Kelly J. Gauger

Polychlorinated biphenyls 105 and 118 form thyroid hormone receptor agonists after cytochrome P4501A1 activation in rat pituitary GH3 cells.

Background Polychlorinated biphenyls (PCBs) may interfere with thyroid hormone (TH) signaling by reducing TH levels in blood, by exerting direct effects on TH receptors (TRs), or both. Objective Our objective was to identify individual PCBs that directly affect TH signaling by acting on the TR. Methods We administered a mixture of six PCB congeners based on their ortho substitution pattern, including PCBs 77 and 126 (non-ortho), PCBs 105 and 118 (mono-ortho), and PCBs 138 and 153 (di-ortho), to pregnant Sprague-Dawley rats from gestational days (G) 6 to 16. This mixture, or various combinations of the components, was also evaluated in a transient transfection system using GH3 cells. Results The mixture reduced serum TH levels in pregnant rats on G16 but simultaneously up-regulated the expression of malic enzyme in liver. It also functioned as a TR agonist in vitro; however, none of the individual PCB congeners comprising this mixture were active in this system. Using the aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone, and the cytochrome P450 (CYP)1A1 antagonist ellipticine, we show that the effect of the mixture on the thyroid hormone response element required AhR and CYP1A1. Conclusions We propose that PCB 126 induces CYP1A1 through the AhR in GH3 cells, and that CYP1A1 activates PCB 105 and/or 118 to a form a compound that acts as a TR agonist. These data suggest that some tissues may be especially vulnerable to PCBs interfering directly with TH signaling due to their capacity to express CYP1A1 in response to coplanar PCBs (or other dioxin-like molecules) if sufficient mono-ortho PCBs are present.

Thyroid hormone exerts site-specific effects on SRC-1 and NCoR expression selectively in the neonatal rat brain

Thyroid hormone receptors (TRs) are ligand-gated transcription factors. Recently, many coregulator proteins have been identified that interact with steroid/TRs and are required for the activation or repression of hormone sensitive genes. We tested whether steroid receptor coactivator-1 (SRC-1) and nuclear corepressor (N-CoR) expression is altered by hypothyroidism in rat brains on gestational day 16 and postnatal day 15. We found that both SRC-1 and N-CoR mRNA levels were decreased in the cortex and dentate gyrus of 6-n-propyl-2 thiouracil treated rats only on P15, while mRNA levels for both genes were increased in the same CA3 region of the brains. These findings do not support the idea that cofactors are involved in the compensatory mechanisms for conserving TH action, but they do suggest that hypothyroidism affects the responsiveness of tissues to steroid hormones by altering the expression of necessary cofactors.

Down-regulation of sfrp1 in a mammary epithelial cell line promotes the development of a cd44high/cd24low population which is invasive and resistant to anoikis.

BackgroundThe Wnt family of secreted proteins is implicated in the regulation of cell fate during development, as well as in cell proliferation, morphology, and migration. Aberrant activation of the Wnt/β-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway by competing with the Frizzled receptor for Wnt ligands resulting in an attenuation of the signal transduction cascade. Loss of SFRP1 expression is observed in breast cancer, along with several other cancers, and is associated with poor patient prognosis. However, it is not clear whether the loss of SFRP1 expression predisposes the mammary gland to tumorigenesis.ResultsWhen SFRP1 is knocked down in a non-malignant immortalized mammary epithelial cell line (76 N TERT), nuclear levels of β-catenin rise and the Wnt pathway is stimulated. The SFRP1 knockdown cells exhibit increased expression of the pro-proliferative Cyclin D1 gene and increased cellular proliferation, undergo a partial epithelial-mesenchymal transition (EMT), are resistant to anchorage-independent cell death, exhibit increased migration, are significantly more invasive, and exhibit a CD24low/CD44high cell surface marker expression pattern.ConclusionOur study suggests that loss of SFRP1 allows non-malignant cells to acquire characteristics associated with breast cancer cells.

Mice deficient in Sfrp1 exhibit increased adiposity, dysregulated glucose metabolism, and enhanced macrophage infiltration.

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Wnt signaling plays an important role in preadipocyte differentiation and adipogenesis. The expression of a Wnt antagonist, secreted frizzled related protein 1 (SFRP1), is increased in response to initial weight gain, then levels are reduced under conditions of extreme obesity in both humans and animals. Here we report that loss of Sfrp1 exacerbates weight gain, glucose homeostasis and inflammation in mice in response to diet induced obesity (DIO). Sfrp1-/- mice fed a high fat diet (HFD) exhibited an increase in body mass accompanied by increases in body fat percentage, visceral white adipose tissue (WAT) mass, and adipocyte size. Moreover, Sfrp1 deficiency increases the mRNA levels of key de novo lipid synthesis genes (Fasn, Acaca, Acly, Elovl, Scd1) and the transcription factors that regulate their expression (Lxr-α, Srebp1, Chreb, and Nr1h3) in WAT. Fasting glucose levels are elevated, glucose clearance is impaired, hepatic gluconeogenesis regulators are aberrantly upregulated (G6pc and Pck1), and glucose transporters are repressed (Slc2a2 and Slc2a4) in Sfrp1-/- mice fed a HFD. Additionally, we observed increased steatosis in the livers of Sfrp1-/- mice. When there is an expansion of adipose tissue there is a sustained inflammatory response accompanied by adipokine dysregulation, which leads to chronic subclinical inflammation. Thus, we assessed the inflammatory state of different tissues and revealed that Sfrp1-/- mice fed a HFD exhibited increased macrophage infiltration and expression of pro-inflammatory markers including IL-6, Nmnat, Tgf-β2, and SerpinE1. Our findings demonstrate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular signaling in response to the onset of obesity.

SFRP1 reduction results in an increased sensitivity to TGF-β signaling

BackgroundTransforming growth factor (TGF)-β plays a dual role during mammary gland development and tumorigenesis and has been shown to stimulate epithelial-mesenchymal transition (EMT) as well as cellular migration. The Wnt/β-catenin pathway is also implicated in EMT and inappropriate activation of the Wnt/β-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway and loss of SFRP1 expression is frequently observed in breast tumors and breast cancer cell lines. We previously showed that when SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells (TERT-siSFRP1) acquire characteristics associated with breast tumor initiating cells. The phenotypic and genotypic changes that occur in response to SFRP1 loss are consistent with EMT, including a substantial increase in the expression of ZEB2. Considering that ZEB2 has been shown to interact with mediators of TGF-β signaling, we sought to determine whether TGF-β signaling is altered in TERT-siSFRP1 cells.MethodsLuciferase reporter assays and real-time PCR analysis were employed to measure TGF-β transcriptional targets. Western blot analysis was used to evaluate TGF-β-mediated ERK1/2 phosphorylation. Migration chamber assays were utilized to quantify cellular migration. TERT-siSFRP1 cells were transfected with Stealth RNAi™ siRNA in order to knock-down the expression of ZEB2.ResultsTERT-siSFRP1 cells exhibit a significant increase in both TGF-β-mediated luciferase activity as well as TGF-β transcriptional targets, including Integrin β3 and PAI-1. Phosphorylation of ERK1/2 is increased in TERT-siSFRP1 cells in response to enhanced TGF-β signaling. Furthermore, when the TGF-β pathway is blocked with a TGF-βR antagonist (LY364947), cellular migration is significantly hindered. Finally, we found that when ZEB2 is knocked-down, there is a significant reduction in the expression of exogeneous and endogenous TGF-β transcriptional targets and cellular migration is impeded.ConclusionsWe demonstrate that down-regulation of SFRP1 renders mammary epithelial cells more sensitive to TGF-β signaling which can be partially ameliorated by blocking the expression of ZEB2.

Loss of sfrp1 promotes ductal branching in the murine mammary gland

BackgroundSecreted frizzled-related proteins (SFRPs) are a family of proteins that block the Wnt signaling pathway and loss of SFRP1 expression is found in breast cancer along with a multitude of other human cancers. Activated Wnt signaling leads to inappropriate mammary gland development and mammary tumorigenesis in mice. When SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells exhibit a malignant phenotype which resembles the characteristics observed in metastatic breast cancer stem-like cells. However, the effects of SFRP1 loss on mammary gland development in vivo are yet to be elucidated. The work described here was initiated to investigate the role of SFRP1 in mammary gland development and whether SFRP1−/− mice exhibit changes in mammary gland morphology and cell signaling pathways shown to be associated with SFRP1 loss in vitro.Results10 week old nulliparous SFRP1−/− mammary glands exhibited branching with clear lobulo-alveolar development, which normally only occurs in hormonally stimulated mid-pregnant wt mammary glands. Explant cultures of SFRP1−/− mammary glands display increased levels of a well known Wnt signaling target gene, Axin2. Histomorphologic evaluation of virgin glands revealed that by 10 weeks of age, the duct profile is markedly altered in SFRP1−/− mice showing a significantly higher density of ducts with distinct alveoli present throughout the mammary gland, and with focal ductal epithelial hyperplasia. These findings persist as the mice age and are evident at 23 weeks of age. Changes in gene expression, including c-Myc, TGFβ-2, Wnt4, RANKL, and Rspo2 early in mammary gland development are consistent with the excessive hyper branching phenotype. Finally, we found that loss of SFRP1 significantly increases the number of mammary epithelial cells capable of mammosphere formation.ConclusionsOur study indicates that SFRP1 gene is critical for maintaining proper mammary gland development, and that reduced levels of SFRP1 results in hyperplastic lesions and its loss may be a critical event in cancer initiation.

The effects of diet induced obesity on breast cancer associated pathways in mice deficient in SFRP1.

BackgroundSecreted frizzled-related proteins (SFRPs) are a family of proteins that block the Wnt signaling pathway and loss of Sfrp1 expression is observed in breast cancer. The molecular mechanisms by which obesity contributes to breast tumorigenesis are not well defined, but involve increased inflammation. Mice deficient in Sfrp1 show enhanced mammary gland inflammation in response to diet induced obesity (DIO). Furthermore, mammary glands from Sfrp1−/− mice exhibit increased Wnt signaling, decreased cell death responses, and excessive hyper branching. The work described here was initiated to investigate whether obesity exacerbates the aforementioned pathways, as they each play a key roles in the development of breast cancer.FindingsWnt signaling is significantly affected by DIO and Sfrp1−/− loss as revealed by analysis of Myc mRNA expression and active β-catenin protein expression. Furthermore, Sfrp1−/− mice fed a high fat diet (HFD) exhibit an increase in mammary cell proliferation. The death response is also impaired in the mammary gland of Sfrp1−/− mice fed a normal diet (ND) as well as a HFD. In response to γ-irradiation, mammary glands from Sfrp1−/− mice express significantly less Bax and Bbc3 mRNA, caspase-3 positive cells, and p53 protein. The expression of Wnt4 and Tnfs11 are critical for normal progesterone mediated mammary gland development and in response to obesity, Sfrp1−/− mice express significantly more Wnt4 and Tnfs11 mRNA expression. Evaluation of progesterone receptor (PR) expression showed that DIO increases the number of PR positive cells.ConclusionsOur data indicate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular homeostasis in response to the onset of obesity.

Abstract 996: SFRP1 reduction results in an increased sensitivity to TGF-β signaling

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Metastasis remains the most deadly aspect of breast cancer. We previously published that when the Wnt/β-catenin signaling antagonist, secreted frizzled-related protein-1 (SFRP1), is knocked down in immortalized non-malignant mammary epithelial cells, the cells (TERT-siSFRP1) exhibit a malignant phenotype which resembles the characteristics observed in metastatic breast cancer. Interestingly, the observed phenotypic and genotypic changes that occur in response to loss of SFRP1 define the EMT process. Among the genes analyzed, the drastic increase in ZEB2 expression was of particular interest to us. This protein is a zinc-finger transcription factor that represses the expression of E-cadherin, which is the mechanism by which ZEB2 is thought to contribute to EMT. Additionally, ZEB2 has been shown to interact with downstream mediators of Transforming Growth Factor (TGF)-β signaling (SMADs). TGF-β signaling is known to actively contribute to the acquisition and development of metastatic phenotypes in part through its ability to stimulate EMT and we wondered whether this cytokine plays a role in the EMT observed in response to SFRP1 loss. Therefore, we tested the hypothesis that TGF-β signaling is augmented due to SFRP1 reduction. First, we used luciferase assays and real-time PCR analysis to show that in response to TGF-β treatment, the expression of TGF-β responsive genes are elevated in TERT-siSFRP1 cells. These downstream targets were also measured after cells were treated with a TGF-βR antagonist (LY364947) to ensure these effects were specifically due to the TGF-β signaling pathway. TGF-β signaling has also been shown to increases the phosphorylation of ERK1/2 which in turn enhances cellular migration. We found that the migratory TERT-siSFRP1 cells exhibit a marked increase in ERK1/2 phosphorylation that is readily blocked by the addition of LY364947. Furthermore, transwell assays revealed that LY364947 blocks cellular migration and this phenomenon is likely due to the inhibition of ERK1/2 because the ERK1/2 inhibitor (U0126) similarly blocks cellular migration. To investigate the contribution of ZEB2, we used siZEB2 oligonucleotides to knock down the expression of this protein. Reduced levels of ZEB2 lowered the expression of exogenous and endogenous TGF-β transcriptional targets and hindered cellular migration. We can conclude from these data that the TGF-β signaling pathway is aberrantly activated in TERT-siSFRP1 cells due to the significant up-regulation of the SMAD-interacting protein, ZEB2. This work has been supported in part by the Rays of Hope Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 996. doi:10.1158/1538-7445.AM2011-996