RANKL inhibition decreases the incidence of mammary adenocarcinomas in wild type (WT) and MMTV-RANK transgenic mice.

Abstract

Abstract Abstract #4167 Purpose: RANK and its ligand (RANKL), key factors for bone remodeling and metastasis, are crucial for the development of mouse mammary gland during pregnancy. Upon treatment with hormone medroxyprogesterone (MPA) and a carcinogen (DMBA), transgenic mice overexpressing RANK in the mammary gland via the MMTV promoter exhibit a higher incidence of ductular hyperplasias, mammary intraepithelial neoplasias (MIN), and mammary adenocarcinomas than WT mice. This study assessed the expression of mouse RANK and RANKL in WT and MMTV-RANK during mammary tumor progression in this model and determined whether RANKL inhibition could inhibit mammary carcinogenesis.
 Methods: At 6 weeks of age WT and MMTV-RANK transgenic mice were implanted with 90-day release 50mg MPA pellets that were replaced at expiration (weeks 19 and 32). DMBA was administered orally at weeks 9, 10, 12, and 13. The expression of RANK and RANKL was determined by immunohistochemistry (IHC). The specificity of the anti-mouse RANKL and anti-mouse RANK antibodies were verified by their lack of reactivity in RANKL -/- or RANK -/- mouse tissues, respectively. Simultaneous with the first DMBA treatment, mice were treated with RANK-Fc (10 mg/kg, 3x/week) or vehicle and the onset of mammary tumor formation was measured. Mammary tumor formation was determined by palpation and confirmed by histologic examination. Preneoplastic lesions were counted by whole mount analysis of the mammary gland at 4 weeks after the last DMBA treatment. Epithelial proliferation was measured by BrdU labeling of the mammary epithelium at multiple timepoints following the last DMBA treatment (2 days after the first DMBA treatment; 4 and 7 weeks following the last).
 Results: In the MPA/DMBA-induced mammary tumor model, MMTV-RANK mice developed palpable mammary tumors earlier than WT mice with a median onset of 86 days for the MMTV-RANK cohort compared with 132 days for the WT mice. At early timepoints, the number of preneoplastic lesions and mammary epithelial proliferation were greater in MMTV-RANK mice relative to WT. IHC demonstrated that MPA increased the number of RANKL-positive epithelial cells and that RANKL was present in preneoplastic MIN lesions and adenocarcinomas in both WT and MMTV-RANK mice. RANK expression was observed in the epithelial component of hyperplasias, MIN lesions, and adenocarcinomas in both WT and MMTV-RANK mice. Inhibition of RANKL with RANK-Fc substantially decreased the presence of preneoplastic lesions and the proliferation of mammary epithelial cells in MMTV-RANK mice. In addition, treatment of either MMTV-RANK or WT mice with RANK-Fc delayed the onset of palpable mammary tumors. Treatment of the MMTV-RANK mice with RANK-Fc decreased the incidence of mammary adenocarcinoma.
 Conclusion: These data suggest an operative role for RANKL in progesterone-dependent mouse mammary tumor development in both transgenic MMTV-RANK and WT mice and support additional studies to determine whether RANKL inhibition will delay tumor progression and metastases. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4167.