Accelerated mammary maturation and differentiation, and delayed MMTVneu-induced tumorigenesis of K303R mutant ERalpha transgenic mice

Abstract

We identified a somatic mutation in estrogen receptor-alpha (ERalpha) in breast cancer causing a lysine to arginine transition (K303R) resulting in hypersensitivity to estrogen, altered associations with coactivators and corepressors and altered posttranslational modifications of ERalpha. We have developed a transgenic mouse expressing the K303R mutant ERalpha under control of the mouse mammary tumor virus (MMTV) promoter. At 4 months of age, K303R ERalpha transgenic animals demonstrate precocious alveolar budding compared with wild-type ERalpha transgenic mice or nontransgenic littermates. Despite these morphologic differences, K303R ERalpha transgenic mice displayed no differences in levels of ERalpha, progesterone receptor or proliferation at this time-point. Pregnancy or chronic estrogen plus progesterone exposure in K303R ERalpha transgenic mice also resulted in significantly more alveolar budding, increased beta-casein production and dilated ducts when compared with nontransgenic littermates. To examine the effects of mutant expression on tumorigenesis, mutant ERalpha mice were crossed with FVB-MMTVneu mice and significantly delayed time to neu-mediated tumorigenesis in bigenic animals. In contrast, mutant expression did not affect carcinogen-induced tumorigenesis. Collectively, these data demonstrate that aberrant estrogenic signaling through the K303R ERalpha mutation may lead to precocious alveolar budding in virgin mice, and to an expedited maturation and differentiation phenotype in the mammary glands of hormonally stimulated animals.

Figure 1

Development of K303R ERα Tsg mice. (A) Diagram of the MMTV-A908G (K303R) ERα construct. (B) Southern blot of A908G (K303R/ mutant ERα founder line, lanes 1–9 contain genomic DNA from mutant Tsg animals. Plasmid DNA dilutions served as the positive control. The difference in migration of genomic and plasmid DNA bands reflects the much higher concentration of nonintegrated plasmid DNA loaded on the gels. (C) Southern blot of WT ERα founder line. Lanes 4–10 contain DNA from WT Tsg animals. Plasmid DNA diltuions served as the control. (D) Representative genomic PCR of Tsg and non-Tsg mutant animals, and the PCR control reaction TSH-β (385 bp). Heterozygous (+/−) HA-tagged mutant, and non-Tsg (−/−) PCR products (345 bp) are shown. (E) Agarose gel analysis of cDNA made from mRNA isolated from Tsg mutant (+/−) or non-Tsg (−/−) mammary glands demonstrating a robust amplification of expression of the introduced ERα transgene in two Tsg animals. Lane 1 is a C57/Bl6 breeder mouse used as a negative control.

Figure 1

Development of K303R ERα Tsg mice. (A) Diagram of the MMTV-A908G (K303R) ERα construct. (B) Southern blot of A908G (K303R/ mutant ERα founder line, lanes 1–9 contain genomic DNA from mutant Tsg animals. Plasmid DNA dilutions served as the positive control. The difference in migration of genomic and plasmid DNA bands reflects the much higher concentration of nonintegrated plasmid DNA loaded on the gels. (C) Southern blot of WT ERα founder line. Lanes 4–10 contain DNA from WT Tsg animals. Plasmid DNA diltuions served as the control. (D) Representative genomic PCR of Tsg and non-Tsg mutant animals, and the PCR control reaction TSH-β (385 bp). Heterozygous (+/−) HA-tagged mutant, and non-Tsg (−/−) PCR products (345 bp) are shown. (E) Agarose gel analysis of cDNA made from mRNA isolated from Tsg mutant (+/−) or non-Tsg (−/−) mammary glands demonstrating a robust amplification of expression of the introduced ERα transgene in two Tsg animals. Lane 1 is a C57/Bl6 breeder mouse used as a negative control.

Figure 1

Development of K303R ERα Tsg mice. (A) Diagram of the MMTV-A908G (K303R) ERα construct. (B) Southern blot of A908G (K303R/ mutant ERα founder line, lanes 1–9 contain genomic DNA from mutant Tsg animals. Plasmid DNA dilutions served as the positive control. The difference in migration of genomic and plasmid DNA bands reflects the much higher concentration of nonintegrated plasmid DNA loaded on the gels. (C) Southern blot of WT ERα founder line. Lanes 4–10 contain DNA from WT Tsg animals. Plasmid DNA diltuions served as the control. (D) Representative genomic PCR of Tsg and non-Tsg mutant animals, and the PCR control reaction TSH-β (385 bp). Heterozygous (+/−) HA-tagged mutant, and non-Tsg (−/−) PCR products (345 bp) are shown. (E) Agarose gel analysis of cDNA made from mRNA isolated from Tsg mutant (+/−) or non-Tsg (−/−) mammary glands demonstrating a robust amplification of expression of the introduced ERα transgene in two Tsg animals. Lane 1 is a C57/Bl6 breeder mouse used as a negative control.

Figure 2

Precocious alveolar budding in 4 month old K303R ERα Tsg mice. (A) Representative pictures of 4 month old virgin mammary glands from non-Tsg (panels a and b), WT-ERα Tsg (panels c and d), and K303R ERα Tsg (panels e and f). Arrows indicate precocious alveolar budding. Glands were stained with carmine alum and photographed at 10× and 50× magnification (as indicated). Scatter plots of ERα (B) and PR (C) and (D) representative immunohistochemistry of ERα (panels a–h) and PR (panels i–p) from 2 month (panels a–d, and i–l) and 6 month (panels e–h, and m–p) old virgin mice. Representative photos are 400× magnification.

Figure 2

Precocious alveolar budding in 4 month old K303R ERα Tsg mice. (A) Representative pictures of 4 month old virgin mammary glands from non-Tsg (panels a and b), WT-ERα Tsg (panels c and d), and K303R ERα Tsg (panels e and f). Arrows indicate precocious alveolar budding. Glands were stained with carmine alum and photographed at 10× and 50× magnification (as indicated). Scatter plots of ERα (B) and PR (C) and (D) representative immunohistochemistry of ERα (panels a–h) and PR (panels i–p) from 2 month (panels a–d, and i–l) and 6 month (panels e–h, and m–p) old virgin mice. Representative photos are 400× magnification.

Figure 2

Precocious alveolar budding in 4 month old K303R ERα Tsg mice. (A) Representative pictures of 4 month old virgin mammary glands from non-Tsg (panels a and b), WT-ERα Tsg (panels c and d), and K303R ERα Tsg (panels e and f). Arrows indicate precocious alveolar budding. Glands were stained with carmine alum and photographed at 10× and 50× magnification (as indicated). Scatter plots of ERα (B) and PR (C) and (D) representative immunohistochemistry of ERα (panels a–h) and PR (panels i–p) from 2 month (panels a–d, and i–l) and 6 month (panels e–h, and m–p) old virgin mice. Representative photos are 400× magnification.

Figure 3

Increased alveolar budding in pregnant Tsg mice. Animals were sacrificed at day 18 of pregnancy and the mammary glands analyzed by whole mount and H&E. Whole mounts of non-Tsg (panels a, b, d, and e) and Tsg (panels g, h, j, and k) mice. H&E of non-Tsg (panels c and f) and Tsg (panels i and l) mice. Single arrows (↑) point to increased alveolar budding and asterisks show dilated ducts. Whole mount glands were stained with carmine alum and photographed at 10× and 50× magnification, while H&E stained slides were photographed at 1000× magnification (as indicated).

Figure 4

Effects of chronic hormonal stimulation on K303R ERα Tsg mice. Mice were chronically treated with E+P as described in Materials and Methods. (A) Representative non-Tsg glands shown were treated for 13 (panel a), or 21 weeks (panel e), and representative Tsg glands shown were treated for 21 weeks (panel i), or 15 weeks (panel m). Whole mounts of non-Tsg (panels a, b, e, and f) and Tsg (panels i, j, m, and n) mice; H&E of non-Tsg (panels c and g) and Tsg (panels k and o) mice. Whole mount glands were stained with carmine alum and photographed at 10× and 50× magnification, H&E stained slides were photographed at 1000× magnification (as indicated). Panels d, h, l, and p are representative pictures of casein stained glands photographed at 1000× magnification. Single arrows point to increased alveolar budding, asterisks show dilated ducts, double arrows show CLD and cell filled lumens, and arrowheads show MFG. Quantitative real-time PCR was performed for β-casein (B) and p21 (C), relative quantization was performed setting the value obtained from one Tsg animal as the reference value.

Figure 4

Effects of chronic hormonal stimulation on K303R ERα Tsg mice. Mice were chronically treated with E+P as described in Materials and Methods. (A) Representative non-Tsg glands shown were treated for 13 (panel a), or 21 weeks (panel e), and representative Tsg glands shown were treated for 21 weeks (panel i), or 15 weeks (panel m). Whole mounts of non-Tsg (panels a, b, e, and f) and Tsg (panels i, j, m, and n) mice; H&E of non-Tsg (panels c and g) and Tsg (panels k and o) mice. Whole mount glands were stained with carmine alum and photographed at 10× and 50× magnification, H&E stained slides were photographed at 1000× magnification (as indicated). Panels d, h, l, and p are representative pictures of casein stained glands photographed at 1000× magnification. Single arrows point to increased alveolar budding, asterisks show dilated ducts, double arrows show CLD and cell filled lumens, and arrowheads show MFG. Quantitative real-time PCR was performed for β-casein (B) and p21 (C), relative quantization was performed setting the value obtained from one Tsg animal as the reference value.

Figure 5

(A) K303R ERα expression delays HER2/neu-mediated tumorigenesis. MMTVneu mice were crossed with K303R ERα or WT-ERα expressing mice and monitored for the formation of tumors. Time to tumor formation (months) was plotted and the median time to tumor formation was calculated with GraphPad Prism Software. Chi-square analysis yielded a significant p-value (p=0.0007). (B) K303R ERα mutation did not alter DMBA-induced tumor formation. After carcinogen administration, time to tumor formation (months) was plotted and the median time to tumor formation was calculated with GraphPad Prism Software. Chi-square analysis yielded a nonsignificant p-value.

Figure 5

(A) K303R ERα expression delays HER2/neu-mediated tumorigenesis. MMTVneu mice were crossed with K303R ERα or WT-ERα expressing mice and monitored for the formation of tumors. Time to tumor formation (months) was plotted and the median time to tumor formation was calculated with GraphPad Prism Software. Chi-square analysis yielded a significant p-value (p=0.0007). (B) K303R ERα mutation did not alter DMBA-induced tumor formation. After carcinogen administration, time to tumor formation (months) was plotted and the median time to tumor formation was calculated with GraphPad Prism Software. Chi-square analysis yielded a nonsignificant p-value.