Targeted therapy with the BRAF inhibitors vemurafenib and dabrafenib is an effective treatment regimen in patients with advanced melanoma carrying the BRAF V600E mutation. A common side effect is an enhanced rate of nonmelanoma skin cancer (NMSC). BRAF inhibition leads to a paradoxical enhanced MAPK signalling in BRAF wild-type cells, which might in part be responsible for the enhanced NMSC burden. It is known that disturbances of DNA repair result in an increased rate of NMSC. In the present study, it was investigated whether BRAF inhibitors might interfere with the repair of ultraviolet radiation-induced DNA damage in vitro. Epidermal keratinocytes of 11 Caucasian donors were treated with vemurafenib or dabrafenib and, 24 h later, exposed to ultraviolet A. DNA damage and repair capacity were analysed using south-western slot blot detecting cyclobutane pyrimidine dimers. Using PCR and DNA sequencing, RAS mutations and human papilloma virus genes were investigated. RNA expression was determined using a Gene Expression Chip and qRT-PCR. In 36% of keratinocytes, vemurafenib hampers the repair of ultraviolet A-induced DNA damage. No changes in DNA repair were observed with dabrafenib, indicating a possible substance-specific effect of vemurafenib. In none of the keratinocytes, pre-existing RAS mutations or human papilloma virus-associated DNA sequences were detected. The expression of the interferon-related damage resistance signature is decreased upon vemurafenib treatment in 36% of donors. The enhanced rate of NMSC in patients treated with vemurafenib might be partly related to a vemurafenib-driven impaired capacity for DNA repair.

Although infrared radiation (IR) represents more than 50% of the solar radiation reaching the Earth's surface, this waveband has been hardly investigated in terms of tumourigenesis. The objective of the present study was to investigate the influence of IR on ultraviolet B (UVB)-induced carcinogenesis in male and female wild type mice. For this purpose, male and female C57BL/6N mice were subjected to a long-term irradiation protocol. Mice were irradiated once neonatally and from the age of eight weeks for 36 weeks with a cumulative dose of 576 kJ m^−2 UVB and/or 78 895 kJ m^−2 IR. In order to resemble natural sun irradiation, exposure to physiological doses of UVB and IR was performed simultaneously. Mice were screened for arising lesions twice a week. Lesions were excised and histologically diagnosed. KaplanMeier analyses were carried out and lesion counts and cumulated hazard rates for the development of lesions in the UVB and IR + UVB-exposed groups in male and female mice were compared. We found that IR-exposure did not change the number of epithelial malignant tumours in UVB-exposed wild type mice. In combination with IR there was a tendency of more tumours with increased malignancy: 23 vs. seven spindle cell shaped sarcomas and seven vs. two MelanA+/S100+ tumours in groups of 35 C57BL/ 6 mice. IR did not influence UVB-induced carcinogenesis differently in male and female mice. However, comparing UVB and sham irradiated animals irrespective of IR exposure, UVB-induced non-epithelial tumours arose significantly earlier in male mice than in female mice.

The exact correlation between melanoma and sun‐light is still a controversially debated issue. Although natural sunlight contains various ratios of UVA and UVB , most investigators so far focused on the effects of single solar wavebands and neglected possible interactions. Therefore, in this study primary human melanocytes of three donors were simultaneously exposed to physiologic doses of UVA 1 and UVB . Effects on apoptosis were analysed using annexin V assays and cell death ELISA s, and effects on DNA damage were investigated using southwestern slot blots. While UVA 1 did not influence UVB ‐induced apoptosis, UVA 1 impaired the repair of UVB ‐induced cyclobutane pyrimidine dimers ( CPD ) as the amount of CPD was 1.8 times higher in UVA 1 + UVB than in UVB only exposed melanocytes six hours after irradiation. We conclude that UVA 1 might contribute to melanomagenesis as it partially inhibits the repair of UVB ‐induced CPD in human melanocytes while it does not affect UVB ‐mediated apoptosis.

Targeted therapy with the BRAF inhibitors Vemurafenib and Dabrafenib are effective treatment regimens in patients with metastasized melanoma carrying the BRAF V600E mutation. A common side effect is an enhanced rate of non-melanoma skin cancer (NMSC). Moreover, about 30% of patients under Vemurafenib (Dabrafenib: 2%) are more sensitive to ultraviolet A radiation (UVA). It has been described that BRAF inhibition leads to a paradoxical enhanced MAPK signaling in BRAF wildtype cells, which might in part be responsible for the enhanced NMSC burden. UV radiation (UVR) induces DNA photoproducts (mainly cyclobutane pyrimidine dimers [CPDs]), which are efficiently removed by the nucleotide excision repair (NER) mechanism. It is known that disturbances in the NER result in an increased rate of NMSC. In the present study we investigated whether Vemurafenib and/or Dabrafenib might interfere with the repair of UVR-induced CPDs and hence with NER. Epidermal keratinocytes of twelve Caucasian donors were treated with Vemurafenib or Dabrafenib and exposed to UVA. South-Western Dot Blot analysis revealed that in three out of twelve donors treated with Vemurafenib the amount of CPDs three hours after exposure to UVA was increased in comparison to UVA only treated controls. This indicates that Vemurafenib partly inhibits NER in one fourth of donors (= "sensitive donors"). In contrast, Dabrafenib in none of the donors affected NER, indicating a substance specific effect of Vemurafenib. In addition, the mutational states of RAS proteins in the keratinocytes were studied. No pre-existing mutations in these genes were detected, indicating that RAS mutational status is not relevant for the effect observed. However, as determined by qRT-PCR, all "sensitive donors" exhibited a reduced expression of the DNA repair genes SUPT5H and GTF2H5 upon Vemurafenib treatment. Hence, the enhanced rate of NMSC in patients treated with Vemurafenib might be partly related to a Vemurafenib-driven impaired NER.

It is generally accepted that chronic sun exposure is a risk factor for the development of non-melanoma skin cancer. In contrast, the exact correlation between melanoma and sun light is still a controversially debated issue. Natural sunlight contains various ratios of UVA and UVB, depending on daytime, season, altitude and latitude. Nevertheless, most investigators focused on the effects of single solar wavebands and neglected possible interactions. Therefore we investigated the impact of UVA1 on UVB-induced apoptosis and the repair of UVB-induced DNA damage in normal human melanocytes of three different Caucasian donors. Cells were simultaneously exposed to physiologic doses of UVA1 (340-400nm) and UVB (390-320). On the next day the apoptotic rate was determined using a cell death detection ELISA and Annexin/Propidium Iodide staining followed by FACS analysis. UVA1 did not influence UVB-induced apoptosis and UVA1 alone did not induce detectable amounts of apoptosis at all. Accordingly, UVA1 did not alter UVB-induced activation of caspases 8 and 9. Likewise, UVA1 alone did not induce caspases 8 and 9. South Western Slot Blot analysis using antibodies against cyclobutane-pyrimidine dimers (CPD) revealed that UVA1 slightly enhanced the amount of UVB-induced CPD immediately and 6 hours after irradiation whereas UVA1 alone did not induce detectable amounts of CPD at all. This indicates that in human melanocytes UVA1 synergistically contributes to UVB-induced CPD while it does not affect the repair of CPD. Taken together, since UVA1 on the one hand enhances the formation of UVB-induced CPD while on the other hand it does not affect UVB-mediated apoptosis and the repair of CPD, UVA1 might contribute to the risk of malignant transformation of melanocytes.

The link between solar radiation and melanoma is still elusive. Although infrared radiation (IR) accounts for over 50% of terrestrial solar energy, its influence on human skin is not well explored. There is increasing evidence that IR influences the expression patterns of several molecules independently of heat. A previous in vivo study revealed that pretreatment with IR might promote the development of UVR-induced non-epithelial skin cancer and possibly of melanoma in mice. To expand on this, the aim of the present study was to evaluate the impact of IR on UVR-induced apoptosis and DNA repair in normal human epidermal melanocytes. The balance between these two effects is a key factor of malignant transformation. Human melanocytes were exposed to physiologic doses of IR and UVR. Compared to cells irradiated with UVR only, simultaneous exposure to IR significantly reduced the apoptotic rate. However, IR did not influence the repair of UVR-induced DNA damage. IR partly reversed the pro-apoptotic effects of UVR via modification of the expression and activity of proteins mainly of the extrinsic apoptotic pathway. In conclusion, IR enhances the survival of melanocytes carrying UVR-induced DNA damage and thereby might contribute to melanomagenesis.

OBJECTIVE:Cingulin is a cytoplasmic component of tight junctions. Although modulation of cingulin levels in cultured epithelial model systems has no significant effect on barrier function, evidence from cingulin knockout mice suggests that cingulin may be involved in the regulation of the behavior of epithelial or endothelial cells. Here, we investigate the role of cingulin in the barrier function of endothelial cells.APPROACH AND RESULTS:We show that cingulin is expressed in human endothelial cells of the skin, brain, and lung in vivo and in vitro. Endothelial cingulin colocalizes and coimmunoprecipitates with the tight junction proteins zonula occludens-1 and guanine nucleotide exchange factor-H1. Cingulin overexpression in human umbilical vein endothelial cell induces tight junction formation, increases transendothelial electric resistance, and strengthens barrier function for low and high molecular weight tracers. In contrast, cultured endothelial cells lacking cingulin are more permeable for low molecular weight tracers. In cingulin knockout mice, neurons of the area postrema and Purkinje cells show an increased uptake of small molecular weight tracers indicating decreased barrier function at these sites.CONCLUSIONS:We demonstrate that cingulin participates in the modulation of endothelial barrier function both in human cultured cells in vitro and in mouse brains in vivo. Understanding the role of cingulin in maintaining tight barriers in endothelia may allow developing new strategies for the treatment of vascular leak syndromes.

BACKGROUND:Extracorporeal photopheresis (ECP) improves skin sclerosis in systemic sclerosis (SSc) patients. SSc is associated with an increased risk of lung cancer. As ECP is supposed to exert immunomodulatory effects, a possible impact of ECP on the incidence of lung cancer in SSc patients was evaluated.METHODS:Seventy-one SSc patients treated with ECP at the Photopheresis Unit of the Department of Dermatology at the Medical University of Vienna between 1991 and 2013 were analyzed retrospectively.RESULTS:We calculated a standardized incidence ratio (SIR) for lung cancer in ECP-treated SSc patients of 2.34 [95% confidence interval (CI) 1.63-2.49]. This is in accordance with recent meta-analyses demonstrating a significantly enhanced risk of lung carcinoma in SSc patients. Comparison of the lung cancer risks of these patients with our ECP-treated patients revealed that ECP has no influence. Each patient with lung carcinoma had previously been diagnosed with lung involvement of the non-specific interstitial pneumonitis (NSIP) type.CONCLUSION:We confirm that SSc patients are at significantly increased risk for lung cancer. However, ECP does not influence this risk. NSIP may be a risk factor for lung cancer in SSc patients.

Detecting serum-autoantibodies by anti-Desmoglein-1 (anti-Dsg1) and anti-Dsg3 ELISAs as well as indirect immunofluorescence (IIF) are established complementary methods to diagnose pemphigus. Whether autoantibody levels also reflect clinical disease activity is still a matter of debate, as head-to-head comparisons of ELISA values and IIF titres with clinical activity over a longer treatment period are scarce. In our retrospective study, we compared aggregated repetitive intra-patient ELISA values and IIF titres with grades of clinical disease (1 = remission, 2 = moderate activity, 3 = exacerbation) in 47 patients suffering from pemphigus vulgaris (PV, n = 36) and pemphigus foliaceus (PF, n=11). We found that anti-Dsg1 ELISA values in PF and mucocutaneous PV as well as anti-Dsg3 ELISA values in PV best reflect disease activity. IIF titres, by contrast, did not show a significant association with disease severity. From these data we conclude that ELISA index values can be a valuable tool to monitor disease in patients with pemphigus, whereas IIF titres reflect clinical activity only insufficiently.

Abstract The FDA-approved BRAF inhibitor vemurafenib achieves outstanding clinical response rates in patients with melanoma, but early resistance is common. Understanding the pathologic mechanisms of drug resistance and identification of effective therapeutic alternatives are key scientific challenges in the melanoma setting. Using proteomic techniques, including shotgun analysis and 2D-gel electrophoresis, we identified a comprehensive signature of the vemurafenib-resistant M24met in comparison with the vemurafenib-sensitive A375 melanoma cell line. The resistant cells were characterized by loss of differentiation, induction of transformation, enhanced expression of the lysosomal compartment, increased potential for metastasis, migration, adherence and Ca2+ ion binding, enhanced expression of the MAPK pathway and extracellular matrix proteins, and epithelial–mesenchymal transformation. The main features were verified by shotgun analysis with QEXACTIVE orbitrap MS, electron microscopy, lysosomal staining, Western blotting, and adherence assay in a VM-1 melanoma cell line with acquired vemurafenib resistance. On the basis of the resistance profile, we were able to successfully predict that a novel resveratrol-derived COX-2 inhibitor, M8, would be active against the vemurafenib-resistant but not the vemurafenib-sensitive melanoma cells. Using high-throughput methods for cell line and drug characterization may thus offer a new way to identify key features of vemurafenib resistance, facilitating the design of effective rational therapeutic alternatives. Mol Cancer Ther; 14(3); 757–68. ©2015 AACR.

Background: Cornification of keratinocytes involves the degradation of intracellular constituents which has led to the hypothesis that autophagy plays a role in this process. Mice, in which essential autophagy-related genes such as Atg7 are deleted systemically, die after birth and have not been characterized for potential epidermal defects.Objective: This study tested whether autophagy is essential for epidermal barrier formation and function.Methods: Atg7 was inactivated in epidermal keratinocytes by the Cre-loxP system under the control of the keratin K14 promoter (Atg7 Delta epi mice). Autophagic activity was detected using the GFP-microtubule-associated protein light chain 3 (GFP-LC3) reporter construct and Western blot analysis of LC3. Epidermal morphology was examined by histological and ultrastructural analyses, and barrier functions were assessed by dye diffusion and water loss assays.Results: Suprabasal epidermal cells of normal mice contained GFP-LC3-labeled autophagosomes and epidermal lysates of these mice showed an excess of lipidated over non-lipidated LC3. These features of active autophagy were efficiently suppressed in Atg7 Delta epi epidermis. Atg7 Delta epi mice survived the perinatal period and were apparently healthy. Histologically, their epidermis was inconspicuous and ultrastructural analysis revealed no significant defect in cornification. There was however, an increase in the thickness of corneocytes in the back skin of mutant mice. Nevertheless, resistance to dye penetration into the skin and transepidermal water loss were normal in Atg7 Delta epi mice.Conclusion: This study demonstrates that autophagy is constitutively active in the epidermis but not essential for the barrier function of the skin. (C) 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

The reverse transmigration (RT) of tissue-resident dendritic cells (DCs) across lymphatic endothelia is prerequisite for the initiation of adaptive immune responses and might be regulated in a manner similar to diapedesis. Specifically, CD31 and CD99, which act as gatekeepers during diapedesis, might have a role in RT of DCs. We found that human lymphatic endothelial cells (LECs) and DCs in vitro and in human skin explants express CD31 and CD99. In human skin, CD31 was enriched along intercellular surfaces of LECs, whereas CD99 was preferentially confined to luminal surfaces as evidenced by immunoelectron microscopy. Confocal microscopy analysis revealed that tumor necrosis factor-alpha (TNF-alpha) and CXCL12 acted as inducers of RT in vitro, but only CXCL12 stimulation resulted in a significant increase in migration rate of DCs. Upon TNF-alpha stimulation, CXCL12 mRNA levels transiently increased in human fibroblasts and LECs, whereas CXCL12 protein expression levels did not significantly change. Blocking mAbs to CD31 and CD99 significantly reduced RT of DCs across cultured human LEC monolayers and blocked CXCL12-induced migration of DCs in whole-skin explants. In sum, this study shows that CD31 and CD99 are involved in the RT of DCs across LECs and that similar mechanisms promote both diapedesis and RT.

The exact correlation between melanoma and sun light is not as clear as it is between non-melanoma skin cancer and solar exposure. So far most investigators studied the effects of particular solar spectra although sun light contains both UVA and UVB in varying proportions. Hence, in the present stud

Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.