Mitochondrial Dysfunction and mtDNA Mutations in Canine Hemangiosarcoma: Novel Biomarkers for Early Diagnosis and Personalized Therapy

Posted on by Crystal Smith

SUMMARY: Researchers will study if dysfunction of the mitochondria, the cell’s “powerhouses,” are drivers of tumor aggressiveness.

THE PROBLEM: Hemangiosarcoma is a highly aggressive and rapidly progressing cancer of dogs. Hemangiosarcoma is usually diagnosed when the tumor is in an advanced stage. Nearly 90% of all dogs die within the first year of diagnosis, and long-term survival is rare. New treatments are desperately needed.

THE PROJECT: Recent findings in human oncology strongly indicate that mitochondrial dysfunction serves as a crucial driver of cancer progression, metastasis, and treatment resistance. Unfortunately, there is a lack of information about how mitochondrial dysfunction could influence cancer progression dogs. This innovative study aims to address this critical gap by investigating mitochondrial dysfunction and associated mitochondrial DNA (mtDNA) mutations within canine HSA tumors. Utilizing advanced techniques, including spatial transcriptomics, cutting-edge mtDNA sequencing and comprehensive bioenergetic profiling, this study will identify regions of tumors exhibiting severe mitochondrial impairment and hyperactivation. Identifying region-specific mitochondrial dysfunction is vital, as it reveals different metabolic and genetic signatures directly linked to tumor aggressiveness. We hypothesize that region-specific mitochondrial dysfunction drives tumor aggressiveness through oxidative stress, mtDNA mutations, and synergy with oncogenic mutations.

POTENTIAL IMPACT: Identifying mitochondrial molecular signatures could lead to non-invasive blood-based biomarkers, enabling earlier detection and personalized therapies, offering new hope for veterinarians, pet owners, and affected dogs alike.

Co-sponsored with the Morris Animal Foundation, Grant Number: 26CAANFT-0005

RESEARCHERS

Krysztof Marycz, Ph.D, CVT; International Institute of Translational Medicine (MIMT)

Amount: $10,000

Building a Portuguese Water Dog Reference Genome to Unlock Breed-Specific Health Insights

Posted on by Janet Boyd

The Portuguese Water Dog Foundation is very excited to join with Drs. Jaime Modiano VMD, PhD and Steven Friedenberg DVM, PhD, DACVECC in a project to improve the lives and well-being of PWDs. The team of Drs. Modiano and Friedenberg will lead a project creating a reference genome specific to PWDs, which will improve our ability to identify genetic drivers of common inherited disorders in the PWD. This project is fully funded by your donations to the PWD Foundation. Once completed, this high-quality reference genome will be made available to the public, allowing researchers from around the world to access this cutting-edge resource that will enhance the breed’s welfare.

Background of Canine Genome

Both Addison’s disease and hemangiosarcoma impose serious health burdens on Portuguese Water Dogs (PWDs). Other conditions – for example hip dysplasia, dilated cardiomyopathy, inflammatory bowel disease, and hypothyroidism – also disproportionately affect the breed. Efforts to pinpoint the genetic drivers of these disorders have been limited by reliance on canine reference genomes built from other dog breeds, such as the Boxer (CanFam 3.0) and German Shepherd (CanFam 4.0), which miss PWD-specific variation, particularly in complex or repetitive regions of the genome. Human genome studies have shown that relying on a single reference can omit up to 10% of a species’ genetic content – hundreds of millions of bases and the majority of structural variants – highlighting just how much PWD-specific information may be lost without a dedicated genome assembly.

How This Project Will Make a Difference

This project will generate a high-quality, telomere-to-telomere reference genome combining the latest technologies, to produce a PWD genome assembly of unprecedented accuracy and completeness. Crucially, this assembly will be released publicly so that researchers everywhere can align existing and future PWD sequencing data directly to the correct reference. Additionally, this assembly will enable researchers to compare PWD genomes with those of other breeds – unlocking insights into inherited variation that governs morphology, disease risk, and longevity.

This breed-specific framework will immediately accelerate discovery of genetic variants underlying Addison’s, hemangiosarcoma, and the full spectrum of PWD-predisposed conditions. Existing whole-genome and targeted sequencing datasets can be reanalyzed at once, and all future studies will benefit from a precise, breed-specific scaffold for variant calling, functional annotation, and the development of diagnostic tests. By funding a PWD reference genome, the Foundation will help create a lasting, community-wide resource that transforms research, informs breeding decisions, and ultimately improves the healthspan of PWDs.

The Team Behind the Project

The research team will be co-led by Dr. Steven Friedenberg, DVM, PhD, and Dr. Jaime Modiano, VMD, PhD. Together, Drs. Friedenberg and Modiano are uniquely positioned to complete this work because of their deep knowledge of PWDs and complementary skill set. Dr. Friedenberg brings years of experience studying Addison’s disease in Portuguese Water Dogs along with deep bioinformatics expertise, while Dr. Modiano, a world-renowned cancer biologist, has spearheaded hemangiosarcoma research in the breed. Bioinformatics analyses will be driven by Dr. Friedenberg’s graduate student Hayden Hamsher, who already has two years of pipeline development experience, and the project will benefit from the guidance of Dr. Jonah Cullen, whose postdoctoral work on the horse pangenome will provide invaluable genome assembly knowledge. They have also enlisted collaborators at UC Santa Cruz, the University of Washington, and the BC Cancer Research Centre – each contributing specialized expertise in long-read sequencing, Omni-C scaffolding, and Strand-seq methodologies. Together, this multidisciplinary team combines breed-specific veterinary insight, advanced computational skills, and world-class sequencing know-how to ensure the success of the PWD reference genome.

Whole-genome Sequencing of non-prcd PRA in PWD

Posted on by Janet Boyd

The Portuguese Water Dog Foundation will contribute $9,400 for Whole Genome Sequencing (WGS) on 4 different dogs [2 affected, 1 carriers, and 1 homozygous normal]. Samples will be tested at the WGS platform at the University of Bern, Switzerland. Dr. Aguirre’s eye lab is part of a consortium at Bern where they have WGS on 465 different dogs (at this point, none are PWDs). Bern will provide the initial filtering and data analysis, allowing Dr. Aguirre’s team to focus their analysis and to analyze the approximately 498 predicted genes in the interval in an expedited manner.

Although this proposal focuses strictly on non-prcd form of PRA, the results of the WGS studies will be critically important to other diseases of importance to PWD, for example microphthalmia/multiple congenital ocular anomalies (aka “puppy eye syndrome”) and possibly other diseases that rely on Genome Wide Association Studies (GWAS) to identify the genomic region of interest.

Any study using GWAS to map a disease interval will, much sooner than later, need high quality genomic sequence formation for the region. This comes from having access to as many different dogs/breeds as possible as well as having specific sequence information for the breed of interest from unaffected animals that can be used as disease controls. As the sequence information generated from this project will be publicly available, this resource will complement and inform research work being done on microphthalmia/multiple congenital ocular anomalies disorder as well as other diseases.

Research funded by the Portuguese Water Dog Foundation, Inc.

RESEARCHERS

Gustavo Aguirre
School of Veterinary Medicine, University of Pennsylvania
Amount: $9,400

Genetic Background and the Angiogenic Phenotype in Cancer

Posted on by PWDF Admin

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

Posted on by PWDF Admin

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

Genetic Analysis of Hypoandrenocorticism in Nova Scotia Duck Tolling Retrievers

Posted on by PWDF Admin

Addison’s disease, also known as hypoadrenocorticism, is a deficiency of hormones that are produced by the adrenal glands and help regulate a dog’s metabolism, blood pressure, electrolyte balance and stress response. Though the disease is relatively uncommon in dogs, certain breeds—including Nova Scotia Duck Tolling Retrievers, Bearded Collies, Great Danes, Leonbergers, Portuguese Water Dogs, Standard Poodles and West Highland White Terriers—have a much higher risk than the general dog population.

Results

Researchers identified a region of the genome that is associated with the development of Addison’s disease in Nova Scotia duck tolling retrievers. Additionally, it appears that dogs that are homozygous (both chromosomes carrying the same genes) with respect to this region are at greater risk of developing Addison’s disease, even at a young age (under 2 years). Although additional genes are likely involved, this information is the first step toward understanding the genetics of this disease and developing a genetic test that will help eliminate Addison’s disease through informed breeding practices. This fellowship training grant also provided hands-on training for a veterinarian who is pursuing a research career.

Co-sponsored with the Morris Animal Foundation, Grant Number: D08CA-402

RESEARCHERS

Angela M. Hughes, DVM
University of California at Davis

Pooled Association Mapping for Canine Hereditary Disorders

Posted on by PWDF Admin

To develop a system that will map genetic traits causing health problems.

More than 450 canine genetic traits are listed on the Online Mendelian Inheritance in Animals list. These traits affect all body systems in dogs and can cause health problems ranging from mild disease susceptibility to severe illness and death. Researchers will use a genetic tool called the Affymetrix Canine SNP Chip to develop a mapping strategy using pooled DMA samples that will map genes for hereditary canine disorders. This method promises to be 10 times more efficient and cost effective than current methods for analyzing canine inherited traits. Once this method is validated, it will be used to map a series of hereditary retinal disorders affecting multiple breeds.

Co-sponsored with the Morris Animal Foundation, Grant Number: D07CA-085

RESEARCHERS

Gregory M. Acland, BVSc
Cornell University

Dr. Acland is a professor of medical genetics at Cornell University and an adjunct p of ophthalmology at the University of Pennsylvania. He received his veterinary degree from the University and completed a residency and postdoctoral work at the University of Pennsylvania.

Reciprocal Relationship of PTEN and p21 in Canine Cancer

Posted on by PWDF Admin

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

Posted on by PWDF Admin

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

Identifying Genes Regulating Addison’s Disease in the Portuguese Water Dog

Posted on by PWDF Admin

Addison’s disease, or primary adrenocortical insufficiency, is characterized by destruction of the adrenal cortex, resulting in the inability to produce cortisone when stimulated with the hormone ACTH. In Portuguese Water Dogs (PWDs), this disease occurs with a frequency of 1-2 percent, and is a heritable autoimmune disease of low penetrance, caused by several interacting genes.

Using both new and existing data, we propose to identify regions of the PWD genome that contain genes regulating the frequency of Addison’s disease. Within those large regions we propose to identify the specific DNA sequence variants that are associated with Addison’s.

Results

To date we have obtained DNA from about 90 Addisonian PWDs, as well as a number of unaffected PWDs, for which no family history of Addison’s is reported. We have already identified two genomic regions, on canine chromosomes 12 and 37, that appear to be associated with the disease. To identify candidate genes, we will make selections using the newly available canine genome sequence, as well as the more detailed human genome sequence. Once affected gene disease frequency is identified, our long term hope is that prognostic tests can be developed that will aid breeders in selecting the most genetically compatible dogs for future.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 00589B

RESEARCHERS

Elaine Ostrander, PhD
National Human Genome Research Institute