GIPR expression is induced by thiazolidinediones in a PPARγ-independent manner and repressed by obesogenic stimuli.

Adipose tissue (AT) dysfunctions are associated with the onset of insulin resistance (IR) and type 2 diabetes mellitus (T2DM). Targeting glucose-dependent insulinotropic peptide receptor (GIPR) is a valid option to increase the efficacy of glucagon-like peptide 1 (GLP-1) receptor agonists in T2DM treatment. Nevertheless, the therapeutic potential of targeting the GIP/GIPR axis and its effect on the AT are controversial. In this work, we explored the expression and regulation of GIPR in precursor cells and mature adipocytes, investigating if and how obesogenic stimuli and thiazolidinediones perturb GIPR expression. Using publicly available gene expression datasets, we assessed that, among white adipose tissue (WAT) cells, adipocytes express lower levels of GIPR compared to cells of mesothelial origin, pericytes, dendritic and NK/T cells. However, we report that GIPR levels markedly increase during the in vitro differentiation of both murine and human adipocytes, from 3T3-L1 and human mesenchymal precursor cells (MSCs), respectively. Notably, we demonstrated that thiazolidinediones - ie. synthetic PPARγ agonists widely used as anti-diabetic drugs and contained in the adipogenic mix - markedly induce GIPR expression. Moreover, using multiple in vitro systems, we assessed that thiazolidinediones induce GIPR in a PPARγ-independent manner. Our results support the hypothesis that PPARγ synthetic agonists may be used to increase GIPR levels in AT, potentially affecting in turn the targeting of GIP system in patients with metabolic dysfunctions. Furthermore, we demonstrate in vitro and in vivo that proinflammatory stimuli, and especially the TNFα, represses GIPR both in human and murine adipocytes, even though discordant results were obtained between human and murine cellular systems for other cytokines. Finally, we demonstrated that GIPR is negatively affected also by the excessive lipid engulfment. Overall, we report that obesogenic stimuli - ie. pro-inflammatory cytokines and the increased lipid accumulation - and PPARγ synthetic ligands oppositely modulate GIPR expression, possibly influencing the effectiveness of GIP agonists.

Transcriptional Regulation and Its Misregulation in Human Diseases.

Transcriptional regulation is a critical biological process that allows the cell or an organism to respond to a variety of intra- and extracellular signals, to define cell identity during development, to maintain it throughout its lifetime, and to coordinate cellular activity [...].

Targeting metabolism by B-raf inhibitors and diclofenac restrains the viability of BRAF-mutated thyroid carcinomas with Hif-1α-mediated glycolytic phenotype.

BACKGROUND: B-raf inhibitors (BRAFi) are effective for BRAF-mutated papillary (PTC) and anaplastic (ATC) thyroid carcinomas, although acquired resistance impairs tumour cells' sensitivity and/or limits drug efficacy. Targeting metabolic vulnerabilities is emerging as powerful approach in cancer. METHODS: In silico analyses identified metabolic gene signatures and Hif-1α as glycolysis regulator in PTC. BRAF-mutated PTC, ATC and control thyroid cell lines were exposed to HIF1A siRNAs or chemical/drug treatments (CoCl2, EGF, HGF, BRAFi, MEKi and diclofenac). Genes/proteins expression, glucose uptake, lactate quantification and viability assays were used to investigate the metabolic vulnerability of BRAF-mutated cells. RESULTS: A specific metabolic gene signature was identified as a hallmark of BRAF-mutated tumours, which display a glycolytic phenotype, characterised by enhanced glucose uptake, lactate efflux and increased expression of Hif-1α-modulated glycolytic genes. Indeed, Hif-1α stabilisation counteracts the inhibitory effects of BRAFi on these genes and on cell viability. Interestingly, targeting metabolic routes with BRAFi and diclofenac combination we could restrain the glycolytic phenotype and synergistically reduce tumour cells' viability. CONCLUSION: The identification of a metabolic vulnerability of BRAF-mutated carcinomas and the capacity BRAFi and diclofenac combination to target metabolism open new therapeutic perspectives in maximising drug efficacy and reducing the onset of secondary resistance and drug-related toxicity.

Diabetic Retinopathy: Are lncRNAs New Molecular Players and Targets?

The growing incidence of diabetes mellitus worldwide implies the increasing prevalence of several related macro- (e.g., hypertension and atherosclerosis) and micro-vascular (e.g., nephropathy and retinopathy) complications. Notably, diabetic retinopathy (DR) is the leading cause of blindness in older diabetic patients and can occur with different degrees of severity. Chronic hyperglycemia is the main determinant of the functional damage of retinal cells. The oxidative stress, inflammatory factors and vascular endothelial growth factor signaling have been widely reported as contributors of DR onset and progression, and an emerging role has been described for different classes of non-coding RNA, including several long non-coding RNAs (lncRNAs). Here, we report the main results of all research articles (i.e., 150) listed on PubMed database from 2014 to 2022 regarding the putative role of lncRNAs in DR, including small nucleolar RNA host genes (SNHGs). Particularly, in this review we describe all lncRNAs and SNHGs with altered expression in DR and related contexts, discussing their association with DR outcomes, their mechanism of action related to DR, the molecular/functional effects, as well as the biological and experimental contexts. Thus, herein we provide an overview of the current state of knowledge regarding the putative involvement of 50 lncRNAs and SNHGs in the pathogenesis of DR, highlighting their potential as therapeutic targets or biomarkers for improving the clinical management of DR.

Hepatic Insulin Resistance in Hyperthyroid Rat Liver: Vitamin E Supplementation Highlights a Possible Role of ROS.

Thyroid hormones are normally involved in glycaemic control, but their excess can lead to altered glucose metabolism and insulin resistance (IR). Since hyperthyroidism-linked increase in ROS results in tissue oxidative stress that is considered a hallmark of conditions leading to IR, it is conceivable a role of ROS in the onset of IR in hyperthyroidism. To verify this hypothesis, we evaluated the effects of vitamin E on thyroid hormone-induced oxidative damage, insulin resistance, and on gene expression of key molecules involved in IR in the rat liver. The factors involved in oxidative damage, namely the total content of ROS, the mitochondrial production of ROS, the activity of antioxidant enzymes, the in vitro susceptibility to oxidative stress, have been correlated to insulin resistance indices, such as insulin activation of hepatic Akt and plasma level of glucose, insulin and HOMA index. Our results indicate that increased levels of oxidative damage ROS content and production and susceptibility to oxidative damage, parallel increased fasting plasma level of glucose and insulin, reduced activation of Akt and increased activation of JNK. This last result suggests a role for JNK in the insulin resistance induced by hyperthyroidism. Furthermore, the variation of the genes Pparg, Ppara, Cd36 and Slc2a2 could explain, at least in part, the observed metabolic phenotypes.

Integrated Network Pharmacology Approach for Drug Combination Discovery: A Multi-Cancer Case Study.

Despite remarkable efforts of computational and predictive pharmacology to improve therapeutic strategies for complex diseases, only in a few cases have the predictions been eventually employed in the clinics. One of the reasons behind this drawback is that current predictive approaches are based only on the integration of molecular perturbation of a certain disease with drug sensitivity signatures, neglecting intrinsic properties of the drugs. Here we integrate mechanistic and chemocentric approaches to drug repositioning by developing an innovative network pharmacology strategy. We developed a multilayer network-based computational framework integrating perturbational signatures of the disease as well as intrinsic characteristics of the drugs, such as their mechanism of action and chemical structure. We present five case studies carried out on public data from The Cancer Genome Atlas, including invasive breast cancer, colon adenocarcinoma, lung squamous cell carcinoma, hepatocellular carcinoma and prostate adenocarcinoma. Our results highlight paclitaxel as a suitable drug for combination therapy for many of the considered cancer types. In addition, several non-cancer-related genes representing unusual drug targets were identified as potential candidates for pharmacological treatment of cancer.

PPARγ and Diabetes: Beyond the Genome and Towards Personalized Medicine.

PURPOSE OF REVIEW: Full and partial synthetic agonists targeting the transcription factor PPARγ are contained in FDA-approved insulin-sensitizing drugs and used for the treatment of metabolic syndrome-related dysfunctions. Here, we discuss the association between PPARG genetic variants and drug efficacy, as well as the role of alternative splicing and post-translational modifications as contributors to the complexity of PPARγ signaling and to the effects of synthetic PPARγ ligands. RECENT FINDINGS: PPARγ regulates the transcription of several target genes governing adipocyte differentiation and glucose and lipid metabolism, as well as insulin sensitivity and inflammatory pathways. These pleiotropic functions confer great relevance to PPARγ in physiological regulation of whole-body metabolism, as well as in the etiology of metabolic disorders. Accordingly, PPARG gene mutations, nucleotide variations, and post-translational modifications have been associated with adipose tissue disorders and the related risk of insulin resistance and type 2 diabetes (T2D). Moreover, PPARγ alternative splicing isoforms-generating dominant-negative isoforms mainly expressed in human adipose tissue-have been related to impaired PPARγ activity and adipose tissue dysfunctions. Thus, multiple regulatory levels that contribute to PPARγ signaling complexity may account for the beneficial as well as adverse effects of PPARγ agonists. Further targeted analyses, taking into account all these aspects, are needed for better deciphering the role of PPARγ in human pathophysiology, especially in insulin resistance and T2D. The therapeutic potential of full and partial PPARγ synthetic agonists underlines the clinical significance of this nuclear receptor. PPARG mutations, polymorphisms, alternative splicing isoforms, and post-translational modifications may contribute to the pathogenesis of metabolic disorders, also influencing the responsiveness of pharmacological therapy. Therefore, in the context of the current evidence-based trend to personalized diabetes management, we highlight the need to decipher the intricate regulation of PPARγ signaling to pave the way to tailored therapies in patients with insulin resistance and T2D.

TNFα Mediates Inflammation-Induced Effects on PPARG Splicing in Adipose Tissue and Mesenchymal Precursor Cells.

Low-grade chronic inflammation and reduced differentiation capacity are hallmarks of hypertrophic adipose tissue (AT) and key contributors of insulin resistance. We identified PPARGΔ5 as a dominant-negative splicing isoform overexpressed in the AT of obese/diabetic patients able to impair adipocyte differentiation and PPARγ activity in hypertrophic adipocytes. Herein, we investigate the impact of macrophage-secreted pro-inflammatory factors on PPARG splicing, focusing on PPARGΔ5. We report that the epididymal AT of LPS-treated mice displays increased PpargΔ5/cPparg ratio and reduced expression of Pparg-regulated genes. Interestingly, pro-inflammatory factors secreted from murine and human pro-inflammatory macrophages enhance the PPARGΔ5/cPPARG ratio in exposed adipogenic precursors. TNFα is identified herein as factor able to alter PPARG splicing-increasing PPARGΔ5/cPPARG ratio-through PI3K/Akt signaling and SRp40 splicing factor. In line with in vitro data, TNFA expression is higher in the SAT of obese (vs. lean) patients and positively correlates with PPARGΔ5 levels. In conclusion, our results indicate that inflammatory factors secreted by metabolically-activated macrophages are potent stimuli that modulate the expression and splicing of PPARG. The resulting imbalance between canonical and dominant negative isoforms may crucially contribute to impair PPARγ activity in hypertrophic AT, exacerbating the defective adipogenic capacity of precursor cells.

In-Vitro-Generated Hypertrophic-Like Adipocytes Displaying PPARG Isoforms Unbalance Recapitulate Adipocyte Dysfunctions In Vivo.

Reduced neo-adipogenesis and dysfunctional lipid-overloaded adipocytes are hallmarks of hypertrophic obesity linked to insulin resistance. Identifying molecular features of hypertrophic adipocytes requires appropriate in vitro models. We describe the generation of a model of human hypertrophic-like adipocytes directly comparable to normal adipose cells and the pathologic evolution toward hypertrophic state. We generate in vitro hypertrophic cells from mature adipocytes, differentiated from human mesenchymal stem cells. Combining optical, confocal, and transmission electron microscopy with mRNA/protein quantification, we characterize this cellular model, confirming specific alterations also in subcutaneous adipose tissue. Specifically, we report the generation and morphological/molecular characterization of human normal and hypertrophic-like adipocytes. The latter displays altered morphology and unbalance between canonical and dominant negative (PPARGΔ5) transcripts of PPARG, paralleled by reduced expression of PPARγ targets, including GLUT4. Furthermore, the unbalance of PPARγ isoforms associates with GLUT4 down-regulation in subcutaneous adipose tissue of individuals with overweight/obesity or impaired glucose tolerance/type 2 diabetes, but not with normal weight or glucose tolerance. In conclusion, the hypertrophic-like cells described herein are an innovative tool for studying molecular dysfunctions in hypertrophic obesity and the unbalance between PPARγ isoforms associates with down-regulation of GLUT4 and other PPARγ targets, representing a new hallmark of hypertrophic adipocytes.

Oncogenic Properties of the Antisense lncRNA COMET in BRAF- and RET-Driven Papillary Thyroid Carcinomas.

RET rearrangements as well as BRAF and RAS mutations drive differential pathway activation in papillary thyroid carcinomas, leading to different tumor phenotypes and prognoses. Although The Cancer Genome Atlas Consortium has identified tumor subgroups based on protein-coding gene signatures, neither expression of long noncoding RNAs (lncRNA) nor their correlation with specific tumor-driving mutations and rearrangements have been systematically assessed. Here, we reanalyzed our RNA-sequencing data using a de novo discovery approach to identify lncRNAs and define tumor subtype-specific signatures of annotated lncRNAs. Among them, we identified COMET (Correlated-to-MET), a natural antisense transcript that was highly expressed in carcinomas harboring BRAF V600E mutation or RET gene rearrangements (i.e., BRAF-like tumors) and induced the downstream MAPK pathway. In papillary thyroid carcinomas, COMET was part of a coexpression network including different oncogenes belonging to the MAPK pathway, and its expression highly correlated with MET expression. Depletion of COMET resulted in reduced expression of genes within this network, including the MET oncogene. COMET repression inhibited viability and proliferation of tumor cells harboring BRAF V600E somatic mutation or RET oncogene rearrangement and dramatically reduced motility and invasiveness of tumor cells. Moreover, silencing COMET markedly increased sensitivity to vemurafenib, a common inhibitor of mutated B-raf. Collectively, our results suggest COMET as a new target to improve drug-based cancer therapies, especially in BRAF-mutated and MET-addicted papillary thyroid carcinomas. SIGNIFICANCE: These results highlight the oncogenic role of lncRNA COMET in thyroid and indicate it as a potential new target to overcome vemurafenib resistance in BRAF-mutated and MET-addicted carcinomas.

Transcriptional Regulation: Molecules, Involved Mechanisms, and Misregulation.

Transcriptional regulation is a critical biological process that allows the cell or an organism to respond to a variety of intra- and extra-cellular signals, to define cell identity during development, to maintain it throughout its lifetime, and to coordinate cellular activity [...].

PPARγΔ5, a Naturally Occurring Dominant-Negative Splice Isoform, Impairs PPARγ Function and Adipocyte Differentiation.

Peroxisome-proliferator-activated receptor γ (PPARγ) regulates glucose and lipid homeostasis, insulin signaling, and adipocyte differentiation. Here, we report the skipping of exon 5 as a legitimate splicing event generating PPARγΔ5, a previously unidentified naturally occurring truncated isoform of PPARγ, which lacks the entire ligand-binding domain. PPARγΔ5 is endogenously expressed in human adipose tissue and, during adipocyte differentiation, lacks ligand-dependent transactivation ability and acts as a dominant-negative isoform reducing PPARγ activity. Ligand-mediated PPARγ activation induces exon 5 skipping in a negative feedback loop, suggesting alternative splicing as a mechanism regulating PPARγ activity. PPARγΔ5 overexpression modifies the PPARγ-induced transcriptional network, significantly impairing the differentiation ability of adipocyte precursor cells. Additionally, PPARγΔ5 expression in subcutaneous adipose tissue positively correlates with BMI in two independent cohorts of overweight or obese and type 2 diabetic patients. From a functional perspective, PPARγΔ5 mimics PPARG dominant-negative mutated receptors, possibly contributing to adipose tissue dysfunction. These findings open an unexplored scenario in PPARG regulation and PPARγ-related diseases.

PR/SET Domain Family and Cancer: Novel Insights from the Cancer Genome Atlas.

The PR/SET domain gene family (PRDM) encodes 19 different transcription factors that share a subtype of the SET domain [Su(var)3-9, enhancer-of-zeste and trithorax] known as the PRDF1-RIZ (PR) homology domain. This domain, with its potential methyltransferase activity, is followed by a variable number of zinc-finger motifs, which likely mediate protein⁻protein, protein⁻RNA, or protein⁻DNA interactions. Intriguingly, almost all PRDM family members express different isoforms, which likely play opposite roles in oncogenesis. Remarkably, several studies have described alterations in most of the family members in malignancies. Here, to obtain a pan-cancer overview of the genomic and transcriptomic alterations of PRDM genes, we reanalyzed the Exome- and RNA-Seq public datasets available at The Cancer Genome Atlas portal. Overall, PRDM2, PRDM3/MECOM, PRDM9, PRDM16 and ZFPM2/FOG2 were the most mutated genes with pan-cancer frequencies of protein-affecting mutations higher than 1%. Moreover, we observed heterogeneity in the mutation frequencies of these genes across tumors, with cancer types also reaching a value of about 20% of mutated samples for a specific PRDM gene. Of note, ZFPM1/FOG1 mutations occurred in 50% of adrenocortical carcinoma patients and were localized in a hotspot region. These findings, together with OncodriveCLUST results, suggest it could be putatively considered a cancer driver gene in this malignancy. Finally, transcriptome analysis from RNA-Seq data of paired samples revealed that transcription of PRDMs was significantly altered in several tumors. Specifically, PRDM12 and PRDM13 were largely overexpressed in many cancers whereas PRDM16 and ZFPM2/FOG2 were often downregulated. Some of these findings were also confirmed by real-time-PCR on primary tumors.

RBPMetaDB: a comprehensive annotation of mouse RNA-Seq datasets with perturbations of RNA-binding proteins.

RNA-binding proteins (RBPs) may play a critical role in gene regulation in various diseases or biological processes by controlling post-transcriptional events such as polyadenylation, splicing and mRNA stabilization via binding activities to RNA molecules. Owing to the importance of RBPs in gene regulation, a great number of studies have been conducted, resulting in a large amount of RNA-Seq datasets. However, these datasets usually do not have structured organization of metadata, which limits their potentially wide use. To bridge this gap, the metadata of a comprehensive set of publicly available mouse RNA-Seq datasets with perturbed RBPs were collected and integrated into a database called RBPMetaDB. This database contains 292 mouse RNA-Seq datasets for a comprehensive list of 187 RBPs. These RBPs account for only ∼10% of all known RBPs annotated in Gene Ontology, indicating that most are still unexplored using high-throughput sequencing. This negative information provides a great pool of candidate RBPs for biologists to conduct future experimental studies. In addition, we found that DNA-binding activities are significantly enriched among RBPs in RBPMetaDB, suggesting that prior studies of these DNA- and RNA-binding factors focus more on DNA-binding activities instead of RNA-binding activities. This result reveals the opportunity to efficiently reuse these data for investigation of the roles of their RNA-binding activities. A web application has also been implemented to enable easy access and wide use of RBPMetaDB. It is expected that RBPMetaDB will be a great resource for improving understanding of the biological roles of RBPs.Database URL: http://rbpmetadb.yubiolab.org.

SFMetaDB: a comprehensive annotation of mouse RNA splicing factor RNA-Seq datasets.

Although the number of RNA-Seq datasets deposited publicly has increased over the past few years, incomplete annotation of the associated metadata limits their potential use. Because of the importance of RNA splicing in diseases and biological processes, we constructed a database called SFMetaDB by curating datasets related with RNA splicing factors. Our effort focused on the RNA-Seq datasets in which splicing factors were knocked-down, knocked-out or over-expressed, leading to 75 datasets corresponding to 56 splicing factors. These datasets can be used in differential alternative splicing analysis for the identification of the potential targets of these splicing factors and other functional studies. Surprisingly, only ∼15% of all the splicing factors have been studied by loss- or gain-of-function experiments using RNA-Seq. In particular, splicing factors with domains from a few dominant Pfam domain families have not been studied. This suggests a significant gap that needs to be addressed to fully elucidate the splicing regulatory landscape. Indeed, there are already mouse models available for ∼20 of the unstudied splicing factors, and it can be a fruitful research direction to study these splicing factors in vitro and in vivo using RNA-Seq. Database URL:http://sfmetadb.ece.tamu.edu/

Transcriptome Profiling in Human Diseases: New Advances and Perspectives.

In the last decades, transcriptome profiling has been one of the most utilized approaches to investigate human diseases at the molecular level. Through expression studies, many molecular biomarkers and therapeutic targets have been found for several human pathologies. This number is continuously increasing thanks to total RNA sequencing. Indeed, this new technology has completely revolutionized transcriptome analysis allowing the quantification of gene expression levels and allele-specific expression in a single experiment, as well as to identify novel genes, splice isoforms, fusion transcripts, and to investigate the world of non-coding RNA at an unprecedented level. RNA sequencing has also been employed in important projects, like ENCODE (Encyclopedia of the regulatory elements) and TCGA (The Cancer Genome Atlas), to provide a snapshot of the transcriptome of dozens of cell lines and thousands of primary tumor specimens. Moreover, these studies have also paved the way to the development of data integration approaches in order to facilitate management and analysis of data and to identify novel disease markers and molecular targets to use in the clinics. In this scenario, several ongoing clinical trials utilize transcriptome profiling through RNA sequencing strategies as an important instrument in the diagnosis of numerous human pathologies.

Pan-Cancer Mutational and Transcriptional Analysis of the Integrator Complex.

The integrator complex has been recently identified as a key regulator of RNA Polymerase II-mediated transcription, with many functions including the processing of small nuclear RNAs, the pause-release and elongation of polymerase during the transcription of protein coding genes, and the biogenesis of enhancer derived transcripts. Moreover, some of its components also play a role in genome maintenance. Thus, it is reasonable to hypothesize that their functional impairment or altered expression can contribute to malignancies. Indeed, several studies have described the mutations or transcriptional alteration of some Integrator genes in different cancers. Here, to draw a comprehensive pan-cancer picture of the genomic and transcriptomic alterations for the members of the complex, we reanalyzed public data from The Cancer Genome Atlas. Somatic mutations affecting Integrator subunit genes and their transcriptional profiles have been investigated in about 11,000 patients and 31 tumor types. A general heterogeneity in the mutation frequencies was observed, mostly depending on tumor type. Despite the fact that we could not establish them as cancer drivers, INTS7 and INTS8 genes were highly mutated in specific cancers. A transcriptome analysis of paired (normal and tumor) samples revealed that the transcription of INTS7, INTS8, and INTS13 is significantly altered in several cancers. Experimental validation performed on primary tumors confirmed these findings.

Heart failure: Pilot transcriptomic analysis of cardiac tissue by RNA-sequencing.

BACKGROUND: Despite left ventricular (LV) dysfunction contributing to mortality in chronic heart failure (HF), the molecular mechanisms of LV failure continues to remain poorly understood and myocardial biomarkers have yet to be identified. The aim of this pilot study was to investigate specific transcriptome changes occurring in cardiac tissues of patients with HF compared to healthy condition patients to improve diagnosis and possible treatment of affected subjects. METHODS: Unlike other studies, only dilated cardiomyopathy (DCM) (n = 2) and restrictive cardiomyopathy (RCM) (n = 2) patients who did not report family history of the disease were selected with the aim of obtaining a homogeneous population for the study. The transcriptome of all patients were studied by RNA-sequencing (RNA-Seq) and the read counts were adequately filtered and normalized using a recently developed user-friendly tool for RNA-Seq data analysis, based on a new graphical user interface (RNA-SeqGUI). RESULTS: By using this approach in a pairwise comparison with healthy donors, we were able to identify DCM- and RCM-specific expression signatures for protein-coding genes as well as for long noncoding RNAs (lncRNAs). Differential expression of 5 genes encoding different members of the mediator complex was disclosed in this analysis. Interestingly, a significant alteration was found for genes which had never been associated with HF until now, and 27 lncRNA/mRNA pairs that were significantly altered in HF patients. CONCLUSIONS: The present findings revealed specific expression pattern of both protein-coding and lncRNAs in HF patients, confirming that new LV myocardial biomarkers could be reliably identified using Next-Generation Sequencing-based approaches.

Glucose impairs tamoxifen responsiveness modulating connective tissue growth factor in breast cancer cells.

Type 2 diabetes and obesity are negative prognostic factors in patients with breast cancer (BC). We found that sensitivity to tamoxifen was reduced by 2-fold by 25 mM glucose (High Glucose; HG) compared to 5.5 mM glucose (Low Glucose; LG) in MCF7 BC cells. Shifting from HG to LG ameliorated MCF7 cell responsiveness to tamoxifen. RNA-Sequencing of MCF7 BC cells revealed that cell cycle-related genes were mainly affected by glucose. Connective Tissue Growth Factor (CTGF) was identified as a glucose-induced modulator of cell sensitivity to tamoxifen. Co-culturing MCF7 cells with human adipocytes exposed to HG, enhanced CTGF mRNA levels and reduced tamoxifen responsiveness of BC cells. Inhibition of adipocyte-released IL8 reverted these effects. Interestingly, CTGF immuno-detection in bioptic specimens from women with estrogen receptor positive (ER+) BC correlated with hormone therapy resistance, distant metastases, reduced overall and disease-free survival. Thus, glucose affects tamoxifen responsiveness directly modulating CTGF in BC cells, and indirectly promoting IL8 release by adipocytes.

E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells.

BACKGROUND: The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. RESULTS: The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. CONCLUSIONS: The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.