Inhibitory Effects of Cathepsin K Inhibitor (ODN-MK-0822) on the Paracrine Pro-Osteoclast Factors of Breast Cancer Cells

Background: Breast cancer (BC) produces bone resorptive cytokines and growth factorsthat accelerate the development of osteoclasts (OCs), leading to osteolytic bone metastases. In theLong-term Odanacatib Fracture Trial (LOFT), the skeletal-metastasized breast cancer subjects whoreceived odanacatib (ODN) had a delayed tumour progression and skeletal tumour burden as a resultof anti-resorptive effects through inhibition of cathepsin K (CTSK). In this study, we exploredthe effect of ODN, a CTSK inhibitor, on the paracrine pro-osteoclast activity of breast cancer cells. Methods: An immunohistochemistry study was performed to demonstrate CTSK and PTHrP expressionin the samples of primary breast carcinoma. Expression of CTSK mRNA and protein wasconfirmed by the reverse transcription PCR and western blotting analysis in two human breast cancercell lines, MDA-MB-231 and MCF-7 BC cell lines. Cells were incubated with sub-lethalamounts of ODN, and their conditioned supernatants were assessed for their capacity to differentiatePBMCs of healthy donors into osteoclast and its interference on bone-resorbing activities. Wealso measured the mRNA levels of major pro-osteoclast (pro-OC) factors in ODN-treated breastcancer cells and their secreted levels by semi-quantitative reverse transcription PCR and protein expressionby immunoblotting. Results: Different staining intensity was observed in samples containing PTHrP and CTSK in varioushistological grades of breast carcinoma. A significant positive relationship was found betweenCTSK expression and histological grade of BC and presence or absence of distant metastasis. Thepresent study results also indicate that ODN has no effects on OCs number, however, ODN decreasesthe mRNA expression of secreted pro-OC factors such as PTHrP, CXCR-4, and TNF-α. Immunoblotindicates that ODN treatment decreased the protein expression of CTSK, IL-6, andIL-1β, and thus lowered protein levels paralleled the defective phosphorylation of NF-κB. Moreover,there was a significant reduction in the level of growth factors such as IGF-1, PDGF, andTGFβ expression at transcriptional level after ODN treatment as compared to control. Conclusion: ODN has shown to prevent osteolytic metastasis by interacting with the NF-κB pathway,inhibiting bone resorptive cytokines and growth factors. This effect can also be taken into accountthe delayed development of metastatic bone disease found in the long-term odanacatib fracturetrial (LOFT) study.