Tumor necrosis factor deficiency inhibits mammary tumorigenesis and a tumor necrosis factor neutralizing antibody decreases mammary tumor growth in neu/erbB2 transgenic mice.

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that is synthesized and secreted by cells of the immune system, as well as by certain epithelia and stroma. Based on our previous studies demonstrating TNF-stimulated proliferation of normal and malignant mammary epithelial cells, we hypothesized that TNF might promote the growth of breast cancer in vivo. To test this, we generated bigenic mice that overexpressed activated neu/erbB2 in the mammary epithelium and whose TNF status was wild-type, heterozygous, or null. Mammary tumorigenesis was significantly decreased in TNF-/- mice (n = 30) compared with that in TNF+/+ mice (n = 27), with a palpable tumor incidence of 10.0% and 44.4%, and palpable tumors/mouse of 0.10 +/- 0.06 and 0.67 +/- 0.17, respectively. Tumorigenesis in the heterozygous group fell between that in the TNF+/+ and TNF-/- groups, but was not significantly different from either of the homozygous groups. The decreased tumor development in the TNF-/- mice was associated with a decreased proliferative index in the lobular and ductal mammary epithelium. To further investigate the role of TNF in breast cancer, mammary tumor-bearing mice whose tumors overexpressed wild-type neu/erbB2 were treated with a TNF-neutralizing antibody or a control antibody for 4 weeks (n = 20/group). Mammary tumor growth was significantly inhibited in mice treated with the anti-TNF antibody compared with the control antibody. Together, these data show a stimulatory role for TNF in the growth of breast tumors and suggest that TNF antagonists may be effective in a subset of patients with breast cancer.

t10,c12-Conjugated linoleic acid stimulates mammary tumor progression in Her2/ErbB2 mice through activation of both proliferative and survival pathways.

The t10,c12 isomer of conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis, metastasis from a transplantable mouse mammary tumor and angiogenesis; however, it stimulates mammary tumorigenesis in transgenic mice overexpressing ErbB2 in the mammary epithelium (ErbB2 transgenic mice). In the current study, we report that a 4-week supplementation of the diet with 0.5% trans-10, cis-12 conjugated linoleic acid (t10,c12-CLA) stimulated the growth of established ErbB2-overexpressing mammary tumors by 30% and increased the number of new tumors from 11% to 82%. Additionally, when t10,c12-CLA supplementation of ErbB2 transgenic mice was initiated at 21 weeks of age, a time just prior to tumor appearance, overall survival was decreased from 46.4 weeks in the control to 39.0 weeks in the CLA group, and survival after detection of a palpable tumor from 7.5 to 4.6 weeks. Short-term supplementation from 10 to 14 weeks or 21 to 25 weeks of age temporarily accelerated tumor development, but over the long term, there was no significant effect on mammary tumorigenesis. Long term as well as a short 4-week supplementation increased mammary epithelial hyperplasia and lobular development, and altered the mammary stroma; this was reversible in mice returned to the control diet. t10,c12-CLA altered proliferation and apoptosis of the mammary epithelium, although this differed depending on the length of administration and/or the age of the mice. The increased tumor development with t10,c12-CLA was associated with increased phosphorylation of the IGF-I/insulin receptor, as well as increased signaling through the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways; however, neither phospho-ErbB2 nor ErbB2 was altered.

The t10,c12 isomer of conjugated linoleic acid stimulates mammary tumorigenesis in transgenic mice over-expressing erbB2 in the mammary epithelium.

Conjugated linoleic acid (CLA), a family of isomers of octadecadienoic acid, inhibits rat mammary carcinogenesis, angiogenesis, and lung metastasis from a transplantable mammary tumor. c9,t11-CLA, the predominant isomer in dairy products, and t10,c12-CLA, a component of CLA supplements, are equally effective. The objective of the current studies was to test the efficacy of these two CLA isomers in a clinically relevant breast cancer model. Transgenic mice over-expressing erbB2 in the mammary epithelium were fed control or 0.5% CLA-supplemented diets continuously from weaning. Unexpectedly, t10,c12-CLA stimulated lobular hyperplasia of the mammary epithelium and accelerated mammary tumor development, decreasing median tumor latency to 168 days of age compared with 256 and 270 days in the c9,t11-CLA and control groups, respectively. Metastasis was also increased by t10,c12-CLA, with percentage of tumor-bearing mice with lung metastasis 73, 14 and 31% in the t10,c12-CLA, c9,t11-CLA and control groups, respectively. A second study, in which CLA administration was initiated after puberty, confirmed the stimulatory effect of t10,c12-CLA on mammary tumor development and metastasis. Additionally, t10,c12-CLA, but not c9,t11-CLA, increased the size of the liver, heart, spleen and mammary lymph node. The effects of t10,c12-CLA were not specific to erbB2 transgenic mice, as t10,c12-CLA supplementation increased proliferation in the mammary epithelium of both wild-type FVB and FVB/erbB2 mice. Moreover, the number of terminal end buds, the mammary epithelial structures most sensitive to a carcinogenic insult, was increased 30-fold in FVB wild-type mice fed t10,c12-CLA. These data suggest that it would be prudent to avoid CLA supplements containing the t10,c12-CLA isomer. However, even though c9,t11-CLA was not efficacious in the erbB2 model, its ability to inhibit mammary tumor development in rat models suggests that it may have activity for prevention of some types of breast cancer.

NFkappaB1/p50 is not required for tumor necrosis factor-stimulated growth of primary mammary epithelial cells: implications for NFkappaB2/p52 and RelB.

Nuclear factor kappaB (NFkappaB) plays an important role in mammary gland development and breast cancer. We previously demonstrated that TNF stimulates growth of mammary epithelial cells (MEC) in a physiologically relevant three-dimensional primary culture system, accompanied by enhanced DNA-binding of the NFkappaB p50 homodimer. To further understand the mechanism of TNF-stimulated growth of primary MEC, the requirement for NFkappaB1/p50, and the role of cyclin D1 in TNF-stimulated growth were examined. TNF induced the formation of DNA-binding complexes of p50 and p52 with their coactivator bcl3 in MEC nuclear extracts. Concomitantly, TNF increased the binding of NFkappaB proteins to the kappaB site on the cyclin D1 promoter, and increased expression of cyclin D1 mRNA and protein. Using MEC from p50 null mice, we found that p50 was not required for TNF-induced growth nor for up-regulation of cyclin D1. However, TNF induced a p52/RelB NFkappaB DNA-binding complex in p50 null MEC nuclear extracts. In addition, we found that in wild-type MEC, TNF stimulated the occupancy of p52 and RelB on the cyclin D1 promoter kappaB site, whereas p50 was present constitutively. These data suggest that in wild-type MEC, TNF stimulates the interaction of bcl3 with p50 and p52, and the binding of p52, as well as RelB, to cyclin D1 promoter kappaB sites, and as a consequence, stimulates the growth of MEC. In the absence of p50, p52 and RelB can compensate for p50 in TNF-stimulated growth and cyclin D1 induction in MEC.

Isomers of conjugated linoleic acid differ in their effects on angiogenesis and survival of mouse mammary adipose vasculature.

Dietary conjugated linoleic acid (CLA) is a cancer chemopreventive agent that has been shown to inhibit angiogenesis in vivo and in vitro, and to decrease vascular endothelial growth factor (VEGF) and Flk-1 concentrations in the mouse mammary gland. To determine which isomer mediates the antiangiogenic effects of CLA in vivo, the effects of diets supplemented with 5 or 10 g/kg c9,t11- or t10,c12-CLA isomers were compared in CD2F1Cr mice. Both isomers inhibited functional vascularization of a matrigel pellet in vivo and decreased serum VEGF concentrations; the t10,c12 isomer also decreased the proangiogenic hormone leptin (P < 0.05). Additionally, the t10,c12 isomer, but not c9,t11-CLA, rapidly induced apoptosis of the white and brown adipocytes as well as the preexisting supporting vasculature of the mammary fat pad (P < 0.05). Independent of this isomer-specific adipose apoptotic effect, both isomers induced a rapid and reversible decrease in the diameter of the unilocular adipocytes (P < 0.05). The ability of both CLA isomers to inhibit angiogenesis in vivo may contribute to their ability to inhibit carcinogenesis. Moreover, we propose that each CLA isomer uniquely modifies the mammary stromal "soil" in a manner that is useful for chemoprevention of breast cancer.