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

<p>Abstract</p> <p>Background</p> <p>The 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.</p> <p>Results</p> <p>When 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 CD24^low/CD44^highcell surface marker expression pattern.</p> <p>Conclusion</p> <p>Our study suggests that loss of SFRP1 allows non-malignant cells to acquire characteristics associated with breast cancer cells.</p

Mice Deficient in SFRP1 Exhibit Increased Adiposity, Dysregulated Glucose Metabolism

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 and glucose homeostasis 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 WAT mass, and adipocyte size. Fasting glucose levels are elevated, glucose clearance is impaired, hepatic gluconeogenesis regulators are aberrantly upregulated, and glucose transporters are repressed in Sfrp1-/- mice fed a HFD. Additionally, we observed increased steatosis in the livers of Sfrp1-/- mice. 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

Gene Expression Profiles Identify Features Common to Lobular and Ductal Premalignant Breast Lesions

Premalignant lesions have been identified in both the ductal and lobular units of the breast epithelium. These lesions have a 4-fold increase in risk of progression to invasive breast cancer, but 80% will remain indolent. This may be due, in part, to the uncertainty of diagnoses as inter-observer reproducibility is poor. When treated with prophylactic hormone therapies blocking the estrogen receptor, up to 40% of women still develop tumors. Therefore the challenge is to develop diagnostic tests that identify the subset of high-risk lesions and provide appropriate prophylactic therapies. We undertook genome-wide expression studies to define sets of genes that show reproducible alterations in atypical hyperplastic lesions. Patients with sporadic atypical hyperplasias and no evidence of breast cancer for at least 2 years following the initial biopsy were selected. RNA quality from formalin-fixed, paraffin-embedded (FFPE) was assessed using the ratio of 150 and 500 bp amplicons of B-actin determined by RT-qPCR. A total of 23 patients were included with diagnoses of pure flattened epithelial atypia (FEA, n=2), atypical lobular lesions (n=9), and atypical ductal lesions (n=12). The atypical lesions and histologically normal breast epithelium were microdissected separately from 6 um thick tissue sections from each patient. RNA was amplified linearly, labeled and hybridized to Affymetrix ST1.0 arrays. Genes differentially expressed by \u3e2-fold between the lesion and normal epithlium within each patient were used to identify gene expression signatures of atypical hyperplasias. Hierarchical clustering of a 512 gene signature yielded 3 major groupings: Benign, Intermediate, Atypia. These results reveal that atypia of the lobular and ductal structures share common underlying transcriptional features. The gene profile provides markers that can improve the reproducibility of diagnoses of atypia. Expression profiling of individuals who subsequently progress to invasive carcinoma will provide biomarkers of high-risk premalignancies and assist selection of therapeutic choices

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

Background Transforming 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. Methods Luciferase 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. Results TERT-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. Conclusions We 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

The effects of polychlorinated biphenyls on thyroid hormone-mediated action in vivo and in vitro

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants routinely found in human and animal tissues. Developmental exposure to PCBs is associated with neuropsychological deficits, which may be related to effects on thyroid hormone (TH) signaling in the developing brain. However, PCBs may interfere with TH signaling solely by reducing circulating levels of thyroid hormone, or they may exert direct effects on thyroid hormone receptors (TRs). Therefore, the goal of this work was to examine the effects of polychlorinated biphenyl (PCB) exposure on TH receptor (TR)-mediated gene expression in vivo, including both dams and their fetuses, as well as in vitro utilizing a cell culture system. The work described in this dissertation demonstrates that PCBs exert TH-like effects both in vivo and in vitro. Specifically, a commercial PCB mixture (Aroclor 1254; A1254) significantly reduced circulating levels of triiodothyronine (T3) and thyroxine (T4) in pregnant rats but increased the expression of several TH-responsive genes in the fetal cortex, including RC3/Neurogranin and Oct-1. These findings are consistent with a direct action of PCBs on TRs. However, I did not identify parent PCB congeners or metabolites that bound to rat TRs isolated from hepatic nuclei. Nonetheless, I showed that a mixture of 6 PCB congeners (PCB Mix 6), chosen based on their ortho substitution pattern, significantly reduced serum TH levels in pregnant rats on G16. Moreover, I found that exposure to this PCB Mix 6 up-regulated the expression malic enzyme (ME), a gene well-known to be positively regulated by TH in the adult liver, and significantly increased TH response element (TRE) driven luciferase reporter activity in vitro. Taken together, these studies suggest that PCBs can act as TH agonists. Finally, I demonstrated that PCB Mix 6 can exert agonistic actions on the TR by a two-step process. In the first step, dioxin-like PCB 126 activates AhR-regulated cytochrome P450 enzymes. In the second step, CYP1A1 metabolizes non-coplanar PCBs 105 and 118 converting them into TR agonists. The present findings clearly indicate that the effects of PCB exposure on circulating levels of TH can only partly explain developmental abnormalities observed in both humans and animals. Moreover, these studies are the first to demonstrate that PCBs increase the transcription of several different TH-responsive genes via a TR dependent mechanism by which one PCB up-regulates the molecular machinery necessary to metabolically activate other PCBs, which ultimately generates a putative TH agonist. The implication for these findings is important because PCBs are likely to exert very different tissue and cellular specific effects which may be attributable to the expression pattern of the molecular machinery involved in TH agonist production. Therefore, depending on the cellular make up of different tissues, PCBs may exert TH-like effects or the tissue response may be solely due to the PCB induced decrease in TH levels

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

Abstract Background Transforming 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. Methods Luciferase 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. Results TERT-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. Conclusions We 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.</p