Disrupting the Differentiation of Cancer Stem Cells to Prevent the Spread of Hemangiosarcoma

Hemangiosarcoma is a rapidly fatal disease. The lifetime risk is alarmingly high for some breeds like Golden Retrievers (~20% will die of this disease) and Portuguese Water Dogs (~15% will die of this disease). The risk of hemangiosarcoma is not limited to just these breeds but is considered a research priority for 40 different breed Parent Clubs and Foundations.

Despite considerable efforts to find effective treatments, the outcome for dogs with hemangiosarcoma has changed very little over the past 30 years. Recent evidence suggests hemangiosarcoma conforms to the “cancer stem cell” model, where a defined subset of cells is responsible for initiating and maintaining the tumor. These cells are resistant to conventional therapies and are very adaptable, being able to survive in a variety of tissues in the body.

For this project, Dr. Modiano proposes to reduce the malignant potential of hemangiosarcoma stem cells by forcing them to terminally differentiate into cells which can no longer self-renew. He further proposes that by disrupting their ability to self-renew he will enhance the sensitivity of these cells to conventional and targeted therapies and improve the outcomes of dogs with this disease.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 01759

RESEARCHERS

Jaime F. Modiano, VMD, PhD
University of Minnesota

Assessing How a Protein Helps Hemangiosarcoma Cells Survive

Canine hemangiosarcoma is a common and highly fatal cancer in dogs. Recent evidence suggests that populations of cancer stem cells give rise to tumors, promote tumor growth and are the main culprits behind drug resistance and disease recurrence.

This study examines how a protein expressed by stem cells contributes to the maintenance and survival of hemangiosarcoma stem cells.

Co-sponsored with the Morris Animal Foundation, Grant Number: D13CA-062

RESEARCHERS

Dr. Erin B. Dickerson, PhD
University of Minnesota

Searching for Ways to Control Hemangiosarcoma Cancer Cells in Dogs

Hemangiosarcoma is a highly metastatic and incurable cancer that can affect dogs at any age. It is particularly prevalent in certain breeds such as Golden Retrievers, German Shepherds and Portuguese Water Dogs. Unfortunately, hemangiosarcoma is a disease that is poorly understood and for which there are currently no good treatment options.

One of the main reasons why therapies fail may be due to the existence of cancer stem cells. These cells are responsible for initiating and maintaining the cells within the tumor and they are also highly resistant to most chemotherapy. Researchers at University of Minnesota, funded by Morris Animal Foundation, are examining the role of small molecules that may serve as signals in the regeneration of hemangiosarcoma stem cells. Specifically, investigators are evaluating the potential to control the activity of hemangiosarcoma stem cells by altering these molecular signals in a way that stops stem cell regeneration and enhances sensitivity to chemotherapy.

So far, preliminary data show that one signaling pathway under study has distinct effects on the regeneration of hemangiosarcoma cancer stem cells in the lab. This signaling pathway seems to affect the stem cells’ efficiency in forming a sphere as part of the self-renewal process. If this signal can be altered, it may reduce the stem cells’ ability to renew and maintain the tumor. Further research is needed to confirm the results.

This study is providing valuable insight into the properties of cancer stem cells that could help in the development of tests to predict a patient’s outcome and appropriate therapies to treat canine hemangiosarcoma. Funding for this project is also supporting the training of a promising, new canine cancer researcher.

Co-sponsored with the Morris Animal Foundation, Grant Number: D13CA-400

RESEARCHERS

Jong Hyuk Kim, DVM, PhD
University of Minnesota

Genetic Background and the Angiogenic Phenotype in Cancer

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

Mechanistic Relationship of IL-8 in Cell Proliferation and Survival of Canine Hemangiosarcoma

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

Making Advanced Discoveries in Golden Cancers

The three-year project will examine genetic traits that contribute to risk and progression of hemangiosarcoma and lymphoma.

Golden Retrievers have been one of the most popular breeds in America for decades, but unfortunately these dogs also have one of the highest incidences of cancer. Hemangiosarcoma and lymphoma account for more than 30 percent of the deaths in this breed. Although breed susceptibility to cancer was first reported 30 years ago, the relationship between inherited traits and susceptibility for these cancers is still not known. The Golden Retriever Foundation and Morris Animal Foundation are funding this study to discover and characterize heritable and somatic cancer mutations in Golden Retrievers.

The three-year project will examine genetic traits that contribute to risk and progression of hemangiosarcoma and lymphoma in Golden Retrievers. The long-term goal is to understand what causes these diseases. Because both cancers occur with such high frequency, reducing their incidence (while retaining the positive phenotypes of the breed) will be a complex task, but the development of reliable genetic tests would allow breeders to build programs whereby high-risk combinations of factors could be avoided. In addition, effective strategies could be developed to control and treat hemangiosarcoma and lymphoma in Golden Retrievers and other dogs. What is learned from this research may also lead to effective prevention and treatment strategies for these diseases in people and other breeds.

Co-sponsored with the Morris Animal Foundation, Grant Number: D10CA-501

RESEARCHERS

Jaime F. Modiano, VMD, PhD
University of Minnesota

Matthew Breen, PhD, CBiol, FSB
North Carolina State University

Kerstin Lindblad-Toh, PhD,
Uppsala University, Sweden

Comment: Because of Dr. Modiano’s work with PWDs in the past, this will definitely benefit our breed.

Evaluating Drugs to Treat Hemangiosarcoma

Tyrosine kinase inhibitors may have the potential to control the growth of hemangiosarcoma.

Hemangiosarcoma remains one of the deadliest canine cancers. Despite treatments such as chemotherapy, immunotherapy and surgery, dogs rarely live beyond six months after diagnosis. New approaches are needed to improve the survival time of dogs afflicted with this devastating disease.

This study will expand on the team’s previous research into a novel class of drugs – tyrosine kinase inhibitors – that may have the potential to control the growth of hemangiosarcoma. The results will help to clarify abnormalities that contribute to hemangiosarcoma proliferation and may ultimately lead to new treatment options for this aggressive cancer.

Co-sponsored with the Morris Animal Foundation, Grant Number: D08CA-050

RESEARCHERS

Stuart C. Helfand, DVM
Oregon State University

Reciprocal Relationship of PTEN and p21 in Canine Cancer

An estimated one out of every two dogs alive today will get cancer in its lifetime, and as many as 50 percent of those will die from the disease. Despite significant gains in cancer treatment, a thorough understanding of why cancers arise and why they behave as they do is essential to improving prevention and treatment. For this project, researchers will investigate two proteins whose interactions appear to be intimately tied to the behavior of two serious cancers, melanoma and hemangiosarcoma. What they learn may help to test targeted therapies for these cancers and significantly improve the lives of affected dogs.

Results

Cancer is the leading cause of disease-related death in dogs, which is why the Morris Animal Foundation and the Portuguese Water Dog Foundation have invested considerable resources to understand this group of diseases to develop more effective treatments. Cancer happens when genes that control the balance of division and survival cease to function normally in a cell and cause it to become malignant. One of these genes is called PTEN. The protein product of this gene generally restrains cell division, in part by controlling another protein called p21.

Scientists at the University of Minnesota and at the University of California, Davis have found that using compounds to lower the levels of p21 in some tumors decreased the resistance to conventional chemotherapy drugs. They also concluded that chemotherapy resistance is sometimes unrelated to abnormalities of PTEN, while it is often associated with elevations of p21. The results have allowed the investigators to justify efforts to move these compounds to the next step of clinical development.

Co-sponsored with the Morris Animal Foundation, Grant Number: D06CA-065

RESEARCHERS

Jaime F. Modiano, VMD, PhD
University of Minnesota

Mapping Genes Associated with Canine Hemangiosarcoma

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

Canine Hemangiosarcoma

The main goal of this project was to develop a diagnostic test that can be used to detect hemangiosarcoma cells in blood samples from dogs. The rationale for the test is based on the fact that, as a tumor of cells that line blood vessels, hemangiosarcoma tumor cells are in intimate contact with blood and are likely to shed cells into the circulation. We can therefore use the identification of those cells to confirm a possible diagnosis in dogs that have clinical signs, and eventually to detect the disease before it becomes clinically evident (early stages when it may be more amenable to treatment) as well as to follow response to therapy (monitor remissions). Less than 50% of dogs treated with standard of care for this tumor (surgery and intensive chemotherapy) survive more than six months. Many dogs die from severe internal bleeding before there is an opportunity to institute treatment.

Results

We have further refined the system to verify we can detect malignant endothelial cells in dog blood when they represent as few as 0.01% of the nucleated cells. We are continuing to work out technical details, including cell enrichment methods, use of “positive” vs. “negative” selection, and the most reliable and reproducible format to offer the test in the field (that is, to “real” patients). The test will be licensed by Idexx Laboratories, Inc., which has now provided support for completion of the development process, including clearing regulatory hurdles and providing nationwide availability. We are much closer to a real product, and the support from the Portuguese Water Dog Club of America and the Portuguese Water Dog Foundation, Inc. allowed us to make significant progress and secure more stable funding for development from Idexx. We are indebted to the Club and the Foundation for their generosity and vision to make this happen. We truly feel this test will make a quantitative improvement in the quality of life for our dogs, by improving our ability to diagnose the disease and monitor the response to therapy.

Co-sponsored research with the Portuguese Water Dog Club of America, Inc.

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Canine Hemangiosarcoma – The Road from Despair to Hope

RESEARCHERS

Jaime F. Modiano, VMD, PhD
University of Colorado

NOTE: Dr. Modiano is now at the University of Minnesota