HDAC inhibition does not induce estrogen receptor in human triple-negative breast cancer cell lines and patient-derived xenografts.

Several publications have suggested that histone deacetylase inhibitors (HDACis) could reverse the repression of estrogen receptor alpha (ERα) in triple-negative breast cancer (TNBC) cell lines, leading to the induction of a functional protein. Using different HDACis, vorinostat, panobinostat, and abexinostat, we therefore investigated this hypothesis in various human TNBC cell lines and patient-derived xenografts (PDXs). We used three human TNBC cell lines and three PDXs. We analyzed the in vitro toxicity of the compounds, their effects on the hormone receptors and hormone-related genes and protein expression both in vitro and in vivo models. We then explored intra-tumor histone H3 acetylation under abexinostat in xenograft models. Despite major cytotoxicity of all tested HDAC inhibitors and repression of deactylation-dependent CCND1 gene, neither ERα nor ERß, ESR1 or ESR2 genes respectively, were re-expressed in vitro. In vivo, after administration of abexinostat for three consecutive days, we did not observe any induction of ESR1 or ESR1-related genes and ERα protein expression by RT-qPCR and immunohistochemical methods in PDXs. This observation was concomitant to the fact that in vivo administration of abexinostat increased intra-tumor histone H3 acetylation. These observations do not allow us to confirm previous studies which suggested that HDACis are able to convert ER-negative (ER-) tumors to ER-positive (ER+) tumors, and that a combination of HDAC inhibitors and hormone therapy could be proposed in the management of TNBC patients.

Applying systems biology in drug discovery and development.

Translational research is a continuum between clinical and basic research where the patient is the center of the research process. It brings clinical research to a starting point for the drug discovery process, permitting the generation of a more robust pathophysiological hypothesis essential for a better selection of drug targets and candidate optimization. It also establishes the basis of early proof for clinical concept studies, preferably in phase I, for which biomarkers and surrogate endpoints can often be used. Systems biology is a prerequisite approach to translational research where technologies and expertise are integrated and articulated to support efficient and productive realization of this concept. The first component of systems biology relies on omics-based technologies and integrates the changes in variables, such as genes, proteins and metabolites, into networks that are responsible for an organism's normal and diseased state. The second component of systems biology is in the domain of computational methods, where simulation and modeling create hypotheses of signaling pathways, transcription networks, physiological processes or even cell- or organism-based models. The simulations aim to show the origin of perturbations of the system that lead to pathological states and what treatment could be achieved to ameliorate or normalize the system. This review discusses how translational research and systems biology together could improve global understanding of drug targets, suggest new targets and approaches for therapeutics, and provide a deeper understanding of drug effects. Taken together, these types of analyses can lead to new therapeutic options while improving the safety and efficacy of new and existing medications.

Shallow Whole-Genome Sequencing from Plasma Identifies FGFR1 Amplified Breast Cancers and Predicts Overall Survival.

Background: Focal amplification of fibroblast growth factor receptor 1 (FGFR1) defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate FGFR1 copy numbers from a cohort of 100 metastatic breast cancer (mBC) patients. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were screened for FGFR1 amplification by FISH, and positive cases were confirmed with a microarray platform (OncoscanTM). Subsequently, cfDNA was evaluated by two approaches, i.e., mFAST-SeqS and shallow whole-genome sequencing (sWGS), to estimate the circulating tumor DNA (ctDNA) allele fraction (AF) and to evaluate the FGFR1 status. Results: Tissue-based analyses identified FGFR1 amplifications in 20/100 tumors. All cases with a ctDNA AF above 3% (n = 12) showed concordance for FGFR1 status between tissue and cfDNA. In one case, we were able to detect a high-level FGFR1 amplification, although the ctDNA AF was below 1%. Furthermore, high levels of ctDNA indicated an association with unfavorable prognosis based on overall survival. Conclusions: Screening for FGFR1 amplification in ctDNA might represent a viable strategy to identify patients eligible for treatment by FGFR inhibition, and mBC ctDNA levels might be used for the evaluation of prognosis in clinical drug trials.

S18986: a positive modulator of AMPA-receptors enhances (S)-AMPA-mediated BDNF mRNA and protein expression in rat primary cortical neuronal cultures.

The present study describes the effect of (S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, on (S)-AMPA-mediated increases in brain-derived neurotrophic factor (BDNF) mRNA and protein expression in rat primary cortical neuronal cultures. (S)-AMPA (0.01-300 microM) induced a concentration-dependent increase in BDNF mRNA and protein expression (EC(50)=7 microM) with maximal increases (50-fold) compared to untreated cultures observed between 5 and 12 h, whereas for cellular protein levels, maximal expression was detected at 24 h. S18986 alone (< or =300 microM) failed to increase basal BDNF expression. However, S18986 (300 microM) in the presence of increasing concentrations of (S)-AMPA maximally enhanced AMPA-induced expression of BDNF mRNA and protein levels (3-5-fold). S18986 (100-300 microM) potentiated BDNF mRNA induced by 3 microM (S)-AMPA (2-3-fold). Under similar conditions, the AMPA allosteric modulator cyclothiazide induced a potent stimulation of (S)-AMPA-mediated BDNF expression (40-fold; EC(50)=18 microM), whereas IDRA-21 was inactive. Kinetic studies indicated that S18986 (300 microM) in the presence of 3 microM (S)-AMPA was capable of enhancing BDNF mRNA levels for up to 25 h, compared to 3 microM (S)-AMPA alone. On the other hand, S18986 only partially enhanced kainate-mediated expression of BDNF mRNA, but failed to significantly enhance N-methyl-D-aspartate-stimulated BDNF expression levels. In support of these observations, the competitive AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) but not the selective NMDA-receptor antagonist, (+)-MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], abrogated S18986-induced effects on BDNF expression. S18986-mediated enhancement of (S)-AMPA-evoked BDNF protein expression was markedly attenuated in Ca(2+)-free culture conditions. Furthermore, from a series of kinase inhibitors only the Calmodulin-Kinase II/IV inhibitor (KN-62, 25 microM) significantly inhibited (-85%, P<0.001) AMPA+S18986 stimulated expression of BDNF mRNA. The present study supports the observations that AMPA receptor allosteric modulators can enhance the expression of BDNF mRNA and protein expression via the AMPA receptor in cultured primary neurones. Consequently, the long-term elevation of endogenous BDNF expression by pharmacological intervention with this class of compounds represents a potentially promising therapeutic approach for behavioural disorders implicating cognitive deficits.

Molecular characterization of the AMPA-receptor potentiator S70340 in rat primary cortical culture: whole-genome expression profiling.

To improve our understanding of the molecular events underlying the effects of positive allosteric modulators of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (S)-AMPA-type glutamate receptors, gene expression profiles of primary cortical culture were measured by Agilent-Microarray technique under (S)-AMPA (1µM) stimulation for 0.5, 6, 24 and 48h in the presence or absence of S70340 (30µM), an allosteric potentiator of AMPA receptors. (S)-AMPA and S70340 treatment alone have little effect on gene expression whereas as early as 6h, their combination induced a large number of genes known to decrease apoptosis and mediate cell survival. Pathway analyses of (S)-AMPA+S70340 treatment-mediated gene expression from 6 to 48h further suggested the activation of cellular functions including neuron differentiation and neurite outgrowth. A proportion of genes implicated in these functions encode proteins involved in environmental cues and are expressed in growth cones, such as extracellular matrix component proteins and filopodia microfilament-associated proteins. Time course analysis of mRNA expression combined with in silico promoter analysis revealed an enrichment in the cAMP response element (CRE) among co-regulated genes. This study demonstrated that S70340-mediated AMPA potentialisation activated genes and functional processes involved in neuroprotective and cognitive effects and describes putative new functional biomarkers.

Coordinate Transcriptomic and Metabolomic Effects of the Insulin Sensitizer Rosiglitazone on Fundamental Metabolic Pathways in Liver, Soleus Muscle, and Adipose Tissue in Diabetic db/db Mice.

Rosiglitazone (RSG), developed for the treatment of type 2 diabetes mellitus, is known to have potent effects on carbohydrate and lipid metabolism leading to the improvement of insulin sensitivity in target tissues. To further assess the capacity of RSG to normalize gene expression in insulin-sensitive tissues, we compared groups of 18-day-treated db/db mice with increasing oral doses of RSG (10, 30, and 100 mg/kg/d) with untreated non-diabetic littermates (db/+). For this aim, transcriptional changes were measured in liver, inguinal adipose tissue (IAT) and soleus muscle using microarrays and real-time PCR. In parallel, targeted metabolomic assessment of lipids (triglycerides (TGs) and free fatty acids (FFAs)) in plasma and tissues was performed by UPLC-MS methods. Multivariate analyses revealed a relationship between the differential gene expressions in liver and liver trioleate content and between blood glucose levels and a combination of differentially expressed genes measured in liver, IAT, and muscle. In summary, we have integrated gene expression and targeted metabolomic data to present a comprehensive overview of RSG-induced changes in a diabetes mouse model and improved the molecular understanding of how RSG ameliorates diabetes through its effect on the major insulin-sensitive tissues.