El tratamiento con 5azadC regula al alza el nivel de miR-375 y reprime la expresión de HPV16 E6

High-risk human papillomaviruses are the etiological agents of cervical cancer and HPV16 is the most oncogenic genotype. Immortalization and transformation of infected cells requires the overexpression of the two viral oncoproteins E6 and E7 following HPV DNA integration into the host cell genome. Integration often leads to the loss of the E2 open reading frame and the corresponding protein can no longer act as a transcriptional repressor on p97 promoter. Recently, it has been proposed that long control region methylation also contributes to the regulation of E6/E7 expression. To determine which epigenetic mechanism is involved in HPV16 early gene regulation, 5-aza-2?-deoxycytidine was used to demethylate Ca Ski and SiHa cell DNA. Decreased expression of E6 mRNA and protein levels was observed in both cell lines in an E2-independent manner. E6 repression was accompanied by neither a modification of the main cellular transcription factor expression involved in long control region regulation, nor by a modification of the E6 mRNA splicing pattern. In contrast, a pronounced upregulation of miR-375, known to destabilize HPV16 early viral mRNA, was observed. Finally, the use of miR-375 inhibitor definitively proved the involvement of miR-375 in E6 repression. These results highlight that cellular DNA methylation modulates HPV16 early gene expression and support a role for epigenetic events in high-risk HPV associated-carcinogenesis

The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD. Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery.This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub. This work was funded by Cancer Research UK (C47718/A16337, O.F.), the Medical Research Council (RG89611, R.B.) and the BBSRC Institute Strategic Programme Gut Health and Food Safety (BB/J004529/1)

Metilación de sitios CpG del virus del papiloma humano tipo 16 en el sitio de unión 1 de E2 (E2BS1), E2BS2 y el sitio de unión de Sp1 en muestras de cáncer de cuello uterino, según lo determinado por análisis de fusión de alta resolución-PCR

High-risk (HR) human papillomavirus (HPV)-associated carcinogenesis is driven mainly by the overexpression of E7 and E6 oncoproteins following viral DNA integration and the concomitant loss of the E2 open reading frame (ORF). However, the integration of HR-HPV DNA is not systematically observed in cervical cancers. The E2 protein acts as a transcription factor that governs viral oncogene expression. The methylation of CpGs in the E2-binding sites (E2BSs) in the viral long control region abrogates E2 binding, thus impairing the E2-mediated regulation of E7/E6 transcription. Here, high-resolution melting (HRM)–PCR was developed to quantitatively analyze the methylation statuses of E2BS1, E2BS2, and the specificity protein 1 (Sp1)-binding site in 119 HPV16-positive cervical smears. This is a rapid assay that is suitable for the analysis of cervical samples. The proportion of cancer samples with methylated E2BS1, E2BS2, and Sp1-binding site CpGs was 47%, whereas the vast majority of samples diagnosed as being within normal limits, low-grade squamous intraepithelial lesions (LSIL), or high-grade squamous intraepithelial lesions (HSIL) harbored unmethylated CpGs. Methylation levels varied widely, since some cancer samples harbored up to 60% of methylated HPV16 genomes. A pyrosequencing approach was used as a confirmation test and highlighted that quantitative measurement of methylation can be achieved by HRM-PCR. Its prognostic value deserves to be investigated alone or in association with other biomarkers. The reliability of this single-tube assay offers great opportunities for the investigation of HPV16 methylation in other HPV-related cancers, such as head and neck cancers, which are a major public health burden

Apoptotic HPV Positive Cancer Cells Exhibit Transforming Properties

Previous studies have shown that DNA can be transferred from dying engineered cells to neighboring cells through the phagocytosis of apoptotic bodies, which leads to cellular transformation. Here, we provide evidence of an uptake of apoptotic-derived cervical cancer cells by human mesenchymal cells. Interestingly, HeLa (HPV 18+) or Ca Ski (HPV16+) cells, harboring integrated high-risk HPV DNA but not C-33 A cells (HPV-), were able to transform the recipient cells. Human primary fibroblasts engulfed the apoptotic bodies effectively within 30 minutes after co-cultivation. This mechanism is active and involves the actin cytoskeleton. In situ hybridization of transformed fibroblasts revealed the presence of HPV DNA in the nucleus of a subset of phagocytosing cells. These cells expressed the HPV16/18 E6 gene, which contributes to the disruption of the p53/p21 pathway, and the cells exhibited a tumorigenic phenotype, including an increased proliferation rate, polyploidy and anchorage independence growth. Such horizontal transfer of viral oncogenes to surrounding cells that lack receptors for HPV could facilitate the persistence of the virus, the main risk factor for cervical cancer development. This process might contribute to HPV-associated disease progression in vivo

Cell-Intrinsic Roles for Autophagy in Modulating CD4 T Cell Functions

The catabolic process of autophagy plays important functions in inflammatory and immune responses by modulating innate immunity and adaptive immunity. Over the last decade, a cell-intrinsic role for autophagy in modulating CD4 T cell functions and differentiation was revealed. After the initial observation of autophagosomes in effector CD4 T cells, further work has shown that not only autophagy levels are modulated in CD4 T cells in response to environmental signals but also that autophagy critically affects the biology of these cells. Mouse models of autophagy deletion in CD4 T cells have indeed shown that autophagy is essential for CD4 T cell survival and homeostasis in peripheral lymphoid organs. Furthermore, autophagy is required for CD4 T cell proliferation and cytokine production in response to T cell receptor activation. Recent developments have uncovered that autophagy controls CD4 T cell differentiation and functions. While autophagy is required for the maintenance of immunosuppressive functions of regulatory T cells, it restrains the differentiation of TH9 effector cells, thus limiting their antitumor and pro-inflammatory properties. We will here discuss these findings that collectively suggest that therapeutic strategies targeting autophagy could be exploited for the treatment of cancer and inflammatory diseases

Biomarkers of human papillomavirus-associated carcinogenesis

La persistance d'une infection par un même papillomavirus humain à haut risque oncogène (HPV-HR) est le principal facteur de risque d'apparition et de progression des lésions précancéreuses et cancéreuses du col de l'utérus. La recherche des lésions précancéreuses et l'introduction des tests basés sur la détection des HPV-HR dans le dépistage ont permis de réduire l'incidence du cancer du col de l'utérus dans les pays développés. Un des enjeux actuels réside dans l'identification de nouveaux marqueurs qui permettraient d'améliorer le dépistage. Dans ce contexte, les objectifs de cette thèse étaient d'explorer l'histoire naturelle de l'infection par HPV-HR au travers de facteurs tels que la charge virale, l'intégration, l'expression des oncogènes viraux ou encore la méthylation du promoteur viral à partir de prélèvements biologiques et d'évaluer l'intérêt de ces facteurs en pratique clinique. Après optimisation et validation d'outils de biologie moléculaire, l'analyse de frottis cervico-utérins représentatifs de la progression des lésions du col de l'utérus a permis de mieux comprendre l'histoire naturelle de l'infection transformante par HPV16. Nos données confirment la valeur diagnostique de la charge virale, indiquent une méthylation tardive du promoteur viral et suggèrent que les niveaux d'expression des gènes viraux ne sont pas suffisamment robustes pour présenter un intérêt clinique. D'autre part, nos travaux ont mis en évidence de potentielles analogies de comportement virologique entre génotypes apparentés. Enfin, la caractérisation de tumeurs du canal anal à l'aide de nos marqueurs viraux a permis d'identifier une signature moléculaire des cancers associés à HPV16.The main risk factor of precancerous and cancerous cervical lesions is a persistent infection with the same high-risk HPV (HR-HPV). Screening based on precancerous lesions and HR-HPV detection considerably reduced cervical cancer incidence in developed countries. A current challenge remains the identification of new biomarkers in order to improve cervical cancer screening. This work was conducted with the purpose of (i) exploring HR-HPV infection natural history through viral factors such as viral load, integration, viral oncogene expression and viral promoter methylation and (ii) evaluating the clinical value of these potential markers. After optimization and validation of several molecular biology methods, the analysis of cervical smears representative of the full spectrum of cervical diseases allowed a better knowledge of transforming HPVI6-infection natural history. Our data confirm the potential diagnostic value of viral load. show that HPVI6 promoter methylation seems to be a late event in cervical carcinogenesis, and suggest that viral oncogene expression levels are not robust enough to be transferred to clinical practice. Moreover, our data highlight similar molecular behavior between closely related HR-HPV genotypes. Finally, the characterization of anal tumors with markers identified a molecular signature pattern of HPVI6-associated tumor

Selective autophagy restricts IL-9 secretion from T H 9 cells: relevance in cancer growth

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Nouveaux biomarqueurs viraux des lésions du col de l'utérus associées aux papillomavirus humains.

International audienceHigh-risk human Papillomaviruses (HR-HPV) - the most carcinogenic infectious agents - are responsible for the development of cervical cancer. The knowledge of HPV infection natural history and viral carcinogenesis led to the investigation of viral biomarkers (genotype, viral load, integration, E6/E7 mRNA expression, viral DNA methylation) from clinical samples representative of the evolution of cervical lesions. Mostly concerning HPV16, the literature data agree on an increase of viral load, proportion of samples harboring integrated HPV genomes and methylation of CpG located in the L1 gene with the lesion severity. Viral load and L1 CpG methylation are interesting for clinical practice since appropriate cutoff values allow the identification of precancerous lesions with a high specificity. Although HPV E6/E7 transcript detection is more specific than HPV DNA detection to identify precancerous cervical lesions, viral transcript quantitation and cutoff value determination are unlikely feasible in clinical practice. Taken together, data highlight promising biomarkers that could be integrated to screening algorithms

Real-time duplex PCR for simultaneous HPV 16 and HPV 18 DNA quantitation

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