Molecular markers to identify osteosarcoma patients at risk for metastatic progression are critical to adopting suitable treatment options. Recently, several molecular markers based on gene expression in tumor tissues have been identified in osteosarcoma. Higher levels of oncogene YY1 expression in primary sites of osteosarcoma are associated with metastasis and poor clinical outcomes [18
]. Similarly, overexpression of markers such as cancer-testis antigens, Cystein Rich Protein (CYR61) and Melanoma Antigen Family A (MAGEA) are implicated in predicting tumor progression and prognosis in osteosarcoma [19
]. Additionally, the presence of tumor-infiltrating macrophages is associated with metastasis suppression in high-grade osteosarcoma [21
]. These studies show that the development of biomarkers for tumor progression and outcomes have clinical significance and may help develop patient specific treatment strategies. Recently, miRNAs from tumor tissues have been proposed for use in the diagnosis, classification and the prognosis of various cancers [22
The cluster of miRNAs at the 14q32 locus show decreased levels in human osteosarcoma [10
]. miRNA profiling of murine osteosarcoma tumors also show decreases in the orthologuos region of chromosome 12 relative to the levels observed in normal bone tissues (personal communication). Here, we report decreases in miRNA in the orthologous region of CFA 8 cluster members in canine osteosarcoma tumors. This is consistent with the presence of a commonly conserved mechanism of tumorigenesis involving the orthologous 14q32 locus associated genes and miRNAs between these species. Thus, the cross species comparison of human and canine expression profiles provides an opportunity to define the existence of evolutionarily conserved molecular signatures and to determine whether those signatures influence the outcome of our 3 patient cohorts (2 human and 1 canine).
In both the human and canine osteosarcoma cases the 14q32 miRNAs and its orthologs appear to be regulated by a common mechanism, based on the high levels of expression correlation observed between the transcript levels of each member. Analyses of the Sarcoma MicroRNA Expression Database (SMED) [9
] data reveal that 14q32 miRNA, as a group, are lost in a subset of GIST (3/14) and MFH (5/29). We conclude from this that the level of 14q32 miRNAs in general can be estimated from the measurement of a single member of the family and that measurement of the majority of the 14q32 miRNAs would provide highly similar data. Polycistronic regulation of these miRNAs has previously been proposed as a regulatory mechanism active at this locus and our data supports this concept [17
In an initial set of human osteosarcoma tumors miRNA microarray profiling data we observed that lower levels of 14q32 miRNA were associated with metastases. We further validated this by qRT-PCR for both miR-382 and miR-154 for osteosarcoma tumors from this initial dataset. We also showed that the lowest levels of miR-382 were significantly associated with metastasis and significantly worse survival in a second independent set of human primary osteosarcoma tumors where full clinical follow-up information was available. Taken together, these results suggest that the level of miR-382 may have prognostic utility for predicting the likelihood of developing metastatic tumors and disease outcomes. This observation is supported by three independent dataset from two different species. While the association between reduced expression of 14q32 miRNAs and overall survival in humans represented a trend, in dogs, the magnitudes of miR-134 and miR-154 reduction were negative predictors of survival. This is likely due to the fact that, unlike humans (and especially children) where preserving life is the guiding principle, companion animal patients are treated with quality of life as the guiding principle. Therefore, metastatic disease is seldom treated aggressively and recurrence often leads to a decision of humane euthanasia, creating little difference between the disease-free interval and overall survival [27
]. It is prudent to note, however, that osteosarcoma is a highly heterogeneous disease and the sample sizes of the cohorts used in this study were relatively small. Thus, our conclusion will require additional validation in a larger cohort of osteosarcoma patients with clinical follow-up.
We have previously shown that decreases in 14q32 miRNA levels stabilize cMYC expression in osteosarcoma and subsequently increase the expression of oncogenic miR-17-92 miRNAs [10
]. Deregulations in 14q32 miRNA-cMYC network is also associated with increased proliferation and apoptotic escape in osteosarcoma [10
]. The prognostic relevance of decreasing 14q32 miRNA levels may be explained 1) by increased activation of the cMYC/miR-17-92 network leading to poor outcomes 2) via regulation of an alternative cancer signaling pathway(s) either directly or indirectly or 3) as a biomarker whereby decreased levels of 14q32 miRNAs are observed in osteosarcoma cases with poor outcomes.
Genes involved in metastasis are significantly enriched in transcripts correlated to 14q32 miRNAs and the direction of change indicates that metastasis function is also significantly increased (Additional file 6
: Table S5 and Additional file 7
: Table S6). CDK5 and TWIST1 have been reported to increase metastasis [28
] and are upregulated in osteosarcoma tumors with low levels of 14q32 miRNAs. IFNB1 [30
], TEK [31
] and COL18A1 [32
] have been reported to decrease metastasis and are downregulated in tumors with low levels of 14q32 miRNAs. We also noticed that TK1
is negatively correlated to 14q32 miRNA levels. TK1
is involved in the catalysis of phosphorylation of thymidine to deoxythymidine monophosphate and expressed at high levels in proliferating cells and appears to correlate with high risk in multiple cancer types [34
]. This suggests that genes correlated with 14q32 miRNA expression patterns may provides molecular rational for the observed association between low levels of 14q32 and metastasis and poor survival outcomes and can be further investigated as markers of prognosis.
We have previously demonstrated that comparative studies of human and canine osteosarcoma patients allow study of evolutionary conserved mechanisms of tumorigenesis associated with outcomes [6
]. A majority of the dogs evaluated in this study (21/25 in the correlating miRNA analysis and 12/16 in the qRT-PCR analysis) were treated with standard of care, which consists of amputation of the limb to remove the primary tumor followed by with adjuvant chemotherapy. Predictably, treatment generally was associated with increased survival, but the negative correlation with miR-134 and miR-154 was not driven by the type of treatment that the dogs received, and in fact, the data suggest that the dogs that received palliative care and amputation (2, 8 and 58
days survival) would have been reasonable candidates for more prolonged survival with standard of care treatment.