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Perfluorobutanesulfonic acid (PFBS) is a replacement for perfluorooctanesulfonic acid (PFOS) that is increasingly detected in drinking water and human serum. Higher PFBS exposure is associated with risk for preeclampsia, the leading cause of maternal and infant morbidity and mortality in the United States. This study investigated relevant maternal and fetal health outcomes after gestational exposure to PFBS in a New Zealand White rabbit model. Nulliparous female rabbits were supplied drinking water containing 0 mg/l (control), 10 mg/l (low), or 100 mg/l (high) PFBS. Maternal blood pressure, body weights, liver and kidney weights histopathology, clinical chemistry panels, and thyroid hormone levels were evaluated. Fetal endpoints evaluated at necropsy included viability, body weights, crown-rump length, and liver and kidney histopathology, whereas placenta endpoints included weight, morphology, histopathology, and full transcriptome RNA sequencing. PFBS-high dose dams exhibited significant changes in blood pressure markers, seen through increased pulse pressure and renal resistive index measures, as well as kidney histopathological changes. Fetuses from these dams showed decreased crown-rump length. Statistical analysis of placental weight via a mixed model statistical approach identified a significant interaction term between PFBS high dose and fetal sex, suggesting a sex-specific effect on placental weight. RNA sequencing identified the dysregulation of angiotensin (AGT) in PFBS high-dose placentas. These results suggest that PFBS exposure during gestation leads to adverse maternal outcomes, such as renal injury and hypertension, and fetal outcomes, including decreased growth parameters and adverse placenta function. These outcomes raise concerns about pregnant women’s exposure to PFBS and pregnancy outcomes.

Introduction Smoking and smoke exposure among pregnant women remain persistent public health issues. Recent estimates suggest that approximately one out of four nonsmokers have measurable levels of cotinine, a marker indicating regular exposure to secondhand smoke. Epidemiological research has attempted to pinpoint individual-level and neighborhood-level factors for smoking during pregnancy. However, most of these studies have relied upon self-reported measures of smoking. Aims and Methods To more accurately assess smoke exposure resulting from both smoking and secondhand exposure in mothers during pregnancy, we used Bayesian regression models to estimate the association of cotinine levels with tobacco retail outlet (TRO) exposure and a neighborhood deprivation index (NDI) in six counties in North Carolina centered on Durham County. Results Results showed a significant positive association between TRO exposure (β = 0.008, 95% credible interval (CI) = [0.003, 0.013]) and log cotinine after adjusting for individual covariates (eg, age, race/ethnicity, education, marital status). TRO exposure was not significant after including the NDI, which was significantly associated with log cotinine (β = 0.143, 95% CI = [0.030, 0.267]). However, in a low cotinine stratum (indicating secondhand smoke exposure), TRO exposure was significantly associated with log cotinine (β = 0.005, 95% CI = [0.001, 0.009]), while in a high cotinine stratum (indicating active smoking), the NDI was significantly associated with log cotinine (β = 0.176, 95% CI = [0.005, 0.372]). Conclusions In summary, our findings add to the evidence that contextual factors are important for active smoking during pregnancy. Implications In this study, we found several significant associations that suggest a more nuanced understanding of the potential influence of environmental- and individual-level factors for levels of prenatal smoke exposure. Results suggested a significant positive association between TRO exposure and cotinine levels, after adjusting for the individual factors such as race, education, and marital status. Individually, NDI was similarly positively associated with cotinine levels as well. However, when combining TRO exposure alongside NDI in the same model, TROs were no longer significantly associated with overall cotinine levels.

Maternal smoking is associated with a host of negative health outcomes, including an increased risk of children developing attention-deficit/hyperactivity disorder (ADHD). This study evaluated the efficacy of health messages disseminated through Facebook Ads focused on reducing tobacco smoke exposure during pregnancy. Two message versions were promoted via post advertisements on Facebook—a static infographic and a video containing an animated version of the infographic. The reach of and engagement with each message version was evaluated. Comments made to the posts were assessed using content analysis. The infographic reached approximately 60,000 people and the video reached about 16,000 people. The average costs were $10.00 and $40.00 per 1,000 people reached for the infographic- and video-based posts, respectively. While there was no engagement with the video, the infographic was liked (n = 157), given alternative likes (n = 59), shared (n = 171 to 341), and commented on (n = 221). About one-quarter of comments contained a personal narrative and mentions of health history related to ADHD and/or smoking. Comments were more often negative (than positive) (16.6% vs 3.9%) and expressed skepticism more often than message acceptance (21.5% vs 12.2%). Facebook users were more responsive to the infographic (compared to the video) and static posts were a preferred channel (i.e., higher engagement at a lower cost) to disseminate messages when using the boost post feature on Facebook for health education. Our review of the comments provided insights into message acceptance and guidance for future social media-based health message campaigns. However, it is not known whether and if so, how, these findings on message exposure would correlate with behavioral intentions or changes in behavior, such as intentions to quit smoking or smoking cessation.

Nicotine is a neuroteratogenic component of tobacco smoke, e-cigarettes, and other products and can exert sex-specific effects in the developing brain, likely mediated through sex hormones. Estradiol modulates expression of nicotinic acetylcholine receptors in rats, and plays critical roles in neurodevelopmental processes, including sexual differentiation of the brain. Here, we examined the effects of developmental nicotine exposure on the sexual differentiation of the preoptic area (POA), a brain region that normally displays robust structural sexual dimorphisms and controls adult mating behavior in rodents. Using a rat model of gestational exposure, developing pups were exposed to nicotine (2 mg/kg/day) via maternal osmotic minipump (subcutaneously, sc) throughout the critical window for brain sexual differentiation. At postnatal day (PND) 4, a subset of offspring was analyzed for epigenetic effects in the POA. At PND40, all offspring were gonadectomized, implanted with a testosterone-releasing capsule (sc), and assessed for male sexual behavior at PND60. Following sexual behavior assessment, the area of the sexually dimorphic nucleus of the POA (SDN-POA) was measured using immunofluorescent staining techniques. In adults, normal sex differences in male sexual behavior and in the SDN-POA area were eliminated in nicotine-treated animals. Using novel analytical approaches to evaluate overall masculinization of the adult POA, we identified significant masculinization of the nicotine-treated female POA. In neonates (PND4), nicotine exposure induced trending alterations in methylation-dependent masculinizing gene expression and DNA methylation levels at sexually-dimorphic differentially methylated regions, suggesting that developmental nicotine exposure is capable of triggering masculinization of the rat POA via epigenetic mechanisms.

Figures S1, S2 and Tables S1,S2 and S7

Legends describing all supplemental figures.

A. ChIP-qPCR on H3.3K27M-GFP, and H3.3WT-GFP (n=3 each) expressing murine tumor lines for DNMT1 localization to p16 promoter region. B. Pyrosequencing analysis of DNA methylation at the p16 promoter after 7days of 1uM GSK343 treatment (n=3 for both groups). C. Pyrosequencing DNA methylation analysis of p16 and p19 promoters on human DIPG lines (SF8628, DIPG IV, and DIPG 007) treated with 72hr decitabine or vehicle at 10uM (*= p< 0.05, n=3). D. Western blot analysis of human DIPG lines treated with 72hr decitabine or vehicle and blotted for p16 (n=2) E. BrdU incorporation IC50 analysis of decitabine and azacitidine DNA methyltransferase inhibitors on mouse tumor cells (top 2 panels). Decitabine IC50 on human DIPG lines (bottom 3 panels). IC50''s for human lines were as follows: SF6828 (8μM), (0.5μM), DIPG IV, and DIPG 007 (2μM).

Functional annotations (from DAVID) for genes with higher expression in cells with low spheroid-forming capacity relative to cells with high spheroid-forming capacity.

A. Positive (Hoxa11) and negative (actin) promoter control loci for H3K27me3 ChIP-qPCR. B. H3K27me3 ChIP-qPCR analysis of murine tumor cells on downstream p16 exon 1α and exon 2. C. Histone extraction western blot of H3.3K27M-GFP and H3.3WT-GFP expressing murine tumor lines treated with 1μM EPZ-6438 for 7 days. D. H3K4me3 ChIP-qPCR analyzing the p16 promoter region with or without 7 day GSK343 treatment (p= 0.035 by paired t-test) E. 21 day treatment with EZH2 inhibitor GSK343 followed by qPCR for p16 expression (p=0.036) F. BrdU incorporation on H3.3K27M-GFP and H3.3WT-GFP expressing murine tumor lines with EZH2 inhibitors GSK343 (p= 0.012), EPZ-6438, and GSK126 (p= 0.002) treatments incubated for 72hrs.

list of all viral infection combinations performed on mice and the median survival (days) of each model.

Percent DNA methylation and p16 expression changes with or without DNA methytransferase inhibition.

Dysregulation of MAL (myelin and lymphocyte protein) has been implicated in several malignancies including esophageal, ovarian, and cervical cancers. The MAL protein functions in apical transport in polarized epithelial cells; therefore, its disruption may lead to loss of organized polarity characteristic of most solid malignancies. Bisulfite sequencing of the MAL promoter CpG island revealed hypermethylation in breast cancer cell lines and 69% of primary tumors analyzed compared with normal breast epithelial cells. Differential methylation between normal and cancer DNA was confined to the proximal promoter region. In a subset of breast cancer cell lines including T47D and MCF7 cells, promoter methylation correlated with transcriptional silencing that was reversible with the methylation inhibitor 5-aza-2′-deoxycytidine. In addition, expression of MAL reduced motility and resulted in a redistribution of lipid raft components in MCF10A cells. MAL protein expression measured by immunohistochemistry revealed no significant correlation with clinicopathologic features. However, in patients who did not receive adjuvant chemotherapy, reduced MAL expression was a significant predictive factor for disease-free survival. These data implicate MAL as a commonly altered gene in breast cancer with implications for response to chemotherapy. (Mol Cancer Res 2009;7(2):199–209)

Supplementary Figures 1 - 3

Supplementary Data from Yin Yang 1 Modulates Taxane Response in Epithelial Ovarian Cancer

Supplementary Movie 1 from GPR54 Is a Target for Suppression of Metastasis in Endometrial Cancer

Representative photomicrographs of ERα staining in a transitional duct from a prepubertal animal (A); ERα staining in post-pubertal, differentiated lobuloalveolar tissue (B); ERβ staining in prepubertal transitional duct (C ); and ERβ staining in mature lobuloalveolar tissue (D). Bar = 50 microns.

lists of antibodies and primer information used in the manuscript.

Immunolocalization of Steroidogenic Enzymes in the Mammary Gland.

List of all significantly differentially expressed genes in empty vector vs H3.3K27M tumor RNA-Seq analysis.

Endogenous estrogens influence mammary gland development during puberty and breast cancer risk during adulthood. Early-life exposure to dietary or environmental estrogens may alter estrogen-mediated processes. Soy foods contain phytoestrogenic isoflavones (IF), which have mixed estrogen agonist/antagonist properties. Here, we evaluated mammary gland responses over time in pubertal female cynomolgus macaques fed diets containing either casein/lactalbumin (n = 12) or soy protein containing a human-equivalent dose of 120 mg IF/day (n = 17) for approximately 4.5 years spanning menarche. We assessed estrogen receptor (ER) expression and activity, promoter methylation of ERs and their downstream targets, and markers of estrogen metabolism. Expression of ERα and classical ERα response genes (TFF1, PGR, and GREB1) decreased with maturity, independent of diet. A significant inverse correlation was observed between TFF1 mRNA and methylation of CpG sites within the TFF1 promoter. Soy effects included lower ERβ expression before menarche and lower mRNA for ERα and GREB1 after menarche. Expression of GATA-3, an epithelial differentiation marker that regulates ERα-mediated transcription, was elevated before menarche and decreased after menarche in soy-fed animals. Soy did not significantly alter expression of other ER activity markers, estrogen-metabolizing enzymes, or promoter methylation for ERs or ER-regulated genes. Our results demonstrate greater ER expression and activity during the pubertal transition, supporting the idea that this life stage is a critical window for phenotypic modulation by estrogenic compounds. Pubertal soy exposure decreases mammary ERα expression after menarche and exerts subtle effects on receptor activity and mammary gland differentiation. Cancer Prev Res; 9(5); 385–95. ©2016 AACR.

Gene expression profile in the post-menarchal macaque breast by microarray. (A) Venn diagram showing number of genes that changed with time (i.e. between 7-12 months to 18-24 months after menarche) in the control (CL) and soy group (SOY), based on unadjusted P-values from a paired t-test in each diet group. (B) The list of top-10 genes that showed significant difference by dietary treatment using empirical Bayes statistics. (C) The top-10 significantly enriched KEGG pathways in CL and SOY groups across time. FC = fold-change; FDR=false discovery rate.

1A. Cynomolgus macaque and human primer probe sets used to generate custom Taqman-based assay for qRT-PCR 1B. Applied Biosystems (ABI) Taqman gene expression assays used in qRT-PCR.

Ovarian cancer is the deadliest gynecologic cancer. Despite recent advances, clinical outcomes remain poor, necessitating novel therapeutic approaches. To investigate metabolic susceptibility, we performed nutrigenetic screens on a panel of clear cell and serous ovarian cancer cells and identified cystine addiction and vulnerability to ferroptosis, a novel form of regulated cell death. Our results may have therapeutic potential, but little is known about the determinants of ferroptosis susceptibility in ovarian cancer. We found that vulnerability to ferroptosis in ovarian cancer cells is enhanced by lower cell confluency. Because the Hippo pathway effectors Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) are recognized as sensors of cell density, and TAZ is the predominant effector in the tested ovarian cancer cell lines, we investigated the role of TAZ in ferroptosis of ovarian cancer. TAZ removal confers ferroptosis resistance, while TAZS89A overexpression sensitizes cells to ferroptosis. In addition, we found that lower TAZ level in chemo-resistant recurrent ovarian cancer is responsible for reduced ferroptosis susceptibility. The integrative genomic analysis identified ANGPTL4 as a direct TAZ-regulated target gene that sensitizes ferroptosis by activating NOX2. Collectively, cell density–regulated ferroptosis in ovarian cancer is mediated by TAZ through the regulation of the ANGPTL4–NOX2 axis, suggesting therapeutic potentials for ovarian cancers and other TAZ-activated tumors.Implications:This study reveals that TAZ promotes ferroptosis in ovarian cancers by regulating ANGPTL4 and NOX, offering a novel therapeutic potential for ovarian tumors with TAZ activation.

A. Gene set enrichment analysis of differentially upregulated genes from RNA-Seq of high grade tumor model with or without H3.3K27M-HA expression shows high association with known PRC2-regulated H3K27me3 target genes in mouse embryonic, and neuronal progenitor cells (1). B. GSEA comparing genes losing H3K27me3 (UPS) and gaining H3K27me3 (DOWN) from Chan et al to our mouse tumor RNA-Seq. C. qPCR analysis of both p16 and p19 alternate transcripts from ex-vivo infected neuronal progenitor cells isolated from neonatal Nestin-tv-a pups and infected with either H3.3K27M-HA, H3.3WT-HA, or empty vector 2 weeks prior to collection.

Cynomolgus macaque primer sets (forward, F; reverse, R; and sequencing, S) for pyrosequencing assay.

A. Analysis of tumor volume of PDGF driven, p53 WT models with expression of H3.3K27M (n= 6), H3.3WT (n= 6), or empty vector (n= 7) shows trend for larger tumors with mutation compared to H3.3WT (p= 0.25) or empty vector (p= 0.28) controls. B. Kaplan-Meier survival curve of murine tumors driven by co-injection of nestin tv-a; p53 floxed mice with RCAS-PDGFB; RCAS-Cre; and either RCAS-H3.3K27M-GFP (n=12) or RCAS-H3.3WT-GFP (n= 18). C. In vivo proliferation quantification of H3.3K27M or H3.3WT expressing tumors driven by PDGFB overexpression and p53 knockout using pH3 (H3.3K27M n= 5, H3.3WT n=4) or D. EdU incorporation IF quantification (n=3 each) E. Histone extraction followed by western blot using total H3 antibody comparing murine tumor cells infected with empty vector, H3.3WT, or H3.3K27M to determine the amount of H3 overexpression.

Serum isoflavonoid concentrations across the pubertal transition. Levels of circulating total isoflavonoids (genistein + daidzein + equol) were significantly higher in soy-fed monkeys. Values represent LSMs for n=11-17 monkeys/group (bars = SEM). Significant main effects and interactions are indicated in the panel.

Spheroids exhibit drug resistance and slow proliferation, suggesting involvement in cancer recurrence. The protein kinase C inhibitor UCN-01 (7-hydroxystaurosporine) has shown higher efficacy against slow proliferating and/or quiescent ovarian cancer cells. In this study, tumorigenic potential was assessed using anchorage-independent growth assays and spheroid-forming capacity, which was determined with ovarian cancer cell lines as well as primary ovarian cancers. Of 12 cell lines with increased anchorage-independent growth, 8 formed spheroids under serum-free culture conditions. Spheroids showed reduced proliferation (P < 0.0001) and Ki-67 immunostaining (8% vs. 87%) relative to monolayer cells. Spheroid formation was associated with increased expression of mitochondrial pathway genes (P ≤ 0.001) from Affymetrix HT U133A gene expression data. UCN-01, a kinase inhibitor/mitochondrial uncoupler that has been shown to lead to Puma-induced mitochondrial apoptosis as well as ATP synthase inhibitor oligomycin, demonstrated effectiveness against spheroids, whereas spheroids were refractory to cisplatin and paclitaxel. By live in vivo imaging, ovarian cancer xenograft tumors were reduced after primary treatment with carboplatin. Continued treatment with carboplatin was accompanied by an increase in tumor signal, whereas there was little or no increase in tumor signal observed with subsequent treatment with UCN-01 or oltipraz. Taken together, our findings suggest that genes involved in mitochondrial function in spheroids may be an important therapeutic target in preventing disease recurrence.

Supplementary Tables S1-S3 from Microarray Analysis of Early Stage Serous Ovarian Cancers Shows Profiles Predictive of Favorable Outcome

Purpose: Cancer/testis (CT) genes predominantly expressed in the testis (germ cells) and generally not in other normal tissues are aberrantly expressed in human cancers. This highly restricted expression provides a unique opportunity to use these CT genes for diagnostics, immunotherapeutic, or other targeted therapies. The purpose of this study was to identify those CT genes with the greatest incidence of expression in uterine cancers.Experimental Design: We queried the expression of known and putative CT gene transcripts (representing 79 gene loci) using whole genome gene expression arrays. Specifically, the global gene expressions of uterine cancers (n = 122) and normal uteri (n = 10) were determined using expression data from the Affymetrix HG-U133A and HG-U133B chips. Additionally, we also examined the brother of the regulator of imprinted sites (BORIS) transcript by reverse transcription-PCR and quantitative PCR because its transcript was not represented on the array.Results: Global microarray analysis detected many CT genes expressed in various uterine cancers; however, no individual CT gene was expressed in more than 25% of all cancers. The expression of the two most commonly expressed CT genes on the arrays, MAGEA9 (24 of 122 cancers and 0 of 10 normal tissues) and Down syndrome critical region 8 (DSCR8)/MMA1 (16 if 122 cancers and 0 of 10 normal tissues), was confirmed by reverse transcription-PCR methods, validating the array screening approach. In contrast to the relatively low incidence of expression of the other CT genes, BORIS expression was detected in 73 of 95 (77%) endometrial cancers and 24 of 31 (77%) uterine mixed mesodermal tumors.Conclusions: These data provide the first extensive survey of multiple CT genes in uterine cancers. Importantly, we detected a high frequency of BORIS expression in uterine cancers, suggesting its potential as an immunologic or diagnostic target for these cancers. Given the high incidence of BORIS expression and its possible regulatory role, an examination of BORIS function in the etiology of these cancers is warranted.

Purpose: Although few women with advanced serous ovarian cancer are cured, detection of the disease at an early stage is associated with a much higher likelihood of survival. We previously used gene expression array analysis to distinguish subsets of advanced cancers based on disease outcome. In the present study, we report on gene expression of early-stage cancers and validate our prognostic model for advanced-stage cancers.Experimental Design: Frozen specimens from 39 stage I/II, 42 stage III/IV, and 20 low malignant potential cancers were obtained from four different sites. A linear discriminant model was used to predict survival based upon array data.Results: We validated the late-stage survival model and show that three of the most differentially expressed genes continue to be predictive of outcome. Most early-stage cancers (38 of 39 invasive, 15 of 20 low malignant potential) were classified as long-term survivors (median probabilities 0.97 and 0.86). MAL, the most differentially expressed gene, was further validated at the protein level and found to be an independent predictor of poor survival in an unselected group of advanced serous cancers (P = 0.0004).Conclusions: These data suggest that serous ovarian cancers detected at an early stage generally have a favorable underlying biology similar to advanced-stage cases that are long-term survivors. Conversely, most late-stage ovarian cancers seem to have a more virulent biology. This insight suggests that if screening approaches are to succeed it will be necessary to develop approaches that are able to detect these virulent cancers at an early stage.