A concomitant action of multiple profibrotic mediators appears crucial in the development and progression of fibrosis. Sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways are both involved in pathogenesis of fibrosis in several organs by controlling differentiation of fibroblasts to myofibroblasts and the epithelial to-mesenchymal transition. However, their direct involvement in chronic colitis-associated fibrosis it is not yet known. In this study we evaluated the immunohistochemical expression of some proteins implicated in sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in Dextrane Sodium Sulphate (DSS)-induced colorectal fibrosis in mice. Compared to control mice, DSS-induced chronic colitis mice developed a marked intestinal fibrosis associated with a concomitant overexpression of TGF-β, p-Smad3, α-SMA, collagen I-III, SPHK1, RhoA, PI3K, Akt, p-Akt, p-mTOR. This study highlights the relationship between the two pathways and the possible role of SPHK1 in the intestinal fibrosis.  These results, if confirmed by in vitro studies, may have important clinical implications in the development of new therapeutical approaches in inflammatory bowel disease.

Glioblastoma (GBM) is the most common and aggressive tumor of the central nervous system. Unfortunately, patients affected by this disease have a very poor prognosis, due to high level of invasiveness and resistance to standard therapies. Although the molecular profile of GBM has been extensively investigated, the events responsible for its pathogenesis and progression remain largely unknown. Histone Deacetylases (HDAC) dependent epigenetic modifications and transforming growth factor (TGF)-β/Smad pathway seem to play an important role in GBM tumorigenesis, resistance to common therapies and poor clinical outcome.  The aim of this study was to evaluate the involvement and the possible interaction between these two molecular cascades in the pathogenesis and prognosis of GBM. Immunohistochemistry (IHC) was performed on microdissected GBM samples, collected from 14 patients (6 men and 8 women) ranging in age from 43 to 74 years. The patients were previously divided, on the basis of their overall survival (OS), into two groups: short and long OS. Patients with poor prognosis showed hyperexpression of HDAC4 and HDAC6, an activation of the TGF-β/Smad pathway, with high levels of IL-13, Smad2, PDGF and MMP3 expression, compared to the long survivors. The short OS group exhibits a decrease in Smad 7 expression and also low levels of p21 immunostaining, which represents a common target of the two pathways. The IHC data was confirmed by quantitative analysis and Immunoblotting. Our preliminary results suggest that both HDAC4 and HDAC6 together with the TGF-β/Smad pathway may be involved in progression of GBM and this cross talking could be a useful prognostic marker in this deadly disease.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease. It can progress to nonalcoholic steatohepatitis (NASH) and, in a percentage of cases, to hepatocarcinogenesis. The strong incidence in western countries of obesity and metabolic syndrome, whose NAFLD is the hepatic expression, is thought to be correlated to consumption of diets characterized by processed food and sweet beverages. Previous studies described high-fat diet-induced liver tumors. Conversely, the involvement of low-fat/high-carbohydrate diet in the progression of liver disease or cancer initiation has not been described yet. Here we show for the first time hepatic cancer formation in low-fat/high-carbohydrate diet fed NAFLD/NASH mouse model. Animals were long term high-fat, low-fat/high-carbohydrate or standard diet fed. We observed progressive liver damage in low-fat/high-carbohydrate and high-fat animals after 12 and, more, 18 months. Tumors were detected in 20% and 50% of high-fat diet fed mice after 12 and 18 months and, interestingly, in 30% of low-fat/high-carbohydrate fed animals after 18 months. No tumors were detected in standard diet fed mice. Global increase of hepatic interleukin-1β, interleukin-6, tumor necrosis factor-α and hepatocyte growth factor was detected in low-fat/high-carbohydrate and high-fat with respect to standard diet fed mice as well as in tumor with respect to non-tumor bearing mice. A panel of 15 microRNAs was analyzed: some of them revealed differential expression in low-fat/high-carbohydrate with respect to high-fat diet fed groups and in tumors. Data here shown provide the first evidence of the involvement of low-fat/high-carbohydrate diet in hepatic damage leading to tumorigenesis.

The use of alkylating agents such as temozolomide in association with radiotherapy (RT) is the therapeutic standard of glioblastoma (GBM). This regimen modestly prolongs overall survival, also if, in light of the still dismal prognosis, further improvements are desperately needed, especially in the patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors, in which the benefit of standard treatment is less. Tinostamustine (EDO-S101) is a first-in-class alkylating deacetylase inhibitor (AK-DACi) molecule that fuses the DNA damaging effect of bendamustine with the fully functional pan-histone deacetylase (HDAC) inhibitor, vorinostat, in a completely new chemical entity.

Cancer therapies are associated with increased infertility risk due to accelerated reproductive aging. Oxidative stress (OS) is a potential mechanism behind ovarian toxicity by cyclophosphamide (CPM), the most ovotoxic anticancer drug. An important sensor of OS is SIRT1, a NAD+-dependent deacetylase which regulates cellular defence and cell fate. This study investigated whether the natural carotenoid crocetin and the synthetic compound AS101 protect the ovary against CPM by modulating SIRT1 and mitochondrial markers. We found that the number of primordial follicles of female CD1 mice receiving crocetin plus CPM increased when compared with CPM alone and similar to AS101, whose protective effects are known. SIRT1 increased in CPM mouse ovaries revealing the occurrence of OS. Similarly, mitochondrial SIRT3 rose, whilst SOD2 and the mitochondrial biogenesis activator PGC1-α decreased, suggesting the occurrence of mitochondrial damage. Crocetin and AS101 administration prevented SIRT1 burst suggesting that preservation of redox balance can help the ovary to counteract ovarian damage by CPM. Decreased SIRT3 and increased SOD2 and PGC1-α in mice receiving crocetin or AS101 prior to CPM provide evidence for mitochondrial protection. Present results improve the knowledge of ovarian damage by CPM and may help to develop interventions for preserving fertility in cancer patients.

Intestinal fibrosis is characterized by abnormal production and deposition of extracellular matrix (ECM) proteins by activated myofibroblasts. The main progenitor cells of activated myofibroblasts are the fibroblasts and the epithelial cells, the latter through the epithelial-mesenchymal transition (EMT).

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a persistent sinonasal mucosa inflammatory disease with still unclear pathophysiologic mechanisms that imply events of tissue repair and structural remodelling. Several cascades seem to have a considerable role in the onset and progression of mucosa hyperproliferation in nasal polyps including transforming growth factor β/Small mother against decapentaplegic (TGFβ/Smads), mitogenactivated protein kinases (MAPKs), advanced glycosylation end-products (AGEs) together with epithelial-tomesenchymal transition (EMT). Since many inflammatory mediators are reported to play important roles in the development of nasal polyps (NP) disease, this study aimed to analyse the correlation between the AGEs/receptor of advanced glycosylation end-products (RAGE)/extracellular signal-regulated kinase (ERK) signalling pathway and the main markers of EMT to better understand the influence that they exert on the remodelling of nasal mucous membranes in patients affected by CRSwNP vs normal controls. A total of 30 patients were enrolled in this study. Immunohistochemical analysis, using AGE, RAGE, p-ERK, MMP-3, TGF-β1, Smad2/3, Collagen I-III, α-SMA, E-cadherin, IL-6 and Vimentin antibodies, was performed. AGE, RAGE, ERK, p-ERK and MMP3 were also evaluated using western blot analysis. We observed an overexpression of the AGE/RAGE/p-ERK and the main mesenchymal markers of EMT (Vimentin and IL-6) in CRSwNP vs controls whereas the TGF-β/Smad3 pathway did not show any significant differences between the two groups of patients. These observations suggest a complex network of processes in the pathogenesis of NP, and the AGE/RAGE/ERK pathway and EMT might work together in promoting tissue remodelling in the formation of CRSwNP.

Low radiation doses can affect and modulate cell responses to various stress stimuli, resulting in perturbations leading to resistance or sensitivity to damage. To explore possible mechanisms taking place at an environmental radiation exposure, we set-up twin biological models, one growing in a low radiation environment (LRE) laboratory at the Gran Sasso National Laboratory, and one growing in a reference radiation environment (RRE) laboratory at the Italian National Health Institute (Istituto Superiore di Sanità, ISS). Studies were performed on pKZ1 A11 mouse hybridoma cells, which are derived from the pKZ1 transgenic mouse model used to study the effects of low dose radiation, and focused on the analysis of cellular/molecular end-points, such as proliferation and expression of key proteins involved in stress response, apoptosis, and autophagy. Cells cultured up to 4 weeks in LRE showed no significant differences in proliferation rate compared to cells cultured in RRE. However, caspase-3 activation and PARP1 cleavage were observed in cells entering to an overgrowth state in RRE, indicating a triggering of apoptosis due to growth-stress conditions. Notably, in LRE conditions, cells responded to growth stress by switching toward autophagy. Interestingly, autophagic signaling induced by overgrowth in LRE correlated with activation of p53. Finally, the gamma component of environmental radiation did not significantly influence these biological responses since cells grown in LRE either in incubators with or without an iron shield did not modify their responses. Overall, in vitro data presented here suggest the hypothesis that environmental radiation contributes to the development and maintenance of balance and defense response in organisms.

Intestinal fibrosis, the most common complication of inflammatory bowel disease (IBD), is characterized by an uncontrolled deposition of extracellular matrix proteins leading to complications resolvable only with surgery. Transforming growth factor is the key player in the epithelial-mesenchymal transition (EMT) and fibrogenesis process, and some molecules modulating its activity, including peroxisome proliferator-activated receptor (PPAR)-γ and its agonists, exert a promising antifibrotic action. The purpose of this study is to evaluate the contribution of signaling other than EMT, such as the AGE/RAGE (advanced glycation end products/receptor of AGEs) and the senescence pathways, in the etiopathogenesis of IBD. We used human biopsies from control and IBD patients, and we used a mouse model of colitis induced by dextran-sodium-sulfate (DSS), without/with treatments with GED (PPAR-gamma-agonist), or 5-aminosalicylic acid (5-ASA), a reference drug for IBD treatment. In patients, we found an increase in EMT markers, AGE/RAGE, and senescence signaling activation compared to controls. Consistently, we found the overexpression of the same pathways in DSS-treated mice. Surprisingly, the GED reduced all the pro-fibrotic pathways, in some circumstances more efficiently than 5-ASA. Results suggest that IBD patients could benefit from a combined pharmacological treatment targeting simultaneously different pathways involved in pro-fibrotic signals. In this scenario, PPAR-gamma activation could be a suitable strategy to alleviate the signs and symptoms of IBD and also its progression.

Machine learning (ML) represents a powerful tool to capture relationships between molecular alterations and cancer types and to extract biological information. Here, we developed a plain ML model aimed at distinguishing cancer types based on genetic lesions, providing an additional tool to improve cancer diagnosis, particularly for tumors of unknown origin.

Maroteaux-Lamy disease, also known as mucopolysaccharidosis (MPS) VI, is an MPS disorder caused by mutations in the ARSB gene encoding for the lysosomal enzyme arysulfatase B (ARSB). Deficient ARSB activity leads to lysosomal accumulation of dermatan sulfate in a wide range of tissues and organs. There are various animal models of MPS VI that have been well characterized from a biochemical and morphological point of view. In this study, we report the sensory-motor characterization of MPS VI rats carrying homozygous null ARSB mutations. We show that adult MPS VI rats are specifically impaired in vertical activity and motor endurance. All together, these data are consistent with biochemical findings that show a major impairment in connective tissues, such as joints and bones. The behavioral abnormalities of MPS VI rats represent fundamental endpoints for studies aimed at testing the pre-clinical safety and efficacy of novel therapeutic approaches for MPS VI.

Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 10(13) genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 10(12) gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients.

Ocular neovascularization associated with proliferative diabetic retinopathy and age-related macular degeneration is the leading cause of severe visual loss in adults in developed countries. Physiological and pathological retinal angiogenesis may occur independently in postnatal life through the complex activation of pro- and antiangiogenic pathways. We report that the Sonic hedgehog (Shh) pathway is activated in the retina in animal models of retinal and choroidal neovascularization. We show that pharmacological inhibition of the Shh signaling pathway significantly reduces physiological retinal angiogenesis and inhibits pathological vascularization in both models. Under retinal hypoxic conditions, inhibition of the Shh pathway results in reduction of vascular endothelial growth factor (VEGF) level, along with that of Patched-1 (Ptch1), a canonical Shh target, thus placing Shh activation upstream of VEGF in experimental retinal neovascularization. Our data demonstrate the requirement of the Shh pathway for retinal angiogenesis and its inhibition as a potential therapeutic strategy targeting ocular neovascular disease.

Since the establishment of the Sanger sequencing method, scientists around the world focused their efforts to progress in the field to produce the utmost technology. The introduction of next-generation sequencing (NGS) represents a revolutionary step and promises to lead to massive improvements in our understanding on the role of nucleic acids functions. Cancer research began to use this innovative and highly performing method, and interesting results started to appear in colorectal cancer (CRC) analysis. Several studies produced high-quality data in terms of mutation discovery, especially about actionable or less frequently mutated genes, epigenetics, transcriptomics. Analysis of results is unveiling relevant perspectives aiding to evaluate the response to therapies. Novel evidences have been presented also in other directions such as gut microbiota or CRC circulating tumor cells. However, despite its unquestioned potential, NGS poses some issues calling for additional studies. This review intends to offer a view of the state of the art of NGS applications to CRC through examination of the most important technologies and discussion of recent published results.

The detection of circulating microRNA (miRNA)-based biomarkers represents an innovative, non-invasive method for the early detection of cancer. However, the low concentration of miRNAs released in body fluids and the difficult identification of the tumor site have limited their clinical use as effective cancer biomarkers. To evaluate if ultrasound treatment could amplify the release of extracellular cancer biomarkers, we treated a panel of prostate cancer (PCa) cell lines with an ultrasound-based prototype and profiled the release of miRNAs in the extracellular space, with the aim of identifying novel miRNA-based biomarkers that could be used for PCa diagnosis and the monitoring of tumor evolution. We provide evidence that US-mediated sonoporation amplifies the release of miRNAs from both androgen-dependent (AD) and -independent (AI) PCa cells. We identified four PCa-related miRNAs, whose levels in LNCaP and DU145 supernatants were significantly increased following ultrasound treatment: mir-629-5p, mir-374-5p, mir-194-5p, and let-7d-5p. We further analyzed a publicly available dataset of PCa, showing that the serum expression of these novel miRNAs was upregulated in PCa patients compared to controls, thus confirming their clinical relevance. Our findings highlight the potential of using ultrasound to identify novel cell-free miRNAs released from cancer cells, with the aim of developing new biomarkers with diagnostic and predictive value.

The number of intestinal mast cells (MC) is increased in several types of colitis, but the mucosa of patients with chronic non-bloody diarrhea has not been studied. The current study sought to determine the relationship between MC counts and degranulation and the severity of symptoms in patients with chronic loose stools. Following a negative laboratory workup for the most common causes of chronic diarrhea, patients with chronic non-bloody loose stools were included in the study. Patients with macroscopic evidence of inflammation or organic disease were excluded after endoscopy with biopsies. Biopsies from the 179 patients in the study were stained with hematoxylin and eosin and anti-CD117 c-kit antibodies. Immunohistochemistry was used to assess the degree of MC degranulation. Out of the 179 patients, 128 had normal histologic findings suggestive of irritable bowel syndrome and were used as controls. Twenty-four presented with abnormally high MC counts (≥40 MC x HPF), 23 with ≥20 intraepithelial lymphocytes x HPF suggesting lymphocytic colitis, and 4 had both (≥40 MC and ≥20 intraepithelial lymphocytes x HPF). In the patients with high MC counts, figures were significantly higher in the right colon versus the left colon (p=0.016), but degranulation did not differ in the right versus the left colon (p=0.125). No age or sex-related difference was observed (p=0.527 and p=0.859 respectively). The prevalence of abdominal pain and bloating did not differ in the three groups (p=0.959 and p=0.140, respectively). Patients with lymphocytic colitis (p=0.008) and those with high MC counts (p=0.025) had significantly higher evacuation rates compared to controls. There was no difference between these two groups (p=0.831). Mast cell degranulation was not associated with the number of evacuations, abdominal pain, or bloating (p=0.51; p=0.41; p=0.42, respectively). The finding that a significantly higher number of evacuations was linked to increased MC in the colonic mucosa of a subset of patients with otherwise normal laboratory and endoscopic findings suggests that "mastocytic colitis" may be a new clinical-pathological entity responsible for chronic non-bloody diarrhea. Prospective studies with a larger number of patients, as well as endoscopic and histological follow-up, are needed to confirm this hypothesis.

The advent of Next Generation Sequencing technologies brought with it the discovery of several microRNA (miRNA) variants of heterogeneous lengths and/or sequences. Initially ascribed to sequencing errors/artifacts, these isoforms, named isomiRs, are now considered non-canonical variants that originate from physiological processes affecting the canonical miRNA biogenesis. To date, accurate IsomiRs abundance, biological activity, and functions are not completely understood; however, the study of isomiR biology is an area of great interest due to their high frequency in the human miRNome, their putative functions in cooperating with the canonical miRNAs, and potential for exhibiting novel functional roles. The discovery of isomiRs highlighted the complexity of the small RNA transcriptional landscape in several diseases, including cancer. In this field, the study of isomiRs could provide further insights into the miRNA biology and its implication in oncogenesis, possibly providing putative new cancer diagnostic, prognostic, and predictive biomarkers as well. In this review, a comprehensive overview of the state of research on isomiRs in different cancer types, including the most common tumors such as breast cancer, colorectal cancer, melanoma, and prostate cancer, as well as in the less frequent tumors, as for example brain tumors and hematological malignancies, will be summarized and discussed.

Despite the growing body of knowledge showing that testosterone (T) may not significantly affect tumor progression in hypogonadal patients treated for prostate cancer (Pca), the use of this hormone in this population still remains controversial. The effects of continuous or pulsed T stimulation were tested in vitro and in vivo on androgen-sensitive Pca cell lines in order to assess the differential biological properties of these two treatment modalities. Pulsed T treatment resulted in a greater inhibition than continuous T supplementation of tumor growth in vitro and in vivo. The effects of pulsed T treatment on tumor growth inhibition, G0/G1 cell cycle arrest, and tumor senescence was more pronounced than those obtained upon continuous T treatments. Mechanistic studies revealed that G0/G1 arrest and tumor senescence upon pulsed T treatment were associated with a marked decrease in cyclin D1, c-Myc and SKp2, CDK4 and p-Rb levels and upregulation of p27 and p-ERK1/2. Pulsed, but not continuous, T supplementation decreased the expression levels of AR, p-ARser81 and CDK1 in both cellular models. The in vitro results were confirmed in an in vivo xenografts, providing evidence of a greater inhibitory activity of pulsed supraphysiological T supplementation than continuous treatment, both in terms of tumor volume and decreased AR, p-ARser81, PSA and CDK1 staining. The rapid cycling from hypogonadal to physiological or supra-physiological T intraprostatic concentrations results in cytostatic and senescence effects in preclinical models of androgen-sensitive Pca. Our preclinical evidence provides relevant new insights in the biology of Pca response to pulsed T supplementation.

MRE11 is a component of the MRE11/RAD50/NBS1 (MRN) complex, whose activity is essential to control faithful DNA replication and to prevent accumulation of deleterious DNA double-strand breaks. In humans, hypomorphic mutations in these genes lead to DNA damage response (DDR)-defective and cancer-prone syndromes. Moreover, MRN complex dysfunction dramatically affects the nervous system, where MRE11 is required to restrain MYCN-dependent replication stress, during the rapid expansion of progenitor cells. MYCN activation, often due to genetic amplification, represents the driving oncogenic event for a number of human tumors, conferring bad prognosis and predicting very poor responses even to the most aggressive therapeutic protocols. This is prototypically exemplified by neuroblastoma, where MYCN amplification occurs in about 25% of the cases. Intriguingly, MRE11 is highly expressed and predicts bad prognosis in MYCN-amplified neuroblastoma. Due to the lack of direct means to target MYCN, we explored the possibility to trigger intolerable levels of replication stress-dependent DNA damage, by inhibiting MRE11 in MYCN-amplified preclinical models. Indeed, either MRE11 knockdown or its pharmacological inhibitor mirin induce accumulation of replication stress and DNA damage biomarkers in MYCN-amplified cells. The consequent DDR recruits p53 and promotes a p53-dependent cell death, as indicated by p53 loss- and gain-of-function experiments. Encapsulation of mirin in nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts in vivo, which resulted in a sharp impairment of tumor growth, associated with DDR activation, p53 accumulation, and cell death. Therefore, we propose that MRE11 inhibition might be an effective strategy to treat MYCN-amplified and p53 wild-type neuroblastoma, and suggest that targeting replication stress with appropriate tools should be further exploited to tackle MYCN-driven tumors.

Cellular senescence and ferroptosis are the two main, fine-tuned processes in tissue damage restraint; however, they can be overactivated in pathologies such as nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH), becoming dangerous stimuli. Senescence is characterized by a decline in cell division and an abnormal release of reactive oxygen species (ROS), and ferroptosis is represented by iron deposition associated with an excessive accumulation of ROS. ROS and cellular stress pathways are also drivers of NAFLD/NASH development. The etiology of NAFLD/NASH lies in poor diets enriched in fat and sugar. This food regimen leads to liver steatosis, resulting in progressive degeneration of the organ, with a late onset of irreversible fibrosis and cirrhosis. Few studies have investigated the possible connection between senescence and ferroptosis in NAFLD/NASH progression, despite the two events sharing some molecular players. We hypothesized a possible link between senescence and ferroptosis in a NAFLD background. To thoroughly investigate this in the context of "Western-style" diet (WSD) abuse, we used an amylin-modified liver NASH mouse model. The main NASH hallmarks have been confirmed in this model, as well as an increase in apoptosis, and Ki67 and p53 expression in the liver. Senescent beta-galactosidase-positive cells were elevated, as well as the expression of the related secretory molecules Il-6 and MMP-1. Features of DNA damage and iron-overload were found in the livers of NASH mice. Gpx4 (glutathione peroxidase 4) expression, counteracting ferroptotic cell death, was increased. Notably, an increased number of senescent cells showing overexpression of gpx4 was also found. Our data seem to suggest that senescent cells acquire a gpx4-mediated mechanism of ferroptosis resistance and thus remain in the liver, fostering the deterioration of liver fitness.