Our cross-species genomic analysis of osteosarcoma is the first to compare global gene signatures of any spontaneously occurring disease state with the same disease in humans. Two important findings have emerged from this work: (1) there are very strong similarities in gene expression patterns between canine and human osteosarcoma. These similarities further support the inclusion of pet dogs as a translational model in studies of osteosarcoma therapy; and (2) that these similarities, coupled with the more aggressive biology of the canine disease provides a new perspective from which genes and pathways integrally related to the metastatic biology of this disease may be assessed. Indeed, we identified four specific genes that were defined as "dog-like" genes to be expressed in humans. Expression levels of two of these "dog-like" genes (IL-8 and SLC1A3) were associated with poor outcome in human osteosarcoma patient samples. It is unlikely that these potential progression associated genes would have been considered without the perspective provided by the cross-species approach.
The public release of a high-quality sequence covering 99% of the canine genome (2.5 billion base pairs) has confirmed remarkable similarities between the genomes of dog and man and has provided the opportunity for the cross-species studies conducted here [18, 19]. The implementation of such approaches is feasible through commercially available canine oligonucleotide and SNP arrays, first by Affymetrix and now by others. It is now possible for high throughput interrogation of canine tissues and disease states, using platforms and processes previously limited to the mouse and human [4, 18, 19, 22]. Work by several groups has begun to use the canine genome as a means to understand basic biology and genetics of health and disease, including cancer [23–25]. Past comParative genomic studies in cancer have included work in rodents and humans [26, 27]. There are many challenges to cross-species comParative genomics that include the dog, including incomplete gene annotation and the lack of methodologies for orthologous gene assessment. We used a comParative genomics methodology, Entrez Gene ID alignment, similar to that described by Sweet-Cordero et al. and Lee et al. to study gene signatures in murine models of cancer, in our canine-human analysis of osteosarcoma progression. Improved cross-species genomic analyses will be possible with further genome annotation and sequence similarity assessments .
Past studies that have sought to define targets linked to metastatic progression in osteosarcoma, a highly metastatic pediatric malignancy, have been hampered by the overwhelming bone signature of osteosarcoma, heterogeneous and karyotypic complexity of the disease, relative paucity of tumor samples available before exposure to chemotherapy, and the small number of patient samples available given the rarity of this disease. Baird et al., used expression profiling to define and distinguish osteosarcoma from other similar pediatric cancers, but this signature was highly influenced by its association with bone . Since initial response to neoadjuvant chemotherapy is a recognized prognostic factor in osteosarcoma. Mintz et al. and Ochi et al. used expression analysis by microarray to define classifiers of a poor response to chemotherapy [30, 31]. The starting material for these studies were tumor samples obtained following chemotherapy at the time of definitive resection of the tumor . The greater number of dogs who are diagnosed yearly with osteosarcoma, along with the more accelerated progression to metastasis compared to pediatric osteosarcoma, provides a greatly needed resource for the study of this rare pediatric cancer and may be necessary for optimal progress to be made.
Using cluster analysis of human-dog orthologous genes that were differentially expressed between canine cancers and canine normal tissues we were unable to segregate the gene expression signatures of canine from human osteosarcoma. Previous candidate studies of the genetics and biology of osteosarcoma in both species have been limited but supported the similarities between the diseases. We were nonetheless surprised that the expression of orthologous genes could not distinguish the cancers by species. It is important to note that the normal canine and normal human tissue expression signatures were clearly distinguished using this same approach. These data suggest that the osteosarcoma gene expression pattern was dominant over the gene expression patterns of species. Since normal bone was not included as a comParator in normal tissue panel for both, it is reasonable that the dominant expression pattern, common to both species is their association to bone. However, the abundantly expressed biological themes found in canine osteosarcoma are similar to the previously published work describing human osteosarcoma [30–33].
Additionally, it is important to note that the canine normal tissue samples used for this cross-species analysis are true biological replicates and not pooled samples or single samples assayed multiple times. This contrasts the normal human gene expression data derived from the online GNF-GEA database that has batch effects that may introduce bias that differs from expression data obtained from canine osteosarcoma, canine normal, and human osteosarcoma tissues. Limitations exist when harvesting normal human tissue for gene expression analysis due to the fact that most, if not all samples, are attained at various times post mortem. The canine normal tissue samples used in this current study were harvested promptly minimizing sample degradation and bias that may negatively influence gene expression quantification. It is possible that previous genomics studies of human osteosarcoma failed to capture the true biological variability between individuals due to normal sample pooling. Therefore, the ability to collect individual, high-quality canine normal and tumor samples may unmask superior and distinct relationships and improve upon the opportunity to detect biological differences between normal and diseased tissues.
The strength of the similarities between the dog and human gene signatures allowed us to extend the use of the comParative data to identify progression-associated genes. Since dogs have a more aggressive course of disease than humans we hypothesized that "dog-like" genes may define a more aggressive phenotype of human osteosarcoma that could not be previously identified in human genomic evaluations. By identifying genes with the highest expression in a population of canine osteosarcoma and marginal expression in a population of human osteosarcoma patients, two of four "dog-like" genes, IL8 and SLC1A3 (IL-8, p = 0.0201; SLC1A3, p = 0.0264), were defined that were negatively associated with survival in a distinct human osteosarcoma data set. IL-8 is a major mediator of the inflammatory response. It functions as both a chemo-attractant for a variety of cell types as well as an angiogenic factor. Both functions have been mechanistically linked to cancer progression in a number of histologies including human melanoma, breast, prostate, pancreatic, head and neck, bladder, ovarian and colorectal carcinomas [34–42]. In a study by Rutkowski et al., elevated serum levels of a variety of cytokines including IL-8, IL-6 (interleukin-6), IL-1 (interleukin-1) and TNFR1 (Tissue Necrosis Factor Receptor 1) were linked to tumor extent and poor prognosis in adult patients with bone sarcomas . IL-8 up-regulation has also been implicated as a possible pathway for Doxorubicin resistance in a drug resistant human osteosarcoma cell line (143B-DR-DOX), although its impact in paclitaxel resistance in less clear in other in vitro assessments [43, 44]. If indeed an indicator of future progression in human osteosarcoma patients, this gene is of particular interest, as it is a druggable target for inhibition because monoclonal IL-8 antibodies are already in clinical development . SLC1A3 (also known as EAAT1) is a high affinity glutamate transporter that normally regulates neurotransmitter concentrations, although it has also been found outside of the CNS. It has been linked to motility and is highly expressed in aggressive glioma cell lines versus less aggressive variants . Interestingly, it has been recently described by Kalaiti et al. to be present in MG-63, an osteoblastic osteosarcoma cell line, and functionally can be up regulated by glucocorticoids [47, 48]. Therefore it may also have implications in bone pathophysiology and as a target for further evaluation in osteosarcoma. The value of the comParative approach (i.e. search for "dog-like" genes) in the studying human osteosarcoma progression associated genes was supported when the predictive value of the dog-genes was compared to candidate non-dog genes previously linked to cancer biology or progression. Furthermore, the demonstration of IL-8 and SLC1A3 expression at the protein level in human osteosarcoma patient (TMA) samples validated the potential relevance of this comParative approach across platforms. Future studies will include functional analysis of these poor outcome genes within in vitro and in vivo models and evaluation of their expression in larger outcome-linked patient datasets. Such data sets are not currently available but are a focus of work by several collaborating groups.