(P 025) INCREASED ALDH1A1 AND ATOX1 GENE EXPRESSION AND COPPER LEVELS IN CANINE OSTEOSARCOMA PATIENTS

Objective: Osteosarcoma (OS) is responsible for a large proportion of human pediatric cancer and outcomes have not improved for patients with metastatic disease for over 30 years. However, it is extraordinary rare and there is an inherent paucity of primary human patient tissue to study. Canine OS is over 20 times more prevalent than human OS and accurately reproduces clinical progression seen in pediatric patients as they exhibit a compressed pulmonary metastatic and chemotherapy-resistant disease. Comparative oncology of pediatric and canine OS provides a unique opportunity to complement and accelerate translational research of metastatic OS. Furthermore, the activity of the enzyme ALDH1A1 and intracellular copper transporters have been predictors of metastatic disease and antineoplastic therapies have been shown both ALDH1A1 and Copper transport in OS. The purpose of our study is to 1. Compare the gene expression ALDH1A1 and copper-regulating proteins in dog OS versus normal bone cells. 2. Examine differences in copper concentrations of OS, normal bone and serum in dogs with non-metastatic disease.

Methods: Methods: This is an animal study in dogs with non-metastatic osteosarcoma that underwent limb amputation surgery by a veterinarian orthopedic surgeon. After informed consent by the owners was obtained, serum, osteosarcoma tumor (tumor), and normal bone (bone) samples were harvested from dogs at the time of surgery. Following harvest, tumor and bone samples were stored at -80ºC and ground into a fine powder. RNA was extracted from powdered samples and gene expression was performed using polymerase chain reaction (PCR). Serum, tumor and bone samples were also sent for measurement of copper concentration. Statistical analysis of gene expression and copper levels were conducted through Mann Whitney tests and ANOVA, respectively.

Results: Twenty-four dogs were included in the study. When compared to bone samples, tumor samples had higher expression of Aldehyde dehydrogenase 1A1 (ALDH1A1). Tumor samples also had higher expression of copper-regulating proteins copper transport receptor 1 (CTR1) and Antioxidant 1 copper chaperone gene (ATOX1). Tumor cells had significantly lower levels of ATPase copper transporting Beta (ATP7B), a copper efflux pump. (Figure 1). For the copper concentration analysis, four values were considered outliers (Over 3 x IQR above the third quartile) and excluded from final analysis. Average copper levels of serum (505 ng/mL, SD 124.62) and bone (576 ng/mg, SD 268.72) were not statistically significant from each other (p=0.724). However, average copper levels found in tumor samples was 809 (SD 452.98), significantly higher than that of both Serum (p< 0.010) and Bone (p=0.038) (Figure 2).

Conclusion: An increase in the gene expression of ALDH1A1 as well as genes regulating the copper transport system were seen, showing a dysregulation in gene expression in the copper pathway of osteosarcoma. It is also worth noting that copper concentrations in these cells is significantly higher in copper concentration than those of normal bone or serum. ALDH1A1 inhibition has been shown to potentiate the cytotoxic effects of the chemotherapeutic agent doxorubicin, and may be a target for lowering toxic chemotherapeutic doses needed for patients. ATOX1 has been shown to play a key role in regulating intracellular levels of copper and inhibition of ATOX1 has been shown to reduce cell viability in both canine and human cancer cell lines. All of the above suggest that both ALDH1A1 and copper dysregulation are key pathways in our samples, similar to that of human OS. Limitations include a low sample size and lack of adequate human control cells. Despite this, canines may provide us with a readily accessible animal model to study copper dysregulation in OS.

CANOS Abstract fig1.docx

CANOS Abstract fig2.docx