Ingeborg Zehbe

The E6 and E7 Proteins of the Cutaneous Human Papillomavirus Type 38 Display Transforming Properties

ABSTRACT Several studies have suggested the involvement of cutaneous human papillomaviruses (HPVs) in the development of nonmelanoma skin cancers. Here we have characterized the in vitro properties of E7 proteins of three cutaneous HPV types, 10, 20, and 38, which are frequently detected in skin specimens. We show that HPV38 E7 is able to inactivate the tumor suppressor pRb and induces loss of G1/S transition control, a key event in carcinogenesis. In contrast, HPV10 and HPV20 E7 proteins do not display these in vitro transforming activities. We also show that the two early proteins E6 and E7 of HPV38 are sufficient to corrupt the cell cycle and senescence programs in primary cells, inducing active and long-lasting proliferation of primary human keratinocytes, the natural host cells. Our study shows that E6 and E7 of this cutaneous HPV type have transforming activity in primary human cells, suggesting a role for HPV38 infection in skin carcinogenesis. In further support of such a role, we detected HPV38 DNA in approximately 50% of nonmelanoma skin cancers, but only in 10% of healthy skin specimens (P < 0.001).

Codon 72 polymorphism of p53 and its association with cervical cancer

Swedish and Italian women with HPV 16-positive cervical disease were checked for codon 72 polymorphisms of p53. In both groups, arginine homozygotes were enriched in cancer compared with controls and precursor lesions.

TP53 codon 72 polymorphism and cervical cancer: a pooled analysis of individual data from 49 studies.

BACKGROUND Cervical cancer is caused primarily by human papillomaviruses (HPV). The polymorphism rs1042522 at codon 72 of the TP53 tumour-suppressor gene has been investigated as a genetic cofactor. More than 80 studies were done between 1998 and 2006, after it was initially reported that women who are homozygous for the arginine allele had a risk for cervical cancer seven times higher than women who were heterozygous for the allele. However, results have been inconsistent. Here we analyse pooled data from 49 studies to determine whether there is an association between TP53 codon 72 polymorphism and cervical cancer. METHODS Individual data on 7946 cases and 7888 controls from 49 different studies worldwide were reanalysed. Odds ratios (OR) were estimated using logistic regression, stratifying by study and ethnic origin. Subgroup analyses were done for infection with HPV, ethnic origin, Hardy-Weinberg equilibrium, study quality, and the material used to determine TP53 genotype. FINDINGS The pooled estimates (OR) for invasive cervical cancer were 1.22 (95% CI 1.08-1.39) for arginine homozygotes compared with heterozygotes, and 1.13 (0.94-1.35) for arginine homozygotes versus proline homozygotes. Subgroup analyses showed significant excess risks only in studies where controls were not in Hardy-Weinberg equilibrium (1.71 [1.21-2.42] for arginine homozygotes compared with heterozygotes), in non-epidemiological studies (1.35 [1.15-1.58] for arginine homozygotes compared with heterozygotes), and in studies where TP53 genotype was determined from tumour tissue (1.39 [1.13-1.73] for arginine homozygotes compared with heterozygotes). Null results were noted in studies with sound epidemiological design and conduct (1.06 [0.87-1.29] for arginine homozygotes compared with heterozygotes), and studies in which TP53 genotype was determined from white blood cells (1.06 [0.87-1.29] for arginine homozygotes compared with heterozygotes). INTERPRETATION Subgroup analyses indicated that excess risks were most likely not due to clinical or biological factors, but to errors in study methods. No association was found between cervical cancer and TP53 codon 72 polymorphism when the analysis was restricted to methodologically sound studies. FUNDING German Research Foundation (DFG).

Human papillomavirus 16 E6 polymorphisms in cervical lesions from different European populations and their correlation with human leukocyte antigen class II haplotypes

Infection with high‐risk human papillomavirus (HPV) is necessary for the development of a cervical lesion, but only a fraction of precursor lesions progress to cancer. Additional factors, other than HPV type per se, are likely to increase the probability for progression. Intratype genome variations have been reported to be associated with viral persistence and the development of a major cervical disease. We have recently shown that the prevalence of specific HPV16‐E6 variants in invasive cervical cancer (ICC) varies between Italian and Swedish women. To extend our initial study we have analyzed E6 variants in cervical lesions from Czech women, ranging from low‐grade cervical intraepithelial neoplasia (LCIN) to ICC and scaled up the sample size of our initial study of Swedish and Italian women. In addition, we have correlated the cases of cancers with human leukocyte antigen (HLA) class II haplotypes. In line with our earlier observation, the distribution of specific HPV16‐E6 genotypes in CIN and ICC varied in the 3 cohorts. For instance, the HPV16‐E6 L83V variant, which has been found to be positively associated with ICC in Swedish women (p = 0.002), was more prevalent in LCIN than in ICC in Italian and Czech women (p = 0.01 and = 0.03, respectively). These data indicate that host genetic factors, such as HLA polymorphism, may determine the potential oncogenicity of the HPV16‐E6 L83V variant. Indeed, the DR04‐DQ03 haplotype, which is approximately 3‐fold more abundant in the normal Swedish population than in those in Italy and the Czech Republic, was found to be positively associated with HPV16‐E6 L83V in the 3 cohorts investigated (p = 0.01). This observation may explain why L83V is a risk factor more in Sweden than in the other 2 countries.

Increased risk for cervical disease progression of French women infected with the human papillomavirus type 16 E6-350G variant.

To test the significance of human papillomavirus (HPV) type 16 and HPV16 E6 variants as risk factors for viral persistence and progression to high-grade lesion, we did a nested case-control study within a cohort study of >15,000 Caucasian French women. Three groups infected with high-risk HPV were compared: (a) women with cleared infection (controls, n = 201), (b) women with persistent infection (cases, n = 87), and (c) women who progressed into high-grade lesion (cases, n = 58). Women with persistent HPV infection and those that progressed into high-grade lesions were likelier to harbor HPV16 than other high-risk HPV types [odds ratio (OR), 2.4; 95% confidence interval (95% CI), 1.3-4.3 and OR, 4.2; 95% CI, 2.2-8.1, respectively]. Notably, especially elevated ORs of persistence (3.0; 95% CI, 1.4-6.7) and progression (6.2; 95% CI, 2.7-14.3) were found among women who harbored the HPV16 350G variant. Thus, HPV type and HPV16 variant seem to be risk factors for viral persistence and progression of infections into high-grade cervical lesions. Cancer Epidemiol Biomarkers Prev 2006:15(4);820–2

The immortalizing and transforming ability of two common human papillomavirus 16 E6 variants with different prevalences in cervical cancer.

Persistent infection with high-risk human papillomaviruses (HPVs), especially type 16 has been undeniably linked to cervical cancer. The Asian-American (AA) variant of HPV16 is more common in the Americas than the prototype in cervical cancer. The different prevalence is based on three amino acid changes within the E6 protein denoted Q14H/H78Y/L83V. To investigate the mechanism(s) behind this observation, both E6 proteins, in the presence of E7, were evaluated for their ability to extend the life span of and transform primary human foreskin keratinocytes (PHFKs). Long-term cell culture studies resulted in death at passage 9 of vector-transduced PHFKs (negative control), but survival of both E6 PHFKs to passage 65 (and beyond). Compared with E6/E7 PHFKs, AA/E7 PHFKs were significantly faster dividing, developed larger cells in monolayer cultures, showed double the epithelial thickness and expressed cytokeratin 10 when grown as organotypic raft cultures. Telomerase activation and p53 inactivation, two hallmarks of immortalization, were not significantly different between the two populations. Both were resistant to anoikis at later passages, but only AA/E7 PHFKs acquired the capacity for in vitro transformation. Proteomic analysis revealed markedly different protein patterns between E6/E7 and AA/E7, particularly with respect to key cellular metabolic enzymes. Our results provide new insights into the reasons underlying the greater prevalence of the AA variant in cervical cancer as evidenced by characteristics associated with higher oncogenic potential.

Overriding of cyclin-dependent kinase inhibitors by high and low risk human papillomavirus types; Evidence for an in vivo role in cervical lesions.

High risk types of human papillomavirus (HPV) are agents in the aetiology of cervical carcinoma. The products of two early genes, E6 and E7, appear to be the principal transforming proteins. Studies of various monolayer cell culture systems have shown that the E7 oncoprotein of human papillomavirus type 16 is able to neutralize or bypass the inhibitory effect of the cell cycle-dependent kinase (CDK) inhibitors (CKIs) p21WAF1/CIP1 and p27KIP1. To understand whether the p21WAF1/CIP1 or p27KIP1 neutralization also plays a role in vivo, we performed studies on clinical specimens. Forty-five cervical biopsies, including HPV-negative mucosa, HPV 16-positive preinvasive (low and high grade lesions) and invasive neoplasia as well as HPV 6-positive condyloma acuminatum were analysed by single and double immunohistology. We examined the positive cell cycle regulator cyclin A and the universal cell cycle marker Ki67 as well as the negative cell cycle regulators p21WAF1/CIP1 and p27KIP1. Here, we show that in a significant fraction of cells the G1 block can be overcome despite high levels of CKIs in HPV lesions. This phenomenon, which was more evident for p21WAF1/CIP1 than for p27KIP1 was most marked in low grade lesions and in condylomata acuminata, in which a high viral productivity is expected. These results indicate that the overriding of CKI inactivation by viral oncoproteins appears to be a conserved property between low and high risk HPV types. We conclude that the CKI neutralization by HPVs is likely to be required for viral DNA replication rather than for malignant transformation of the host cell.

Toll-Like Receptor Transcriptome in the HPV-Positive Cervical Cancer Microenvironment

The human papillomavirus (HPV) directly infects cervical keratinocytes and interferes with TLR signalling. To shed light on the effect of HPV on upstream receptors, we evaluated TLRs 1–9 gene expression in HPV-negative normal and HPV-positive pre-malignant and malignant ex vivo cervical tissue. Quantitative real-time polymerase chain reaction was performed separately for epithelial and stromal tissue compartments. Differences in gene expression were analyzed by the Jonckheere-Terpstra trend test or the Students t-test for pairwise comparison. Laser capture microdissection revealed an increase in TLR3 and a decrease in TLR1 mRNA levels in dysplastic and carcinoma epithelium, respectively. In the stroma, a trend of increasing TLR 1, 2, 5, 6, and 9 mRNA levels with disease severity was found. These findings implicate the involvement of TLR3 and TLR1 in early and late cervical carcinogenesis, respectively, suggesting that stromal upregulation of TLRs may play a role in cervical disease progression.

Hypoxia-inducible factor 1 and its role in viral carcinogenesis

The advent of modern molecular biology has allowed for the discovery of several mechanisms by which oncoviruses promote carcinogenesis. Remarkably, nearly all human oncogenic viruses increase levels of the transcription factor hypoxia-inducible factor 1 (HIF-1). In this review, we highlight HIF-1׳s significance in viral oncogenesis, while providing an in-depth analysis of its activation mechanisms by the following oncoviruses: human papillomaviruses (HPVs), hepatitis B/C viruses (HBV/HCVs), Epstein-Barr virus (EBV), Kaposi׳s sarcoma-associated herpes virus (KSHV), and human T-cell lymphotropic virus (HTLV-1). We discuss virus-induced HIF-1׳s role in transcriptional upregulation of metabolic, angiogenic, and microenvironmental factors that are integral for oncogenesis. Admittedly, conclusive evidence is lacking as to whether activation of HIF-1 target genes is necessary for malignant transformation or merely a result thereof. In addition, a complete understanding of host-virus interactions, the effect of viral genomic variation, and the clinical (and potential therapeutic) relevance of HIF-1 in viral oncogenesis warrant further investigation.

Feasibility of self-sampling and human papillomavirus testing for cervical cancer screening in First Nation women from Northwest Ontario, Canada: a pilot study

Background The incidence of cervical cancer is up to sixfold higher among First Nation women in Canada than in the general population. This is probably due to lower participation rates in cervical cancer prevention programmes. Objective To raise screening participation in this underserved population by launching an alternative approach to (Pap)anicolaou testing in a clinic—namely, vaginal self-sampling followed by human papillomavirus (HPV) diagnostics. Methods Good relationships were established with a First Nation community of the Northern Superior region in Northwest Ontario, and then 49 community women, aged 25–59, were recruited, who provided a vaginal self-sample and answered a questionnaire. Frequency distributions and cross-tabulations were used to summarise the data. Associations between categorical variables were assessed using the χ2 test of association, or the Goodman–Kruskal γ if both variables had ordered categories. Self-collected samples were tested for integrity and HPV using optimised molecular biological methods. Results The majority of participants (87.2%) were amenable to future HPV screening by self-sampling. This finding was independent of age, educational level and a previous history of abnormal Pap tests. Interestingly, the preferred way to learn about sexual health remained through interaction with healthcare professionals. As defined by the presence of a housekeeping gene, self-sample integrity was high (96%). Using polymerase chain reaction-based Luminex typing, the overall HPV positivity was 28.6% (ie, with either a low- or high-risk type) and 16.3% were infected with a high-risk type such as HPV16. Conclusion In this pilot study of First Nation women, self-sampling and HPV testing was well received and self-sample quality was excellent. A larger survey to be conducted in other Northern Superior communities in Northwest Ontario will determine whether this approach could become a viable screening strategy for First Nation women.