DPPA4 increases aggressiveness of pituitary neuroendocrine tumors by enhancing cell stemness.

BACKGROUND: Pituitary neuroendocrine tumors, PitNETs, are often aggressive and precipitate in distant metastases that are refractory to current therapies. However, the molecular mechanism in PitNETs' aggressiveness is not well understood. Developmental pluripotency-associated 4 (DPPA4) is known as a stem cell regulatory gene and overexpressed in certain cancers, but its function in the context of PitNETs' aggressiveness is not known. METHODS: We employed both rat and human models of PitNETs. In the rat pituitary tumor model (RPT), we used prenatal-alcohol-exposed (PAE) female Fischer rats which developed aggressive PitNETs following estrogen treatment, while in the human pituitary tumor (HPT) model, we used aggressively proliferative cells from pituitary tumors of patients undergone surgery. Various molecular, cellular, and epigenetic techniques were used to determine the role of DPPA4 in PitNETs' aggressiveness. RESULTS: We show that DPPA4 is overexpressed in association with increased cell stemness factors in aggressive PitNETs of PAE rats and of human patients. Gene-editing experiments demonstrate that DPPA4 increases the expression of cell stemness and tumor aggressiveness genes and promotes proliferation, colonization, migration, and tumorigenic potential of PitNET cells. ChIP assays and receptor antagonism studies reveal that DPPA4 binds to canonical WINTs promoters and increases directly or indirectly the Wnt/ß-catenin control of cell stemness, tumor growth, and aggressiveness of PitNETs. Epigenetic studies show involvement of histone methyltransferase in alcohol activation of DPPA4. CONCLUSIONS: These findings support a role of DPPA4 in tumor stemness and aggressiveness and provide a preclinical rationale for modulating this stemness regulator for the treatment of PitNETs.

Epigenetic insight into effects of prenatal alcohol exposure on stress axis development: Systematic review with meta-analytic approaches.

We conducted a systematic review with meta-analytic elements using publicly available Gene Expression Omnibus (GEO) datasets to determine the role of epigenetic mechanisms in prenatal alcohol exposure (PAE)-induced hypothalamic-pituitary-adrenal (HPA) axis dysfunctions in offspring. Several studies have demonstrated that PAE has long-term consequences on HPA axis functions in offspring. Some studies determined that alcohol-induced epigenetic alterations during fetal development persist in adulthood. However, additional research is needed to understand the major epigenetic events leading to alcohol-induced teratogenesis of the HPA axis. Our network analysis of GEO datasets identified key pathways relevant to alcohol-mediated histone modifications, DNA methylation, and miRNA involvement associated with PAE-induced alterations of the HPA axis. Our analysis indicated that PAE perturbated the epigenetic machinery to activate corticotrophin-releasing hormone, while it suppressed opioid, glucocorticoid receptor, and circadian clock genes. These results help to further our understanding of the epigenetic basis of alcohol's effects on HPA axis development.

Beta 2 adrenergic receptor and mu opioid receptor interact to potentiate the aggressiveness of human breast cancer cell by activating the glycogen synthase kinase 3 signaling.

BACKGROUND: Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control the growth and proliferation of breast cancer cells. However, the underlying cell signaling mechanism remained unclear. METHODS: To determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, CRISPR or chemical inhibition or activation of beta-adrenergic receptors (B2AR) and mu-opioid receptors (MOR) gene, and PCR array technology and studied aggressive tumor phenotype and signaling cascades. RESULTS: We show here that in triple-negative breast cancer cells, the reduction in expression B2AR and MOR by genetic and pharmacological tools leads to a less aggressive phenotype of triple-negative breast cancer cells in vitro and in animal xenografts. Genomic analysis indicates the glycogen synthase kinase 3 (GSK3) pathway as a possible candidate messenger system involved in B2AR and MOR cross talk. GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation. CONCLUSIONS: These data confirm a role of B2AR and MOR interaction in the control of breast cancer cell growth and identify a possible role of the GSK3 signaling system in mediation of these two receptors' cross talk. Screening for ligands targeting B2AR and MOR interaction and/or the GSK3 system may help to identify novel drugs for the prevention of triple-negative breast cancer cell growth and metastasis.

Beta 2 Adrenergic Receptor Antagonist Propranolol and Opioidergic Receptor Antagonist Naltrexone Produce Synergistic Effects on Breast Cancer Growth Prevention by Acting on Cancer Cells and Immune Environment in a Preclinical Model of Breast Cancer.

Cancer progression is known to be promoted by increased body stress caused by elevated beta-adrenergic and opioidergic nervous system activities. The effects of ß2-adrenergic blocker propranolol (PRO) and µ-opioid receptor antagonist naltrexone (NTX) were tested using a preclinical model of human breast cancer. These drugs, individually, and more potently when combined, inhibited the cell growth and progression of breast cancer cells in vitro in cultures, and in vivo in rat xenografts. The antitumor activities of these drugs were associated with direct cell intrinsic effects, including increased cell growth arrest, elevated levels of apoptotic proteins, and reduced production of epithelial-mesenchymal transition factors by the tumor cells, as well as effects on innate immune activation and reduced inflammatory cytokine levels in plasma. These data suggest that the combined treatments of PRO and NTX produce impressive antitumor effects in the preclinical breast cancer model, and thereby may provide a new combinatorial treatment strategy with more clinical treatment modalities.

Preconception Alcohol Exposure Increases the Susceptibility to Diabetes in the Offspring.

Heavy alcohol drinking alters glucose metabolism, but the inheritability of this effect of alcohol is not well understood. We used an animal model of preconception alcohol exposure in which adult female rats were given free access to 6.7% alcohol in a liquid diet and water for about 4 weeks, went without alcohol for 3 weeks, and then were bred to generate male and female offspring. Control animals were either ad lib-fed rat chow or pair-fed an isocaloric liquid diet during the time of alcohol-feeding in the experimental animals. Our results show that the female rats fed with alcohol in the liquid diet, but not with the isocaloric liquid diet, prior to conception had an altered stress gene network involving glucose metabolism in oocytes when compared with those in ad lib-fed chow diet controls. The offspring born from preconception alcohol-fed mothers showed significant hyperglycemia and hypoinsulinemia when they were adults. These rats also showed increased levels of inflammatory cytokines and cellular apoptosis in the pancreas, altered insulin production and actions in the liver, and a reduced number of proopiomelanocortin neurons in the hypothalamus. Replenishment of proopiomelanocortin neurons in these animals normalized the abnormal glucose to restore homeostasis. These data suggest that preconception alcohol exposures alter glucose homeostasis by inducing proopiomelanocortin neuronal functional abnormalities. Our findings provide a novel insight into the impact of high doses of alcohol on the female gamete that may cause inheritance of an increased susceptibility to diabetes.

Alcohol Increases Exosome Release from Microglia to Promote Complement C1q-Induced Cellular Death of Proopiomelanocortin Neurons in the Hypothalamus in a Rat Model of Fetal Alcohol Spectrum Disorders.

Microglia, a type of CNS immune cell, have been shown to contribute to ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing ß-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined whether the microglial extracellular vesicle exosome is involved in the ethanol-induced neuronal death of the ß-endorphin neuron. Extracellular vesicles were prepared from hypothalamic tissues collected from postnatal rats (both males and females) fed daily with 2.5 mg/kg ethanol or control milk formula for 5 d or from hypothalamic microglia cells obtained from postnatal rats, grown in cultures for several days, and then challenged with ethanol or vehicle for 24 h. Nanoparticle tracking analysis and transmission electron microscopy indicated that these vesicles had the size range and shape of exosomes. Ethanol treatments increased the number and the ß-endorphin neuronal killing activity of microglial exosomes both in vivo and in vitro Proteomics analyses of exosomes of cultured microglial cells identified a large number of proteins, including various complements, which were elevated following ethanol treatment. Proteomics data involving complements were reconfirmed using quantitative protein assays. Ethanol treatments also increased deposition of the complement protein C1q in ß-endorphin neuronal cells in both in vitro and in vivo systems. Recombinant C1q protein increased while C1q blockers reduced ethanol-induced C3a/b, C4, and membrane attack complex/C5b9 formations; ROS production; and ultimately cellular death of ß-endorphin neurons. These data suggest that the complement system involving C1q-C3-C4-membrane attack complex and ROS regulates exosome-mediated, ethanol-induced ß-endorphin neuronal death.SIGNIFICANCE STATEMENT Neurotoxic action of alcohol during the developmental period is recognized for its involvement in fetal alcohol spectrum disorders, but the lack of clear understanding of the mechanism of alcohol action has delayed the progress in therapeutic intervention of this disease. Proopiomelanocortin neurons known to regulate stress, energy homeostasis, and immune functions are reported to be killed by developmental alcohol exposure because of activation of microglial immune cells in the brain. While microglia are known to use extracellular vesicles to communicate with neurons for maintaining homeostasis, we show here that ethanol exposure during the developmental period hijacks this system to spread apoptotic factors, including complement protein C1q, to induce the membrane attack complex and reactive super-oxygen species for proopiomelanocortin neuronal killing.

Discovery of Novel (Imidazo[1,2-a]pyrazin-6-yl)ureas as Antiproliferative Agents Targeting P53 in Non-small Cell Lung Cancer Cell Lines.

A series of (imidazo[1,2-a]pyrazin-6-yl)ureas were synthesized through 6-aminoimidazo[1,2-a]pyrazine as a key intermediate. 1-(Imidazo[1,2-a]pyrazin-6-yl)-3-(4-methoxy - phenyl)urea displayed a cytostatic activity against a non-small cell lung cancer cell line and was chosen for further mechanistic studies. Growth kinetics highlighted a selective dose-dependent response of P53-mutant NSCLC-N6-L16 cell line and overexpression of TP53 gene induced by this compound. These pharmacological data suggest a promising reactivation of p53 mutant in NSCLC-N6-L16 cell line.

TP53 transcription factor for the NEDD9/HEF1/Cas-L gene: potential targets in Non-Small Cell Lung Cancer treatment.

Lung cancer is a serious public health problem. Although there has been significant progress in chemotherapy, non-small cell lung cancer is still resistant to current treatments, primarily because of the slow rate of cell development. It is thus important to find new molecules directed against targets other than proliferation agents. Considering the high proportion of mutant proteins in tumor cells, and the high rate of mutation of the TP53 gene in all cancers, and in NSCLC in particular, this gene is a perfect target. Certain new molecules have been shown to restore the activity of mutated p53 protein, for example PRIMA-1, which reactivates the His273 mutant p53. In a previous study, we presented triazine A190, a molecule with a cytostatic activity that blocks cells in the G1 phase and induces apoptosis. Here, we show that A190 not only restores mutant p53 activity, but also induces an overexpression of the NEDD9 gene, leading to apoptotic death. These findings might offer hope for the development of new targeted therapies, specific to tumor cells, which spare healthy cells.

Cucurbitacin-D-induced CDK1 mRNA up-regulation causes proliferation arrest of a non-small cell lung carcinoma cell line (NSCLC-N6).

Despite progress in chemotherapeutic agents, non-small cell lung cancers (NSCLC) still have a poor survival rate. Thus, development of new therapeutic strategies, specifically against cancer cells is still required. For this purpose, we treated the non-small cell lung cancer cell line NSCLC-N6 with the natural product cucurbitacin D (CucD) - extracted from the plant Ecballium elaterium in order first to assess its in vitro cytotoxicity, but also to study the genetic changes that it could bring out. CucD has shown a blocking in the G1 phase of the cell cycle in NSCLC-N6 cells prior to apoptotic cell death. The reverse transcriptase-polymerase chain reaction-differential display (RT-PCR-DD) technique was also applied on treated cells to elucidate the genetic mechanisms involved. We revealed an overexpression of Cyclin-dependent kinase 1 (CDK1) mRNA after treatment and, with the use of antisense oligonucleotides, an effective role in the proliferation arrest of NSCLC-N6 cells. The present study provides new insights about the mechanisms of proliferation arrest in tumor cells and open new ways of treatment to target tumor growth.

Screening and discovery of nitro-benzoxadiazole compounds activating epidermal growth factor receptor (EGFR) in cancer cells.

Peptide ligand-induced dimerization of the extracellular region of the epidermal growth factor receptor (sEGFR) is central to the signal transduction of many cellular processes. A small molecule microarray screen has been developed to search for non-peptide compounds able to bind to sEGFR. We describe the discovery of nitro-benzoxadiazole (NBD) compounds that enhance tyrosine phosphorylation of EGFR and thereby trigger downstream signaling pathways and other receptor tyrosine kinases in cancer cells. The protein phosphorylation profile in cells exposed to NBD compounds is to some extent reminiscent of the profile induced by the cognate ligand. Experimental studies indicate that the small compounds bind to the dimerization domain of sEGFR, and generate stable dimers providing allosteric activation of the receptor. Moreover, receptor phosphorylation is associated with inhibition of PTP-1B phosphatase. Our data offer a promising paradigm for investigating new aspects of signal transduction mediated by EGFR in cancer cells exposed to electrophilic NBD compounds.

From policy making to service use. Down's syndrome antenatal screening in England, France and the Netherlands.

In industrialised countries, certain biomedical innovations have come into general use, but the ways they are used vary considerably. Prenatal screening techniques for Down's syndrome are a perfect example of this. In 2010, screening rates stood at 61% in England and 84% in France; the previous year the rate was 26% in the Netherlands. The objective of our research, which took place in these three countries between 2008 and 2011, was to explain these differences. In these countries, public authorities focus on women's free access to innovations and on receiving their informed consent. But other aspects of screening policy vary, as do the health systems in which they are implemented. Our study shows that the sociotechnical settings which vary from country to country affected the interactions during the consultations we observed and thus impacted the decision of whether or not to screen.

Synthesis and antiproliferative activity of benzofuran-based analogs of cercosporamide against non-small cell lung cancer cell lines.

A novel series of 3-methyl-1-benzofuran derivatives were synthesized and screened in vitro for their antiproliferative activity against two human NSCLC cell lines (NSCLC-N6 mutant p53 and A549 wild type p53). Most promising compounds presented a structural analogy with the west part of cercosporamide, a natural product of biological interest. In particular, compounds 10, 12 and 31 showed cytotoxic activities at micromolar concentrations (IC50 < 9.3 µM) and compounds 13, 18 and 32 displayed moderate IC50 values (25-40 µM).

miRNAs, a potential target in the treatment of Non-Small-Cell Lung Carcinomas.

Lung cancer is a serious public health problem and Non Small Cell Lung Carcinoma, NSCLC, is particularly resistant to current treatments. So it is important to find new strategies that are active against NSCLC. miRNA is implicated in cancer and may be implicated in NSCLC. Our team has been working on two genes HEF1, a gene implicated in different functions of cell cycle and B2, a large non-coding RNA (nc RNA). These two genes have the same localisation: chromosome 6 and locus p24-25. nc RNA B2 may be involved in the regulation of HEF1. Firstly, we examine a bank of different human miRNAs known to interact with exons of HEF1. HEF1 and B2 were overexpressed in vitro by treating NSCLC-N6 with the cytostatic molecule A190, and carried out qRT-PCR for the expression of miRNA. Secondly, using specific software, we sought for structures originating from the B2 RNA sequence which might interact with HEF1 and assessed their expression. This strategy enabled us to confirm firstly that known miRNAs that can interact with exons of HEF1 are expressed in NSCLC-N6 cells. More precisely this strategy highlighted overexpression of one miRNA, hsa-miR-146b, listed in miRbase. The second step of the studies highlighted the expression of miRNA, potentially sequences originating from B2 in the NSCLC-N6. This miRNA overexpressed might be one of the regulators of the gene HEF1 and consequently implies on the carcinogenesis of lung cancer. So in the future it could be a potential and an innovative way to find a new strategy for the treatment of lung cancer.

Study of antiproliferative effects of synthetic substances against lens epithelial cell line (SRA 01/04).

A cataract is a clouded area of the eye, which impairs vision. Cataracts can be caused by a natural hardening of the lens in the elderly, or may be the result of eye injury. However there is a treatment by extracapsular surgery, almost 50% of operations are followed by another posterior capsule opacification. This secondary cataract is due to abnormal cellular proliferation. Pharmacologic inhibition of this cellular proliferation would be a very promising treatment. The objective of our study is to test some antiproliferative drugs, less toxic than those currently used such as 5-FU or mytomycin C. We have investigated the in vitro effects of several molecules (V0 and its derivatives) on a proliferative human lens epithelial cell line (SRA 01/04). During a first step, we have measured the IC50 of each molecule. After this first screening, we have studied the kinetic of the cell growth with or without the molecules at different concentration. Then, flow cytometry was used to determine the phase of the cell cycle at which the proliferation stopped. This study has shown that 3 molecules V19, V1, and A190 have an interesting profile in vitro and were selected to analyze their mechanism of action.