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Background The kidney is the main organ in the pathophysiology of essential hypertension. Although most bicarbonate reabsorption occurs in the proximal tubule, the medullary thick ascending limb (mTAL) of the nephron also maintains acid–base balance by contributing to 25% of bicarbonate reabsorption. A crucial element in this regulation is the sodium-hydrogen exchanger 1 (NHE1), a ubiquitous membrane protein controlling intracellular pH, where proton extrusion is driven by the inward sodium flux. MicroRNA (miRNA) expression of hypertensive patients significantly differs from that of normotensive subjects. The aim of this study was to determine the functional role of miRNA alterations at the mTAL level. Methods By miRNA microarray analysis, we identified miRNA expression profiles in isolated mTALs from high sodium intake–induced hypertensive rats (HSD) versus their normotensive counterparts (NSD). In vitro validation was carried out in rat mTAL cells. Results Five miRNAs involved in the onset of salt-sensitive hypertension were identified, including miR-23a, which was bioinformatically predicted to target NHE1 mRNA. Data demonstrated that miRNA-23a is downregulated in the mTAL of HSD rats while NHE1 is upregulated. Consistently, transfection of an miRNA-23a mimic in an mTAL cell line, using a viral vector, resulted in NHE1 downregulation. Conclusion NHE1, a protein involved in sodium reabsorption at the mTAL level and blood pressure regulation, is upregulated in our model. This was due to a downregulation of miRNA-23a. Expression levels of this miRNA are influenced by high sodium intake in the mTALs of rats. The downregulation of miRNA-23a in humans affected by essential hypertension corroborate our data and point to the potential role of miRNA-23a in the regulation of mTAL function following high salt intake.

Early life exposure to Endocrine Disruptor Chemicals (EDCs), such as the organophosphate pesticide Chlorpyrifos (CPF), affects the thyroid activity and dependent process, including the glucose metabolism. The damage of thyroid hormones (THs) as a mechanism of action of CPF is underestimated because the studies rarely consider that TH levels and signaling are customized peripherally. Here, we investigated the impairment of metabolism/signaling of THs and lipid/glucose metabolism in the livers of 6-month-old mice, developmentally and lifelong exposed to 0.1, 1, and 10 mg/kg/die CPF (F1) and their offspring similarly exposed (F2), analyzing the levels of transcripts of the enzymes involved in the metabolism of T3 (Dio1), lipids (Fasn, Acc1), and glucose (G6pase, Pck1). Both processes were altered only in F2 males, affected by hypothyroidism and by a systemic hyperglycemia linked to the activation of gluconeogenesis in mice exposed to 1 and 10 mg/kg/die CPF. Interestingly, we observed an increase in active FOXO1 protein due to a decrease in AKT phosphorylation, despite insulin signaling activation. Experiments in vitro revealed that chronic exposure to CPF affected glucose metabolism via the direct modulation of FOXO1 activity and T3 levels in hepatic cells. In conclusion, we described different sex and intergenerational effects of CPF exposure on the hepatic homeostasis of THs, their signaling, and, finally, glucose metabolism. The data points to FOXO1-T3-glucose signaling as a target of CPF in liver.

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer–mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy. Mol Cancer Ther; 13(3); 752–62. ©2013 AACR.

PDF file - 470K, Figure S1. UN1 mAb did not affect the proliferation rate, cell cycle, viability and apoptosis of HPB-ALL cells. Figure S2. The 2/165 phage clone and synthetic peptides do not inhibit the binding of mAb W6/32 to HPB-ALL cells. Supplementary Figure S3. Competition of the UN1 mAb-binding to UN1-positive breast and gastric cancer tissues by the 2/165 phage and G23 peptide. Figure S4. Characterization of affinity-purified antibodies from sera of 2/165 phagotopeimmunized mice.

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STING is a transmembrane ER resident protein that was initially described as a regulator of innate immune response triggered by viral DNA and later found to be involved in a broader range of immune processes. Here, we assessed its role in the antigen presentation by generating a STING KO macrophage cell line. In the absence of STING, we observed an impaired OVA-derived SIINFEKL peptide presentation together with a decreased level of MHC-I complex on the plasma membrane, likely due to a decreased mRNA expression of β2 m light chain as no relevant alterations of the peptide-loading complex (TAPs) were found. Moreover, JAK-STAT signaling resulted in impaired STING KO cells following OVA and LPS treatments, suggesting a dampened activation of immune response. Our data revealed a new molecular role of STING in immune mechanisms that could elucidate its role in the pathogenesis of autoimmune disorders and cancer.

Although the imbalance of circulating levels of Thyroid Hormones (THs) affects female fertility in vertebrates, its involvement in the promotion of Premature Ovarian Aging (POA) is debated. Therefore, altered synthesis of THs in both thyroid and ovary can be a trait of POA. We investigated the relationship between abnormal TH signaling, dysthyroidism, and POA in evolutionary distant vertebrates: from zebrafish to humans. Ovarian T3 signaling/metabolism was evaluated by measuring T3 levels, T3 responsive transcript, and protein levels along with transcripts governing T3 availability (deiodinases) and signaling (TH receptors) in distinct models of POA depending on genetic background and environmental exposures (e.g., diets, pesticides). Expression levels of well-known (Amh, Gdf9, and Inhibins) and novel (miR143/145 and Gas5) biomarkers of POA were assessed. Ovarian dysthyroidism was slightly influenced by genetics since very few differences were found between C57BL/6J and FVB/NJ females. However, diets exacerbated it in a strain-dependent manner. Similar findings were observed in zebrafish and mouse models of POA induced by developmental and long-life exposure to low-dose chlorpyrifos (CPF). Lastly, the T3 decrease in follicular fluids from women affected by diminished ovarian reserve, as well as of the transcripts modulating T3 signaling/availability in the cumulus cells, confirmed ovarian dysthyroidism as a common and evolutionary conserved trait of POA.

Thyroid hormones (THs) regulate many biological processes in vertebrates, including reproduction. Testicular somatic and germ cells are equipped with the arrays of enzymes (deiodinases), transporters, and receptors necessary to locally maintain the optimal level of THs and their signalling, needed for their functions and spermatogenesis. Pesticides, as chlorpyrifos (CPF) and ethylene thiourea (ETU), impair the function of thyroid and testis, affecting male fertility. However, their ability to disarrange testicular T3 (t-T3) metabolism and signalling is poorly considered. Here, a multi-species analysis involving zebrafish and mouse suggests the damage of t-T3 metabolism and signalling as a mechanism of gonadic toxicity of low-doses CPF and ETU. Indeed, the developmental exposure to both compounds reduces Dio2 transcript in both models, as well as in ex-vivo cultures of murine seminiferous tubules, and it is linked to alteration of steroidogenesis and germ cell differentiation. A major impact on spermatogonia was confirmed molecularly by the expression of their markers and morphologically evidenced in zebrafish. The results reveal that in the adopted models, exposure to both pesticides alters the t-T3 metabolism and signalling, affecting the reproductive capability. Our data, together with previous reports suggest zebrafish as an evaluable model in assessing the action of compounds impairing locally T3 signalling.

Thyroid hormone levels are usually genetically determined. Thyrocytes produce a unique set of enzymes that are dedicated to thyroid hormone synthesis. While thyroid transcriptional regulation is well-characterized, post-transcriptional mechanisms have been less investigated. Here, we describe the involvement of ZFP36L2, a protein that stimulates degradation of target mRNAs, in thyroid development and function, by in vivo and in vitro gene targeting in thyrocytes. Thyroid-specific Zfp36l2-/- females were hypothyroid, with reduced levels of circulating free Thyroxine (cfT4) and Triiodothyronine (cfT3). Their hypothyroidism was due to dyshormonogenesis, already evident one week after weaning, while thyroid development appeared normal. We observed decreases in several thyroid-specific transcripts and proteins, such as Nis and its transcriptional regulators (Pax8 and Nkx2.1), and increased apoptosis in Zfp36l2-/- thyroids. Nis, Pax8, and Nkx2.1 mRNAs were also reduced in Zfp36l2 knock-out thyrocytes in vitro (L2KO), in which we confirmed the increased apoptosis. Finally, in L2KO cells, we showed an altered response to TSH stimulation regarding both thyroid-specific gene expression and cell proliferation and survival. This result was supported by increases in P21/WAF1 and p-P38MAPK levels. Mechanistically, we confirmed Notch1 as a target of ZFP36L2 in the thyroid since its levels were increased in both in vitro and in vivo models. In both models, the levels of Id4 mRNA, a potential inhibitor of Pax8 activity, were increased. Overall, the data indicate that the regulation of mRNA stability by ZFP36L2 is a mechanism that controls the function and survival of thyrocytes.

Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs.

Background: HER2-based retargeted viruses are in advanced phases of preclinical development of breast cancer models. Mesothelin (MSLN) is a cell-surface tumor antigen expressed in different subtypes of breast and non-breast cancer. Its recent identification as a marker of some triple-negative breast tumors renders it an attractive target, presently investigated in clinical trials employing antibody drug conjugates and CAR-T cells. The availability of MSLN-retargeted oncolytic viruses may complement the current immunotherapeutic panel of biological drugs against HER2-negative breast and non-breast tumors. Methods: A fully virulent, tumor-targeted oncolytic Herpes simplex virus-1 (MSLN-THV) with a selectivity for mesothelin-expressing cancer cells was generated. Recombineering technology was used to replace an essential moiety of the viral glycoprotein D with antibody fragments derived from clinically validated MSLN monoclonal antibodies, and to allow IL12 cargo expression in infected cells. Panels of breast and female reproductive system cell lines were used to verify the oncolytic potential of the viral constructs. A platform for production of the retargeted viruses was developed in HEK 293 cells, providing stable expression of a suitable chimeric receptor. Results: We demonstrated the selectivity of viral infection and cytotoxicity by MSLN-retargeted viruses in a panel of mesothelin-positive cancer cells, originating from breast and female reproductive system tumors. We also developed a second-generation oncolytic MSLN-THV, encoding IL12, to enhance the immunotherapeutic potential of the viral backbone. A non-tumor cell line expressing a chimeric MSLN/Nectin-1 receptor, de-sensitized from antiviral responses by genetic inactivation of the Stimulator of Interferon Genes (STING)-dependent pathway was engineered, to optimize viral yields. Conclusions: Our proof-of-concept study proposes MSLN-retargeted herpesviruses as potential cancer immunotherapeutics for assessments in preclinical models of MSLN-positive tumors, complementing the available panel of oncolytic viruses to HER2-negative breast tumors.

Simple Summary Oncolytic viruses are emerging immunotherapeutics in cancer treatments. The conflicting role of innate immunity in the antitumor activity of oncolytic viruses is still a matter of debate. The STING-dependent DNA sensing axis is considered detrimental for viral replication and cancer cell clearance. Accordingly, we observed that STING loss in tumor cells was associated with improved lytic potential by a herpes-based oncolytic virus. However, STING-knockout cancer cells infected with the oncolytic virus showed impaired immunogenicity, as immunogenic cell death was improperly triggered. In agreement with these observations, STING-knockout tumors raised in a murine syngeneic model were more resistant to a combined treatment of the oncolytic virus with PD-1 blockade. The present study demonstrates the antitumor benefit of antiviral immunity and sheds lights on the mechanisms of immune resistance to oncovirotherapy exerted by STING-loss in tumor cells. Abstract The dichotomic contribution of cancer cell lysis and tumor immunogenicity is considered essential for effective oncovirotherapy, suggesting that the innate antiviral immune response is a hurdle for efficacy of oncolytic viruses. However, emerging evidence is resizing this view. By sensing cytosolic DNA, the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) axis can both counteract viral spread and contribute to the elicitation of adaptive immunity via type I interferon responses. In this paper, we analyzed the tumor-resident function of Sting-mediated DNA sensing in a combined approach of oncovirotherapy and PD-1 immune checkpoint blockade, in an immunocompetent murine model. While supporting increased lytic potential by oncolytic HER2-retargeted HSV-1 in vitro and in vivo, Sting-knockout tumors showed molecular signatures of an immunosuppressive tumor microenvironment. These signatures were correspondingly associated with ineffectiveness of the combination therapy in a model of established tumors. Results suggest that the impairment in antiviral response of Sting-knockout tumors, while favoring viral replication, is not able to elicit an adequate immunotherapeutic effect, due to lack of immunogenic cell death and the inability of Sting-knockout cancer cells to promote anti-tumor adaptive immune responses. Accordingly, we propose that antiviral, tumor-resident Sting provides fundamental contributions to immunotherapeutic efficacy of oncolytic viruses.

The intra-tissue levels of thyroid hormones (THs) regulate organ functions. Environmental factors can impair these levels by damaging the thyroid gland and/or peripheral TH metabolism. We investigated the effects of embryonic and/or long-life exposure to low-dose pesticides, ethylene thiourea (ETU), chlorpyrifos (CPF) and both combined on intra-tissue T4/T3 metabolism/signaling in zebrafish at different life stages. Hypothyroidism was evident in exposed larvae that showed reduced number of follicles and induced tshb mRNAs. Despite that, we found an increase in free T4 (fT4) and free T3 (fT3) levels/signaling that was confirmed by transcriptional regulation of TH metabolic enzymes (deiodinases) and T3-regulated mRNAs (cpt1, igfbp1a). Second-generation larvae showed that thyroid and TH signaling was affected even when not directly exposed, suggesting the role of parental exposure. In adult zebrafish, we found that sex-dependent damage of hepatic T3 level/signaling was associated with liver steatosis, which was more pronounced in females, with sex-dependent alteration of transcripts codifying the key enzymes involved in ‘de novo lipogenesis’ and β-oxidation. We found impaired activation of liver T3 and PPARα/Foxo3a pathways whose deregulation was already involved in mammalian liver steatosis. The data emphasizes that the intra-tissue imbalance of the T3 level is due to thyroid endocrine disruptors (THDC) and suggests that the effect of a slight modification in T3 signaling might be amplified by its direct regulation or crosstalk with PPARα/Foxo3a pathways. Because T3 levels define the hypothyroid/hyperthyroid status of each organ, our findings might explain the pleiotropic and site-dependent effects of pesticides.

Thyroid hormones (THs) exert pleiotropic effects in different mammalian organs, including gonads. Genetic and non-genetic factors, such as ageing and environmental stressors (e.g., low-iodine intake, exposure to endocrine disruptors, etc.), can alter T4/T3 synthesis by the thyroid. In any case, peripheral T3, controlled by tissue-specific enzymes (deiodinases), receptors and transporters, ensures organ homeostasis. Conflicting reports suggest that both hypothyroidism and hyperthyroidism, assessed by mean of circulating T4, T3 and Thyroid-Stimulating Hormone (TSH), could affect the functionality of the ovarian reserve determining infertility. The relationship between ovarian T3 level and functional ovarian reserve (FOR) is poorly understood despite that the modifications of local T3 metabolism and signalling have been associated with dysfunctions of several organs. Here, we will summarize the current knowledge on the role of TH signalling and its crosstalk with other pathways in controlling the physiological and premature ovarian ageing and, finally, in preserving FOR. We will consider separately the reports describing the effects of circulating and local THs on the ovarian health to elucidate their role in ovarian dysfunctions.

T-cell development in the thymus is a complex and highly regulated process, involving a wide variety of cells and molecules which orchestrate thymocyte maturation into either CD4+ or CD8+ single-positive (SP) T cells. Here, we briefly review the process regulating T-cell differentiation, which includes the latest advances in this field. In particular, we highlight how, starting from a pool of hematopoietic stem cells in the bone marrow, the sequential action of transcriptional factors and cytokines dictates the proliferation, restriction of lineage potential, T-cell antigen receptors (TCR) gene rearrangements, and selection events on the T-cell progenitors, ultimately leading to the generation of mature T cells. Moreover, this review discusses paradigmatic examples of viral infections affecting the thymus that, by inducing functional changes within this lymphoid gland, consequently influence the behavior of peripheral mature T-lymphocytes.

The antigen-mediated triggering of B cell receptor (BCR) activates the transcription factor NF-κB that regulates the expression of genes involved in B cell differentiation, proliferation, and survival. The tyrosine kinase Btk is essentially required for the activation of NF-κB in BCR signaling through the canonical pathway of IKK-dependent phosphorylation and proteasomal degradation of IκB-α, the main repressor of NF-κB. Here, we provide the evidence of an additional mechanism of NF-κB activation in BCR signaling that is Btk-dependent and IKK-independent. In DeFew B lymphoma cells, the anti-IgM stimulation of BCR activated Btk and NF-κB p50/p65 within 0.5 min in absence of IKK activation and IκB-α degradation. IKK silencing did not affect the rapid activation of NF-κB. Within this short time, Btk associated and phosphorylated IκB-α at Y289 and Y305, and, concomitantly, p65 translocated from cytosol to nucleus. The mutant IκB-α Y289/305A inhibited the NF-κB activation after BCR triggering, suggesting that the phosphorylation of IκB-α at tyrosines 289 and 305 was required for NF-κB activation. In primary chronic lymphocytic leukemia cells, Btk was constitutively active and associated with IκB-α, which correlated with Y305-phosphorylation of IκB-α and increased NF-κB activity compared with healthy B cells. Altogether, these results describe a novel mechanism of NF-κB activation in BCR signaling that could be relevant for Btk-targeted therapy in B-lymphoproliferative disorders. KEY MESSAGES: Anti-IgM stimulation of BCR activates NF-κB p50/p65 within 30 s by a Btk-dependent and IKK-independent mechanism. Btk associates and phosphorylates IκB-α at Y289 and Y305, promoting NF-κB activation. In primary CLLs, the binding of Btk to IκB-α correlates with tyrosine phosphorylation of IκB-α and increased NF-κB activity.

Acute administration of a high level of extracellular citrate displays an anti-proliferative effect on both in vitro and in vivo models. However, the long-term effect of citrate treatment has not been investigated yet. Here, we address this question in PC3 cells, a prostate-cancer-derived cell line. Acute administration of high levels of extracellular citrate impaired cell adhesion and inhibited the proliferation of PC3 cells, but surviving cells adapted to grow in the chronic presence of 20 mM citrate. Citrate-resistant PC3 cells are significantly less glycolytic than control cells. Moreover, they overexpress short-form, citrate-insensitive phosphofructokinase 1 (PFK1) together with full-length PFK1. In addition, they show traits of mesenchymal-epithelial transition: an increase in E-cadherin and a decrease in vimentin. In comparison with PC3 cells, citrate-resistant cells display morphological changes that involve both microtubule and microfilament organization. This was accompanied by changes in homeostasis and the organization of intracellular organelles. Thus, the mitochondrial network appears fragmented, the Golgi complex is scattered, and the lysosomal compartment is enlarged. Interestingly, citrate-resistant cells produce less total ROS but accumulate more mitochondrial ROS than control cells. Consistently, in citrate-resistant cells, the autophagic pathway is upregulated, possibly sustaining their survival. In conclusion, chronic administration of citrate might select resistant cells, which could jeopardize the benefits of citrate anticancer treatment.

Epidemiological and experimental studies emphasize the link between environmental chemicals exposure and thyroid cancer. However, this association is strongly debated and the mechanisms of action of environmental thyroid carcinogens still need to be identified. The analysis of in vitro transcriptomic data developed to investigate the effects of chlorpyrifos on immortalized thyrocytes highlighted the impaired expression of genes involved in endodermal carcinogenesis. This endodermal carcinogenic gene-network (ECGN, including Zfp36l2, Dmbt1, Ddit4), was validated in cellular and mouse models of thyroid carcinogenesis, characterized by the constitutive activation of the mitogen-activated protein kinase (MAPK) pathway and in immortalized thyrocytes exposed to tetrachlorodibenzo-p-dioxin (TCDD) and chlorpyrifos (CPF). The mRNA levels of Zfp36l2, Dmbt1 and Ddit4 were increased in models characterized by MAPK activation or following TCDD exposure, whereas they were inhibited by CPF exposure. Overall, the ECGN transcripts identify a novel gene-regulatory network associated with thyroid carcinogenesis promoted by genetic mutation or by environmental carcinogens. The latter have opposite effects on the modulation of the ECGN transcripts according to their mechanisms of action in promoting carcinogenesis. Therefore, the analyses of ECGN might be helpful in discriminating compounds that promote cellular survival associated or not to proliferation of thyrocytes.

Introduction to the Special issue “Advances in Molecular Oncology and personalized medicine: individuating molecular targets for intervention and amelioration of patients status”. Editorial. Seminars in Cancer Biology, 2019. Scope of the thematic issue is to provide an update on the effects of modifications (at single nucleotide changes or chromosome translocation), gene silencing by chromatin repression or by mRNA translation block by microRNA activation, and to discuss the possible amplification effects and collaboration between different oncogenic pathways to the development and progression of cancerogenesis process. Driver mutations, epigenetic deregulation, DNA damage, telomere control escape, and changes in RNAs levels affect the cell differentiation state, potentially contributing to the stemness and the proliferation at an embryo-like state, as for cancer stem cells.

Several studies associate foetal human exposure to bisphenol A (BPA) to metabolic/endocrine diseases, mainly diabesity. They describe the role of BPA in the disruption of pancreatic beta cell, adipocyte and hepatocyte functions. Indeed, the complexity of the diabesity phenotype is due to the involvement of different endoderm-derived organs, all targets of BPA. Here, we analyse this point delineating a picture of different mechanisms of BPA toxicity in endoderm-derived organs leading to diabesity. Moving from epidemiological data, we summarize the in vivo experimental data of the BPA effects on endoderm-derived organs (thyroid, pancreas, liver, gut, prostate and lung) after prenatal exposure. Mainly, we gather molecular data evidencing harmful effects at low-dose exposure, pointing to the risk to human health. Although the fragmentation of molecular data does not allow a clear conclusion to be drawn, the present work indicates that the developmental exposure to BPA represents a risk for endoderm-derived organs development as it deregulates the gene expression from the earliest developmental stages. A more systematic analysis of BPA impact on the transcriptomes of endoderm-derived organs is still missing. Here, we suggest in vitro toxicogenomics approaches as a tool for the identification of common mechanisms of BPA toxicity leading to the diabesity in organs having the same developmental origin.

The progressive and physiological decline in ovarian function depends on the rate of follicular loss by atresia, contributing to the reduction in ovarian reserve. Genetics and environmental factors play important roles in ovarian senescence and in the onset of ovarian dysfunctions such as diminished ovarian reserve. A better understanding of the mechanisms underlying ovarian aging and their regulation by genetic and environmental factors is needed to evaluate ovarian reserve and to predict fertility potential by identification of more accurate and less invasive markers. We report transcriptomic data (i) implicating novel (e.g. EIF2 signalling) and well-known pathways (e.g. TGFβ signalling), and (ii) defining a unique set of non-coding RNA (ncRNA), both associated with ovarian function. The latter includes miRNAs (e.g. Mir143 and Mir145), snoRNAs (e.g. Snord16a and Snora34), and one lncRNA (Gas5), which are differentially expressed in middle-aged ovaries (12 months) vs young-aged (3 months) from CD1 mice. Experimental analysis confirms that ovary lifespan varies across genetic backgrounds in mice and, genetics influences the response to environmental perturbations such as diet. Moreover, the identified ncRNAs were verified in a model of reproductive dysfunction promoted by the environmental toxicant ethylenthiourea. We also report the increase of miRNA143 and miRNA145 in follicular fluid of women with diminished ovarian reserve. Their levels inversely correlate with the hormonal profile and with the number of the oocytes recruited upon hormonal stimulation. Overall, we report a transcriptomic signature for ovarian dysfunction in vivo that provides a valuable resource for translational research in human reproductive aging.

Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy with a variable clinical outcome. Biomarkers of CLL progression are required for optimising prognosis and therapy. The Inhibitor of Bruton’s tyrosine kinase—isoform α (IBTKα) gene encodes a substrate receptor of Cullin 3-dependent E3 ubiquitin ligase, and promotes cell survival in response to the reticulum stress. Searching for novel markers of CLL progression, we analysed the expression of IBTKα in the peripheral blood B-cells of CLL patients, before and after first line therapy causing remission. The expression of IBTKα was significantly increased in disease progression, and decreased in remission after chemotherapy. Consistently with a pro-survival action, RNA interference of IBTKα increased the spontaneous and Fludarabine-induced apoptosis of MEC-1 CLL cells, and impaired the cell cycle of DeFew B-lymphoma cells by promoting the arrest in G0/G1 phase and apoptosis. Consistently, RNA interference of IBTKα up regulated the expression of pro-apoptotic genes, including TNF, CRADD, CASP7, BNIP3 and BIRC3. Our results indicate that IBTKα is a novel marker of CLL progression promoting cell growth and resistance to apoptosis. In this view, IBTKα may represent an attractive cancer drug target for counteracting the therapy-resistance of tumour cells.

Since of their discovery in mammalians, microRNAs (miRNAs) have been associated to almost every physiological function within cells, tissues and organs. miRNAs are small non-coding RNAs that regulate gene expression by targeting messenger RNAs for translational repression or, at lesser extent, mRNAs degradation. Within the several functions controlled by miRNAs there are the control of cell proliferation, apoptosis, differentiation, cell migration, autophagy and metabolism. Thus, the uncontrolled expression of miRNAs has been associated with cancer onset, progression and cancer spreading into metastasis. miRNAs up- or down-regulation has been linked to oncogenic and tumor-suppressive roles in all type of cancers. Altered expression of many miRNAs has been reported many human malignant tumors, participating in various cellular processes accordingly with its broad range of potential mRNAs target. Here, we want to briefly discuss the mechanisms underlying miRNA-mediated tumorigenesis in different human cancers, presenting, as an example, the oncogenic and tumor-suppressive function of miR-25. Moreover, we summarize the possible future as a potential diagnostic and prognostic parameter as well as therapeutic target in clinical applications and the main techniques to study miRNAs.

The review intends to present and recapitulate the current knowledge on the roles and importance of regulatory RNAs, such as microRNAs and small interfering RNAs, RNA binding proteins and enzymes processing RNAs or activated by RNAs, in cells infected by RNA viruses. The review focuses on how non-coding RNAs are involved in RNA virus replication, pathogenesis and host response, especially in retroviruses HIV, with examples of the mechanisms of action, transcriptional regulation, and promotion of increased stability of their targets or their degradation.

Upon binding, ligands can chaperone their protein targets by preventing them from misfolding and aggregating. Thus, an organic molecule that works as folding chaperone for a protein might be its specific ligand, and, similarly, the chaperone potential could represent an alternative readout in a molecular screening campaign toward the identification of new hits. Here we show that small molecules selected for acting as pharmacological chaperones on a misfolded mutant of the Frizzled4 (Fz4) receptor bind and modulate wild-type Fz4, representing what are to our knowledge the first organic ligands of this until-now-undruggable GPCR. The novelty and the advantages of the screening platform, the allosteric binding site addressed by these new ligands and the mechanism they use to modulate Fz4 suggest new avenues for development of inhibitors of the Wnt-beta-catenin pathway and for drug discovery.

Genetic and environmental factors contribute to thyroid diseases. Although still debated, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to induce thyroid dysfunction in humans and rodents. The data here reported point out the contribution of the exposure window and genetic background in mediating the low-dose TCDD effects on thyroid. Indeed, early (from E0.5 to PND30) and low-dose (0,001 μg/kg/day) TCDD exposure reduced the circulating fT4 and altered the expression of thyroid specific transcripts. The role of genetic components was estimated monitoring the same markers in Pax8+/− and Nkx2-1+/− mice, susceptible to thyroid dysfunction, exposed to 0, 1 μg/kg/day TCDD from E15.5 to PND60. Haploinsufficiency of either Pax8 or Nkx2-1 genes exacerbated the effects of the exposure impairing the thyroid enriched mRNAs in sex dependent manner. Such effect was mediated by mechanisms involving the Nkx2-1/p53/p65/IĸBα pathway in vitro and in vivo. Foetal exposure to TCDD impaired both thyroid function and genes expression while thyroid development and differentiation did not appear significantly affected. In mouse, stronger effects were related to earlier exposure or specific genetic background such as either Pax8 or Nkx2-1 haploinsufficiency, both associated to hypothyroidism in humans. Furthermore, our data underline that long exposure time are needed to model in vitro and in vivo results.

In the last 10 years, the discovery that the human genome is able to generate a large number of RNAs non coding for proteins (ncRNAs) has changed our way to approach the understanding and studying of biology. In particular, the discovery of several Long ncRNAs (lncRNAs) and microRNAs (miRNAs) with tumor suppressor or oncogenic function, opened new horizons in molecular oncology research, diagnosis and potential therapies.

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer–mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy. Mol Cancer Ther; 13(3); 752–62. ©2013 AACR . This article is highlighted in the In This Issue feature, [p. 555][1] [1]: /lookup/volpage/13/555?iss=3

Bisphenol A (BPA) is an endocrine-disrupting chemical that leads to low-dose human exposure due to its ability to leach from chemically derived products, as polycarbonate plastics and epoxy resin. In addition to its known xeno-endocrine action, BPA exerts a wide range of metabolic effects. Despite the documented BPA exposure outcomes on synthesis of thyroid hormones, there are not any data available on its actions on the thyroid follicular cells, site of synthesis of the thyroid hormones. Recently, it has been shown that several environmental pollutants, as BPA, can exert a thyroid disrupting activity. In this study, we employed in vitro and in vivo (zebrafish) models to examine the effects of BPA in regulating the expression of genes involved in the thyroid hormone synthesis and of their transcriptional regulators at BPA doses as low as 10(-9)M, a dose that is environmentally pertinent and far below the one detected in infants plasma. In both systems we could detect an altered expression of the genes involved in thyroid hormones synthesis and of thyroid specific transcriptional factors in BPA dose and time dependent manner. Our results suggest that BPA exerts a direct effect on thyroid follicular cell. We show that these cells can "sense" very low amount of BPA. Thus they, potentially, represent an ideal in vitro system to develop assays to detect BPA and other pollutants with thyroid disrupting activity at level far below the ones considered to be environmental relevant. Moreover, this report may provide new insight into the mode of BPA-induced deregulation of physiological processes as well as on the extensively debated molecular pathways underlying its biological activities.

In vitro Omics analysis (i.e. transcriptome) is suggested to predict in vivo toxicity and adverse effects in humans, although the causal link between high-throughput data and effects in vivo is not easily established. Indeed, the chemical-organism interaction can involve processes, such as adaptation, not established in cell cultures. Starting from this consideration we investigate the transcriptomic response of immortalized thyrocytes to ethylenthiourea and chlorpyrifos. In vitro data revealed specific and common genes/mechanisms of toxicity, controlling the proliferation/survival of the thyrocytes and unrelated hematopoietic cell lineages. These results were phenotypically confirmed in vivo by the reduction of circulating T4 hormone and the development of pancytopenia after long exposure. Our data imply that in vitro toxicogenomics is a powerful tool in predicting adverse effects in vivo, experimentally confirming the vision described as Tox21c (Toxicity Testing in the 21st century) although not fully recapitulating the biocomplexity of a living animal.

Epidemiological and experimental evidence associates the exposure to Bisphenol A with the increase of cancer risk in several organs, including prostate. BPA targets different pathways involved in carcinogenicity including the Nuclear Receptors (i.e. estrogen and androgen receptors), stress regulated proteins and, finally, epigenetic changes. Here, we analyse BPA-dependent carcinogenesis in endoderm-derived glands, thyroid, liver, pancreas and prostate focusing on cell signalling, DNA damage repair pathways and epigenetic modifications. Mainly, we gather molecular data evidencing harmful effects at doses relevant for human risk (low-doses). Since few molecular data are available, above all for the pancreas, we analysed transcriptomic data generated in our laboratory to suggest possible mechanisms of BPA carcinogenicity in endoderm-derived glands, discussing the role of nuclear receptors and stress/NF-kB pathways. We evidence that an in vitro toxicogenomic approach might suggest mechanisms of toxicity applicable to cells having the same developmental origin. Although we cannot draw firm conclusions, published data summarized in this review suggest that exposure to BPA, primarily during the developmental stages, represents a risk for carcinogenesis of endoderm-derived glands.

MicroRNAs (miRNAs) are non-coding RNA molecules, with sequence length of 19-24 nucleotides, which can induce mRNA degradation and regulate protein translation repression. Recently plenty of reports showed that miRNAs increase or decrease in the serum (circulating miRNAs) and in PBMC of Human immunodeficiency virus type 1 (HIV-1) infected individuals to affect the replication of HIV-1 through regulating HIV-1 proteins or HIV-1 replication related host factors. Many of miRNAs can suppress HIV-1 replication, but do not affect the integrated viral DNA. Low or no viral protein expression could result in a block of virus and its replication to induce HIV-1 latency, which is the great obstacle of the cure of HIV-1 infection. In the HIV-1 latency reservoir, the integrated provirus can reactivate under appropriate stimulus, which results in HIV-1 reproduction. Factors imply that cellular miRNAs may promote the establishment of HIV-1 latency. Further studies on the mechanisms of miRNAs affecting viral protein expression will provide new approaches to clear the viral reservoir.

Epidemiological and experimental data highlighted the thyroid-disrupting activity of bisphenol A (BPA). Although pivotal to identify the mechanisms of toxicity, direct low-dose BPA effects on thyrocytes have not been assessed. Here, we report the results of microarray experiments revealing that the transcriptome reacts dynamically to low-dose BPA exposure, adapting the changes in gene expression to the exposure duration. The response involves many genes, enriching specific pathways and biological functions mainly cell death/proliferation or DNA repair. Their expression is only slightly altered but, since they enrich specific pathways, this results in major effects as shown here for transcripts involved in the DNA repair pathway. Indeed, even though no phenotypic changes are induced by the treatment, we show that the exposure to BPA impairs the cell response to further stressors. We experimentally verify that prolonged exposure to low doses of BPA results in a delayed response to UV-C-induced DNA damage, due to impairment of p21-Tp53 axis, with the BPA-treated cells more prone to cell death and DNA damage accumulation. The present findings shed light on a possible mechanism by which BPA, not able to directly cause genetic damage at environmental dose, may exert an indirect genotoxic activity.

microRNAs (miRNAs) are small noncoding RNAs able to suppress gene expression by targeting messenger RNAs for translational repression or, at lesser extent, degradation. miRNAs are widely expressed in tissues and organs and play fundamental roles in controlling cell proliferation, apoptosis, differentiation, cell migration, autophagy and metabolism. Uncontrolled expression of miRNAs has been associated with cancer progression, and miRNA up- or down-regulation has been linked to oncogenic and tumor-suppressive roles in cancers such as breast cancer, colorectal cancer, lung cancer, gastric cancer and glioblastoma. Altered expression of the miRNA mir-25 has been reported in many human malignant tumors, participating in various cellular processes accordingly with its broad range of potential mRNAs target. In the present review, we briefly discuss the mechanisms underlying miR-25-mediated tumorigenesis in six different human cancers and its possible future as a potential diagnostic and prognostic parameter as well as therapeutic target in clinical applications.

Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4+ and CD8+ T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation.

Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10−9M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions.

p38 MAPKs (mitogen-activated protein kinases) play important roles in the regulation of cellular responses to environmental stress. Recently, this signalling pathway has also been implicated in the regulation of processes unrelated to stress, for example, in T lymphocytes and cardiomyocytes. In order to identify molecular targets responsible for the housekeeping functions of p38 MAPKs, we have analysed the differences in the transcriptomes of normally proliferating wild-type and p38alpha knockout immortalized embryonic cardiomyocytes. Interestingly, many potential components of the myocardium extracellular matrix were found to be upregulated in the absence of p38alpha. Further analysis of the microarray data identified TEF-1 (transcriptional enhancer factor-1), a known regulator of heart-specific gene expression, and C/EBPbeta (CCAAT/enhancer-binding protein beta), as the two transcription factors the binding sites of which were most enriched in the promoters of p38alpha-regulated genes. We have focused on the study of the extracellular matrix component COL1A1 (alpha1 chain of type I collagen) and found evidence for the involvement of both TEF-1 and C/EBPbeta in the p38alpha-dependent inhibition of COL1A1 transcription. Our data therefore show that p38 MAPKs regulate TEF-1 and C/EBPbeta transcriptional activity in the absence of environmental stress and suggests a role for p38alpha in the expression of extracellular matrix components that maintain organ architecture.

Localized mRNAs are thought to be transported in defined particles to their final destination. These particles represent large protein complexes that may be involved in recognizing, transporting, and anchoring localized messages. Few components of these ribonucleoparticles, however, have been identified yet. We chose the strategy to biochemically enrich native RNA–protein complexes involved in RNA transport to identify the associated RNAs and proteins. Because Staufen proteins were implicated in intracellular RNA transport, we chose mammalian Staufen proteins as markers for the purification of RNA transport particles. Here, we present evidence that Staufen proteins exist in two different complexes: ( i ) distinct large, ribosome- and endoplasmic reticulum-containing granules preferentially found in the membrane pellets during differential centrifugation and ( ii ) smaller particles in the S100 from rat brain homogenates. On gel filtration of the S100, we identified soluble 670-kDa Staufen1-containing and 440-kDa Staufen2-containing particles. They do not cofractionate with ribosomes and endoplasmic reticulum but rather coenrich with kinesin heavy chain. Furthermore, the fractions containing the Staufen1 particles show a 15-fold enrichment of mRNAs compared with control fractions. Most importantly, these fractions are highly enriched in BC1, and, to a lesser extent, in the α-subunit of the Ca 2+ /calmodulin-dependent kinase II, two dendritically localized RNAs. Finally, both RNAs colocalize with Staufen1–hemagglutinin in particles in dendrites of transfected hippocampal neurons. We therefore propose that these Staufen1-containing particles may represent RNA transport intermediates that are in transit to their final destination within neurons.

Background Comparison of toxicogenomic data facilitates the identification of deregulated gene patterns and maximizes health risk prediction in human. Results Here, we performed phenotypic anchoring on the effects of acute exposure to low-grade polluted groundwater using mouse and zebrafish. Also, we evaluated two windows of chronic exposure in mouse, starting in utero and at the end of lactation. Bioinformatic analysis of livers microarray data showed that the number of deregulated biofunctions and pathways is higher after acute exposure, compared to the chronic one. It also revealed specific profiles of altered gene expression in all treatments, pointing to stress response/mitochondrial pathways as major players of environmental toxicity. Of note, dysfunction of steroid hormones was also predicted by bioinformatic analysis and verified in both models by traditional approaches, serum estrogens measurement and vitellogenin mRNA determination in mice and zebrafish, respectively. Conclusions In our report, phenotypic anchoring in two vertebrate model organisms highlights the toxicity of low-grade pollution, with varying susceptibility based on exposure window. The overlay of zebrafish and mice deregulated pathways, more than single genes, is useful in risk identification from chemicals implicated in the observed effects. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1067) contains supplementary material, which is available to authorized users.

Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immunodeficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of accessory genes such as nef) has been forwarded as a primary approach for creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence of virulent mutants. New strategies that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a novel approach for attenuating HIV-1. We have constructed an HIV-1 genome carrying the cDNA of a proteolysis-resistant nuclear factor-kappaB inhibitor (IkappaB-alphaS32/36A) in the nef region. HIV-1 expressing IkappaB-alphaS32/36A down-regulates viral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenuating characteristics of IkappaB-alphaS32/36A permit its stable maintenance in a live, replicating HIV-1 despite 180 days of forced ex vivo passaging in tissue culture. As compared with other open-reading frames embedded into HIV/SIV genome, this degree of stability is unprecedented. Thus, IkappaB-alphaS32/36A offers proof-of-principle that artifactually gained functions, when used to attenuate the replication of live HIV-1, can be stable. These findings illustrate gain-of-function as a feasible strategy for developing safer live-attenuated HIVs to be tested as candidates for AIDS vaccine.