BRCA1-associated protein 1: Tumor predisposition syndrome and Kury-Isidor syndrome, from genotype-phenotype correlation to clinical management.

The BAP1 tumor suppressor gene encodes a deubiquitinase enzyme involved in several cellular activities, including DNA repair and apoptosis. Germline pathogenic variants in BAP1 have been associated with heritable conditions including BAP1 tumor predisposition syndrome 1 (BAP1-TPDS1) and a neurodevelopmental disorder known as Kury-Isidor syndrome (KURIS). Both these conditions are caused by monoallelic, dominant alterations of BAP1 but have never been reported in the same subject or family, suggesting a mutually exclusive genotype-phenotype correlation. This distinction is extremely important considering the early onset and aggressive nature of the types of cancer reported in individuals with TPDS1. Genetic counseling in subjects with germline BAP1 variants is fundamental to predicting the effect of the variant and the expected phenotype, assessing the potential risk of developing cancer for the tested subject and the family members who may carry the same variant and providing the multidisciplinary clinical team with the proper information to establish precise surveillance and management protocols.

Spatial Distribution of Immune Cells Drives Resistance to Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

Neoadjuvant chemotherapy (NAC) alone or combined with target therapies represents the standard of care for localized triple-negative breast cancer (TNBC). However, only a fraction of patients have a response, necessitating better understanding of the complex elements in the TNBC ecosystem that establish continuous and multidimensional interactions. Resolving such complexity requires new spatially-defined approaches. Here, we used spatial transcriptomics to investigate the multidimensional organization of TNBC at diagnosis and explore the contribution of each cell component to response to NAC. Starting from a consecutive retrospective series of TNBC cases, we designed a case-control study including 24 patients with TNBC of which 12 experienced a pathologic complete response (pCR) and 12 no-response or progression (pNR) after NAC. Over 200 regions of interest (ROI) were profiled. Our computational approaches described a model that recapitulates clinical response to therapy. The data were validated in an independent cohort of patients. Differences in the transcriptional program were detected in the tumor, stroma, and immune infiltrate comparing patients with a pCR with those with pNR. In pCR, spatial contamination between the tumor mass and the infiltrating lymphocytes was observed, sustained by a massive activation of IFN-signaling. Conversely, pNR lesions displayed increased pro-angiogenetic signaling and oxygen-based metabolism. Only modest differences were observed in the stroma, revealing a topology-based functional heterogeneity of the immune infiltrate. Thus, spatial transcriptomics provides fundamental information on the multidimensionality of TNBC and allows an effective prediction of tumor behavior. These results open new perspectives for the improvement and personalization of therapeutic approaches to TNBCs.

Genes selection using deep learning and explainable artificial intelligence for chronic lymphocytic leukemia predicting the need and time to therapy.

Analyzing gene expression profiles (GEP) through artificial intelligence provides meaningful insight into cancer disease. This study introduces DeepSHAP Autoencoder Filter for Genes Selection (DSAF-GS), a novel deep learning and explainable artificial intelligence-based approach for feature selection in genomics-scale data. DSAF-GS exploits the autoencoder's reconstruction capabilities without changing the original feature space, enhancing the interpretation of the results. Explainable artificial intelligence is then used to select the informative genes for chronic lymphocytic leukemia prognosis of 217 cases from a GEP database comprising roughly 20,000 genes. The model for prognosis prediction achieved an accuracy of 86.4%, a sensitivity of 85.0%, and a specificity of 87.5%. According to the proposed approach, predictions were strongly influenced by CEACAM19 and PIGP, moderately influenced by MKL1 and GNE, and poorly influenced by other genes. The 10 most influential genes were selected for further analysis. Among them, FADD, FIBP, FIBP, GNE, IGF1R, MKL1, PIGP, and SLC39A6 were identified in the Reactome pathway database as involved in signal transduction, transcription, protein metabolism, immune system, cell cycle, and apoptosis. Moreover, according to the network model of the 3D protein-protein interaction (PPI) explored using the NetworkAnalyst tool, FADD, FIBP, IGF1R, QTRT1, GNE, SLC39A6, and MKL1 appear coupled into a complex network. Finally, all 10 selected genes showed a predictive power on time to first treatment (TTFT) in univariate analyses on a basic prognostic model including IGHV mutational status, del(11q) and del(17p), NOTCH1 mutations, ß2-microglobulin, Rai stage, and B-lymphocytosis known to predict TTFT in CLL. However, only IGF1R [hazard ratio (HR) 1.41, 95% CI 1.08-1.84, P=0.013), COL28A1 (HR 0.32, 95% CI 0.10-0.97, P=0.045), and QTRT1 (HR 7.73, 95% CI 2.48-24.04, P<0.001) genes were significantly associated with TTFT in multivariable analyses when combined with the prognostic factors of the basic model, ultimately increasing the Harrell's c-index and the explained variation to 78.6% (versus 76.5% of the basic prognostic model) and 52.6% (versus 42.2% of the basic prognostic model), respectively. Also, the goodness of model fit was enhanced (χ2 = 20.1, P=0.002), indicating its improved performance above the basic prognostic model. In conclusion, DSAF-GS identified a group of significant genes for CLL prognosis, suggesting future directions for bio-molecular research.

Medullary Thyroid Carcinomas Classified According to the International Medullary Carcinoma Grading System and a Surveillance, Epidemiology, and End Results-Based Metastatic Risk Score: A Correlation With Genetic Profile and Angioinvasion.

Due to the lack of a standardized tool for risk-based stratification, the International Medullary Carcinoma Grading System (IMTCGS) has been proposed for medullary thyroid carcinomas (MTCs) based on necrosis, mitosis, and Ki67. Similarly, a risk stratification study using the Surveillance, Epidemiology, and End Results (SEER) database highlighted significant differences in MTCs in terms of clinical-pathological variables. We aimed to validate both the IMTCGS and SEER-based risk table on 66 MTC cases, with special attention to angioinvasion and the genetic profile. We found a significant association between the IMTCGS and survival because patients classified as high-grade had a lower event-free survival probability. Angioinvasion was also found to be significantly correlated with metastasis and death. Applying the SEER-based risk table, patients classified either as intermediate- or high-risk had a lower survival rate than low-risk patients. In addition, high-grade IMTCGS cases had a higher average SEER-based risk score than low-grade cases. Moreover, when we explored angioinvasion in correlation with the SEER-based risk table, patients with angioinvasion had a higher average SEER-based score than patients without angioinvasion. Deep sequencing analysis found that 10 out of 20 genes frequently mutated in MTCs belonged to a specific functional class, namely chromatin organization, and function, which may be responsible for the MTC heterogeneity. In addition, the genetic signature identified 3 main clusters; cases belonging to cluster II displayed a significantly higher number of mutations and higher tumor mutational burden, suggesting increased genetic instability, but cluster I was associated with the highest number of negative events. In conclusion, we confirmed the prognostic performance of the IMTCGS and SEER-based risk score, showing that patients classified as high-grade had a lower event-free survival probability. We also underline that angioinvasion has a significant prognostic role, which has not been incorporated in previous risk scores.

BETi enhance ATGL expression and its lipase activity to exert their antitumoral effects in triple-negative breast cancer (TNBC) cells.

BACKGROUND: Triple-Negative Breast Cancer (TNBC) is a subtype of breast cancer that differs from other types of breast cancers in the faster spread and worse outcome. TNBC presented limited treatment options. BET (Bromodomain and extra-terminal domain) proteins are epigenetic readers that control the expression of different oncogenic proteins, and their inhibition (BETi) is considered a promising anti-cancer strategy. Recent evidence demonstrated the involvement of BET proteins in regulation of metabolic processes. METHODS: MDA-MB231 cells treated with JQ1 followed by RNA-sequencing analysis showed altered expression of lipid metabolic genes; among these, we focused on ATGL, a lipase required for efficient mobilization of triglyceride. Different in vitro approaches were performed to validate the RNA-sequencing data (qRT-PCR, immunofluorescence and flow cytometry). NMR (Nuclear Magnetic Resonance) was used to analyze the lipid reprogramming upon treatment. ATGL expression was determined by immunoblot and qRT-PCR, and the impact of ATGL function or protein knockdown, alone and in combination with BETi, was assessed by analyzing cell proliferation, mitochondrial function, and metabolic activity in TNBC and non-TNBC cells culture models. RESULTS: TNBC cells treated with two BETi markedly increased ATGL expression and lipolytic function and decreased intracellular lipid content in a dose and time-dependent manner. The intracellular composition of fatty acids (FAs) after BETi treatment reflected a significant reduction in neutral lipids. The short-chain FA propionate entered directly into the mitochondria mimicking ATGL activity. ATGL KD (knockdown) modulated the levels of SOD1 and CPT1a decreasing ROS and helped to downregulate the expression of mitochondrial ß-oxidation genes in favor of the upregulation of glycolytic markers. The enhanced glycolysis is reflected by the increased of the mitochondrial activity (MTT assay). Finally, we found that after BETi treatment, the FoxO1 protein is upregulated and binds to the PNPLA2 promoter leading to the induction of ATGL. However, FoxO1 only partially prompted the induction of ATGL expression by BETi. CONCLUSIONS: The anti-proliferative effect achieved by BETi is helped by ATGL mediating lipolysis. This study showed that BETi altered the mitochondrial dynamics taking advantage of ATGL function to induce cell cycle arrest and cell death. Schematic representation of BETi mechanism of action on ATGL in TNBC cells. BETi induce the expression of FoxO1 and ATGL, lowering the expression of G0G2, leading to a switch in metabolic status. The induced expression of ATGL leads to increased lipolysis and a decrease in lipid droplet content and bioavailability of neutral lipid. At the same time, the mitochondria are enriched with fatty acids. This cellular status inhibits cell proliferation and increases ROS production and mitochondrial stress. Interfering for ATGL expression, the oxidative phenotypic status mildly reverted to a glycolytic status where neutral lipids are stored into lipid droplets with a consequent reduction of oxidative stress in the mitochondrial.

Long Noncoding RNA NEAT1 Acts as a Molecular Switch for BRD4 Transcriptional Activity and Mediates Repression of BRD4/WDR5 Target Genes.

BRD4 is an epigenome reader known to exert key roles at the interface between chromatin remodeling and transcriptional regulation, and is primarily known for its role in promoting gene expression. In selective contexts, however, BRD4 may work as negative regulator of transcription. Here, we reported that BRD4 binds several long noncoding RNAs (lncRNA). Among these, the lncRNA NEAT1 was found to interfere with BRD4 transcriptional activity. Mechanistically, lncNEAT1 forms a complex with BRD4 and WDR5 and maintains them in a low-activity state. Treatment with Bromodomains and Extraterminal (BET) inhibitor caused the lncRNA NEAT1 to dissociate from the BRD4/WDR5 complex, restored the acetyl-transferase capacity of BRD4, and restored the availability of WDR5 to promote histone trimethylation, thereby promoting BRD4/WDR5 transcriptional activity and activation of target gene expression. In addition, the lncRNA NEAT1 then became available to bind and to inhibit EZH2, cooperatively increasing transcriptional activation. IMPLICATIONS: Our results revealed an epigenetic program that involves the interaction between the lncRNA NEAT1 and BRD4, functioning as a molecular switch between BRD4's activator and repressor chromatin complexes.

RAIN Is a Novel Enhancer-Associated lncRNA That Controls RUNX2 Expression and Promotes Breast and Thyroid Cancer.

Enhancer (ENH)-associated long noncoding RNAs (lncRNA) are a peculiar class of RNAs produced by transcriptionally active ENHs, owning potential gene-regulatory function. Here, we characterized RAIN, a novel ENH-associated lncRNA. Analysis of RAIN expression in a retrospective cohort of human thyroid cancers showed that the expression of this lncRNA is restricted to cancer cells and strongly correlates with the expression of the cancer-promoting transcription factor RUNX2. We showed that RAIN, serving as a cis-regulatory element, promotes RUNX2 expression by two mechanisms. Binding WDR5 and facilitating its localization on the RUNX2 promoter, RAIN modifies the transcriptional status of the RUNX2 locus facilitating transcription initiation. In parallel, RAIN acts as decoy for negative elongation factor complex, restraining its inhibitory function on transcription elongation. In both thyroid and breast cancer cells, RAIN promotes oncogenic features. Using RNA-sequencing profiling, we showed that RAIN orchestrates the expression of a network of cancer-promoting transcription regulators, suggesting that RAIN affects cancer cell phenotype by coordinating the expression of a complex transcriptional network. IMPLICATIONS: Our data contribute to understand lncRNA function in gene regulation and to consolidate their role in cancer.

Expression of NOTCH1 in thyroid cancer is mostly restricted to papillary carcinoma.

The NOTCH signaling is an evolutionarily conserved signaling pathway that regulates cell-cell interactions. NOTCH family members play a fundamental role in a variety of processes during development in particular in cell fate decisions. As other crucial factors during embryogenesis, NOTCH signaling is aberrantly reactivated in cancer where it has been linked to context-dependent effects. In thyroid cancer, NOTCH1 expression has been associated to aggressive features even if its in vivo expression within the entire spectrum of thyroid tumors has not definitively established. A series of 106 thyroid specimens including non-neoplastic lesions, benign and malignant tumors of common and rare histotypes, were investigated by immunohistochemistry to assess NOTCH1 expression. Extent of positivity and protein localization were investigated and correlated with clinical and morphological parameters. NOTCH1 positivity was predominantly associated with papillary carcinomas and only occasionally found in follicular carcinomas. Poorly differentiated and undifferentiated thyroid carcinomas showed only a partial positivity. NOTCH1 expression pattern also seemed differently distributed according to histotype. Our data confirm a role of NOTCH1 in thyroid cancer and highlight for the first time the specific involvement of this pathway in papillary carcinomas. Our data also indicate that other thyroid malignancies do not rely on NOTCH1 signaling for development and progression.

RUNX2 expression in thyroid and breast cancer requires the cooperation of three non-redundant enhancers under the control of BRD4 and c-JUN.

Aberrant reactivation of embryonic pathways is a common feature of cancer. RUNX2 is a transcription factor crucial during embryogenesis that is aberrantly reactivated in many tumors, including thyroid and breast cancer, where it promotes aggressiveness and metastatic spreading. Currently, the mechanisms driving RUNX2 expression in cancer are still largely unknown. Here we showed that RUNX2 transcription in thyroid and breast cancer requires the cooperation of three distantly located enhancers (ENHs) brought together by chromatin three-dimensional looping. We showed that BRD4 controls RUNX2 by binding to the newly identified ENHs and we demonstrated that the anti-proliferative effects of bromodomain inhibitors (BETi) is associated with RUNX2 transcriptional repression. We demonstrated that each RUNX2 ENH is potentially controlled by a distinct set of TFs and we identified c-JUN as the principal pivot of this regulatory platform. We also observed that accumulation of genetic mutations within these elements correlates with metastatic behavior in human thyroid tumors. Finally, we identified RAINs, a novel family of ENH-associated long non-coding RNAs, transcribed from the identified RUNX2 regulatory unit. Our data provide a new model to explain how RUNX2 expression is reactivated in thyroid and breast cancer and how cancer-driving signaling pathways converge on the regulation of this gene.

Nutrigenomics of extra-virgin olive oil: A review.

Nutrigenomics data on the functional components of olive oil are still sparse, but rapidly increasing. Olive oil is the main source of fat and health-promoting component of the Mediterranean diet. Positive effects have been observed on genes involved in the pathobiology of most prevalent age- and lifestyle-related human conditions, such as cancer, cardiovascular disease and neurodegeneration. Other effects on health-promoting genes have been identified for bioactive components of olives and olive leafs. Omics technologies are offering unique opportunities to identify nutritional and health biomarkers associated with these gene responses, the use of which in personalized and even predictive protocols of investigation, is a main breakthrough in modern medicine and nutrition. Gene regulation properties of the functional components of olive oil, such as oleic acid, biophenols and vitamin E, point to a role for these molecules as natural homeostatic and even hormetic factors with applications as prevention agents in conditions of premature and pathologic aging. Therapeutic applications can be foreseen in conditions of chronic inflammation, and particularly in cancer, which will be discussed in detail in this review paper as major clinical target of nutritional interventions with olive oil and its functional components. © 2016 BioFactors, 43(1):17-41, 2017.

Efficacy of NEDD8 Pathway Inhibition in Preclinical Models of Poorly Differentiated, Clinically Aggressive Colorectal Cancer.

Background: The NEDD8 conjugation pathway modulates the ubiquitination and activity of a wide range of intracellular proteins, and its blockade by pevonedistat is emerging as a promising therapeutic approach in various cancer settings. However, systematic characterization of pevonedistat efficacy in specific tumor types and definition of response predictors are still missing. Methods: We investigated in vitro sensitivity to pevonedistat in 122 colorectal cancer (CRC) cell lines by an ATP-based proliferation assay and evaluated apoptosis and DNA content by flow cytometry. Associations between pevonedistat sensitivity and CRC molecular features were assessed by Student's t test. A 184-gene transcriptional predictor was generated in cell lines and applied to 87 metastatic CRC samples for which patient-derived xenografts (PDXs) were available. In vivo reponse to pevonedistat was assessed in PDX models (≥5 mice per group). All statistical tests were two-sided. Results: Sixteen (13.1%) cell lines displayed a marked response to pevonedistat, featuring DNA re-replication, proliferative block, and increased apoptosis. Pevonedistat sensitivity did not statistically significantly correlate with microsatellite instability or mutations in KRAS or BRAF and was functionally associated with low EGFR pathway activity. While ineffective on predicted resistant PDXs, in vivo administration of pevonedistat statistically significantly impaired growth of five out of six predicted sensitive models (P < .01). In samples from CRC patients, transcriptional prediction of pevonedistat sensitivity was associated with poor prognosis after surgery (hazard ratio [HR] = 2.49, 95% confidence interval [CI] = 1.34 to 4.62, P = .003) and early progression under cetuximab treatment (HR = 3.59, 95% CI = 1.60 to 8.04, P < .001). Histological and immunohistochemical analyses revealed that the pevonedistat sensitivity signature captures transcriptional traits of poor differentiation and high-grade mucinous adenocarcinoma. Conclusions: These results highlight NEDD8-pathway inhibition by pevonedistat as a potentially effective treatment for poorly differentiated, clinically aggressive CRC.

Hop derived flavonoid xanthohumol inhibits endothelial cell functions via AMPK activation.

Angiogenesis, a process characterized by the formation of new blood vessels from pre-existing ones, is a crucial step in tumor growth and dissemination. Recently, increased attention has been addressed to the ability of flavonoids to prevent cancer by suppressing angiogenesis, strategy that we named "angioprevention". Several natural compounds exert their anti-tumor properties by activating 5' adenosine monophosphate-activated protein kinase (AMPK), a key regulator of metabolism in cancer cells. Drugs with angiopreventive activities, in particular metformin, regulate AMPK in endothelial cells. Here we investigated the involvement of AMPK in the anti-angiogenic effects of xanthohumol (XN), the major prenylated flavonoid of the hop plant, and mechanisms of action. The anti-angiogenic activity of XN was more potent than epigallocatechin-3-gallate (EGCG). Treatment of endothelial cells with XN led to increased AMPK phosphorylation and activity. Functional studies using biochemical approaches confirmed that AMPK mediates XN anti-angiogenic activity. AMPK activation by XN was mediated by CAMMKß, but not LKB1. Analysis of the downstream mechanisms showed that XN-induced AMPK activation reduced nitric oxide (NO) levels in endothelial cells by decreasing eNOS phosphorylation. Finally, AKT pathway was inactivated by XN as part of its anti-angiogenic activity, but independently from AMPK, suggesting that these two signaling pathways proceed autonomously. Our study dissects the molecular mechanism by which XN exerts its potent anti-angiogenic activity, pointing out AMPK as a crucial signal transducer.

Aspirin and atenolol enhance metformin activity against breast cancer by targeting both neoplastic and microenvironment cells.

Metformin can induce breast cancer (BC) cell apoptosis and reduce BC local and metastatic growth in preclinical models. Since Metformin is frequently used along with Aspirin or beta-blockers, we investigated the effect of Metformin, Aspirin and the beta-blocker Atenolol in several BC models. In vitro, Aspirin synergized with Metformin in inducing apoptosis of triple negative and endocrine-sensitive BC cells, and in activating AMPK in BC and in white adipose tissue (WAT) progenitors known to cooperate to BC progression. Both Aspirin and Atenolol added to the inhibitory effect of Metformin against complex I of the respiratory chain. In both immune-deficient and immune-competent preclinical models, Atenolol increased Metformin activity against angiogenesis, local and metastatic growth of HER2+ and triple negative BC. Aspirin increased the activity of Metformin only in immune-competent HER2+ BC models. Both Aspirin and Atenolol, when added to Metformin, significantly reduced the endothelial cell component of tumor vessels, whereas pericytes were reduced by the addition of Atenolol but not by the addition of Aspirin. Our data indicate that the addition of Aspirin or of Atenolol to Metformin might be beneficial for BC control, and that this activity is likely due to effects on both BC and microenvironment cells.

Preliminary Evidence on the Diagnostic and Molecular Role of Circulating Soluble EGFR in Non-Small Cell Lung Cancer.

Assessment of biological diagnostic factors providing clinically-relevant information to guide physician decision-making are still needed for diseases with poor outcomes, such as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a promising molecule in the clinical management of NSCLC. While the EGFR transmembrane form has been extensively investigated in large clinical trials, the soluble, circulating EGFR isoform (sEGFR), which may have a potential clinical use, has rarely been considered. This study investigates the use of sEGFR as a potential diagnostic biomarker for NSCLC and also characterizes the biological function of sEGFR to clarify the molecular mechanisms involved in the course of action of this protein. Plasma sEGFR levels from a heterogeneous cohort of 37 non-advanced NSCLC patients and 54 healthy subjects were analyzed by using an enzyme-linked immunosorbent assay. The biological function of sEGFR was analyzed in vitro using NSCLC cell lines, investigating effects on cell proliferation and migration. We found that plasma sEGFR was significantly decreased in the NSCLC patient group as compared to the control group (median value: 48.6 vs. 55.6 ng/mL respectively; p = 0.0002). Moreover, we demonstrated that sEGFR inhibits growth and migration of NSCLC cells in vitro through molecular mechanisms that included perturbation of EGF/EGFR cell signaling and holoreceptor internalization. These data show that sEGFR is a potential circulating biomarker with a physiological protective role, providing a first approach to the functional role of the soluble isoform of EGFR. However, the impact of these data on daily clinical practice needs to be further investigated in larger prospective studies.

Drink your prevention: beverages with cancer preventive phytochemicals.

Specific alimentary habits, including oriental and Mediterranean diets characterized by high consumption of vegetables, fruits, cereals and, for the Mediterranean diet, olive oil, are associated with a reduction of risk of cardiovascular diseases, type 2 diabetes, neurodegenerative diseases, and some cancers. Numerous beverages contain diverse natural compounds, termed phytochemicals, that have been reported to exert antitumor, antiangiogenic, and antioxidant properties. Here we review the chemopreventive and angiopreventive properties of selected phytochemicals found in common beverages: epigallocatechin(green tea), triterpenoids (citrus juices), resveratrol (red wine), xanthohumol (beer), procyanidin (chocolate), and caffeine (coffee), focusing on their molecular mechanisms, providing "ready to drink" prevention approaches.

Inflammatory angiogenesis and the tumor microenvironment as targets for cancer therapy and prevention.

In addition to aberrant transformed cells, tumors are tissues that contain host components, including stromal cells, vascular cells (ECs) and their precursors, and immune cells. All these constituents interact with each other at the cellular and molecular levels, resulting in the production of an intricate and heterogeneous complex of cells and matrix defined as the tumor microenvironment. Several pathways involved in these interactions have been investigated both in pathological and physiological scenarios, and diverse molecules are currently targets of chemotherapeutic and preventive drugs. Many phytochemicals and their derivatives show the ability to inhibit tumor progression, angiogenesis, and metastasis, exerting effects on the tumor microenvironment. In this review, we will outline the principal players and mechanisms involved in the tumor microenvironment network and we will discuss some interesting compounds aimed at interrupting these interactions and blocking tumor insurgence and progression. The considerations provided will be crucial for the design of new preventive approaches to the reduction in cancer risk that need to be applied to large populations composed of apparently healthy individuals.