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

Gauger KJ, Bassa LM, Henchey EM, Wyman J, Ser-Dolansky J, Shimono A, Schneider SS - Mol. Cancer (2014)

Bottom Line: Secreted 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.Wnt signaling is significantly affected by DIO and Sfrp1-/- loss as revealed by analysis of Myc mRNA expression and active β-catenin protein expression.Evaluation of progesterone receptor (PR) expression showed that DIO increases the number of PR positive cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pioneer Valley Life Sciences Institute, Baystate Medical Center, 3601 Main St, Springfield, MA 01199, USA. sallie.schneider@baystatehealth.org.

ABSTRACT

Background: Secreted 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.

Findings: Wnt 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.

Conclusions: Our data indicate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular homeostasis in response to the onset of obesity.

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Related in: MedlinePlus

Loss of Sfrp1 increases Wnt signaling and cellular proliferation in response to DIO the murine mammary gland. (A) Total RNA was harvested from 5th inguinal mammary glands and employed for real-time PCR analysis of Myc gene expression (n = 6/genotype). The results shown represent experiments performed in duplicate and are normalized to the amplification of β-Actin mRNA. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (B)Upper panel, Mammary gland lysates were analyzed for non-phospho (active) β-catenin and β-actin protein expression by western blot. Lower panel, Band density was quantified and bars represent mean ± SEM of % control ND fed mice. (C)Left panel, 3rd & 4th inguinal mammary gland sections were subjected to immunohistochemical analysis, stained for BrdU (brown chromogen), and representative images were captured at 400X are displayed for mice in each treatment group (scale bar 50 μm). Right panel, BrDU-stained cells were counted out for each mammary gland (n = 6/genotype) and bars represent mean ± SEM % BrdU-positive cells. (*p < 0.05, significantly different from control mice fed a ND using Bonferroni’s t test after a two-way ANOVA).
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Figure 1: Loss of Sfrp1 increases Wnt signaling and cellular proliferation in response to DIO the murine mammary gland. (A) Total RNA was harvested from 5th inguinal mammary glands and employed for real-time PCR analysis of Myc gene expression (n = 6/genotype). The results shown represent experiments performed in duplicate and are normalized to the amplification of β-Actin mRNA. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (B)Upper panel, Mammary gland lysates were analyzed for non-phospho (active) β-catenin and β-actin protein expression by western blot. Lower panel, Band density was quantified and bars represent mean ± SEM of % control ND fed mice. (C)Left panel, 3rd & 4th inguinal mammary gland sections were subjected to immunohistochemical analysis, stained for BrdU (brown chromogen), and representative images were captured at 400X are displayed for mice in each treatment group (scale bar 50 μm). Right panel, BrDU-stained cells were counted out for each mammary gland (n = 6/genotype) and bars represent mean ± SEM % BrdU-positive cells. (*p < 0.05, significantly different from control mice fed a ND using Bonferroni’s t test after a two-way ANOVA).

Mentions: 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 [7]. The best characterized Wnt pathway is the canonical Wnt/β-catenin pathway whereby Wnt signaling leads to the stabilization of β-catenin and activation of β-catenin-responsive gene expression. Sfrp1 antagonizes Wnt signaling by binding to Wnt ligands and preventing ligand-receptor interactions and signal transduction [8]. Indeed, loss of SFRP1 increases Wnt signaling in mammary epithelial cells [3], a deleterious effect considering that inappropriate activation of the Wnt/β-catenin pathway contributes to the development of breast cancer [7]. To determine whether increased adiposity exacerbates the effect of Sfrp1 loss on Wnt/β-catenin signaling, we measured the mRNA expression of the β-catenin target gene, Myc, in control and Sfrp1−/− mice [9] (Additional file 1: Figure S1) fed a normal diet (ND) and HFD. A two-way ANOVA revealed that Myc was significantly affected in response to Sfrp1 loss on the HFD (F1,17 = 5.17; P < 0.05; F1,17 = 5.23; P < 0.05). In addition, there was a significant interaction between these two main effects (F1,17 = 7.34; P < 0.05) (Figure 1A). These findings are consistent with our recently published results demonstrating that Axin2, a hallmark Wnt target gene, is significantly elevated in the mammary gland of Sfrp1−/− mice fed a HFD [6]. To investigate whether Wnt signaling is activated in the absence of Sfrp1, we employed western blot analysis with a non-phospho (active) β-catenin antibody (Figure 1B, upper panel). Densitometry measurements revealed that the active form of β-catenin was significantly upregulated in response to Sfrp1 loss (F1,10 = 8.50; P < 0.05) as well as the HFD (F1,10 = 5.94; P < 0.05), but there was no interaction between these two main effects (F1,10 = 1.15; P > 0.05) (Figure 1B). We show that in response to DIO, β-catenin activity was significantly increased, but the absence of Sfrp1 did not further enhance the expression of active β-catenin. These data may be partially explained by published findings and our previous results which demonstrate that adiposity increases the expression of other Wnt signaling antagonists, including Sfrp5, and thus may act to diminish the effect of Sfrp1 loss on β-catenin activity [10,11]. Given the role Wnt/β-catenin plays in cellular proliferation, mice were injected with BrdU to evaluate the effect of Sfrp1 loss and diet induced obesity (DIO) on proliferation. We reveal that the percentage of BrdU positive epithelial cells was significantly increased in response to Sfrp1 loss (F1,18 = 7.02; P < 0.05) as well as the HFD (F1,18 = 5.10; P < 0.05), but there was no interaction between these two main effects (F1,18 = 1.13; P > 0.05) (Figure 1C). Although both DIO and Sfrp1 loss exhibited effects on their own that could participate in an increased risk for cancer, the expression of Myc was enhanced by the two main effects together suggesting that a HFD and Sfrp1 loss, through methylation or mutation, could drive the expression of Myc to very high levels and thus work together to promote cancer risk. Thus, in the context of obesity, Sfrp1 expression is especially important in preventing aberrant Wnt signaling.


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

Gauger KJ, Bassa LM, Henchey EM, Wyman J, Ser-Dolansky J, Shimono A, Schneider SS - Mol. Cancer (2014)

Loss of Sfrp1 increases Wnt signaling and cellular proliferation in response to DIO the murine mammary gland. (A) Total RNA was harvested from 5th inguinal mammary glands and employed for real-time PCR analysis of Myc gene expression (n = 6/genotype). The results shown represent experiments performed in duplicate and are normalized to the amplification of β-Actin mRNA. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (B)Upper panel, Mammary gland lysates were analyzed for non-phospho (active) β-catenin and β-actin protein expression by western blot. Lower panel, Band density was quantified and bars represent mean ± SEM of % control ND fed mice. (C)Left panel, 3rd & 4th inguinal mammary gland sections were subjected to immunohistochemical analysis, stained for BrdU (brown chromogen), and representative images were captured at 400X are displayed for mice in each treatment group (scale bar 50 μm). Right panel, BrDU-stained cells were counted out for each mammary gland (n = 6/genotype) and bars represent mean ± SEM % BrdU-positive cells. (*p < 0.05, significantly different from control mice fed a ND using Bonferroni’s t test after a two-way ANOVA).
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Figure 1: Loss of Sfrp1 increases Wnt signaling and cellular proliferation in response to DIO the murine mammary gland. (A) Total RNA was harvested from 5th inguinal mammary glands and employed for real-time PCR analysis of Myc gene expression (n = 6/genotype). The results shown represent experiments performed in duplicate and are normalized to the amplification of β-Actin mRNA. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (B)Upper panel, Mammary gland lysates were analyzed for non-phospho (active) β-catenin and β-actin protein expression by western blot. Lower panel, Band density was quantified and bars represent mean ± SEM of % control ND fed mice. (C)Left panel, 3rd & 4th inguinal mammary gland sections were subjected to immunohistochemical analysis, stained for BrdU (brown chromogen), and representative images were captured at 400X are displayed for mice in each treatment group (scale bar 50 μm). Right panel, BrDU-stained cells were counted out for each mammary gland (n = 6/genotype) and bars represent mean ± SEM % BrdU-positive cells. (*p < 0.05, significantly different from control mice fed a ND using Bonferroni’s t test after a two-way ANOVA).
Mentions: 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 [7]. The best characterized Wnt pathway is the canonical Wnt/β-catenin pathway whereby Wnt signaling leads to the stabilization of β-catenin and activation of β-catenin-responsive gene expression. Sfrp1 antagonizes Wnt signaling by binding to Wnt ligands and preventing ligand-receptor interactions and signal transduction [8]. Indeed, loss of SFRP1 increases Wnt signaling in mammary epithelial cells [3], a deleterious effect considering that inappropriate activation of the Wnt/β-catenin pathway contributes to the development of breast cancer [7]. To determine whether increased adiposity exacerbates the effect of Sfrp1 loss on Wnt/β-catenin signaling, we measured the mRNA expression of the β-catenin target gene, Myc, in control and Sfrp1−/− mice [9] (Additional file 1: Figure S1) fed a normal diet (ND) and HFD. A two-way ANOVA revealed that Myc was significantly affected in response to Sfrp1 loss on the HFD (F1,17 = 5.17; P < 0.05; F1,17 = 5.23; P < 0.05). In addition, there was a significant interaction between these two main effects (F1,17 = 7.34; P < 0.05) (Figure 1A). These findings are consistent with our recently published results demonstrating that Axin2, a hallmark Wnt target gene, is significantly elevated in the mammary gland of Sfrp1−/− mice fed a HFD [6]. To investigate whether Wnt signaling is activated in the absence of Sfrp1, we employed western blot analysis with a non-phospho (active) β-catenin antibody (Figure 1B, upper panel). Densitometry measurements revealed that the active form of β-catenin was significantly upregulated in response to Sfrp1 loss (F1,10 = 8.50; P < 0.05) as well as the HFD (F1,10 = 5.94; P < 0.05), but there was no interaction between these two main effects (F1,10 = 1.15; P > 0.05) (Figure 1B). We show that in response to DIO, β-catenin activity was significantly increased, but the absence of Sfrp1 did not further enhance the expression of active β-catenin. These data may be partially explained by published findings and our previous results which demonstrate that adiposity increases the expression of other Wnt signaling antagonists, including Sfrp5, and thus may act to diminish the effect of Sfrp1 loss on β-catenin activity [10,11]. Given the role Wnt/β-catenin plays in cellular proliferation, mice were injected with BrdU to evaluate the effect of Sfrp1 loss and diet induced obesity (DIO) on proliferation. We reveal that the percentage of BrdU positive epithelial cells was significantly increased in response to Sfrp1 loss (F1,18 = 7.02; P < 0.05) as well as the HFD (F1,18 = 5.10; P < 0.05), but there was no interaction between these two main effects (F1,18 = 1.13; P > 0.05) (Figure 1C). Although both DIO and Sfrp1 loss exhibited effects on their own that could participate in an increased risk for cancer, the expression of Myc was enhanced by the two main effects together suggesting that a HFD and Sfrp1 loss, through methylation or mutation, could drive the expression of Myc to very high levels and thus work together to promote cancer risk. Thus, in the context of obesity, Sfrp1 expression is especially important in preventing aberrant Wnt signaling.

Bottom Line: Secreted 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.Wnt signaling is significantly affected by DIO and Sfrp1-/- loss as revealed by analysis of Myc mRNA expression and active β-catenin protein expression.Evaluation of progesterone receptor (PR) expression showed that DIO increases the number of PR positive cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pioneer Valley Life Sciences Institute, Baystate Medical Center, 3601 Main St, Springfield, MA 01199, USA. sallie.schneider@baystatehealth.org.

ABSTRACT

Background: Secreted 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.

Findings: Wnt 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.

Conclusions: Our data indicate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular homeostasis in response to the onset of obesity.

Show MeSH
Related in: MedlinePlus