Abstract
Abstract Breast cancer metastasis to bone is a major source of morbidity and mortality in patients. Local erosion of bone increases risk of fracture and decreases mean survival time. Currently patients are treated with bisphosphonates and denosumab. Breast cancer bone metastases secrete high local concentrations of a parathyroid hormone (PTH) analog termed parathyroid hormone-related peptide (PTHrP). PTHrP stimulates bone turnover creating cavities in the bone allowing tumor growth in the resulting space. PTH antagonists directed towards bone metastatic breast cancer have failed clinically due to drug turnover and failure to concentrate in bone. We synthesized two novel classes of PTHrP antagonists that consist of modifications to the native PTH peptide fused to the inert collagen binding domain (CBD) of ColG collagenase from Clostridium histolyticum. Fusion with the CBD directs PTH analog delivery to the bone matrix and blocks PTHrP action in vitro. Antagonist PTH(7-33)-CBD is an N-terminally truncated analog of PTH, and PTH([-1]-33)-CBD is an N-terminally extended analog of PTH. PTH(7-33)-CBD and PTH([-1]-33)-CBD bound to collagen type 1 in vitro. Neither compound induced cAMP accumulation in SaOS-2 osteosarcoma cells indicating no PTH agonist action. Both compounds partially antagonized PTH(1-34) agonist-induced cAMP accumulation in SaOS-2 cells. We used an established mouse model of breast cancer invasion of bone using a bone-trophic variant of estrogen receptor negative MDA-MB-231 breast cancer cells expressing luciferase (MDA-MB-231-BM/luc+) injected into the tibia marrow of nude mice (Day 1) to assess in vivo efficacy of PTH(7-33)-CBD. PTH(7-33)-CBD (1000 μg/kg administered IP on Day 7 and Day 28) reduced overall tumor burden in bone by 78% (P<0.0001) and reduced cortical bone destruction by 30% (P<0.05) compared to vehicle at 5 weeks post-tumor cell injection. Tartrate resistant acid phosphatase (TRAP) is released by osteoclasts and mediates bone resorption. PTH(7-33)-CBD reduced serum TRAP by 18% (P<0.05) compared to vehicle 12 weeks post-tumor cell injection. Importantly, PTH(7-33)-CBD treatment did not alter serum calcium levels or alter mouse body weight indicating no hypocalcemia or general toxicity on body weight. MDA-MB-231 cells and other breast cancer cells express the PTH/PTHrP receptor and PTHrP functions as an autocrine trophic factor in these cell lines. PTH(7-33)-CBD (30 nM) exhibited a direct cytotoxic effect against MDA-MB-231 cells in-vitro reducing cell number by 73% (P<0.0001) at 48 h with no effect on a series of non-tumor cells including MCF10A non-tumorigenic mammary cells, normal human fibroblasts (BJ5TA), and normal human T-cells (THP-1 monocytes). Taken together, these data demonstrate that PTH(7-33)-CBD reduces osteolysis and metastatic tumor burden in bone, and exhibits selective cytotoxicity against breast cancer cells in vitro. Support: Breast Cancer Research Program (BCRP) Breakthrough Award, BC150685 awarded to BGR and RCG. Citation Format: Brian G. Rowan, Tulasi Ponnapakkam, Murali Anbalagan, Yibin Kang, Robert C. Gensure. Bone targeted parathyroid hormone antagonist for prevention of breast cancer bone metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-259.
Published Version
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