HumanIslets: An integrated platform for human islet data access and analysis.

Comprehensive molecular and cellular phenotyping of human islets can enable deep mechanistic insights for diabetes research. We established the Human Islet Data Analysis and Sharing (HI-DAS) consortium to advance goals in accessibility, usability, and integration of data from human islets isolated from donors with and without diabetes at the Alberta Diabetes Institute (ADI) IsletCore. Here we introduce HumanIslets.com , an open resource for the research community. This platform, which presently includes data on 547 human islet donors, allows users to access linked datasets describing molecular profiles, islet function and donor phenotypes, and to perform various statistical and functional analyses at the donor, islet and single-cell levels. As an example of the analytic capacity of this resource we show a dissociation between cell culture effects on transcript and protein expression, and an approach to correct for exocrine contamination found in hand-picked islets. Finally, we provide an example workflow and visualization that highlights links between type 2 diabetes status, SERCA3b Ca 2+ -ATPase levels at the transcript and protein level, insulin secretion and islet cell phenotypes. HumanIslets.com provides a growing and adaptable set of resources and tools to support the metabolism and diabetes research community.

Characterizing the effects of Dechlorane Plus on ß-cells: a comparative study across models and species.

Epidemiological studies consistently link environmental toxicant exposure with increased Type 2 diabetes risk. Our study investigated the diabetogenic effects of a widely used flame retardant, Dechlorane Plus (DP), on pancreatic ß-cells using rodent and human model systems. We first examined pancreas tissues from male mice exposed daily to oral gavage of either vehicle (corn oil) or DP (10, 100, or 1000 µg/kg per day) and fed chow or high fat diet for 28-days in vivo. DP exposure did not affect islet size or endocrine cell composition in either diet group. Next, we assessed the effect of 48-hour exposure to vehicle (DMSO) or DP (1, 10, or 100 nM) in vitro using immortalized rat ß-cells (INS-1 832/3), primary mouse and human islets, and human stem-cell derived islet-like cells (SC-islets). In INS-1 832/3 cells, DP did not impact glucose-stimulated insulin secretion (GSIS) but significantly decreased intracellular insulin content. DP had no effect on GSIS in mouse islets or SC-islets but had variable effects on GSIS in human islets depending on the donor. DP alone did not affect insulin content in mouse islets, human islets, or SC-islets, but mouse islets co-exposed to DP and glucolipotoxic (GLT) stress conditions (28.7 mM glucose + 0.5 mM palmitate) had reduced insulin content compared to control conditions. Co-exposure of mouse islets to DP + GLT amplified the upregulation of Slc30a8 compared to GLT alone. Our study highlights the importance and challenges of using different in vitro models for studying chemical toxicity.

A 50-gene biomarker identifies estrogen receptor-modulating chemicals in a microarray compendium.

High throughput transcriptomics (HTTr) profiling has the potential to rapidly and comprehensively identify molecular targets of environmental chemicals that can be linked to adverse outcomes. We describe here the construction and characterization of a 50-gene expression biomarker designed to identify estrogen receptor (ER) active chemicals in HTTr datasets. Using microarray comparisons, the genes in the biomarker were identified as those that exhibited consistent directional changes when ER was activated (4 ER agonists; 4 ESR1 gene constitutively active mutants) and opposite directional changes when ER was suppressed (4 antagonist treatments; 4 ESR1 knockdown experiments). The biomarker was evaluated as a predictive tool using the Running Fisher algorithm by comparison to annotated gene expression microarray datasets including those evaluating the transcriptional effects of hormones and chemicals in MCF-7 cells. Depending on the reference dataset used, the biomarker had a predictive accuracy for activation of up to 96%. To demonstrate applicability for HTTr data analysis, the biomarker was used to identify ER activators in a set of 15 chemicals that are considered potential bisphenol A (BPA) alternatives examined at up to 10 concentrations in MCF-7 cells and analyzed by full-genome TempO-Seq. Using benchmark dose (BMD) modeling, the biomarker genes stratified the ER potency of BPA alternatives consistent with previous studies. These results demonstrate that the ER biomarker can be used to accurately identify ER activators in transcript profile data derived from MCF-7 cells.

Empirical Characterization of False Discovery Rates of Differentially Expressed Genes and Transcriptomic Benchmark Concentrations in Zebrafish Embryos.

High-throughput transcriptomics (HTTr) is increasingly applied to zebrafish embryos to survey the toxicological effects of environmental chemicals. Before the adoption of this approach in regulatory testing, it is essential to characterize background noise in order to guide experimental designs. We thus empirically quantified the HTTr false discovery rate (FDR) across different embryo pool sizes, sample sizes, and concentration groups for toxicology studies. We exposed zebrafish embryos to 0.1% dimethyl sulfoxide (DMSO) for 5 days. Pools of 1, 5, 10, and 20 embryos were created (n = 24 samples for each pool size). Samples were sequenced on the TempO-Seq platform and then randomly assigned to mock treatment groups before differentially expressed gene (DEG), pathway, and benchmark concentration (BMC) analyses. Given that all samples were treated with DMSO, any significant DEGs, pathways, or BMCs are false positives. As expected, we found decreasing FDRs for DEG and pathway analyses with increasing pool and sample sizes. Similarly, FDRs for BMC analyses decreased with increasing pool size and concentration groups, with more stringent BMC premodel filtering reducing BMC FDRs. Our study provides foundational data for determining appropriate experiment designs for regulatory toxicity testing with HTTr in zebrafish embryos.

Tetra methyl bisphenol F: another potential obesogen.

BACKGROUND/OBJECTIVES: Obesity and its associated metabolic diseases are increasing globally. Sedentary lifestyle, high caloric diet, and genetic predisposition are known to contribute to the onset of obesity. It is increasingly recognized that exposure to environmental chemicals such as Bisphenol A (BPA) may also play a significant role. BPA has been correlated with an array of adverse health effects, including obesity and metabolic disorders. Due to public concern, manufacturers are replacing BPA with structural analogues for which there is limited toxicological data. The objective of this study was to assess the effects of these BPA analogues on adipogenesis. METHODS: The adipogenic effects of Tetra Methyl Bisphenol F (TMBPF), Bisphenol F (BPF), Bisphenol AP (BPAP), and fluorine-9-bisphenol (BHPF) were evaluated in murine 3T3-L1 cells. The cells were treated with BPA and its analogues at concentrations from 0.01 µM to 20 µM, throughout differentiation, in the absence of Dexamethasone (Dex). Lipid accumulation, mRNA and protein levels of adipogenic markers was assessed. RESULTS: We found that TMBPF, BPF and BPA increased 3T3-L1 lipid accumulation and the expression levels of adipogenic markers lipoprotein lipase (Lpl), fatty acid binding protein 4 (Fabp4) and perilipin (Plin) (1-20 µM; p < 0.05), whereas BHPF and BPAP had no effect in this model. Further, TMBPF induced adipogenesis to a greater extent than all the other chemicals including BPA (1-20 µM; p < 0.05). The effect mediated by TMBPF on expression levels of Fabp4, but not Plin, is likely mediated via peroxisome proliferator-activated receptor (PPAR) γ activation. CONCLUSIONS: Of the BPA analogues tested, BPF was most similar to BPA in its effects, while TMBPF was most adipogenic. In addition, TMBPF is likely a PPARγ agonist, it is likely an obesogenic chemical and may be a metabolic disruptor.

From vision toward best practices: Evaluating in vitro transcriptomic points of departure for application in risk assessment using a uniform workflow.

The growing number of chemicals in the current consumer and industrial markets presents a major challenge for regulatory programs faced with the need to assess the potential risks they pose to human and ecological health. The increasing demand for hazard and risk assessment of chemicals currently exceeds the capacity to produce the toxicity data necessary for regulatory decision making, and the applied data is commonly generated using traditional approaches with animal models that have limited context in terms of human relevance. This scenario provides the opportunity to implement novel, more efficient strategies for risk assessment purposes. This study aims to increase confidence in the implementation of new approach methods in a risk assessment context by using a parallel analysis to identify data gaps in current experimental designs, reveal the limitations of common approaches deriving transcriptomic points of departure, and demonstrate the strengths in using high-throughput transcriptomics (HTTr) to derive practical endpoints. A uniform workflow was applied across six curated gene expression datasets from concentration-response studies containing 117 diverse chemicals, three cell types, and a range of exposure durations, to determine tPODs based on gene expression profiles. After benchmark concentration modeling, a range of approaches was used to determine consistent and reliable tPODs. High-throughput toxicokinetics were employed to translate in vitro tPODs (µM) to human-relevant administered equivalent doses (AEDs, mg/kg-bw/day). The tPODs from most chemicals had AEDs that were lower (i.e., more conservative) than apical PODs in the US EPA CompTox chemical dashboard, suggesting in vitro tPODs would be protective of potential effects on human health. An assessment of multiple data points for single chemicals revealed that longer exposure duration and varied cell culture systems (e.g., 3D vs. 2D) lead to a decreased tPOD value that indicated increased chemical potency. Seven chemicals were flagged as outliers when comparing the ratio of tPOD to traditional POD, thus indicating they require further assessment to better understand their hazard potential. Our findings build confidence in the use of tPODs but also reveal data gaps that must be addressed prior to their adoption to support risk assessment applications.

Per- and polyfluoroalkyl substances (PFAS) in mixtures show additive effects on transcriptomic points of departure in human liver spheroids.

Per- and polyfluoroalkyl substances (PFAS) are a wide range of chemicals that are used in a variety of consumer and industrial products leading to direct human exposure. Many PFAS are chemically nonreactive and persistent in the environment, resulting in additional exposure from water, soil, and dietary intake. While some PFAS have documented negative health effects, data on simultaneous exposures to multiple PFAS (PFAS mixtures) are inadequate for making informed decisions for risk assessment. The current study leverages data from previous work in our group using Templated Oligo-Sequencing (TempO-Seq) for high-throughput transcriptomic analysis of PFAS-exposed primary human liver cell spheroids; herein, we determine the transcriptomic potency of PFAS in mixtures. Gene expression data from single PFAS and mixture exposures of liver cell spheroids were subject to benchmark concentration (BMC) analysis. We used the 25th lowest gene BMC as the point of departure to compare the potencies of single PFAS to PFAS mixtures of varying complexity and composition. Specifically, the empirical potency of 8 PFAS mixtures were compared to predicted mixture potencies calculated using the principal of concentration addition (ie, dose addition) in which mixture component potencies are summed by proportion to predict mixture potency. In this study, for most mixtures, empirical mixture potencies were comparable to potencies calculated through concentration addition. This work supports that the effects of PFAS mixtures on gene expression largely follow the concentration addition predicted response and suggests that effects of these individual PFAS in mixtures are not strongly synergistic or antagonistic.

Obesogens and Obesity: State-of-the-Science and Future Directions Summary from a Healthy Environment and Endocrine Disruptors Strategies Workshop.

On September 7 and 8, 2022, Healthy Environment and Endocrine Disruptors Strategies, an Environmental Health Sciences program, convened a scientific workshop of relevant stakeholders involved in obesity, toxicology, or obesogen research to review the state of the science regarding the role of obesogenic chemicals that might be contributing to the obesity pandemic. The workshop's objectives were to examine the evidence supporting the hypothesis that obesogens contribute to the etiology of human obesity; to discuss opportunities for improved understanding, acceptance, and dissemination of obesogens as contributors to the obesity pandemic; and to consider the need for future research and potential mitigation strategies. This report details the discussions, key areas of agreement, and future opportunities to prevent obesity. The attendees agreed that environmental obesogens are real, significant, and a contributor at some degree to weight gain at the individual level and to the global obesity and metabolic disease pandemic at a societal level; moreover, it is at least, in theory, remediable.

In vitro transcriptomic analyses reveal pathway perturbations, estrogenic activities, and potencies of data-poor BPA alternative chemicals.

Since initial regulatory action in 2010 in Canada, bisphenol A (BPA) has been progressively replaced by structurally related alternative chemicals. Unfortunately, many of these chemicals are data-poor, limiting toxicological risk assessment. We used high-throughput transcriptomics to evaluate potential hazards and compare potencies of BPA and 15 BPA alternative chemicals in cultured breast cancer cells. MCF-7 cells were exposed to BPA and 15 alternative chemicals (0.0005-100 µM) for 48 h. TempO-Seq (BioSpyder Inc) was used to examine global transcriptomic changes and estrogen receptor alpha (ERα)-associated transcriptional changes. Benchmark concentration (BMC) analysis was conducted to identify 2 global transcriptomic points of departure: (1) the lowest pathway median gene BMC and (2) the 25th lowest rank-ordered gene BMC. ERα activation was evaluated using a published transcriptomic biomarker and an ERα-specific transcriptomic point of departure was derived. Genes fitting BMC models were subjected to upstream regulator and canonical pathway analysis in Ingenuity Pathway Analysis. Biomarker analysis identified BPA and 8 alternative chemicals as ERα active. Global and ERα transcriptomic points of departure produced highly similar potency rankings with bisphenol AF as the most potent chemical tested, followed by BPA and bisphenol C. Further, BPA and transcriptionally active alternative chemicals enriched similar gene sets associated with increased cell division and cancer-related processes. These data provide support for future read-across applications of transcriptomic profiling for risk assessment of data-poor chemicals and suggest that several BPA alternative chemicals may cause hazards at similar concentrations to BPA.

The association between blood PFAS concentrations and clinical biochemical measures of organ function and metabolism in participants of the Canadian Health Measures Survey (CHMS).

Per- and poly-fluoroalkyl substances (PFAS) are ubiquitous and may persist in human tissue for several years. Only a small proportion of PFAS have been studied for human health effects. We tested the association between human blood levels of six PFAS and several clinical measures of organ and metabolic function in a nationally representative sample of 6768 participants aged 3-79 years old who participated in the Canadian Health Measures Survey. Cross-sectional associations were assessed by generalized linear mixed models incorporating survey-specific sampling weights. An increase in perfluorooctanoic acid (PFOA) equivalent to the magnitude of its geometric mean (GM) of 2.0 µg/L was associated with percentage (95% CI) increases in serum enzymes reflecting liver function: aspartate aminotransferase (AST) 3.7 (1.1, 6.4), gamma-glutamyl transferase (GGT) 11.8 (2.5, 21.8), alanine aminotransferase (ALT) 3.2 (0.5, 5.9), and bilirubin 3.6 (2.7, 4.5). A GM increase in perfluorodecanoic acid (PFDA) of 0.2 µg/L was positively associated with percentage increases in GGT, triglycerides, low-density lipoprotein (LDL) cholesterol, total cholesterol, and calcium with respective increases of 15.5 (2.2, 30.4), 7.0 (1.0, 13.2), 10.7 (5.5, 16.1), 2.8 (0.2, 5.3), and 0.8 (0.3, 1.3). PFOA, perfluorooctane sulfonate (PFOS), PFDA and perfluorononanoic acid (PFNA) were positively associated with GGT. All six congeners were positively associated with at least one biomarker of lipid metabolism, and 5 of 6, PFOA, PFOS, PFDA, perfluorohexane sulfonate (PFHxS) and PFNA were positively associated with serum calcium. Exposure to selected PFAS is associated with clinical blood tests reflecting metabolism and the function of several organ systems. These relatively small changes may possibly indicate early pathology that is clinically inapparent and may possibly be of significance in a general population or in individuals exposed to very high levels of PFAS.

Potency Ranking of Per- and Polyfluoroalkyl Substances Using High-Throughput Transcriptomic Analysis of Human Liver Spheroids.

Per- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood. Although the toxicological implications of human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on lesser-understood PFAS are limited. New approach methodologies (NAMs) that apply bioinformatic tools to high-throughput data are being increasingly considered to inform risk assessment for data-poor chemicals. The aim of this study was to compare the potencies (ie, benchmark concentrations: BMCs) of PFAS in primary human liver microtissues (3D spheroids) using high-throughput transcriptional profiling. Gene expression changes were measured using TempO-seq, a templated, multiplexed RNA-sequencing platform. Spheroids were exposed for 1 or 10 days to increasing concentrations of 23 PFAS in 3 subgroups: carboxylates (PFCAs), sulfonates (PFSAs), and fluorotelomers and sulfonamides. PFCAs and PFSAs exhibited trends toward increased transcriptional potency with carbon chain-length. Specifically, longer-chain compounds (7-10 carbons) were more likely to induce changes in gene expression and have lower transcriptional BMCs. The combined high-throughput transcriptomic and bioinformatic analyses support the capability of NAMs to efficiently assess the effects of PFAS in liver microtissues. The data enable potency ranking of PFAS for human liver cell spheroid cytotoxicity and transcriptional changes, and assessment of in vitro transcriptomic points of departure. These data improve our understanding of the possible health effects of PFAS and will be used to inform read-across for human health risk assessment.

Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study.

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30-70 % for pre-adipocyte proliferation; this was 40-80 % for triglyceride accumulation induced by pyraclostrobin, 80-100 % for tributyltin, and 80-100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30-70 % active determinations for pyraclostrobin, 30-50 % for tributyltin, and 20-40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.

High-Throughput Transcriptomic Analysis of Human Primary Hepatocyte Spheroids Exposed to Per- and Polyfluoroalkyl Substances as a Platform for Relative Potency Characterization.

Per- and poly-fluoroalkyl substances (PFAS) are widely found in the environment because of their extensive use and persistence. Although several PFAS are well studied, most lack toxicity data to inform human health hazard and risk assessment. This study focused on 4 model PFAS: perfluorooctanoic acid (PFOA; 8 carbon), perfluorobutane sulfonate (PFBS; 4 carbon), perfluorooctane sulfonate (PFOS; 8 carbon), and perfluorodecane sulfonate (PFDS; 10 carbon). Human primary liver cell spheroids (pooled from 10 donors) were exposed to 10 concentrations of each PFAS and analyzed at 4 time points. The approach aimed to: (1) identify gene expression changes mediated by the PFAS, (2) identify similarities in biological responses, (3) compare PFAS potency through benchmark concentration analysis, and (4) derive bioactivity exposure ratios (ratio of the concentration at which biological responses occur, relative to daily human exposure). All PFAS induced transcriptional changes in cholesterol biosynthesis and lipid metabolism pathways, and predicted PPARα activation. PFOS exhibited the most transcriptional activity and had a highly similar gene expression profile to PFDS. PFBS induced the least transcriptional changes and the highest benchmark concentration (ie, was the least potent). The data indicate that these PFAS may have common molecular targets and toxicities, but that PFOS and PFDS are the most similar. The transcriptomic bioactivity exposure ratios derived here for PFOA and PFOS were comparable to those derived using rodent apical endpoints in risk assessments. These data provide a baseline level of toxicity for comparison with other known PFAS using this testing strategy.

Transcriptomic pathway and benchmark dose analysis of Bisphenol A, Bisphenol S, Bisphenol F, and 3,3',5,5'-Tetrabromobisphenol A in H9 human embryonic stem cells.

Bisphenol A (BPA) is a chemical used in the manufacturing of plastics to which human exposure is ubiquitous. Numerous studies have linked BPA exposure to many adverse health outcomes prompting the replacement of BPA with various analogues including bisphenol-F (BPF) and bisphenol S (BPS). Other bisphenols are used in various consumer applications, such as 3,3',5,5'-Tetrabromobisphenol A (TBBPA), which is used as a flame retardant. Few studies to date have examined the effects of BPA and its analogues in stem cells to explore potential developmental impacts. Here we used transcriptomics to investigate similarities and differences of BPA and three of its analogues in the estrogen receptor negative, human embryonic stem cell line H9 (WA09). H9 cells were exposed to increasing concentrations of the bisphenols and analyzed using RNA-sequencing. Our data indicate that BPA, BPF, and BPS have similar potencies in inducing transcriptional changes and perturb many of the same pathways. TBBPA, the least structurally similar bisphenol of the group, exhibited much lower potency. All bisphenols robustly impacted gene expression in these cells, albeit at concentrations well above those observed in estrogen-positive cells. Overall, we provide a foundational data set against which to explore the transcriptional similarities of other bisphenols in embryonic stem cells, which may be used to assess the suitability of chemical grouping for read-across and for preliminary potency evaluation.

Effects of PCB126 on Adipose-to-Muscle Communication in an in Vitro Model.

BACKGROUND: Exposure to coplanar polychlorinated biphenyls (PCBs) is linked to the development of insulin resistance. Previous studies suggested PCB126 alters muscle mitochondrial function through an indirect mechanism. Given that PCBs are stored in fat, we hypothesized that PCB126 alters adipokine secretion, which in turn affects muscle metabolism. OBJECTIVES: We determined a) the impacts of PCB126 exposure on adipocyte cytokine/adipokine secretion in vitro; b) whether adipocyte-derived factors alter glucose metabolism and mitochondrial function in myotubes when exposed to PCB126; and c) whether preestablished insulin resistance alters the metabolic responses of adipocytes exposed to PCB126 and the communication between adipocytes and myotubes. METHODS: 3T3-L1 adipocytes were exposed to PCB126 (1-100 nM) in two insulin sensitivity conditions [insulin sensitive (IS) and insulin resistant (IR) adipocytes], followed by the measurement of secreted adipokines, mitochondrial function, and insulin-stimulated glucose uptake. Communication between adipocytes and myotubes was reproduced by exposing C2C12 myotubes or mouse primary myotubes to conditioned medium (CM) derived from IS or IR 3T3-L1 adipocytes exposed to PCB126. Mitochondrial function and insulin-stimulated glucose uptake were then determined in myotubes. RESULTS: IR 3T3-L1 adipocytes treated with PCB126 had significantly higher adipokine (adiponectin, IL-6, MCP-1, TNF-α) secretion and lower mitochondrial function, glucose uptake, and glycolysis. However, PCB126 did not significantly alter these parameters in IS adipocytes. Altered energy metabolism in IR 3T3-L1 adipocytes was linked to lower phosphorylation of AMP-activated protein kinase (p-AMPK) and higher superoxide dismutase 2 levels, an enzyme involved in reactive oxygen species detoxification. Myotubes exposed to the CM from PCB126-treated IR adipocytes had lower glucose uptake, with no alteration in glycolysis or mitochondrial function. Interestingly, p-AMPK levels were higher in myotubes exposed to the CM of PCB126-treated IR adipocytes. DISCUSSION: Taken together, these data suggest that increased adipokine secretion from IR adipocytes exposed to PCB126 might explain impaired glucose uptake in myotubes. https://doi.org/10.1289/EHP7058.

Fatty Acid Synthase Is a Key Enabler for Endocrine Resistance in Heregulin-Overexpressing Luminal B-Like Breast Cancer.

HER2 transactivation by the HER3 ligand heregulin (HRG) promotes an endocrine-resistant phenotype in the estrogen receptor-positive (ER+) luminal-B subtype of breast cancer. The underlying biological mechanisms that link them are, however, incompletely understood. Here, we evaluated the putative role of the lipogenic enzyme fatty acid synthase (FASN) as a major cause of HRG-driven endocrine resistance in ER+/HER2-negative breast cancer cells. MCF-7 cells engineered to stably overexpress HRG (MCF-7/HRG), an in vitro model of tamoxifen/fulvestrant-resistant luminal B-like breast cancer, showed a pronounced up-regulation of FASN gene/FASN protein expression. Autocrine HRG up-regulated FASN expression via HER2 transactivation and downstream activation of PI-3K/AKT and MAPK-ERK1/2 signaling pathways. The HRG-driven FASN-overexpressing phenotype was fully prevented in MCF-7 cells expressing a structural deletion mutant of HRG that is sequestered in a cellular compartment and lacks the ability to promote endocrine-resistance in an autocrine manner. Pharmacological inhibition of FASN activity blocked the estradiol-independent and tamoxifen/fulvestrant-refractory ability of MCF-7/HRG cells to anchorage-independently grow in soft-agar. In vivo treatment with a FASN inhibitor restored the anti-tumor activity of tamoxifen and fulvestrant against fast-growing, hormone-resistant MCF-7/HRG xenograft tumors in mice. Overall, these findings implicate FASN as a key enabler for endocrine resistance in HRG+/HER2- breast cancer and highlight the therapeutic potential of FASN inhibitors for the treatment of endocrine therapy-resistant luminal-B breast cancer.

Heregulin Drives Endocrine Resistance by Altering IL-8 Expression in ER-Positive Breast Cancer.

Sustained HER2/HER3 signaling due to the overproduction of the HER3 ligand heregulin (HRG) is proposed as a key contributor to endocrine resistance in estrogen receptor-positive (ER+) breast cancer. The molecular mechanisms linking HER2 transactivation by HRG-bound HER3 to the acquisition of a hormone-independent phenotype in ER+ breast cancer is, however, largely unknown. Here, we explored the possibility that autocrine HRG signaling drives cytokine-related endocrine resistance in ER+ breast cancer cells. We used human cytokine antibody arrays to semi-quantitatively measure the expression level of 60 cytokines and growth factors in the extracellular milieu of MCF-7 cells engineered to overexpress full-length HRGß2 (MCF-7/HRG cells). Interleukin-8 (IL-8), a chemokine closely linked to ER inaction, emerged as one the most differentially expressed cytokines. Cytokine profiling using structural deletion mutants lacking both the N-terminus and the cytoplasmic-transmembrane region of HRGß2-which is not secreted and cannot transactivate HER2-or lacking a nuclear localization signal at the N-terminus-which cannot localize at the nucleus but is actively secreted and transactivates HER2-revealed that the HRG-driven activation of IL-8 expression in ER+ cells required HRG secretion and transactivation of HER2 but not HRG nuclear localization. The functional blockade of IL-8 with a specific antibody inversely regulated ERα-driven transcriptional activation in endocrine-sensitive MCF-7 cells and endocrine-resistant MCF-7/HRG cells. Overall, these findings suggest that IL-8 participates in the HRG-driven endocrine resistance program in ER+/HER2- breast cancer and might illuminate a potential clinical setting for IL8- or CXCR1/2-neutralizing antibodies.

Exposure to Low Doses of Dechlorane Plus Promotes Adipose Tissue Dysfunction and Glucose Intolerance in Male Mice.

The prevalence of type 2 diabetes (T2D) continues to increase worldwide. It is well established that genetic susceptibility, obesity, overnutrition and a sedentary life style are risk factors for the development of T2D. However, more recently, studies have also proposed links between exposure to endocrine-disrupting chemicals (EDCs) and altered glucose metabolism. Human exposure to environmental pollutants that are suspected to have endocrine disruptor activity is ubiquitous. One such chemical is Dechlorane Plus (DP), a flame retardant, that is now detected in humans and the environment. Here we show that exposure of mice to low, environmentally relevant doses of DP promoted glucose intolerance in mice fed a high-fat diet independent of weight gain. Furthermore, DP had pronounced effects on the adipose tissue, where it induced the development of hypertrophied white adipose tissue (WAT), and increased serum levels of resistin, leptin, and plasminogen activator inhibitor-1. In addition, DP exposure induced "whitening" of brown adipose tissue (BAT), and reduced BAT uncoupling protein 1 expression. Importantly, some of these effects occurred even when the mice were fed a regular, low-fat, diet. Finally, WAT adipogenic markers were reduced with DP treatment in the WAT. We also show that DP directly inhibited insulin signaling in murine adipocytes and human primary subcutaneous adipocytes in vitro. Taken together, our results show that the exposure to low and environmentally relevant levels of DP may contribute to the development of T2D.

Depot-Specific Analysis of Human Adipose Cells and Their Responses to Bisphenol S.

Exposure to endocrine-disrupting chemicals (EDCs) is associated with adverse health outcomes including obesity and diabetes. Obesity, and more specifically visceral obesity, is correlated with metabolic disease. The adipose tissue is an endocrine organ and a potential target for many environmental pollutants including bisphenols. The subcutaneous (Sc) and the omental (Om, visceral) depots are composed of mature adipocytes and residing progenitors, which may be different between the depots and may be EDCs targets. Bisphenol A (BPA) is a suspected metabolic disruptor, and is being replaced with structurally similar compounds such as bisphenol S (BPS). Like BPA, BPS induces adipogenesis in murine and primary human Sc preadipocytes. However, the effect of BPS on Om preadipocytes is not known. In this study, we show that human primary progenitors from Om depots have a distinct transcriptomic signature as compared to progenitors derived from donor-matched Sc depots. Furthermore, we show that BPS increases adipogenesis both of Om and Sc preadipocytes and can mimic the action of glucocorticoids or peroxisome proliferator-activated receptor γ (PPARγ) agonists. We also show that BPS treatment, at 0.1 µM and 25 µM, modifies the adipokine profiles both of Om- and Sc-derived adipocytes in a depot-specific manner. Taken together our data show distinct gene expression profiles in the Om vs Sc progenitors and similar responses to the BPA analogue, BPS.

Bisphenol S and Bisphenol A disrupt morphogenesis of MCF-12A human mammary epithelial cells.

Breast cancer is one of the most common cancers diagnosed in women worldwide. Genetic predisposition, such as breast cancer 1 (BRCA1) mutations, account for a minor percentage of the total breast cancer incidences. And thus, many life style factors have also been linked to the disease such as smoking, alcohol consumption and obesity. Emerging studies show that environmental pollutants may also play a role. Bisphenol-A (BPA) has been suspected to contribute to breast cancer development, and has been shown to affect mammary gland development amongst other effects. This prompted its replacement with other bisphenol analogs such as, bisphenol-S (BPS). In this study we used the human mammary epithelial cells, MCF-12A, grown in extracellular matrix to investigate the ability of BPA and BPS to disrupt mammary epithelial cells organization. We show that both BPA and BPS were equipotent in disrupting the organization of the acinar structures, despite BPS being less oestrogenic by other assays. Further, treatment with both compounds enabled the cells to invade the lumen of the structures. This study shows that BPS and BPA are environmental pollutants that may affect mammary development and may contribute to the development of breast cancer.