P3-04-05: Expression Profiles of RANK and RANKL mRNA and Protein in the Mammary Gland of Female Cynomolgus Monkeys after Long-Term Treatment with Different Menopausal Hormone Therapies.
Authors: Branstetter D , Wood CE , Rohrbach K , Borgerink H , Dougall WC 
Published: June 2012

Abstract
Background: RANK and its ligand (RANKL), key factors for bone remodeling and metastasis, are crucial for the development of the mouse mammary gland during pregnancy. OPG also binds RANKL and inhibits RANKL action. Treatment of mice with progesterone increases RANKL expression in mammary epithelium. RANKL functions as a major paracrine effector of both progesterone-dependent mitogenic action in mammary tissue, and also in ovarian hormone-dependent expansion and regenerative potential of mammary stem cells. RANKL inhibition in vivo reduces progestin-induced mammary preneoplasias and adenocarcinomas. In postmenopausal women, the addition of a progestin to estrogen therapy has been associated with significant increases in mammary proliferation, breast density, and breast cancer risk and recurrence. The goal of this study was to characterize the expression of RANK, RANKL, and OPG in the mammary gland following estrogen+progestin therapy in comparison with estrogen-alone therapy or placebo in a postmenopausal primate model.
Methods: Ovariectomized female cynomolgus monkeys (Macaca fascicularis) were randomized into 3 treatment groups: placebo, oral conjugated equine estrogens at 0.625 mg/d (CEE) and CEE with medroxyprogesterone acetate (MPA) at 2.5 mg/d (CEE+MPA). Treatments were administered in the diet for 2 years. RANK, RANKL, and OPG mRNA were measured in mammary gland samples (n = 28 to 31/group) using quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). Antibodies against human RANK (N-2B10 and N-1H8; Amgen) and human RANKL (M366; Amgen) were used for immunohistochemistry (IHC) of formalin-fixed tissues (n = 20 to 23/group) along with an isotype control. The intensity of IHC staining was scored on a semiquantitative scale (0 = absent, 1 = weak, 2 = moderate, 3 = intense). Incidence was scored as a positive IHC signal (any intensity).
Results: Analysis of mRNA revealed that treatment with CEE+MPA resulted in a significantly greater ratio of RANKL to OPG compared to treatment with placebo (P &lt; 0.01) or CEE (P &lt; 0.05). IHC demonstrated RANK protein was predominately localized to basal cells of ducts and acinar cells, while RANKL protein was localized to luminal cells. RANK protein was detected in mammary glands of animals in all groups: placebo (74%), CEE (55%), and CEE +MPA (53%). RANKL protein was expressed in the animals treated with CEE+MPA (74%) and CEE (15%), but not in placebo-treated animals. Quantitative expression of specific mRNA markers for epithelial proliferation (MKI67) and density (KRT19) was highest for CEE+MPA, intermediate for CEE, and lowest for placebo.
Discussion: Key components of the RANKL/RANK pathway are expressed in the normal postmenopausal primate mammary gland, modulated by long-term estrogen+progestin exposure at clinically relevant doses, and associated with changes in estrogen+progestin-driven proliferation of mammary tissue.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-04-05.