Given the strong association between breed and risk, we predicted that gene expression profiles in tumors such as hemangiosarcoma also would reflect features uniquely associated with the breed. Furthermore, we anticipated that breed-related gene expression profiles would uncover biologically and therapeutically significant pathways that would inform etiology and identify therapeutic targets.
Specifically, the central hypothesis was that naturally occurring hemangiosarcomas of Golden Retrievers would be distinguishable from histologically similar hemangiosarcomas of dogs from other breeds non-Golden Retrievers based on the overexpression or underexpression of genes preferentially concentrated in one or a few metabolic pathways, thus providing insights into the pathogenesis of this disease. To test this hypothesis, we used gene expression arrays and gene set enrichment analysis GSEA to identify genes that vary according to breed, as a proxy for heritability, in naturally occurring canine hemangiosarcoma.
We hypothesized this would outline the potential influence of genetic background on cancer susceptibility and progression in a more unique way than simply comparing cancer cells to normal cells. For the first time, our data uncover unique gene sets that are peculiar to hemangiosarcoma tumors from a single dog breed sharing a common genetic background. Overall, this study emphasizes the potential benefits of gene expression analysis and bioinformatics to study different biological aspects unique to a cancer susceptible dog breed and can fill gaps in our knowledge of disease susceptibility, heritability and progression.
While many human cancer cells have been shown to harbor different gene expression signatures compared to their normal counterpart cells e. What is more, nothing has been done to outline how these phenotypes are influenced by heritable factors in any species. We showed elsewhere that hemangiosarcoma cells separate from non-malignant splenic hematoma cells based on gene expression profiles Tamburini et al, manuscript in preparation.
Before we addressed potential differences in these two groups, however, we sought to ensure there were no hidden biases in the sample population. During the course of our study, we received blood samples from 76 dogs with pathologically confirmed hemangiosarcoma, including 48 Golden Retrievers and 28 non-Golden Retrievers. The characteristics of the population were similar to those previously described both for Golden Retrievers  and for all dogs independent of breed  , .
Age and gender as variables did not account for the observed clustering of the samples: when we segregated the 10 tumor samples into groups where affected dogs were younger than 7 years vs. Nevertheless, a pattern remained when the nine tumor samples from purebred dogs excluding the sample from the F1 separated according to breed.
We anticipated that differences among hemangiosarcomas from dogs of different breeds would be subtle, thus the relatively small number of genes was not surprising given the relatively low expected discovery rate for this sample size. Figure 1A is a heat map illustrating hierarchical clusters defined by 12 known genes, 4 unknown genes and 1 repeated gene acid ceramidase isolated by two different probes. The list of known genes includes an additional MHC gene, genes involved in DNA replication and maintenance, and genes that regulate cellular metabolism Table 2.
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When gene differences were plotted according to their cytogenetic location, there were few notable changes. Predictably, since samples from Golden Retrievers included 3 females, this group also showed a net increase in the sum of expression of genes in the X chromosome. Figure 1B shows the location of individual genes that were recurrently and significantly overexpressed or underexpressed in the Golden Retriever samples. Increasing green intensity indicates increased gene expression, increasing red intensity indicates decreased gene expression, and black indicates no change.
Bars represent groups that cluster together. Gene differences between Golden Retrievers GR and non-Golden Retrievers nGR were plotted according to their cytogenetic location along the 38 autosomes and the X chromosome. Tick marks represent individual genes that show differential regulation, with the color intensity green to black to red representing expression changes as described in part A. One sample originating from a non Golden Retriever dog Dal-4 was normalized to 1. The genes were chosen because they may define MHC haplotypes or because of their relevance to tumor biology; i.
Student's T-test for equal variance was used to calculate p-values as an indication of statistical significance Table 2. When we included this sample in the hierarchical clustering, the features that separated the two groups were less distinguishable.
Thus, the expression of genes in the tumor was predictably modulated by the dog's Golden Retriever and non-Golden Retriever background. One possible explanation for why Golden Retrievers separate from non-Golden Retrievers in this analysis is that hierarchical clustering by breed reflected unique properties of genetic variants within the breed, rather than a particular influence of breed on tumor phenotypes. To our knowledge, there is no reported association between breed and MHC haplotypes, so this was unlikely.
Samples analyzed included blood leukocytes from healthy Golden Retrievers and non-Golden Retrievers, blood leukocytes from Golden Retrievers and non-Golden Retrievers that did not have hemangiosarcoma, but were diagnosed with another cancer melanoma, non-Hodgkin lymphoma, or osteosarcoma , and the hemangiosarcoma cells from each affected dog Table 3.
One interesting observation is that the range of expression for these genes in the hemangiosarcoma samples and in blood samples from healthy dogs were narrow, but they were relatively wide in blood samples from dogs that had non-hemangiosarcoma tumors. This suggests the differences were not due to variants in the breed, and instead were due to the influence of genetic background breed itself on hemangiosarcoma phenotypes.
Another possibility was that this difference would be reflected only on tumor samples, so we assessed whether these genes had significantly different calls when comparing our hemangiosarcoma Golden Retriever expression arrays to expression arrays from lymphoma and leukemia 30 Golden Retrievers and from osteosarcoma 9 Golden Retrievers.
The association between hemangiosarcoma and overexpression of acid ceramidase was reinforced in these analyses, but neither TSP-3, nor DLA, nor SMARCA1 showed differential expression according to breed in lymphoma and leukemia or in osteosarcoma, although those samples also appear to have different and unique sets of genes whose expression varies as a function of breed T. Phang, K. Gavin, A. Sarver, and J.
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Modiano, unpublished data. The single largest category where genes differed between the two groups was genes involved in transcription.
GSEA is designed to identify categories, families, or sets of genes where there are potentially small but coordinated changes in gene expression. Yet, immunologic analysis verified the GSEA data. One recently developed line that had not been arrayed Emma was included as a means to provide validation of the data. Increasing red intensity reflects higher enrichment scores.
The genes enriched in the highest number of gene sets are identified by name. Staining was visualized using epifluorescence. Conditions were optimized for linearity. Densitometric band quantification was done using Image J 1. Finally, we examined if these expression patterns had functional correlates.
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As would be predicted from the data in Figures 3A and 3B , we noticed some variation in the levels of phosphorylated VEGF receptors in the cells, mostly related to the overall steady state expression of these proteins. Figure 4 shows that Drug 1 did not significantly affect any of the seven cell lines tested. These responses were dose dependent and peaked at concentrations of 0.
Drug 2, which has lower affinity for both receptors, did not significantly alter proliferation of hemangiosarcoma cells, but it is compelling that there was a trend for greater proliferation by the Golden Retriever tumor lines at higher concentrations 1 to nM. Additionally, VEGFR1 did not appear over-represented in any of the other tumor types we examined from Golden Retrievers suggesting these changes are specific to Golden Retrievers with hemangiosarcoma.
Together, the data indicate that gene expression patterns identified by gene set enrichment analysis across distinct subgroups are biologically significant, and in this case, they suggest VEGFR1 is not a decoy receptor, but rather it is an active growth inhibitor in hemangiosarcoma cells derived from Golden Retrievers.
The effect of three VEGFR inhibitors on proliferation and viability of hemangiosarcoma cells was tested in vitro.
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The selectivity and half maximal inhibitory concentrations for Drugs 1, 2, and 3 are listed in the Materials and Methods. Cells were then cultured for 72 hr, and the number of viable cells was determined using the MTS assay. The mean of two independent experiments is shown at drug concentrations of nM.
P-values were calculated using Student's T-test. The relevance of naturally occurring canine tumors to improve our understanding of cancer biology and genetics has been increasingly recognized in recent years  ,  , . Canine tumors can be utilized as a system to understand how genetic background can influence the susceptibility of an individual to non-inherited cancers.
Due to the homogeneity among dog breeds, we can study frequently occurring cancers within groups in a way that would be difficult within the genetically diverse human population or in laboratory animals, where most tumors are induced chemically or by genetic manipulation. We studied naturally occurring canine hemangiosarcoma to test the hypothesis that patterns of gene expression could outline biological differences between tumor cells originating from dogs of a distinct breed that have a higher lifetime risk for hemangiosarcoma. Hemangiosarcoma is ontogenetically related to human angiosarcoma and Kaposi sarcoma, as all three are presumed to arise from hemangioblastic or endothelial progenitors and they share signaling abnormalities  ,  , .
The highly metastatic behavior and modest response to chemotherapy distinguish canine hemangiosarcoma and human angiosarcoma from other common soft tissue sarcomas that are locally invasive and generally unresponsive to chemotherapy. We uncovered a set of hemangiosarcoma-associated genes peculiar to a single dog breed suggesting these are modulated by or with heritable traits that may influence risk for this cancer.
We considered carefully the choice of low passage cell lines vs. Tumors are in essence tissues . Tumor cells modify the microenvironment and are themselves responsive to environmental cues. Nevertheless, to understand the contribution of the tumor cells to biological and pathological processes, it is important to be able to examine the response on isolated cells. One approach to do this is microdissection, but in a vascular tumor, it is difficult to microdissect malignant tissue without retaining normal angiogenic components, which are morphologically indistinguishable in many cases, and blood elements.
On the other hand, cell lines provide a homogeneous, unlimited resource that can be extensively characterized with regard to ontogeny.
The potential limitations of cell lines such as their restricted origin, possible in vitro evolution or drift, and adaptation for growth in culture, can be mitigated by use of controls that replicate culture conditions so that adaptation to ex vivo growth is filtered from responsive transcript lists, and by use of more than one sample. Our results show that despite the different origin, isolation, and establishment of the cell lines we used for these experiments, hemangiosarcomas retained unique characteristics that distinguished them from other cultured or primary cells, and that the recurrent finding of genes that are over- or under-expressed in the samples is significant and represents differences that can be traced to the developmental process of the sample ontogeny or pathological progression , rather than to selection in culture.
Ongoing experiments are designed to define the correlation of these findings in intact tumor samples where extracellular matrix associations are maintained. Among genes whose expression differed between Golden Retrievers and non-Golden Retrievers, a disproportionately high number of genes encode transcription factors. This suggests that transcriptional regulation might play a key role in disease susceptibility and progression.
Upregulation of SMARCA1 in Golden Retrievers with hemangiosarcoma was intriguing since changes in expression of a single transcriptional regulator can create genome-wide disruption of a variety of genes, possibly resulting in faster progression of the disease.
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The downregulation of MHC class I genes in hemangiosarcoma from Golden Retrievers added a level of confidence, as these genes represent the likely targets to define individual or breed-specific differences. This pattern is rather unique to hemangiosarcoma, as normal blood leukocytes and other tumors from Golden Retrievers for example, leukemias show robust expression of MHC class I. The organization and control of genes in the canine MHC class I locus remains poorly understood, and our data will undoubtedly spur further study of how genetic variants within breed and transforming factors might influence MHC class I expression.
In fact, breed-related polymorphisms or changes in expression level have not been identified in normal canine somatic cells; thus, downregulation of MHC class I genes at least MHC DLA and DLA in hemangiosarcoma cells from Golden Retrievers might reflect selective pressure to evade immune responses, or perhaps a response to autocrine or paracrine factors such as interferons or other inflammatory mediators.