(P 351) TARGETING ANDROGEN RECEPTOR IN OSTEOSARCOMA CELLS WITH VARYING METASTATIC POTENTIAL

Objective: Lung metastasis (LM) is the primary predictor of mortality in osteosarcoma (OS) patients. Because nearly all cases of metastases (~90%) occur in the lungs, there is a significant need for effective treatment options for this patient population. Previous studies have shown that metastatic OS displays increased expression of aldehyde dehydrogenase 1A1 (ALDH1A1), however, treatment approaches targeting this biomarker alone have not been successful clinically. Androgen Receptor (AR) may be an upstream target of ALDH1A1 based on previous pathway analyses in cohorts of OS patients. Currently FDA-approved for prostate cancer treatment, enzalutamide, is a clinically available AR-inhibitor that can be repurposed for use in OS. In the present study, we aim to validate this hypothesis by evaluating the effect of enzalutamide on the migration of human OS cell lines.

Methods: OS cell lines SaOS-2 (low metastatic potential) and LM2 (high metastatic potential) and dedifferentiated chondrosarcoma cell line HT-1080 (positive control) were cultured. Cells were plated in 60 mm dishes and serum starved for 24 hours during which cells were treated with enzalutamide at their respective IC50 concentration. Next, cells were plated on the transwell insert of a Boyden chamber and incubated for 18 hours, at which point the migrated cells were stained with crystal violet and images were taken. The viability of the OS cell lines when treated with enzalutamide was evaluated using a WST-1 assay (SaOS2) and MTT assay (LM2) per manufacturer’s protocol. Briefly, each cell line was plated into a 96-well plate at a density of 25,000 cells. Cells were allowed to adhere overnight. Once adhered, cells were treated with enzalutamide ranging from 300 µM to 1.2 µM or not treated (control). Once incubation with viability assay reagents was complete, the absorbance of each well was measured at 460 nm (SaOS-2) or 570 nm (LM2).

Results: Our preliminary results suggest that treatment with enzalutamide decreased migration in both OS cell lines, regardless of their metastatic potential. However, the highly metastatic LM2 cells displayed a larger magnitude of sensitivity to enzalutamide (Figure 1). Similar to previous observations, all serum-free (negative controls) displayed significantly lower migration potential than their experimental counterparts. HT-1080 displayed the highest migration potential under normal culture conditions, as expected. Enzalutamide decreased OS cell viability at all concentrations tested. LM2 displayed particular sensitivity to enzalutamide with all tested concentrations yielding less than 50% cell viability. However, SaOS-2 cell viability was greater than 50% when treated with 3.7 µM (mean = 57%) and 33.3 µM (mean = 55%) enzalutamide. SaOS-2 cells displayed more variability to treatment with enzalutamide than did LM2 (Figure 2). Repeat and validation experiments are ongoing.

Conclusion: The presence of LM remains the most important clinical determinant of OS prognosis, nevertheless few treatment options are available for these patients. Recent studies suggest that upregulation of the AR pathway in human LM tissue samples may provide a therapeutic target for patients with LM resistant to conventional chemotherapy. Similar to what has been observed in prostate cancer, AR overexpression may result in aberrant ALDH1A1 expression in OS patients presenting with LM. This opens the possibility of repurposing FDA-approved prostate treatment such as enzalutamide, for the treatment of metastatic OS. Indeed, preliminary results suggest that treatment with enzalutamide has the potential to reduce OS cell migration. While these results are encouraging, additional studies are needed to elucidate the likely complex relationship between AR and ALDH1A1 and their impact on OS metastasis.