Novel interaction partners of cutaneous HPV types and their role in skin cancer

Ultraviolet (UV) irradiation and UVB in particular, is the major environmental risk factor for the development of skin cancer. Furthermore, an increasing body of evidence supports a role for cutaneous human papillomaviruses (HPVs) in combination with UV irradiation in the development of cutaneous squamous cell carcinoma. Moreover, cutaneous HPV E6 proteins have been described to indirectly influence pathways controlled by p53, to inhibit UV-induced apoptosis and to prolong the life span of keratinocytes, the natural host of all HPVs. The present study identified the LIM protein Ajuba as a novel interaction partner of the E6 protein of the beta2 HPV23, the gamma1 HPV4 and the gamma11 HPV148. These E6 proteins were shown to directly interact with Ajuba both in vitro and in vivo as well as to co-localise with Ajuba in the cytoplasm. Furthermore, the E6 proteins of HPV23 and HPV148 showed a coincidental decrease in protein expression together with their interaction partner Ajuba upon DNA damage induction and siRNA-mediated Ajuba knockdown. This knockdown also revealed that the p53 protein was involved in the regulation of Ajuba and HPV E6 protein levels. On the other hand, Ajuba co-accumulated following overexpression of the cutaneous E6 proteins, further pointing to a close interaction of both proteins in vivo. A trimeric complex formation between p53, Ajuba and HPV148 E6 was additionally discovered, which did not take place with HPV23 E6. Moreover, p53 was shown to co-localise and to interact directly with HPV148 E6 in vitro and in vivo, even in the absence of Ajuba. A functional luciferase reporter assay revealed a strong repression of p53 activity in the presence of HPV148 E6, demonstrating an impairment of the transactivation activity of p53. This observation could however not be reported for HPV23 E6. In summary, the present study identified the LIM protein Ajuba as a novel interaction partner of cutaneous E6 proteins. Additionally, it demonstrated for the first time that the p53 protein not only binds to cutaneous HPV E6 proteins in vitro, but also interacts and co-localises with HPV148 E6 in vivo, subsequently repressing p53 activity. These findings suggest a novel regulatory mechanism elicited by cutaneous E6 proteins and further strengthen the hypothesis that cutaneous HPVs can act as co-factors in the development of skin cancer

Eyebrow hairs from actinic keratosis patients harbor the highest number of cutaneous human papillomaviruses

BACKGROUND: Cutaneous human papillomavirus (HPV) infections seem to be associated with the onset of actinic keratosis (AK). This study compares the presence of cutaneous HPV types in eyebrow hairs to those in tissues of normal skin and skin lesions of 75 immunocompetent AK patients. METHODS: Biopsies from AK lesions, normal skin and plucked eyebrow hairs were collected from each patient. DNA from these specimens was tested for the presence of 28 cutaneous HPV (betaPV and gammaPV) by a PCR based method. RESULTS: The highest number of HPV prevalence was detected in 84% of the eyebrow hairs (63/75, median 6 types) compared to 47% of AK lesions (35/75, median 3 types) (p< 0.001) and 37% of normal skin (28/75, median 4 types) (p< 0.001), respectively. A total of 228 HPV infections were found in eyebrow hairs compared to only 92 HPV infections in AK and 69 in normal skin. In all three specimens HPV20, HPV23 and/or HPV37 were the most prevalent types. The highest number of multiple types of HPV positive specimens was found in 76% of the eyebrow hairs compared to 60% in AK and 57% in normal skin. The concordance of at least one HPV type in virus positive specimens was 81% (three specimens) and 88-93% of all three combinations with two specimens. CONCLUSIONS: Thus, eyebrow hairs revealed the highest number of cutaneous HPV infections, are easy to collect and are an appropriate screening tool in order to identify a possible association of HPV and AK

The management of desmoid tumours: A joint global consensus-based guideline approach for adult and paediatric patients

Abstract Desmoid tumor (DT; other synonymously used terms: Desmoid-type fibromatosis, aggressive fibromatosis) is a rare and locally aggressive monoclonal, fibroblastic proliferation characterised by a variable and often unpredictable clinical course. Previously surgery was the standard primary treatment modality; however, in recent years a paradigm shift towards a more conservative management has been introduced and an effort to harmonise the strategy amongst clinicians has been made. We present herein an evidence-based, joint global consensus guideline approach to the management of this disease focussing on: molecular genetics, indications for an active treatment, and available systemic therapeutic options. This paper follows a one-day consensus meeting held in Milan, Italy, in June 2018 under the auspices of the European Reference Network for rare solid adult cancers, EURACAN, the European Organisation for Research and Treatment of Cancer (EORTC) Soft Tissue and Bone Sarcoma Group (STBSG) as well as Sarcoma Patients EuroNet (SPAEN) and The Desmoid tumour Research Foundation (DTRF). The meeting brought together over 50 adult and pediatric sarcoma experts from different disciplines, patients and patient advocates from Europe, North America and Japan

Post-translational control of IL-1β via the human papillomavirus type 16 E6 oncoprotein: a novel mechanism of innate immune escape mediated by the E3-ubiquitin ligase E6-AP and p53.

Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1β) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1β production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1β processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1β regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1β was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1β that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1β is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1β levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1β regulation which ultimately inhibits the secretion of IL-1β in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1β towards cervical cancer could be discerned. Hence, attenuation of IL-1β by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy

Expression and secretion of IL-1β in human primary keratinocytes and HPV-positive cell lines.

<p>A) Quantification of secreted IL-1β (expressed as pg/ml) by ELISA in human primary keratinocytes (PK), keratinocytes immortalized by individual oncogenes (E6, E7, E6/E7) and HPV16/18-positive cervical carcinoma cell lines (SiHa, CaSki, HeLa, respectively) 24 h after infection with a recombinant GFP-expressing adenovirus 5 (Ad) in comparison to uninfected cells. B) Quantification of basal intracellular IL-1β levels by ELISA. C) Western blot analysis of pro-IL-1β in human primary keratinocytes (PK) and HPV-positive cells. Actin was used as a loading control. D) qPCR analysis of IL-1β cDNA obtained from PK and HPV-positive cells (Ordinate: expressed as fold changes using the average IL-1β steady state level of 3 different primary human keratinocyte preparations PK cells as a reference which was arbitrarily set as 1. The graphs in A, B and D represent the mean values (± SEM) of three independent experiments. ANOVA ***p<0.05.</p

Analysis of IL-1β expression in normal and HPV16-positive cervical tissues.

<p>A) Immunohistochemical analysis of IL-1β expression in normal epithelium and HPV-positive lesions differing in their progression grades CIN I to CIN III and cervical tumors; scale bars represent 25 µm. B) qPCR analysis of IL-1β cDNA derived from samples negative for intraepithelial lesion and malignancy (NT), different HPV-positive lesions (CIN I, II, III) and cervical tumors (CxCa); ordinate: expression as fold changes using SiHa cells as reference which was arbitrarily set as 1. The pictures in A are a representative example of 25 biopsies analyzed from normal tissue (n = 5) and HPV-positive lesions of different donors (n = 25). The box-and-whisker blot in B represents the mean values from three to five samples for each group depicted in the graph (± SEM). ANOVA *p<0.01.</p

Caspase-1 activity did not account for decreased IL-1β in E6-positive cells.

<p>A) RT-PCR analysis of caspase-1 mRNA in comparison to the GAPDH steady state levels in PK and HPV-positive immortalized keratinocytes. B) Fluorometric measurement of caspase-1 activity was performed incubating the cells for 4 h at 37°C with 20 µM of the specific caspase-1 substrate R110-YVAD. RFU: Relative fluorescence units. C) Quantification of intracellular IL-1β levels by ELISA after 5 h of caspase-1 inhibition using 250 nM of caspase-1 inhibitor. The graphs in B and C represent the mean values (± SEM) of three independent experiments. ANOVA **p<0.01, ***p<0.001.</p

Proteasome inhibition or knock-down of E6-AP increases the levels of pro-IL-1β in immortalized E6-positive cells.

<p>A) ELISA of intracellular IL-1β from untreated immortalized HPV-positive cells and cells incubated with 10 µM of the proteasome inhibitor MG132 for 6 h. B) Western blot analysis of pro-IL1β and p53 in immortalized keratinocytes after inhibition of deubiquitinases using PR-619 (10 µM) for 6 h prior to protein extraction. C) Detection of poly-ubiquitinated pro-IL-1β in immortalized keratinocytes by Western blotting. Cells were treated with MG132 for 6 h prior to protein extraction and pull-down of ubiquitinated proteins was performed by the tandem ubiquitin-binding entities technique (TUBE-PD, right panel). Left panel: input, representing 2.5% of the total protein extract. D) Confocal microscopy analysis of IL-1β (green) in immE6 cells after knock-down of E6-AP by siRNA or scrambled siRNA delivery used as a control. Nuclei (blue) were stained using Hoechst dye solution; scale bars represent 10 µm. E) Western blot analysis of pro-IL-1β after the knock-down of E6-AP. F) ELISA of intracellular IL-1β from immortalized HPV-positive cells after the knock-down of E6-AP by siRNA (+) or control knock-down.(−) G) ELISA of IL-1β secretion from immortalized HPV-positive cells after E6-AP knock-down and/or after NALP3 inflammasome activation using 50 µM of nigericine for 6 h. H) Knock-down of p53 and/or E6-AP in immortalized cells. Cells were transfected with 30 pmoles of the respective siRNA against E6-AP or p53 and incubated for 72 h prior to protein extraction and Western blot analysis. The bar graphs shown in A (ANOVA ***p<0.05), D (ANOVA ***p<0.001, **p<0.01) F and G (ANOVA ***p<0.001) represent the mean levels (± SEM) of three independent experiments.</p