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A substantial part of cutaneous malignant melanomas develops from benign nevi. However, the precise molecular events driving the transformation from benign to malignant melanoma are not well-understood. We used laser microdissection and mass spectrometry to analyze the proteomes of melanoma subtypes, including superficial spreading melanomas (n = 17), nodular melanomas (n = 17), and acral melanomas (n = 15). Furthermore, we compared the proteomes of nevi cells with those of melanoma cells within the same specimens (nevus-associated melanoma (n = 14)). In total, we quantified 7935 proteins. Despite the genomic and clinical differences of the melanoma subtypes, our analysis revealed relatively similar proteomes, except for the upregulation of proteins involved in immune activation in nodular melanomas versus acral melanomas. Examining nevus-associated melanoma versus nevi, we found 1725 differentially expressed proteins (false discovery rate < 0.05). Among these proteins were 140 that overlapped with cancer hallmarks, tumor suppressors, and regulators of metabolism and cell cycle. Pathway analysis indicated aberrant activation of the phosphoinositide 3-kinase-protein kinase B-mTOR pathways and the Hippo-YAP pathway. Using a classifier, we identified six proteins capable of distinguishing melanoma from nevi samples. Our study represents a comprehensive comparative analysis of the proteome in melanoma subtypes and associated nevi, offering insights into the biological behavior of these distinct entities., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Patients with Sézary syndrome (SS), a leukemic variant of cutaneous T-cell lymphoma (CTCL), are prone to Staphylococcus aureus infections and have a poor prognosis due to treatment resistance. Here, we report that S aureus and staphylococcal enterotoxins (SE) induce drug resistance in malignant T cells against therapeutics commonly used in CTCL. Supernatant from patient-derived, SE-producing S aureus and recombinant SE significantly inhibit cell death induced by histone deacetylase (HDAC) inhibitor romidepsin in primary malignant T cells from patients with SS. Bacterial killing by engineered, bacteriophage-derived, S aureus-specific endolysin (XZ.700) abrogates the effect of S aureus supernatant. Similarly, mutations in major histocompatibility complex (MHC) class II binding sites of SE type A (SEA) and anti-SEA antibody block induction of resistance. Importantly, SE also triggers resistance to other HDAC inhibitors (vorinostat and resminostat) and chemotherapeutic drugs (doxorubicin and etoposide). Multimodal single-cell sequencing indicates T-cell receptor (TCR), NF-κB, and JAK/STAT signaling pathways (previously associated with drug resistance) as putative mediators of SE-induced drug resistance. In support, inhibition of TCR-signaling and Protein kinase C (upstream of NF-κB) counteracts SE-induced rescue from drug-induced cell death. Inversely, SE cannot rescue from cell death induced by the proteasome/NF-κB inhibitor bortezomib. Inhibition of JAK/STAT only blocks rescue in patients whose malignant T-cell survival is dependent on SE-induced cytokines, suggesting 2 distinct ways SE can induce drug resistance. In conclusion, we show that S aureus enterotoxins induce drug resistance in primary malignant T cells. These findings suggest that S aureus enterotoxins cause clinical treatment resistance in patients with SS, and antibacterial measures may improve the outcome of cancer-directed therapy in patients harboring S aureus., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Tissue transcriptomics is used to uncover molecular dysregulations underlying diseases. However, the majority of transcriptomics studies focus on single diseases with limited relevance for understanding the molecular relationship between diseases or for identifying disease-specific markers. In this study, we used a normalization approach to compare gene expression across nine inflammatory skin diseases. The normalized datasets were found to retain differential expression signals that allowed unsupervised disease clustering and identification of disease-specific gene signatures. Using the NS-Forest algorithm, we identified a minimal set of biomarkers and validated their use as diagnostic disease classifier. Among them, PTEN was identified as being a specific marker for cutaneous lupus erythematosus and found to be strongly expressed by lesional keratinocytes in association with pathogenic type I IFNs. In fact, PTEN facilitated the expression of IFN-β and IFN-κ in keratinocytes by promoting activation and nuclear translocation of IRF3. Thus, cross-comparison of tissue transcriptomics is a valid strategy to establish a molecular disease classification and to identify pathogenic disease biomarkers., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Atopic dermatitis (AD) is a common inflammatory skin disorder characterized by a heterogeneous and fluctuating disease course. To obtain a detailed molecular understanding of both the temporal and spatial variation in AD, we conducted a longitudinal case-control study, in which we followed a population, the GENAD (Gentofte AD) cohort, of mild-to-moderate patients with AD and matched healthy controls for more than a year. By the use of 1.5 mm minipunch biopsies, we obtained 393 samples from lesional, nonlesional, and healthy skin from multiple anatomical regions at different time points for transcriptomic profiling. We observed that the skin transcriptome was remarkably stable over time, with the largest variation being because of disease, individual, and skin site. Numerous AD-specific, differentially expressed genes were identified and indicated a disrupted skin barrier and activated immune response as the main features of AD. We also identified potentially novel targets in AD, including IL-37, MAML1, and several long noncoding RNAs. We envisage that the application of small biopsies, such as those introduced in this study, combined with omics technologies, will enable future skin research, in which multiple sampling from the same individual will give a more detailed, dynamic picture of how a disease fluctuates in time and space., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms that hamper quality of life and are entry sites for bacterial infection, a major cause of morbidity and mortality in advanced diseases. The mechanism driving the pathological processes that compromise the skin barrier remains unknown. Here, we report increased transepidermal water loss and compromised expression of the skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX-positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as interleukin 13 [IL-13], IL-22, and oncostatin M) that activate STAT3 signaling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, the repression of filaggrins can be counteracted by a cocktail of antibodies targeting these cytokines/receptors, small interfering RNA-mediated knockdown of JAK1/STAT3, and JAK1 inhibitors. Notably, we show that treatment with a clinically approved JAK inhibitor, tofacitinib, increases filaggrin expression in lesional skin from patients with mycosis fungoides. Taken together, these findings indicate that malignant T cells secrete cytokines that induce skin barrier defects via a JAK1/STAT3-dependent mechanism. As clinical grade JAK inhibitors largely abrogate the negative effect of malignant T cells on skin barrier proteins, our findings suggest that such inhibitors provide novel treatment options for patients with CTCL with advanced disease and a compromised skin barrier., (© 2023 by The American Society of Hematology.)

Staphylococcal enterotoxins are believed to fuel disease activity in cutaneous T-cell lymphoma. Recent data support this by showing that antibiotics inhibit malignant T cells in skin lesions in mycosis fungoides and Sézary syndrome, the most common forms of cutaneous T-cell lymphoma. Yet, it remains incompletely characterized how staphylococcal enterotoxins fuel disease activity. In this study, we show that staphylococcal enterotoxins induce the expression of the oncogenic microRNA miR-155 in primary malignant T cells. Thus, staphylococcal enterotoxins and Staphyloccocus aureus isolates from lesional skin of patients induce miR-155 expression at least partly through the IL-2Rg‒Jak‒signal transducer and activator of transcription 5 pathway, and the effect is augmented by the presence of nonmalignant T cells. Importantly, mycosis fungoides lesions harbor S. aureus, express Y-phosphorylated signal transducer and activator of transcription 5, and display enhanced miR-155 expression, when compared with nonlesional and healthy skin. Preliminary data show that aggressive antibiotic therapy is associated with decreased Y-phosphorylated signal transducer and activator of transcription 5 and miR-155 expression in lesional skin in two patients with Sézary syndrome. In conclusion, we show that S. aureus and its enterotoxins induce enhanced expression of oncogenic miR-155, providing mechanistic insight into the role of S. aureus in cutaneous T-cell lymphoma. Our findings support that environmental stimuli such as bacteria can fuel disease progression in cutaneous T-cell lymphoma., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Skin biopsies represent a gold standard in skin immunology and pathology but can cause pain and induce scarring. Non-invasive techniques will facilitate study recruitment of e.g. patients with paediatric atopic dermatitis (AD), hand eczema or facial dermatitis., Objective: By RNA sequencing, we examined whether the stratum corneum transcriptome in AD skin can be assessed by tape stripping, as compared to the epidermal transcriptome of AD in skin biopsies. To make the procedure clinically relevant tape strips were stored and shipped at room temperature for up to 3 days., Methods: Nine adult Caucasian AD patients and three healthy volunteers were included. Tape samples were collected from non-lesional and lesional skin. Biopsies were collected from lesional skin and were split into epidermis and dermis. Total RNA was extracted, and shotgun sequencing was performed., Results: Shotgun sequencing could be performed on skin cells obtained from two consecutive tape strips which had been stored and shipped at room temperature for up to three days. The most prominent differences between the tape strip and biopsy derived transcriptome were due to structural genes, while established molecular markers of AD, including CCL17, CCL22, IL17A and S100A7-S100A9, were also identified in tape strip samples. Furthermore, the tape strip derived transcriptome showed promise in also analysing the skin microbiome., Conclusion: Our study shows that the stratum corneum (SC) transcriptome of AD can be assessed by tape stripping the skin, supporting that this method may be central in future skin biomarker research. NCBI GEO data accession: GSE160501., Competing Interests: Declaration of Competing Interest Thomas Litman is employed both by KU and by LEO Pharma. The other authors declare no conflict of interest., (Copyright © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

It has been proposed that CD4 T-cell responses to Staphylococcus aureus (SA) can inadvertently enhance neoplastic progression in models of skin cancer and cutaneous T-cell lymphoma (CTCL). In this prospective study, we explored the effect of transient antibiotic treatment on tumor cells and disease activity in 8 patients with advanced-stage CTCL. All patients experienced significant decrease in clinical symptoms in response to aggressive, transient antibiotic treatment. In some patients, clinical improvements lasted for more than 8 months. In 6 of 8 patients, a malignant T-cell clone could be identified in lesional skin, and a significant decrease in the fraction of malignant T cells was observed following antibiotics but an otherwise unchanged treatment regimen. Immunohistochemistry, global messenger RNA expression, and cell-signaling pathway analysis indicated that transient aggressive antibiotic therapy was associated with decreased expression of interleukin-2 high-affinity receptors (CD25), STAT3 signaling, and cell proliferation in lesional skin. In conclusion, this study provides novel evidence suggesting that aggressive antibiotic treatment inhibits malignant T cells in lesional skin. Thus, we provide a novel rationale for treatment of SA in advanced CTCL.

Mycosis fungoides (MF) is the most frequent form of cutaneous T-cell lymphoma. The disease often takes an indolent course, but in approximately one-third of the patients, the disease progresses to an aggressive malignancy with a poor prognosis. At the time of diagnosis, it is impossible to predict which patients develop severe disease and are in need of aggressive treatment. Accordingly, we investigated the prognostic potential of microRNAs (miRNAs) at the time of diagnosis in MF. Using a quantitative reverse transcription polymerase chain reaction platform, we analyzed miRNA expression in diagnostic skin biopsies from 154 Danish patients with early-stage MF. The patients were subdivided into a discovery cohort (n = 82) and an independent validation cohort (n = 72). The miRNA classifier was built using a LASSO (least absolute shrinkage and selection operator) Cox regression to predict progression-free survival (PFS). We developed a 3-miRNA classifier, based on miR-106b-5p, miR-148a-3p, and miR-338-3p, which successfully separated patients into high-risk and low-risk groups of disease progression. PFS was significantly different between these groups in both the discovery cohort and the validation cohort. The classifier was stronger than existing clinical prognostic factors and remained a strong independent prognostic tool after stratification and adjustment for these factors. Importantly, patients in the high-risk group had a significantly reduced overall survival. The 3-miRNA classifier is an effective tool to predict disease progression of early-stage MF at the time of diagnosis. The classifier adds significant prognostic value to existing clinical prognostic factors and may facilitate more individualized treatment of these patients., (© 2018 by The American Society of Hematology.)

Background: Topical glucocorticoids (GCs) are known to induce atrophy of human skin including thinning of epidermal and dermal compartments by influencing keratinocyte proliferation and synthesis of extracellular matrix proteins. GCs are also known to reduce skin barrier integrity but little is known about the changes in lipid composition in human skin following topical administration of GCs., Objective: This study investigated the effects of GCs on stratum corneum (SC) function and lipid profile of human skin in vivo., Method: Over a period of 4 weeks, 16 healthy volunteers were treated on the forearms once daily with topical clobetasol proprionate (CP), betamethasone diproprionate (BDP) or vehicle. One day after last application (Day 29) SC lipids were collected by tape stripping and analysed by a high sensitivity liquid chromatography-mass spectrometry method. Gene expression was analysed in skin biopsies. The full skin, epidermal and SC thickness were assessed by ultrasound, optical coherence tomography and confocal microscopy, respectively, and barrier integrity was assessed by measuring transepidermal water loss (TEWL)., Results: Compared to vehicle controls, GCs induced significant alterations in SC lipid profiles. CP caused a reduction in 98 lipids of 226 analysed while BDP treatment only resulted in a significant change of 29 lipids. Most pronounced changes occurred among long chain, ester-linked, ceramide classes while other ceramide classes were much less affected. Almost the complete profile of triacylglycerols (TGs) was significantly decreased by CP while more modest changes were observed in free fatty acids. Topical GCs reduced the thickness of skin layers and increased TEWL. GC treatment also induced changes in expression of genes coding for extracellular markers and enzymes involved in lipid synthesis., Conclusions: This study shows a reduction in specific SC lipid classes following topical GC treatment of human skin and contributes to the characterisation of the barrier disruption in human skin induced by topical steroids., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Carcinomas of unknown primary origin constitute 3% to 5% of all newly diagnosed metastatic cancers, with the primary source difficult to classify with current histological methods. Effective cancer treatment depends on early and accurate identification of the tumor; patients with metastases of unknown origin have poor prognosis and short survival. Because miRNA expression is highly tissue specific, the miRNA profile of a metastasis may be used to identify its origin. We therefore evaluated the potential of miRNA profiling to identify the primary tumor of known metastases. Two hundred eight formalin-fixed, paraffin-embedded samples, representing 15 different histologies, were profiled on a locked nucleic acid-enhanced microarray platform, which allows for highly sensitive and specific detection of miRNA. On the basis of these data, we developed and cross-validated a novel classification algorithm, least absolute shrinkage and selection operator, which had an overall accuracy of 85% (CI, 79%-89%). When the classifier was applied on an independent test set of 48 metastases, the primary site was correctly identified in 42 cases (88% accuracy; CI, 75%-94%). Our findings suggest that miRNA expression profiling on paraffin tissue can efficiently predict the primary origin of a tumor and may provide pathologists with a molecular diagnostic tool that can improve their capability to correctly identify the origin of hitherto unidentifiable metastatic tumors and, eventually, enable tailored therapy., (Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.

Background: Psoriasis is a chronic inflammatory skin disease often seen in patients with a genetic susceptibility. MicroRNAs (miRNA) are endogenous, short RNA molecules that can bind to parts of mRNA target genes, thus inhibiting their translation and causing accelerated turnover or transcript degradation. MicroRNAs are important in the pathogenesis of human diseases such as immunological disorders, as they regulate a broad range of biological processes., Objective: We investigated miRNA-mRNA interactions in involved (PP) and non-involved (PN) psoriatic skin compared with healthy skin (NN)., Methods: Biopsies were obtained from PP, PN and NN, the miRNA and mRNA expression was analyzed by microarray techniques and a subset of miRNAs and mRNAs were validated by q-RT-PCR. Novel target interactions in psoriasis were found using PubMed, miRBase and RNAhybrid. In addition, TIMP3 protein expression was studied in PP, PN and NN. Finally, the miR-221/2-TIMP3 target interaction was studied in primary human keratinocytes by endogenous overexpression of the miRNAs., Results: We identified 42 upregulated miRNAs and 5 downregulated miRNAs in PP compared with NN, and only few deregulated miRNAs in PN compared with NN. Based on the miRNA and mRNA profiles miR-21, -205, -221 and -222 were found to have the following potential mRNA targets in psoriatic skin: PDCD4, TPM1, P57, C-KIT, RTN4, SHIP2, TIMP3, RECK and NFIB. The identified target mRNAs were likely to be involved in cellular growth, proliferation, apoptosis and degradation of the extracellular matrix. Finally we found that TIMP3 is downregulated in psoriatic skin. In vitro overexpression of miR-221 and miR-222 lead to degradation of TIMP3 resulting in decreased TIMP3 protein level., Conclusion: Our data indicate several novel important associations for miRNAs in psoriasis and in particular the miR-221/2-TIMP3 target interaction could among others play a role in the psoriasis pathogenesis., (Copyright 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

In most living cells, K(+) channels are important for the generation of the membrane potential and for volume regulation. The parasite Plasmodium falciparum, which causes malignant malaria, must be able to deal with large variations in the ambient K(+) concentration: it is exposed to high concentrations of K(+) when inside the erythrocyte and low concentrations when in plasma. In the recently published genome of P. falciparum, we have identified a gene, pfkch1, encoding a potential K(+) channel, which to some extent resembles the big-conductance (BK) K(+) channel. We have cloned the approximately 6000 nucleotide (nt) fragment from cDNA, studied the pattern of expression of pfkch1 throughout the intraerythrocytic part of the parasite's life-cyclus, and characterized the channel on the basis of similarity to other K(+) channels from pro- and eukaryotic organisms. This P. falciparum K(+) channel could be a potential drug target.