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Malignant transformation of T-cell progenitors causes T-cell acute lymphoblastic leukemia (T-ALL), an aggressive childhood lymphoproliferative disorder. Activating mutations of Notch, Notch1 and Notch3, have been detected in T-ALL patients. In this study, we aimed to deeply characterize hyperactive Notch3-related pathways involved in T-cell dynamics within the thymus and bone marrow to propose these processes as an important step in facilitating the progression of T-ALL. We previously generated a transgenic T-ALL mouse model (N3-ICtg) demonstrating that aberrant Notch3 signaling affects early thymocyte maturation programs and leads to bone marrow infiltration by CD4 + CD8 + (DP) T cells that are notably, Notch3 high CXCR4 high . Newly, our in vivo results suggest that an anomalous immature thymocyte subpopulation, such as CD4 - CD8 - (DN) over-expressing CD3ɛ, but with low CXCR4 expression, dominates N3-ICtg thymus-resident DN subset in T-ALL progression. MicroRNAs might be of significance in T-ALL pathobiology, however, whether required for leukemia maintenance is not fully understood. The selection of specific DN subsets demonstrates the inverse correlation between CXCR4 expression and a panel of Notch3-deregulated miRNAs. Interestingly, we found that within DN thymocyte subset hyperactive Notch3 inhibits CXCR4 expression through the cooperative effects of miR-139-5p and miR-150-5p, thus impinging on thymocyte differentiation with accumulation of DNCD3ɛ + CXCR4 - cells. These data point out that deregulation of Notch3 in T-ALL, besides its role in sustaining dissemination of abnormal DP T cells, as we previously demonstrated, could play a role in selecting specific DN immature T cells within the thymus, thus impeding T cell development, to facilitate T-ALL progression inside the bone marrow., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Bisphenol-A (BPA), a synthetic compound ubiquitously present in the environment, can act as an endocrine disruptor by binding to both canonical and non-canonical estrogen receptors (ERs). Exposure to BPA has been linked to various cancers, in particular, those arising in hormone-targeted tissues such as the breast. In this study, we evaluated the effect of BPA intake through drinking water on ErbB2/ neu -driven cancerogenesis in BALB- neu T mice, transgenic for a mutated ErbB2/ neu receptor gene, which reproducibly develop carcinomas in all mammary glands. In this model, BPA accelerated mammary cancerogenesis with an increase in the number of tumors per mouse and a concurrent decrease in tumor-free and overall survival. As assessed by immunohistochemistry, BALB- neu T tumors were ER-negative but expressed high levels of the alternative estrogen receptor GPR30, regardless of BPA exposure. On the other hand, BPA exposure resulted in a marked upregulation of progesterone receptors in preinvasive tumors and of Ki67, CD31, and phosphorylated Akt in invasive tumors. Moreover, based on several infiltration markers of immune cells, BPA favored an immunosuppressive tumor microenvironment. Finally, in vitro cell survival studies performed on a cell line established from a BALB- neu T breast carcinoma confirmed that BPA's impact on cancer progression can be particularly relevant after chronic, low-dose exposure.

In vitro models of pediatric brain tumors (pBT) are instrumental for better understanding the mechanisms contributing to oncogenesis and testing new therapies; thus, ideally, they should recapitulate the original tumor. We applied DNA methylation (DNAm) and copy number variation (CNV) profiling to characterize 241 pBT samples, including 155 tumors and 86 pBT-derived cell cultures, considering serum vs serum-free conditions, late vs early passages, and dimensionality (2D vs 3D cultures). We performed a t-SNE classification and identified differentially methylated regions in tumors compared to cell models. Early cell cultures recapitulate the original tumor, but serum media and 2D culturing were demonstrated to significantly contribute to the divergence of DNAm profiles from the parental ones. All divergent cells clustered together acquiring a common deregulated epigenetic signature suggesting a shared selective pressure. We identified a set of hypomethylated genes shared among unfaithful cells converging on response to growth factors and migration pathways, such as signaling cascade activation, tissue organization, and cellular migration. In conclusion, DNAm and CNV are informative tools that should be used to assess the recapitulation of pBT-cells from parental tumors., (© 2024. The Author(s).)

Background: Breast cancer (BC) treatment has recently been revolutionized by the introduction of newer targeted agents, that helped tailoring therapies around the single patient. Along with increased survival rates, a careful evaluation of diet, lifestyle habits, physical activity, emotional and psychological experiences linked to the treatment journey, is now mandatory. However, a true proposal for an omnicomprehensive and "integrative" approach is still lacking in literature., Methods: A scientific board of internationally recognized specialists throughout different disciplines designed a shared proposal of holistic approach for BC patients., Results: A narrative review, containing information on BC treatment, endocrinological and diet aspects, physical activity, rehabilitation, integrative medicine, and digital narrative medicine, was developed., Conclusions: In the context of a patient-centered care, BC treatment cannot be separated from a patient's long-term follow-up and care, and an organized interdisciplinary collaboration is the future in this disease's cure, to make sure that our patients will live longer and better., Trial Registration: NCT05893368: New Model for Integrating Person-based Care (PbC) in the Treatment of Advanced HER2-negative Breast Cancer (PERGIQUAL). Registration date: 29th May 2023., (© 2024. The Author(s).)

Melanoma is the most aggressive form of skin cancer, representing approximately 4% of all cutaneous neoplasms and accounting for up to 80% of deaths. Advanced stages of melanoma involve metastatic processes and are associated with high mortality and morbidity, mainly due to the rapid dissemination and heterogeneous responses to current therapies, including immunotherapy. Immune checkpoint inhibitors (ICIs) are currently used in the treatment of metastatic melanoma (MM) and despite being linked to an increase in patient survival, a high percentage of them still do not benefit from it. Accordingly, the number of therapeutic regimens for MM patients using ICIs either alone or in combination with other therapies has increased, together with the need for reliable biomarkers that can both predict and monitor response to ICIs. In this context, circulating biomarkers, such as DNA, RNA, proteins, and cells, have emerged due to their ability to reflect disease status. Moreover, blood tests are minimally invasive and provide an attractive option to detect biomarkers, avoiding stressful medical procedures. This systematic review aims to evaluate the possibility of a non-invasive biomarker signature that can guide therapeutic decisions. The studies reported here offer valuable insight into how circulating biomarkers can have a role in personalized treatments for melanoma patients receiving ICIs therapy, emphasizing the need for rigorous clinical trials to confirm findings and establish standardized procedures., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Breast cancer (BC) is the most common malignancy that affects women, and it is, to date, their leading cause of death. Luminal A molecular subtype accounts for 40% of BC and is characterized by hormone receptors positive/human epidermal growth factor 2 expression and current treatment consists of surgery plus aromatase inhibitor therapy. Interestingly, several studies demonstrated that the heavy metal cadmium (Cd), classified as a group 1 human carcinogen and widely spread in the environment, exerts estrogen-like activities in several tissues and suggested an intriguing relationship between increased Cd exposure and BC incidence. Thus, aim of this study was to evaluate effects of Cd on Luminal A BC estrogen receptor (ER) positive/progesterone receptor positive cell models in vitro to characterize the mechanism(s) involved in breast cell homeostasis disruption., Methods: T47D and MCF7 were exposed to Cd (0.5-1 µM) for 6-24 h to evaluate potential alterations in: cells viability, steroid receptors and intracellular signaling by western blot. Moreover, we evaluated the expression of inflammatory cytokines interleukin by RT-PCR., Results: Our results showed a significant induction of androgen receptor (AR) and an increased AR/ER ratio. Further, Cd exposure increased pro-inflammatory cytokines interleukin (IL)6, IL8 and tumor necrosis factor α levels. Finally, as previously demonstrated by our group, Cd alters pathways such as mitogen-activated protein kinase family and protein kinase B., Conclusion: In conclusion, our study demonstrates that Cd modifies the expression and pattern of ERs and AR in BC cell lines, suggesting an alteration of BC cells homeostasis, likely predisposing to a carcinogenetic microenvironment., (© 2023. The Author(s).)

Background: Prostate cancer (PCa) is the most diagnosed cancer in men, with an increasing need to integrate noninvasive imaging and circulating microRNAs beyond prostate-specific antigen for screening and early detection., Objective: To validate magnetic resonance imaging (MRI) biomarkers and circulating microRNAs as triage tests for patients directed to prostate biopsy, and to test different diagnostic pathways to compare their performance on patients' outcome, in terms of unnecessary biopsy avoidance., Design, Setting, and Participants: A prospective single-center cohort study, enrolling patients with PCa suspicion who underwent MRI, MRI-directed fusion biopsy (MRDB), and circulating microRNAs, was conducted. A network-based analysis was used to identify MRI biomarkers and microRNA drivers of clinically significant PCa., Intervention: MRI, MRDB, and blood sampling., Outcome Measurements and Statistical Analysis: The decision curve analysis was exploited to assess the performance of the proposed diagnostic pathways and to quantify their benefit in terms of biopsy avoidance., Results and Limitations: Overall, 261 men were enrolled and underwent MRDB for PCa detection. A total of 178 patients represented the entire cohort: 55 (30.9%) were negative for PCa, 39 (21.9%) had grade group (GG) 1 PCa, and 84 (47.2%) had GG >1 PCa. The proposed integrated pathway, including clinical data, MRI biomarkers, and microRNAs, provided the best net benefit with a biopsy avoidance rate of about 20% at a low disease probability. The main limitation is the monocentric design in a referral center., Conclusions: The integrated pathway represents a validated model that sees MRI biomarkers and microRNAs as a prebiopsy triage of patients at a risk for clinically significant PCa. The proposed pathway showed the highest net benefit in terms of unnecessary biopsy avoidance., Patient Summary: The proposed integrated pathway for early detection of prostate cancer (PCa) allows accurate patient allocation to biopsy and patients' stratification into risk group categories, reducing overdiagnosis and overtreatment of clinically insignificant PCa., (Copyright © 2023 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Purpose/methods: The determination of tumour biomarkers is paramount to advancing personalized medicine, more so in rare tumours like medullary thyroid carcinoma (MTC), whose diagnosis is still challenging. The aim of this study was to identify non-invasive circulating biomarkers in MTC. To achieve this goal, paired MTC tissue and plasma extracellular vesicle samples were collected from multiple centres and microRNA (miRNA) expression levels were evaluated., Results: The samples from a discovery cohort of 23 MTC patients were analysed using miRNA arrays. Lasso logistic regression analysis resulted in the identification of a set of circulating miRNAs as diagnostic biomarkers. Among them, miR-26b-5p and miR-451a, were highly expressed and their expression decreased during follow-up in disease-free patients in the discovery cohort. Circulating miR-26b-5p and miR-451a were validated using droplet digital PCR in a second independent cohort of 12 MTC patients., Conclusion: This study allowed the identification and validation of a signature of two circulating miRNAs, miR-26b-5p and miR-451a, in two independent cohorts reporting a significant diagnostic performance for MTC. The results of this study offer advancements in molecular diagnosis of MTC proposing a novel non-invasive tool to use in precision medicine., (© 2023. The Author(s).)

Obesity is the main risk factor for many non-communicable diseases. In clinical practice, unspecific markers are used for the determination of metabolic alterations and inflammation, without allowing the characterization of subjects at higher risk of complications. Circulating microRNAs represent an attractive approach for early screening to identify subjects affected by obesity more at risk of developing connected pathologies. The aim of this study was the identification of circulating free and extracellular vesicles (EVs)-embedded microRNAs able to identify obese patients at higher risk of type 2 diabetes (DM2). The expression data of circulating microRNAs derived from obese patients (OB), with DM2 (OBDM) and healthy donors were combined with clinical data, through network-based methodology implemented by weighted gene co-expression network analysis. The six circulating microRNAs overexpressed in OBDM patients were evaluated in a second group of patients, confirming the overexpression of miR-155-5p in OBDM patients. Interestingly, the combination of miR-155-5p with serum levels of IL-8, Leptin and RAGE was useful to identify OB patients most at risk of developing DM2. These results suggest that miR-155-5p is a potential circulating biomarker for DM2 and that the combination of this microRNA with other inflammatory markers in OB patients can predict the risk of developing DM2., (© 2023. The Author(s).)

The Sonic Hedgehog (Hh) signal transduction pathway plays a critical role in many developmental processes and, when deregulated, may contribute to several cancers, including basal cell carcinoma, medulloblastoma, colorectal, prostate, and pancreatic cancer. In recent years, several Hh inhibitors have been developed, mainly acting on the Smo receptor. However, drug resistance due to Smo mutations or non-canonical Hh pathway activation highlights the need to identify further mechanisms of Hh pathway modulation. Among these, deacetylation of the Hh transcription factor Gli1 by the histone deacetylase HDAC1 increases Hh activity. On the other end, the KCASH family of oncosuppressors binds HDAC1, leading to its ubiquitination and subsequent proteasomal degradation, leaving Gli1 acetylated and not active. It was recently demonstrated that the potassium channel containing protein KCTD15 is able to interact with KCASH2 protein and stabilize it, enhancing its effect on HDAC1 and Hh pathway. KCTD15 and KCTD1 proteins share a high homology and are clustered in a specific KCTD subfamily. We characterize here KCTD1 role on the Hh pathway. Therefore, we demonstrated KCTD1 interaction with KCASH1 and KCASH2 proteins, and its role in their stabilization by reducing their ubiquitination and proteasome-mediated degradation. Consequently, KCTD1 expression reduces HDAC1 protein levels and Hh/Gli1 activity, inhibiting Hh dependent cell proliferation in Hh tumour cells. Furthermore, analysis of expression data on publicly available databases indicates that KCTD1 expression is reduced in Hh dependent MB samples, compared to normal cerebella, suggesting that KCTD1 may represent a new putative target for therapeutic approaches against Hh-dependent tumour., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Medullary thyroid carcinoma (MTC) is a malignant tumor with challenging management. Multi-targeted kinase inhibitors (MKI) and tyrosine-kinase inhibitors (TKI) with high specificity for RET protein are approved for advanced MTC treatment. However, their efficacy is hindered by evasion mechanisms of tumor cells. Thus, the aim of this study was the identification of an escape mechanism in MTC cells exposed to a highly selective RET TKI. TT cells were treated with TKI, MKI, and/or the HH-Gli inhibitors, GANT61 and Arsenic Trioxide (ATO), in the presence or absence of hypoxia. RET modifications, oncogenic signaling activation, proliferation and apoptosis were assessed. Additionally, cell modifications and HH-Gli activation were also evaluated in pralsetinib-resistant TT cells. Pralsetinib inhibited RET autophosphorylation and RET downstream pathways activation in normoxic and hypoxic conditions. Additionally, pralsetinib impaired proliferation, induced the activation of apoptosis and, in hypoxic cells, downregulated HIF-1α. Focusing on escape molecular mechanisms associated with therapy, we observed increased Gli1 levels in a subset of cells. Indeed, pralsetinib stimulated the re-localization of Gli1 into the cell nuclei. Treatment of TT cells with both pralsetinib and ATO resulted in Gli1 down-regulation and impaired cell viability. Moreover, pralsetinib-resistant cells confirmed Gli1 activation and up-regulation of its transcriptionally regulated target genes. Altogether, we showed that pralsetinib impairs MTC cell growth and induces cell death, also in hypoxic conditions. The HH-Gli pathway is a new molecular mechanism of escape to pralsetinib therapy that can be overcome through combined therapy., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Gastrointestinal (GI) cancers are the most frequent neoplasm, responsible for half of all cancer-related deaths. Metastasis is the leading cause of death from GI cancer; thus, studying the processes that regulate cancer cell migration is of paramount importance for the development of new therapeutic strategies. In this review, we summarize the mechanisms adopted by cancer cells to promote cell migration and the subsequent metastasis formation by highlighting the key role that tumor microenvironment components play in deregulating cellular pathways involved in these processes. We, therefore, provide an overview of the role of different microRNAs in promoting tumor metastasis and their role as potential biomarkers for the prognosis, monitoring, and diagnosis of GI cancer patients. Finally, we relate the possible use of nutraceuticals as a new strategy for targeting numerous microRNAs and different pathways involved in GI tumor invasiveness.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality and chemoresistance is a major medical issue. The epithelial-to-mesenchymal transition (EMT) is the primary step in the emergence of the invasive phenotype and the Hedgehog-GLI (HH-GLI) and NOTCH signaling pathways are associated with poor prognosis and EMT in CRC. CRC cell lines harboring KRAS or BRAF mutations, grown as monolayers and organoids, were treated with the chemotherapeutic agent 5-Fluorouracil (5-FU) alone or combined with HH-GLI and NOTCH pathway inhibitors GANT61 and DAPT, or arsenic trioxide (ATO) to inhibit both pathways. Treatment with 5-FU led to the activation of HH-GLI and NOTCH pathways in both models. In KRAS mutant CRC, HH-GLI and NOTCH signaling activation co-operate to enhance chemoresistance and cell motility, while in BRAF mutant CRC, the HH-GLI pathway drives the chemoresistant and motile phenotype. We then showed that 5-FU promotes the mesenchymal and thus invasive phenotype in KRAS and BRAF mutant organoids and that chemosensitivity could be restored by targeting the HH-GLI pathway in BRAF mutant CRC or both HH-GLI and NOTCH pathways in KRAS mutant CRC. We suggest that in KRAS-driven CRC, the FDA-approved ATO acts as a chemotherapeutic sensitizer, whereas GANT61 is a promising chemotherapeutic sensitizer in BRAF-driven CRC.

The definition of metabolic syndrome (MetS) has undergone several changes over the years due to the difficulty in establishing universal criteria for it. Underlying the disorders related to MetS is almost invariably a pro-inflammatory state related to altered glucose metabolism, which could lead to elevated cardiovascular risk. Indeed, the complications closely related to MetS are cardiovascular diseases (CVDs) and type 2 diabetes (T2D). It has been observed that the predisposition to metabolic syndrome is modulated by complex interactions between human microbiota, genetic factors, and diet. This review provides a summary of the last decade of literature related to three principal aspects of MetS: (i) the syndrome's definition and classification, pathophysiology, and treatment approaches; (ii) prediction and diagnosis underlying the biomarkers identified by means of advanced methodologies (NMR, LC/GC-MS, and LC, LC-MS); and (iii) the role of foods and food components in prevention and/or treatment of MetS, demonstrating a possible role of specific foods intake in the development of MetS., Competing Interests: The authors declare no conflict of interest.

Background: Melanoma is the deadliest form of skin cancer and metastatic disease is associated with a significant survival rate drop. There is an urgent need for consistent tumor biomarkers to scale precision medicine and reduce cancer mortality. Here, we aimed to identify a melanoma-specific circulating microRNA signature and assess its value as a diagnostic tool., Methods: The study consisted of a discovery phase and two validation phases. Circulating plasma extracellular vesicles (pEV) associated microRNA profiles were obtained from a discovery cohort of metastatic melanoma patients and normal subjects as controls. A pEV-microRNA signature was obtained using a LASSO penalized logistic regression model. The pEV-microRNA signature was subsequently validated both in a publicly available dataset and in an independent internal cohort., Results: We identified and validated in three independent cohorts a panel of melanoma-specific circulating microRNAs that showed high accuracy in differentiating melanoma patients from healthy subjects with an area under the curve (AUC) of 1.00, 0.94 and 0.75 respectively. Investigation of the function of the pEV-microRNA signature evidenced their possible immune suppressive role in melanoma patients., Conclusions: We demonstrate that a blood test based on circulating microRNAs can non-invasively detect melanoma, offering a novel diagnostic tool for improving standard care. Moreover, we revealed an immune suppressive role for melanoma pEV-microRNAs., (© 2022. The Author(s).)

Type 2 diabetes (T2D), a chronic metabolic disease, has attained the status of a global epidemic with steadily increasing incidence worldwide. Improved diagnosis, stratification and prognosis of T2D patients and the development of more effective treatments are needed. In this era of personalized medicine, the discovery and evaluation of innovative circulating biomarkers can be an effective tool for better stratification, prognosis and therapeutic selection/management of T2D patients. MicroRNAs (miRNAs), a class of small non-coding RNAs that modulate gene expression, have been investigated as potential circulating biomarkers in T2D. Several studies have investigated the expression of circulating miRNAs in T2D patients from various biological fluids, including plasma and serum, and have demonstrated their potential as diagnostic and prognostic biomarkers, as well as biomarkers of response to therapy. In this review, we provide an overview of the current state of knowledge, focusing on circulating miRNAs that have been consistently expressed in at least two independent studies, in order to identify a set of consistent biomarker candidates in T2D. The expression levels of miRNAs, correlation with clinical parameters, functional roles of miRNAs and their potential as biomarkers are reported. A systematic literature search and assessment of studies led to the selection and review of 10 miRNAs (miR-126-3p, miR-223-3p, miR-21-5p, miR-15a-5p, miR-24-3p, miR-34a-5p, miR-146a-5p, miR-148a-3p, miR-30d-5p and miR-30c-5p). We also present technical challenges and our thoughts on the potential validation of circulating miRNAs and their application as biomarkers in the context of T2D., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tuberous sclerosis complex (TSC) is a rare multi-system genetic disorder characterized by a high incidence of epilepsy and neuropsychiatric manifestations known as tuberous-sclerosis-associated neuropsychiatric disorders (TANDs), including autism spectrum disorder (ASD) and intellectual disability (ID). MicroRNAs (miRNAs) are small regulatory non-coding RNAs that regulate the expression of more than 60% of all protein-coding genes in humans and have been reported to be dysregulated in several diseases, including TSC. In the current study, RNA sequencing analysis was performed to define the miRNA and isoform (isomiR) expression patterns in serum. A Receiver Operating Characteristic (ROC) curve analysis was used to identify circulating molecular biomarkers, miRNAs, and isomiRs, able to discriminate the development of neuropsychiatric comorbidity, either ASD, ID, or ASD + ID, in patients with TSC. Part of our bioinformatics predictions was verified with RT-qPCR performed on RNA isolated from patients' serum. Our results support the notion that circulating miRNAs and isomiRs have the potential to aid standard clinical testing in the early risk assessment of ASD and ID development in TSC patients.

Background: Pediatric low-grade gliomas (pLGGs), particularly incompletely resected supratentorial tumours, can undergo progression after surgery. However to date, there are no predictive biomarkers for progression. Here, we aimed to identify pLGG-specific microRNA signatures and evaluate their value as a prognostic tool., Methods: We identified and validated supratentorial incompletey resected pLGG-specific microRNAs in independent cohorts from four European Pediatric Neuro-Oncology Centres., Results: These microRNAs demonstrated high accuracy in differentiating patients with or without progression. Specifically, incompletely resected supratentorial pLGGs with disease progression showed significantly higher miR-1248 combined with lower miR-376a-3p and miR-888-5p levels than tumours without progression. A significant (p < 0.001) prognostic performance for miR-1248 was reported with an area under the curve (AUC) of 1.00. We also highlighted a critical oncogenic role for miR-1248 in gliomas tumours. Indeed, high miR-1248 levels maintain low its validated target genes (CDKN1A (p21)/FRK/SPOP/VHL/MTAP) and consequently sustain the activation of oncogenic pathways., Conclusions: Altogether, we provide a novel molecular biomarker able to successfully identify pLGG patients associated with disease progression that could support the clinicians in the decision-making strategy, advancing personalized medicine., (© 2022. The Author(s).)

Colorectal cancer (CRC) is one of most common cancers worldwide, with high rates of mortality. Epidemiological findings demonstrate that coffee consumption reduces the risk of developing CRC by ~13%. In general, in vivo and in vitro findings demonstrate the antiproliferative, antioxidant and proapoptotic effects of brewed coffee or major bioavailable coffee compounds. Thus, it was assessed whether caffeine (CAF) and/or chlorogenic acid (CGA) attenuates the early-stage of chemically induced mouse colon carcinogenesis. Male Swiss mice were submitted to a 1,2-dimethylhydrazine/deoxycholic acid (DMH/DCA)-induced colon carcinogenesis model. These animals received CAF (50 mg/kg), CGA (25 mg/kg) or CAF+CGA (50 + 25 mg/kg) intragastrically for five times/week for ten weeks. CAF+CGA had the most pronounced effects on decreasing epithelial cell proliferation (Ki-67) and increasing apoptosis (cleaved caspase-3) in colonic crypts. This treatment also decreased the levels of proinflammatory cytokines IL-6, IL-17 and TNF-α, and downregulated the oncomiR miR-21a-5p in the colon. Accordingly, the analysis of miR-21a-5p targets demonstrated the genes involved in the negative regulation of proliferation and inflammation, and the positive regulation of apoptosis. Ultimately, CAF+CGA attenuated preneoplastic aberrant crypt foci (ACF) development. Our findings suggest that a combination of coffee compounds reduces early-stage colon carcinogenesis by the modulation of miR-21a-5p expression, highlighting the importance of coffee intake to prevent CRC.

The number of aged individuals is increasing worldwide, rendering essential the comprehension of pathophysiological mechanisms of age-related alterations, which could facilitate the development of interventions contributing to "successful aging" and improving quality of life. Cardiovascular diseases (CVD) include pathologies affecting the heart or blood vessels, such as hypertension, peripheral artery disease and coronary heart disease. Indeed, age-associated modifications in body composition, hormonal, nutritional and metabolic factors, as well as a decline in physical activity are all involved in the increased risk of developing atherogenic alterations that raise the risk of CVD development. Several factors have been reported to play a role in the alterations observed in muscle and endothelial cells and that lead to increased CVD, such as genetic pattern, smoking and unhealthy lifestyle. Moreover, a difference in the risk of these diseases in women and men has been reported. Interestingly, in the past decades attention has been focused on a potential role of several pollutants that disrupt human health by interfering with hormonal pathways, and more specifically in non-communicable diseases such as obesity, diabetes and CVD. This review will focus on the potential alteration induced by Endocrine Disruptors (Eds) in the attempt to characterize a potential role in the cellular and molecular mechanisms involved in the atheromatous degeneration process and CVD progression.

The MRI of the prostate is the gold standard for the detection of clinically significant prostate cancer (csPCa). Nonetheless, MRI still misses around 11% of clinically significant disease. The aim was to comprehensively integrate tissue and circulating microRNA profiling, MRI biomarkers and clinical data to implement PCa early detection. In this prospective cohort study, 76 biopsy naïve patients underwent MRI and MRI directed biopsy. A sentinel sample of 15 patients was selected for a pilot molecular analysis. Weighted gene coexpression network analysis was applied to identify the microRNAs drivers of csPCa. MicroRNA-target gene interaction maps were constructed, and enrichment analysis performed. The ANOVA on ranks test and ROC analysis were performed for statistics. Disease status was associated with the underexpression of the miRNA profiled; a correlation was found with ADC (r = -0.51, p = 0.02) and normalized ADC values (r = -0.64, p = 0.002). The overexpression of miRNAs from plasma was associated with csPCa (r = 0.72; p = 0.02), and with PI-RADS assessment score (r = 0.73; p = 0.02); a linear correlation was found with biomarkers of diffusion and perfusion. Among the 800 profiled microRNA, eleven were identified as correlating with PCa, among which miR-548a-3p, miR-138-5p and miR-520d-3p were confirmed using the RT-qPCR approach on an additional cohort of ten subjects. ROC analysis showed an accuracy of >90%. Provided an additional validation set of the identified miRNAs on a larger cohort, we propose a diagnostic paradigm shift that sees molecular data and MRI biomarkers as the prebiopsy triage of patients at risk for PCa. This approach will allow for accurate patient allocation to biopsy, and for stratification into risk group categories, reducing overdiagnosis and overtreatment.

Cadmium (Cd), a highly toxic heavy metal, is found in soil, environment and contaminated water and food. Moreover, Cd is used in various industrial activities, such as electroplating, batteries production, fertilizers, while an important non-occupational source is represented by cigarette smoking, as Cd deposits in tobacco leaves. Since many years it is clear a strong correlation between Cd body accumulation and incidence of many diseases. Indeed, acute exposure to Cd can cause inflammation and affect many organs such as kidneys and liver. Furthermore, the attention has focused on its activity as environmental pollutant and endocrine disruptor able to interfere with metabolic and energy balance of living beings. Both in vitro and in vivo experiments have demonstrated that the Cd-exposure is related to metabolic diseases such as obesity, diabetes and osteoporosis even if human studies are still controversial. Recent data show that Cd-exposure is associated with atherosclerosis, hypertension and endothelial damage that are responsible for cardiovascular diseases. Due to the large environmental diffusion of Cd, in this review, we summarize the current knowledge concerning the role of Cd in the incidence of metabolic and cardiovascular diseases.

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor comprising hereditary or sporadic form with frequent mutations in the rearranged during transfection (RET) or RAS genes. Diagnosis is based on the presence of thyroid tumor mass with altered levels of calcitonin (Ctn) and carcinoembryonal antigen (CEA) in the serum and/or in the cytological smears from fine needle aspiration biopsies. Treatment consists of total thyroidectomy, followed by tyrosine kinase inhibitors (TKi) in case of disease persistence. During TKi treatment, Ctn and CEA levels can fluctuate regardless of tumor volume, metastasis or response to therapy. Research for more reliable non-invasive biomarkers in MTC is still underway. In this context, circulating nucleic acids, namely circulating microRNAs (miRNAs) and cell free DNA (cfDNA), have been evaluated by different research groups. Aiming to shed light on whether miRNAs and cfDNA are suitable as MTC biomarkers we searched three different databases, PubMed, Scopus, WOS and reviewed the literature. We classified 83 publications fulfilling our search criteria and summarized the results. We report data on miRNAs and cfDNA that can be evaluated for validation in independent studies and clinical application., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Pediatric low-grade gliomas (pLGGs), the most frequent pediatric brain tumors, include different entities harboring distinct histological and molecular features. A major limitation in the development of treatments for these tumors is the absence of reliable in vitro models that would allow a better understanding of the molecular mechanisms that support their growth. Surgical excision is the primary treatment method and the extent of resection represents one of the strongest prognostic factors. pLGGs that cannot be completely resected are prone to recur and associated with relapses and extensive morbidities, thus remaining a major clinical challenge., Methods: We established a protocol to successfully derive primary patient-derived pLGG cells and to fully characterize them from a molecular point of view., Results: Primary patients-derived pLGG cells were extensively analyzed in order to confirm their reliability as cellular models. Specifically, we evaluated the growth rate, senescence, and molecular features, such as BRAF mutational status, methylation, and protein expression profile., Conclusion: This study extensively describes pLGG primary cellular models in terms of isolation, culture method, and molecular characterization that can be used to investigate pLGG biology.

Persistent mortality rates of medulloblastoma (MB) and severe side effects of the current therapies require the definition of the molecular mechanisms that contribute to tumor progression. Using cultured MB cancer stem cells and xenograft tumors generated in mice, we show that low expression of miR-326 and its host gene β-arrestin1 (ARRB1) promotes tumor growth enhancing the E2F1 pro-survival function. Our models revealed that miR-326 and ARRB1 are controlled by a bivalent domain, since the H3K27me3 repressive mark is found at their regulatory region together with the activation-associated H3K4me3 mark. High levels of EZH2, a feature of MB, are responsible for the presence of H3K27me3. Ectopic expression of miR-326 and ARRB1 provides hints into how their low levels regulate E2F1 activity. MiR-326 targets E2F1 mRNA, thereby reducing its protein levels; ARRB1, triggering E2F1 acetylation, reverses its function into pro-apoptotic activity. Similar to miR-326 and ARRB1 overexpression, we also show that EZH2 inhibition restores miR-326/ARRB1 expression, limiting E2F1 pro-proliferative activity. Our results reveal a new regulatory molecular axis critical for MB progression., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Medulloblastoma is the most common malignant brain tumor in children. In addition to sporadic cases, medulloblastoma may occur in association with cancer predisposition syndromes. This review aims to provide a complete description of inherited cancer syndromes associated with medulloblastoma. We examine their epidemiological, clinical, genetic, and diagnostic features and therapeutic approaches, including their correlation with medulloblastoma. Furthermore, according to the most recent molecular advances, we describe the association between the various molecular subgroups of medulloblastoma and each cancer predisposition syndrome. Knowledge of the aforementioned conditions can guide pediatric oncologists in performing adequate cancer surveillance. This will allow clinicians to promptly diagnose and treat medulloblastoma in syndromic children, forming a team with all specialists necessary for the correct management of the other various manifestations/symptoms related to the inherited cancer syndromes., (Copyright © 2020 Carta, Del Baldo, Miele, Po, Besharat, Nazio, Colafati, Piccirilli, Agolini, Rinelli, Lodi, Cacchione, Carai, Boccuto, Ferretti, Locatelli and Mastronuzzi.)

Unfolded protein response (UPR) is a conserved adaptive response that tries to restore protein homeostasis after endoplasmic reticulum (ER) stress. Recent studies highlighted the role of UPR in acute leukemias and UPR targeting has been suggested as a therapeutic approach. Aberrant Notch signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), as downregulation of Notch activity negatively affects T-ALL cell survival, leading to the employment of Notch inhibitors in T-ALL therapy. Here we demonstrate that Notch3 is able to sustain UPR in T-ALL cells, as Notch3 silencing favored a Bip-dependent IRE1α inactivation under ER stress conditions, leading to increased apoptosis via upregulation of the ER stress cell death mediator CHOP. By using Juglone, a naturally occurring naphthoquinone acting as an anticancer agent, to decrease Notch3 expression and induce ER stress, we observed an increased ER stress-associated apoptosis. Altogether our results suggest that Notch3 inhibition may prevent leukemia cells from engaging a functional UPR needed to compensate the Juglone-mediated ER proteotoxic stress. Notably, in vivo administration of Juglone to human T-ALL xenotransplant models significantly reduced tumor growth, finally fostering the exploitation of Juglone-dependent Notch3 inhibition to perturb the ER stress/UPR signaling in Notch3-dependent T-ALL subsets.

Colorectal cancer (CRC) is a leading cause of cancer death. Chemoresistance is a pivotal feature of cancer cells leading to treatment failure and ATP-binding cassette (ABC) transporters are responsible for the efflux of several molecules, including anticancer drugs. The Hedgehog-GLI (HH-GLI) pathway is a major signalling in CRC, however its role in chemoresistance has not been fully elucidated. Here we show that the HH-GLI pathway favours resistance to 5-fluorouracil and Oxaliplatin in CRC cells. We identified potential GLI1 binding sites in the promoter region of six ABC transporters, namely ABCA2, ABCB1, ABCB4, ABCB7, ABCC2 and ABCG1. Next, we investigated the binding of GLI1 using chromatin immunoprecipitation experiments and we demonstrate that GLI1 transcriptionally regulates the identified ABC transporters. We show that chemoresistant cells express high levels of GLI1 and of the ABC transporters and that GLI1 inhibition disrupts the transporters up-regulation. Moreover, we report that human CRC tumours express high levels of the ABCG1 transporter and that its expression correlates with worse patients' prognosis. This study identifies a new mechanism where HH-GLI signalling regulates CRC chemoresistance features. Our results indicate that the inhibition of Gli1 regulates the ABC transporters expression and therefore should be considered as a therapeutic option in chemoresistant patients.

Background: Colorectal cancer (CRC) is a heterogeneous disease with a complex biology and a wide number of altered genes such as BRAF, KRAS and PIK3CA. Advances with new-targeted therapies have been achieved and available treating options have prolonged patient's survival. However, BRAF-mutated CRC patients remain unresponsive to available therapies with RAF inhibitors (RAFi) alone or combined with ErbB inhibitors (ErbBi). These unmet needs require further exploitation of oncogenic signaling in order to set up individualized treatments., Methods: To this end, we tested the efficacy of single agent or combined treatments using the BRAFi, vemurafenib and two different ErbBi: panitumumab and afatinib in CRC cells characterized by different molecular phenotypes., Results: Combination strategies with BRAFi and ErbBi achieved a better response in BRAF V600E mutated cells expressing high levels of ErbB2., Conclusions: Our findings support the importance of ErbB2 evaluation in BRAF-mutated CRC patients and its role as a positive predictor factor of response to BRAFi/ErbBi combination.

Colorectal cancer is characterized by well-known genetic defects and approximately 50% of cases harbor oncogenic Ras mutations. Increased expression of Notch ligand Jagged1 occurs in several human malignancies, including colorectal cancer, and correlates with cancer progression, poor prognosis, and recurrence. Herein, we demonstrated that Jagged1 was constitutively processed in colorectal cancer tumors with mutant Kras, which ultimately triggered intrinsic reverse signaling via its nuclear-targeted intracellular domain Jag1-ICD. This process occurred when Kras/Erk/ADAM17 signaling was switched on, demonstrating that Jagged1 is a novel target of the Kras signaling pathway. Notably, Jag1-ICD promoted tumor growth and epithelial-mesenchymal transition, enhancing colorectal cancer progression and chemoresistance both in vitro and in vivo . These data highlight a novel role for Jagged1 in colorectal cancer tumor biology that may go beyond its effect on canonical Notch activation and suggest that Jag1-ICD may behave as an oncogenic driver that is able to sustain tumor pathogenesis and to confer chemoresistance through a noncanonical mechanism. SIGNIFICANCE: These findings present a novel role of the transcriptionally active Jag1-ICD fragment to confer and mediate some of the activity of oncogenic KRAS., (©2019 American Association for Cancer Research.)

Medulloblastoma is one of the most frequent among pediatric brain tumors, and it has been classified in various subgroups. Some of them already benefit from quite good therapeutic options, whereas others urgently need novel therapeutic approaches. Epigenetic modulators have long been studied in various types of cancer. Within this review, we summarize the main preclinical studies regarding epigenetic targets (such as HDAC, SIRT, BET, EZH2, G9a, LSD1, and DNMT) inhibitors in medulloblastoma. Furthermore, we shed light on the increasing number of applications of drug combinations as well as hybrid compounds involving epigenetic mechanisms. Nevertheless, in the studies published so far, mainly un-specific or old modulators have been used, and the PKs (brain permeability) have not been well-evaluated. Thus, these findings should be considered as a starting point for further improvement and not as a final result.

Ovarian cancer is a leading cause of death among gynecologic malignancies. This disappointing prognosis is closely related to intrinsic or acquired resistance to conventional platinum-based chemotherapy, which can affect a third of patients. As such, investigating relevant molecular targets is crucial in the fight against this disease. So far, many mutations involved in ovarian cancer pathogenesis have been identified. Among them, a few pathways were implicated. One such pathway is the P13K/AKT/mTOR with abnormalities found in many cases. This pathway is considered to have an instrumental role in proliferation, migration, invasion and, more recently, in chemotherapy resistance. Many miRNAs have been found to influence P13K/AKT/mTOR pathway with different potential role in tumor genesis and ovarian cancer behaviour. In particular, their biological function was recently investigated as regards chemoresistance, therefore, leading to the identification of potential specific indirect biomarker of platinum sensitivity in ovarian cancer.

Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying "pre-leukemic" cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4 + CD8 + thymocytes, possibly contributing to "pre-leukemic" cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4 + CD8 + cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4 + CD8 + cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4 + CD8 + cells and such an expression is regulated by Notch3 through β-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4 + CD8 + cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4 + CD8 + cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of "pre-leukemic" CD4 + CD8 + cells, by a "thymus-autonomous" mechanism.

Aims: Paediatric low-grade gliomas (pLGGs) are a heterogeneous group of brain tumours associated with a high overall survival: however, they are prone to recur and supratentorial lesions are difficult to resect, being associated with high percentage of disease recurrence. Our aim was to shed light on the biology of pLGGs., Methods: We performed microRNA profiling on 45 fresh-frozen grade I tumour samples of various histological classes, resected from patients aged ≤16 years. We identified 93 microRNAs specifically dysregulated in tumours as compared to non-neoplastic brain tissue. Pathway analysis of the microRNAs signature revealed PI3K/AKT signalling as one of the centrally enriched oncogenic signalling. To date, activation of the PI3K/AKT pathway in pLGGs has been reported, although activation mechanisms have not been fully investigated yet., Results: One of the most markedly down-regulated microRNAs in our supratentorial pLGGs cohort was miR-139-5p, whose targets include the gene encoding the PI3K's (phosphatidylinositol 3-kinase) catalytic unit, PIK3CA. We investigated the role of miR-139-5p in regulating PI3K/AKT signalling by the use of human cell cultures derived from supratentorial pLGGs. MiR-139-5p overexpression inhibited pLGG cell proliferation and decreased the phosphorylation of PI3K target AKT and phosphorylated-p70 S6 kinase (p-p70 S6K), a hallmark of PI3K/AKT/mTORC1 signalling activation. The effect of miR-139-5p was mediated by PI3K inhibition, as suggested by the decrease in proliferation and phosphorylation of AKT and p70 S6K after treatment with the direct PI3K inhibitor LY294002., Conclusions: These findings provide the first evidence that down-regulation of miR-139-5p in supratentorial pLGG drives cell proliferation by derepressing PI3K/AKT signalling., (© 2018 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

High-throughput analysis has improved the knowledge of medulloblastoma (MB), the leading cause of cancer related death in children, allowing a better comprehension of the key molecular pathways in MB pathogenesis. However, despite these advances, 30% of patients still die from the disease and survivors face severe long-term side effects. Cancer stem cells (CSCs) represent a subset of cells that not only drive tumorigenesis, but are also one of the main determinants of chemoresistance. Epithelial mesenchymal transition (EMT) is a hallmark of cancer and up to now few data is available in MB. To give insight into the role of the EMT process in maintaining the mesenchymal phenotype of CSCs, we analyzed the expression of EMT related transcripts and microRNAs in these cells. We firstly isolated CSCs from Sonic Hedgehog (SHH) MB derived from Ptch1 heterozygous mice and compared their expression level of EMT-related transcripts and microRNAs with cerebellar NSCs. We identified two molecules linked to SHH and EMT, Vegfa and its receptor Nrp2, over-expressed in SHH MB CSCs. Inhibition of Vegfa showed impairment of cell proliferation and self-renewal ability of CSCs concurrent with an increase of the expression of the EMT gene, E-cadherin, and a decrease of the EMT marker, Vimentin. Moreover, among deregulated microRNAs, we identified miR-466f-3p, a validated inhibitor of both Vegfa and Nrp2. These results allowed us to describe a new EMT molecular network, involving the down-regulation of miR-466f-3p together with the concordant up-regulation of Vegfa and Nrp2, that sustains the mesenchymal phenotype of SHH MB CSCs.

Numb is an intracellular protein with multiple functions. The two prevalent isoforms, Numb p66 and Numb p72, are regulators of differentiation and proliferation in neuronal development. Additionally, Numb functions as cell fate determinant of stem cells and cancer stem cells and its abnormal expression has been described in several types of cancer. Involvement of deregulated Numb expression has been described in the malignant childhood brain tumor medulloblastoma, while Numb isoforms in these tumors and in cancer stem-like cells derived from them, have not been studied to date. Here we show that medulloblastoma stem-like cells and cerebellar neuronal stem cells (NSCs) express Numb p66 where its expression tampers stemness features. Furthermore, medulloblastoma samples evaluated in this study express decreased levels of Numb p66 while overexpressed Numb p72 compared with normal tissues. Our results uncover different roles for the two major Numb isoforms examined in medulloblastoma and a critical role for Numb p66 in regulating stem-like cells and NSCs maintenance.

Molecular classification has improved the knowledge of medulloblastoma (MB), the most common malignant brain tumour in children, however current treatments cause severe side effects in patients. Cancer stem cells (CSCs) have been described in MB and represent a sub population characterised by self-renewal and the ability to generate tumour cells, thus representing the reservoir of the tumour. To investigate molecular pathways that characterise this sub population, we isolated CSCs from Sonic Hedgehog Medulloblastoma (SHH MB) arisen in Patched 1 ( Ptch1 ) heterozygous mice, and performed miRNA- and mRNA-sequencing. Comparison of the miRNA-sequencing of SHH MB CSCs with that obtained from cerebellar Neural Stem Cells (NSCs), allowed us to obtain a SHH MB CSC miRNA differential signature. Pathway enrichment analysis in SHH MB CSCs mirnome and transcriptome was performed and revealed a series of enriched pathways. We focused on the putative targets of the SHH MB CSC miRNAs that were involved in the enriched pathways of interest, namely pathways in cancer, PI3k-Akt pathway and protein processing in endoplasmic reticulum pathway. In silico analysis was performed in SHH MB patients and identified several genes, whose expression was associated with worse overall survival of SHH MB patients. This study provides novel candidates whose functional role should be further investigated in SHH MB.

Remote damage is a secondary phenomenon that usually occurs after a primary brain damage in regions that are distant, yet functionally connected, and that is critical for determining the outcomes of several CNS pathologies, including traumatic brain and spinal cord injuries. The understanding of remote damage-associated mechanisms has been mostly achieved in several models of focal brain injury such as the hemicerebellectomy (HCb) experimental paradigm, which helped to identify the involvement of many key players, such as inflammation, oxidative stress, apoptosis and autophagy. Currently, few interventions have been shown to successfully limit the progression of secondary damage events and there is still an unmet need for new therapeutic options. Given the emergence of the novel concept of resolution of inflammation, mediated by the newly identified ω3-derived specialized pro-resolving lipid mediators, such as resolvins, we reported a reduced ability of HCb-injured animals to produce resolvin D1 (RvD1) and an increased expression of its target receptor ALX/FPR2 in remote brain regions. The in vivo administration of RvD1 promoted functional recovery and neuroprotection by reducing the activation of Iba-1+ microglia and GFAP+ astrocytes as well as by impairing inflammatory-induced neuronal cell death in remote regions. These effects were counteracted by intracerebroventricular neutralization of ALX/FPR2, whose activation by RvD1 also down-regulated miR-146b- and miR-219a-1-dependent inflammatory markers. In conclusion, we propose that innovative therapies based on RvD1-ALX/FPR2 axis could be exploited to curtail remote damage and enable neuroprotective effects after acute focal brain damage.

Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment.

The circulating microRNAs (miRNAs) associated with type 2 diabetes (T2D) in elderly patients are still being defined. To identify novel miRNA biomarker candidates for monitoring responses to sitagliptin in such patients, we prospectively studied 40 T2D patients (age > 65) with HbA1c levels of 7.5-9.0% on metformin. After collection of baseline blood samples ( t 0 ), the dipeptidyl peptidase-IV (DPP-IV) inhibitor (DPP-IVi) sitagliptin was added to the metformin regimen, and patients were followed for 15 months. Patients with HbA1c < 7.5% or HbA1c reduction > 0.5% after 3 and 15 months of therapy were classified as "responders" (group R, n = 34); all others were classified as "nonresponders" (group NR, n = 6). Circulating miRNA profiling was performed on plasma collected in each group before and after 15 months of therapy ( t 0 and t 15 ). Intra- and intergroup comparison of miRNA profiles pinpointed three miRNAs that correlated with responses to sitagliptin: miR-378, which is a candidate biomarker of resistance to this DPP-IVi, and miR-126-3p and miR-223, which are associated with positive responses to the drug. The translational implications are as immediate as evident, with the possibility to develop noninvasive diagnostic tools to predict drug response and development of chronic complications.

Cerebellar neural stem cells (NSCs) require Hedgehog-Gli (Hh-Gli) signalling for their maintenance and Nanog expression for their self-renewal. To identify novel molecular features of this regulatory pathway, we used next-generation sequencing technology to profile mRNA and microRNA expression in cerebellar NSCs, before and after induced differentiation (Diff-NSCs). Genes with higher transcript levels in NSCs (vs. Diff-NSCs) included Foxm1, which proved to be directly regulated by Gli and Nanog. Foxm1 in turn regulated several microRNAs that were overexpressed in NSCs: miR-130b, miR-301a, and members of the miR-15~16 and miR-17~92 clusters and whose knockdown significantly impaired the neurosphere formation ability. Our results reveal a novel Hh-Gli-Nanog-driven Foxm1-microRNA network that controls the self-renewal capacity of NSCs.

The mechanisms by which microRNAs control pediatric high-grade gliomas (pHGGs) have yet to be fully elucidated. Our studies of patient-derived pHGG tissues and of the pHGG cell line KNS42 revealed down-regulation in these tumors of three microRNAs, specifically miR-107, miR-181c, and miR-29a-3p. This down-regulation increases the proliferation of KNS42 cells by de-repressing expression of the Notch2 receptor (Notch2), a validated target of miR-107 and miR-181c and a putative target of miR-29a-3p. Inhibition (either pharmacologic or genetic) of Notch2 or re-expression of the implicated microRNAs (all three combined but also individually) significantly reduced KNS42 cell proliferation. These findings suggest that Notch2 pathway activation plays a critical role in pHGGs growth and reveal a direct epigenetic mechanism that controls Notch2 expression, which could potentially be targeted by novel forms of therapy for these childhood tumors characterized by high-morbidity and high-mortality., Competing Interests: The authors declare no conflicts of interest.

Aberrant Hedgehog/GLI signaling has been implicated in a diverse spectrum of human cancers, but its role in lung adenocarcinoma (LAC) is still under debate. We show that the downstream effector of the Hedgehog pathway, GLI1, is expressed in 76% of LACs, but in roughly half of these tumors, the canonical pathway activator, Smoothened, is expressed at low levels, possibly owing to epigenetic silencing. In LAC cells including the cancer stem cell compartment, we show that GLI1 is activated noncanonically by MAPK/ERK signaling. Different mechanisms can trigger the MAPK/ERK/GLI1 cascade including KRAS mutation and stimulation of NRP2 by VEGF produced by the cancer cells themselves in an autocrine loop or by stromal cells as paracrine cross talk. Suppression of GLI1, by silencing or drug-mediated, inhibits LAC cells proliferation, attenuates their stemness and increases their susceptibility to apoptosis in vitro and in vivo. These findings provide insight into the growth of LACs and point to GLI1 as a downstream effector for oncogenic pathways. Thus, strategies involving direct inhibition of GLI1 may be useful in the treatment of LACs.

The histone methyltransferase EZH2 plays a role in maintenance of the stem component of cancer, and its overexpression and/or mutation typically drives tumor aggressiveness, drug resistance and patients' poor prognosis. In this study, we use mouse and human medulloblastoma stem-like cells belonging to the Sonic Hedgehog subgroup (SHH MB-SLCs) and demonstrate that genetic suppression of EZH2 reduces the level of its histone mark H3K27me3 and lowers proliferation and self-renewal. We designed an EZH2 inhibitor (EZH2i) as a simplified analog of EPZ005687 and GSK2816126, MC3629, and we tested its biological activity in SHH MB-SLCs. Pharmacological inhibition of EZH2 impairs SHH MB cells proliferation and self-renewal, and induces apoptosis in vitro . Finally, we generated xenograft MB-SLCs orthotopic tumors in nude mice to test MC3629 in vivo . In treated mice, we observed impairment of tumor growth, together with induction of apoptosis and reduction of proliferation and stemness. Overall, these findings describe EZH2 as a druggable target in MB and provide insight into the biological activity of MC3629 as an EZH2i., Competing Interests: CONFLICTS OF INTEREST No potential conflicts of interest were disclosed.