Unravelling the landscape of skin cancer through single-cell transcriptomics.
2022; 27: 101557
The human skin is a complex organ that forms the first line of defense against pathogens and external injury. It is composed of a wide variety of cells that work together to maintain homeostasis and prevent disease, such as skin cancer. The exponentially rising incidence of skin malignancies poses a growing public health challenge, particularly when the disease course is complicated by metastasis and therapeutic resistance. Recent advances in single-cell transcriptomics have provided a high-resolution view of gene expression heterogeneity that can be applied to skin cancers to define cell types and states, understand disease evolution, and develop new therapeutic concepts. This approach has been particularly valuable in characterizing the contribution of immune cells in skin cancer, an area of great clinical importance given the increasing use of immunotherapy in this setting. In this review, we highlight recent skin cancer studies utilizing bulk RNA sequencing, introduce various single-cell transcriptomics approaches, and summarize key findings obtained by applying single-cell transcriptomics to skin cancer.
View details for DOI 10.1016/j.tranon.2022.101557
View details for PubMedID 36257209
MAB21L4 deficiency drives squamous cell carcinoma via activation of RET.
Epithelial squamous cell carcinomas (SCC) most commonly originate in the skin, where they display disruptions in the normally tightly regulated homeostatic balance between keratinocyte proliferation and terminal differentiation. We performed a transcriptome-wide screen for genes of unknown function that possess inverse expression patterns in differentiating keratinocytes compared to cutaneous SCC (cSCC), leading to the identification of MAB21L4 (C2ORF54) as an enforcer of terminal differentiation that suppresses carcinogenesis. Loss of MAB21L4 in human cSCC organoids increased expression of RET to enable malignant progression. In addition to transcriptional upregulation of RET, deletion of MAB21L4 preempted recruitment of the CacyBP-Siah1 E3 ligase complex to RET and reduced its ubiquitylation. In SCC organoids and in vivo tumor models, genetic disruption of RET or selective inhibition of RET with BLU-667 (pralsetinib) suppressed SCC growth while inducing concomitant differentiation. Overall, loss of MAB21L4 early during SCC development blocks differentiation by increasing RET expression. These results suggest that targeting RET activation is a potential therapeutic strategy for treating SCC.
View details for DOI 10.1158/0008-5472.CAN-22-0047
View details for PubMedID 35705526
Cross-talk between IFN-γ and TWEAK through miR-149 amplifies skin inflammation in psoriasis.
The Journal of allergy and clinical immunology
2021; 147 (6): 2225-2235
Psoriasis is a chronic inflammatory skin disease with disturbed interplay between immune cells and keratinocytes. A strong IFN-γ signature is characteristic for psoriasis skin, but the role of IFN-γ has been elusive. MicroRNAs are short RNAs regulating gene expression.Our aim was to investigate the role of miR-149 in psoriasis and in the inflammatory responses of keratinocytes.miR-149 expression was measured by quantitative RT-PCR in keratinocytes isolated from healthy skin and lesional and nonlesional psoriasis skin. Synthetic miR-149 was injected intradermally into the back skin of mice, and imiquimod was applied to induce psoriasis-like skin inflammation, which was then evaluated at the morphologic, histologic, and molecular levels. miR-149 was transiently overexpressed or inhibited in keratinocytes in combination with IFN-γ- and/or TNF-related weak inducer of apoptosis (TWEAK)-treatment.Here we report a microRNA-mediated mechanism by which IFN-γ primes keratinocytes to inflammatory stimuli. Treatment with IFN-γ results in a rapid and long-lasting suppression of miR-149 in keratinocytes. Depletion of miR-149 in keratinocytes leads to widespread transcriptomic changes and induction of inflammatory mediators with enrichment of the TWEAK pathway. We show that IFN-γ-mediated suppression of miR-149 leads to amplified inflammatory responses to TWEAK. TWEAK receptor (TWEAKR/Fn14) is identified as a novel direct target of miR-149. The in vivo relevance of this pathway is supported by decreased miR-149 expression in psoriasis keratinocytes, as well as by the protective effect of synthetic miR-149 in the imiquimod-induced mouse model of psoriasis.Our data define a new mechanism, in which IFN-γ primes keratinocytes for TWEAK-induced inflammatory responses through suppression of miR-149, promoting skin inflammation.
View details for DOI 10.1016/j.jaci.2020.12.657
View details for PubMedID 33705829
MicroRNA-146a suppresses IL-17-mediated skin inflammation and is genetically associated with psoriasis
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2017; 139 (2): 550-561
Psoriasis is an immune-mediated inflammatory skin disease with a strong genetic background in which activation of IL-17 signaling is central in the pathogenesis. Little has been known about the role of noncoding RNAs, including microRNAs (miRNAs), in predisposition to the disease.We sought to investigate the genetic association of single nucleotide polymorphisms in microRNA-146a (miR-146a) to psoriasis and to explore its function in the initiation and resolution of the disease.Analysis of the genetic association of miR-146a rs2910164 and psoriasis was carried out on 1546 patients with psoriasis and 1526 control subjects. The role of miR-146a in patients with psoriasis was assessed by using miR-146a-/- mice in conjunction with the imiquimod-induced mouse model of psoriasis. The severity of psoriasis-like skin inflammation was evaluated at morphologic, histologic, and molecular levels. miR-146a was ectopically overexpressed and inhibited in keratinocytes treated with IL-17. Synthetic miR-146a was injected intradermally into mice.Here we report protective association of a functional polymorphism in the miR-146a precursor (rs2910164). Genetic deficiency in miR-146a leads to earlier onset and exacerbated pathology of skin inflammation, with increased expression of IL-17-induced keratinocyte-derived inflammatory mediators, epidermal hyperproliferation, and increased neutrophil infiltration. Moreover, miR-146a-deficient mice do not resolve inflammation after discontinuation of imiquimod challenge. The overexpression of miR-146a suppressed, whereas its inhibition enhanced, IL-17-driven inflammation in keratinocytes. Functionally, miR-146a impairs the neutrophil chemoattractant capacity of keratinocytes. Finally, delivery of miR-146a mimics into the skin leads to amelioration of psoriasiform skin inflammation, decreased epidermal proliferation, and neutrophil infiltration.Our results define a crucial role for miR-146a in modulating IL-17-driven inflammation in the skin.
View details for DOI 10.1016/j.jaci.2016.07.025
View details for Web of Science ID 000397002400021
View details for PubMedID 27568078
The long non-coding RNA LINC00958 is induced in psoriasis epidermis and modulates epidermal proliferation.
The Journal of investigative dermatology
Psoriasis is a common immune-mediated skin disease characterized by epidermal hyperproliferation and chronic skin inflammation. Long non-coding RNAs (lncRNAs) are >200 nucleotide long transcripts, which possess important regulatory functions. To date, little is known about the contribution of lncRNAs to psoriasis. Here, we identify LINC00958 as a lncRNA overexpressed in keratinocytes from psoriasis skin lesions, in a transcriptomic screen performed on keratinocytes sorted from psoriasis and healthy skin. Increased levels of LINC00958 in psoriasis keratinocytes were confirmed by RT-qPCR and single molecule in situ hybridization. Confocal microscopy and analysis of subcellular fractions showed that LINC00958 is mainly localized in the cytoplasm of keratinocytes. IL-17A, a key psoriasis cytokine, induced LINC00958 in keratinocytes through C/EBP-β and the p38 pathway. Inhibition of LINC00958 led to decreased proliferation as measured by Ki67 expression, IncuCyte imaging and EdU assays. Transcriptomic analysis of LINC00958-depleted keratinocytes revealed enrichment of proliferation and cell cycle-related genes among differentially expressed transcripts. Moreover, LINC00958-depletion led to decreased basal and IL-17A-induced phosphorylation of p38. Furthermore, IL-17A-induced keratinocyte proliferation was counteracted by the inhibition of LINC00958. In summary, our data support a role for the IL-17A-induced lncRNA, LINC00958, in the pathological circuits in psoriasis by reinforcing IL-17A-induced epidermal hyperproliferation.
View details for DOI 10.1016/j.jid.2022.12.011
View details for PubMedID 36641130
miR-378a regulates keratinocyte responsiveness to IL-17A in psoriasis.
The British journal of dermatology
BACKGROUND: Psoriasis is an immune-mediated inflammatory skin disease, in which an interplay between infiltrating immune cells and keratinocytes sustains chronic skin inflammation. IL-17A is a key inflammatory cytokine in psoriasis and its main cellular targets are keratinocytes.OBJECTIVES: To explore the role of miR-378a in psoriasis.METHODS: Keratinocytes were separated by magnetic sorting from psoriasis and healthy epidermis, and the expression of miR-378a was analysed by qPCR. The regulation and function of miR-378a was studied using primary human keratinocytes. MiR-378a expression was modulated by synthetic mimics, and NF-kappaB activity and transcriptomic changes were studied. Synthetic miR-378a was delivered to mouse skin in conjunction with induction of psoriasiform skin inflammation by imiquimod.RESULTS: We show that miR-378a is induced by IL-17A in keratinocytes through NF-kappaB, C/EBP-beta and IkappaBzeta, and it is overexpressed in psoriasis epidermis. In cultured keratinocytes, ectopic expression of miR-378a resulted in the nuclear translocation of p65 and enhanced NF-kappaB-driven promoter activity even in the absence of inflammatory stimuli. Moreover, miR-378a potentiated the effect of IL-17A on NF-kappaB nuclear translocation and downstream activation of the NF-kappaB pathway. Finally, injection of miR-378a into mouse skin augmented psoriasis-like skin inflammation with increased epidermal proliferation and induction of inflammatory mediators. Mechanistically, miR-378a acts as a suppressor of NFKBIA/IkappaBalpha, an important negative regulator of the NF-kappaB pathway in keratinocytes.CONCLUSIONS: Collectively, our findings identify miR-378a as an amplifier of IL-17A-induced NF-kappaB signalling in keratinocytes and suggest that increased miR-378a levels contribute to the amplification of IL-17A-driven skin inflammation in psoriasis.
View details for DOI 10.1111/bjd.21232
View details for PubMedID 35257359
IL-22 downregulates peptidylarginine deiminase-1 in human keratinocytes: adding another piece to the IL-22 puzzle in epidermal barrier formation.
The Journal of investigative dermatology
Increased presence of interleukin (IL)-22+ cells in the skin is a characteristic finding in skin barrier defects, such as atopic dermatitis (AD) and psoriasis. However, mechanistic insights into effects of IL-22 on epidermal functioning is yet to be elucidated. One crucial step during epidermal differentiation is deimination or citrullination. Here we show reduced levels of peptidylarginine deiminase 1(PAD1), enzyme that converts peptidyl-arginine into citrulline in lesional psoriatic skin. IL-22 signaling through the IL-22 receptor complex was found to suppress expression of PAD1 in epidermal keratinocytes. Subsequently, the total PAD activity and the extent of protein deimination in keratinocytes treated with IL-22 was reduced together with a significant decrease in deimination of keratin 1 (KRT1) and filaggrin (FLG), both important for epidermal differentiation. Importantly, vitamin D and acitretin partly restored the PAD1 defect caused by IL-22. Collectively, we show that IL-22 downregulates deimination, thus identifying a potential target for treatment of skin barrier defects.
View details for DOI 10.1016/j.jid.2021.07.155
View details for PubMedID 34352263
Chromatin interactions in differentiating keratinocytes reveal novel atopic dermatitis- and psoriasis-associated genes
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2021; 147 (5): 1742-1752
Hundreds of variants associated with atopic dermatitis (AD) and psoriasis, 2 common inflammatory skin disorders, have previously been discovered through genome-wide association studies (GWASs). The majority of these variants are in noncoding regions, and their target genes remain largely unclear.We sought to understand the effects of these noncoding variants on the development of AD and psoriasis by linking them to the genes that they regulate.We constructed genomic 3-dimensional maps of human keratinocytes during differentiation by using targeted chromosome conformation capture (Capture Hi-C) targeting more than 20,000 promoters and 214 GWAS variants and combined these data with transcriptome and epigenomic data sets. We validated our results with reporter assays, clustered regularly interspaced short palindromic repeats activation, and examination of patient gene expression from previous studies.We identified 118 target genes of 82 AD and psoriasis GWAS variants. Differential expression of 58 of the 118 target genes (49%) occurred in either AD or psoriatic lesions, many of which were not previously linked to any skin disease. We highlighted the genes AFG1L, CLINT1, ADO, LINC00302, and RP1-140J1.1 and provided further evidence for their potential roles in AD and psoriasis.Our work focused on skin barrier pathology through investigation of the interaction profile of GWAS variants during keratinocyte differentiation. We have provided a catalogue of candidate genes that could modulate the risk of AD and psoriasis. Given that only 35% of the target genes are the gene nearest to the known GWAS variants, we expect that our work will contribute to the discovery of novel pathways involved in AD and psoriasis.
View details for DOI 10.1016/j.jaci.2020.09.035
View details for Web of Science ID 000647680800023
View details for PubMedID 33069716
Circulating microRNAs in extracellular vesicles as potential biomarkers for psoriatic arthritis in patients with psoriasis.
Journal of the European Academy of Dermatology and Venereology : JEADV
2020; 34 (6): 1248-1256
Psoriatic arthritis (PsA) develops in ~30% of patients with psoriasis. The diagnosis of PsA is challenging, and there are no reliable molecular markers in clinical use. MicroRNAs are short non-coding regulatory RNAs, which can be actively packaged into extracellular vesicles (EVs) and secreted to the circulation.To explore whether plasma-derived EV microRNAs may serve as biomarkers for PsA in patients with psoriasis.Plasma samples were obtained from patients with cutaneous-only psoriasis (PsC) and patients with psoriasis and PsA. Plasma EVs were isolated using miRCURY™ Exosome Isolation Kit. RNA sequencing was used to identify differentially expressed EV miRNAs in the discovery phase (PsC, n = 15; PsA, n = 14). In the validation phase (PsC, n = 29; PsA, n = 28), 41 selected miRNAs were analysed in plasma EVs by qPCR. The association of the identified miRNAs with PsA was assessed by logistic regression analysis.RNA sequencing identified 19 plasma EV miRNAs with significantly different levels between PsA and PsC in the discovery cohort. Significantly lower levels of plasma EV let-7b-5p and miR-30e-5p in PsA vs. PsC were confirmed in the validation cohort, and their decreased levels were found to be associated with the presence of PsA. ROC analysis revealed an AUC of 0.68 (95% CI 0.53-0.83) for let-7b-5p and 0.69 (95% CI 0.55-0.84) for miR-30e-5p.Circulating EV microRNA levels are altered in patients with PsA as compared with PsC. Findings of this exploratory study suggest that circulating EV microRNAs may serve as biomarkers for arthritis in psoriasis patients.
View details for DOI 10.1111/jdv.16203
View details for PubMedID 31954077
- Next-Generation Sequencing Identifies the Keratinocyte-Specific miRNA Signature of Psoriasis JOURNAL OF INVESTIGATIVE DERMATOLOGY 2019; 139 (12): 2547-+
The Keratinocyte Transcriptome in Psoriasis: Pathways Related to Immune Responses, Cell Cycle and Keratinization
2019; 99 (2): 196-205
Psoriasis is a common immune-mediated disease resulting from altered cross-talk between keratinocytes and immune cells. Previous transcriptomic studies have identified thousands of deregulated genes in psoriasis skin; however, the transcriptomic changes confined to the epidermal compartment remained poorly characterized. The aim of this study was to characterize the transcriptomic landscape of psoriatic keratinocytes, using sorted CD45neg epidermal cells. Genes with functions in innate immunity, type I interferon response, cell cycle and keratinization were enriched among deregulated genes in psoriatic keratinocytes. Gene set enrichment analysis indicated the dominance of interleukin (IL)-22/IL-17A signatures in the epidermal psoriasis-signature. A set of deregulated genes overlapped with psoriasis-associated genetic regions, suggesting that genetic variations affecting gene expression in keratinocytes contribute to susceptibility to psoriasis. Several psoriasis-susceptibility genes, which were previously believed to be expressed preferentially or exclusively in immune cells, were identified as having altered expression in psoriatic keratinocytes. These results highlight the role of keratinocytes in the pathogenesis of psoriasis, and indicate that both genetic factors and an inflammatory microenvironment contribute to epidermal alterations in psoriasis.
View details for DOI 10.2340/00015555-3066
View details for Web of Science ID 000456794300012
View details for PubMedID 30320872
Tofacitinib Represses the Janus Kinase-Signal Transducer and Activators of Transcription Signalling Pathway in Keratinocytes
2018; 98 (8): 772-775
Tofacitinib is a Janus kinase (JAK) inhibitor, which has shown efficacy in treating psoriasis. The mode of action of tofacitinib is not completely understood but it has been thought to be mediated by the inhibition of CD4+ T-cell activation. Here, we investigated whether the molecular targets of tofacitinib are expressed in keratinocytes, and whether tofacitinib can modulate the activity of the JAK/Signal Transducer and Activators of Transcription (STAT)-pathway in keratinocytes. Transcriptomic profiling of human keratinocytes treated with IL-22 in combination with tofacitinib revealed that tofacitinib could prevent the majority of IL-22-mediated gene expression changes. Pathway analysis of tofacitinib-regulated genes in keratinocytes revealed enrichment of genes involved in the JAK/STAT signalling pathway. Quantitative real-time-PCR confirmed the upregulation of S100A7 and downregulation of EGR1 expression by IL-22, which was prevented by tofacitinib pre-treatment. These results indicate a direct effect of tofacinitib on keratinocytes, which can have relevance for systemic as well as for topical treatment of psoriasis with tofacitinib.
View details for DOI 10.2340/00015555-2960
View details for Web of Science ID 000442511400010
View details for PubMedID 29738047
Identification of chronological and photoageing-associated microRNAs in human skin
2018; 8: 12990
MicroRNAs are short non-coding RNAs that play key roles in regulating biological processes. In this study, we explored effects of chronological and photoageing on the miRNome of human skin. To this end, biopsies were collected from sun-exposed (outer arm, n = 45) and sun-protected (inner arm, n = 45) skin from fair-skinned (phototype II/III) healthy female volunteers of three age groups: young, 18-25 years, middle age, 40-50 years and aged, > 70 years. Strict inclusion criteria were used for photoageing scoring and for chronological ageing. Microarray analysis revealed that chronological ageing had minor effect on the human skin miRNome. In contrast, photoageing had a robust impact on miRNAs, and a set of miRNAs differentially expressed between sun-protected and sun-exposed skin of the young and aged groups was identified. Upregulation of miR-383, miR-145 and miR-34a and downregulation of miR-6879, miR-3648 and miR-663b were confirmed using qRT-PCR in sun-exposed skin compared with sun-protected skin. qRT-PCR analysis revealed that miR-383, miR-34a and miR-134 were differentially expressed in all three age groups both in chronological and photoageing, suggesting a synergetic effect of intrinsic and extrinsic ageing on their expression. In conclusion, our study identifies a unique miRNA signature which may contribute to skin ageing.
View details for DOI 10.1038/s41598-018-31217-8
View details for Web of Science ID 000442918300001
View details for PubMedID 30154427
View details for PubMedCentralID PMC6113407
Long term impact of the endocrine disruptor tributyltin on male fertility following a single acute exposure
2017; 32 (10): 2295-2304
Declining rate of human fertility is a growing concern, where lifestyle and environmental factors play an important role. We recently demonstrated that tributyltin (TBT), an omnipresent endocrine disruptor, affects testicular cells in vitro. In this study, male Wistar rats were gavaged a single dose of 10, 20, and 30 mg/kg TBT-chloride (TBTC) (to mimic accidental exposure in vivo) and sacrificed on day 3 and day 7, respectively. TBT bioavailability was evaluated by estimating total tin content, and essential metal levels were analyzed along with redox molecules (ROS and GSH/GSSG) to understand the effect on physiological conditions. Blood-testicular barrier (BTB) disruption, levels of associated proteins and activity of proteolytic enzymes were evaluated to understand the effect on BTB. Histological analysis of tissue architecture and effect on protein expression of steroidogenic, stress and apoptotic markers were also evaluated. Widespread TBTC pollution can be an eventual threat to male fertility worldwide.
View details for DOI 10.1002/tox.22446
View details for Web of Science ID 000410635200008
View details for PubMedID 28707438
Consequences of tributyltin chloride induced stress in Leydig cells: An ex-vivo approach
ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY
2014; 37 (2): 850-860
Tributyltin (TBT), a member of the organotin family, is a known endocrine disruptor. It persists long in the environment and is widely used in various industrial applications. This study was planned to understand its toxic influence on Leydig cells isolated from 28 day old wistar rats. In-vitro exposure to TBT-Chloride (TBTC) (300-3000 nM) reduced cell viability (DNA fragmentation, nuclear condensation and MTT assay) and affected testosterone production. TBTC induced both apoptotic and necrotic cell death (AnnexinV/PI binding assay). Involvement of calcium (Ca(2+)), redox imbalance (ROS, GSH and TBARS) and mitochondria in TBTC toxicity was evaluated by using Ca(2+) inhibitors (BAPTA-AM, EGTA, Ruthenium Red), free radical scavengers (NAC, C-Phycocyanin) and mitochondrial permeability transition pore inhibitor (Cyclosporine A). Protein expression analysis of phosphorylated MAPKinases (ERK1/2, JNK1/2, & p38), steroidogenic proteins (3β-HSD, StAR & TSPO) and apoptotic proteins (Bax, Bcl2) illustrates the cytotoxic and anti-steroidogenic activity of TBTC.
View details for DOI 10.1016/j.etap.2014.02.018
View details for Web of Science ID 000335636600041
View details for PubMedID 24657357
Tributyltin chloride induced testicular toxicity by JNK and p38 activation, redox imbalance and cell death in sertoli-germ cell co-culture
2013; 314 (1): 39-50
The widespread use of tributyltin (TBT) as biocides in antifouling paints and agricultural chemicals has led to environmental and marine pollution. Human exposure occurs mainly through TBT contaminated seafood and drinking water. It is a well known endocrine disruptor in mammals, but its molecular mechanism in testicular damage is largely unexplored. This study was therefore, designed to ascertain effects of tributyltin chloride (TBTC) on sertoli-germ cell co-culture in ex-vivo and in the testicular tissue in-vivo conditions. An initial Ca(2+) rise followed by ROS generation and glutathione depletion resulted in oxidative damage and cell death. We observed p38 and JNK phosphorylation, stress proteins (Nrf2, MT and GST) induction and mitochondrial depolarization leading to caspase-3 activation. Prevention of TBTC reduced cell survival and cell death by Ca(2+) inhibitors and free radical scavengers specify definitive role of Ca(2+) and ROS. Sertoli cells were found to be more severely affected which in turn can hamper germ cells functionality. TBTC exposure in-vivo resulted in increased tin content in the testis with enhanced Evans blue leakage into the testicular tissue indicating blood-testis barrier disruption. Tesmin levels were significantly diminished and histopathological studies revealed marked tissue damage. Our data collectively indicates the toxic manifestations of TBTC on the male reproductive system and the mechanisms involved.
View details for DOI 10.1016/j.tox.2013.09.003
View details for Web of Science ID 000209429700005
View details for PubMedID 24055800