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Veterinary Pathology

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Review article

First published online February 1, 2010

Effect of Obesity on Breast Cancer Development

M. P. Cleary [email protected], M. E. Grossmann, and A. RayView all authors and affiliations

https://doi.org/10.1177/0300985809357753

Abstract

In recent years, obesity has been identified as a risk factor for the development of breast cancer in postmenopausal women, and it has been associated with a poor outcome. Many factors appear to be important in the mechanism of this increased risk, including estrogen, estrogen receptors, and the adipokines leptin and adiponectin. Estrogen, a potent mitogen for mammary cells, has long been implicated in the development of mammary tumors. Because adipose-associated aromatase activity increases the conversion of androgen to estrogen, mammary adipose tissue is thought to be an important source of local estrogen production. Leptin, which increases in the circulation in proportion to body fat stores, has been demonstrated in vitro to promote breast cancer cell growth. Animal models have also identified leptin as an important factor for the development of mammary tumors. In contrast to leptin, serum adiponectin concentrations are inversely related to body fat stores, and the addition of adiponectin to human breast cancer cells reduces cell proliferation and enhances apoptosis. This review explores the relationship between these factors and the development of mammary cancer in humans and mouse models.

Obesity has become a significant health issue in the USA and elsewhere, and it is associated with increased risk for a myriad of diseases, including breast cancer. Obesity is now recognized as a risk factor for the development of postmenopausal breast cancer,^14,17,28,127 as well as with a poor prognosis following breast cancer diagnosis, regardless of menopausal status.^1,16,94 The increased incidence of obesity in adulthood before menopause has been important in illuminating the relationship between body weight and breast cancer development.^40,55,57 In addition, not only the amount of body fat⁷¹ but also the distribution of body fat may have a significant effect on the relationship of body weight and breast cancer.^99,102 Many animal studies (primarily in rodents^{52,68,122,124-126} and dogs¹⁰⁷) suggest that obesity shortens the latency time for mammary tumor detection and/or increases tumor incidence; however, many reports have been descriptive, with little focus on the mechanisms by which body fat mediates effects on mammary tumorigenesis.

Estrogens promote cell proliferation and growth, and are important in developing and maintaining the normal breast. However, studies have shown that estrogen also has important roles in the induction and maintenance of breast cancer.³³ Because of this, estrogen has long been considered to be a major link between body fat and breast tumor induction.⁶⁷ Adipose tissue is an important source of estrogen owing to its notable aromatase activity, which converts androgens to estrone, of which some is converted to 17β-estradiol, a potent estrogen. Thus, increased mammary adipose tissue could contribute to increased mammary gland estrogen exposure.^39,103 In vitro studies using human breast cancer cell lines have provided evidence that estrogen is mitogenic in mammary cancer cells.^24,32,37,108 In addition, estrogen receptor (ER) status is closely related to prognosis; that is, ER-negative tumors tend to have a worse prognosis than do ER-positive tumors,^85,116 in part because ER-negative tumors do not respond to important anticancer therapies such as tamoxifen. Additional factors that are implicated as important links between obesity and mammary tumorigenesis include insulin and insulinlike growth factor 1^56,73,79 for which elevated levels have been associated with premenopausal breast cancer.³⁸ Recently, leptin and adiponectin have been identified as being important in the relationship between obesity and the development and progression of breast cancer in humans,^8,59,95 and it is these factors that are the focus of this review.

Adipokines and Mammary Cancer

The concentration of the adipokines leptin and adiponectin are altered with changes in body weight but in opposite directions.^10,30,100 Leptin, a protein synthesized primarily in adipose tissue, was identified in 1994 and found to be absent in the genetically obese Lep^ob mouse.¹³⁰ This mouse strain and the Lepr^db strain have been frequently used as models of obesity.⁷ This second genetically obese mouse strain was identified as having a mutation in the leptin receptor.²³ These mice have high-circulating levels of leptin, which is more representative of the relationship between leptin and its positive association with body weight, body mass index, and/or body fat levels.^{43,51,101,114} Leptin is present in the serum of almost all human beings, usually in the range of 5 to 50 ng/ml. Initial interest focused on leptin and its receptors in the hypothalamus; as such, leptin was thought to be an important regulatory hormone for signaling body fat status. Over time, however, it became apparent that leptin receptors were expressed in many normal cell types throughout the body as well as in malignant cells, and the addition of leptin to cells in culture was found to promote proliferation and to inhibit apoptosis.^6,44,69,113 Whereas leptin is localized to the cytoplasm of breast cancer cells, the leptin receptor is both cytoplasmic and membrane bound.^62,84

Leptin receptors (OB-Rb and OB-R) have been identified in human breast tumors,^62,72 and increased expression of the leptin receptor in mammary tumors may be associated with a poor prognosis.^45,62 Measurement of serum leptin levels in women in relationship to breast cancer has not provided consistent results, given that most investigations combined premenopausal and postmenopausal cases and had small cohorts.^{31,54,83,90,97,110} Interestingly, Ishikawa et al observed that overexpression of both leptin and leptin receptors in breast cancer tissue was associated with distant metastasis.⁶² Similarly, Garofalo et al reported that overexpression of these two biomarkers were most abundant in grade III tumors.⁴⁵ In addition, in a recent study, leptin receptor expression in breast carcinoma was positively correlated with ER expression and tumor size.⁶⁴ However, when serum leptin levels were assessed in relation with breast cancer outcome, no overall impact was found.⁸³

The mechanism by which leptin influences cell growth and apoptosis are under investigation (Table 1 ). However, Cyclin D1, a significant cell cycle regulatory protein, appears to be an important mediator for leptin-induced growth stimulation. Cyclin D1 signals through the JAK2-PI3K/Akt-MEK/ERK or STAT3 (signal transducer and activator of transcription 3) signaling pathways.^21,98 In vitro studies of MCF-7 and T47-D mammary tumor cells suggest that STAT3 signaling in leptin-treated breast cancer cells is important in promoting cell proliferation.^60,65,129 Frankenberry et al reported that high concentrations of leptin can contribute to breast cancer cell proliferation by activation of MAPK and PI3K signaling pathways that are known to be involved in cell growth and survival.⁴² In a recent study, leptin was shown to transactivate HER2/neu (human epidermal growth factor receptor 2) and enhance the growth of breast cancer cells with HER2/neu overexpression.¹⁰⁵ HER2/neu is a tyrosine kinase receptor that is homologous to the epidermal growth factor receptor, and its overexpression in breast tumors is associated with a poor prognosis and invasive breast cancers.^12,41,104 To date, the human breast cancer cell line most responsive to leptin’s ability to enhance cell proliferation is the MDA-MB-361 line, which is ER-positive and overexpresses HER2/neu. The addition of leptin to this cell line increases expression of a number of cellular proteins, including the leptin receptor, Cox-2, vascular endothelial growth factor, insulin, and proliferating cell nuclear antigen.⁹²

Table 1. Summary of In Vitro Studies of Human Breast Cancer Cells Treated With Leptin

Investigators	Effects of Leptin on Breast Cancer Cells
Hu et al⁶⁰	In T47-D breast cancer cells and HBL100 breast epithelial cells, leptin increased the expression of pSTAT3 (phosphorylated signal transducer and activator of transcription 3), p-ERK (phosphorylated extracellular signal-regulated kinase), and AP-1 (activator protein 1). Leptin enhanced anchorage-dependent proliferation in both cell lines but only anchorage-independent proliferation in T47-D cells.
Laud et al⁷²	Leptin stimulated proliferation of T47-D cells and induced a time-dependent activation of mitogen-activated protein kinase (MAPK).
Dieudonne et al³⁵	MCF-7 breast cancer cells showed response to leptin administration by STAT3 (signal transducer and activator of transcription 3) and p42/p44 MAPK activation and by increased cell proliferation.
Ray et al⁹²	Estrogen-dependent MCF-7, T47-D, and MDA-MB-361 and estrogen-independent MDA-MB-231 and SK-BR-3 breast cancer lines were studied. Leptin stimulated proliferation in all lines except MCF-7 cells. Leptin enhanced expression of signaling proteins Janus-activated kinase 2 (JAK2), phosphatidylinositol 3-kinase (PI3K), and STAT3 in MCF-7 cells; JAK2 and STAT3 in T47-D; PI3K and STAT3 in MDA-MB-361; and JAK2 and PI3K in SK-BR-3. Cyclin D1 levels were increased by leptin in MCF-7, T47-D, MDA-MB-231, and SK-BR-3 cells, whereas higher PCNA (proliferating cell nuclear antigen) levels were found in T47-D, MDA-MB-361, and MDA-MB-231 cells.
Perera et al⁸⁹	MCF-7 cells with 500 ng/ml leptin for 24 hours resulted in a 40% increase in cell number and a fivefold increase in protein secretion.
Soma et al¹⁰⁵	In sum, 500 ng/ml leptin increased proliferation of SK-BR-3 cells that overexpress HER2/neu; phosphorylation of HER2/neu was detected at 2 minutes and continued up to 120 minutes after the start of stimulation.
Jiang et al⁶⁵	In MCF-7 cells, leptin up-regulated survivin, an inhibitor of apoptosis.
Mauro et al⁸¹	Leptin promoted proliferation of MCF-7 cells and cell–cell adhesion via an increased E-cadherin expression.
Saxena et al⁹⁸	Leptin increased MCF-7 cell population along with an increase in Cyclin D1 expression.
Chen et al²¹	Proliferative effect of leptin on ZR-75-1 breast cancer cells was associated with the up-regulation of Cyclin D1 and c-Myc.
Frankenberry et al⁴²	ZR-75-1 and HTB-26 breast cancer cell lines showed activation of MAPK and PI3K in the presence of leptin.
Garofalo et al⁴⁶	MCF-7 cells responded to leptin with cell growth and activation of STAT3, ERK, and Akt/GSK3 antiapoptotic pathways.
Yin et al¹²⁹	Leptin induced STAT3 phosphorylation in association with steroid receptor coactivator 1 in proliferation of MCF-7 cells.
Catalano et al¹⁹	In MCF-7 cells, leptin activated ERα through MAPK pathway and potentiated the estradiol-induced activation of ERα.
Somasundar et al¹⁰⁶	Leptin caused growth potentiation in ZR-75-1 and MCF-7 cell lines.
Catalano et al¹⁸	Leptin enhanced aromatase expression and its enzymatic activity in MCF-7 cells.
Okumura et al⁸⁶	In MCF-7 cells, leptin increased cell proliferation through accelerated cell cycle progression with up-regulation of cdk2 and Cyclin D1 levels.

Adiponectin, like leptin, is secreted from adipose tissue. Adiponectin is measured in human serum in the range of 2 to 20 μg/ml.^5,96 However, in contrast to leptin, adiponectin decreases with increasing body mass index, and circulating levels have been found to decrease in obese individuals as compared with nonobese individuals. In the circulation, adiponectin is present in three oligomeric forms: a high-molecular-weight multimer, a middle-molecular-weight multimer, and a low-molecular-weight trimer (but may be further cleaved to a globular form in the serum; gArcp30).¹¹¹ Circulating adiponectin concentrations have been reported to be lower in women with postmenopausal mammary cancer, compared with controls.^66,70,80 In addition, women with aggressive breast tumors are more likely to be those with low serum adiponectin concentrations.⁸² In vitro studies using the MDA-MB-231 breast cancer cell line have demonstrated that adiponectin treatment reduced cell proliferation and promoted apoptosis; furthermore, adiponectin has been shown to block phosphorylation of Akt as well as decrease CyclinD1 expression in mammary cancer cell lines.^4,34,109,119 Some mammary cancer cell lines have been reported to have adiponectin-associated growth inhibition under specific conditions and so include MCF-7, T47-D, and SK-BR-3.^4,35,47,66 Adiponectin has two receptors, AdipoR1 and AdipoR2, which have been identified in human breast cancer cell lines, including MCF-7, T47-D, MDA-MB-231, SK-BR-3, and MDA-MB-361 cells (Table 2 ).⁴⁷ In addition to having direct growth inhibitory effects, adiponectin binds and inhibits a number of serum growth factors (bFGF, platelet-derived growth factor BB, heparin-binding growth factor–like growth factor), and the increase in proliferation that breast cancer cells undergo in response to the growth factors found in serum is blocked by adiponectin.¹²⁰ The ratio of leptin to adiponectin may also be important in understanding obesity-associated breast cancer development.

Table 2. Breast Cancer Cell Line Expression of Leptin and Adiponectin Receptors and Regulation of Proliferation by Leptin and Adiponectin^a

Cell Line	Expression			Effect on Proliferation
	OB-R	AdipoR1	AdipoR2	Leptin	Adiponectin
MCF-7	Yes	Yes	Yes	No change^b	Decreased
T47-D	Yes	Yes	Yes	Increased	Decreased
SK-Br3	Yes	Yes	Yes	Increased	Decreased
MDA-MB-231	Yes	Yes	Yes	Increased	Mixed results
MDA-ERα7	Yes	Yes	Yes	No change^b	Decreased
MDA-MB-361	Yes	No	Yes	Increased	No change^b

^a Summarized from Grossman et al⁴⁷ and from a poster presentation at the American Association for Cancer Research meeting “Advances in Breast Cancer Research: Genetics, Biology and Clinical Applications,” San Diego, California, October 17–20, 2007 (abstract No. B50).

^b No statistically significant change.

In one study, women with breast cancer had a higher leptin-to-adiponectin ratio when compared with controls.²² Finally, it is important to remember that these studies were performed with recombinant adiponectin (Met1-Asn244)⁷⁸ purified from the NS0 mouse myeloma cell line with unknown multimeric distributions. Adiponectin is capable of associating into trimers, medium-molecular-weight hexamers, and high-molecular-weight oligomers,^112,117,121 and different forms of adiponectin exhibit distinct tissue- and sex-specific activities,^88,121 thereby illustrating that additional work needs to be done to fully characterize the effects of adiponectin.

The effect of adiponectin and leptin in ER-positive and ER-negative tumors in vitro has been evaluated using the ER-negative MDA-MB-231 cells and a subclone of the MDA-MB-231 cell line transfected with ERα (MDA-ERα7).⁴⁸ These cells were treated with physiological combinations of leptin, adiponectin, and globular adiponectin equivalent to that found in a woman with a normal body mass index, and led to a reduction in breast cancer cell proliferation.⁴⁷ Interestingly, the ER-positive MDA-ERα7 cells were more sensitive to the growth inhibitory effects of leptin and gArcp30 than the ER-negative MDA-MB-231 cells were, thus indicating that adiponectin, leptin, and ERα all influence breast cancer growth.

The difference in sensitivity of the ER-negative MDA-MB-231 versus the ER-positive MDA-ERα7 cells to gArcp30 is of interest given that the two cell lines are similar except for the presence of ERα. Evaluation of apoptotic markers revealed that caspase-8 and PARP were activated by adiponectin only in ER-positive MDA-ERα7 cells but not in the ER-negative cells, thereby suggesting that ER-negative cells were less susceptible to adiponectin-induced apoptosis. The effect of serum on differences in sensitivity to adiponectin was tested in both these cell lines. The ER-negative MDA-MB-231 cells activated the JNK and Akt signaling pathways, but only the JNK pathway was used by the ER-positive MDA-ERα7 cells.⁴⁷ Thus, the inactivation of serum-activated JNK pathway by gArcp30 may be important in the associated growth inhibition in MDA-ERα7 cells. Future investigations will seek to provide additional and more specific links between the in vivo development of breast cancer and the balance of adiponectin, leptin, and ER status.

Animal Models, Obesity, and Mammary Carcinogenesis

To better understand the effects of body fat on mammary tumor progression in relationship to estrogen, ER status, and leptin expression, we utilized combinations of xenograft models and genetically engineered mice in a number of different studies. Numerous genetically engineered animal models of mammary cancer utilize the MMTV (mouse mammary tumor virus) or WAP (whey acidic protein) promoter, which are primarily expressed in the mammary gland, to drive the local expression of the gene of interest. These include the insulinlike growth factor 1 receptor (Fcgr1) mouse (tumors by 8 weeks), the MMTV-PyVmT [Tg(MMTV-PyVT)634Mul] mouse (tumors by 6 to 8 weeks), as well as the CyclinD1-CdK2, MMTV-HER2/neu [Tg(MMTV-Erbb2)1Pv], MMTV-TGF-α [Tg(MMTV-TGFA)], MMTV-TGF-β [Tg(MMTV-TGFB1)46Hlm], and WAP-p53 [Tg(WAP-Trp53^172H)] mice, to name only a few.^{2,3,15,49,50,53,61,76,118} For our studies, 2 of these genetically engineered mice were of particular interest: the transgenic mice MMTV-TGF-α (which develops hormone responsive mammary tumors) and MMTV-HER2/neu (which develops ER-negative mammary tumors). TGF-α is a growth factor whose overexpression has been associated with human breast carcinomas.^9,77 HER2/neu is a membrane-bound tyrosine kinase that is commonly overexpressed in human mammary tumors, and its overexpression is associated with a poor prognosis (as cited above), although our reason for choosing these mice was primarily for their ER-negative status.

The transgenic mouse MMTV-TGF-α is of particular value for studying the effect of age on mammary tumor initiation and incidence, given that these mice have been reported to develop mammary tumors in the second year of life, with a 30% incidence rate at 15 to 16 months of age.⁵³ Because MMTV-TGF-α mammary tumors are ER-positive, they are considered to model hormone-responsive breast cancer development in postmenopausal women. Mammary tumors from MMTV-TGF-α transgenic female mice express the leptin receptor OB-Rb (Fig. 1A) primarily on the membrane, whereas the leptin receptor OB-R, which includes several different isoforms of the leptin receptor, was found in both the cytoplasm and the membrane of the tumor cells (Fig. 1B). Leptin itself was expressed in the cytoplasm of the mammary tumor cells in quantities that varied from cell to cell (Fig. 1C). MMTV-TGF-α mice mammary tumors also express the adiponectin receptors AdipoR1 and AdipoR2 (Fig. 1D, E). Note, however, the mammary tumor sections from MMTV-HER2/neu transgenic female mice, as shown in Fig. 2. These mice develop ER-negative high-grade tumors²⁴ and thus represent a second type of mammary tumors. The tumors from these mice are characterized by highly vascularized and undifferentiated tissue with sheetlike structures of cytoplasm studded with nuclei. The cells have ill-defined membranes and few glandular structures. Expression of OB-Rb (Fig. 2A), OB-R (Fig. 2B), and leptin (Fig. 2C) was found; however, the nature of the tumors made distinguishing between cytoplasmic and membranous localization difficult. In MMTV-HER2/neu mice, we found that these tumor cells express AdipoR1 and AdipoR2, as shown in Fig. 2D and 2E, respectively. We used these mouse models to study the relationship among estrogen, ER status, obesity, and mammary tumor development. In an attempt to evaluate the role of adipose and leptin signaling in the development of mammary tumors, we crossed the TGF-α mice with both the Lep^ob⁸⁸ mice and the Lepr^db mice.²³ We followed the mouse strains for 2 years to assess mammary tumor development. Neither obese Lep^ob nor obese Lepr^db double-mutant TGF-α mice developed mammary tumors, whereas the lean mice developed mammary tumors at rates consistent with the published results for this strain.^27,29Table 3 shows a summary of the results. Although these genetically obese mice exhibit many problems associated with their obesity syndrome (eg, the mammary glands do not develop normally), note that supplementation of the Lep^ob mice with leptin restores fertility and allows the mice to lactate.²⁰

Figure 1. Mammary gland; MMTV-TGFα mice (estrogen receptor–positive low-grade tumors). Immunohistochemistry of mammary tumor tissue examined for the presence of Acrp30 and leptin receptors (arrows): 1A, OB-Rb; 1B, OB-R; 1C, leptin; 1D, AdipoR1; 1E, AdipoR2; and 1F, control with goat serum instead of primary antibody. Optimally diluted primary antibodies against the above-mentioned parameters were used on 4-μm-thick paraffin-embedded tissue sections from tumors. After washing, the tissue sections were incubated with biotinylated secondary antibody, followed by incubation with horseradish peroxidase complex. Immunoreactivity was visualized by the substrate diaminobenzidine. Hematoxylin was utilized for the blue counterstaining of the nuclei.

Figure 2. Mammary gland; MMTV-neu mice (estrogen receptor-negative high-grade tumors). Expression of leptin, leptin receptors, and adiponectin (Acrp30) receptors in mammary tumor tissue sections from immunohistochemistry: 2A, OB-Rb; 2B, OB-R; 2C, leptin; 2D, AdipoR1; 2E, AdipoR2; and 2F, control with goat serum instead of primary antibody. Immunohistochemical staining was performed on paraffin-embedded tissue sections with primary antibodies, followed by incubation with secondary antibody and horseradish peroxidase. The reaction was detected by diaminobenzidine. The presence of reddish brown color indicates a positive reaction for the specific primary antibodies. Hematoxylin was utilized for the blue counterstaining of the nuclei.

Table 3. Summary of Mammary Tumor Incidence in Lean and Obese MMTV-TGF-α Mice^a

	Lean^b	Obese Lep^ob	Obese Lepr^db
Mice, no.	74	59	42
Mice with mammary tumors, no.	44	0	0
Mice with mammary tumors, %	59	0	0

^a Data summarized from Cleary et al.^27,29

^b Mice with no obesity genes.

The findings of the lack of mammary tumor development in the 2 genetically obese mouse strains were clearly unexpected given the hypothesis—that obesity was a risk factor for mammary tumor development. One possible explanation was that the TGF-α oncogene is not affected by obesity; a more likely explanation is that intact leptin signaling is important in the pathogenesis of breast cancer development in MMTV-TGF-α mice. To evaluate the effect of obesity in the presence of intact leptin signaling, we undertook studies of MMTV-TGF-α mice with dietary-induced obesity. Mice were fed a high-fat diet and stratified by body weight gain based on previously described protocols.^74,75,87,128 The heaviest mice were classified as obesity prone (average weight, 46 g), the intermediate weight mice as overweight (average weight, 37 g), and the lightest mice as obesity resistant (average weight, 32 g).²⁶ The obesity-resistant mice had similar average weights as the lean mice fed a low-fat diet (29 g). In these groups, mammary tumors developed most rapidly in the obesity-prone mice.^26,36 In addition, obesity-prone mice tended to develop greater numbers of high-grade adenocarcinomas when compared with the lean mice regardless of whether they were in the low-fat diet group or the obesity-resistant group. Serum leptin levels were measured and found to correlate with body weight and body fat.³⁶ Thus, in the presence of a complete leptin axis, tumor development in MMTV-TGF-α mice is apparently affected by body weight.

The HER2/neu mouse, as stated previously, develops ER-negative mammary tumors (Fig. 2). We used this model to evaluate the effect of dietary-induced obesity on ER-negative tumors. We determined that there was little effect of body weight or serum leptin levels on age of tumor onset, incidence, or burden in this mouse model.²⁵ Several factors may have contributed to the lack of any apparent effect of obesity on mammary tumors in these mice, the most important of which may be their genetic background. On the same high-fat diet, the MMTV-HER2/neu mice, which were on a FVB/N background, did not get as obese and did not have as high serum leptin as did the MMTV-TGF-α mice, which were on the C57BL6 background. FVB mice are highly susceptible to tumor development, which may be the reason that the addition of obesity did not alter tumor formation.

Xenograft Models, Obesity, and Mammary Cancer

To better study the influence of obesity on the growth and development of mammary tumors, human mammary cancer cell lines have been studied in xenograft models. The importance of ER status on tumor development in obese versus nonobese animals was studied using ER-positive MCF-7 cells and ER-negative MDA-MB-231 cells implanted into B6.129S7-Rag1^tm1M^om/J. Mice were then either fed a normal or high-fat diet. Interestingly, there were no notable effects of obesity on tumor development with either cell line, regardless of body weight.⁹³ Despite no effect on tumor size or latency, there were molecular alterations in the tumors relative to body weight in the ER-negative MDA-MB-231 cells. The expression of OB-Rb, the signaling isoform of the leptin receptor, was twofold higher in MDA-MB-231 tumors from mice on a high-fat diet as compared with mice fed a low-fat diet. Furthermore, there was a significantly higher level of the antiapoptotic protein Bcl-2 in MDA-MB-231 tumors from the high-fat diet group compared with those from the low-fat diet group. Unlike MDA-MB-231 mammary tumors, MCF-7-derived tumors (which were notably smaller than MDA-MB-231 tumors) had no change in OB-Rb levels regardless of diet group. In MCF-7 tumors, OB-Rb expression was about half that obtained from MDA-MB-231 high-fat tumors but was in the range of the MDA-MB-231 low-fat group. Leptin signaling protein concentrations (ie, STAT3 and JAK2), however, were similar in tumors derived from MCF-7 and MDA-MB-231. ER-negative MDA-MB-231 cells have highly aggressive behavior; in this study, 94% of MDA-MB-231 tumors were classified as grade III adenocarcinomas, whereas the majority of MCF-7 tumors were classified as either grade I (57%) or grade II (40%). The findings of this study indicate an association of OB-Rb expression and intake of a high-fat diet in aggressive mammary tumors, thus suggesting that the presence of OB-Rb may aggravate the pathological process.

To specifically evaluate the importance of estrogen in obesity-associated mammary tumor growth, ER-positive T47-D cells (leptin responsive in vitro^60,72) were implanted into ovariectomized CD-1 nude mice that were made obese by administration of goldthioglucose.⁸⁴ This compound damages the hypothalamus and leads to obesity in a portion of the treated animals and is a way to induce obesity without consumption of a high-fat diet.^11,63,123 One group of goldthioglucose-treated mice was implanted with estrogen pellets, whereas the other group received placebo pellets. Results indicate that obese mice implanted with the estrogen pellets did not develop tumors, whereas obese mice without estrogen had a 100% mammary tumor development. Serum leptin concentrations were significantly elevated in the obese mice regardless of the group.⁸⁴ Estrogens mediate their functions through two receptors: ERα is responsible for proliferative effects whereas ERβ mediates antiproliferative effects. ERβ is more highly expressed in T47-D cells as compared with MCF-7 cells. Interestingly, estradiol induces up-regulation of ERβ in T47-D cells.^91,115 Therefore, the presence of estrogen was protective against the development of T47-D mammary tumors, possibly through the induction of ERβ.⁸⁴

In contrast to these findings, a recent study using MCF-7 cells inoculated into Crl:NU(Ico)-Foxn1^nu mice implanted with estrogen pellets reported that mice receiving intraperitioneal injections of leptin (231 μg/kg 5 days a week for 13 weeks) had larger tumors than did mice who did not receive leptin.⁸¹ The tumors from the estrogen-plus-leptin-treated mice had higher tumor expression of pMAPK and pSTAT3 compared with those receiving estrogen only, suggesting that estrogen and leptin may work together to mediate tumor growth.

The effects of adiponectin in a xenograft model using nude mice inoculated with either ER-negative MDA-MB-231 cells or ER-positive T47-D cells demonstrated that adiponectin treatment markedly inhibited tumor growth and distant metastasis of the MDA-MB-231 cells. This research also demonstrated that in MDA-MB-231 cells, adiponectin can inhibit the GSK-3β/β-catenin signaling and can down-regulate Cyclin D1.¹¹⁹ Studies have demonstrated that stabilization of β-catenin is associated with mammary tumorigenesis and that overexpression of Cyclin D1 is common in human breast tumors.⁵⁸ In addition, the antiproliferative effects of adiponectin on endothelial cells may reduce the ingrowth of vessels into the tumors, thereby reducing tumor growth and metastatic potential.¹³

Conclusions

Integration of in vivo and in vitro findings indicates that adipokines, estrogen, and ER expression act in symphony to link obesity and breast cancer. The past 10 years have seen an increased awareness of the role of lifestyle factors and body weight in breast cancer development. Research in our laboratory and others is expanding the understanding of the impact that adipose tissue–secreted factors play in mammary tumorigenesis. Figure 3 presents a scheme of what may occur with respect to the interrelationship of leptin and adiponectin and mammary tumorigenesis. It is becoming increasingly clear that multiple cell characteristics in relationship to the environment play important roles in determining how cells respond to different stimuli. Furthermore, it now appears important to look at not only single serum growth factors but their interactions with one another. Further work is needed to better define these interactions and for the potential development of ancillary treatments for breast cancer.

Figure 3. Proposed relation of body weight, adipokines, and breast cancer development.

Acknowledgments

This research would not have been possible without the initial input of Drs Xin Hu and Nita J. Maihle. The technical assistance of Mr Katai J. Nkhata and Ms Nancy K. Mizuno was critical to the completion of recent studies.

Competing Interests

The authors declared that they had no conflicts of interests with respect to their authorship or the publication of this article.

Funding Information

Funding for these studies was received from the DOD Breast Cancer Program, Eagles Cancer Telethon, the Breast Cancer Research Foundation, and the Hormel Foundation.

References

1. Abrahamson PE, Gammon MD, Lund MJ, Flagg EW, Porter PL, Stevens J, Swanson CA, Brinton LA, Eley JW, Coates RJ: General and abdominal obesity and survival among young women with breast cancer. Cancer Epidemiol Biomarkers Prev 15: 1871–1877, 2006.

Abstract

Adipokines and Mammary Cancer

Animal Models, Obesity, and Mammary Carcinogenesis

Xenograft Models, Obesity, and Mammary Cancer

Conclusions

Acknowledgments

Competing Interests

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