Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes

Background  

Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features.     

Bias in effect size of systemic lupus erythematosus susceptibility loci across Europe: a case-control study

Introduction

Prevalence of an abnormal Papanicolaou smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk human papillomavirus (HPV) infection. The nucleic acid-specific Toll-like receptors (TLRs) locate at the endolysosomal compartments and trigger the induction of cytokines for the innate immune response. This study evaluated whether abnormal host innate immune response in lupus patients may enhance HPV persistence.

Methods

Protein levels of TLRs 3, 7, 8 and 9 in cervical epithelial cells of lupus patients and controls with or without HPV infection were assessed using flow cytometry. Characteristics associated with the differential expression of TLRs in systemic lupus erythematosus (SLE) were elucidated. The effect and interferon-stimulated genes (ISGs) (ISG15 and Mx-1) gene expressions were then measured in oncogenic HeLa (HPV18), CaSki (HPV) and C33A (HPV negative) cell lines using flow cytometry and quantitative real-time PCR. Ex vivo productions of cytokines and interferon-gamma (IFN-??) upon TLR ligands stimulations were subsequently measured using cytometric bead array and ELISA.

Results

For subjects with HPV infection, levels of TLR3 and TLR7 were significantly lower in lupus patients compared with controls. Significantly decreased TLRs 7, 8 and 9 levels were observed in HPV-negative SLE compared to healthy controls. For SLE with and without HPV infection, TLR7 and 9 levels were significantly lower in infected SLE than those in HPV-negative patients. Independent explanatory variables associated with down-regulation of TLR7 level included HPV infection and a higher cumulative dose of prednisolone; while a higher cumulative dose of hydroxychloroquine and HPV infection were associated with down-regulation of TLR9 level. In cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral ISG15 and Mx-1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands (R837, ssRNA and ODN2395) was greatly impaired in CaSki and HeLa than C33A cells.

Conclusions

In conclusion, prednisolone and TLR antagonist (hydroxychloroquine) may down-regulate protein levels of TLR7 and TLR9 in lupus patients, thereby decreasing the innate immune response against HPV infection. Upon infection, HPV further down-regulate TLR7 and 9 levels for viral persistence. Furthermore, reduction of nucleic acid-sensing TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs (ISG15 and Mx-1) on a biologically relevant antiviral response.     

tigaR: integrative significance analysis of temporal differential gene expression induced by genomic abnormalities

Background

To determine which changes in the host cell genome are crucial for cervical carcinogenesis, a longitudinal in vitro model system of HPV-transformed keratinocytes was profiled in a genome-wide manner. Four cell lines affected with either HPV16 or HPV18 were assayed at 8 sequential time points for gene expression (mRNA) and gene copy number (DNA) using high-resolution microarrays. Available methods for temporal differential expression analysis are not designed for integrative genomic studies.

Results

Here, we present a method that allows for the identification of differential gene expression associated with DNA copy number changes over time. The temporal variation in gene expression is described by a generalized linear mixed model employing low-rank thin-plate splines. Model parameters are estimated with an empirical Bayes procedure, which exploits integrated nested Laplace approximation for fast computation. Iteratively, posteriors of hyperparameters and model parameters are estimated. The empirical Bayes procedure shrinks multiple dispersion-related parameters. Shrinkage leads to more stable estimates of the model parameters, better control of false positives and improvement of reproducibility. In addition, to make estimates of the DNA copy number more stable, model parameters are also estimated in a multivariate way using triplets of features, imposing a spatial prior for the copy number effect.

Conclusion

With the proposed method for analysis of time-course multilevel molecular data, more profound insight may be gained through the identification of temporal differential expression induced by DNA copy number abnormalities. In particular, in the analysis of an integrative oncogenomics study with a time-course set-up our method finds genes previously reported to be involved in cervical carcinogenesis. Furthermore, the proposed method yields improvements in sensitivity, specificity and reproducibility compared to existing methods. Finally, the proposed method is able to handle count (RNAseq) data from time course experiments as is shown on a real data set.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-327) contains supplementary material, which is available to authorized users.     

Expression of cellular genes in HPV16-immortalized and cigarette smoke condensate-transformed human endocervical cells

We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H-ras, c-myc, B-myb, p53, p16^INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC-transformed counterparts of HPV16-immortalized cells had up-regulated levels of B-myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis. J. Cell. Biochem. 66: 309–321, 1997. © 1997 Wiley-Liss, Inc.     

Prevalence of Human Papillomavirus Genotypes among African Women with Normal Cervical Cytology and Neoplasia: A Systematic Review and Meta-Analysis

Background

Several meta-analyses confirmed the five most prevalent human papillomavirus (HPV) strains in women with and without cervical neoplastic diseases are HPV16, 18, 31, 52, and 58. HPV16/18 are the predominant oncogenic genotypes, causing approximately 70% of global cervical cancer cases. The vast majority of the women studied in previous analyses were from Europe, North America, Asia, and most recently Latin America and the Caribbean. Despite the high burden of cervical cancer morbidity and mortality in Africa, a robust meta-analysis of HPV genotype prevalence and distribution in African women is lacking.

Methods and Findings

We systematically searched 14 major databases from inception to August 2013 without language restriction, following the Meta-Analysis of Observational Studies in Epidemiology and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Seventy-one studies from 23 African countries were identified after screening 1162 citations and data abstracted and study quality appraised from 195 articles. HPV type-specific prevalence and distribution was estimated from 17,273 cases of women with normal cervical cytology; 1019 women with atypical squamous cells of undetermined significance (ASCUS); 1444 women with low-grade squamous intraepithelial lesion (LSIL); 1571 women with high-grade squamous intraepithelial lesion (HSIL); and 4,067 cases of invasive cervical carcinoma (ICC). Overall prevalence of HPV16/18 were 4.4% and 2.8% of women with normal cytology, 12.0% and 4.4% with ASCUS, 14.5% and 10.0% with LSIL, 31.2% and 13.9% with HSIL, and 49.7% and 18.0% with ICC, respectively. Study limitations include the lack of adequate data from Middle and Northern African regions, and variations in the HPV type-specific sensitivity of different genotyping protocols.

Conclusions

To our knowledge, this study is the most comprehensive assessment of the overall prevalence and distribution of HPV genotypes in African women with and without different cervical neoplasias. We have established that HPV16/18 account for 67.7% of ICC cases among African women. Based on our findings, we highly recommend the administration of existing prophylactic vaccines to younger women not infected with HPV16/18 and an increase in HPV screening efforts for high-risk genotypes to prevent cervical cancer. Review registration: International Prospective Register of Systematic Reviews CRD42013006558.     

Inhibition of human papillomavirus type 16 E7 phosphorylation by the S100 MRP-8/14 protein complex

The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related factor protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G1 phase of the cell cycle. Analysis of MRP expression in primary keratinocytes and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed keratinocytes and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized keratinocytes was approximately fourfold higher than in HPV-negative primary keratinocytes. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell growth within 2 weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed 25 and 40% growth inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.     

High-Risk Cervical Human Papillomavirus Infections among Human Immunodeficiency Virus-Positive Women in the Bahamas

Background

High-risk (HR) HPV genotypes other than 16 and 18 have been detected in a significant proportion of immunocompromised females. We aim to evaluate the frequency of HR HPV genotypes in a population of HIV-positive Caribbean women.

Methods

One hundred sixty-seven consecutive, non-pregnant, HIV-positive females ≥18 years were recruited in this study. Each participant received a vaginal examination, PAP smear, and completed a questionnaire. DNA was extracted for HPV testing in 86 patients.

Results

Mean age was 39.1 years for women positive for HR HPV and 43.1 years for women negative for HR HPV (P value  = 0.040). 78% (130/167) of the women had HR HPV infections; the prevalence of abnormal cervical cytology was 38% among women who were HR HPV-positive compared to women who were HR HPV-negative (22%). Fifty-one percent of the 86 women with available genotype carried infections with HPV 16 and/or HPV 18; genotypes of unknown risk were also frequently observed. Women who had a CD4+ count of ≤200 had 7 times increased odds of carrying HR HPV infection in comparison to women with CD4+>200.

Conclusions

HR HPV infections in HIV infected females may consist of more than just HPV 16 and 18, but also HPV 52 and 58. Further studies are needed to determine whether HPV 52 and 58 play a significant role in the development of cervical cytological abnormalities in HIV+ women.     

H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

Background

Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.

Methodology/Principal Findings

Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras), were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63), basement-membrane formation (collagen IV, laminin-5), and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin) were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes.

Conclusions/Significance

The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.     

Human Papillomavirus Types Distribution in Organised Cervical Cancer Screening in France

Background

Knowledge of prevalence rates and distribution of human papillomavirus (HPV) genotypes prior high HPV vaccine coverage is necessary to assess its expected impact on HPV ecology and on cervical lesions and cancers.

Methods

Residual specimens of cervical cytology (N = 6,538) were obtained from 16 sites participating in organised cervical cancer screening pilot programs throughout France, anonymised and tested for HPV DNA using the PapilloCheck® genotyping test. Samples were stratified according to age of women and cytological grades.

Results

The age-standardised prevalence rates of HPV 16 and/or 18 (with or without other high-risk types) was 47.2% (95% Confidence Interval, CI: 42.4–52.1) in high-grade squamous intraepithelial lesions (HSILs), 20.2% in low-grade SIL (95% CI: 16.7–23.7) and 3.9% (95% CI: 2.8–5.1) in normal cytology. Overall HR HPV were detected in 13.7% (95%I CI: 11.7–15.6) of normal cytology. In women below 30 years of age, 64% of HSILs were associated with HPV16 and/or 18. In our study population, HPV16 was the most commonly detected type in all cervical grades with prevalence rates ranking from 3.0% in normal cytology to 50.9% in HSILs. HPV16 was also detected in 54% (27/50) of invasive cervical cancers including 5 adenocarcinomas.

Conclusion

HPV16 was strongly associated with cervical precancer and cancer. The high prevalence rates of HPV16/18 infection among women below 30 years of age with HSILs suggests that the impact of vaccination would be primarily observed among young women.     

Epidemiologic characterization of human papillomavirus infection in rural Chaozhou, eastern Guangdong Province of China

Background

Human papilloma virus (HPV) infection was the main cause of cervical cancer. There were only a few reports and detailed data about epidemiological research of HPV infection in rural population of China.

Materials and Methods

The cervical cells of rural Chaozhou women were collected, and multiplex real time PCR was firstly performed to detect high-risk HPV (HR-HPV) infection, which could detect 13 types of HR-HPV (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Then, HPV-positive samples were typed by HPV GenoArray test.

Results

HR-HPV DNA was detected by multiplex real time-PCR in 3830 of 48559 cases (7.89%). There was a peak incidence in age of 55–60 years group, and a lower incidence in who lived in plain group compared with suburban, mountain and seashore group. 3380 cases of HPV positive sample were genotyped, 11.01% (372/3380) cases could not be classified, among the typed 3008 cases, 101 cases were identified without HR-HPV type infection, 2907 cases were infected with one HR-HPV type at least, the 6 most common HR-HPV types in descending order of infection, were type 52 (33.4%, 16 (20.95%), 58 (15.93%), 33 (9.94%), 68 (9.22%) and 18 (8.36%). The combined prevalence of HPV types 16 and 18 accounted for 28.52% of total infection. However, type 52 plus 58 presented 48.23% of total infection. 2209/2907 cases were infected with a single HPV type and 698/2907 cases were infected with multiple types, and multiple infection constituent ratio increased with age, with a peak incidence in age 55–60 years group.

Conclusions

Our findings showed low prevalence of HPV vaccine types (16 and 18) and relatively high prevalence of HPV-52 and -58, support the hypothesis that the second-generation HPV vaccines including HPV-52 and -58 may offer higher protection for women in rural Guangdong Province.     

In vitro activities of novel 4-HPR derivatives on a panel of rhabdoid and other tumor cell lines

Background

Natural killer (NK) cells are an important resource of the innate immune system directly involved in the spontaneous recognition and lysis of virus-infected and tumor cells. An exquisite balance of inhibitory and activating receptors tightly controls the NK cell activity. At present, one of the best-characterized activating receptors is NKG2D, which promotes the NK-mediated lysis of target cells by binding to a family of cell surface ligands encoded by the MHC class I chain-related (MIC) genes, among others. The goal of this study was to describe the expression pattern of MICA and MICB at the molecular and cellular levels in human cervical cancer cell lines infected or not with human papillomavirus, as well as in a non-tumorigenic keratinocyte cell line.

Results

Here we show that MICA and MICB exhibit differential expression patterns among HPV-infected (SiHa and HeLa) and non-infected cell lines (C33-A, a tumor cell line, and HaCaT, an immortalized keratinocyte cell line). Cell surface expression of MICA was higher than cell surface expression of MICB in the HPV-positive cell lines; in contrast, HPV-negative cells expressed lower levels of MICA. Interestingly, the MICA levels observed in C33-A cells were overcome by significantly higher MICB expression. Also, all cell lines released higher amounts of soluble MICB than of soluble MICA into the cell culture supernatant, although this was most pronounced in C33-A cells. Additionally, Real-Time PCR analysis demonstrated that MICA was strongly upregulated after genotoxic stress.

Conclusions

This study provides evidence that even when MICA and MICB share a high degree of homology at both genomic and protein levels, differential regulation of their expression and cell surface appearance might be occurring in cervical cancer-derived cells.     

Single nucleotide polymorphisms in the PRDX3 and RPS19 and risk of HPV persistence and cervical precancer/cancer

Background

Host genetic factors might affect the risk of progression from infection with carcinogenic human papillomavirus (HPV), the etiologic agent for cervical cancer, to persistent HPV infection, and hence to cervical precancer and cancer.

Methodology/Principal Findings

We assessed 18,310 tag single nucleotide polymorphisms (SNPs) from 1113 genes in 416 cervical intraepithelial neoplasia 3 (CIN3)/cancer cases, 356 women with persistent carcinogenic HPV infection (median persistence of 25 months) and 425 randomly selected women (non-cases and non-HPV persistent) from the 10,049 women from the Guanacaste, Costa Rica HPV natural history cohort. For gene and SNP associations, we computed age-adjusted odds ratio and p-trend. Three comparisons were made: 1) association with CIN3/cancer (compared CIN3/cancer cases to random controls), 2) association with persistence (compared HPV persistence to random controls), and 3) progression (compared CIN3/cancers with HPV-persistent group). Regions statistically significantly associated with CIN3/cancer included genes for peroxiredoxin 3 PRDX3, and ribosomal protein S19 RPS19. The single most significant SNPs from each gene associated with CIN3/cancer were PRDX3 rs7082598 (P _trend<0.0001), and RPS19 rs2305809 (P _trend=0.0007), respectively. Both SNPs were also associated with progression.

Conclusions/Significance

These data suggest involvement of two genes, RSP19 and PRDX3, or other SNPs in linkage disequilibrium, with cervical cancer risk. Further investigation showed that they may be involved in both the persistence and progression transition stages. Our results require replication but, if true, suggest a role for ribosomal dysfunction, mitochondrial processes, and/or oxidative stress, or other unknown function of these genes in cervical carcinogenesis.     

TriVax-HPV: an improved peptide-based therapeutic vaccination strategy against human papillomavirus-induced cancers

Background

Therapeutic vaccines for cancer are an attractive alternative to conventional therapies, since the later result in serious adverse effects and in most cases are not effective against advanced disease. Human papillomavirus (HPV) is responsible for several malignancies such as cervical carcinoma. Vaccines targeting oncogenic viral proteins like HPV16-E6 and HPV16-E7 are ideal candidates to elicit strong immune responses without generating autoimmunity because: (1) these products are not expressed in normal cells and (2) their expression is required to maintain the malignant phenotype. Our group has developed peptide vaccination strategy called TriVax, which is effective in generating vast numbers of antigen-specific T cells in mice capable of persisting for long time periods.

Materials and methods

We have used two HPV-induced mouse cancer models (TC-1 and C3.43) to evaluate the immunogenicity and therapeutic efficacy of TriVax prepared with the immunodominant CD8 T-cell epitope HPV16-E7_49-57, mixed with poly-IC adjuvant and costimulatory anti-CD40 antibodies.

Results

TriVax using HPV16-E7_49-57 induced large and persistent T-cell responses that were therapeutically effective against established HPV16-E7 expressing tumors. In most cases, TriVax was successful in attaining complete rejections of 6–11-day established tumors. In addition, TriVax induced long-term immunological memory, which prevented tumor recurrences. The anti-tumor effects of TriVax were independent of NK and CD4 T cells and, surprisingly, did not rely to a great extent on type-I or type-II interferon.

Conclusions

These findings indicate that the TriVax strategy is an appealing immunotherapeutic approach for the treatment of established viral-induced tumors. We believe that these studies may help to launch more effective and less invasive therapeutic vaccines for HPV-mediated malignancies.