In this study we described that, in mammary gland of overweight female Aromhum mice fed a high fat diet compared with lean littermates fed normal chow, human aromatase mRNA levels were increased via activation of promoters I.4 and II associated with increased levels of their respective regulators TNFα and C/EBPβ. The sites of human aromatase expression in the mammary glands of lean and overweight Aromhum mice were identical, and included undifferentiated adipose fibroblasts and myoepithelial cells. Our findings suggest that aromatase may be a molecular link between adulthood weight gain and increased breast cancer risk among pre- and postmenopausal women.
Subbaramaiah et al. (2011)
recently reported increased endogenous aromatase expression in the mammary gland of C57BL/6J mice fed a high fat diet as compared to mice fed a low fat diet, but did not identify the mouse aromatase promoters that were activated (Subbaramaiah et al., 2011
). Although we were not able to detect endogenous aromatase expression in adipose tissue and mammary gland of female Aromhum
mice (unpublished observations), possibly due to difference in the mouse strains used (FNB/N vs. C57BL/6J), our results are consistent with their findings. Moreover, because the female Aromhum
mouse model has similar human aromatase tissue distribution as women, our results support a causal relationship between body weight gain and breast cancer risk through local aromatase in humans. One indication of this relationship may be the effect of obesity on the clinical benefit from aromatase inhibitors in estrogen receptor (ER)-positive breast cancer treatment. Sestak et al (2010)
demonstrated that women with a higher BMI had more recurrences of breast cancer after treatment with an aromatase inhibitor (Sestak et al., 2010
). The mechanism for this important clinical observartion may be complex and is unclear at the moment. Our findings suggest that high breast tissue aromatase expression associated with overweight and obesity may account, at least in part, for the lower efficacy of aromatase inhibitor treatment in high BMI women. Thus, higher doses or more potent aromatase inhibitors may be required for overweight or obese women with breast cancer.
By real-time PCR, we found that overweight female Aromhum mice had 3-fold higher human aromatase mRNA levels in their mammary gland compared with their lean littermates. The levels of human aromatase mRNA quantified were relative amounts per unit mass of mRNA, normalized to mRNA levels of the endogenous housekeeping gene GAPDH. Because overweight female Aromhum mice had twice the mammary gland mass as lean controls, in absolute terms, the overweight Aromhum mice could have up to 6-fold higher human aromatase mRNA in their mammary gland compared with lean counterparts. However, immunohistochemistry did not show a difference in human aromatase protein level between the mammary glands of normal chow and high fat diet-fed Aromhum mice, likely due to its limited quantification power.
Both semi-quantitative and quantitative real-time PCRs revealed that promoters I.4 and II were activated in mammary gland of overweight female Aromhum
mice, correlating with marked increases in TNFα and C/EBPβ expression levels. In the presence of dexamethasone, TNFα stimulates aromatase promoter I.4 in primary human breast adipose fibroblasts (Zhao et al., 1996
). Additionally, Ghosh et al. (2009)
found that high cell density of primary human adipose fibroblasts increases the expression of C/EBPβ, which in turn mediates density-induced aromatase promoter II activation, suggesting that C/EBPβ may play a role in mammographic density- and obesity-associated breast cancer risk by stimulating aromatase expression (Ghosh et al., 2009
The higher fold increases in promoter I.4- and II-specific aromatase mRNA than that in total aromatase mRNA suggests a possibility that the promoter I.4- and II-specific transcripts make up a minority of total aromatase transcripts present in the mammary gland of high fat diet-fed Aromhum mice, i.e. that additional aromatase promoters may be activated, although the cellular sites of human aromatase expression remain unchanged. However, such a direct comparison among the results of these disparate real-time PCRs needs caution, because of the differences in the assay methodology used (Taqman-based for total and promoter I.4-specific aromatase mRNA vs. SYBR green-based for promoter II-specific aromatase mRNA) and assay sensitivity affected by the real-time PCR mechanism, the primer pair and probe selections, and the absolute amount of target mRNA species. Absolute real-time quantification may overcome at least some of the above limitations, however, the internal standards it requires for constructing standard curves are not available.
In contrast to mammary tissue, gonadal white adipose tissue of overweight female Aromhum
mice exhibited no difference in total human aromatase or TNFα mRNA levels compared with those of their lean littermates, despite increased C/EBPβ mRNA levels, suggesting that C/EBPβ is not sufficient for aromatase induction. Hotamisligil et al (1993)
showed that TNFα was strongly induced in epididymal white adipose tissue of many obese male rodent models, such as db/db, ob/ob, and tub/tub mice and fa/fa rats, with the exception of monosodium glutamate (MSG)-induced obese mice, likely because obesity in the MSG model was less severe than that of the genetic models as the authors concluded (Hotamisligil et al., 1993
). Thus, the absence of TNFα induction in the gonadal white adipose tissue of our high fat diet-fed Aromhum
mice is not too surprising, because the mice were only moderately obese, and more precisely, overweight, as compared to the lean littermates on normal chow; at 6 months of age when they were sacrificed, the high fat diet-fed female Aromhum
mice weighed 37.3±6.2 g (mean±SD), whereas their littermates on normal chow weighed 28.0±4.0 g (mean±SD) (). The body weight differential between the two groups was 9.3 g, or 2.3 SDs of the control group. In comparison, according to data from the Jackson Laboratory, female db/db mice weighed 52.8±4.7 g (mean±SD) at 5 months (the oldest age when the body weight was recorded), whereas wild type littermates of the same age weighed 25.1±1.9 g (mean±SD). The body weight differential increased to 27.7 g, or 14.6 SDs of the control group.
The increase in human aromatase expression in mammary gland was relatively modest in our high fat diet-fed, ovary-intact Aromhum
mice, which mimic overweight, premenopausal women. Normal to slightly elevated circulating levels of estrogen in the mice probably suppress aromatase expression in the mammary gland via feedback inhibition (see the attached unpublished manuscript). Subbaramaiah et al. (2011)
observed more severe body weight gain, higher levels of TNFα, and stronger induction of aromatase in mammary gland of ovariectomized wild type C57BL/6J mice fed a high fat diet than in ovary-intact littermates fed the same high fat diet (Subbaramaiah et al., 2011
). This may explain why adulthood weight gain-associated breast cancer risk is more pronounced and more prevalent in postmenopausal women of all ethnicities (Huang et al., 1997
; Wenten et al., 2002
). Therefore, participation in diet and weight loss programs may benefit premenopausal overweight women by reducing breast cancer risk via a reduction in local aromatase production in the breast, and aromatase inhibitors may be particularly effective in breast cancer prevention among postmenopausal overweight and obese women.