Cytokeratin-17 expression is commonly observed in keratinocytic skin tumours and controls tissue homeostasis impacting HPV protein expression.

BACKGROUND: The structured expression of several keratins in the skin is associated with differentiation status of the epidermal layers, whereas others are upregulated only during wound healing, in skin disorders and in cancers. One of these stress keratins, K17, is correlated with poor prognosis in various cancer types and its loss has been shown to decelerate tumour growth. K17 expression can also be detected in cutaneous squamous cell carcinomas (SCCs), where UV-irradiation and infection with cutaneous human papillomaviruses (HPVs) are important co-factors. It was previously reported that K17 is upregulated in papillomavirus (PV)-induced benign skin lesions in mice and induces an immunological status that is beneficial for tumour growth. OBJECTIVES: In order to investigate whether K17 upregulation is induced by PVs, we analysed K17 levels in skin tumour specimens of different animal models and humans. METHODS: Various immunofluorescence stainings were performed to identify K17 expression as well as levels of E-Cadherin, vimentin and CD271. Tissues were further analysed by PCRs, qPCRs and ELISA to control for PV activity. K17knockdown cells were generated and effects on viral life cycle were investigated by infection assays, qPCR and Western blotting. RESULTS: We could show that K17 is commonly expressed in skin tumours and that its presence is not directly linked to viral oncoprotein expression. Rather, K17 expression seems to be a marker of epithelial differentiation and its absence in tumour tissue is associated with an epithelial-to-mesenchymal transition. We further showed that the absence of K17 in skin tumours increases markers of cancer stem-like cells and negatively affects viral protein synthesis. CONCLUSIONS: Collectively, our data indicate that K17 expression is a common feature in skin tumourigenesis. While it is not primarily targeted by PV oncoproteins, our in vivo and in vitro data suggest that it is an important regulator of epithelial differentiation and thus may play a role in controlling viral protein synthesis.

Viruses and phospholipids: Friends and foes during infection.

Viruses have evolved complex and dynamic interactions with their host cells to enable viral replication. In recent years, insights have been gained into the increasingly important role of the host cell lipidome in the life cycle of several viruses. In particular, viruses target phospholipid signaling, synthesis, and metabolism to remodel their host cells into an optimal environment for their replication cycle. Conversely, phospholipids and their associated regulatory enzymes can interfere with viral infection or replication. This review highlights examples of different viruses that illustrate the importance of these diverse virus-phospholipid interactions in different cellular compartments, particularly the role of nuclear phospholipids and their association with human papillomavirus (HPV)-mediated cancer development.

MAML1-induced HPV E6 oncoprotein stability is required for cellular proliferation and migration of cervical tumor-derived cells.

While a small proportion of high-risk (HR) alpha (α) human papillomaviruses (HPVs) is associated with numerous human malignancies, of which cervical cancer is the most prevalent, beta (ß) HPVs predominantly act as co-factors in skin carcinogenesis. A characteristic feature of both α- and ß-E6 oncoproteins is the presence of the LXXLL binding motif, which α-E6s utilize to form a complex with E6AP and which enables ß-E6s to interact with MAML1. Here we show that multiple α-E6 oncoproteins bind to MAML1 via the LXXLL binding motif and that this results in increased protein stability. Moreover, ß-E6 oncoprotein stability is also dependent on the interaction with MAML1. Additionally, in the absence of MAML1, endogenous HPV-8 E6 and HPV-18 E6 are rapidly degraded at the proteasome. Ablation of both E6AP and MAML1 leads to an even more profound downregulation of α-E6 protein expression, whereas this is not observed with ß-E6. This highly suggests that there is one cellular pool for most of ß-E6 that interacts solely with MAML1, whereas there are two cellular pools of HR α-E6, one forming a complex with MAML1 and the other interacting with E6AP. Furthermore, MAML1 induces HPV-8 E6 shuttling from the nucleus to the cytosolic fraction, while MAML1 interaction with HR E6 induces a drastic nuclear and membrane upregulation of E6. Interestingly, the HR α-E6/MAML1 complex does not affect targeting of some of the known HR E6 cellular substrates such as p53 and DLG1. However, MAML1 and E6AP joint co-expression with HR α-E6 leads to a significant increase in cellular proliferation, whereas silencing MAML1 decreases wound closure in HeLa cells. These results demonstrate that HR α-E6 interaction with MAML1 results in a stable form of E6, which likely modulates MAML1's normal cellular activities, one consequence of which being an increased proliferative capacity of HPV-transformed cancer cells. Thus, this study shows a novel function of the α-E6 oncoprotein and how it's activity might affect HPV-induced pathogenesis.

Subacute thyroiditis after SARS-Cov2 vaccination: A review of the cases being described and personal experience.

Objective. The present study evaluates the occurrence of subacute thyroiditis in temporal connection with SARS-Cov2 vaccinations described in the literature last year and confirmed by our clinical routine. Methods. Systematic literature search in Medline for studies reporting diagnosis of subacute thyroiditis in temporal connection with vaccinations against Covid 19. Results. The literature search yielded 24 relevant references out of which 22 were "case reports" and two "Letters to the Editor" and encompassed 37 patient cases, in total. They had received a SARS-Cov2 vaccination shortly before the diagnosis (median interval to vaccination six days). In none of these cases, infection of the upper respiratory tract had previously been identified as a classic trigger of the disease. Newly occurring hyperthyroidism and increased laboratory signs of inflammation were described in 78% and 74% of cases, respectively. Atypical clinical pictures (asymptomatic, euthyroid, no inflammation marks) have been observed in both the literature and our patients suspected of thyroid cancer referred to surgery. Conclusions. In times of pandemics and the resulting vaccination, new rapidly occurring sonographic changes in the thyroid gland should be revaluated after 2-3 weeks, or recommended to undergo a fine-needle biopsy, in order to avoid unnecessary surgical interventions.

[Role of human papillomavirus (HPV) in the development of skin cancer]. / Rolle von humanen Papillomviren (HPV) in der Entwicklung von Hautkarzinomen.

The incidence of nonmelanoma skin cancer, the most common cancer in humans, continues to rise. The development of precancerous actinic keratoses and cutaneous squamous cell carcinoma (cSCC) is associated with infection with human papillomavirus (HPV) of genus beta (betaHPV). Persistent betaHPV infections in immunocompetent individuals are generally very well controlled by the immune system and largely asymptomatic. However, immunosuppression results in high levels of betaHPV in the skin and consequently increased viral oncoprotein activity, which in turn leads to a significantly increased risk for skin cancer. However, even in immunocompetent individuals, the risk of cSCC increases with age as a result of accumulated UV-induced DNA damage in the skin. In these patients, the mechanism of betaHPV-dependent carcinogenesis seems to be different from that observed in immunocompromised patients. The underlying mechanism of oncogenesis in immunocompetent patients is currently less well understood. This review summarizes the current research data, which provide compelling evidence that cutaneous papillomaviruses, particularly in interaction with UV light, promote skin carcinogenesis via a "hit-and-run" mechanism by enhancing the genotoxic effects of UV light in the initial phases of this multistep process. Furthermore, an overview of novel vaccination strategies against papillomaviruses that are currently tested in clinical trials is provided, which could significantly improve the treatment options for high-risk patients in the future.

Syphilis seroprevalence among HIV-infected males in Istanbul, Turkey.

Syphilis has become a serious issue for human immunodeficiency virus (HIV)-infected patients worldwide in recent years; however, the studies related to HIV coinfection and syphilis reinfections in Istanbul, Turkey, are limited. Our objective was to determine the seroprevalence of syphilis among HIV-infected men in the city which has one of the highest HIV prevalence rates in Turkey. Two hundred and forty four (244) HIV-positive men were evaluated at Istanbul Medical Faculty, Department of Medical Microbiology from March to June 2018. Serum samples were screened for the presence of antibodies against Treponema pallidum using the chemiluminescent microparticle immunoassay (CMIA). Samples found to be positive were investigated with the rapid plasma reagin (RPR) test and the T. pallidum hemagglutination assay (TPHA). The patients completed a questionnaire for sociodemographic data. The mean age was found to be 41.8 years; 35.6% were men who have sex with men (MSM). The overall seroprevalence of syphilis among the patients was 19.3%. MSM had a significantly higher seroprevalence than heterosexual patients (28.7%). In Turkey, there is a high seroprevalence of syphilis in HIV-infected patients, MSM being the most affected group. Therefore, HIV-infected patients should be screened for syphilis at least annually and should be informed about sexually transmitted diseases (STDs).

No Evidence for Role of Cutavirus in Malignant Melanoma.

Cutavirus was previously found in cutaneous melanoma. We detected cutavirus DNA in only 2/185 melanoma biopsies and in 0/52 melanoma metastases from patients in Germany. Viral DNA was localized in the upper epidermal layers. Swab specimens from healthy skin were cutavirus positive for 3.8% (9/237) of immunocompetent and 17.1% (35/205) of HIV-positive men.

Human papillomavirus type 8 oncoproteins E6 and E7 cooperate in downregulation of the cellular checkpoint kinase-1.

Human papillomavirus 8 (HPV8) is associated with the development of squamous cell carcinoma (SCC) of the skin. HPV-infected keratinocytes are able to override normal checkpoint control mechanisms and sustain cell cycle activity, allowing for synthesis of cellular proteins necessary for viral genome amplification. To study how HPV8 may disrupt cell cycle control, we analyzed the impact of HPV8 early gene expression on one of the key regulators of cell cycle and DNA damage response, checkpoint kinase-1 (CHK1). We found that expression of E1, E1̂E4, E2, E6 or E7 individually did not affect CHK1; however, keratinocytes expressing the complete early genome region (CER) of HPV8 showed a profound loss of CHK1 protein levels, that proved to be mediated by E6E7 co-expression. Neither CHK1 promoter regulation nor the ubiquitin-proteasome pathway are involved in HPV8-mediated CHK1 repression. However, CHK1 protein repression in organotypic skin cultures was paralleled by downregulation of the autophagy marker LC3B. Treatment of HPV8-CER expressing cells with the autophagy inhibitor Bafilomycin A1 rescued CHK1 expression and led to LC3B accumulation. Taken together, our data implicate that CHK1 autophagic degradation is enhanced by HPV8, which may contribute to the oncogenic potential of the virus.

The interplay of UV and cutaneous papillomavirus infection in skin cancer development.

Cutaneous human papillomaviruses (HPVs) are considered as cofactors for non-melanoma skin cancer (NMSC) development, especially in association with UVB. Extensively studied transgenic mouse models failed to mimic all aspects of virus-host interactions starting from primary infection to the appearance of a tumor. Using the natural model Mastomys coucha, which reflects the human situation in many aspects, we provide the first evidence that only UVB and Mastomys natalensis papillomavirus (MnPV) infection strongly promote NMSC formation. Using UVB exposures that correspond to UV indices of different geographical regions, irradiated animals developed either well-differentiated keratinizing squamous cell carcinomas (SCCs), still supporting productive infections with high viral loads and transcriptional activity, or poorly differentiated non-keratinizing SCCs almost lacking MnPV DNA and in turn, early and late viral transcription. Intriguingly, animals with the latter phenotype, however, still showed strong seropositivity, clearly verifying a preceding MnPV infection. Of note, the mere presence of MnPV could induce γH2AX foci, indicating that viral infection without prior UVB exposure can already perturb genome stability of the host cell. Moreover, as shown both under in vitro and in vivo conditions, MnPV E6/E7 expression also attenuates the excision repair of cyclobutane pyrimidine dimers upon UVB irradiation, suggesting a viral impact on the DNA damage response. While mutations of Ras family members (e.g. Hras, Kras, and Nras) were absent, the majority of SCCs harbored-like in humans-Trp53 mutations especially at two hot-spots in the DNA-binding domain, resulting in a loss of function that favored tumor dedifferentiation, counter-selective for viral maintenance. Such a constellation provides a reasonable explanation for making continuous viral presence dispensable during skin carcinogenesis as observed in patients with NMSC.

BetaHPV E6 and E7 colocalize with NuMa in dividing keratinocytes.

Human papillomaviruses (HPVs) of genus betapapillomavirus (betaHPV) are implicated in skin carcinogenesis, but their exact role in keratinocyte transformation is poorly understood. We show an interaction of HPV5 and HPV8 oncoproteins E6 and E7 with the nuclear mitotic apparatus protein 1 (NuMA). Binding of E6 or E7 to NuMA induces little aneuploidy, cell cycle alterations, or aberrant centrosomes. Intracellular localization of NuMA is not altered by E6 and E7 expression in 2D cultures. However, the localization profile is predominantly cytoplasmic in 3D organotypic skin models. Both viral proteins colocalize with NuMA in interphase cells, while only E7 colocalizes with NuMA in mitotic cells. Intriguingly, a small subset of cells shows E7 at only one spindle pole, whereas NuMA is present at both poles. This dissimilar distribution of E7 at the spindle poles may alter cell differentiation, which may in turn be relevant for betaHPV-induced skin carcinogenesis.

HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer.

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV+ OPSCC metastasize faster than HPV- tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV+ OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44high /EpCAMhigh ) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44high /EpCAMlow ) CSC pool. This increase in migratory CSCs could also be confirmed in HPV+ OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44high /EpCAMlow CSCs were validated by RT-qPCR and in situ hybridization on HPV16+ OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16+ OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV+ and HPV- metastasis. As inhibition of this miRNA led to a markedly reduction of CD44high /EpCAMlow cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC.

Human polyomavirus and human papillomavirus prevalence and viral load in non-malignant tonsillar tissue and tonsillar carcinoma.

Human papillomaviruses (HPVs) are an acknowledged cause of a subset of oropharyngeal cancers, especially of tonsillar cancer. Similar to HPV, some human polyomaviruses (HPyVs), such as Merkel cell polyomavirus (MCPyV), have an oncogenic potential. Recently, several novel HPyVs have been discovered. The aim of our study was to determine viral DNA prevalence and viral DNA load of 13 different HPyVs in benign and malignant tonsillar tissue and to compare the data with those found for HPV. A total of 78 biopsies of palatine tonsils with a histologic diagnosis of non-malignant disease (chronic tonsillitis, tonsillar hyperplasia, n = 40) or tonsillar squamous cell carcinoma (n = 38) were included in the study. HPyV DNA prevalence and viral load were determined by virus-specific quantitative real-time PCRs. JCPyV (1/40, 2.5%) and WUPyV (3/40, 7.5%) were only found in non-malignant tonsillar tissue. HPyV7 and HPyV10 were only detected in one (2.6%) and seven (18.4%) of the 38 cancer biopsies, respectively. Both MCPyV (8/38, 21.1 vs. 4/40, 10.0%) and HPyV6 (2/38, 5.3 vs. 1/40, 2.5%) were found more frequently in cancer samples than in non-malignant tissue, but the differences were not significant. BKPyV, KIPyV, TSPyV, HPyV9, STLPyV, HPyV12 and NJPyV were not discovered in any of the samples. HPyV loads found in HPyV DNA-positive biopsies were very low with no difference between non-malignant and malignant samples (median load <0.0001 HPyV DNA copies per beta-globin gene copy, respectively). In contrast to HPyV, high-risk HPV types (HPV16/HPV18) were found significantly more frequently in tonsillar cancers than in non-malignant tonsillar tissue (17/38, 44.7 vs. 2/40, 5.0%, p < 0.001). Furthermore, high-risk HPV DNA loads were significantly higher in the cancer compared to the non-malignant samples (median load 11.861 vs. 7 × 10-6 HPV DNA copies per beta-globin gene copy, p = 0.012). While both HPV and HPyV may persist in tonsillar tissue, our data on HPyV DNA prevalence and load do not support a role of HPyV in tonsillar carcinogenesis, neither alone nor as co-infecting agents of HPV.

The levels of epithelial anchor proteins ß-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

Infection with viruses of the genus Betapapillomavirus, ß-human papillomaviruses (ß-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins ß-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that ß-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of ß-catenin was elevated, no signs of ß-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of ß-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that ß-catenin and ZO-1 are direct targets of E7 of the oncogenic ß-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin.

Human papillomavirus mediated inhibition of DNA damage sensing and repair drives skin carcinogenesis.

BACKGROUND: The failure to mount an effective DNA damage response to repair UV induced cyclobutane pyrimidine dimers (CPDs) results in an increased propensity to develop cutaneous squamous cell carcinoma (cSCC). High-risk patient groups, such as organ transplant recipients (OTRs) frequently exhibit field cancerization at UV exposed body sites from which multiple human papillomavirus (HPV)-associated cSCCs develop rapidly, leading to profound morbidity and increased mortality. In vitro molecular evidence indicates that HPV of genus beta-papillomavirus (ß-PV) play an important role in accelerating the early stages of skin tumorigenesis. METHODS: We investigated the effects of UV induced DNA damage in murine models of ß-PV E6 oncoprotein driven skin tumorigenesis by crossing K14-HPV8-E6wt mice (developing skin tumors after UV treatment) with K14-CPD-photolyase animals and by generating the K14-HPV8-E6-K136N mutant mouse strain. Thymine dimers (marker for CPDs) and γH2AX (a marker for DNA double strand breaks) levels were determined in the murine skin and organotypic skin cultures of E6 expressing primary human keratinocytes after UV-irradiation by immunohistochemistry and in cell lines by In Cell Western blotting. Phosphorylation of ATR/Chk1 and ATM were assessed in cell lines and organotypic skin cultures by Western blots and immunohistochemistry. RESULTS: Skin tumor development after UV-irradiation in K14-HPV8-E6wt mice could completely be blocked through expression of CPD-photolyase. Through quantification of thymine dimers after UV irradiation in cells expressing E6 proteins with point mutations at conserved residues we identified a critical lysine in the C-terminal part of the protein for prevention of DNA damage repair and p300 binding. Whereas all K14-HPV8-E6wt animals develop skin tumors after UV expression of the HPV8-E6-K136N mutant significantly blocked skin tumor development after UV treatment. The persistence of CPDs in hyperproliferative epidermis K14-HPV8-E6wt skin resulted in the accumulation of γH2AX foci. DNA damage sensing was impaired in E6 positive cells grown as monolayer culture and in organotypic cultures, due to lack of phosphorylation of ATM, ATR and Chk1. CONCLUSION: We showed that cells expressing E6 fail to sense and mount an effective response to repair UV-induced DNA lesions and demonstrated a physiological relevance of E6-mediated inhibition of DNA damage repair for tumor initiation. These are the first mechanistical in vivo data on the tumorigenicity of HPV8 and demonstrate that the impairment of DNA damage repair pathways by the viral E6 protein is a critical factor in HPV-driven skin carcinogenesis.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia.

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

HIV-1 subtypes and drug resistance profiles in a cohort of heterosexual patients in Istanbul, Turkey.

Turkey is seeing a steady rise in rates of HIV infection in the country. The number of individuals with HIV/AIDS was greater than 7000 in 2014 according to data released by the Ministry of Health, and heterosexual contacts were reported to be the main transmission routes. Istanbul has the highest number of reported cases of HIV infection. The aim of the study was to determine the prevalence of HIV-1 drug resistance in 50 heterosexual patients from Istanbul. The most prevalent subtype was found to be subtype B (56.2 %). Resistance-associated mutations were found in 14 patients with 6/14 patients being therapy-experienced and 8/14 therapy naive at the time point of analysis. With increasing number of patients who require treatment and the rapid up-scaling of the antiretroviral therapy in Turkey, HIV-1 drug resistance testing is recommended before starting treatment in order to achieve better clinical outcomes.

Expression of betapapillomavirus oncogenes increases the number of keratinocytes with stem cell-like properties.

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a "stem cell-like" state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44(high) EpCAM(high) cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.