The formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation. Here, we report the reciprocal effect on keratin expression of ΔNp63, pivotal in normal epidermal morphogenesis and maintenance, and Skn-1a/Oct-11, a POU transcription factor that triggers and regulates the differentiation of keratinocytes. The expression of Skn-1a markedly downregulated ΔNp63-driven K14 expression in luciferase reporter assays. The extent of downregulation was comparable to the inhibition of Skn-1a-mediated K10 expression upon expression of ΔNp63. ΔNp63, mutated in the protein-protein interaction domain (SAM domain; mutated in human ectodermal dysplasia syndrome), was significantly less effecting in downregulating K10, raising the possibility of a direct interaction among Skn-1a and ΔNp63. Immunolocalization in human skin biopsies revealed that the expression of the two transcription factors is partially overlapping. Co-immunoprecipitation experiments did not, however, demonstrate a direct interaction between ΔNp63 and Skn-1a, suggesting that the antagonistic effects of Skn-1a and p63 on keratin promoter transactivation is probably through competition for overlapping binding sites on target gene promoter or through an indirect interaction.

Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells. The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy.

Transcribed-ultraconserved regions (T-UCRs) are long non-coding RNAs (lncRNA) encoded by a subset of long ultraconserved stretches in the human genome. Recent studies revealed that the expression of several T-UCRs is altered in cancer and growing evidences underline the importance of T-UCRs in oncogenesis, offering also potential new strategies for diagnosis and prognosis. We found that overexpression of one specific T-UCRs named uc.63 is associated with bad outcome in luminal A subtype of breast cancer patients. uc.63 is localized in the third intron of exportin-1 gene (XPO1) and is transcribed in the same orientation of its host gene. Interestingly, silencing of uc.63 induces apoptosis in vitro. However, silencing of host gene XPO1 does not cause the same effect suggesting that the transcription of uc.63 is independent of XPO1. Our results reveal an important role of uc.63 in promoting breast cancer cells survival and offer the prospect to identify a signature associated with poor prognosis.

Accumulating evidence has proved that deregulation of ΔNp63 expression plays an oncogenic role in head and neck squamous cell carcinomas (HNSCCs). Besides p63, the type 1-insulin-like growth factor (IGF) signalling pathway has been implicated in HNSCC development and progression. Most insulin/IGF1 signalling converges intracellularly onto the protein adaptor insulin receptor substrate-1 (IRS-1) that transmits signals from the receptor to downstream effectors, including the PI3K/AKT and the MAPK kinase pathways, which, ultimately, promote proliferation, invasion, and cell survival. Here we report that p63 directly controls IRS1 transcription and cellular abundance and fosters the PI3K/AKT and MAPK downstream signalling pathways. Inactivation of ΔNp63 expression indeed reduces tumour cell responsiveness to IGF1 stimulation, and inhibits the growth potential of HNSCC cells. In addition, a positive correlation was observed between p63 and IRS1 expression in human HNSCC tissue arrays and in publicly available gene expression data. Our findings indicate that aberrant expression of ΔNp63 in HNSSC may act as an oncogenic stimulus by altering the IGF signalling pathway.

In the past few decades, obesity has reached pandemic proportions. Obesity is among the main risk factors for cardiovascular diseases, since chronic fat accumulation leads to dysfunction in vascular endothelium and to a precocious arterial stiffness. So far, not all the mechanisms linking adipose tissue and vascular reactivity have been explained. Recently, novel findings reported interesting pathological link between endothelial dysfunction with gut hormones and gut microbiota and energy homeostasis. These findings suggest an active role of gut secretome in regulating the mediators of vascular function, such as nitric oxide (NO) and endothelin-1 (ET-1) that need to be further investigated. Moreover, a central role of brain has been suggested as a main player in the regulation of the different factors and hormones beyond these complex mechanisms. The aim of the present review is to discuss the state of the art in this field, by focusing on the processes leading to endothelial dysfunction mediated by obesity and metabolic diseases, such as insulin resistance. The role of perivascular adipose tissue (PVAT), gut hormones, gut microbiota dysbiosis, and the CNS function in controlling satiety have been considered. Further understanding the crosstalk between these complex mechanisms will allow us to better design novel strategies for the prevention of obesity and its complications.

Non-melanoma skin cancer (NMSC) is a tumor that arises from human keratinocytes, showing abnormal control of cell proliferation and aberrant stratification. Cutaneous basal cell carcinoma (cBCC) and cutaneous squamous cell carcinoma (cSCC) are the most common sub-types of NMSC. From a molecular point of view, we are still far from fully understanding the molecular mechanisms behind the onset and progression of NMSC and to unravel targetable vulnerabilities to leverage for their treatment, which is still essentially based on surgery. Under this assumption, it is still not elucidated how the central cellular metabolism, a potential therapeutical target, is involved in NMSC progression. Therefore, our work is based on the characterization of the serine anabolism/catabolism and/or one-carbon metabolism (OCM) role in NMSC pathogenesis. Expression and protein analysis of normal skin and NMSC samples show the alteration of the expression of two enzymes involved in the serine metabolism and OCM, the Serine Hydroxy-Methyl Transferase 2 (SHMT2) and Methylen-ThetraHydroFolate dehydrogenase/cyclohydrolase 2 (MTHFD2). Tissues analysis shows that these two enzymes are mainly expressed in the proliferative areas of cBCC and in the poorly differentiated areas of cSCC, suggesting their role in tumor proliferation maintenance. Moreover, in vitro silencing of SHMT2 and MTHFD2 impairs the proliferation of epidermoid cancer cell line. Taken together these data allow us to link the central cellular metabolism (serine and/or OCM) and NMSC proliferation and progression, offering the opportunity to modulate pharmacologically the involved enzymes activity against this type of human cancer.

Keratinocyte replicative senescence has an important role in time-related changes of epidermis. Previous studies demonstrated that miRNAs play key roles in inhibiting proliferation and in the acquisition of the keratinocyte senescent phenotype as well as in individual ageing. Kruppel-like factor 4 is a transcription factor with dual functions in keratinocytes, being a stemness factor and a pro-differentiation factor. Interestingly, in skin squamous cell carcinomas KLF4 expression is strongly down-regulated or absent. While KLF4 involvement in senescence and ageing has not been investigated yet. Here, we show that Klf4 protein decreases during keratinocyte replicative senescence and during physiological skin aging, while its mRNA level does not change. We demonstrated that the senescence-associated miR-34a regulates post-transcriptionally Klf4 expression. KLF4 silencing is sufficient to induce a senescent phenotype in primary keratinocytes and ectopic miR-34a over-expression phenocopies this result. Our findings identify a novel regulatory loop between miR-34a and KLF4 during keratinocytes replicative senescence. This regulatory loop, beside aging, may play a role in age-related pathologies.

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by progressive pontobulbar palsy, sensory and motor neuron degeneration, sensorineural hearing loss, and optic atrophy. As riboflavin (RF) is the precursor of FAD and FMN, we hypothesize that both mitochondrial and peroxisomal energy metabolism pathways involving flavoproteins could be directly affected in RTD, thus impacting cellular redox status. In the present work, we used induced pluripotent stem cells (iPSCs) from RTD patients to investigate morphofunctional features, focusing on mitochondrial and peroxisomal compartments. Using this model, we document the following RTD-associated alterations: (i) abnormal colony-forming ability and loss of cell-cell contacts, revealed by light, electron, and confocal microscopy, using tight junction marker ZO-1; (ii) mitochondrial ultrastructural abnormalities, involving shape, number, and intracellular distribution of the organelles, as assessed by focused ion beam/scanning electron microscopy (FIB/SEM); (iii) redox imbalance, with high levels of superoxide anion, as assessed by MitoSOX assay accompanied by abnormal mitochondrial polarization state, evaluated by JC-1 staining; (iv) altered immunofluorescence expression of antioxidant systems, namely, glutathione, superoxide dismutase 1 and 2, and catalase, as assessed by quantitatively evaluated confocal microscopy; and (v) peroxisomal downregulation, as demonstrated by levels and distribution of fatty acyl β-oxidation enzymes. RF supplementation results in amelioration of cell phenotype and rescue of redox status, which was associated to improved ultrastructural features of mitochondria, thus strongly supporting patient treatment with RF, to restore mitochondrial- and peroxisomal-related aspects of energy dysmetabolism and oxidative stress in RTD syndrome.

Several transcription factors, including the master regulator of the epidermis, p63, are involved in controlling human keratinocyte proliferation and differentiation. Here, we report that in normal keratinocytes, the expression of FOXM1, a member of the Forkhead superfamily of transcription factors, is controlled by p63. We observe that, together with p63, FOXM1 strongly contributes to the maintenance of high proliferative potential in keratinocytes, whereas its expression decreases during differentiation, as well as during replicative-induced senescence. Depletion of FOXM1 is sufficient to induce keratinocyte senescence, paralleled by an increased ROS production and an inhibition of ROS-scavenger genes (SOD2, CAT, GPX2, PRDX). Interestingly, FOXM1 expression is strongly reduced in keratinocytes isolated from old human subjects compared with young subjects. FOXM1 depletion sensitizes both normal keratinocytes and squamous carcinoma cells to apoptosis and ROS-induced apoptosis. Together, these data identify FOXM1 as a key regulator of ROS in normal dividing epithelial cells and suggest that squamous carcinoma cells may also use FOXM1 to control oxidative stress to escape premature senescence and apoptosis.

SignificanceΔNp63 is a master regulator of skin homeostasis since it finely controls keratinocyte differentiation and proliferation. Here, we provide cellular and molecular evidence demonstrating the functional role of a ΔNp63 interactor, the R-loop-resolving enzyme Senataxin (SETX), in fine-tuning keratinocyte differentiation. We found that SETX physically binds the p63 DNA-binding motif present in two early epidermal differentiation genes, Keratin 1 (KRT1) and ZNF750, facilitating R-loop removal over their 3' ends and thus allowing efficient transcriptional termination and gene expression. These molecular events translate into the inability of SETX-depleted keratinocytes to undergo the correct epidermal differentiation program. Remarkably, SETX is dysregulated in cutaneous squamous cell carcinoma, suggesting its potential involvement in the pathogenesis of skin disorders.

Four transglutaminase (TG) isoforms have been detected in epidermal keratinocytes: TG1, TG2, TG3, and TG5. Except for TG1 and TG3, their contribution to keratinocyte development and structure remains undefined. In this paper, we focused on the roles of TG2 and TG3 in imiquimod-induced psoriasis in mouse skin. We evaluated the severity of psoriasis markers in the skin of imiquimod-treated TG3 null and TG2 null mice. Our results showed that compromised TG3KO mouse skin was more responsive than WT or TG2KO mouse skin to the action of the pro-inflammatory drug imiquimod.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of mammary carcinoma. Here, we describe a case of an 81-year-old female diagnosed with ductal triple negative breast cancer with a germline pathogenic variant in BReast CAncer gene2 (BRCA2). Genetic testing also revealed the presence of four somatic mutations in the ephrin type-A receptor 3 (EphA3), TP53, BRCA1-associated protein (BAP1), and MYB genes. The BRCA2, TP53, and BAP1 gene mutations are highly predictive of a defective homologous recombination repair system and subsequent chromosomal instability in this patient. Coherently, the patient displayed a strong homologous recombination deficiency signature and high tumor mutational burden status, which are generally associated with increased probability of immune neoantigens formation and presentation, and with tumor immunogenicity. Analysis of immune checkpoint revealed high expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), programmed death 1 (PD1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA 4), suggesting that the patient might likely benefit from immunotherapies. Altogether, these findings support an unveiled link between BRCA2 inactivation, HR deficiency and increased expression of immune checkpoints in TNBC. This clinical case highlights the importance of screening TNBC patients for genetic mutations and TMB biomarkers in order to predict the potential efficacy of immunotherapy.

The uc.291 transcript controls keratinocytes differentiation by physical interaction with ACTL6A and subsequent induction of transcription of the genes belonging to the epidermal differentiation complex (EDC). Uc.291 is also implicated in the dedifferentiation phenotype seen in poorly differentiated cutaneous squamous cell carcinomas. Here, we would like to investigate the contribution of uc.291 to the unbalanced differentiation state of keratinocytes observed in hyperproliferative skin disorders, e. g., psoriasis. Psoriasis is a multifactorial inflammatory disease, caused by alteration of keratinocytes homeostasis. The imbalanced differentiation state, triggered by the infiltration of immune cells, represents one of the events responsible for this pathology. In the present work, we explore the role of uc.291 and its interactor ACTL6A in psoriasis skin, using quantitative real-time PCR (RT-qPCR), immunohistochemistry and bioinformatic analysis of publicly available datasets. Our data suggest that the expression of the uc.291 and of EDC genes loricrin and filaggrin (LOR, FLG) is reduced in lesional skin compared to nonlesional skin of psoriatic patients; conversely, the mRNA and protein level of ACTL6A are up-regulated. Furthermore, we provide evidence that the expression of uc.291, FLG and LOR is reduced, while ACTL6A mRNA is up-regulated, in an in vitro psoriasis-like model obtained by treating differentiated keratinocytes with interleukin 22 (IL-22). Furthermore, analysis of a publicly available dataset of human epidermal keratinocytes treated with IL-22 (GSE7216) confirmed our in vitro results. Taken together, our data reveal a novel role of uc.291 and its functional axis with ACTL6A in psoriasis disorder and a proof of concept that biological inhibition of this molecular axis could have a potential pharmacological effect against psoriasis and, in general, in skin diseases with a suppressed differentiation programme.

ΔNp63 has been recently involved in self-renewal potential of breast cancer stem cells. Although the p63 transcriptional profile has been extensively characterized, our knowledge of the p63-binding partners potentially involved in the regulation of breast tumour progression is limited. Here, we performed the yeast two hybrid approach to identify p63α interactors involved in breast tumorigenesis and we found that SETDB1, a histone lysine methyl transferases, interacts with ΔNp63α and that this interaction contributes to p63 protein stability. SETDB1 is often amplified in primary breast tumours, and its depletion confers to breast cancer cells growth disadvantage. We identified a list of thirty genes repressed by ΔNp63 in a SETDB1-dependent manner, whose expression is positively correlated to survival of breast cancer patients. These results suggest that p63 and SETDB1 expression, together with the repressed genes, may have diagnostic and prognostic potential.

Piroxicam (PXM), a nonsteroidal anti-inflammatory drug, is an enolic benzothiazine and a potent member of the oxicam series. The drug suppresses the synthesis of proinflammatory enzymes, such as cyclo-oxygenases-1 and -2 (COX-1 and 2), downregulates the production of prostaglandins (PGs) and tromboxanes, and inhibits polyamines production by blocking ornithine decarboxylase induction involved in nonmelanoma skin carcinogenesis. In addition, PXM is able to induce tumor cell apoptosis and suppresses metalloproteinase 2 activities. Skin carcinogenesis is a multistep process in which the accumulation of genetic events leads to a gradually dysplastic cellular expression, deregulation of cell growth, and carcinomatous progression. COX-1 upregulation plays a significant role in PG and vascular epidermal growth factor production supporting tumor growth. Increased level of PGs in premalignant and/or malignant cutaneous tumors is also favored by upregulation of COX-2 and downregulation of the tumor suppressor gene 15-hydroxy-prostaglandin dehydrogenase. Chemoprevention can be a hopeful approach to inhibit carcinoma occurrence before an invasive tumor develops. The chemopreventive effect of nonsteroidal anti-inflammatory drugs on nonmelanoma skin cancers has been established. In this study, we highlighted the different modalities of action of PXM on the pathogenesis of nonmelanoma skin cancer, analyzing and evaluating binding modes and energies between COX-1 or COX-2 and PXM by protein-ligand molecular docking. Our clinical experience about the local use of PXM on actinic keratoses and field cancerization is also reported, confirming its efficacy as target therapy.

TP63, an important epithelial developmental gene, has significant homology to p53. Unlike p53, the expression of p63 is regulated by two different promoters resulting in proteins with opposite functions: the full-length transcriptionally active TAp63 and the dominant-negative DeltaNp63. We investigated the downstream mechanisms by which TAp63alpha elicits apoptosis. TAp63alpha directly transactivates the CD95 gene via the p53 binding site in the first intron resulting in upregulation of a functional CD95 death receptor. Stimulation and blocking experiments of the CD95, TNF-R and TRAIL-R death receptor systems revealed that TAp63alpha can trigger expression of each of these death receptors. Furthermore, our findings demonstrate a link between TAp63alpha and the mitochondrial apoptosis pathway. TAp63alpha upregulates expression of proapoptotic Bcl-2 family members like Bax and BCL2L11 and the expression of RAD9, DAP3 and APAF1. Of clinical relevance is the fact that TAp63alpha is induced by many chemotherapeutic drugs and that inhibiting TAp63 function leads to chemoresistance. Thus, beyond its importance in development and differentiation, we describe an important role for TAp63alpha in the induction of apoptosis and chemosensitivity.

Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy is a rare, genetic, premature aging disease named MDPL Syndrome, due to almost always a de novo variant in POLD1 gene, encoding the DNA polymerase δ. In previous in vitro studies, we have already described several hallmarks of aging, including genetic damage, telomere shortening, cell senescence and proliferation defects. Since a clear connection has been reported between telomere shortening and mitochondria malfunction to initiate the aging process, we explored the role that mitochondrial metabolism and activity play in pathogenesis of MDPL Syndrome, an aspect that has not been addressed yet. We thus evaluated mtDNA copy number, assessing a significant decrease in mutated cells. The expression level of genes related to mitochondrial biogenesis and activity also revealed a significant reduction, highlighting a mitochondrial dysfunction in MDPL cells. Even the expression levels of mitochondrial marker SOD2, as assessed by immunofluorescence, were reduced. The decrease in this antioxidant enzyme correlated with increased production of mitochondrial ROS in MDPL cells, compared to WT. Consistent with these data, Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) analysis revealed in MDPL cells fewer mitochondria, which also displayed morphological abnormalities. Accordingly, we detected autophagic vacuoles containing partially digested mitochondria. Overall, our results demonstrate a dramatic impairment of mitochondrial biogenesis and activity in MDPL Syndrome. Administration of Metformin, though unable to restore mitochondrial impairment, proved efficient in rescuing nuclear abnormalities, suggesting its use to specifically ameliorate the premature aging phenotype.

Mesenchymal stromal/stem cells (MSCs) possess multi-lineage differentiation and self-renewal potentials. MSCs-based therapies have been widely utilized for the treatment of diverse inflammatory diseases, due to the potent immunoregulatory functions of MSCs. An increasing body of evidence indicates that MSCs exert their therapeutic effects largely through their paracrine actions. Growth factors, cytokines, chemokines, extracellular matrix components, and metabolic products were all found to be functional molecules of MSCs in various therapeutic paradigms. These secretory factors contribute to immune modulation, tissue remodeling, and cellular homeostasis during regeneration. In this review, we summarize and discuss recent advances in our understanding of the secretory behavior of MSCs and the intracellular communication that accounts for their potential in treating human diseases.

Cancer genomes have been explored from the early 2000s through massive exome sequencing efforts, leading to the publication of The Cancer Genome Atlas in 2013. Sequencing techniques have been developed alongside this project and have allowed scientists to bypass the limitation of costs for whole-genome sequencing (WGS) of single specimens by developing more accurate and extensive cancer sequencing projects, such as deep sequencing of whole genomes and transcriptomic analysis. The Pan-Cancer Analysis of Whole Genomes recently published WGS data from more than 2600 human cancers together with almost 1200 related transcriptomes. The application of WGS on a large database allowed, for the first time in history, a global analysis of features such as molecular signatures, large structural variations and noncoding regions of the genome, as well as the evaluation of RNA alterations in the absence of underlying DNA mutations. The vast amount of data generated still needs to be thoroughly deciphered, and the advent of machine-learning approaches will be the next step towards the generation of personalized approaches for cancer medicine. The present manuscript wants to give a broad perspective on some of the biological evidence derived from the largest sequencing attempts on human cancers so far, discussing advantages and limitations of this approach and its power in the era of machine learning.

Epidermolysis bullosa simplex (EBS) with cardiomyopathy (EBS-KLHL24) is an EBS subtype caused by dominantly inherited, gain-of-function mutations in the gene encoding for the ubiquitin-ligase KLHL24, which addresses specific proteins to proteasomal degradation. EBS-KLHL24 patients are born with extensive denuded skin areas and skin fragility. Whilst skin fragility rapidly ameliorates, atrophy and scarring develop over time, accompanied by life-threatening cardiomyopathy. To date, pathogenetic mechanisms underlying such a unique disease phenotype are not fully characterized. The basal keratin 14 (K14) has been indicated as a KLHL24 substrate in keratinocytes. However, EBS-KLHL24 pathobiology cannot be determined by the mutation-enhanced disruption of K14 alone, as K14 is similarly expressed in foetal and postnatal epidermis and its protein levels are preserved both in vivo and in vitro disease models. In this study, we focused on foetal keratins as additional KLHL24 substrates. We showed that K7, K8, K17 and K18 protein levels are markedly reduced via proteasome degradation in normal foetal keratinocytes transduced with the mutant KLHL24 protein (ΔN28-KLHL24) as compared to control cells expressing the wild-type form. In addition, heat stress led to keratin network defects and decreased resilience in ΔN28-KLHL24 cells. The KLHL24-mediated degradation of foetal keratins could contribute to congenital skin defects in EBS-KLHL24. Furthermore, we observed that primary keratinocytes from EBS-KLHL24 patients undergo accelerated clonal conversion with reduced colony forming efficiency (CFE) and early replicative senescence. Finally, our findings pointed out a reduced CFE in ΔN28-KLHL24-transduced foetal keratinocytes as compared to controls, suggesting that mutant KLHL24 contributes to patients' keratinocyte clonogenicity impairment.