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Association of cellular and molecular responses in the rat mammary gland to 17β-estradiol with susceptibility to mammary cancer.

Ding L, Zhao Y, Warren CL, Sullivan R, Eliceiri KW, Shull JD - BMC Cancer (2013)

Bottom Line: Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM.Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

View Article: PubMed Central - HTML - PubMed

Affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin Madison, 1400 University Avenue, Madison, WI 53706, USA. shull@oncology.wisc.edu.

ABSTRACT

Background: We are using ACI and BN rats, which differ markedly in their susceptibility to 17β-estradiol (E2)-induced mammary cancer, to identify genetic variants and environmental factors that determine mammary cancer susceptibility. The objective of this study was to characterize the cellular and molecular responses to E2 in the mammary glands of ACI and BN rats to identify qualitative and quantitative phenotypes that associate with and/or may confer differences in susceptibility to mammary cancer.

Methods: Female ACI and BN rats were treated with E2 for 1, 3 or 12 weeks. Mammary gland morphology and histology were examined by whole mount and hematoxylin and eosin (H&E) staining. Cell proliferation and epithelial density were evaluated by quantitative immunohistochemistry. Apoptosis was evaluated by quantitative western blotting and flow cytometry. Mammary gland differentiation was examined by immunohistochemistry. Gene expression was evaluated by microarray, qRT-PCR and quantitative western blotting assays. Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.

Results: The luminal epithelium of ACI rats exhibited a rapid and sustained proliferative response to E2. By contrast, the proliferative response exhibited by the mammary epithelium of BN rats was restrained and transitory. Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM. Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

Conclusions: We propose that these cellular and molecular phenotypes are heritable and may underlie, at least in part, the differences in mammary cancer susceptibility exhibited by ACI and BN rats.

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Rat strain-specific effects of 17β-estradiol on protein expression. Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 1, 3 or 12 weeks, and probed with antibody to Spp1 (Panel A), Lcn2 (Panel B) or β-actin (Panel C). The amounts of Spp1 (Panel D) and Lcn2 (Panel E) were quantified using a LI-COR Odyssey system and expressed relative to the amount of β-actin in the same lysate. Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 12 weeks, and probed with antibody to Mmp7 (Panel F), Mmp9 (Panel G) or β-actin (Panel H). The amounts of the proenzymes and active forms of Mmp7 (Panel I) and Mmp9 (Panel J) were quantified using a Bio-Rad ChemiDoc XRS + imaging system and expressed relative to the amount of β-actin in the same lysate. Each data bar represents the mean ± SEM, n = 3 biological replicates. 1, p < 0.05, E2 treated vs. sham treated rats of same strain. 2, p < 0.05, E2 treated BN rats compared to E2 treated ACI rats.
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Figure 6: Rat strain-specific effects of 17β-estradiol on protein expression. Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 1, 3 or 12 weeks, and probed with antibody to Spp1 (Panel A), Lcn2 (Panel B) or β-actin (Panel C). The amounts of Spp1 (Panel D) and Lcn2 (Panel E) were quantified using a LI-COR Odyssey system and expressed relative to the amount of β-actin in the same lysate. Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 12 weeks, and probed with antibody to Mmp7 (Panel F), Mmp9 (Panel G) or β-actin (Panel H). The amounts of the proenzymes and active forms of Mmp7 (Panel I) and Mmp9 (Panel J) were quantified using a Bio-Rad ChemiDoc XRS + imaging system and expressed relative to the amount of β-actin in the same lysate. Each data bar represents the mean ± SEM, n = 3 biological replicates. 1, p < 0.05, E2 treated vs. sham treated rats of same strain. 2, p < 0.05, E2 treated BN rats compared to E2 treated ACI rats.

Mentions: Expression of a subset of the genes that are potentially of functional significance in relation to mammary development, ECM and/or ECM remodeling and mammary cancer susceptibility was further evaluated at the protein level. Although Spp1 was expressed at similar levels in control ACI and BN rats, expression increased in response to E2 treatment in mammary glands of BN but not ACI rats, resulting in significantly higher levels of Spp1 in treated BN rats at the 3 (5.5-fold) and 12 (4.1-fold) week time points, relative to treated ACI rats (Figure 6A and 6D). Lcn2 was virtually undetectable in mammary glands of control and E2 treated ACI rats. By contrast, Lcn2 was highly expressed in mammary glands of control and E2 treated BN rats (Figure 6B and 6E). Mmp7 was undetectable in mammary glands of control ACI and BN rats at each of the three time points examined (data not shown), remained undetectable in the mammary glands of ACI and BN rats treated with E2 for 1 week, but was detected in glands from ACI and BN rats treated with E2 for 3 (data not shown) and 12 weeks (Figure 6F and 6I). Moreover, the active 18kDa form of Mmp7 predominated over the 25kDa proenzyme in mammary glands from BN rats treated with E2 for 12 weeks (Figure 6F and 6I). Mmp9 was expressed at similar levels in mammary glands of control and E2 treated ACI and BN rats at the 1 and 3 week time points (data not shown). At the 12 week time point, Mmp9 was expressed at a higher level in E2 treated BN rats, relative to treated ACI rats, and the active form of Mmp9 was observed only in mammary glands from the treated BN rats (Figure 6G and 6J).


Association of cellular and molecular responses in the rat mammary gland to 17β-estradiol with susceptibility to mammary cancer.

Ding L, Zhao Y, Warren CL, Sullivan R, Eliceiri KW, Shull JD - BMC Cancer (2013)

Rat strain-specific effects of 17β-estradiol on protein expression. Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 1, 3 or 12 weeks, and probed with antibody to Spp1 (Panel A), Lcn2 (Panel B) or β-actin (Panel C). The amounts of Spp1 (Panel D) and Lcn2 (Panel E) were quantified using a LI-COR Odyssey system and expressed relative to the amount of β-actin in the same lysate. Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 12 weeks, and probed with antibody to Mmp7 (Panel F), Mmp9 (Panel G) or β-actin (Panel H). The amounts of the proenzymes and active forms of Mmp7 (Panel I) and Mmp9 (Panel J) were quantified using a Bio-Rad ChemiDoc XRS + imaging system and expressed relative to the amount of β-actin in the same lysate. Each data bar represents the mean ± SEM, n = 3 biological replicates. 1, p < 0.05, E2 treated vs. sham treated rats of same strain. 2, p < 0.05, E2 treated BN rats compared to E2 treated ACI rats.
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Related In: Results  -  Collection

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Figure 6: Rat strain-specific effects of 17β-estradiol on protein expression. Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 1, 3 or 12 weeks, and probed with antibody to Spp1 (Panel A), Lcn2 (Panel B) or β-actin (Panel C). The amounts of Spp1 (Panel D) and Lcn2 (Panel E) were quantified using a LI-COR Odyssey system and expressed relative to the amount of β-actin in the same lysate. Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 12 weeks, and probed with antibody to Mmp7 (Panel F), Mmp9 (Panel G) or β-actin (Panel H). The amounts of the proenzymes and active forms of Mmp7 (Panel I) and Mmp9 (Panel J) were quantified using a Bio-Rad ChemiDoc XRS + imaging system and expressed relative to the amount of β-actin in the same lysate. Each data bar represents the mean ± SEM, n = 3 biological replicates. 1, p < 0.05, E2 treated vs. sham treated rats of same strain. 2, p < 0.05, E2 treated BN rats compared to E2 treated ACI rats.
Mentions: Expression of a subset of the genes that are potentially of functional significance in relation to mammary development, ECM and/or ECM remodeling and mammary cancer susceptibility was further evaluated at the protein level. Although Spp1 was expressed at similar levels in control ACI and BN rats, expression increased in response to E2 treatment in mammary glands of BN but not ACI rats, resulting in significantly higher levels of Spp1 in treated BN rats at the 3 (5.5-fold) and 12 (4.1-fold) week time points, relative to treated ACI rats (Figure 6A and 6D). Lcn2 was virtually undetectable in mammary glands of control and E2 treated ACI rats. By contrast, Lcn2 was highly expressed in mammary glands of control and E2 treated BN rats (Figure 6B and 6E). Mmp7 was undetectable in mammary glands of control ACI and BN rats at each of the three time points examined (data not shown), remained undetectable in the mammary glands of ACI and BN rats treated with E2 for 1 week, but was detected in glands from ACI and BN rats treated with E2 for 3 (data not shown) and 12 weeks (Figure 6F and 6I). Moreover, the active 18kDa form of Mmp7 predominated over the 25kDa proenzyme in mammary glands from BN rats treated with E2 for 12 weeks (Figure 6F and 6I). Mmp9 was expressed at similar levels in mammary glands of control and E2 treated ACI and BN rats at the 1 and 3 week time points (data not shown). At the 12 week time point, Mmp9 was expressed at a higher level in E2 treated BN rats, relative to treated ACI rats, and the active form of Mmp9 was observed only in mammary glands from the treated BN rats (Figure 6G and 6J).

Bottom Line: Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM.Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

View Article: PubMed Central - HTML - PubMed

Affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin Madison, 1400 University Avenue, Madison, WI 53706, USA. shull@oncology.wisc.edu.

ABSTRACT

Background: We are using ACI and BN rats, which differ markedly in their susceptibility to 17β-estradiol (E2)-induced mammary cancer, to identify genetic variants and environmental factors that determine mammary cancer susceptibility. The objective of this study was to characterize the cellular and molecular responses to E2 in the mammary glands of ACI and BN rats to identify qualitative and quantitative phenotypes that associate with and/or may confer differences in susceptibility to mammary cancer.

Methods: Female ACI and BN rats were treated with E2 for 1, 3 or 12 weeks. Mammary gland morphology and histology were examined by whole mount and hematoxylin and eosin (H&E) staining. Cell proliferation and epithelial density were evaluated by quantitative immunohistochemistry. Apoptosis was evaluated by quantitative western blotting and flow cytometry. Mammary gland differentiation was examined by immunohistochemistry. Gene expression was evaluated by microarray, qRT-PCR and quantitative western blotting assays. Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.

Results: The luminal epithelium of ACI rats exhibited a rapid and sustained proliferative response to E2. By contrast, the proliferative response exhibited by the mammary epithelium of BN rats was restrained and transitory. Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM. Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

Conclusions: We propose that these cellular and molecular phenotypes are heritable and may underlie, at least in part, the differences in mammary cancer susceptibility exhibited by ACI and BN rats.

Show MeSH
Related in: MedlinePlus