Kelli E. Valdez

Ovarian Endocrine Disruption Underlies Premature Reproductive Senescence Following Environmentally Relevant Chronic Exposure to the Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Abstract The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. While previous work has focused on mechanisms of short-term endocrine disruption and ovotoxicity in response to AHR ligands, we have shown recently that chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces premature reproductive senescence in female rats without depletion of ovarian follicular reserves. In the current study, premature reproductive senescence was induced using a range of low-dose exposure to TCDD (0, 1, 5, 50, and 200 ng kg−1 wk−1) beginning in utero and continuing until the transition to reproductive senescence. Doses of 50 and 200 ng TCDD kg−1 wk−1 delayed the age at vaginal opening and accelerated the loss of normal reproductive cyclicity with age without depletion of follicular reserves. Serum estradiol concentrations were decreased in a dose-dependent fashion (≥5 ng kg−1 wk−1) across the estrous cycle in perisenescent rats still displaying normal cyclic vaginal cytology. Serum FSH, LH, and progesterone profiles were unchanged by TCDD. The loss of reproductive cyclicity following chronic exposure to TCDD was not accompanied by decreased responsiveness to GnRH. Ovarian endocrine disruption is the predominant functional change preceding the premature reproductive senescence induced by chronic exposure to low doses of the AHR-specific ligand TCDD.

Human primary ductal carcinoma in situ (DCIS) subtype-specific pathology is preserved in a mouse intraductal (MIND) xenograft model

Ductal carcinoma in situ (DCIS) is a non‐obligate precursor of invasive breast cancer. The current recognition that DCIS lesions exhibit inter‐ and intra‐lesion diversity suggests that the process of evolution to invasive breast cancer is more complex than previously recognized. Here we demonstrate the reproducible growth of primary DCIS cells derived from patients surgical and biopsy samples by the mouse intraductal (MIND) model. MIND involves injection of cells into the NOD‐SCID IL2Rgamma

Effects of Acute and Chronic Exposure to the Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the Transition to Reproductive Senescence in Female Sprague-Dawley Rats

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Effect of chronic exposure to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin in female rats on ovarian gene expression

(NSG) mouse mammary ducts. Twelve (eight unique and four repeats) DCIS and two atypical hyperplasia specimens, heterogeneous with respect to biomarker expression and histology, were injected into 48 mouse mammary glands and analysed for successful xenotransplantation. Overall, 14/34 and 11/14 MIND xenotransplanted glands contained human DCIS and atypical hyperplastic cells, respectively, after 8 weeks, which formed single and multi‐layered epithelium inside the ducts, and were heterogeneous with respect to expression of human cytokeratins, oestrogen receptor α (ER), and HER2. ER protein expression was recapitulated in MIND xenografts at ratios similar to the corresponding patient biopsies. In both patient biopsies and corresponding MIND xenografts, HER2 protein expression and nuclear HER2 gene overexpression were restricted to the DCIS lesions and were not found in the surrounding stroma or normal ducts. The xenografted DCIS lesions recapitulate the pathology and heterogeneity of human disease, thus providing a powerful tool for the characterization of the distinct cellular and molecular basis of inter‐ and intra‐tumoural heterogeneity and the processes of DCIS to early invasive breast cancer progression. Copyright

Temporal and anatomical sensitivities to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin leading to premature acyclicity with age in rats.

Abstract Activation of the aryl hydrocarbon receptor (AHR) can occur in polluted environments, either from smoking-related toxicants or from endogenous ligands. We tested whether acute or chronic exposure to the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the transition to reproductive senescence in female Sprague-Dawley rats. In experiment 1, rats (n = 6 per experimental group) received a single dose of 0 or 10 μg/kg of TCDD orally (p.o.) on Postnatal Day 29. Vaginal cytology was monitored for 1 wk each month until rats were killed at 1 yr of age. The single prepubertal exposure to TCDD hastened the transition to reproductive senescence in female rats and was associated with delayed puberty, abnormal cyclicity, and premature reproductive senescence. In a second experiment, rats were exposed to TCDD chronically through weekly dosing (0, 50, or 200 ng kg−1 wk−1 p.o., n = 7 each dose) beginning in utero. Lifelong exposure to these lower doses of TCDD induced a dose- and time-dependent loss of normal cyclicity and significantly hastened the onset of the transition to reproductive senescence (P < 0.05). This premature transition to reproductive senescence was associated with prolonged estrous cycles and, at the highest dose of TCDD, persistent estrus or diestrus. The number and size of ovarian follicles were not altered by TCDD. Diestrous concentrations of LH in rats exposed chronically to TCDD were similar to those in controls, whereas progesterone tended to be elevated at both doses of the dioxin (P < 0.08). Serum FSH was elevated in the group exposed to 50 ng/kg of TCDD (P < 0.02), whereas estradiol was decreased at both doses of dioxin (P < 0.01). Data thus far support endocrine disruption rather than depletion of follicular reserves as a primary mechanism of the premature transition to reproductive senescence following activation of the AHR pathway by TCDD in female rats.

Expression profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion

The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. Female rats exposed chronically to environmentally relevant doses of the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) across their lifespan experience accelerated reproductive senescence preceded by ovarian endocrine disruption. The purpose of this study was to determine the changes in ovarian gene expression that accompany the loss of ovarian function caused by chronic exposure to TCDD. Beginning in utero, female Sprague-Dawley rats received TCDD (1, 5, 50, or 200 ng/kg-week; n=4 per group) or vehicle weekly throughout their lifespan, and were sacrificed on diestrus just prior to loss of reproductive cyclicity at 11 months of age. Microarray analysis was used to determine differences in ovarian gene expression between control and TCDD-treated (200 ng/kg-week) animals. To confirm microarray results, real-time PCR was used to assess changes in gene expression among treatment groups. TCDD treatment decreased (p<0.05) proestrus serum estradiol concentrations with no effect on serum progesterone. In ovaries from rats treated with 200 ng/kg-week TCDD compared to controls, 19 genes of known function were found to be up-regulated, while 31 ovarian genes were found to be down-regulated >or=1.5-fold (p<or=0.05). Gene expression of 17 alpha-hydroxylase decreased following chronic TCDD treatment, suggesting the decrease in estradiol biosynthesis may be a consequence of decreased substrate. Taken together with past studies indicating a lack of effect on hypothalamus or pituitary function, the apparent regulation of key ovarian genes support the hypothesis that chronic TCDD exposure directly affects ovarian function.

A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates diverse dioxin toxicities. While the acute effects of activation of the AhR pathway by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been a focus of past study, the role of this pathway in normal physiology and ageing is unclear. The purpose of this study was to identify the portion of the reproductive axis [ovary vs. hypothalamus and pituitary gland (H-H axis)] and the stages of the reproductive lifespan (foetal and early post-natal life vs. adolescence and adulthood) that are particularly sensitive to the effects of TCDD during female reproductive ageing. Adult pregnant Lewis rat dams were dosed with corn oil vehicle or TCDD (50 ng/kg-week by gavage) on days 14 and 21 of gestation and post-natal days 7 and 14 to provide in utero and lactational (IUL) exposure to pups. Female pups (n = 96) were weaned on post-natal day 21 and dosed with TCDD or vehicle weekly. Half of the pups were used as donors for ovary transplantation while the remainder were recipients. Following ovary transplantation, rats (n = 6-8 per group) received weekly TCDD or vehicle again until sacrifice at 8 months of age. Beginning at vaginal opening, reproductive cycles were monitored by vaginal cytology for 10 days each month. Blood samples were collected at 22.00 h on proestrus to measure concentration of 17beta-oestradiol in serum. Real-time PCR was used to determine differences in Cyp1a1, Cyp19a1, Cyp17a1, LH receptor (LHR), FoxA2 and FoxJ1 genes expression between control and remaining groups. IUL exposure of the H-H axis plus adult exposure of the whole body to TCDD significantly delayed puberty in females rats. Data analysis revealed an accelerated onset of acyclicity by 5 months in all groups involving IUL exposure of the developing ovary to TCDD. 17beta-oestradiol was significantly decreased in animals receiving TCDD during IUL exposure of the H-H axis. CYP1a1 expression was markedly greater in the liver than in ovarian tissue and correlated with ongoing TCDD exposure. Aromatase, 17alpha-hydroxylase and LHR gene expressions were largely unchanged (or occasionally elevated) by TCDD. FoxA2 and FoxJ1 mRNAs were similarly of limited value mechanistically, although FoxJ1 was much higher in TTT females (receiving TCDD as donor, recipient and adult). This study reveals a particular sensitivity of the developing ovary to TCDD leading to early loss of reproductive function with age.

Emerging Functions of microRNA-146a/b in Development and Breast Cancer

IntroductionThere are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized.MethodsMicroarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers.ResultsAnalysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification.ConclusionBCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.

Targeting the Perpetrator: Breast Cancer Stem Cell Therapeutics

Summary In this report, we have shown that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. MiR146b expression was significantly higher in the mammary glands of pregnant and lactating mice than in virgin mice. Furthermore, miR146b levels were significantly higher in mouse mammary glands exposed to the sex hormones, estrogen and progesterone, compared with those of untreated control animals. Pregnancy-derived primary mouse mammary epithelial cells in which miR146b was knocked down showed a significant reduction in the number of hollow acinar organoid structures formed on three-dimensional Matrigel and in &bgr;-casein expression. This demonstrates that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. It has been shown that mouse mammary luminal progenitors give rise to hollow organoid structures, whereas solid organoid structures are derived from stem cells. Among several miR146b targets, miR146b knockdown resulted in preferential STAT3&bgr; overexpression. In the primary mouse mammary epithelial cells, overexpression of STAT3&bgr; isoform caused mammary epithelial cell death and a significant reduction in &bgr;-casein mRNA expression. Therefore, we conclude that during pregnancy miR146b is involved in luminal alveolar progenitor cell maintenance, at least partially, by regulating STAT3&bgr;.

The aryl hydrocarbon receptor agonist 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) alters early embryonic development in a rat IVF exposure model

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression through translational repression or mRNA degradation. These molecules play critical roles in regulating normal developmental processes, but when deregulated, are causally linked to the pathogenesis of numerous diseases, including cancer. MicroRNA-146a and -146b are encoded by two different genes, but differ by only two bases and appear to function redundantly in many systems. Initial studies branded miR-146a/b as important mediators of inflammatory signaling, documenting the ability of these miRNAs to influence differentiation, proliferation, apoptosis and effector immune mechanisms within the hematopoietic system. Numerous contemporary studies now implicate miR-146a/b as pleiotropic regulators of tumorigenesis, as a polymorphism in miR-146a and altered expression of both miR-146a/b have been linked with cancer risk, tumor histogenesis and invasive and metastatic capacity in diverse cancers. Despite the numerous reports concerning miR-146a/b in human cancers, the mechanistic contributions of these miRNAs in both normal and neoplastic mammary gland development and biology remains poorly characterized.