The role of Microenvironment in tumorigenesis. Focus on dendritic cells in human papillomavirus E6, E7-transformed keratinocytes

The inception of tumor microenvironment (TME), a complex and dynamic system constituted by different types of cells engaged by tumor and extracellular matrix surrounding cancer cells, is a way for them to elude the immune surveillance. Dendritic cells (DCs), as a sentinel, are able to recognize alteration in the microenvironment and predispose the immune system response. The relationship between cancer and virus infection is well documented. High-risk Human Papillomavirus (HR-HPV) has a well-characterized transforming property and has been associated with squamous cell carcinoma of the ano-genital and oral tracts. Transforming ability of HR-HPVs is based on the function of E6 and E7 viral oncoproteins, which interact and inactivate p53 and pRb oncosuppressors, respectively. Recently, it was demonstrated that HPV oncoproteins are also able to affect a number of microRNAs (miRNAs) regulating the expression of genes involved in proliferative control. For these reasons DC-based vaccines targeting oncogenic E6 and E7 are ideal candidates to elicit strong immune responses. Here we summarize significant data about the analysis of TME in HPV-induced tumorigenesis. We also report original results produced by cytotoxic T lymphocyte (CTL) in vitro priming against E6 and E7 HPV16 antigens, performed using human monocyte-derived dendritic cells. Dendritic cells, maturated by the exposition to necrotic or apoptotic keratinocytes expressing both oncoproteins of HPV16, show different expression levels of specific maturation markers. Evidence indicating the ability of necrotic keratinocytes to alter the microRNA profile in DCs, macrophages (MΦ) and Langerhans cells (LCs) compared to prototypical stimuli as bacterial lipopolysaccharide was also provided. We can speculate that, based on transformed cells death pathway (i.e. apoptosis versus necrosis), virus-induced immune alterations might show different results in creating an immunotolerogenic microenvironment during the carcinogenesis process

Rare human papillomavirus 16 E6 variants reveal significant oncogenic potential

The aim of this study was to determine whether low prevalence human papillomavirus (HPV) 16 E6 variants differ from high prevalence types in their functional abilities. We evaluated functions relevant to carcinogenesis for the rarely-detected European variants R8Q, R10G and R48W as compared to the commonly detected L83V. Human immortalized keratinocytes (NIKS) stably transduced with the E6 variants were used in most functional assays. Low and high prevalence E6 variants displayed similar abilities in abrogation of growth arrest and inhibition of p53 elevation induced by actinomycin D. Differences were detected in the abilities to dysregulate stratification and differentiation of NIKS in organotypic raft cultures, modulate detachment induced apoptosis (anoikis) and hyperactivate Wnt signaling. No distinctive phenotype could be assigned to include all rare variants. Like L83V, raft cultures derived from variants R10G and R48W similarly induced hyperplasia and aberrantly expressed keratin 5 in the suprabasal compartment with significantly lower expression of keratin 10. Unlike L83V, both variants, and particularly R48W, induced increased levels of anoikis upon suspension in semisolid medium. R8Q induced a unique phenotype characterized by thin organotypic raft cultures, low expression of keratin 10, and high expression of keratins 5 and 14 throughout all raft layers. Interestingly, in a reporter based assay R8Q exhibited a higher ability to augment TCF/β-catenin transcription. The data suggests that differences in E6 variant prevalence in cervical carcinoma may not be related to the carcinogenic potential of the E6 protein

HPV as a marker for molecular characterization in head and neck oncology: Looking for a standardization of clinical use and of detection method(s) in clinical practice

Background: A consensus about the most appropriate diagnostic method(s) for head and neck human papillomavirus (HPV)\u2010induced carcinogenesis is still lacking because most of the commercially available assays have been designed for the cervix. Methods: This article summarizes current data and trends concerning HPV diagnostic strategies in oropharyngeal squamous cell carcinoma (OPSCC). Six main approaches are described. Results: The diagnostic gold standard for HPV\u2010related OPSCC, focusing on E6/E7 mRNA detection, requires fresh samples. Because most frequently available samples are formalin\u2010fixed paraffin\u2010embedded (FFPE), the pros and cons of the different approaches were analyzed. Conclusions: In the FFPE samples, the immunohistochemistry of p16, which is considered appropriate to assess HPV\u2010driven carcinogenesis in OPSCC according to the 8th American Joint Committee on Cancer TNM classification, may not be specific enough to become the diagnostic standard in the perspective of treatment deintensification. p16 may play a safer role in combination with another highly sensible assay. Other promising approaches are based on DNA detection through real\u2010time polymerase chain reaction and RNAscope

Complete genome sequence of a novel human gammapapillomavirus isolated from a cervical swab in Luxembourg

A novel human papillomavirus genotype was detected in a cervical swab specimen by next-generation sequencing after rolling circular amplification. It was fully cloned and characterized. The L1 open reading frame showed 77% nucleotide similarity with the closest genotype, HPV101, belonging to the gamma-6 species

Epstein-Barr virus down-regulates tumor suppressor DOK1 expression

The DOK1 tumor suppressor gene encodes an adapter protein that acts as a negative regulator of several signaling pathways. We have previously reported that DOK1 expression is up-regulated upon cellular stress, via the transcription factor E2F1, and down-regulated in a variety of human malignancies due to aberrant hypermethylation of its promoter. Here we show that Epstein Barr virus (EBV) infection of primary human B-cells leads to the down-regulation of DOK1 gene expression via the viral oncoprotein LMP1. LMP1 alone induces recruitment to the DOK1 promoter of at least two independent inhibitory complexes, one containing E2F1/pRB/DNMT1 and another containing at least EZH2. These events result in tri-methylation of histone H3 at lysine 27 (H3K27me3) of the DOK1 promoter and gene expression silencing. We also present evidence that the presence of additional EBV proteins leads to further repression of DOK1 expression with an additional mechanism. Indeed, EBV infection of B-cells induces DNA methylation at the DOK1 promoter region including the E2F1 responsive elements that, in turn, lose the ability to interact with E2F complexes. Treatment of EBV-infected B-cell-lines with the methyl-transferase inhibitor 5-aza-2′-deoxycytidine rescues DOK1 expression. In summary, our data show the deregulation of DOK1 gene expression by EBV and provide novel insights into the regulation of the DOK1 tumor suppressor in viral-related carcinogenesis.Fil: Siouda, Maha. World Health Organization; FranciaFil: Frecha, Cecilia Ariana. World Health Organization; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Accardi, Rosita. World Health Organization; FranciaFil: Yue, Jiping. World Health Organization; FranciaFil: Cuenin, Cyrille. World Health Organization; FranciaFil: Grufat, Henri. Inserm; Francia. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Ecole Normale Supérieure; FranciaFil: Manet, Evelyne. Inserm; Francia. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Ecole Normale Supérieure; FranciaFil: Herceg, Zdenko. World Health Organization; FranciaFil: Sylla, Bakary S.. World Health Organization; FranciaFil: Tommasino, Massimo. World Health Organization; Franci

Benign proliferative epithelial lesions of oral mucosa are infrequently associated with α‐, β‐, or γ human papillomaviruses

BackgroundOral papillomas and verruca vulgaris have been associated with human papillomavirus (HPV) infection. However, approximately half of these have remained HPV‐negative when tested for mucosal HPV genotypes. In this study, we evaluated presence of α‐, β‐, and γ‐HPVs in benign papillary and verrucous lesions.MethodsEighty‐three clinical lesions with suspected HPV etiology were analyzed for HPV types of genus α (n = 24), β (n = 46), and γ (n = 52). Immunohistochemistry was used for p16 as a possible surrogate marker of high‐risk HPV, accompanied by Ki‐67 proliferation marker.ResultsAltogether, α‐HPVs were detected in 6.4%, β‐HPVs in 2.4%, and γ‐HPV in 4.8%. The following genotypes were identified: HPV6, 8, 11, 16, 22, 161, and 170. Neither Ki‐67 nor p16 positivity alone were associated with HPV but combined staining showed significant inverse association (P = .042).ConclusionHPV infection is found only in a minority of benign verrucous and papillary oral lesions, with the predominance of α‐HPVs.</div

Prognostic significance of non-HPV16 genotypes in oropharyngeal squamous cell carcinoma

Recent studies have found that cases with oropharyngeal squamous cell carcinoma (OPSCC) positive for HPV16 genotype have better overall survival compared with cases positive for other HPV genotypes. We sought to further replicate these studies and determine if this relationship is modified by expression of p16 tumor suppressor protein

Bariatric surgery-induced weight loss and associated genome-wide DNA-methylation alterations in obese individuals

Obesity is a multifactorial and chronic condition of growing universal concern. It has recently been reported that bariatric surgery is a more successful treatment for severe obesity than other noninvasive interventions, resulting in rapid significant weight loss and associated chronic disease remission. The identification of distinct epigenetic patterns in patients who are obese or have metabolic imbalances has suggested a potential role for epigenetic alterations in causal or mediating pathways in the development of obesity-related pathologies. Specific changes in the epigenome (DNA methylome), associated with metabolic disorders, can be detected in the blood. We investigated whether such epigenetic changes are reversible after weight loss using genome-wide DNA methylome analysis of blood samples from individuals with severe obesity (mean BMI ~ 45) undergoing bariatric surgery

The E6E7 oncoproteins of cutaneous human papillomavirus type 38 interfere with the interferon pathway

Non-melanoma skin cancer is the most frequent malignancy in Caucasian populations. Evidence suggests the involvement of cutaneous Human Papillomavirus (HPV) of the genus beta () in this disease. The ability of E6 and E7 of mucosal HPV to promote cellular transformation and inhibit immune response-related pathways plays a key role in cervical carcinogenesis. HPV-38 E6 and E7 display transforming activities in in vitro and in vivo models, but their impact on immune surveillance is unknown. Here we show that HPV-38 E6 and E7 affect the IFN-induced up-regulation of MHC class I. Expression of the two viral proteins in HaCaT keratinocytes led to a decrease of MHC I levels. This down-regulation is associated with a reduction of expression of MHC I heavy chain, of the peptide chaperone TAP and of the STAT-1 downstream effector IRF-1. The down-regulation of these proteins is ultimately due to the inhibition of STAT-1 expression. Analysis of cells expressing either HPV-38 E6 or E7 suggests that these effects are primarily the result of E6 expression, although a contribution by E7 cannot be excluded. We conclude that HPV-38 encodes oncoproteins that potentially contribute to the evasion of host immune surveillance

Comparison between urine and cervical samples for HPV DNA detection and typing in young women in Colombia

Urine sampling for HPV DNA detection has been proposed as an effective method for monitoring the impact of HPV vaccination programs; however, conflicting results have been reported. The goal of this study was to evaluate the performance of optimized urine HPV DNA testing in women aged 19 to 25 years. Optimization process included the use of first void urine, immediate mixing of urine with DNA preservative, and the concentration of all HPV DNA, including cell-free DNA fragments. Urine and cervical samples were collected from 535 young women attending cervical screening at health centers from two Colombian cities. HPV DNA detection and genotyping was performed using an HPV type-specific multiplex genotyping assay, which combines multiplex polymerase chain reaction with bead-based Luminex technology. Concordance between HPV DNA detection in urine and cervical samples was determined using kappa statistics and McNemar tests. The accuracy of HPV DNA testing in urine samples was evaluated measuring sensitivity and specificity using as reference the results obtained from cervical samples. Statistical analysis was performed using STATA11.2 software. The findings revealed an overall HPV prevalence of 60.00% in cervical samples and 64.72% in urine samples, HPV-16 being the most frequent HPV type detected in both specimens. Moreover, our results indicate that detection of HPV DNA in first void urine provides similar results to those obtained with cervical samples and can be used to monitor HPV vaccination trials and programs as evidenced by the substantial concordance found for the detection of the four vaccine type