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Our understanding of the regulatory processes of reepithelialization during wound healing is incomplete. In an attempt to map the genes involved in epidermal regeneration and differentiation, we measured gene expression in formalin-fixed, paraffin-embedded standardized epidermal wounds induced by the suction-blister technique with associated nonwounded skin using NanoString technology. The transcripts of 139 selected genes involved in clotting, immune response to tissue injury, signaling pathways, cell adhesion and proliferation, extracellular matrix remodeling, zinc transport and keratinocyte differentiation were evaluated. We identified 22 upregulated differentially expressed genes (DEGs) in descending order of fold change (MMP1, MMP3, IL6, CXCL8, SERPINE1, IL1B, PTGS2, HBEGF, CXCL5, CXCL2, TIMP1, CYR61, CXCL1, MMP12, MMP9, HGF, CTGF, ITGB3, MT2A, FGF7, COL4A1 and PLAUR). The expression of the most upregulated gene, MMP1, correlated strongly with MMP3 followed by IL6 and IL1B. rhIL-1β, but not rhIL-6, exposure of cultured normal human epidermal keratinocytes and normal human dermal fibroblasts increased both MMP1 mRNA and MMP-1 protein levels, as well as TIMP1 mRNA levels. The increased TIMP1 in wounds was validated by immunohistochemistry. The six downregulated DEGs (COL7A1, MMP28, SLC39A2, FLG1, KRT10 and FLG2) were associated with epidermal maturation. KLK8 showed the strongest correlation with MKI67 mRNA levels and is a potential biomarker for keratinocyte proliferation. The observed gene expression changes correlate well with the current knowledge of physiological reepithelialization. Thus, the gene expression panel described in this paper could be used in patients with impaired healing to identify possible therapeutic targets. [ABSTRACT FROM AUTHOR]

Altered miRNA expressions are assigned pathogenic properties in several cancers including mycosis fungoides and could play a role in the early onset of the disease. The aim of this study was to examine disease-specific miRNA expression in early-stage mycosis fungoides patch and plaque lesions. A quantitative real-time PCR platform of 384 human miRNAs was used to study miRNA expression in 154 diagnostic mycosis fungoides biopsies. A total of 110 miRNAs were significantly differentially expressed (>2-fold, p < 0.05) between plaque lesions and healthy controls, and 90 miRNAs (>2-fold, p < 0.05) differed between patch lesions and healthy controls. Moreover, 13 miRNAs differed in expression between patch and plaque lesions. Early-stage mycosis fungoides exhibited miRNA features that overlapped with those of psoriasis. However, 39 miRNAs, including miR-142-3p, miR-150 and miR-146b, were specific to mycosis fungoides. In conclusion, early-stage mycosis fungoides expresses a distinct miRNA profile, indicating that miRNAs could play a role in the early development of mycosis fungoides. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) is a chronic, pruritic skin disease characterized by type 2 immune-mediated inflammation and skin barrier dysfunction (1). The impaired skin barrier in AD is related to decreased expression of epidermal barrier proteins and to changes in lipid composition in the stratum corneum. AD lesions are characterized by increased expression of pro-inflammatory mediators, such as chemokines, leading to recruitment of immune cells, which may further exacerbate the inflammation. Interleukin (IL)-13 and IL-4 are the signature type 2 cytokines involved in AD. These cytokines both signal through the type II receptor, composed of IL-4Ra and IL-13Ra1, and display functional redundancy in cells harbouring this receptor, including keratinocytes and fibroblasts. This is in contrast to cells, such as T lymphocytes, containing the type I receptor (IL-4Ra combined with the common gamma chain), which is only used by IL-4 for signalling. Monoclonal antibodies targeting IL-4Ra (dupilumab) or IL-13 (tralokinumab and lebrikizumab) have demonstrated clinical efficacy in AD (2-4). Interestingly, protein levels of IL-13, but not IL-4, are consistently detected and shown to be increased in AD skin across studies (5, 6). Furthermore, 3 recently published studies using RNA-sequencing of AD biopsies found increased levels of IL13 in lesional and non-lesional AD skin, whereas expression of IL4 was undetectable or very low (7-9). Independent studies also found a correlation between expression levels of IL-13 at mRNA or protein level in lesional AD skin and disease severity (6, 7). The aim of the current study is to further investigate effects of IL-13 on AD-associated genes in human skin cells and to provide molecular insights into the mechanism of action of tralokinumab, a fully human IgG4 monoclonal antibody that specifically neutralizes IL-13. [ABSTRACT FROM AUTHOR]

The current state, tools, and applications of personalized medicine with special emphasis on inflammatory skin diseases like psoriasis and atopic dermatitis are discussed. Inflammatory pathways are outlined as well as potential targets for monoclonal antibodies and small‐molecule inhibitors. [ABSTRACT FROM AUTHOR]

Simple Summary: The screening of experimental and repurposing drugs has enormous potential for rapid clinical impact, particularly when new therapeutic strategies are sought. By screening two drug libraries, nicotinamide phosphoribosyltransferase (NAMPT) and histone deacetylase (HDAC) inhibitors were identified as agents with the highest activity against neuroendocrine tumor-derived cell lines. An evaluation of different NAMPT inhibitors revealed a reduction in basal oxidative phosphorylation and energy production to be partially but not completely responsible for cell death following the exposure to the drug, with the yes-associated protein 1 (YAP1) playing a pivotal role in the sensitivity to the NAMPT inhibitors. Insight into the contribution of YAP1 guided the evaluation of combinations of HDAC and NAMPT inhibitors for the treatment of neuroendocrine neoplasms. Background: Over the last few decades of treatment, the outcomes for at least some subsets of neuroendocrine neoplasms (NENs) have improved. However, the identification of new vulnerabilities for this heterogeneous group of cancers remains a priority. Methods: Using two libraries of compounds selected for potential repurposing, we identified the inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) and histone deacetylases (HDAC) as the agents with the highest activity. We validated the hits in an expanded set of neuroendocrine cell lines and examined the mechanisms of action. Results: In Kelly, NH-6, and NCI-H82, which are two neuroblastoma and one small cell lung cancer cell lines, respectively, metabolic studies suggested that cell death following NAMPT inhibition is the result of a reduction in basal oxidative phosphorylation and energy production. NAMPT is the rate-limiting enzyme in the production of NAD+, and in the three cell lines, NAMPT inhibition led to a marked reduction in the ATP and NAD+ levels and the catalytic activity of the citric acid cycle. Moreover, comparative analysis of the mRNA expression in drug-sensitive and -insensitive cell lines found less dependency of the latter on oxidative phosphorylation for their energy requirement. Further, the analysis of HDAC and NAMPT inhibitors administered in combination found marked activity using low sub-lethal concentrations of both agents, suggesting a synergistic effect. Conclusion: These data suggest NAMPT inhibitors alone or in combination with HDAC inhibitors could be particularly effective in the treatment of neuroendocrine neoplasms. [ABSTRACT FROM AUTHOR]

Atopic dermatitis ( AD) is a common inflammatory skin disease with underlying defects in epidermal function and immune responses. In this study, we used microarray analysis to investigate differences in gene expression in lesional skin from patients with mild extrinsic or intrinsic AD compared to skin from healthy controls and from lesional psoriasis skin. The primary aim was to identify differentially expressed genes involved in skin barrier formation and inflammation, and to compare our results with those reported for patients with moderate and severe AD. In contrast to severe AD, expression of the majority of genes associated with skin barrier formation was unchanged or upregulated in patients with mild AD compared to normal healthy skin. Among these, no significant differences in the expression of filaggrin ( FLG) and loricrin at both mRNA and protein level were found in lesional skin from patients with mild AD, despite the presence of heterozygous FLG mutations in the majority of patients with mild extrinsic AD. Several inflammation-associated genes such as S100A9, MMP12, CXCL10 and CCL18 were highly expressed in lesional skin from patients with mild psoriasis and were also increased in patients with mild extrinsic and intrinsic AD similar to previous reports for severe AD. Interestingly, expression of genes involved in inflammatory responses in intrinsic AD resembled that of psoriasis more than that of extrinsic AD. Overall, differences in expression of inflammation-associated genes found among patients with mild intrinsic and extrinsic AD correlated with previous findings for patients with severe intrinsic and extrinsic AD. [ABSTRACT FROM AUTHOR]

Systemic treatment with hedgehog inhibitors (HHis) is available to treat basal cell carcinomas but their utility is limited by adverse effects. Topical delivery methods may reduce adverse effects, but successful topical treatment depends on sufficient skin uptake, biological response, and time in tumor tissue. The aim of this review was to evaluate the current status of topical HHi delivery for BCCs and discuss barriers for translating systemic HHis into topical treatments. A literature search identified 16 preclinical studies and 7 clinical trials on the topical delivery of 12 HHis that have been clinically tested on BCCs. Preclinical studies on drug uptake demonstrated that novel formulations, and delivery- and pre-treatment techniques enhanced topical HHi delivery. Murine studies showed that the topical delivery of sonidegib, itraconazole, vitamin D₃ and CUR-61414 led to biological responses and tumor remission. In clinical trials, only topical patidegib and sonidegib led to at least a partial response in 26/86 BCCs and 30/34 patients, respectively. However, histological clearance was not observed in the samples analyzed. In conclusion, the incomplete clinical response could be due to poor HHi uptake, biodistribution or biological response over time. Novel topical delivery techniques may improve HHi delivery, but additional research on cutaneous pharmacokinetics and biological response is needed. [ABSTRACT FROM AUTHOR]

Altered miRNA expressions are assigned pathogenic properties in several cancers including mycosis fungoides (MF) and could play a role in the early onset of the disease. To examine disease specific miRNA expression in early-stage MF patch and plaque lesions. We used a qRT-PCR platform of 384 human miRNAs to study the miRNA expression in 154 diagnostic MF biopsies. One-hundred-and-ten miRNAs were significantly differentially expressed (>2-fold, P<0.05) between plaque lesions and healthy controls, and 90 miRNAs (>2-fold, P<0.05) differed between patch lesions and healthy controls. Moreover, 13 miRNAs differed in expression between patch and plaque lesions. Early-stage MF exhibited miRNA features overlapping with psoriasis. However, 39 miRNAs, including miR-142-3p, miR-150 and miR-146b, were MF specific. Early-stage MF express a distinct miRNA profile indicating that miRNAs play a role in the early development of MF. [ABSTRACT FROM AUTHOR]

Abstract: A published microarray gene expression database containing data on 174 tumor samples from ten tissues was mined, enabling the identification of classes of genes whose expression correlates significantly with the intractability, or tractability, to therapy of tumors derived from such tissues. As a measure of tractability, the 5-year survival of patients presenting with distant (metastatic) tumors was used. Genes that encode proteins related to cell adhesion, and enzymes involved in metabolic oxidation or reduction, were upregulated in intractable cancers. Genes that encode proteins implicated in the control of DNA transcription were downregulated in the intractable cancers. We describe hypotheses with regard to cell functions that may help in designing new therapeutic modalities, aimed at improving survival of cancer patients. [Copyright &y& Elsevier]

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport. [Copyright &y& Elsevier]

Secukinumab and Dead Sea treatment result in clear skin for many psoriasis patients, through distinct mechanisms. However, recurrence in the same areas after treatments suggests the existence of a molecular scar. We aimed to compare the molecular and genetic differences in psoriasis patients who achieved complete response from secukinumab and Dead Sea climatotherapy treatments. We performed quantitative immunohistochemical and transcriptomic analysis, in addition to digital spatial profiling of skin punch biopsies. Histologically, both treatments resulted in a normalization of the lesional skin to a level resembling nonlesional skin. Interestingly, the transcriptome was not normalized by either treatments. We revealed 479 differentially expressed genes between secukinumab and Dead Sea climatotherapy at the end of treatment, with a psoriasis panel identifying SERPINB4, SERPINB13, IL36G, IL36RN, and AKR1B10 as upregulated in Dead Sea climatotherapy compared with secukinumab. Using digital spatial profiling, pan-RAS was observed to be differentially expressed in the microenvironment surrounding CD103+ cells, and IDO1 was differentially expressed in the dermis when comparing the two treatments. The differences observed between secukinumab and Dead Sea climatotherapy suggest the presence of a molecular scar, which may stem from mechanistically different pathways and potentially contribute to disease recurrence. This may be important for determining treatment response duration and disease memory. [ABSTRACT FROM AUTHOR]

The oncogene PIM2 is upregulated in several malignancies but has never been investigated in mycosis fungoides (MF), the most common type of cutaneous T‐cell lymphoma (CTCL). PIM2 is a well‐known oncogene and is regulated by cell signaling pathways like the JAK/STAT‐ and NF‐kB‐pathway, key regulators in the pathogenesis of CTCL. The aim of this study was to examine the role of PIM2 in MF. PIM2 gene expression was measured in 81 formalin‐fixed paraffin‐embedded skin biopsies from patients with MF and 46 control biopsies from healthy skin (HS) and benign inflammatory skin disease (BID). Validation of PIM2 protein expression was performed on selected biopsies with immunohistochemical staining. We found a significant difference in gene expression levels between both early stage MF and HS (p < 0.0001), and BID (p < 0.0001). In addition, the PIM2 gene expression was higher in advanced‐stage MF compared to early stage disease (p = 0.0001). No significant difference in gene expression levels was found between patients with and without disease progression. In conclusion, we found PIM2 expression is significantly increased in MF compared to controls, and in advanced‐stage MF compared to early stage MF. These findings could potentially have diagnostic value in discriminating early stage MF from BID. [ABSTRACT FROM AUTHOR]

Neuroinflammation is an essential part of neurodegeneration. Yet, the current understanding of neuroinflammation-associated molecular events in distinct brain regions of prion disease patients is insufficient to lay the ground for effective treatment strategies targeting this complex neuropathological process. To address this problem, we analyzed the expression of 800 neuroinflammation-associated genes to create a profile of biological processes taking place in the frontal cortex and cerebellum of patients who suffered from sporadic Creutzfeldt–Jakob disease. The analysis was performed using NanoString nCounter technology with human neuroinflammation panel+. The observed gene expression patterns were regionally and sub-regionally distinct, suggesting a variable neuroinflammatory response. Interestingly, the observed differences could not be explained by the molecular subtypes of sporadic Creutzfeldt–Jakob disease. Furthermore, analyses of canonical pathways and upstream regulators based on differentially expressed genes indicated an overlap between biological processes taking place in different brain regions. This suggests that even smaller-scale spatial data reflecting subtle changes in brain cells' functional heterogeneity and their immediate pathologic microenvironments are needed to explain the observed differential gene expression in a greater detail. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the impact of ablative fractional laser (AFL) on hedgehog pathway gene expression in murine microscopic basal cell carcinomas (BCCs) and compare these results to the effect of topical treatment with vismodegib, an FDA-approved hedgehog inhibitor. In 25 mice, 1 cm2 skin test sites (n = 44) containing microscopic BCCs were exposed to one of three interventions: a single CO2 AFL treatment (1 pulse, 40 mJ/microbeam, wavelength 10.6 μm, 5% density, pulse rate 250 Hz, n = 12), eight topical vismodegib treatments (3.8 mg/mL, n = 8), or combination of AFL and vismodegib treatments (n = 9). Untreated controls were included for comparison (n = 15). After 4 days, skin samples were analyzed for hedgehog gene expression (Gli1, Gli2, and Ptch1) by qPCR and vismodegib concentrations by liquid chromatography mass spectrometry (data analyzed with two-tailed t-tests and linear regression). A single treatment with AFL monotherapy significantly reduced hedgehog gene expression compared to untreated controls (Gli1 72.4% reduction, p = 0.003; Gli2 55.2%, p = 0.010; Ptch1 70.9%, p < 0.001). Vismodegib treatment also reduced hedgehog gene expression (Gli1 91.6%; Gli2 83.3%; Ptch1 83.0%), significantly surpassing AFL monotherapy for two out of three genes (Gli1, p = 0.017; Gli2, p = 0.007; Ptch1, p = 0.15). AFL and vismodegib combination mirrored the effects of vismodegib monotherapy (Gli1, p = 0.424; Gli2, p = 0.289; Ptch1, p = 0.593), possibly due to comparable cutaneous vismodegib concentrations (mean ± SD, vismodegib monotherapy 850 ± 475 µmol/L; combination 1036 ± 824 µmol/L; p = 0.573). In conclusion, a single AFL treatment significantly reduced hedgehog gene expression in murine BCCs mimicking the effects of eight topical applications of vismodegib. Further studies are needed to assess whether AFL can be utilized for BCC treatment, either as monotherapy or in combination with other drugs. [ABSTRACT FROM AUTHOR]

There has been considerable research into the understanding of the healthy skin microbiome. Similarly, there is also a considerable body of research into whether specific microbes contribute to skin disorders, with atopic dermatitis (AD) routinely linked to increased Staphylococcus aureus (S. aureus) colonisation. In this study, the epidermal surface of participants was sampled using swabs, while serial tape-stripping (35 tapes) was performed to sample through the stratum corneum. Samples were taken from AD patients and healthy controls, and the bacterial communities were profiled by metabarcoding the universal V3-V4 16S rRNA region. Results show that the majority of bacterial richness is located within the outermost layers of the stratum corneum, however there were many taxa that were found almost exclusively at the very outermost layer of the epidermis. We therefore hypothesise that tape-stripping can be performed to investigate the 'core microbiome' of participants by removing environmental contaminants. Interestingly, significant community variation between AD patients and healthy controls was only observable at the epidermal surface, yet a number of individual taxa were found to consistently differ with AD status across the entire epidermis (i.e. both the epidermal surface and within the epidermis). Sampling strategy could therefore be tailored dependent on the hypothesis, with sampling for forensic applications best performed using surface swabs and outer tapes, while profiling sub-surface communities may better reflect host genome and immunological status. [ABSTRACT FROM AUTHOR]

Tape stripping is a non-invasive skin sampling technique, which has recently gained use for the study of the transcriptome of atopic dermatitis (AD), a common inflammatory skin disorder characterized by a defective epidermal barrier and perturbated immune response. Here, we performed BRB-seq—a low cost, multiplex-based, transcriptomic profiling technique—on tape-stripped skin from 30 AD patients and 30 healthy controls to evaluate the methods' ability to assess the epidermal AD transcriptome. An AD signature consisting of 91 differentially expressed genes, specific for skin barrier and inflammatory response, was identified. The gene expression in the outermost layers, stratum corneum and stratum granulosum, of the skin showed highest correlation between tape-stripped skin and matched full-thickness punch biopsies. However, we observed that low and highly variable transcript counts, probably due to low RNA yield and RNA degradation in the tape-stripped skin samples, were a limiting factor for epidermal transcriptome profiling as compared to punch biopsies. We conclude that deep BRB-seq of tape-stripped skin is needed to counteract large between-sample RNA yield variation and highly zero-inflated data in order to apply this protocol for population-wide screening of the epidermal transcriptome in inflammatory skin diseases. [ABSTRACT FROM AUTHOR]

Background: No biomarkers have been identified that can classify subtypes of hand eczema (HE). Although skin biopsies represent the gold standard for investigations of the skin, the invasive technique is not favorable when investigating skin from sensitive areas. Recent advances in the use of skin‐tape strips for molecular investigations enable noninvasive investigations of HE. Objective: By using whole transcriptome sequencing (WTS), the molecular profile of HE according to different localizations on the hands, etiologies, and clinical/morphological subtypes was investigated. Methods: Thirty adult, Danish HE patients, 12 with and 18 without concurrent atopic dermatitis (AD), as well as 16 controls were included. Tape strip samples were collected from lesional, nonlesional, and healthy skin. Total RNA was extracted and WTS was performed. Results: The largest molecular difference of HE patients with and without AD was found in nonlesional skin areas and included a downregulation of CXCL8 for HE patients without AD. Differences between allergic and irritant contact dermatitis included epidermal biomarkers such as EPHA1. Conclusion: Skin tape strip samples could be used to assess the gene expression profile of HE on different localizations of the hands. The skin tape strip method identified new molecular markers that showed promising result for the identification of HE subtypes. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the early-life development of the skin microbiome in atopic dermatitis. Nineteen infants with atopic dermatitis and 19 healthy infants were evaluated 3 times, at 3 months intervals, within the first 30 months of life. Tape-strips were collected from volar forearms, cheeks, and eczema lesions, and the skin microbiome was assessed by 16S rRNA sequencing. Both the community structure and richness of the skin microbiome of infants with atopic dermatitis differed significantly from that of healthy infants, with greater richness in healthy infants. For infants with atopic dermatitis, the community composition was not dominated by Staphylococci. For healthy infants, community composition and richness correlated significantly with age, while such a pattern was not revealed in infants with atopic dermatitis. This suggests a slower maturation of the skin microbiome in atopic dermatitis, which precedes the staphylococcal predominance observed in older children and adults. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

ABCG2 is recognized as an important efflux transporter in clinical pharmacology and is potentially important in resistance to chemotherapeutic drugs. To identify epigenetic mechanisms regulating ABCG2 mRNA expression at its 3' untranslated region (3'UTR), we performed 3' rapid amplification of cDNA ends with the S1 parental colon cancer cell line and its drug-resistant ABCG2-overexpressing counterpart. We found that the 3'UTR is >1,500 bp longer in parental cells and, using the miRBase TARGETs database, identified a putative microRNA (miRNA) binding site, distinct from the recently reported hsa-miR520h site, in the portion of the 3'UTR missing from ABCG2 mRNA in the resistant cells. We hypothesized that the binding of a putative miRNA at the 3'UTR of ABCG2 suppresses the expression of ABCG2. In resistant S1MI80 cells, the miRNA cannot bind to ABCG2 mRNA because of the shorter 3'UTR, and thus, mRNA degradation and/or repression on protein translation is relieved, contributing to overexpression of ABCG2. This hypothesis was rigorously tested by reporter gene assays, mutational analysis at the miRNA binding sites, and forced expression of miRNA inhibitors or mimics. The removal of this epigenetic regulation by miRNA could be involved in the overexpression of ABCG2 in drug-resistant cancer cells. [ABSTRACT FROM AUTHOR]

Background: Histone acetylation is an epigenetic modification involved in the regulation of gene expression, balanced by histone acetyl transferases and histone deacetylase (HDAC) enzymes. HDAC inhibitors (HDACi) induce growth arrest and cell death in transformed cells, and are currently in many clinical cancer trials. The transcriptional response to HDACi is complex, as is the response to HDAC isoform knockdown (KD). Here, we describe for the first time in a human cancer cell line, a transcriptional comparison of treatment by two structurally unrelated HDACi; belinostat and valproic acid with the KD of HDAC1, 2 and 3 isoforms. Results: HDAC KD showed anti-proliferative effects, although to a lesser extent than HDACi treatment. Moreover, we found a 2-fold increased resistance of HDAC1 knockdown cells to belinostat, suggesting this isoenzyme as a selective target. While both HDACi treatment and individual class I HDAC KD produce significant transcriptional effects, three-times higher for HDACi, the gene-expression profiles of class I HDAC KD compared with that obtained by HDACi treatment exhibited less than 4% of altered genes in common between the two modes of inhibition. Further, cell-specific effects of HDAC KD are evident by comparison with a recent study in a different cell line. Conclusion: The increased resistance to belinostat in response to HDAC1 depletion indicates the possibility of using this isoform as a predictive biomarker of response to HDACi treatment. Further, the transcriptional response to chemical inhibition of HDACs is very different from that of KD of individual class I HDAC isoforms. These data suggest that the anti-tumor effect of HDACi is indeed linked to class I inhibition, but may be more complex than simply targeting individual HDAC enzymes. [ABSTRACT FROM AUTHOR]

Abstract: Reduced drug accumulation due to overexpression of individual members of the ATP binding cassette (ABC) superfamily of membrane transporters has been investigated as a cause of multidrug resistance and treatment failure in oncology. This study was designed to develop an immunohistochemical assay to determine the expression and localization of the 72kDa ABC half-transporter ABCG2 in normal tissues. Formalin-fixed, paraffin embedded archival tissue from 31 distinct normal tissues with an average of eight separate tissue samples of each were immunostained with rabbit-anti-ABCG2 antibody 405 using a modified avidin–biotin procedure. As a negative control, each sample was also stained with antibody pre-adsorbed with peptide to assess background staining. As a means of verification, selected tissues were also stained with the commercially available monoclonal antibody 5D3. ABCG2 positivity was consistently found in alveolar pneumocytes, sebaceous glands, transitional epithelium of bladder, interstitial cells of testes, prostate epithelium, endocervical cells of uterus, squamous epithelium of cervix, small and large intestinal mucosa/epithelial cells, islet and acinar cells of pancreas, zona reticularis layer of adrenal gland, kidney cortical tubules and hepatocytes. Placental syncytiotrophoblasts showed both cytoplasmic and surface staining. Our results support a hypothesis concluding that ABCG2 plays a role in the protection of organs from cytotoxins. However, many of the cell types expressing ABCG2 have a significant secretory function. These data suggest a dual function for ABCG2 in some tissues: the excretion of toxins and xenobiotics including anti-cancer agents and a potential, as-yet undefined role in the secretion of endogenous substrates. [Copyright &y& Elsevier]

ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/ C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2. [ABSTRACT FROM AUTHOR]

The ATP binding cassette (ABC) half-transporter ABCG2 (MXRIBCRP/ABCP) is associated with mitoxantrone resistance accompanied by cross-resistance to a broad spectrum of cytotoxic drugs. Here we investigate the functional consequences of mutating a highly conserved lysine in the Walker A motif of the nucleotide binding domain (NBD) known to be critical for ATP binding and/or hydrolysis in ABC transporters. The mutant (ABCG2- K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. The mutation did not affect the predicted oligomerization properties of the transporter; hence, co-immunoprecipitation experiments using differentially tagged transporters showed evidence for oligomerization of both ABCG2-wt and of ABCG2-wt with ABC G2-KS6M. We also obtained evidence that both ABC G2-wt and ABCG2-KS6M exist in the cells as disulfide-linked dimers. Moreover, measurement of prazosin-stimulated ATPase activity revealed a dominant-negative effect of ABCG2-KS6M on ABCG2-wt function in co-transfected HEK293 cells. This is consistent with the requirement for at least two active NBDs for transporter activity and suggests that the transporter is a functional dimer. Finally, we analyzed targeting of ABCG2-wt and ABCG2-K86M and observed that they localize to two distinct subcellular compartments: ABC G2-wt targets the cell surface whereas ABC G2-K86M is targeted to the Golgi apparatus followed by retrieval to the endoplasmic reticulum. This suggests an as yet unknown role of the NBDs in assisting proper surface targeting of ABC transporters. [ABSTRACT FROM AUTHOR]

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 ∼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. [Copyright &y& Elsevier]

Background: Several cancers, including mycosis fungoides (MF), have reported dysregulation of miR‐195‐5p. miR‐195‐5p plays a role in cell cycle regulation in several malignant diseases. Objectives: This study aimed to investigate: (a) the expression level of miR‐195‐5p in lesional MF skin biopsies and (b) the potential regulatory roles of miR‐195‐5p in MF. Methods: Quantitative real‐time polymerase chain reaction (RT‐qPCR) was used to determine miR‐195‐5p expression in MF skin biopsies and cell lines. The effect of miR‐195‐5p and ADP‐ribosylation factor‐like protein 2 (ARL2) on cell cycle and apoptosis was measured by flow cytometry assays. Changes in ARL2 expression were determined by RT‐qPCR and Western blotting (WB). Results: We found lower expression levels of miR‐195‐5p in lesional skin from MF patients compared with non‐lesional MF skin and skin from healthy volunteers. Additionally, miR‐195‐5p showed lower expression levels in the skin from patients with disease progression compared with patients with stable disease. In vitro studies showed that overexpression of miR‐195‐5p induced a cell cycle arrest in G0G1. Using microarray analysis, we identified several genes that were regulated after miR‐195‐5p overexpression. The most downregulated gene after miR‐195‐5p mimic transfection was ARL2. RT‐qPCR and WB analyses confirmed downregulation of ARL2 following transfection with miR‐195‐5p mimic. Lastly, transfection with siRNA against ARL2 also induced a G0G1 arrest. Conclusion: Upregulation of miR‐195‐5p in MF inhibits cycle arrest by downregulation of ARL2. miR‐195‐5p may thus function as a tumor suppressor in MF and low miR‐195‐5p expression in lesional MF skin may promote disease progression. [ABSTRACT FROM AUTHOR]

Focuses on the binding of Zinc (Zn) to the endogenous Zn binding site in the human dopamine transporter. Generation of an activating Zn switch in the dopamine transporter; Effect of the mutation of an intracellular tyrosine on the conformational equilibrium of the transport cycle.

Mycosis fungoides is the most common type of cutaneous T-cell lymphoma. The inflammatory microenvironment in mycosis fungoides is complex. There is accumulating evidence that the neoplastic T-cells take control of the microenvironment and thereby promote their own expansion by suppressing cellular immunity. B-cells have proved to be upregulated in large-cell transformed mycosis fungoides, and could potentially play a role in disease progression. To investigate the presence of B-cells in mycosis fungoides compared with controls, this study analysed 85 formalin-fixed and paraffin-embedded mycosis fungoides biopsies. MS4A1 gene expression was significantly upregulated in mycosis fungoides compared with controls (p < 0.0001) and further upregulated in disease progression, (p = 0.001). Digital quantification of PAX5+/CD20+ cells confirmed the increased presence of Bcells in mycosis fungoides compared with controls. No co-labelling of CD3/CD20 was observed in the neoplastic T-cells. This study found a significantly increased presence of B-cells in the tumour-associated microenvironment in mycosis fungoides. These findings could potentially lead to new treatment strategies for mycosis fungoides. [ABSTRACT FROM AUTHOR]

Background: Adrenocortical carcinoma (ACC) is a rare, often-aggressive neoplasm of the adrenal cortex, with a 14–17 month median overall survival. We asked whether tumors from patients with advanced or metastatic ACC would offer clues as to putative genes that might have critical roles in disease progression or in more aggressive disease biology. Methods: We conducted comprehensive genomic and expression analyses of ACCs from 43 patients, 30 female, and 42 from metastatic sites, including deep sequencing, copy number analysis, mRNA expression and microRNA arrays. Results: Copy number gains and losses were similar to that previously reported for ACC. We identified a median mutation rate of 3.38 per megabase (Mb). The mutational signature was characterized by a predominance of C > T, C > A and T > C transitions. Only cancer genes TP53 (26%) and beta-catenin (CTNNB1, 14%) were mutated in more than 10% of samples. The TCGA-identified putative cancer genes MEN1 and PRKAR1A were found in low frequency—4.7 and 2.3%, respectively. The majority of the mutations were in genes not implicated in the etiology or maintenance of cancer. Specifically, amongst the 38 genes that were mutated in more than 9% of samples, only four were represented in Tier 1 of the 576 COSMIC Cancer Gene Census (CCGC). Thus, 82% of genes found to have mutations likely have no role in the etiology or biology of ACC; while the role of the other 18%, if any, remains to be proven. Finally, the transcript length for the 38 most frequently mutated genes in ACC is statistically longer than the average of all coding genes, raising the question of whether transcript length in part determined mutation probability. Conclusions: We conclude that the mutational and expression profiles of advanced and metastatic tumors are very similar to those from newly diagnosed patients—with very little in the way of genomic aberration to explain differences in biology. With relatively low mutation rates, few major oncogenic drivers, and loss of function mutations in several epigenetic regulators, an epigenetic basis for ACC may be postulated and serve as the basis for future studies. [ABSTRACT FROM AUTHOR]

T cell activation is intimately linked to metabolism, as distinct metabolic requirements support the functional and phenotypical differences between quiescent and activated T cells. Metabolic transition from mitochondrial oxidative phosphorylation to aerobic glycolysis is crucial for a proper T cell activation. However, the role of tricarboxylic acid cycle (TCA), and in particular succinate dehydrogenase (SDH) in activated T cells needs further elucidation. Here we show that inhibition of SDH during activation of T cells results in strong impairment of proliferation, expression of activation markers, and production of key inflammatory cytokines, despite a concomitant increase in glycolytic metabolic activity. Similar effect of SDH inhibition were demonstrated in pre-activated T cell. Interestingly, itaconic acid, an endogenous SDH inhibitor released from activated macrophages and dendritic cells, had no immunomodulator effect. Taken together, our findings demonstrate that SDH enzyme fitness is critical for mounting and maintaining appropriate activation and function of human T cells. [ABSTRACT FROM AUTHOR]

• Tape strips, stored at room temperature for up to 3 days, can assess the transcriptome of SC. • Known markers of AD can be assessed by RNA from tape strip samples. • Tape strips have clinically potential for outpatient sampling. • The global difference of RNA from SC and epidermis is driven by structural genes. 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. 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. 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. 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. 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 [ABSTRACT FROM AUTHOR]

Connexin (Cx) gap junction channels comprise two hemi-channels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel. [ABSTRACT FROM AUTHOR]

It is difficult to distinguish erythrodermic mycosis fungoides from Sézary syndrome due to their similar clinical and histological features. The main purpose of this study was to investigate whether microRNA expression profiles in lesional skin could discriminate patients with erythrodermic mycosis fungoides from those with Sézary syndrome. A further aim was to assess whether the microRNA expression profiles in erythrodermic mycosis fungoides skin was more comparable to microRNA expression profiles of Sézary syndrome or early-stage mycosis fungoides. RNA was extracted from diagnostic skin biopsies, followed by quantitative reverse transcription polymerase chain reaction analysis of 383 microRNAs. Twenty-seven microRNAs were significantly differentially expressed between erythrodermic mycosis fungoides and Sézary syndrome. Moreover, erythrodermic mycosis fungoides showed microRNA features overlapping with Sézary syndrome and early-stage mycosis fungoides, although hierarchical cluster analysis co-clustered erythrodermic mycosis fungoides with early-stage mycosis fungoides rather than with Sézary syndrome. These findings underscore that erythrodermic mycosis fungoides and Sézary syndrome are different diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The gene expression time-course of repeated challenge of contact allergy ( CA) remains largely unknown. Therefore, using diphenylcyclopropenone ( DPCP) as model allergen in healthy humans we set out to examine: (i) the monotonous and complex gene expression time-course trajectories following repeated DPCP challenges to find the predominant gene expression pattern, (ii) the time-course of cell infiltration following repeated DPCP challenges and (iii) the transcriptome of a repeated CA exposure model. We obtained punch biopsies from control and DPCP-exposed skin from ten DPCP sensitized individuals at 5-6 monthly elicitation challenges. Biopsies were used for microarray gene expression profiling, histopathology and immunohistochemical staining. Validation of microarray data by qRT- PCR was performed on 15 selected genes. Early gene expression time points were also validated in an independent data set. An increasing and decreasing trend in gene expression followed by a plateau was predominantly observed during repeated DPCP challenges. Immune responses reached a plateau after two challenges histopathologically, immunohistochemically and in the time-course gene expression analysis. Transcriptional responses over time revealed a Th1/Th17 polarization as three upstream regulators ( IFN-γ, IL-1 and IL-17) activated most of the top upregulated genes. Of the latter genes, 9 of 10 were the same throughout the time course. Excellent correlations between array and PCR data were observed. The transcriptional responses to DPCP over time followed a monotonous pattern. This response pattern confirms and supports the newly reported clinical time-course observations in de novo-sensitized individuals showing a plateau response, and thus, there is concordance between clinical response, histopathology, immunohistochemistry and microarray gene expression in volunteers de novo-sensitized to DPCP. [ABSTRACT FROM AUTHOR]

The rapid and strong clinical efficacy of the first-in-class, ingenol mebutate, against actinic keratosis (AK) has resulted in its recent approval. We conducted the first comprehensive analysis of the cellular and molecular mode of action of topical ingenol mebutate 0.05% gel in both AK and uninvolved skin of 26 patients in a phase I, single-center, open-label, within-patient comparison. As early as 1 day after application, ingenol mebutate induced profound epidermal cell death, along with a strong infiltrate of CD4+ and CD8+ T-cells, neutrophils, and macrophages. Endothelial ICAM-1 activation became evident after 2 days. The reaction pattern was significantly more pronounced in AK compared with uninvolved skin, suggesting a tumor-preferential mode of action. Extensive molecular analyses and transcriptomic profiling of mRNAs and microRNAs demonstrated alterations in gene clusters functionally associated with epidermal development, inflammation, innate immunity, and response to wounding. Ingenol mebutate reveals a unique mode of action linking directly to anti-tumoral effects. Trial Registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Background: Atopic dermatitis (AD) is a common inflammatory skin disease with limited treatment options. Several microarray experiments have been conducted on lesional/LS and non-lesional/NL AD skin to develop a genomic disease phenotype. Although these experiments have shed light on disease pathology, inter-study comparisons reveal large differences in resulting sets of differentially expressed genes (DEGs), limiting the utility of direct comparisons across studies. Methods: We carried out a meta-analysis combining 4 published AD datasets to define a robust disease profile, termed meta-analysis derived AD (MADAD) transcriptome. Results: This transcriptome enriches key AD pathways more than the individual studies, and associates AD with novel pathways, such as atherosclerosis signaling (IL-37, selectin E/SELE). We identified wide lipid abnormalities and, for the first time in vivo, correlated Th2 immune activation with downregulation of key epidermal lipids (FA2H, FAR2, ELOVL3), emphasizing the role of cytokines on the barrier disruption in AD. Key AD "classifier genes" discriminate lesional from nonlesional skin, and may evaluate therapeutic responses. Conclusions: Our meta-analysis provides novel and powerful insights into AD disease pathology, and reinforces the concept of AD as a systemic disease. [ABSTRACT FROM AUTHOR]

Purpose: Therapeutic decisions in breast cancer are increasingly guided by prognostic and predictive biomarkers. Non-protein-coding microRNAs (miRNAs) have recently been found to be deregulated in breast cancers and, in addition, to be correlated with several clinico-pathological features. One of the most consistently up-regulated miRNAs is miR-21. Here, we specifically searched for differentially expressed miRNAs in high-risk breast cancer patients as compared to low-risk breast cancer patients. In the same patients, we also compared miR-21 expression with the expression of its presumed target PTEN. Methods: Both microarray and RT-qPCR techniques were used to assess miRNA expression levels in lymph node-positive and -negative human invasive ductal carcinoma tissues. Simultaneously, PTEN protein expression levels were assessed using immunohistochemistry. Results: miR-486-5p and miR-139-5p were found to be down-regulated in patients with lymph node metastases, whereas miR-21 was found to be up-regulated in patients with a positive lymph node status. miR-21 expression levels were found to significantly correlate with tumour size ( r = 0.403, p = 0.009; Spearman's rank), whereas no relation was found between miR-21 and PTEN expression levels (Kruskal-Wallis test). Conclusion: Down-regulation of miR-486-5p and miR-139-5p, in conjunction with up-regulation of miR-21, may represent a useful signature for the identification of high-risk breast cancer patients. [ABSTRACT FROM AUTHOR]

Purpose: Despite the benefits of estrogen receptor (ER)-targeted endocrine therapies in breast cancer, many tumors develop resistance. MicroRNAs (miRNAs) have been suggested as promising biomarkers and we here evaluated whether a miRNA profile could be identified, sub-grouping ER+ breast cancer patients treated with adjuvant Tamoxifen with regards to probability of recurrence. Experimental Design: Global miRNA analysis was performed on 152 ER+ primary tumors from high-risk breast cancer patients with an initial discovery set of 52 patients, followed by two independent test sets (N = 60 and N = 40). All patients had received adjuvant Tamoxifen as mono-therapy (median clinical follow-up: 4.6 years) and half had developed distant recurrence (median time-to-recurrence: 3.5 years). MiRNA expression was examined by unsupervised hierarchical clustering and supervised analysis, including clinical parameters as co-variables. Results: The discovery set identified 10 highly significant miRNAs that discriminated between the patient samples according to outcome. However, the subsequent two independent test sets did not confirm the predictive potential of these miRNAs. A significant correlation was identified between miR-7 and the tumor grade. Investigation of the microRNAs with the most variable expression between patients in different runs yielded a list of 31 microRNAs, eight of which are associated with stem cell characteristics. Conclusions: Based on the large sample size, our data strongly suggests that there is no single miRNA profile predictive of outcome following adjuvant Tamoxifen treatment in a broad cohort of ER+ breast cancer patients. We identified a subgroup of Tamoxifen-treated breast cancer patients with miRNA-expressing tumors associated with cancer stem cell characteristics. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2011) 119, 262-274. Abstract Cone-rod homeobox ( Crx) encodes Crx, a transcription factor expressed selectively in retinal photoreceptors and pinealocytes, the major cell type of the pineal gland. In this study, the influence of Crx on the mammalian pineal gland was studied by light and electron microscopy and by use of microarray and qRTPCR technology, thereby extending previous studies on selected genes (). Deletion of Crx was not found to alter pineal morphology, but was found to broadly modulate the mouse pineal transcriptome, characterized by a > 2-fold down-regulation of 543 genes and a > 2-fold up-regulation of 745 genes ( p < 0.05). Of these, one of the most highly up-regulated (18-fold) was Hoxc4, a member of the Hox gene family, members of which are known to control gene expression cascades. During a 24-h period, a set of 51 genes exhibited differential day/night expression in pineal glands of wild-type animals; only eight of these were also day/night expressed in the Crx −/− pineal gland. However, in the Crx −/− pineal gland 41 genes exhibited differential night/day expression that was not seen in wild-type animals. These findings indicate that Crx broadly modulates the pineal transcriptome and also influences differential night/day gene expression in this tissue. Some effects of Crx deletion on the pineal transcriptome might be mediated by Hoxc4 up-regulation. [ABSTRACT FROM AUTHOR]

Abstract: Recently it has become clear that interferon (IFN)-α, a type I interferon produced rapidly in response to infection, not only plays a key role in innate immunity, but also promotes adaptive immune responses by influencing the production or function of other cytokines. During infections IFN-α fosters the production of IL-15, which plays a pivotal role in the development, survival and function of NK cells and recruitment and activation of T cells. Since these two cytokines exert overlapping functions during infections, this investigation was undertaken to study the priming effect of IFN-α on the effect of IL-15 on human T and NK cells. We show that IFN-α induces an increased expression of IL-15Rα in human activated peripheral T cells, and in CD8+ and CD4+ T-cell lines. Functionally, the IFN-α-enhanced IL-15Rα expression resulted in an enhanced IL-15-mediated phosphorylation of STAT5 and STAT3 followed by a further increase in IL-15Rα expression. Moreover, IFN-α significantly increased the IL-15-induced cytotoxic activity of freshly isolated T and NK cells. Taken together, our data show that IFN-α boosts signaling and functional effects of IL-15, at least in part by fostering the increased IL-15R expression, thus add new facet to the emerging role of IFN-α as an important primer of adaptive immune responses. [Copyright &y& Elsevier]

Background: MicroRNAs are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level and play an important role in the control of developmental and physiological processes. In particular, the developing brain contains an impressive diversity of microRNAs. Most microRNA expression profiling studies have been performed in human or rodents and relatively limited knowledge exists in other mammalian species. The domestic pig is considered to be an excellent, alternate, large mammal model for human-related neurological studies, due to its similarity in both brain development and the growth curve when compared to humans. Considering these similarities, studies examining microRNA expression during porcine brain development could potentially be used to predict the expression profile and role of microRNAs in the human brain. Methodology/Principal Findings: MicroRNA expression profiling by use of microRNA microarrays and qPCR was performed on the porcine developing brain. Our results show that microRNA expression is regulated in a developmentally stage-specific, as well as a tissue-specific manner. Numerous developmental stage or tissue-specific microRNAs including, miR-17, miR-18a, miR-29c, miR-106a, miR-135a and b, miR-221 and miR-222 were found by microarray analysis. Expression profiles of selected candidates were confirmed by qPCR. Conclusions/Significance: The differential expression of specific microRNAs in fetal versus postnatal samples suggests that they likely play an important role in the regulation of developmental and physiological processes during brain development. The data presented here supports the notion that microRNAs act as post-transcriptional switches which may regulate gene expression when required. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Cutaneous T-cell lymphoma (CTCL) represents a heterogeneous group of potentially devastating primary skin malignancies. Despite decades of intense research efforts, the pathogenesis is still not fully understood. In the early stages, both clinical and histopathological diagnosis is often difficult due to the ability of CTCL to masquerade as benign skin inflammatory dermatoses. Due to a lack of reliable biomarkers, it is also difficult to predict which patients will respond to therapy or progress towards severe recalcitrant disease. In this review, we discuss recent discoveries concerning dysregulated microRNA (miR) expression and putative pathological roles of oncogenic and tumor suppressive miRs in CTCL. We also focus on the interplay between miRs, histone deacetylase inhibitors, and oncogenic signaling pathways in malignant T cells as well as the impact of miRs in shaping the inflammatory tumor microenvironment. We highlight the potential use of miRs as diagnostic and prognostic markers, as well as their potential as therapeutic targets. Finally, we propose that the combined use of miR-modulating compounds with epigenetic drugs may provide a novel avenue for boosting the clinical efficacy of existing anti-cancer therapies in CTCL. [ABSTRACT FROM AUTHOR]

The HER2 oncogene and its truncated form p95HER2 play central roles in breast cancer. Here, we show that although HER2 and p95HER2 generally elicit qualitatively similar changes in miRNA profile in MCF-7 breast cancer cells, a subset of changes are distinct and p95HER2 shifts the miRNA profile towards the basal breast cancer subtype. High-throughput miRNA profiling was carried out 15, 36 and 60 h after HER2 or p95HER2 expression and central hits validated by RT-qPCR. miRNAs strongly regulated by p95HER2 yet not by HER2, included miR-221, miR-222, miR-503, miR-29a, miR-149, miR-196 and miR-361. Estrogen receptor-α (ESR1) expression was essentially ablated by p95HER2 expression, in a manner recapitulated by miR-221/-222 mimics. c-Myb family transcription factors MYB and MYBL1, but not MYBL2, were downregulated by p95HER2 and by miR-503 or miR-221/-222 mimics. MYBL1 3′UTR inhibition by miR-221/222 was lost by deletion of a single putative miR-221/222 binding sites. p95HER2 expression, or knockdown of either MYB protein, elicited upregulation of tissue inhibitor of matrix metalloprotease-2 (TIMP2). miR-221/222 and -503 mimics increased, and TIMP2 knockdown decreased, cell migration and invasion. A similar pathway was operational in T47D- and SKBr-3 cells. This work reveals important differences between HER2- and p95HER2- mediated miRNA changes in breast cancer cells, provides novel mechanistic insight into regulation of MYB family transcription factors by p95HER2, and points to a role for a miR-221/222- MYB family-TIMP2 axis in regulation of motility in breast cancer cells. [ABSTRACT FROM AUTHOR]