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. 2012 Dec;51(12):973-83.
doi: 10.1002/mc.20868. Epub 2011 Oct 17.

Transgenic insulin-like growth factor-1 stimulates activation of COX-2 signaling in mammary glands

Jie Tian  1 , Isabel Lambertz, Thomas R Berton, Joyce E Rundhaug, Kaoru Kiguchi, Stephanie H Shirley, John Digiovanni, Claudio J Conti, Susan M Fischer, Robin Fuchs-Young
Affiliations

Transgenic insulin-like growth factor-1 stimulates activation of COX-2 signaling in mammary glands

Jie Tian et al. Mol Carcinog. 2012 Dec.

Abstract

Studies show that elevated insulin-like growth factor-1 (IGF-1) levels are associated with an increased risk of breast cancer; however, mechanisms through which IGF-1 promotes mammary tumorigenesis in vivo have not been fully elucidated. To assess the possible involvement of COX-2 signaling in the pro-tumorigenic effects of IGF-1 in mammary glands, we used the unique BK5.IGF-1 mouse model in which transgenic (Tg) mice have significantly increased incidence of spontaneous and DMBA-induced mammary cancer compared to wild type (WT) littermates. Studies revealed that COX-2 expression was significantly increased in Tg mammary glands and tumors, compared to age-matched WTs. Consistent with this, PGE(2) levels were also increased in Tg mammary glands. Analysis of expression of the EP receptors that mediate the effects of PGE(2) showed that among the four G-protein-coupled receptors, EP3 expression was elevated in Tg glands. Up-regulation of the COX-2/PGE(2) /EP3 pathway was accompanied by increased expression of VEGF and a striking enhancement of angiogenesis in IGF-1 Tg mammary glands. Treatment with celecoxib, a selective COX-2 inhibitor, caused a 45% reduction in mammary PGE(2) levels, attenuated the influx of mast cells and reduced vascularization in Tg glands. These findings indicate that the COX-2/PGE(2) /EP3 signaling pathway is involved in IGF-1-stimulated mammary tumorigenesis and that COX-2-selective inhibitors may be useful in the prevention or treatment of breast cancer associated with elevated IGF-1 levels in humans. © 2011 Wiley Periodicals, Inc.

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Figures

Figure 1
Figure 1
COX-2 expression in mammary glands and tumors from WT and BK5.IGF-1 Tg mice. Protein expression levels of COX-2 in whole prepubertal or postpubertal (A and B) mammary glands and mammary tumors (C) from WT and BK5.IGF-1 Tg mice were examined by Western analysis. GAPDH was used as a loading control. Numbers of samples analyzed are noted within each bar. Representative blots of 3 different samples per group are shown. Values are expressed as mean ± S.D. * indicates a p value of < 0.05 as determined by Student’s t test.
Figure 2
Figure 2
Location of COX-2-expressing cells in mammary tumors from BK5.IGF-1 Tg mice. Double-immunofluorescent staining with anti-COX-2 (green) and anti-K5 (red, marker of myoepithelial cells) were performed on mammary tumors from BK5.IGF-1 Tg mice as described in Methods. COX-1 and K5 signals co-localized, as indicated by yellow fluorescence. The far images are of DAPI staining, which detects nuclei. Original magnification, 40x.
Figure 3
Figure 3
PGE2 expression levels in mammary glands from pre- and postpubertal WT and BK5.IGF-1 Tg mice. PGE2 levels were quantified using ELISA. Numbers of samples analyzed are noted within each bar. Values are expressed as mean ± S.D. * indicates a p value of < 0.05 as determined by Student’s t test.
Figure 4
Figure 4
Expression levels of EP receptors in mammary glands from pre- and postpubertal WT and BK5.IGF-1 Tg mice. A. QPCR analysis of EP-3 mRNA expression in prepubertal or postpubertal mammary glands from WT and BK5.IGF-1 Tg mice. WT and EP3 KO mouse pancreas were used as positive or negative controls, respectively. Numbers of samples analyzed are noted within each bar. All mRNA levels were normalized to the expression of TBP mRNA of the same sample. The values are expressed as mean ± SEM. * indicates a p value of < 0.05 as determined by Student’s t test. (B) Western blot analysis of EP3 protein levels in mammary glands from pre- and postpubertal WT and BK5.IGF-1 Tg mice. GAPDH was used as a loading control. An extract from mammary tumors of BK5.IGF-1 Tg mice was included as a positive control in right panels. Numbers of samples analyzed are noted within each bar. Representative images of 3 different samples per group are shown. Values are expressed as mean ± S.D. * indicates a p value of < 0.05 as determined by Student’s t test. nd= not detectable. (C) QPCR analysis of EP1 (left panel), EP2 (middle panel) and EP4 (right panel). Number of analyzed samples is indicated within each bar. All mRNA levels were normalized to the expression of TBP mRNA of the same sample. The values are expressed as mean ± SEM. * indicates a p value of < 0.05 as determined by Student’s t test.
Figure 5
Figure 5
Angiogenesis in mammary glands from pre- and postpubertal WT and BK5.IGF-1 Tg mice. Expression levels of VEGF in prepubertal (A) or postpubertal (B) mammary glands from WT and BK5.IGF-1 Tg mice were examined by Western analysis. GAPDH was used as a loading control. Numbers of samples analyzed are noted within each bar. Representative blots of 3 different samples per group are shown. Values are expressed as mean ± S.D. * indicates a p value of < 0.05 as determined by Student’s t test. (C) Immunohistochemical staining with anti-CD31 in paraffin-embedded pre- and postpubertal glands from WT and Tg mice. Original magnification, ×20.
Figure 6
Figure 6
Effect of celecoxib treatment on PGE2 levels and mammary phenotype in BK5.IGF-1 Tg mice. Female IGF-1 Tg mice were treated with daily oral gavage of celecoxib at a dose of 100 mg/kg, 5 days per week for 4 wks, starting on the day of weaning. (A) PGE2 levels, in Tg mammary glands, were determined by ELISA following treatment with celecoxib or vehicle. (B) Toluidine blue staining showing mast cell infiltration in the mammary glands of celecoxib– treated and vehicle-treated Tg mice. Quantitation, as described in the Methods, is shown the right panel. (C) CD31 immunostaining of vehicle- and celecoxib–treated Tg glands. Quantitation, as described in the Methods, is shown the right panel. The number of samples analyzed is noted within each bar. Values are expressed as mean ± S.D. * indicates a p value of < 0.05 as determined by Student’s t test. Original magnification in B and C, 20x.
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