Weight gain increases human aromatase expression in mammary gland

Abstract

Adulthood weight gain predicts estrogen receptor-positive breast cancer. Because local estrogen excess in the breast likely contributes to cancer development, and aromatase is the key enzyme in estrogen biosynthesis, we investigated the role of local aromatase expression in weight gain-associated breast cancer risk in a humanized aromatase (Arom(hum)) mouse model containing the coding region and the 5'-regulatory region of the human aromatase gene. Compared with littermates on normal chow, female Arom(hum) mice on a high fat diet gained more weight, and had a larger mammary gland mass with elevated total human aromatase mRNA levels via promoters I.4 and II associated with increased levels of their regulators TNFα and C/EBPβ. There was no difference in total human aromatase mRNA levels in gonadal white adipose tissue. Our data suggest that diet-induced weight gain preferentially stimulates local aromatase expression in the breast, which may lead to local estrogen excess and breast cancer risk.

Figure 1

High fat diet induces weight gain in female Arom^hum mice. Three-month-old female Arom^hum mice were fed a normal chow (NC, n=8) diet or a high fat diet (HFD, n=7) for 3 months, and their body weights were measured on days 0, 18, 39, 53, 67, and 90. **, P<0.01 (paired t test).

Figure 2

High fat diet increases gonadal white adipose tissue and mammary gland mass disproportionate to weight gain in female Arom^hum mice. At the end of the 3-month diet treatment period, female Arom^hum mice were sacrificed and their intact gonadal adipose tissue and fourth mammary glands were removed and weighed. (A) Average absolute mass of gonadal white adipose tissue. (B) Average mass of gonadal white adipose tissue relative to total body mass. Normal chow group, n=4; high fat group, n=4. *, P<0.05 (paired t test). (C) Average absolute mass of mammary glands. (D) Average mass of mammary glands relative to total body mass. Normal chow group, n=8; high fat group, n=7. **, P<0.01 (paired t test).

Figure 3

High fat diet induces human aromatase mRNA expression via activation of promoters II and I.4 in mammary gland of overweight female Arom^hum mice. After the 3-month diet treatment, mammary glands of female Arom^hum mice were removed and subjected to RNA extraction, followed by (A) real-time RT-PCR to quantify total human aromatase mRNA levels, (B) semi-quantitative PCR to quantify promoter II (PII) and I.4 (PI.4)-specific human aromatase and mouse GAPDH mRNA levels, and real-time RT-PCR to verify PII (C) and PI.4-specific (D) human aromatase mRNA levels. The arrows indicate the specific PCR product bands with the expected sizes shown. Normal chow group, n=4; high fat diet group, n=5. *, P<0.05; **, P<0.01 (paired t test). (E) Immunohistochemical localization of human aromatase in the mammary glands of Arom^hum mice on normal chow or a high fat diet. The images shown are representative of 3 mice in each group. Arrows indicate human aromatase positive cells.

Figure 4

High fat diet induces TNFα and C/EBPβ expression in mammary gland of overweight female Arom^hum mice. After the 3-month diet treatments, mammary glands of female Arom^hum mice were removed and subjected to (A) RNA extraction and real-time RT-PCR to quantify mouse TNFα, C/EBPβ, JunB, and JunD mRNA levels, and (B) tissue protein extract preparation and immunoblotting with anti-C/EBPβ and anti-actin antibodies. Normal chow group, n=4; high fat diet group, n=5. **, P<0.01 (paired t test).

Figure 5

High fat diet does not increase human aromatase mRNA levels in gonadal white adipose tissue of overweight female Arom^hum mice. After the 3-month diet treatments, gonadal white adipose tissue of female Arom^hum mice was removed and subjected to real-time RT-PCR to quantify total human aromatase, mouse TNFα, and mouse C/EBPβ mRNA levels. Normal chow group, n=3; high fat diet group, n=3. **, P<0.01 (paired t test).