Project Categories: Genetics

This project will continue the researchers’ observations on gene appearance profiles in hemangiosarcoma from Golden Retrievers to German Shepherd Dogs and Portuguese Water Dogs, and it also will define how new targeted therapies may effectively control the disease in these and other dog breeds.

Results

Certain dog breeds are prone to develop certain types of cancer; yet, there has been little progress to define genes or other factors that account for this risk. Our recent work on hemangiosarcoma was the first to demonstrate that a dog’s genetic background, defined by “breed,” can influence the profile of genes that are expressed by tumors. Among other important implications, this implies that certain breeds are diagnosed with specific cancers more frequently than others because of the behavior of tumors after they arise, and not simply because they arise more frequently. Specifically, this may apply to the observed predisposition for hemangiosarcoma seen in Golden Retrievers, German Shepherd Dogs, and Portuguese Water Dogs. Here, we continued to test this premise by evaluating genome-wide gene expression profiles in samples from dogs of various breeds.

Our results suggest that, while there are subtle differences that are influenced or modulated differently in tumors from dogs of different breeds, these differences may disappear when tumors are considered in their context as “tissues” that include microenvironment constituents. Rather, there appear to be distinct subtypes of hemangiosarcoma (perhaps with different biological behavior and prognosis?), which might arise from different cells of origin, or more likely, which develop in response to adaptation of the hemangiosarcoma cells to environments that show different patterns of inflammation, angiogenesis, coagulation, and hypoxia, each of which alters not only the predominant or favored differentiation of the tumor cells themselves, but also the way they instruct microenvironment cells to create a favorable niche.

This underscores the importance of looking at these tumors in their context as “new tissues” or “new growths” rather than at the cells in isolation as we work to develop more effective strategies for detection, diagnosis, and therapy. To follow on this premise, we evaluated new therapy approaches that target both tumor and microenvironment compartments. Specifically, one such approach also shows efficacy to kill tumor-initiating cells.

Data funded by this project grant and others allowed us to validate the therapy and move it to the clinic. Angiosarcoma Awareness, Inc. provided the initial funds to support a dose finding and efficacy trial where we will treat ~20 dogs with hemangiosarcoma using a bispecific ligand targeted toxin. We completed production of the molecule under “Good Manufacturing Practices” (i.e., suitable for use in human patients) and enrollment is ongoing. Finally, we identified other potential drugs to treat this disease – or perhaps more likely, the pathways they disrupt as potential targets for development of new therapies.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 01131

RESEARCHERS

Jaime F. Modiano, VMD, PhD
University of Minnesota

Characterize the direct effects of IL-8 on HSA cells.

Results

The hypothesis tested in this project was that interleukin-8 (IL-8) promotes growth and survival of hemangiosarcoma cells. This hypothesis was based on our previous results showing significant enrichment of IL-8 gene expression in hemangiosarcoma cells compared to normal endothelial cells isolated from non-malignant hematomas. Here, we confirmed that IL-8 is constitutively expressed by canine hemangiosarcoma cells in laboratory culture, as well as by primary tumors (fresh frozen samples). However, the levels of IL-8 are moderately variable among tumors.

Hemangiosarcoma cells in culture and primary hemangiosarcoma tumors also express IL-8 receptors (IL-8Rs). The receptors are expressed at comparable levels by virtually all the cultured cells and all the tumors, suggesting changes in expression of the receptor are unlikely to contribute to malignant behavior. We also confirmed that IL-8 binds to IL-8 receptors, and this interaction has functional consequences: IL-8 promotes signal transduction (calcium mobilization) in cultured HSA cells, and when we added IL-8 to cultured cells, they were able to “sense” this IL-8 excess and downregulated the expression of their own IL-8 gene. In contrast, if we blocked the interaction of their own secreted IL-8 with the receptor, they increased the amount of IL-8 gene expression. This is a classic response of compensatory regulation to negative feedback. Expression of a gene whose protein product turns on IL-8 gene expression followed the same pattern. It was downregulated when IL-8 was present in excess and induced when IL-8 was prevented from interacting with its receptor.

Despite its biological activity, IL-8 did not promote growth of hemangiosarcoma cells in culture, and IL-8 blockade did not hinder IL-8 growth in culture. When cells were deprived of nutrients and growth factors, they did not compensate by increasing production of IL-8; instead, IL-8 expression was reduced. And the addition of IL-8 did not prevent these nutrient-deprived cells from dying, and neither did it prevent cells treated with chemotherapeutic drugs from dying. Together, the data suggested that IL-8 did not directly mediate growth or survival of hemangiosarcoma cells in culture, refuting the initial hypothesis.

We then compared the gene expression profiles of cells and tumors that expressed high levels of IL-8 (and thus were adapted to growing in an environment rich in IL-8) with those of cells and tumors that expressed lower levels of IL-8 (adapted to growing in environments with relatively scant IL-8). The data show that cells adapted to high IL-8 environments had gene expression profiles indicative of greater inflammation, coagulation, fibrosis, and angiogenesis. These data suggested that IL-8 could be important to modulate the microenvironment and provide a suitable tumor niche. Experiments from an independent, complementary project funded by the National Canine Cancer Foundation showed that indeed, blocking IL-8 hindered the ability of hemangiosarcoma cells to establish a tumor niche in vivo. Finally, preliminary data suggest that IL-8 also may be necessary to maintain the tumor-initiating populations of canine hemangiosarcoma, by enhancing self-renewal. This hypothesis is under investigation in our newly funded project supported by AKC CHF.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 01429

RESEARCHERS

Jaime F. Modiano, VMD, PhD
University of Minnesota

Hemangiosarcoma (HSA), a malignant tumor of vascular endothelial cells, is a significant health concern in dogs, with an incidence of ~2% of all tumors. A national health survey of Golden Retriever reported that neoplasia accounted for >60% of all reported deaths and HAS was the most common malignant tumor affecting >15% of Golden Retrievers. A particularly high disease incidence of hemangiosarcoma in Golden Retriever suggests a genetic susceptibility.

The purpose of this study is to identify the mutations causing the increased risk for hemangiosarcoma in Golden Retriever. To do this, we have proposed to compare the genotypes of dogs diagnosed with HSA with healthy older dogs using a statistical analysis.

Results

To date, we have collected 125 blood samples from Golden Retrievers diagnosed with HSA and more than 400 healthy Golden Retrievers over 8 years old. We have identified six regions in chromosomes associated with HSA and are have narrowed these to precise regions (a few hundred thousand base). We now need to find the precise mutations that cause the disease and link them to functionality. In the long term, this work should allow the development of specific genetic tests for carriers of HSA. Ultimately, understanding of the disease biology, which will lead to identification of target genes for prevention, early detection and novel treatments of this malignancy.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 00593A

RESEARCHERS

Kerstin Lindblad-Toh, PhD
Broad Institute at MA