In Vivo Identification of H3K9me2/H3K79me3 as an Epigenetic Barrier to Carcinogenesis.

The highly dynamic nature of chromatin's structure, due to the epigenetic alterations of histones and DNA, controls cellular plasticity and allows the rewiring of the epigenetic landscape required for either cell differentiation or cell (re)programming. To dissect the epigenetic switch enabling the programming of a cancer cell, we carried out wide genome analysis of Histone 3 (H3) modifications during osteogenic differentiation of SH-SY5Y neuroblastoma cells. The most significant modifications concerned H3K27me2/3, H3K9me2, H3K79me1/2, and H3K4me1 that specify the process of healthy adult stem cell differentiation. Next, we translated these findings in vivo, assessing H3K27, H3K9, and H3K79 methylation states in biopsies derived from patients affected by basalioma, head and neck carcinoma, and bladder tumors. Interestingly, we found a drastic decrease in H3K9me2 and H3K79me3 in cancer specimens with respect to their healthy counterparts and also a positive correlation between these two epigenetic flags in all three tumors. Therefore, we suggest that elevated global levels of H3K9me2 and H3K79me3, present in normal differentiated cells but lost in malignancy, may reflect an important epigenetic barrier to tumorigenesis. This suggestion is further corroborated, at least in part, by the deranged expression of the most relevant H3 modifier enzymes, as revealed by bioinformatic analysis. Overall, our study indicates that the simultaneous occurrence of H3K9me2 and H3K79me3 is fundamental to ensure the integrity of differentiated tissues and, thus, their combined evaluation may represent a novel diagnostic marker and potential therapeutic target.

The long non-coding RNA NEAT1 is a ΔNp63 target gene modulating epidermal differentiation.

The transcription factor ΔNp63 regulates epithelial stem cell function and maintains the integrity of stratified epithelial tissues by acting as transcriptional repressor or activator towards a distinct subset of protein-coding genes and microRNAs. However, our knowledge of the functional link between ∆Np63 transcriptional activity and long non-coding RNAs (lncRNAs) expression is quite limited. Here, we show that in proliferating human keratinocytes ∆Np63 represses the expression of the lncRNA NEAT1 by recruiting the histone deacetylase HDAC1 to the proximal promoter of NEAT1 genomic locus. Upon induction of differentiation, ∆Np63 down-regulation is associated by a marked increase of NEAT1 RNA levels, resulting in an increased assembly of paraspeckles foci both in vitro and in human skin tissues. RNA-seq analysis associated with global DNA binding profile (ChIRP-seq) revealed that NEAT1 associates with the promoter of key epithelial transcription factors sustaining their expression during epidermal differentiation. These molecular events might explain the inability of NEAT1-depleted keratinocytes to undergo the proper formation of epidermal layers. Collectively, these data uncover the lncRNA NEAT1 as an additional player of the intricate network orchestrating epidermal morphogenesis.

High mobility group box 1 in women with unexplained recurrent pregnancy loss.

OBJECTIVES: To investigate whether high mobility group box 1 (HMGB1) is involved in unexplained recurrent pregnancy loss (uRPL). METHODS: Plasma levels of HMGB1 were measured by ELISA in non-pregnant women with (n=44) and without (n=53 controls) uRPL. Their platelets and plasma-derived microvesicles (MVs) were also assayed for HMGB1. Endometrial biopsies were taken in selected uRPL (n=5) and control women (n=5) and the tissue expression of HMGB1 was determined by western blot and immunohistochemistry (IHC). RESULTS: plasma levels of HMGB1 were significantly higher in women with uRPL than in control women. HMGB1 content in platelets and MVs obtained from women with uRPL was significantly higher than that obtained from control women. HMGB1 expression in endometrium was higher in tissues obtained from women with uRPL than in tissues obtained from control women. IHC analysis revealed that HMGB1 is expressed in endometrium with different patterns between uRPL and control women. CONCLUSIONS: HMGB1 could be involved in uRPL.

ZNF750: A Novel Prognostic Biomarker in Metastatic Prostate Cancer.

Prostate cancer is the most frequently diagnosed cancer and the fifth leading cause of cancer death among men in 2020. The clinical decision making for prostate cancer patients is based on the stratification of the patients according to both clinical and pathological parameters such as Gleason score and prostate-specific antigen levels. However, these tools still do not adequately predict patient outcome. The aim of this study was to investigate whether ZNF750 could have a role in better stratifying patients, identifying those with a higher risk of metastasis and with the poorest prognosis. The data reported here revealed that ZNF750 protein levels are reduced in human prostate cancer samples, and this reduction is even higher in metastatic samples. Interestingly, nuclear positivity is significantly reduced in patients with metastatic prostate cancer, regardless of both Gleason score and grade group. More importantly, the bioinformatics analysis indicates that ZNF750 expression is positively correlated with better prognosis. Overall, our findings suggest that nuclear expression of ZNF750 may be a reliable prognostic biomarker for metastatic prostate cancer, which lays the foundation for the development of new biological therapies.

No Time to Die: How Kidney Cancer Evades Cell Death.

The understanding of the pathogenesis of renal cell carcinoma led to the development of targeted therapies, which dramatically changed the overall survival rate. Nonetheless, despite innovative lines of therapy accessible to patients, the prognosis remains severe in most cases. Kidney cancer rarely shows mutations in the genes coding for proteins involved in programmed cell death, including p53. In this paper, we show that the molecular machinery responsible for different forms of cell death, such as apoptosis, ferroptosis, pyroptosis, and necroptosis, which are somehow impaired in kidney cancer to allow cancer cell growth and development, was reactivated by targeted pharmacological intervention. The aim of the present review was to summarize the modality of programmed cell death in the pathogenesis of renal cell carcinoma, showing in vitro and in vivo evidence of their potential role in controlling kidney cancer growth, and highlighting their possible therapeutic value.

The ETS Homologous Factor (EHF) Represents a Useful Immunohistochemical Marker for Predicting Prostate Cancer Metastasis.

The main aim of this study was to investigate the risk of prostate cancer metastasis formation associated with the expression of ETS homologous factor (EHF) in a cohort of bioptic samples. To this end, the expression of EHF was evaluated in a cohort of 152 prostate biopsies including primary prostate cancers that developed metastatic lesions, primary prostate cancers that did not develop metastasis, and benign lesions. Data here reported EHF as a candidate immunohistochemical prognostic biomarker for prostate cancer metastasis formation regardless of the Gleason scoring system. Indeed, our data clearly show that primary lesions with EHF positive cells ≥40% had a great risk of developing metastasis within five years from the first diagnosis. Patients with these lesions had about a 40-fold increased risk of developing metastasis as compared with patients with prostate lesions characterized by a percentage of EHF positive cells ≤30%. In conclusion, the immunohistochemical evaluation of EHF could significantly improve the management of prostate cancer patients by optimizing the diagnostic and therapeutic health procedures and, more important, ameliorating the patient's quality of life.

ΔNp63-Senataxin circuit controls keratinocyte differentiation by promoting the transcriptional termination of epidermal genes.

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.

ITCH E3 ubiquitin ligase downregulation compromises hepatic degradation of branched-chain amino acids.

OBJECTIVE: Metabolic syndrome, obesity, and steatosis are characterized by a range of dysregulations including defects in ubiquitin ligase tagging proteins for degradation. The identification of novel hepatic genes associated with fatty liver disease and metabolic dysregulation may be relevant to unravelling new mechanisms involved in liver disease progression METHODS: Through integrative analysis of liver transcriptomic and metabolomic obtained from obese subjects with steatosis, we identified itchy E ubiquitin protein ligase (ITCH) as a gene downregulated in human hepatic tissue in relation to steatosis grade. Wild-type or ITCH knockout mouse models of non-alcoholic fatty liver disease (NAFLD) and obesity-related hepatocellular carcinoma were analyzed to dissect the causal role of ITCH in steatosis RESULTS: We show that ITCH regulation of branched-chain amino acids (BCAAs) degradation enzymes is impaired in obese women with grade 3 compared with grade 0 steatosis, and that ITCH acts as a gatekeeper whose loss results in elevation of circulating BCAAs associated with hepatic steatosis. When ITCH expression was specifically restored in the liver of ITCH knockout mice, ACADSB mRNA and protein are restored, and BCAA levels are normalized both in liver and plasma CONCLUSIONS: Our data support a novel functional role for ITCH in the hepatic regulation of BCAA metabolism and suggest that targeting ITCH in a liver-specific manner might help delay the progression of metabolic hepatic diseases and insulin resistance.

Macrophage Activation and M2 Polarization in Wound Bed of Diabetic Patients Treated by Dermal/Epidermal Substitute Nevelia.

Clinical evidences have shown good results using dermal/epidermal substitutes (DESs) to treat diabetic foot ulcers. Recent studies suggest that, in addition to their scaffold action, DESs may favor wound healing by influencing wound bed inflammatory cells. This study aims to investigate whether DES may influence the inflammatory infiltrate and macrophages polarization toward a reparative phenotype. Fifteen diabetic patients with chronic foot ulcers have been randomly enrolled: 5 treated only by standard of care, served as control group (CG), and 10 treated with DES composed of type 1 bovin collagen (Nevelia, SYMATESE) considered as test group (TG). A biopsy was taken at baseline (T0) and after 30 days (T1). From bioptic paraffin specimen histological, immunohistochemical, and immunofluorescence analysis was performed. Immunohistochemistry reactions evaluated the number of M1 macrophage (CD38+) and M2 macrophage (CD163+). TG patients displayed general macrophage activation and their greater polarization toward M2 subpopulation 30 days after DES implant, compared with CG. From T0 to T1 there was a significant decrease of CD38+ (230 ± 42 and 135 ± 48 mm2, respectively; P < .001) and significant increase of CD163+ (102 ± 21 positive cells/mm2 and 366 ± 42 positive cells/mm2, respectively; P < .001). Confocal microscopy confirmed an increase of M2 cells as expressed by the reduced CD68+/CD163+ ratio. After 6 months of observation 6 patients (60%) of the TG completely healed, while only 1 patient (20%) healed in the CG (P < .01). The tested DES makes possible to treat diabetic foot ulcers inducing tissue reparative processes through macrophage activation and M2 reparative polarization.

High Sensitivity C-Reactive Protein Increases the Risk of Carotid Plaque Instability in Male Dyslipidemic Patients.

BACKGROUND: The aim of this study was to evaluate how the high sensitivity C-reactive protein (hs-CRP) values influence the risk of carotid plaque instability in association with other cardiovascular risk factors. METHODS: One hundred and fifty-six carotid plaques from both symptomatic and asymptomatic patients requiring surgical carotid endarterectomy were retrospectively collected. According to the modified American Heart Association, atherosclerosis plaques have been histologically distinguished into unstable and stable. The following anamnestic and hematochemical data were also considered: age, gender, hypertension, diabetes mellitus, smoking habit, therapy, low-density lipoprotein (LDL)-C, kidney failure and hs-CRP. RESULTS: The results of our study clearly show that high levels of hs-CRP significantly increase the carotid plaque instability in dyslipidemic patients. Specifically, a 67% increase of the risk of carotid plaque instability was observed in patients with high LDL-C. Therefore, the highest risk was observed in male dyslipidemic patients 2333 (95% CI 0.73-7.48) and in aged female patients 2713 (95% CI 0.14-53.27). DISCUSSION: These data strongly suggest a biological relationship between the hs-CRP values and the alteration of lipidic metabolism mostly in male patients affected by carotid atherosclerosis. The measurement of hs-CRP might be useful as a potential screening tool in the prevention of atheroscletotic disease.

Global mapping of cancers: The Cancer Genome Atlas and beyond.

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.

Effects of Risk Factors on In Situ Expression of Proinflammatory Markers Correlated to Carotid Plaque Instability.

BACKGROUND AND AIMS: Several studies demonstrated a role of active chronic inflammatory infiltrate in carotid plaques progression suggesting a possible link between cardiovascular risk factors and inflammation-related plaque instability. The aim of this study is therefore to evaluate the possible effects of cardiovascular risk factors on in situ expression of proinflammatory markers associated with carotid plaque instability. METHODS AND RESULTS: A tissue microarray containing carotid plaques from 36 symptomatic (major stroke or transient ischemic attack) and 37 asymptomatic patients was built. Serial sections were employed to evaluate the expression of some inflammatory markers by immunohistochemistry [CD3, CD4a, CD8, CD20, CD86, CD163, interleukin (IL)-2, IL-6, IL-17]. Immunohistochemical data were analyzed to study the possible associations between in situ expression of inflammatory biomarker and the main cardiovascular risk factors. Our data demonstrated that plaque instability is associated with the high in situ expression of some cytokines, such as IL-2, IL-6, IL-17. Besides the female sex, none of the risk factors analyzed showed a significant association between the in situ expression of these markers and unstable plaques. A significant increase of IL-6-positive and IL-17-positive cells was observed in unstable atheromatous plaques of female patients, as compared with unstable plaques of male patients. CONCLUSIONS: Plaque destabilization is certainly correlated with the presence of the major cardiovascular risk factors, however, our results showed that, with the exception of sex, their action in the evolutive process of plaque instability seems rather nonspecific, favoring a general release of proinflammatory cytokines.

The risk of carotid plaque instability in patients with metabolic syndrome is higher in women with hypertriglyceridemia.

BACKGROUND: Metabolic syndrome certainly favors growth of carotid plaque; however, it is uncertain if it determines plaque destabilization. Furthermore, it is likely that only some components of metabolic syndrome are associated with increased risk of plaque destabilization. Therefore, we evaluated the effect of different elements of metabolic syndrome, individually and in association, on carotid plaques destabilization. METHODS: A total of 186 carotid endarterectomies from symptomatic and asymptomatic patients were histologically analysed and correlated with major cardiovascular risk factors. RESULTS: Metabolic syndrome, regardless of the cluster of its components, is not associated with a significant increase in risk of plaque destabilization, rather with the presence of stable plaques. The incidence of unstable plaques in patients with metabolic syndrome is quite low (43.9 %), when compared with that seen in the presence of some risk factors, but significantly increases in the subgroup of female patients with hypertriglyceridemia, showing an odds ratio of 3.01 (95% CI, 0.25-36.30). CONCLUSIONS: Our data may help to identify patients with real increased risk of acute cerebrovascular diseases thus supporting the hypothesis that the control of hypertriglyceridemia should be a key point on prevention of carotid atherosclerotic plaque destabilization, especially in post-menopausal female patients.

Persistence of SARS-CoV-2 Viral RNA in Nasopharyngeal Swabs after Death: An Observational Study.

The aim of this study was to investigate the persistence of SARS-CoV-2 in post-mortem swabs of subjects who died from SARS-CoV-2 infection. The presence of the virus was evaluated post-mortem from airways of 27 SARS-CoV-2 positive patients at three different time points (T1 2 h; T2 12 h; T3 24 h) by real-time PCR. Detection of antibodies to SARS-CoV-2 was performed by Maglumi 2019-nCoV IgM/IgG chemiluminescence assay. SARS-CoV-2 viral RNA was still detectable in 70.3% of cases within 2 h after death and in 66,6% of cases up to 24 h after death. Our data showed an increase of the viral load in 78,6% of positive individuals 24 h post-mortem (T3) in comparison to that evaluated 2 h after death (T1). Noteworthy, we detected a positive T3 post-mortem swab (24 h after death) from 4 subjects who were negative at T1 (2 h after death). The results of our study may have an important value in the management of deceased subjects not only with a suspected or confirmed diagnosis of SARS-CoV-2, but also for unspecified causes and in the absence of clinical documentation or medical assistance.

The Paradox Effect of Calcification in Carotid Atherosclerosis: Microcalcification is Correlated with Plaque Instability.

BACKGROUND: this study aims to investigate the possible association among the histopathologic features of carotid plaque instability, the presence of micro- or macrocalcifications, the expression of in situ inflammatory biomarkers, and the occurrence of the major risk factors in this process in a large series of carotid plaques. METHODS: a total of 687 carotid plaques from symptomatic and asymptomatic patients were collected. Histological evaluation was performed to classify the calcium deposits in micro or macrocalcifications according to their morphological features (location and size). Immunohistochemistry was performed to study the expression of the main inflammatory biomarkers. RESULTS: results here reported demonstrated that calcifications are very frequent in carotid plaques, with a significant difference between the presence of micro- and macrocalcifications. Specifically, microcalcifications were significantly associated to high inflamed unstable plaques. Paradoxically, macrocalcifications seem to stabilize the plaque and are associated to a M2 macrophage polarization instead. DISCUSSION: the characterization of mechanisms involved in the formation of carotid calcifications can lay the foundation for developing new strategies for the management of patients affected by carotid atherosclerosis. Data of this study could provide key elements for an exhaustive evaluation of carotid plaque calcifications allowing to establish the risk of associated clinical events.

New immunological potential markers for triple negative breast cancer: IL18R1, CD53, TRIM, Jaw1, LTB, PTPRCAP.

Breast cancer (BC) is the second leading cause of cancer death in women worldwide, and settings of specific prognostic factors and efficacious therapies are made difficult by phenotypic heterogeneity of BC subtypes. Therefore, there is a current urgent need to define novel predictive genetic predictors that may be useful for stratifying patients with distinct prognostic outcomes. Here, we looked for novel molecular signatures for triple negative breast cancers (TNBCs). By a bioinformatic approach, we identified a panel of genes, whose expression was positively correlated with disease-free survival in TNBC patients, namely IL18R1, CD53, TRIM, Jaw1, LTB, and PTPRCAP, showing specific immune expression profiles linked to survival prediction; most of these genes are indeed expressed in immune cells and are required for productive lymphocyte activation. According to our hypothesis, these genes were not, or poorly, expressed in different TNBC cell lines, derived from either primary breast tumours or metastatic pleural effusions. This conclusion was further supported in vivo, as immuno-histochemical analysis on biopsies of TNBC invasive ductal carcinomas highlighted differential expression of these six genes in cancer cells, as well as in intra- and peri-tumoral infiltrating lymphocytes. Our data open to the possibility that inter-tumour heterogeneity of immune markers might have predictive value; further investigations are recommended in order to establish the real power of cancer-related immune profiles as prognostic factors.

Serine and one-carbon metabolisms bring new therapeutic venues in prostate cancer.

Serine and one-carbon unit metabolisms are essential biochemical pathways implicated in fundamental cellular functions such as proliferation, biosynthesis of important anabolic precursors and in general for the availability of methyl groups. These two distinct but interacting pathways are now becoming crucial in cancer, the de novo cytosolic serine pathway and the mitochondrial one-carbon metabolism. Apart from their role in physiological conditions, such as epithelial proliferation, the serine metabolism alterations are associated to several highly neoplastic proliferative pathologies. Accordingly, prostate cancer shows a deep rearrangement of its metabolism, driven by the dependency from the androgenic stimulus. Several new experimental evidence describes the role of a few of the enzymes involved in the serine metabolism in prostate cancer pathogenesis. The aim of this study is to analyze gene and protein expression data publicly available from large cancer specimens dataset, in order to further dissect the potential role of the abovementioned metabolism in the complex reshaping of the anabolic environment in this kind of neoplasm. The data suggest a potential role as biomarkers as well as in cancer therapy for the genes (and enzymes) belonging to the one-carbon metabolism in the context of prostatic cancer.

Liquid biopsies and cancer omics.

The development of the sequencing technologies allowed the generation of huge amounts of molecular data from a single cancer specimen, allowing the clinical oncology to enter the era of the precision medicine. This massive amount of data is highlighting new details on cancer pathogenesis but still relies on tissue biopsies, which are unable to capture the dynamic nature of cancer through its evolution. This assumption led to the exploration of non-tissue sources of tumoral material opening the field of liquid biopsies. Blood, together with body fluids such as urines, or stool, from cancer patients, are analyzed applying the techniques used for the generation of omics data. With blood, this approach would allow to take into account tumor heterogeneity (since the circulating components such as CTCs, ctDNA, or ECVs derive from each cancer clone) in a time dependent manner, resulting in a somehow "real-time" understanding of cancer evolution. Liquid biopsies are beginning nowdays to be applied in many cancer contexts and are at the basis of many clinical trials in oncology.

Cancer predictive studies.

The identification of individual or clusters of predictive genetic alterations might help in defining the outcome of cancer treatment, allowing for the stratification of patients into distinct cohorts for selective therapeutic protocols. Neuroblastoma (NB) is the most common extracranial childhood tumour, clinically defined in five distinct stages (1-4 & 4S), where stages 3-4 define chemotherapy-resistant, highly aggressive disease phases. NB is a model for geneticists and molecular biologists to classify genetic abnormalities and identify causative disease genes. Despite highly intensive basic research, improvements on clinical outcome have been predominantly observed for less aggressive cancers, that is stages 1,2 and 4S. Therefore, stages 3-4 NB are still complicated at the therapeutic level and require more intense fundamental research. Using neuroblastoma as a model system, here we herein outline how cancer prediction studies can help at steering preclinical and clinical research toward the identification and exploitation of specific genetic landscape. This might result in maximising the therapeutic success and minimizing harmful effects in cancer patients.