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Keywords:

  • apoptosis;
  • mammary gland;
  • p53;
  • SFRP1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement and funding
  8. Conflict of interest
  9. References

The most frequently occurring cancer in women, and the second leading cause of cancer death among women, is breast cancer. Cancer results from cellular mutations that enhance proliferation and decrease programmed cell death (apoptosis). Secreted frizzled-related proteins (SFRPs) are a family of proteins known for their ability to negatively modulate the Wnt signalling cascade. SFRP1 expression is lost in a multitude of cancers, including breast cancer, and SFRP1 down regulation reduces apoptosis in vitro but the mechanisms remain unclear, as also the effect of Sfrp1 deficiency on apoptosis on mammary epithelial cells in vivo. Our data show that mammary glands from Sfrp1−/− mice express significantly less Bcl2l11 (Bim) and Bax mRNA in response to DNA damage. The effect of Sfrp1 loss in reducing γ-irradiation induced apoptosis was examined by TUNEL staining and cleaved-caspase-3 immunostaining. The findings show that Sfrp1−/− mice have less DNA fragmentation, whilst caspase-3 expression is decreased, and that p53 expression is generally diminished. Recombinant SFRP1 could replace endogenous expression and elevate the levels of pro-apoptotic and p53-mediated gene expression (Bcl2l, Bax, Cdkn1a and Bbc3) in mammary epithelial cells derived from Sfrp1−/− mice. Thus Sfrp1 plays an important role in mediating mammary epithelial apoptotic response to DNA damage in vivo. The role SFRP1 plays in p53 target gene expression was also noted, which suggests that this pathway may be worth exploiting for novel therapies.


Abbreviations
Sfrp1

secreted frizzled related protein 1

Bim

Bcl2 interacting mediator

Bcl2l11

Bcl2-like 11

Bax

Bcl2 Associated X protein

Cdkn1a

cyclin-dependent kinase inhibitor 1

PUMA

p53 upregulated modulator of apoptosis

Bbc3

Bcl2 binding component 3

TUNEL

terminal deoxynucleotidyl transferase dUTP nick end labeling

Caspase

cysteine aspartic acid specific protease

MMEC

mouse mammary epithelial cell

rSFRP1

recombinant Secreted frizzled related protein 1

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement and funding
  8. Conflict of interest
  9. References

Although death rates have fallen steadily since 1990, breast cancer remains the second most common cause of cancer-related deaths in women (Jemal et al., ). Tumours can result from an alteration in the function of cell-cycle genes and decreased sensitivity to cell death (Hartwell and Kastan, 1994). Resistance to death triggers due to mutations or loss of attachment results in metastasis.

A key transcription factor in the programmed cell death response is the p53 tumour suppressor gene (Schmitt et al., 2002). P53 senses a plethora of stress signals originating from deregulated expression of oncogenes, DNA damage, metabolic deprivation or telomere erosion (Horn and Vousden, 2007). When DNA is damaged and the cell is unable to carry out repair, p53 is responsible for arresting growth and activating pro-apoptotic gene expression. On average, p53 is mutated in 31% of all tumours and is mutated in ∼23% of breast cancers, where it is the second most frequently mutated gene (Forbes et al., 2011). Thus, extensive research has been directed towards modulating the p53 response in an attempt to prevent cancer or improve the outcome of conventional breast cancer therapies.

Secreted frizzled related protein members are Wnt signalling antagonists that have been implicated in carcinogenesis (Lacher et al., 2003; Seol et al., 2005; Roman-Gomez et al., 2006; Saran et al., 2010). SFRP1 is a member that is significantly downregulated in breast tumours and their cell lines (Lo et al., 2006; Veeck et al., 2006). Loss of SFRP1 expression is associated with poor overall survival in patients with early breast cancer (Klopocki et al., 2004). However, it is not known whether SFRP1 loss is simply associated with increased breast cancer risk or plays a causative role in rendering the mammary gland more susceptible to breast cancer development.

Suppression of SFRP1 expression is also associated with early changes in human premalignant breast lesions (Dumont et al., 2009). Loss of Sfrp1 in the mouse mammary gland results in hyperplastic lesions, whereas there appears to be increased Wnt-induced proliferation (Gauger et al., 2012), the role of SFRP1 in regulating apoptosis in the mammary gland has not been investigated. SFRP1 downregulation leads to a resistance to anoikis (apoptosis triggered by loss of attachment). Our investigation into the role of Sfrp1 in mammary epithelial cell death in vivo show that that (1) loss of Sfrp1 diminishes DNA damage induced apoptosis and p53 expression, and (2) SFRP1 treatment increases p53-gene expression.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement and funding
  8. Conflict of interest
  9. References

Animals

Procedures were done in accordance with the NIH guidelines for the ethical treatment of animals and approved by the Baystate Medical Center Institutional Animal Care and Use Committee. Female Balb/c mice (n = 6/genotype) were individually housed in plastic cages with food and water provided ad libitum, and maintained on a 12:12 light cycle. Ten-week-old virgin mice were whole body γ-irradiated with 5 Gy to cause DNA damage and mammary glands were excised 6 h later.

RNA Isolation and real-time PCR analysis

Total RNA was extracted from the fifth inguinal mammary glands or mouse mammary epithelial cells (Gauger et al., 2012). Relative expression levels of mRNA was determined by quantitative real-time PCR using the Mx3005P™ real-time PCR system (Stratagene, La Jolla, CA) and all values were normalised to the amplification of β-Actin. The PCR primer sequences are as follows: β-Actin forward: 5′-CTAAGGCCAACCGTGAAAAG-3′, β-Actin reverse: 5′-ACCAGAGGCATACAGGGACA-3′; Bcl2l11 forward: 5′-AACAGTTGTAAGATAACCATTTGA-3′; Bcl2l11 reverse: 5′-GGAGACGAGTTCAACGAAACTT-3′; Bax forward: 5′- GTGAGCGGCTGCTTGTCT-3′; Bax reverse: 5′-GGTCCCGAAGTAGGAGAGGA-3′; Cdkn1a forward: 5′-GGCACACTTTGCTCCTGTG-3′; Cdkn1a reverse: 5′-CAGATCCACAGCGATATCCA-3′; Bbc3 forward: 5′-TACAGCGGAGGGCATCAG-3′; Bbc3 reverse: 5′-TTCTCCGGAGTGTTCATGC-3′. The assays were performed using the 1-Step Brilliant® SYBRIII® Green QRT-PCR Master Mix Kit (Stratagene) containing 200 nM forward primer, 200 nM reverse primer, and 100 ng total RNA. The conditions for cDNA synthesis and target mRNA amplification were performed as follows: 1 cycle of 50°C for 30 min; 1 cycle of 95°C for 10 min; and 35 cycles each of 95°C for 30 s, 55°C for 1 min, and 72°C for 30 s.

Immunohistochemsitry and TUNEL assay

Mammary tissue blocks were cut at 4 µm on a graded slide, deparaffinised in xylene, rehydrated in graded ethanols, and rinsed in phosphate-buffered saline (PBS). Apoptotic cells were detected using the ApopTag® Peroxidase in situ Apoptosis Detection Kit (Millipore, Billerica, MA). TUNEL-positive cells were scored by a blinded assessor in 10 fields (400×) per section, and >1,000 cells were counted for each section. The apoptotic index (AI) is expressed as the total number of TUNEL-positive cells/epithelial cell nuclei. For immunohistochemical analysis, sections were incubated with the primary rabbit polyclonal anti-cleaved caspase-3 (Asp 175) antibody (1:100, Cell Signalling, 9661) or the primary rabbit polyclonal anti-p53 antibody (1:1,000, Leica, NCL-p53-CM5p) for 45 min. Images were taken with an Olympus BX41 light microscope using SPOT Software 5.1 (SPOT™ Imaging Solutions, Detroit, MI).

Primary mouse mammary cell culture

Ten week-old virgin Sfrp1−/− mice (n = 12) were euthanised with carbon dioxide and the fourth mammary glands excised, minced, and dissociated (Gauger et al., 2012). Primary cells were seeded onto rat tail collagen-1 (BD Biosciences, San Jose, CA) coated tissue culture dishes in 10% serum containing mammary growth medium (EpiCult®B for Mouse Mammary Epithelial Cell Culture, Vancouver, BC) supplemented with10 ng/mL EGF (Sigma), 10 ng/mL FGF (Sigma), 4 μg/mlheparin, 100 U/mL pen/strep (Gibco) and 100 μg/mL gentamicin (Gibco) (Dontu et al., 2003). Cells were cultivated in growth medium with or without 1 μg/mL rSFRP1 (Sigma). After 24 h, cells were irradiated and collected 6 h later.

Statistical analysis

Group means were compared using Student's t-tests (Graphpad Prism) and results were considered significantly different at P < 0.05. Outliers were tested on all data sets using a Grubbs' test (GraphPad QuickCalcs), and those statistical outliers verified were excluded from the analysis.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement and funding
  8. Conflict of interest
  9. References

Loss of Sfrp1 represses pro-apoptotic gene expression in the mammary gland

Mammary gland ducts are formed by epithelial cell extension into the fat pad during puberty and death of the internal cells creates a hollow lumen for eventual passage of milk. Normal developmental associated apoptosis occurs in the mammary gland following lactation due to remodelling, cellular turnover, and in response to irreparable DNA damage. Whilst we previously demonstrated that knock down of SFRP1 in an immortalised mammary epithelial cell line resulted in resistance to anoikis, we did not detect any changes in the lumen of the epithelial ducts in Sfrp1−/− mice which suggests that normal apoptosis was occurring (Gauger et al., 2012). However, the mammary fat pads from these mice had excessive ductal branches and occasional hyperplastic lesions (Gauger et al., 2012), which suggests that there may be an increase in proliferation in association with a decrease in cell death, thus altering the homeostatic balance of cell turnover. SFRP1 induces apoptosis in numerous tissues (Bodine et al., 2005; Seol et al., 2005; Gauger et al., 2009; Jiang et al., 2010; Cooper et al., 2012) but no studies to date have described the role SFRP1 plays in apoptosis within the mammary gland.

In a 3D in vitro model, mammary lumen formation is controlled by caspase activation which is regulated by induction of the pro-apoptotic factor BIM (Reginato et al., 2005). Considering the abnormal ductal development observed in Sfrp1−/− mice, we determined whether Bim expression is affected by Sfrp1 loss. Real-time PCR analysis of Bcl2l11 (Bim) showed that mRNA levels were significantly reduced in mammary glands from Sfrp1−/− mice (Fig. 1). These findings are important since repopulation of the luminal space with cancer cells is a hallmark of early mammary tumourigenesis.

image

Figure 1. Expression analysis of pro-apoptotic genes affected by Sfrp1 loss in the mouse mammary gland. For real-time PCR analysis of Bcl2l11, Bax, Cdkn1a and Bbc3 gene expression, total RNA was isolated from the mammary glands of 10 weeks virgin control and Sfrp1−/− female mice 6 h following 5 Gy whole body irradiation (n = 6/geneotype). Real-time PCR experiments were carried out twice. The results are representative experiments performed in duplicate and normalised to the amplification of β-Actin mRNA. Bars represent mean ± SEM of the fold change with respect to control mice. *P < 0.05 (student's t-test).

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Because aberrant cell death signalling in response to DNA damage contributes to tumourigenesis, control and Sfrp1−/− mice were treated with γ-radiation to damage the DNA and induce p53-mediated apoptosis. Bax expression, a major mediator of p53-induced pro-apoptotic activity (Oltvai et al., 1993), was significantly reduced in Sfrp1−/− mice compared with control mice (Fig. 1). mRNA expression of both Cdkn1 (p21) and Bbc3 (Puma) was measured, since they are direct transcriptional targets of p53 (Gartel and Tyner, 1999; Nakano and Vousden, 2001; Yu, Zhang et al., 2001), but there was no difference from control mice (Fig. 1). These data represent tissue responses to DNA damage and are not indicative of cellular responses. Specifically, p53 expression analysis should suggest whether the decrease in pro-apoptotic gene expression is dependent or independent of p53 signalling.

Sfrp1−/− mice have reduced apoptosis and p53 protein expression

The effect of Sfrp1 loss on cell death at the cellular level examined by TUNEL and cleaved caspase analysis of tissue sections clearly indicate that Sfrp1−/− mice have less TUNEL-positive cells than in control mice, notably so when seen at high magnification (Fig. 2A). In agreement, immunohistochemical analysis of the cleaved (activated) form of caspase-3 showed that the immune cells within the lymph node of both genotypes underwent apoptosis, thereby serving as an excellent internal positive control (Fig. 2B, left panel). However, activated caspase-3 was significantly reduced in the epithelial cells of Sfrp1−/− mice. Thus, reduced DNA fragmentation in the mammary epithelial cells of Sfrp1−/− mice may be due to a reduction in activated caspase-3.

image

Figure 2. Histochemical evaluation of γ-irradiation induced cell death in Sfrp1−/− mammary glands. (A) Left panel, third and fourth inguinal mammary gland sections were subjected to TUNEL analysis and images were captured at 100× (scale bar 200 μm) and 400× (scale bar 50 μm). Representative pictures are of mice from each genotype. Right panel, Measurement of percent TUNEL-positive cells was carried out for each mammary gland (n = 6/genotype), and was used to calculate the apoptotic index; bars represent mean ± SEM %TUNEL-positive cells. (B) Left panel, third and fourth inguinal mammary gland sections from immunohistochemical analysis, stained for cleaved caspase-3 (brown chromogen). Images were taken at 100× (scale bar 200 μm) and 400× (scale bar 50 μm). Inset, In addition to capturing 100× photographs of mammary glands, lymph nodes were imaged as a positive control. Representative pictures are displayed for mice from each genotype. Right panel, Total number of cleaved caspase-3 positive cells was counted for each mammary gland (n = 6/genotype); bars represent mean ± SEM cell number. *P < 0.05 (student's t-test).

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Although changes did not occur in all p53-reponsive targets measured in whole mammary tissue derived from Sfrp1−/− mice, we determined whether the level of p53 protein itself was affected by Sfrp1 loss. Low power (100×; Fig. 3A) and high power (400×; Fig. 3B) images show that p53 is indeed diminished in Sfrp1−/− mice. Moreover, there are distinctly less intensely stained nuclei in Sfrp1−/− mice (Fig. 3B). These results are significant in that 50% of mice heterozygous for p53 develop breast tumours in 12 months (Kuperwasser et al., 2000). Further research is required to determine whether similar to Trp53+/− mice, Sfrp1−/− mice are more susceptible to mammary tumour formation.

image

Figure 3. Increased p53 expression in control and Sfrp1−/− mice in response γ-irradiation. Third and fourth inguinal mammary gland sections were immunohistochemically analysis, staining for p53 (brown chromogen). Representative images were taken at (A) 100× (bar 200 μm) and (B) 400× (bar 50 μm). Pictures illustrate the staining results obtained from each γ-irradiated mouse in the study.

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SFRP1 increases the mRNA expression of pro-apoptotic and p53-mediated gene expression

Since SFRP1 elicits both autocrine and paracrine effects in regulating key processes involved in tumourigenesis, including epithelial to mesenchymal transition (EMT) and acquisition of stem cell characteristics (Scheel et al., 2011), it might sensitise cells to death, opening up the possibility of increasing SFRP1 protein levels in cancer therapeutics. Thus, we determined whether recombinant SFRP1 could replace endogenous expression and raise the levels of pro-apoptotic and p53-mediated gene expression in mammary epithelial cells (MMECs) derived from Sfrp1−/− mice. Real-time PCR analysis of Bcl2l11, Bax, Cdkn1a and Bbc3 gene expression showed that each of these genes was significantly increased in response to rSFRP1 (Fig. 4).

image

Figure 4. The effect of SFRP1 treatment on pro-apoptotic and p53 responsive gene expression in Sfrp1−/− mouse mammary cells. For real-time PCR analysis of Bcl2l11, Bax, Cdkn1a and Bbc3 gene expression, total RNA was isolated from mouse mammary epithelial cells derived from Sfrp1−/− mice and treated with or without rSFRP1 6 h following 5 Gy irradiation (n = 3/treatment group). Real-time PCR experiments were carried out twice. The results shown represent experiments performed in duplicate and normalised to the amplification of β-Actin mRNA. Bars represent mean ± SEM of the fold change with respect to control mice. *P < 0.05, **P < 0.01 (significantly different from untreated MMECs using student's t-test).

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The literature describing the connection between SFRP1 family members and cell death is intriguing in that SFRPs exhibit an apoptotic function in several different tissues. (Bodine et al., 2005; Seol et al., 2005; Gauger et al., 2009; Jiang et al., 2010; Saran et al., 2010). The data we report adds further knowledge, with the clear and novel demonstration that loss of Sfrp1 in the murine mammary gland decreases the apoptotic response to DNA damage. Moreover, a mechanistic role SFRP1 played in p53 target gene expression has been elucidated.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement and funding
  8. Conflict of interest
  9. References

SFRP1 expression is frequently lost in human mammary tumours (Lo et al., 2006; Veeck et al., 2006) and is associated with poor survival in patients with early breast cancer (Klopocki et al., 2004). Suppression of SFRP1 expression is also linked to early changes in human premalignant breast lesions (Dumont et al., 2009), and may represent a novel diagnostic marker in women thought to have atypical ductal hyperplasia and/or lobular carcinoma in situ. Nonetheless, whether SFRP1 loss is merely associated with breast cancer development or definitively leads to breast cancer development is unclear. The data clearly show that loss of Sfrp1 significantly affects p53 expression and apoptotic related gene expression as well as activity, which indicate a causative role for lower SFRP1 in premalignant breast changes leading to tumour progression. Moreover, the finding that addition of rSFRP1 increases the expression of pro-apoptotic and p53-mediated genes, in agreement with rSFRP inhibiting EMT and stem cell like activity (Scheel et al., 2011), suggest that SFRP1 represents an interesting preventive target for women with early breast lesions.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement and funding
  8. Conflict of interest
  9. References

The authors do not have any financial or personal relationships with other people or organisations that could inappropriately influence the work described in this manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgement and funding
  8. Conflict of interest
  9. References
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