Involvement of transcribed lncRNA uc.291 and SWI/SNF complex in cutaneous squamous cell carcinoma.

While non-melanoma skin cancers (NMSCs) are the most common tumours in humans, only the sub-type cutaneous squamous cell carcinoma (cSCC), might become metastatic with high lethality. We have recently identified a regulatory pathway involving the lncRNA transcript uc.291 in controlling the expression of epidermal differentiation complex genes via the interaction with ACTL6A, a component of the chromatin remodelling complex SWI/SNF. Since transcribed ultra-conserved regions (T-UCRs) are expressed in normal tissues and are deregulated in tumorigenesis, here we hypothesize a potential role for dysregulation of this axis in cSCC, accounting for the de-differentiation process observed in aggressive poorly differentiated cutaneous carcinomas. We therefore analysed their expression patterns in human tumour biopsies at mRNA and protein levels. The results suggest that by altering chromatin accessibility of the epidermal differentiation complex genes, down-regulation of uc.291 and BRG1 expression contribute to the de-differentiation process seen in keratinocyte malignancy. This provides future direction for the identification of clinical biomarkers in cutaneous SCC. Analysis of publicly available data sets indicates that the above may also be a general feature for SCCs of different origins.

Emerging roles of long non-coding RNAs in breast cancer biology and management.

Breast cancer is the most common cancer in women with the highest mortality among this gender. Despite treatment strategies including surgery, hormone therapy and targeted therapy have recently advanced, innovative biomarkers are needed for the early detection, treatment and prognosis. An increasing number of non-coding RNAs (ncRNAs) have shown great potential as crucial players in different stages of the breast cancer tumorigenesis, influencing cell death, metabolism, epithelial-mesenchymal transition (EMT), metastasis and drug resistance. Long non-coding RNAs (lncRNAs), specifically, are a class of RNA transcripts with a length greater than 200 nucleotides, which have also been shown to exerts oncogenic or tumour suppressive roles in the pathogenesis of breast cancer. LncRNAs are implicated in different molecular mechanisms by regulating gene expressions and functions at transcriptional, translational, and post-translational levels. Here, we aim to briefly discuss the latest existing body of knowledge regarding the key functions and the molecular mechanisms of some of the most relevant lncRNAs in the pathogenesis, treatment and prognosis of breast cancer.

Free-amino acid metabolic profiling of visceral adipose tissue from obese subjects.

Interest in adipose tissue pathophysiology and biochemistry have expanded considerably in the past two decades due to the ever increasing and alarming rates of global obesity and its critical outcome defined as metabolic syndrome (MS). This obesity-linked systemic dysfunction generates high risk factors of developing perilous diseases like type 2 diabetes, cardiovascular disease or cancer. Amino acids could play a crucial role in the pathophysiology of the MS onset. Focus of this study was to fully characterize amino acids metabolome modulations in visceral adipose tissues (VAT) from three adult cohorts: (i) obese patients (BMI 43-48) with metabolic syndrome (PO), (ii) obese subjects metabolically well (O), and (iii) non obese individuals (H). 128 metabolites identified as 20 protein amino acids, 85 related compounds and 13 dipeptides were measured by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) and gas chromatography-/mass spectrometry GC/MS, in visceral fat samples from a total of 53 patients. Our analysis indicates a probable enhanced BCAA (leucine, isoleucine, valine) degradation in both VAT from O and PO subjects, while levels of their oxidation products are increased. Also PO and O VAT samples were characterized by: elevated levels of kynurenine, a catabolic product of tryptophan and precursor of diabetogenic substances, a significant increase of cysteine sulfinic acid levels, a decrease of 1-methylhistidine, and an up regulating trend of 3-methylhistidine levels. We hope this profiling can aid in novel clinical strategies development against the progression from obesity to metabolic syndrome.

Allele-specific silencing of EEC p63 mutant R304W restores p63 transcriptional activity.

EEC (ectrodactily-ectodermal dysplasia and cleft lip/palate) syndrome is a rare genetic disease, autosomal dominant inherited. It is part of the ectodermal dysplasia disorders caused by heterozygous mutations in TP63 gene. EEC patients present limb malformations, orofacial clefting, skin and skin's appendages defects, ocular abnormalities. The transcription factor p63, encoded by TP63, is a master gene for the commitment of ectodermal-derived tissues, being expressed in the apical ectodermal ridge is critical for vertebrate limb formation and, at a later stage, for skin and skin's appendages development. The ΔNp63α isoform is predominantly expressed in epithelial cells and it is indispensable for preserving the self-renewal capacity of adult stem cells and to engage specific epithelial differentiation programs. Small interfering RNA (siRNA) offers a potential therapy approach for EEC patients by selectively silencing the mutant allele. Here, using a systemic screening based on a dual-luciferase reported gene assay, we have successfully identified specific siRNAs for repressing the EEC-causing p63 mutant, R304W. Upon siRNA treatment, we were able to restore ΔNp63-WT allele transcriptional function in induced pluripotent stem cells that were derived from EEC patient biopsy. This study demonstrates that siRNAs approach is promising and, may pave the way for curing/delaying major symptoms, such as cornea degeneration and skin erosions in young EEC patients.

ΔNp63 targets cytoglobin to inhibit oxidative stress-induced apoptosis in keratinocytes and lung cancer.

During physiological aerobic metabolism, the epidermis undergoes significant oxidative stress as a result of the production of reactive oxygen species (ROS). To maintain a balanced oxidative state, cells have developed protective antioxidant systems, and preliminary studies suggest that the transcriptional factor p63 is involved in cellular oxidative defence. Supporting this hypothesis, the ΔNp63α isoform of p63 is expressed at high levels in the proliferative basal layer of the epidermis. Here we identify the CYGB gene as a novel transcriptional target of ΔNp63 that is involved in maintaining epidermal oxidative defence. The CYGB gene encodes cytoglobin, a member of the globin protein family, which facilitates the diffusion of oxygen through tissues and acts as a scavenger for nitric oxide or other ROS. By performing promoter activity assays and chromatin immunoprecipitation, reverse transcriptase quantitative PCR and western blotting analyses, we confirm the direct regulation of CYGB by ΔNp63α. We also demonstrate that CYGB has a protective role in proliferating keratinocytes grown under normal conditions, as well as in cells treated with exogenous hydrogen peroxide. These results indicate that ΔNp63, through its target CYGB has an important role in the cellular antioxidant system and protects keratinocytes from oxidative stress-induced apoptosis. The ΔNp63-CYGB axis is also present in lung and breast cancer cell lines, indicating that CYGB-mediated ROS-scavenging activity may also have a role in epithelial tumours. In human lung cancer data sets, the p63-CYGB interaction significantly predicts reduction of patient survival.

The interplay between inflammation and metabolism in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by extensive synovitis resulting in erosions of articular cartilage and marginal bone that lead to joint destruction. The autoimmune process in RA depends on the activation of immune cells, which use intracellular kinases to respond to external stimuli such as cytokines, immune complexes, and antigens. An intricate cytokine network participates in inflammation and in perpetuation of disease by positive feedback loops promoting systemic disorder. The widespread systemic effects mediated by pro-inflammatory cytokines in RA impact on metabolism and in particular in lymphocyte metabolism. Moreover, RA pathobiology seems to share some common pathways with atherosclerosis, including endothelial dysfunction that is related to underlying chronic inflammation. The extent of the metabolic changes and the types of metabolites seen may be good markers of cytokine-mediated inflammatory processes in RA. Altered metabolic fingerprints may be useful in predicting the development of RA in patients with early arthritis as well as in the evaluation of the treatment response. Evidence supports the role of metabolomic analysis as a novel and nontargeted approach for identifying potential biomarkers and for improving the clinical and therapeutical management of patients with chronic inflammatory diseases. Here, we review the metabolic changes occurring in the pathogenesis of RA as well as the implication of the metabolic features in the treatment response.

TAp63gamma is required for the late stages of myogenesis.

p53 family members, p63 and p73, play a role in controlling early stage of myogenic differentiation. We demonstrated that TAp63gamma, unlike the other p53 family members, is markedly up-regulated during myogenic differentiation in murine C2C7 cell line. We also found that myotubes formation was inhibited upon TAp63gamma knock-down, as also indicated by atrophyic myotubes and reduction of myoblasts fusion index. Analysis of TAp63gamma-dependend transcripts identified several target genes involved in skeletal muscle contractility energy metabolism, myogenesis and skeletal muscle autocrine signaling. These results indicate that TAp63gamma is a late marker of myogenic differentiation and, by controlling different sub-sets of target genes, it possibly contributes to muscle growth, remodeling, functional differentiation and tissue homeostasis.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

MicroRNAs and p63 in epithelial stemness.

MicroRNAs (miRs) are a class of small noncoding RNAs that suppress the expression of protein-coding genes by repressing protein translation. Although the roles that miRs and the miR processing machinery have in regulating epithelial stem cell biology are not fully understood, their fundamental contributions to these processes have been demonstrated over the last few years. The p53-family member p63 is an essential transcription factor for epidermal morphogenesis and homeostasis. p63 functions as a determinant for keratinocyte cell fate and helps to regulate the balance between stemness, differentiation and senescence. An important factor that regulates p63 function is the reciprocal interaction between p63 and miRs. Some miRs control p63 expression, and p63 regulates the miR expression profile in the epidermis. p63 controls miR expression at different levels. It directly regulates the transcription of several miRs and indirectly regulates their processing by regulating the expression of the miR processing components Dicer and DGCR8. In this review, we will discuss the recent findings on the miR-p63 interaction in epidermal biology, particularly focusing on the ΔNp63-dependent regulation of DGCR8 recently described in the ΔNp63(-/-) mouse. We provide a unified view of the current knowledge and discuss the apparent discrepancies and perspective therapeutic opportunities.

MicroRNAs, miR-154, miR-299-5p, miR-376a, miR-376c, miR-377, miR-381, miR-487b, miR-485-3p, miR-495 and miR-654-3p, mapped to the 14q32.31 locus, regulate proliferation, apoptosis, migration and invasion in metastatic prostate cancer cells.

miRNAs act as oncogenes or tumor suppressors in a wide variety of human cancers, including prostate cancer (PCa). We found a severe and consistent downregulation of miRNAs, miR-154, miR-299-5p, miR-376a, miR-376c, miR-377, miR-381, miR-487b, miR-485-3p, miR-495 and miR-654-3p, mapped to the 14q32.31 region in metastatic cell lines as compared with normal prostatic epithelial cells (PrEC). In specimens of human prostate (28 normals, 99 primary tumors and 13 metastases), lower miRNA levels correlated significantly with a higher incidence of metastatic events and higher prostate specific antigen (PSA) levels, with similar trends observed for lymph node invasion and the Gleason score. We transiently transfected 10 members of the 14q32.31 cluster in normal prostatic epithelial cell lines and characterized their affect on malignant cell behaviors, including proliferation, apoptosis, migration and invasion. Finally, we identified FZD4, a gene important for epithelial-to-mesenchymal transition in (PCa), as a target of miR-377.

miR-24 affects hair follicle morphogenesis targeting Tcf-3.

During embryonic development, hair follicles (HFs) develop from an epidermal-mesenchymal cross talk between the ectoderm progenitor layer and the underlying dermis. Epidermal stem cell activation represents a crucial point both for HF morphogenesis and for hair regeneration. miR-24 is an anti-proliferative microRNA (miRNA), which is induced during differentiation of several cellular systems including the epidermis. Here, we show that miR-24 is expressed in the HF and has a role in hair morphogenesis. We generated transgenic mice ectopically expressing miR-24 under the K5 promoter. The K5::miR-24 animals display a marked defect in HF morphogenesis, with thinning of hair coat and altered HF structure. Expression of miR-24 alters the normal process of hair keratinocyte differentiation, leading to altered expression of differentiation markers. MiR-24 directly represses the hair keratinocyte stemness regulator Tcf-3. These results support the notion that microRNAs, and among them miR-24, have an important role in postnatal epidermal homeostasis.

PIR2/Rnf144B regulates epithelial homeostasis by mediating degradation of p21WAF1 and p63.

ΔNp63 is a transcription factor that is critical for the development of stratified epithelia and is overexpressed or amplified in >80% of squamous cell carcinomas (SCCs). We identified the RING finger E3 ubiquitin ligase PIR2/Rnf144b as a direct transcriptional target of ΔNp63α and showed that its expression parallels that of ΔNp63α in keratinocytes, SCC cell lines and SCCs. We used primary keratinocytes as a model system to investigate the function of PIR2/Rnf144b in stratified epithelia. Depletion of PIR2/Rnf144b severely impaired keratinocyte proliferation and differentiation, associated with accumulation of p21(WAF1/CIP1); a known target of PIR2/Rnf144b. More importantly, we found that PIR2/Rnf144b binds and mediates proteasomal degradation of ΔNp63α, generating a hitherto unknown auto-regulatory feedback loop. These findings substantiate PIR2/Rnf144b as a potentially critical component of epithelial homeostasis, acting downstream of ΔNp63α to regulate cellular levels of p21(WAF1/CIP1) and ΔNp63α.

miR-143 regulates hexokinase 2 expression in cancer cells.

Tumor cells activate pathways that facilitate and stimulate glycolysis even in the presence of adequate levels of oxygen in order to satisfy their continuous need of molecules, such as nucleotides, ATP and fatty acids, necessary to support their rapid proliferation. Accordingly, a variety of human tumors are characterized by elevated expression levels of the hexokinase 2 isoform (HK2). Although different molecular mechanisms, including genetic and epigenetic mechanisms, have been suggested to account for the altered expression of HK2 in tumors, the potential role of microRNAs (miRNAs) in the regulation of HK2 expression has not been evaluated. Here, we report that miR-143 inhibits HK2 expression via a conserved miR-143 recognition motif located in the 3'-untranslated region (3'UTR) of HK2 mRNA. We demonstrate that miR143 inhibits HK2 expression both in primary keratinocytes and in head and neck squamous cell carcinoma (HNSCC)-derived cell lines. Importantly, we found that miR-143 inversely correlates with HK2 expression in HNSCC-derived cell lines and in primary tumors. We also report that the miRNA-dependent regulation of hexokinase expression is not limited to HK2 as miR-138 targets HK1 via a specific recognition motif located in its 3'UTR. All these data unveil a new miRNA-dependent mechanism of regulation of hexokinase expression potentially important in the regulation of glucose metabolism of cancer cells.

DNA methylation silences miR-132 in prostate cancer.

Silencing of microRNAs (miRNAs) by promoter CpG island methylation may be an important mechanism in prostate carcinogenesis. To screen for epigenetically silenced miRNAs in prostate cancer (PCa), we treated prostate normal epithelial and carcinoma cells with 5-aza-2'-deoxycytidine (AZA) and subsequently examined expression changes of 650 miRNAs by megaplex stemloop reverse transcription-quantitative PCR. After applying a selection strategy, we analyzed the methylation status of CpG islands upstream to a subset of miRNAs by methylation-specific PCR. The CpG islands of miR-18b, miR-132, miR-34b/c, miR-148a, miR-450a and miR-542-3p showed methylation patterns congruent with their expression modulations in response to AZA. Methylation analysis of these CpG islands in a panel of 50 human prostate carcinoma specimens and 24 normal controls revealed miR-132 to be methylated in 42% of human cancer cases in a manner positively correlated to total Gleason score and tumor stage. Expression analysis of miR-132 in our tissue panel confirmed its downregulation in methylated tumors. Re-expression of miR-132 in PC3 cells induced cell detachment followed by cell death (anoikis). Two pro-survival proteins-heparin-binding epidermal growth factor and TALIN2-were confirmed as direct targets of miR-132. The results of this study point to miR-132 as a methylation-silenced miRNA with an antimetastatic role in PCa controlling cellular adhesion.

MicroRNA-203 contributes to skin re-epithelialization.

Keratinocyte proliferation and migration are crucial steps for the rapid closure of the epidermis during wound healing, but the molecular mechanisms involved in this cellular response remain to be completely elucidated. Here, by in situ hybridization we characterize the expression pattern of miR-203 after the induction of wound in mouse epidermis, showing that its expression is downregulated in the highly proliferating keratinocytes of the 'migrating tongue', whereas it is strongly expressed in the differentiating cells of the skin outside the wound. Furthermore, subcutaneous injections of antagomiR-203 in new born mice dorsal skin strengthened, in vivo, the inverse correlation between miR-203 expression and two new target mRNAs: RAN and RAPH1. Our data suggest that miR-203, by controlling the expression of target proteins that are responsible for both keratinocyte proliferation and migration, exerts a specific role in wound re-epithelialization and epidermal homeostasis re-establishment of injured skin.

Novel transglutaminase 1 mutations in patients affected by lamellar ichthyosis.

Lamellar Ichthyosis (LI) is a form of congenital ichthyosis that is caused by mutations in the TGM1 gene that encodes for the transglutaminase 1 (TG1) enzyme. Functional inactivation of TG1 could be due to mutations, deletion or insertions. In this study, we have screened 16 patients affected by LI and found six new mutations: two transition/transversion (R37G, V112A), two nonsense mutations and two putative splice site both leading to a premature stop codon. The mutations are localized in exons 2 (N-terminal domain), 5, 11 (central catalytic domain), and none is located in the two beta-barrel C-terminal domains. In conclusion, this study expands the current knowledge on TGM1 mutation spectrum, increasing the characterization of mutations would provide more accurate prenatal genetic counselling for parents at-risk individuals.