Α Humanized RANKL Transgenic Mouse Model of Progestin-Induced Mammary Carcinogenesis for Evaluation of Novel Therapeutics

SIMPLE SUMMARY: Targeting receptor activator of nuclear factor-κB ligand (RANKL) with the monoclonal antibody Denosumab decreases osteoclast-mediated bone resorption and is approved for the treatment of postmenopausal osteoporosis. Since RANKL is also implicated in mammary gland homeostasis and breast tumorigenesis, Denosumab is being currently pursued as a candidate for drug repurposing in oncology, including breast cancer, while its efficacy remains controversial. In this study, by developing a humanized transgenic mouse model of human RANKL overexpression, we demonstrated that RANKL mediated hormone-induced mammary carcinogenesis, while its prophylactic inhibition by Denosumab prevented tumorigenesis. Our humanized transgenic mice provide a unique genetic tool for investigating the involvement of human RANKL in breast cancer pathogenesis and can serve as a preclinical platform for anticancer therapies. ABSTRACT: Receptor activator of nuclear factor-κB ligand (RANKL) is critically involved in mammary gland pathophysiology, while its pharmaceutical inhibition is being currently investigated in breast cancer. Herein, we investigated whether the overexpression of human RANKL in transgenic mice affects hormone-induced mammary carcinogenesis, and evaluated the efficacy of anti-RANKL treatments, such as OPG-Fc targeting both human and mouse RANKL or Denosumab against human RANKL. We established novel MPA/DMBA-driven mammary carcinogenesis models in TgRANKL mice that express both human and mouse RANKL, as well as in humanized humTgRANKL mice expressing only human RANKL, and compared them to MPA/DMBA-treated wild-type (WT) mice. Our results show that TgRANKL and WT mice have similar levels of susceptibility to mammary carcinogenesis, while OPG-Fc treatment restored mammary ductal density, and prevented ductal branching and the formation of neoplastic foci in both genotypes. humTgRANKL mice also developed MPA/DMBA-induced tumors with similar incidence and burden to those of WT and TgRANKL mice. The prophylactic treatment of humTgRANKL mice with Denosumab significantly prevented the rate of appearance of mammary tumors from 86.7% to 15.4% and the early stages of carcinogenesis, whereas therapeutic treatment did not lead to any significant attenuation of tumor incidence or tumor burden compared to control mice, suggesting the importance of RANKL primarily in the initial stages of tumorigenesis. Overall, we provide unique genetic tools for investigating the involvement of RANKL in breast carcinogenesis, and allow the preclinical evaluation of novel therapeutics that target hormone-related breast cancers.