Investigating causal biologic mechanisms involved in differential serum bone-specific alkaline phosphatase activities in canine appendicular osteosarcoma

In dogs with osteosarcoma (OS), pre-treatment serum bone alkaline phosphatase (BALP) activity is recognized as a prognostic factor; however, biologically-linked causation for this clinical observation requires further exploration. Recent data demonstrated that BALP correlates with OS burden in dogs; yet, every dog with advanced macroscopic disease will not present with BALP elevations, indicating additional factors contribute to BALP activities. Given that BALP exists as a glycosylphosphatidylinositol (GPI)-anchored membrane protein and serves as an osteoblast differentiation marker, the purpose of this study is to investigate alternative biologic mechanisms that could explain an elevation in BALP in dogs with OS. We hypothesize that 1) canine osteosarcoma cells will possess the endothelin (ET)-1/endothelin A receptor (ETAR) signaling pathway; 2) blockade of endothelin signaling will attenuate pro-tumorigenic activities in canine OS cells; 3) blockade of endothelin signaling will attenuate BALP activity; and 4) serum BALP activity in naturally-occurring OS-bearing dogs will correlate with tumors that are more osteoblastic in nature than osteolytic as measured via DEXA scan. The expression of the endothelin axis in canine OS cells was investigated by demonstrating protein expression by western blot analysis. The effects of ETAR antagonism using ABT-627 on canine OS cells was investigated by characterizing perturbations in 1) cell proliferation by colony forming assays; 2) cell survival using changes in phosphorylation of Akt pathway and Jun/AP-1 pathway by western blot analysis; 3) cell migration by quantitative analysis; 4) BALP activity by cytochemistry; and 5) BALP activity via ELISA and corresponding western blot analysis. Naturally-occurring OS-bearing patients were utilized to determine if serum BALP activity correlated with osteoblastic tumors as determined via DEXA scan. The endothelin axis is expressed in canine OS cells indicating a possible autocrine/paracrine signaling pathway. Exposing canine OS cells to ABT-627 results in decreased proliferation, survival, and migration of canine OS cells in vitro. Exposure to ABT-627 also resulted in decreased BALP expression and activity in canine OS cells. Upper and lower quartile relative bone mineral densities, when accounted for tumor size, correlate with BALP with a propensity for more osteoblastic tumors to have an elevation in BALP. In conclusion, the endothelin axis is expressed in canine OS and, upon attenuation of the pathway, tumor-derived growth and survival advantages are inhibited and BALP expression and activity is decreased. Clinically, patients with more osteoblastic tumors as measured via DEXA scan, have elevated serum BALP activity. This study’s findings are the first to provide a link between increased BALP activity and a possible underlying biologic mechanism explaining the poor prognosis associated with this biomarker