Grade Squamous Intraepithelial Lesion Triage Study, in which women were followed semiannually for HPV and cervical intraepithelial neoplasia (CIN). Case patients (n = 62) were women diagnosed with CIN3 following HPV-16–positive detection at a follow-up visit. HPV-16–positive controls (n = 152) without CIN2 or CIN3 were matched to cases based on the follow-up visit in which viral load was measured. Real-time polymerase chain reaction was used for HPV-16 DNA quantification.
Results. The risk of CIN3 increased with increasing HPV-16 DNA load at the follow-up visit (odds ratio, 1.63; 95% confidence interval, 1.33–1.99 per 1 log10 unit increase); the association was not affected by whether HPV-16 was present at enrollment. When HPV-16 was present at both enrollment and follow-up, viral load remained high among cases (P = .77) but decreased substantially among controls (P = .004). Among women with HPV-16 found initially during follow-up, viral load in the first HPV-16–positive sample was associated with short-term persistence; load was higher in those with infection, compared with those without infection, 1 visit after the initial positivity (P = .001).
Conclusions. Viral load of newly detected infections and changes in viral load predict persistence and progression of HPV-16 infections.
PMCID: PMC3080901  PMID: 21415020
21.  Transcriptome-Wide Studies of Merkel Cell Carcinoma and Validation of Intratumoral CD8+ Lymphocyte Invasion As an Independent Predictor of Survival 
Journal of Clinical Oncology  2011;29(12):1539-1546.
Merkel cell carcinoma (MCC) is a polyomavirus-associated skin cancer that is frequently lethal and lacks established prognostic biomarkers. This study sought to identify biomarkers that improve prognostic accuracy and provide insight into MCC biology.
Patients and Methods
Gene expression profiles of 35 MCC tumors were clustered based on prognosis. The cluster of genes overexpressed in good-prognosis tumors was tested for biologic process enrichment. Relevant mRNA expression differences were confirmed by quantitative polymerase chain reaction and immunohistochemistry. An independent set of 146 nonoverlapping MCC tumors (median follow-up, 25 months among 116 living patients) was employed for biomarker validation. Univariate and multivariate Cox regression analyses were performed.
Immune response gene signatures were prominent in patients with good prognoses. In particular, genes associated with cytotoxic CD8+ lymphocytes were overexpressed in tumors from patients with favorable prognoses. In the independent validation set, cases with robust intratumoral CD8+ lymphocyte infiltration had improved outcomes (100% MCC-specific survival, n = 26) compared with instances characterized by sparse infiltration (60% survival, n = 120). Only stage and intratumoral CD8 infiltration (but not age, sex, or CD8+ lymphocytes localized to the tumor-stroma interface) were significant in both univariate and multivariate Cox regression analyses. Notably, traditional histologic identification of tumor-infiltrating lymphocytes was not a significant independent predictor of survival.
Intratumoral CD8+ lymphocyte infiltration can be readily assessed on paraffin-embedded tissue, is independently associated with improved MCC-specific survival, and therefore, may provide prognostic information that enhances established MCC staging protocols.
PMCID: PMC3082974  PMID: 21422430
22.  Risk of cervical cancer associated with allergies and polymorphisms in genes in the chromosome 5 cytokine cluster 
Human papillomavirus is the acknowledged cause of cervical cancer. We hypothesized that allergies, characterized by hyperimmune reaction to common allergens andwhich have been associated with various cancers, may be related to cervical cancer, and that genetic variation in cytokine genes related to allergies might impact cervical cancer risk.
We investigated the risk of invasive squamous cell cervical cancer (SCC) associated with self-reported allergies and with variation in allergy-related cytokine genes using data from a case-control study (561 cases, 1258 controls) conducted in Washington State. Logistic regression models yielded odds ratios (OR) and 95% confidence intervals (CI).
Pollen allergy, the most commonly reported allergy, was associated with reduced SCC risk (OR 0.6, 95% CI 0.5–0.8). Of 60 tagging single nucleotide polymorphisms covering eight genes (CSF2, IL3, IL4, IL13, CSF2RB, IL4R, IL13RA1, IL13RA2), several were related to pollen allergies among controls: IL4R rs3024647 (dominant OR 1.5 95% CI 1.0–2.3, p=0.04), CSF2RB rs16997517 (dominant OR 2.2 95% CI 1.0–4.7, p=0.04), and IL13 rs1800925 (per-allele OR 1.7, 95% CI 1.3–2.4, p=0.0007). Two variants were inversely associated with SCC risk: IL4R rs3024656 (per-allele OR 0.8, 95% CI 0.6–1.0, p=0.03) and CSF2RB rs16997517 (dominant OR 0.4, 95% CI 0.2–0.9, p=0.04).
Pollen allergies were related to reduced SCC risk. CSF2RB rs16997517 was directly related to pollen allergies in controls and to reduced SCC risk.
If other studies confirm these results, the mechanism behind allergy-associated immune response associated with SCC risk may be worth exploring in the context of therapeutic or prophylactic vaccines.
PMCID: PMC3057941  PMID: 21071541
Cervical cancer; HPV; allergy; cytokines
23.  Antibodies to Merkel cell polyomavirus T-antigen oncoproteins reflect tumor burden in Merkel cell carcinoma patients 
Cancer research  2010;70(21):8388-8397.
Merkel cell polyomavirus (MCPyV) is a common infectious agent that is likely involved in the etiology of most Merkel cell carcinomas (MCCs). Serum antibodies recognizing the MCPyV capsid protein, VP1, are detectable at high titer in nearly all MCC patients, and remain stable over time. Although antibodies to the viral capsid indicate prior MCPyV infection, they provide limited clinical insight into MCC because they are also detected in more than half of the general population. We investigated whether antibodies recognizing MCPyV large and small tumor-associated antigens (T-Ags) would be more specifically associated with MCC. Among 530 population control subjects, these antibodies were present in only 0.9% and were of low titer. In contrast, among 205 MCC cases, 40.5% had serum IgG antibodies that recognize a portion of T-Ag shared between small and large T-Ags. Among cases, titers of T-Ag antibodies fell rapidly (approximately 8 fold/year) in patients whose cancer did not recur, while they rose rapidly in those with progressive disease. Importantly, in several patients who developed metastases, the rise in T-Ag titer preceded clinical detection of disease spread. These results suggest that antibodies recognizing T-Ag are relatively specifically associated with MCC, do not effectively protect against disease progression, and may serve as a clinically useful indicator of disease status.
PMCID: PMC2970647  PMID: 20959478
Merkel cell carcinoma; Merkel cell polyomavirus; MCPyV; antibody; biomarker
24.  Beta-HPV 5 and 8 E6 Promote p300 Degradation by Blocking AKT/p300 Association 
PLoS Pathogens  2011;7(8):e1002211.
The E6 oncoprotein from high-risk genus alpha human papillomaviruses (α-HPVs), such as HPV 16, has been well characterized with respect to the host-cell proteins it interacts with and corresponding signaling pathways that are disrupted due to these interactions. Less is known regarding the interacting partners of E6 from the genus beta papillomaviruses (β-HPVs); however, it is generally thought that β-HPV E6 proteins do not interact with many of the proteins known to bind to α-HPV E6. Here we identify p300 as a protein that interacts directly with E6 from both α- and β-HPV types. Importantly, this association appears much stronger with β-HPV types 5 and 8-E6 than with α-HPV type 16-E6 or β-HPV type 38-E6. We demonstrate that the enhanced association between 5/8-E6 and p300 leads to p300 degradation in a proteasomal-dependent but E6AP-independent manner. Rather, 5/8-E6 inhibit the association of AKT with p300, an event necessary to ensure p300 stability within the cell. Finally, we demonstrate that the decreased p300 protein levels concomitantly affect downstream signaling events, such as the expression of differentiation markers K1, K10 and Involucrin. Together, these results demonstrate a unique way in which β-HPV E6 proteins are able to affect host-cell signaling in a manner distinct from that of the α-HPVs.
Author Summary
Human papillomaviruses (HPVs) are a family of more than 100 different viruses that cause a wide range of pathologies, from benign warts to cervical cancer. One subgroup of HPVs, the beta-HPVs, have recently become a topic of interest due to their potential involvement in squamous cell skin cancer. However, unlike the HPVs involved in cervical cancer, little is known with regards to how the beta-HPVs may facilitate cellular changes that would allow cancerous lesions to develop. Here we have identified a host-cell protein, p300, which interacts strongly with the E6 oncoprotein from two beta-HPVs, HPV 5 and HPV 8. We show that this interaction subsequently blocks another cellular protein, AKT, from binding to and stabilizing p300. By blocking this association, p300 is targeted for degradation, and thus is present in lower amounts than in normal cells. Importantly, because p300 is involved in numerous cell processes such as DNA repair, cell growth, and differentiation, the potential for E6 disrupting a number of cellular signaling pathways is vast. Taken together, our findings shed new light on how the beta-HPVs may facilitate carcinogenesis.
PMCID: PMC3161984  PMID: 21901101
25.  Cytoplasmic Poly(A) Binding Proteins Regulate Telomerase Activity and Cell Growth in Human Papillomavirus Type 16 E6-Expressing Keratinocytes▿  
Journal of Virology  2010;84(24):12934-12944.
The high-risk human papillomavirus (HPV) E6 and E7 oncoproteins are critical to the immortalization of keratinocytes. HPV type 16 (HPV16) E6 interacts with endogenous proteins to activate hTERT, the catalytic subunit of telomerase, thus avoiding cellular senescence signals. NFX1-123, the longer splice variant of NFX1, interacts with HPV16 E6, as well as cytoplasmic poly(A) binding proteins 1 and 4 (PABPC1 and PABPC4). HPV16 E6 affects hTERT expression posttranscriptionally through NFX1-123, as NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater telomerase activity. The PAM2 motif of NFX1-123, with which it binds PABPCs, is required for the posttranscriptional regulation of hTERT by HPV16 E6 and NFX1-123. There is increasing evidence that RNA and DNA viruses utilize RNA-processing proteins, and specifically PABPCs, in the normal virus life cycle, and there is also evidence that RNA-processing proteins are perturbed in cancers. Here, we show that PABPCs are critical in hTERT regulation by HPV16 E6. Although the amount and cellular localization of PABPCs were largely unchanged in cervical cancer cell lines with or without HPV16 and in human foreskin keratinocytes (HFKs) with or without HPV16 E6, knockdown of PABPCs decreased hTERT mRNA and telomerase activity and overexpression of PABPC4 increased these in HPV16 E6-expressing HFKs. In contrast, knockdown of PABPCs in C33A cells had no effect on hTERT mRNA or telomerase activity. Additionally, overexpression of PABPC4 and hTERT led to greater growth of cultured HPV16 E6-expressing HFKs. This is the first evidence that PABPCs have a targeted role in hTERT regulation leading to a growth advantage in cells expressing HPV16 E6.
PMCID: PMC3004306  PMID: 20943973

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