RNA-seq analysis of Lgr6+ stem cells and identification of an Lgr6 isoform.

We studied Lgr6+ stem cells in experimental UV carcinogenesis in hairless mice. For further characterization through RNA-seq, these stem cells were isolated by FACS from transgenic hairless mice bearing an EGFP-Ires-CreERT2 reporter cassette inserted into exon 1 of the Lgr6 gene (purity confirmed by human ERT2 expression). Between Lgr6/EGFP+ and Lgr6/EGFP- basal cells (Tg/wt), 682 RNAs were differentially expressed, indicating stemness and expression of cancer-related pathways in Lgr6/EGFP+ cells. We discovered that suspected "Lgr6 null" mice (Tg/Tg) expressed RNA of an Lgr6 isoform (delta-Lgr6, lacking 74 N-terminal aa) which could be functional and explain the lack of a phenotype.

Low wintertime pre-diagnostic vitamin D status is associated with an increased risk of internal malignancies in kidney transplant recipients.

Low serum 25-hydroxyvitamin D (25OHD) concentrations have been associated with increased cancer risk, but the relative importance of seasonality, i.e. high summer concentrations versus low winter concentrations, is unclear. We investigated this issue in a high risk group: kidney transplant recipients with known increased risk of cancer and low vitamin D statuses. We examined the relationship between registered concentrations of 25OHD binned by quarter and subsequent risk of internal malignancy or cutaneous squamous cell carcinoma in 1112 kidney transplant recipients. Hazard ratios for internal malignancies were significantly increased with lower pre-diagnostic 25OHD concentrations in the first quarter of the year (January-March); a 1.4 fold increase (95%CI 1.1;1.7) per 10 nmol L-1 decrease in 25OHD. Except for women in April-June (1.3 (1.01;1.7) per 10 nmol L-1 decrease) pre-diagnostic 25OHD concentrations in the other quarters were not statistically significantly associated with internal malignancies. Higher 25OHD concentrations tended to be associated with the development of cutaneous squamous cell carcinomas, independent of the time of the year. Our study indicates that low wintertime 25OHD concentrations are associated with an increased risk of internal malignancies and that transplant recipients may benefit from wintertime vitamin D supplementation. Our findings need further corroboration, but suggest that the lowest concentrations of vitamin D, which occur in winter, are important for the risk of internal malignancies.

UV adaptation: Pigmentation and protection against overexposure.

The skin is known to adapt to UV exposures, that is become less sensitive to sunburn. Reported decreases in sensitivity vary widely from well over 10-fold down to a negligible 10%. This appears to depend importantly on the UV irradiation spectrum to which the skin adapts and on the UV irradiation spectrum that is used to test the sensitivity. The sensitivity is conventionally and generally assessed by the reciprocal of the minimal erythema dose (MED): the UV dose causing a just perceptible reddening of the skin after 8-24 hours. However, MED is much too subtle for everyday life: people will not notice a minimal reddening and commonly consider themselves sunburnt at considerably higher UV doses causing an intense reddening. Levels of adaptation of a well-tanned skin may be substantially higher at these more intense levels of reddening than MED levels. This expectation is based on the fact that people with a constitutively coloured skin may show moderate differences in MED compared with fair-skinned people but far less steep increases in reddening with overexposures to solar-simulated radiation (SSR). UVA exposure is known to enhance pigmentation and may thus be important in protection against overexposure to SSR.

p16 immunostaining in keratinocytic neoplasia in organ transplant recipients: Bowen's disease shows a characteristic pattern.

BACKGROUND: For selecting therapy, it is important to distinguish different types of keratinocytic neoplasia. It is sometimes difficult to make histopathologic diagnosis, especially in organ transplant recipients (OTR) who develop numerous lesions. METHODS: To investigate p16 immunostaining in different types of keratinocytic neoplasia in OTR, we studied 59 actinic keratoses (AK), 51 Bowen' s disease (BD), 63 squamous cell carcinomas (SCC), 16 benign keratotic lesions (BKL) from 31 OTR patients and 25 controls (eczema and psoriasis). Tissue sections were stained for H&E and p16. We scored intensity, proportion and distribution of p16 positive lesional cells. RESULTS: In 19% of AK, 92% of BD, 35% of SCC and 12% of BKL more than 15% of lesional cells were p16-positive. In 16% of AK, 80% of BD, 18% of SCC and 13% of BKL strong p16 staining was observed. BKL, AK and SCC showed focal and patchy staining, BD showed diffuse pattern with strong staining of all atypical cells. Sparing of the basal layer was predominantly seen in BD. No control specimen showed p16-overexpression. CONCLUSIONS: p16 immunostaining shows a characteristic pattern in BD, but not in AK, SCC and BKL. It appears useful in recognizing BD, but not in differentiating between other keratinocytic neoplasia.

UV exposure inhibits intestinal tumor growth and progression to malignancy in intestine-specific Apc mutant mice kept on low vitamin D diet.

Mortality from colorectal cancer increases with latitude and decreases with ambient UV radiation. We investigated whether moderate UV dosages could inhibit intestinal tumor development and whether this corresponded with UV-induced vitamin D. FabplCre;Apc(15lox/+) mice, which develop intestinal tumors, and their parents were put on a vitamin D-deficient diet. Next to a control group, one group was vitamin D supplemented and another one group was daily UV irradiated from 6 weeks of age. Vitamin D statuses after 6 weeks of treatment were markedly increased: mean ± SD from 7.7 ± 1.9 in controls to 75 ± 15 nmol/l with vitamin D supplementation (no gender difference), and to 31 ± 13 nmol/l in males and 85 ± 17 nmol/l in females upon UV irradiation. The tumor load (area covered by tumors) at 7.5 months of age was significantly reduced in both the vitamin D-supplemented group (130 ± 25 mm(2), p = 0.018) and the UV-exposed group (88 ± 9 mm(2), p < 0.0005; no gender differences) compared to the control group (202 ± 23 mm(2)). No reductions in tumor numbers were found. Only UV exposure appeared to reduce progression to malignancy (p = 0.014). Our experiments clearly demonstrate for the first time an inhibitory effect of moderate UV exposure on outgrowth and malignant progression of primary intestinal tumors, which at least in part can be attributed to vitamin D.

"Next top" mouse models advancing CTCL research.

This review systematically describes the application of in vivo mouse models in studying cutaneous T-cell lymphoma (CTCL), a complex hematological neoplasm. It highlights the diverse research approaches essential for understanding CTCL's intricate pathogenesis and evaluating potential treatments. The review categorizes various mouse models, including xenograft, syngeneic transplantation, and genetically engineered mouse models (GEMMs), emphasizing their contributions to understanding tumor-host interactions, gene functions, and studies on drug efficacy in CTCL. It acknowledges the limitations of these models, particularly in fully replicating human immune responses and early stages of CTCL. The review also highlights novel developments focusing on the potential of skin-targeted GEMMs in studying natural skin lymphoma progression and interactions with the immune system from onset. In conclusion, a balanced understanding of these models' strengths and weaknesses are essential for accelerating the deciphering of CTCL pathogenesis and developing treatment methods. The GEMMs engineered to target specifically skin-homing CD4+ T cells can be the next top mouse models that pave the way for exploring the effects of CTCL-related genes.

Molecular profiling of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients.

BACKGROUND: The risk of developing cutaneous squamous cell carcinoma (SCC) is markedly increased in organ transplant recipients (OTRs) compared to the normal population. Next to sun exposure, the immunosuppressive regimen is an important risk factor for the development of SCC in OTRs. Various gene mutations (e.g. TP53) and genetic alterations (e.g. loss of CDKN2A, amplification of RAS) have been found in SCCs. The aim of this genome-wide study was to identify pathways and genomic alterations that are consistently involved in the formation of SCCs and their precursor lesions, actinic keratoses (AKs). METHODS: To perform the analysis in an isogenic background, RNA and DNA were isolated from SCC, AK and normal (unexposed) epidermis (NS) from each of 13 OTRs. Samples were subjected to genome-wide expression analysis and genome SNP analysis using Illumina's HumanWG-6 BeadChips and Infinium II HumanHap550 Genotyping BeadChips, respectively. mRNA expression results were verified by quantitative PCR. RESULTS: Hierarchical cluster analysis of mRNA expression profiles showed SCC, AK and NS samples to separate into three distinct groups. Several thousand genes were differentially expressed between epidermis, AK and SCC; most upregulated in SCCs were hyperproliferation related genes and stress markers, such as keratin 6 (KRT6), KRT16 and KRT17. Matching to oncogenic pathways revealed activation of downstream targets of RAS and cMYC in SCCs and of NFκB and TNF already in AKs. In contrast to what has been reported previously, genome-wide SNP analysis showed very few copy number variations in AKs and SCCs, and these variations had no apparent relationship with observed changes in mRNA expression profiles. CONCLUSION: Vast differences in gene expression profiles exist between SCC, AK and NS from immunosuppressed OTRs. Moreover, several pathways activated in SCCs were already activated in AKs, confirming the assumption that AKs are the precursor lesions of SCCs. Since the drastic changes in gene expression appeared unlinked to specific genomic gains or losses, the causal events driving SCC development require further investigation. Other molecular mechanisms, such as DNA methylation or miRNA alterations, may affect gene expression in SCCs of OTRs. Further study is required to identify the mechanisms of early activation of NFκB and TNF, and to establish whether these pathways offer a feasible target for preventive intervention among OTRs.

Socs1-knockout in skin-resident CD4+ T cells in a protracted contact-allergic reaction results in an autonomous skin inflammation with features of early-stage mycosis fungoides.

Recent detailed genomic analysis of mycosis fungoides (MF) identified suppressor of cytokine signaling 1 (SOCS1), an inhibitor of JAK/STAT signaling, as one of the frequently deleted tumor suppressors in MF, and one-copy deletion of SOCS1 was confirmed in early-stage MF lesions. To better understand the functional role of SOCS1 in the genesis of MF, we used a genetically engineered mouse model emulating heterozygous SOCS1 loss in skin resident CD4+ T cells. In these mice an experimentally induced contact-allergic reaction was maintained for 20 weeks. Ten weeks after discontinuing contact-allergic challenges, only the skin with locally one-copy deletion of Socs1 in CD4+ T cells still showed high numbers of CD3+/CD4+ Socs1 k.o. cells in the dermis (p ＜ 0.0001) with prevalent Stat3 activation (p ＜0.001). And in one out of 9 mice, this had progressed to far more dramatic increases, including the thickened epidermis, and with an explosive growth of Socs1 k.o. T cells in circulation; indicative of cutaneous lymphoma. Hence, we show that Socs1 mono-allelic loss in CD4+ T cells locally in protractedly inflamed skin results in autonomous skin inflammation with features of early-stage MF.

Assessing the safety of new germicidal far-UVC technologies.

The COVID-19 pandemic underscored the crucial importance of enhanced indoor air quality control measures to mitigate the spread of respiratory pathogens. Far-UVC is a type of germicidal ultraviolet technology, with wavelengths between 200 and 235 nm, that has emerged as a highly promising approach for indoor air disinfection. Due to its enhanced safety compared to conventional 254 nm upper-room germicidal systems, far-UVC allows for whole-room direct exposure of occupied spaces, potentially offering greater efficacy, since the total room air is constantly treated. While current evidence supports using far-UVC systems within existing guidelines, understanding the upper safety limit is critical to maximizing its effectiveness, particularly for the acute phase of a pandemic or epidemic when greater protection may be needed. This review article summarizes the substantial present knowledge on far-UVC safety regarding skin and eye exposure and highlights research priorities to discern the maximum exposure levels that avoid adverse effects. We advocate for comprehensive safety studies that explore potential mechanisms of harm, generate action spectra for crucial biological effects and conduct high-dose, long-term exposure trials. Such rigorous scientific investigation will be key to determining safe and effective levels for far-UVC deployment in indoor environments, contributing significantly to future pandemic preparedness and response.

Epidermal growth factor receptor activation and inhibition in 3D in vitro models of normal skin and human cutaneous squamous cell carcinoma.

The transmembrane tyrosine kinase epidermal growth factor receptor (EGFR) is considered a key player in the development of cutaneous squamous cell carcinoma (SCC), which is the second most common malignancy in white populations. Inhibition of EGFR with the small molecule tyrosine kinase inhibitor erlotinib is currently under clinical investigation in cutaneous SCC patients. In this study, we investigated the effects of EGFR activation and inhibition on normal and malignant in vitro human skin equivalents (HSEs). In healthy HSEs, increasing EGF concentrations ranging from 5 to 50 ng/mL resulted in a dramatic decrease in epidermal proliferation as immunohistochemically assessed by Ki67 and increased epidermal stress as assessed by K17 after 2 weeks of air-exposed culture. Also, higher concentrations of EGF induced remarkable epidermal disorganization with loss of proper stratification. Similar effects were observed in HSEs generated with cutaneous SCC cell lines SCC-12B2 and SCC-13. Treatment of both healthy and SCC-HSEs with 10 µM erlotinib resulted in efficient reduction of epidermal thickness from 10 to 3 viable cell layers and counteracted EGF-induced epidermal stress. Remarkably, erlotinib treatment caused severe desquamation in healthy HSEs, reminiscent of xerosis as a known side-effect in patients treated with erlotinib. The presented three-dimensional organotypic SCC models appear suitable for further investigations on the morphological and functional impacts of modifying EGFR signaling in cutaneous SCC, without burdening patients or mice. The effective inhibition of epidermal growth by erlotinib in our HSEs confirms the therapeutic potential of this tyrosine kinase inhibitor for cutaneous SCC patients.

The effects of a mid-winter 8-week course of sub-sunburn sunbed exposures on tanning, vitamin D status and colds.

Like UV irradiation, which generates vitamin D(3) in the skin, the hormonally active metabolite, 1,25-dihydroxyvitamin D(3), boosts innate immunity against viruses and bacteria. Epidemiologic studies have found high vitamin D levels to be associated with lower risk of infections of the upper respiratory tract (colds). We have therefore performed an intervention study in 105 young adults (ages 18-30 years; 91% female) over a mid-winter 8-week period (January-March 2010). The participants were randomised to 3 groups: (A) subjected to 3 times a week sub-sunburn sunbed exposure (n = 35), (B) daily vitamin D supplementation, @ 1000 IU (n = 37), and (C) a control group without any intervention (n = 33). The mean serum level of 25-hydroxyvitamin D (25(OH)D) dropped from 62 to 55 nmol l(-1) in group C, while these levels rose from 62 to 109 and from 58 to 93 nmol l(-1) in groups A and B, respectively (p < 0.001). The skin on the chest darkened significantly in group A (mean difference in lightness, L*, equalled -5.7, p < 0.001), correlating significantly, but weakly, with increases in 25(OH)D (3.3 nmol l(-1) per unit drop in L*, R(2) = 0.17, p = 0.014). The percentage of self-reported colds with proper signs and symptoms was only slightly and not significantly reduced in groups A and B in comparison to group C: 57 and 51 versus 67%, respectively. Hence, the sub-sunburn sunbed treatment was effective in tanning and increasing the 25(OH)D serum level, more so than 1000 IU per day, but had no appreciable effect on colds.

Environmental effects of ozone depletion and its interactions with climate change: progress report, 2011.

The parties to the Montreal Protocol are informed by three panels of experts. One of these is the Environmental Effects Assessment Panel (EEAP), which deals with two focal issues. The first focus is the effects of increased UV radiation on human health, animals, plants, biogeochemistry, air quality, and materials. The second focus is on interactions between UV radiation and global climate change and how these may affect humans and the environment. When considering the effects of climate change, it has become clear that processes resulting in changes in stratospheric ozone are more complex than believed previously. As a result of this, human health and environmental problems will be longer-lasting and more regionally variable. Like the other panels, the EEAP produces a detailed report every four years; the most recent was published in 2010 (Photochem. Photobiol. Sci., 2011, 10, 173-300). In the years in between, the EEAP produces less detailed and shorter progress reports, which highlight and assess the significance of developments in key areas of importance to the parties. The next full quadrennial report will be published in 2014-2015.

Error-prone translesion replication of damaged DNA suppresses skin carcinogenesis by controlling inflammatory hyperplasia.

The induction of skin cancer involves both mutagenic and proliferative responses of the epidermis to ultraviolet (UV) light. It is believed that tumor initiation requires the mutagenic replication of damaged DNA by translesion synthesis (TLS) pathways. The mechanistic basis for the induction of proliferation, providing tumor promotion, is poorly understood. Here, we have investigated the role of TLS in the initiation and promotion of skin carcinogenesis, using a sensitive nucleotide excision repair-deficient mouse model that carries a hypomorphic allele of the error-prone TLS gene Rev1. Despite a defect in UV-induced mutagenesis, skin carcinogenesis was accelerated in these mice. This paradoxical phenotype was caused by the induction of inflammatory hyperplasia of the mutant skin that provides strong tumor promotion. The induction of hyperplasia was associated with mild and transient replicational stress of the UV-damaged genome, triggering DNA damage signaling and senescence. The concomitant expression of Interleukin-6 (IL-6) is in agreement with an executive role for IL-6 and possibly other cytokines in the autocrine induction of senescence and the paracrine induction of inflammatory hyperplasia. In conclusion, error-prone TLS suppresses tumor-promoting activities of UV light, thereby controlling skin carcinogenesis.

Cutaneous Melanoma: Sheep in Wolves Clothing?

Cutaneous melanoma incidence in European-origin populations has risen steeply, however, mortality has not, as 73 years of Danish cancer data strikingly show. It has been suggested that such divergent trends in the US are due to overdiagnosis from increasing diagnostic scrutiny and lowering diagnostic threshold. Alternatively, the increase in melanoma incidence may be largely due to increased sun exposure, which would imply that most of these new, sun-caused, melanomas are non-lethal. Consistent with this hypothesis, Icelandic data show an increase in melanoma incidence, predominantly in young women (<50 years), which paralleled increasing sunbed use that remitted after a campaign against sunbeds. Meanwhile, melanoma mortality in young people remained virtually zero. The increase in mortality was mainly in the elderly (>50 years) and dictated by year of birth. This transient excess of melanoma in young people is most likely attributable to skin burns from sunbeds which, like sunburns, carry a high risk of melanoma. High exposure of naevi to UV radiation can induce transient clinical and pathological features of melanoma, which might explain some of the apparent rise in incidence. Ways of distinguishing non-lethal from potentially lethal thin melanomas are sorely needed.

An Appraisal to Address Health Consequences of Vitamin D Deficiency With Food Fortification and Supplements: Time to Act!

A symposium entitled "Vitamin D in Prevention and Therapy" was held on May 4-5, 2022, in Homburg, Germany to discuss important new advances in the field, including identification of new vitamin D signaling pathways, of new biologic effects of vitamin D-compounds (e.g., on the microbiome), and convincing proof of the relevance of vitamin D deficiency for the risk and outcome of many chronic diseases, including cancer, cardio-vascular, auto-immune, metabolic, and infectious diseases. Concerning the COVID-19-pandemic, an inverse association between 25(OH)D serum concentrations and SARS-CoV-2-infections, morbidity, and mortality was shown. In relation to cancer, several meta-analyses recently demonstrated an association of vitamin D-supplementation with significantly decreased mortality rates, which presumably would reduce health care costs. Considering the impressive body of evidence and the high safety of oral supplementation and food fortification with vitamin D, it was concluded that there is now an urgent need to act. In many countries worldwide, health care authorities need to increase efforts to address vitamin D deficiency, e.g., via food fortification and/or supplementation with vitamin D, and/or promoting moderate UV-exposure. It was estimated that in many countries, vitamin D intakes of the order of appr. 1,000 IE (25 µg)/day would be needed to bring and/or keep the vast majority of people over a serum 25(OH)D threshold of 20 ng/ml (50 nmol/l), which would be difficult to obtain alone from food fortification. New developments in personalized medicine may represent helpful tools to identify populations at risk for vitamin D deficiency and their responsiveness to vitamin D treatment.

In vivo modelling of cutaneous T-cell lymphoma: The role of SOCS1.

Introduction: Mycosis fungoides (MF), the most common type of Cutaneous T cell Lymphoma (CTCL), is characterized by an inflamed skin intermixed with proliferating malignant mature skin-homing CD4+ T cells. Detailed genomic analyses of MF skin biopsies revealed several candidate genes possibly involved in genesis of these tumors and/or potential targets for therapy. These studies showed, in addition to common loss of cell cycle regulator CDKN2A, activation of several oncogenic pathways, most prominently and consistently involving JAK/STAT signaling. SOCS1, an endogenous inhibitor of the JAK/STAT signaling pathway, was identified as a recurrently deleted gene in MF, already occurring in the earliest stages of the disease. Methods: To explore the mechanisms of MF, we create in vivo mouse models of autochthonous CTCLs and these genetically engineered mouse models (GEMMS) can also serve as valid experimental models for targeted therapy. We describe the impact of allelic deletion of Socs1 in CD4 T cells of the skin. To achieve this, we crossed inducible Cre-transgenic mice in the CD4 lineage with transgenic mice carrying floxed genes of Socs1. We first determined optimal conditions for Socs1 ablation with limited effects on circulating CD4 T-cells in blood. Next, we started time-course experiments mimicking sustained inflammation, typical in CTCL. FACS analysis of the blood was done every week. Skin biopsies were analyzed by immunocytochemical staining at the end of the experiment. Results: We found that the Socs1 knockout transgenic group had thicker epidermis of treated skin compared with the control group and had more CD3 and CD4 in the skin of the transgenic group compared to the control group. We also noted more activation of Stat3 by staining for P-Stat3 in Socs1 knockout compared to wt CD4+T cells in the skin. The results also indicated that single copy loss of Socs1 in combination with sustained inflammation is insufficient to start a phenotype resembling early stage mycosis fungoides within eight weeks in these mice. Conclusion: In sum, we developed and optimized an autochthonous murine model permitting selective knockout of Socs1 in skin infiltrating CD4 T-cells. This paves the way for more elaborate experiments to gain insight in the oncogenesis of CTCL.

Functional characterization of cancer-associated fibroblasts of human cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (SCC) is the second most common type of skin cancer in the Caucasian population worldwide, having a propensity for invasion, local recurrence and metastasis. Stromal cancer-associated fibroblasts (CAFs) are suspected to play an important role in SCC carcinogenesis. In this study, we characterized CAFs isolated from primary cutaneous SCCs and compared them to normal fibroblasts (NFs) isolated from healthy dermis. Human skin CAFs in monolayers displayed different morphology, increased proliferation and migration compared to NFs. CAFs caused strong contraction of collagen matrices in which they were seeded and released high levels of the extracellular matrix component pro-collagen I. CAFs decreased proliferation and differentiation in the epidermis of human skin equivalents (HSEs) seeded with SCC cell lines, without affecting basement membrane composition. Finally, CAFs significantly increased invasion and dermal-epidermal detachment of SCC cell lines SCC-12B2 and SCC-13, respectively, when cultured in HSEs. These distinct features of CAFs point out a specific role in cutaneous SCC development.

Differential activity of UV-DDB in mouse keratinocytes and fibroblasts: impact on DNA repair and UV-induced skin cancer.

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers (CPD) and accelerates repair of 6-4 photoproducts (6-4PP). The high UV-induced skin cancer susceptibility of mice compared to man has been attributed to low expression of the UV-DDB subunit DDB2 in mouse skin cells. However, DDB2 knockout mice exhibit enhanced UVB skin carcinogenesis indicating that DDB2 protects mice against UV-induced skin cancer. To resolve these apparent contradictory findings, we systematically investigated the NER capacity of mouse fibroblasts and keratinocytes. Compared to fibroblasts, keratinocytes exhibited an increased level of UV-DDB activity, contained significantly higher levels of other NER proteins (i.e. XPC and XPB) and displayed efficient repair of CPD. At low UVB dosages, the difference in skin cancer susceptibility between DDB2 KO and wild type mice was even much more pronounced than previously reported with high dose UVB exposures. Hence, our observations show that mouse keratinocytes express sufficient levels of UV-DDB for efficient repair of photolesions and efficient protection against UV-induced skin cancer at physiological relevant UV exposure.