Canine tumor cross-species genomics uncovers targets linked to osteosarcoma progression

Abstract

Background: Pulmonary metastasis continues to be the most common cause of death in osteosarcoma. Indeed, the 5-year survival for newly diagnosed osteosarcoma patients has not significantly changed in over 20 years. Further understanding of the mechanisms of metastasis and resistance for this aggressive pediatric cancer is necessary. Pet dogs naturally develop osteosarcoma providing a novel opportunity to model metastasis development and progression. Given the accelerated biology of canine osteosarcoma, we hypothesized that a direct comparison of canine and pediatric osteosarcoma expression profiles may help identify novel metastasis-associated tumor targets that have been missed through the study of the human cancer alone.

Results: Using parallel oligonucleotide array platforms, shared orthologues between species were identified and normalized. The osteosarcoma expression signatures could not distinguish the canine and human diseases by hierarchical clustering. Cross-species target mining identified two genes, interleukin-8 (IL-8) and solute carrier family 1 (glial high affinity glutamate transporter), member 3 (SLC1A3), which were uniformly expressed in dog but not in all pediatric osteosarcoma patient samples. Expression of these genes in an independent population of pediatric osteosarcoma patients was associated with poor outcome (p = 0.020 and p = 0.026, respectively). Validation of IL-8 and SLC1A3 protein expression in pediatric osteosarcoma tissues further supported the potential value of these novel targets. Ongoing evaluation will validate the biological significance of these targets and their associated pathways.

Conclusions: Collectively, these data support the strong similarities between human and canine osteosarcoma and underline the opportunities provided by a comparative oncology approach as a means to improve our understanding of cancer biology and therapies.

Figure 1

Single species cluster dendrograms define canine and human osteosarcoma as distinct from normal organs and osteosarcoma cell lines. Cancer defining gene signatures were generated by calculating the differential expression between canine and human osteosarcoma samples and their respective normal organs using limma (Linear Models for Microarray Data, adjusted p value < 0.01). A. The canine cancer signature consists of 3471 genes and B. human cancer signature 2705. High level, unsupervised hierarchical clustering conducted in each species separately resulted in osteosarcoma samples clustering together and distinctly from normal tissues and their respective cancer cell lines.

Figure 2

Cross species analysis of canine and human osteosarcoma are not distinguishable by global gene expression signature. Comparative genomic analysis was performed by defining the differentially expressed genes between osteosarcoma and normal tissues (adjusted p value < 0.01) and by establishing orthologues between species. After Entrez Gene ID alignment, 265 genes were used to cluster the human and canine osteosarcomas, normal tissues and cell lines. Hierarchical clustering resulted in complete branching of normal and tumor samples, and normal organs could be further defined based on species of origin. Among the 30 primary tumor samples, branching of human and canine osteosarcoma is not divided by species. This suggests that similarities in gene expression signatures in osteosarcoma are due to shared biology across species.

Figure 3

Algorithm depicting the selection process for dog specific osteosarcoma genes using a fold-expression methodology. In order to define a list of dog specific osteosarcoma genes that are variably expressed in human osteosarcoma, probe sets with matching gene names or symbols across both species were evaluated (14,391 probe sets). An initial list of dog osteosarcoma defining genes was generated by identifying those probe sets with the highest fold expression differentials between the canine tumors and their normal tissues and present expression in the human tumors and their normal tissues (dog: > 8-fold up-regulation in tumors versus normal; human: <2-fold upregulation in tumors versus normal). This yielded 27 probe sets, representing 15 unique genes. Those genes that also had representative probe sets upregulated in both dog and man (> 8 fold expression) were then excluded, leaving 10 genes. This was further filtered by retaining only those genes with consistent expression across all their Affymetrix probe sets; using these stringent criteria 4 dog-like specific osteosarcoma genes were defined.

Figure 4

Canine osteosarcoma can predict genes linked to an aggressive phenotype in human osteosarcoma. High expression of two of four "dog-like" genes A. IL-8 (p = 0.0201) and B. SLC1A3 (p = 0.0264) were linked to poor outcome in a distinct population of 34 human osteosarcoma patient samples using Kaplan Meier analysis. IL-8's impact on outcome was evaluated according its median expression (Low (0-50) equivalent to < median expression; High (51-100) equivalent to > median expression); whereas SLC1A3 was assessed according to quartile expression (Lower (0-74) equivalent to < highest quartile expression; Highest (75-100) equivalent to highest quartile expression).