RANK ligand (RANKL), a TNF-related molecule, is essential for osteoclast formation, function and survival through interaction with its receptor RANK. Mammary glands of RANK- and RANKL-deficient mice develop normally during sexual maturation, but fail to form lobuloalveolar structures during pregnancy because of defective proliferation and increased apoptosis of mammary epithelium. It has been shown that RANKL is responsible for the major proliferative response of mouse mammary epithelium to progesterone during mammary lactational morphogenesis, and in mouse models, manipulated to induce activation of the RANK/RANKL pathway in the absence of strict hormonal control, inappropriate mammary proliferation is observed. However, there is no evidence so far of a functional contribution of RANKL to tumorigenesis. Here we show that RANK and RANKL are expressed within normal, pre-malignant and neoplastic mammary epithelium, and using complementary gain-of-function (mouse mammary tumour virus (MMTV)-RANK transgenic mice) and loss-of function (pharmacological inhibition of RANKL) approaches, define a direct contribution of this pathway in mammary tumorigenesis. Accelerated pre-neoplasias and increased mammary tumour formation were observed in MMTV-RANK transgenic mice after multiparity or treatment with carcinogen and hormone (progesterone). Reciprocally, selective pharmacological inhibition of RANKL attenuated mammary tumour development not only in hormone- and carcinogen-treated MMTV-RANK and wild-type mice, but also in the MMTV-neu transgenic spontaneous tumour model. The reduction in tumorigenesis upon RANKL inhibition was preceded by a reduction in pre-neoplasias as well as rapid and sustained reductions in hormone- and carcinogen-induced mammary epithelial proliferation and cyclin D1 levels. Collectively, our results indicate that RANKL inhibition is acting directly on hormone-induced mammary epithelium at early stages in tumorigenesis, and the permissive contribution of progesterone to increased mammary cancer incidence is due to RANKL-dependent proliferative changes in the mammary epithelium. The current study highlights a potential role for RANKL inhibition in the management of proliferative breast disease.
#2057 Purpose: Bone metastases are commonly observed in patients with advanced breast cancer. Tumor cells interact with the bone microenvironment to induce osteoclastogenesis via local bone stromal expression of receptor activator of NF-kB ligand (RANKL), leading to bone destruction and release of growth factors. In addition to its critical role in tumor-induced osteolysis, RANKL has been demonstrated to enhance the invasive behavior of epithelial tumor cells that express RANK, and RANK over-expression in transgenic mice using the breast-specific MMTV promoter results in increased mammary carcinoma. This study assessed the expression of human RANK and its ligand (RANKL) in invasive ductal carcinoma (IDC).
 Methods: We studied a total of 57 IDC specimens. Antibodies against human RANK (AF683) and human RANKL (M366; Amgen) were used for immunohistochemistry (IHC) cell staining along with an isotype control. The specificity of the 2 antibodies was substantiated by IHC, flow cytometry and Western blot analysis of positive and negative control cells. In addition, for RANK, mass spectrometry/protein sequencing of immunoprecipitated proteins was performed. The intensity of IHC staining was scored on a semiquantitative scale (1=weak, 2=moderate, 3=intense) or a complex scale (sum of percentages of stained tumor cells x staining intensity (0-3)).
 Results: Using a complex score with a threshold of 10, 20/57 IDC (35%) expressed minimal level of RANK (range 0 to <10, median 1) while 37/57 IDC (65%) expressed RANK protein with a score ranging from 10 to 100 (median 50). The expression in the tumor was heterogeneous, with cells having no expression and cells having intense expression. In addition, RANK was intensely expressed in the basal layer of all normal ducts and lobules. RANKL was observed in rare tumors (7/57; 11%), in rare lymphocytes of most tumor stroma (42/62; 68%), and in rare fibroblast-like cells in the stroma of rare tumors (3/62; 4.8%).
 Conclusion: In this study, RANK expression was observed by IHC in the majority (65%) of IDC. RANKL expression was observed in the epithelial carcinoma element of some tumors (11%) and was detected in 68% of tumors in rare lymphocytes infiltrating the tumor stroma. Correlation studies between prognosis markers ER, PR, Her2, and CK5/6 expression and RANK or RANKL expression in those IDC have been initiated. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2057.
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