P3-01-14: RANK and RANK Ligand (RANKL) Expression in Invasive Breast Carcinoma and Human Breast Cancer Cell Lines.
Authors: Jacob A , Branstetter D , Rohrbach K , Winters A , Erwert R , Allred S , Jones J , Miller R , Tometsko M , Blake M , Dougall WC 
Published: June 2012

Abstract
Purpose: RANK and its ligand (RANKL), key factors for bone remodeling and metastasis, are crucial for the development of mouse mammary gland during pregnancy. RANKL functions as a major paracrine effector of the mitogenic action of progesterone in mouse mammary epithelium and has a role in ovarian hormone-dependent expansion and regenerative potential of mammary stem cells (MaSC). RANKL inhibition has been shown to reduce mammary tumor formation and pulmonary metastases in mouse models. Many published expression analyses of RANK and RANKL have been performed using immunohistochemistry (IHC) without documented validation of antibody specificity. This study assessed the expression of human RANK and RANKL in human invasive breast carcinoma (IBC) and human breast cancer cell lines using specific, monoclonal antibodies validated and optimized for IHC or flow cytometry.
Methods: RANK and RANKL expression was analyzed in a panel of human breast cancer cell lines representing luminal or basal breast subtypes using qPCR, flow cytometry and surface receptor quantitation. Antibodies against human RANK (N-1H8, N-2B10; Amgen) and human RANKL (M366, AMG161; Amgen) were used for flow cytometry, surface receptor quantitation or IHC staining. For human IBC, 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). In vitro responses of cell lines to RANKL were also tested.
Results: The specificity of the antibodies was substantiated by concordant signals observed using multiple independent analyses, including IHC, flow cytometry and Western blots of positive and negative control cells and xenograft samples. Analysis of primary human IBC using IHC demonstrated that 25/114 (22%) IBC samples expressed RANK and 18/97 IBC (19%) expressed RANKL protein within the tumor epithelium. RANK protein was observed in monocytic cells infiltrating the tumor in 87/114 (76%) and within normal mammary epithelium adjacent to tumors in 35/79 (44%) of samples. RANKL was observed in infiltrating monocytic cells within the tumor in 60/115 (52%) and within normal mammary epithelium adjacent to tumors in 15/68 (22%) of samples. Both mRNA and RANK surface protein were detected in multiple breast cancer cell lines, including basal and luminal subtypes. Functional RANK expression on cell lines was confirmed in vitro by the observation of RANKL-dependent increases in mRNAs (e.g. MMP-9, IL-6 or IL-8) or proteins in conditioned media (e.g. IL-6, IL-8), despite the relatively low surface expression of RANK observed (range = 1240–9120 sites/cell).
Conclusion: RANK and RANKL expression was observed in the epithelial carcinoma element in human IBC using IHC. RANK and RANKL expression was also observed in normal mammary epithelium and monocytic cells adjacent to breast tumors. Functional RANK expression was observed in human breast cancer cell lines, including both basal and luminal subtypes.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-14.