P2-01-02: Pharmacological Inhibition of RANKL Attenuates Mammary Tumor Development and Lung Metastases in the MMTV-neu Transgenic Spontaneous Tumor Model.
Authors: Branstetter D , Jacob A , Soriano R , Allred S , Jones J , Miller R , Dougall WC 
Published: June 2012

Background: RANK and its ligand (RANKL), key factors for bone remodeling and metastasis, are crucial for the development of mouse mammary glands during pregnancy. In a mouse model of mammary carcinogenesis induced by the hormone medroxyprogesterone (MPA) and a carcinogen (DMBA), treatment with a RANKL inhibitor RANK-Fc reduces hormone-induced mammary epithelial proliferation, incidence of preneoplasias, and mammary adenocarcinomas. This study assessed the expression of mouse RANK and RANKL in FVB wild-type (WT) and MMTV-neu mice and whether RANKL inhibition could inhibit mammary tumor formation and lung metastases in this model.

Materials and Methods: MMTV-neu mice were treated with the RANKL inhibitor RANK-Fc (10 mg/kg, 3x/week) or vehicle (muFc) beginning at 20 weeks. Mammary tumor formation was determined by palpation and confirmed by histologic examination. The presence of preneoplastic lesions and histotype of tumors was determined on H&E-stained tissues. Entire lungs from tumor-bearing MMTV-neu mice were step-sectioned at 75 μm and assessed histologically for the presence of metastases. Epithelial proliferation was measured by BrdU labeling of the mammary epithelium. The expression of RANK, RANKL, cyclin D1, progesterone receptor (PR), F4/80, FOXP3, and CD3 was determined by immunohistochemistry (IHC).

Results: RANK-Fc treatment of MMTV-neu mice resulted in a significant reduction in total number of mammary tumors and lung metastases quantified at necropsy but did not impact median time to spontaneous mammary tumor onset. RANKL inhibition resulted in reduced pre-neoplasias and corresponding reductions in proliferation and cyclin D1 within normal mammary epithelia but not in adenocarcinomas. Treatment of MMTV-neu mice with RANK-Fc also reduced the incidence and number of lung metastases per mouse. RANK protein was detected in normal mammary epithelium, preneoplastic mammary intraepithelial neoplasia (MIN), the epithelial component of adenocarcinomas and lung metastases, and monocytic element adjacent to primary MMTV-neu tumors. RANKL protein was detected in normal mammary epithelium but not in the epithelia, stromal component or infiltrating cells of adenocarcinomas or lung metastases. Progesterone receptor expression was observed in normal mammary epithelium of MMTV-neu and FVB WT mice but decreased over time in MMTV-neu mice and was not detected in preneoplasias, adenocarcinomas, or lung metastases. CD3 was detected in mammary tumors and lung metastases but neither RANKL nor FOXP3 were present. Staining of lymph nodes was used as a positive control for RANKL and FOXP3. Expression of FOXP3 and RANKL were not measurably colocalized in dual staining of lymph nodes.

Discussion: RANKL is expressed in normal mammary epithelia of MMTV-neu mice but is not detected in any stage of atypia, including MIN, mammary adenocarcinoma, or lung metastases. Selective pharmacological inhibition of RANKL by RANK-Fc treatment attenuated mammary tumor development and lung metastases in the MMTV-neu transgenic spontaneous tumor model.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-02.