RANK-Fc INHIBITS LEIOMYOMA TUMOR GROWTH IN VIVO

Introduction: Rank-Fc is a recombinant protein containing the murine extracellular domain of receptor activator of nuclear factor kappa-B (RANK) fused to the Fc portion of murine immunoglobulin G1. It acts by binding to RANK ligand (RANKL), thus inhibiting RANK/RANKL downstream effects. We previously demonstrated that RANKL acts as a paracrine signal from estrogen/progesterone receptor-rich mature cells to activate the RANKL/RANK pathway in leiomyoma stem cells. Here we aim to determine whether blocking RANK/RANK signaling pathway with RANK-Fc can inhibit leiomyoma growth. Methods: Fibroid tissues were obtained from pre-menopausal women aged 18-45 who did not report a history of menstrual irregularities. We isolated leiomyoma smooth muscle cells from fresh fibroid tissues and maintained them in primary culture (n=3). The cells were treated with vehicle, RANKL (30ng/ml), RANK-Fc (50ng/ml) or both for 48 hours. Protein was extracted and Cyclin D1 (a cell cycle protein required for cell proliferation) protein expression levels were measured. For in vivo tumor xenograft experiments, freshly isolated smooth muscle cells were suspended in rat-tail collagen (type 1) at 106cells/10µl and cultured for 48 hours in floating cultures. Cell pellets were then grafted underneath kidney capsules of NOD-SCID gamma (NSG) ovariectomized (OVX) mouse hosts supplemented with subcutaneous (s.c.) implantation of estradiol and progesterone releasing pellets (n=20 mice). The mice were treated with or without RANK-Fc (s.c, 10mg/kg, twice a week) for 4 weeks. Then the mice were sacrificed and the tumors were harvested for the measurement of tumor volumes as well as immunohistochemistry (IHC) staining for Ki67 (a marker of cell proliferation). Results: In primary cultured cells, RANKL was preferentially expressed by leiomyoma when compared to myometrium. Furthermore, leiomyomas express RANKL in response to activation of the progesterone receptor by progesterone agonist, R5020. Treatment of leiomyoma cells with RANKL led to increased expression of cyclin D1 and Bcl-2; known downstream targets of RANKL. Conversely, treatment with RANK-Fc, even in the presence of RANKL led to decreased expression of these proteins. Importantly, in a murine model, RANK-FC treatment led to decreased expression of cell cycle proteins associated with cell growth as well as markers of proliferation. Treatment with RANK-Fc was also associated with decreased tumor growth and subsequently decreased tumor volume in vivo (p<0.01, n=4 patients). Conclusions: RANK-Fc is an effective inhibitor of leiomyoma cell proliferation and growth, both in the in vitro cell culture and in the in vivo tumor implantation settings. These findings implicate RANKL blockade as a potential strategy in the treatment of uterine leiomyoma.