Establishment and characterization of a canine soft tissue sarcoma patient‐derived xenograft model

Spontaneously occurring soft tissue sarcoma (STS) is relatively common in canine cancer patients. Because of the similarities to human disease, canine STSs are a valuable and readily available resource for the study of new therapeutics. In this study, a canine patient‐derived xenograft (PDX) model, CDX‐STS2, was established. The CDX‐STS2 model was engrafted and expanded for systemic administration studies with chemotherapeutic agents commonly used to treat STS, including doxorubicin, docetaxel and gemcitabine. Immunohistochemistry for drug‐specific biomarkers and tumour growth measurement revealed tumour sensitivity to doxorubicin and docetaxel, whereas gemcitabine had no effect on tumour growth. Although many human PDX tumour models have been established, relatively few canine PDX models have been reported to date. CDX‐STS2 represents a new STS PDX research model of canine origin that will be useful in bridging preclinical research with clinical studies of STS in pet dogs.     

Telomerase: a potential diagnostic and therapeutic tool in canine oncology

In recent years there has been considerable interest in telomerase as a target for therapeutic intervention in oncology. This largely stems from the vast number of studies that have demonstrated expression and activity of the enzyme telomerase in the majority of human cancer tissues with little or no activity detectable in normal somatic tissues. These studies have led to an interest in the role of telomerase in cancers associated with domesticated species, in particular tumors that affect dogs. This article reviews the biology of telomerase and the biological significance of telomerase activity in canine tumors and discusses the clinical implications of telomerase expression in canine cancers with regard to therapeutics and diagnostics.     

Telomerase activity in canine osteosarcoma

Appendicular osteosarcoma (OSA) is the most common primary bone tumour in dogs, and the prognosis with standard of care therapy of amputation and adjunctive chemotherapy is generally poor, with median survival times of 1 year. The ability of neoplastic cells to maintain their telomere length, by either telomerase activity or alternate methods, is an important step in tumour development and malignancy. The purpose of this study was to determine the presence of telomerase activity in canine OSA. To evaluate the frequency of alternative lengthening of telomeres in canine OSA, we have used the telomeric repeat amplification protocol in five canine cell lines and in six samples taken from clinical patients at the time of amputation. Our results reveal the presence of telomerase activity in 100% of canine OSA cell lines and 83% of clinical samples evaluated. This is in contrast to human OSA where 25–40% expression levels of telomerase are reported. Importantly, our results not only suggest that canine OSA may serve as a good model for aggressive telomerase‐positive forms of human OSA but also that antitelomerase therapy strategies for treatment of canine OSA may be more successful than in the treatment of majority of human patients with OSA.     

Inhibition of canine telomerase in vitro and in vivo using RNAi: further development of a natural canine model for telomerase-based cancer therapies

Despite advances in cancer therapy, cancer related morbidity and mortality among humans and companion animals remains high, and there is a clear need to develop novel targeted therapies. Expression of the enzyme telomerase has emerged as a central unifying mechanism underlying the immortal phenotype of canine cancer cells and has thus become a candidate for targeted molecular therapies. In this study, the value of telomerase inhibition to target telomerase expressing cancer cells was explored using the novel mechanism of RNA interference (RNAi). Using a Lentiviral expression construct, targeting the RNA component of canine telomerase was effective at inhibiting telomerase in vitro and tumour growth in vivo, but possible resistance mechanisms are highlighted. As canine telomerase biology is more closely related to human telomerase biology than the murine system, it is proposed that this study highlights the value of natural canine models to study anti-telomerase therapies for human patients.     

Leading the pack: Best practices in comparative canine cancer genomics to inform human oncology

Pet dogs develop spontaneous cancers at a rate estimated to be five times higher than that of humans, providing a unique opportunity to study disease biology and evaluate novel therapeutic strategies in a model system that possesses an intact immune system and mirrors key aspects of human cancer biology. Despite decades of interest, effective utilization of pet dog cancers has been hindered by a limited repertoire of necessary cellular and molecular reagents for both in vitro and in vivo studies, as well as a dearth of information regarding the genomic landscape of these cancers. Recently, many of these critical gaps have been addressed through the generation of a highly annotated canine reference genome, the creation of several tools necessary for multi-omic analysis of canine tumours, and the development of a centralized repository for key genomic and associated clinical information from canine cancer patients, the Integrated Canine Data Commons. Together, these advances have catalysed multidisciplinary efforts designed to integrate the study of pet dog cancers more effectively into the translational continuum, with the ultimate goal of improving human outcomes. The current review summarizes this recent progress and provides a guide to resources and tools available for comparative study of pet dog cancers.     

Inhibition of telomerase in canine cancer cells following telomestatin treatment

Telomere shortening in normal somatic cells has been proposed as a major barrier to unlimited cellular proliferation. Telomerase is an enzyme capable of maintaining telomere length, and thus bypassing this barrier. In human beings, telomerase activity is restricted to cancer cells and cells of stem or germ cell lineages. Dogs represent a potentially useful clinical model for the development of telomerase‐based therapies because telomerase activity is also restricted to cancer cells and stem cells in this species. We examined the ability of telomestatin to inhibit telomerase activity in telomerase‐positive D17 and CMT7 canine cancer cell lines. At a concentration of 2 μM, telomestatin treatment resulted in a decrease in telomerase activity, telomere shortening, growth inhibition and apoptosis in telomerase‐positive cancer cells. These effects were not seen in telomerase‐negative skin fibroblasts or negative controls. These results confirm that telomestatin specifically inhibits telomerase activity in canine cancer cells and strengthens the usefulness of dogs as a model for testing telomerase‐based therapies.     

Impact of telomerase status on canine osteosarcoma patients

Background: We demonstrated previously that canine osteosarcoma (OSA) cell lines and samples from clinical patients are predominantly telomerase positive. In contrast, the majority of OSA samples from human patients appear to be telomerase negative, maintaining telomere length by an alternative lengthening of telomeres (ALT) mechanism. The purpose of the current study was to examine the telomerase status of a large number of OSA samples from dogs and determine if telomerase status can serve as a prognostic factor. Hypothesis: The majority of clinical canine OSA appendicular lesions will be telomerase positive, and telomerase positivity will negatively impact disease outcome. Animals: Sixty‐seven dogs with appendicular OSA presenting to the Colorado State University Animal Cancer Center for treatment. Methods: The Telomeric Repeat Amplification Protocol was performed on tissue samples from primary canine appendicular OSA to determine the presence of telomerase activity. Telomere restriction fragment (TRF) analysis was utilized to determine telomere length and detect ALT. Outcome data were obtained in a retrospective manner and correlated with telomerase status. Results: Seventy‐three percent of canine OSA samples were telomerase positive. Telomerase status did not have an impact on disease‐free interval or survival time. Nine of 10 telomerase‐negative samples examined were consistent with an ALT phenotype, based on TRF analysis. Conclusions and Clinical Importance: These results are consistent with the hypothesis that the majority of canine OSA are telomerase positive, suggesting that telomerase may be a valuable target for canine OSA therapy. Additionally, telomerase status does not appear to be a prognostic factor in canine OSA.     

Effects of cyclosporin A and cilomilast on activated canine, murine and human keratinocytes

The calcineurin inhibitor cyclosporin A and the phosphodiesterase 4 inhibitor cilomilast exhibit potent immunomodulatory properties which make them interesting therapeutics for the treatment of skin disorders like canine and human atopic dermatitis. Cyclosporin A and phosphodiesterase 4 inhibitors have already demonstrated clinical efficacy in the therapy of canine and human atopic dermatitis. Their direct impact on keratinocytes, especially canine keratinocytes, is less obvious. Thus, an investigation was carried out to ascertain whether cyclosporin A and cilomilast modulate keratinocyte proliferation and secretion of proinflammatory mediators. Cyclosporin A inhibited canine and murine keratinocyte proliferation, whereas cilomilast had no affect. Cyclosporin A and cilomilast reduced the lipopolysaccharide-induced prostaglandin E2 synthesis in canine and murine keratinocytes. Both immunomodulators also inhibited the production of the CXC chemokine KC and CCL2 in the murine keratinocyte cell line MSC-P5. The two immunomodulators also significantly reduced the interferon-gamma-induced production of interferon-gamma-inducible protein 10 in human keratinocytes (HaCaT cells). Thus, cyclosporin A and cilomilast directly modulate keratinocyte functions which might contribute to the anti-inflammatory and immunomodulatory action of these compounds in the treatment of allergic skin diseases.     

Viral vectors for targeting the canine retina: a review

Clinical trials are currently underway using gene therapy to treat retinal disease such as Leber congenital amaurosis (LCA). Viral vectors that have been utilized to target retinal cells include adenoviruses, lentiviruses, and recombinant adeno-associated viruses (rAAV). Of the three classes, rAAV vectors show the greatest promise for retinal gene therapy. Recent developments in virus technology such as the development of hybrid and capsid mutant rAAV vectors mean that specific retinal cells can be targeted and faster stronger transgene expression is now possible compared to that achieved with the first generation of vectors. Gene therapy trials in dogs have been very important in the development of therapy for RPE65 LCA which is currently in phase I/II clinical trials in humans. Recent successes in using gene therapy to treat canine achromatopsia, X-linked progressive retinal atrophy (PRA) and the more severe rapid degenerations such as rod-cone dysplasia type 3 may lead also to the translation to human clinical trials. Dogs have played and continue to play an important role as animal models for proof-of-concept studies of retinal gene therapy. As modifications and improvements in gene therapy protocols are made from experience gathered from human clinical trials perhaps gene therapy for the treatment of canine clinical patients will become available to veterinary ophthalmologists.     

Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma

The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer‐related signalling pathways despite differences in activating mutations.     

Clinical trials in spontaneous disease in dogs: a new paradigm for investigations of sepsis

Objective: To provide evidence that naturally occurring sepsis in dogs provides a unique opportunity to test new therapies in clinically relevant settings. Data sources: Human and veterinary literature. Human data synthesis: Sepsis is a devastating condition responsible for most intensive care unit deaths. Most clinical trials targeting inflammatory mediators of sepsis have failed to improve outcome in clinical patients despite promising results in laboratory animal models. Animal models of sepsis fail to reproduce the clinical syndrome and therefore lead to conclusions that may not be relevant to clinical care. Veterinary data synthesis: Sepsis is recognized but not well‐characterized in companion animal species. Despite some species variability, the cardiopulmonary response to sepsis in dogs is similar to humans. Additionally, inflammatory and coagulation changes that accompany canine sepsis are consistent with those documented in humans. Sepsis secondary to canine parvoviral infection offers the advantages of relative population homogeneity, predictable course, and easy early diagnosis. The disadvantages of canine parvovirus are that it affects a predominantly young and healthy population and results in low mortality with aggressive supportive care. Septic peritonitis and pneumonia have high mortality but can be challenging to diagnose, have a variable course, and affect a heterogeneous population, which can be an advantage or a disadvantage. Conclusions: Similar to trials currently being performed in canine cancer patients, veterinary clinical trials of new sepsis therapeutics may provide a unique opportunity to advance the treatment of sepsis in dogs, humans, and other species. Spontaneous sepsis from canine parvovirus, peritonitis, and pneumonia are common clinical conditions in which therapeutics can be tested.     

Standing in the canine precision medicine knowledge gap: Improving annotation of canine cancer genomic biomarkers through systematic comparative analysis of human cancer mutations in COSMIC

The accrual of cancer mutation data and related functional and clinical associations have revolutionised human oncology, enabling the advancement of precision medicine and biomarker-guided clinical management. The catalogue of cancer mutations is also growing in canine cancers. However, without direct high-powered functional data in dogs, it remains challenging to interpret and utilise them in research and clinical settings. It is well-recognised that canine and human cancers share genetic, molecular and phenotypic similarities. Therefore, leveraging the massive wealth of human mutation data may help advance canine oncology. Here, we present a structured analysis of sequence conservation and conversion of human mutations to the canine genome through a ‘caninisation’ process. We applied this analysis to COSMIC, the Catalogue of Somatic Mutations in Cancer, the most prominent human cancer mutation database. For the project's initial phase, we focused on the subset of the COSMIC data corresponding to Cancer Gene Census (CGC) genes. A total of 670 canine orthologs were found for 721 CGC genes. In these genes, 365 K unique mutations across 160 tumour types were converted successfully to canine coordinates. We identified shared putative cancer-driving mutations, including pathogenic and hotspot mutations and mutations bearing similar biomarker associations with diagnostic, prognostic and therapeutic utility. Thus, this structured caninisation of human cancer mutations facilitates the interpretation and annotation of canine mutations and helps bridge the knowledge gap to enable canine precision medicine.     

Molecular biological aspects on canine and human mammary tumors

The high incidence of mammary tumor disease reported in certain canine breeds suggests a significant genetic component, as has already been described in human familial breast cancer-in BRCA1- and BRCA2-associated breast cancer in particular. The identification of genetic risk factors is critical to improvements in the prevention, diagnosis, and treatment of these tumors. In recent years, there has been significant progress in developing the tools and reagents necessary to analyze the canine genome. This work has culminated in a high-quality draft genome sequence, as well as a single-nucleotide polymorphism map and single-nucleotide polymorphism arrays for genomewide association analysis. These tools provide an unprecedented opportunity to characterize the genetic influences in canine diseases such as cancer, eventually allowing for exploration of more effective therapies. Given the high homology between the canine genome sequence and its human counterpart--as well as the many similarities regarding the morphology, biological behavior, and clinical course of mammary tumors in both species--the dog has proven to be an excellent comparative model. This review highlights the comparative aspects regarding certain areas within molecular biology, and it discusses future perspectives. The findings in larger genomewide association analyses and cDNA expression arrays are described, and the BRCA1/BRCA2 complex is compared in detail between the 2 species.     

Telomeres and telomerase: Biological and clinical importance in dogs

In recent years in human oncology the enzyme telomerase has emerged as an ideal target for cancer therapy. This has led to the assessment of telomerase in cancers in companion animals, mainly dogs and these studies confirm that in dogs, like humans, telomere maintenance by telomerase is the primary mechanism by which cancer cells overcome their mortality and extend their lifespan. This review aims to provide an introduction to the biology of telomeres and telomerase and to discuss some of the telomere/telomerase directed therapeutic methodologies currently under development which may be of benefit to the canine cancer patient.     

Evaluation of telomerase-targeted therapies in canine cancer cell lines

Despite advances in conventional therapeutics, cancer remains an invariably fatal disease, the major challenge being to develop tumour‐specific cancer treatment strategies. Current treatments such as chemotherapy and radiotherapy rely on a crude distinction between cancer cells and normal cells. However, with an increased understanding of the molecular events in the development of cancer, it is possible that far more innovative and targeted approaches can be developed. From studies on humans and dogs, the enzyme telomerase has emerged as a central unifying mechanism underlying the immortal phenotype of cancer and has thus become a candidate for differentiating between normal and cancer cells. The level and frequency of telomerase activity and component gene expression in cancers reinforces this as a potential target for cancer therapies. This article describes two approaches to target cancer by capitalizing on the expression of this enzyme. In the first approach, we target the enzyme itself, the goal being to cause cancer cell death. In the second approach, we utilize the respective gene promoters for telomerase component enzymes to drive expression of a reporter gene in cancer cell lines. The results demonstrated that targeted gene expression using promoter elements can be achieved specifically in telomerase‐positive cell lines. However, targeting the enzyme itself proved less successful and warrants investigations into alternative approaches.     

The canine prostate cancer cell line CHP‐1 shows over‐expression of the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α

Although androgen therapy resistance and poor clinical outcomes are seen in most canine prostate cancer cases, there are only a few tools for analysing canine prostate cancer by using a cell biological approach. Therefore, to evaluate androgen‐independent neoplastic cell growth, a new canine prostate cancer cell line (CHP‐1) was established in this study. CHP‐1 over‐expressed the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α (SGTA), which is over‐expressed in human androgen‐independent prostate cancer. The CHP‐1 xenograft also showed SGTA over‐expression. Although CHP‐1 shows poor androgen receptor (AR) signalling upon dihydrotestosterone stimulation, forced expression of AR enabled evaluation of AR signalling. Taken together, these results suggest that CHP‐1 will be a useful model for investigating the pathogenesis of androgen‐dependent and androgen‐independent canine prostate cancer.     

Oncolytic gene therapy for canine cancers: teaching old dog viruses new tricks

The use of viruses to treat cancer has been studied for decades. With the advancement of molecular biology, viruses have been modified and genetically engineered to optimize their ability to target cancer cells. Canine viruses, such as distemper virus and adenovirus, are being exploited for the treatment of canine cancer as the dog has proven to be a good comparative model for human cancer research and proof of concept investigations. In this review, we introduce the concept of oncolytic viruses and describe some of the preliminary attempts to use oncolytic viruses for the treatment of canine cancer.     

Identification of cancer stem cells derived from a canine lung adenocarcinoma cell line

Accumulating evidence supporting the cancer stem cell (CSC) hypothesis is based on the finding that tumors contain a small population of self-renewing cells that generate differentiated progeny and thereby contribute to tumor heterogeneity. CSCs are reported to exist in several human cancers, yet only a few reports demonstrate the existence of CSCs in primary lung cancer in dogs. In this study, the authors established a cancer cell line derived from a canine primary lung adenocarcinoma and identified a side population (SP) of cells that displayed drug-resistant features. To confirm the characteristics of these SP cells, the authors investigated the tumorigenicity of the cells in vivo by using a nude mouse xenograft model. Only 100 SP cells were able to give rise to new tumors, giving a 10-fold enrichment over the main population (MP) of cells, suggesting that these cells have the cancer-initiating ability of CSCs. Further studies characterizing CSCs in canine lung adenocarcinoma might contribute to the elucidation of the mechanisms of tumorigenesis and to the establishment of novel therapeutic strategies.