Publications by authors named "Dawn L Duval"

25 Publications

  • Page 1 of 1

Characterizing the molecular and immune landscape of canine bladder cancer.

Vet Comp Oncol 2021 May 22. Epub 2021 May 22.

Flint Animal Cancer Center, Colorado State University, Fort Collins, Colorado, USA.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.
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http://dx.doi.org/10.1111/vco.12740DOI Listing
May 2021

MicroRNA Expression Changes and Integrated Pathways Associated With Poor Outcome in Canine Osteosarcoma.

Front Vet Sci 2021 15;8:637622. Epub 2021 Apr 15.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, United States.

MicroRNAs (miRNA) are small non-coding RNA molecules involved in post-transcriptional gene regulation. Deregulation of miRNA expression occurs in cancer, and miRNA expression profiles have been associated with diagnosis and prognosis in many cancers. Osteosarcoma (OS), an aggressive primary tumor of bone, affects ~10,000 dogs each year. Though survival has improved with the addition of chemotherapy, up to 80% of canine patients will succumb to metastatic disease. Reliable prognostic markers are lacking for this disease. miRNAs are attractive targets of biomarker discovery efforts due to their increased stability in easily obtained body fluids as well as within fixed tissue. Previous studies in our laboratory demonstrated that dysregulation of genes in aggressive canine OS tumors that participate in miRNA regulatory networks is reportedly disrupted in OS or other cancers. We utilized RT-qPCR in a 384-well-plate system to measure the relative expression of 190 miRNAs in 14 canine tumors from two cohorts of dogs with good or poor outcome (disease-free interval >300 or <100 days, respectively). Differential expression analysis in this subset guided the selection of candidate miRNAs in tumors and serum samples from larger groups of dogs. We ultimately identified a tumor-based three-miR Cox proportional hazards regression model and a serum-based two-miR model, each being able to distinguish patients with good and poor prognosis via Kaplan-Meier analysis with log rank test. Additionally, we integrated miRNA and gene expression data to identify potentially important miRNA-mRNA interactions that are disrupted in canine OS. Integrated analyses of miRNAs in the three-miR predictive model and disrupted genes from previous expression studies suggest the contribution of the primary tumor microenvironment to the metastatic phenotype of aggressive tumors.
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http://dx.doi.org/10.3389/fvets.2021.637622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081964PMC
April 2021

Role of Periostin Expression in Canine Osteosarcoma Biology and Clinical Outcome.

Vet Pathol 2021 Mar 9:300985821996671. Epub 2021 Mar 9.

3447Colorado State University, Fort Collins, CO, USA.

Periostin is a matricellular protein important in regulating bone, tooth, and cardiac development. In pathologic conditions, periostin drives allergic and fibrotic inflammatory diseases and is also overexpressed in certain cancers. Periostin signaling in tumors has been shown to promote angiogenesis, metastasis, and cancer stem cell survival in rodent models, and its overexpression is associated with poor prognosis in human glioblastoma. However, the role of periostin in regulating tumorigenesis of canine cancers has not been evaluated. Given its role in bone development, we sought to evaluate mRNA and protein expression of periostin in canine osteosarcoma (OS) and assess its association with patient outcome. We validated an anti-human periostin antibody cross-reactive to canine periostin via western blot and immunohistochemistry and evaluated periostin expression in microarray data from 49 primary canine OS tumors and 8 normal bone samples. Periostin mRNA was upregulated greater than 40-fold in canine OS tumors compared to normal bone and was significantly correlated with periostin protein expression based on quantitative image analysis. However, neither periostin mRNA nor protein expression were associated with time to metastasis in this cohort. Gene Set Enrichment Analysis demonstrated significant enhancement of pro-tumorigenic pathways including canonical signaling, epithelial-mesenchymal transition, and angiogenesis in periostin-high tumors, while periostin-low tumors demonstrated evidence of heightened antitumor immune responses. Overall, these data identify a novel antibody that can be used as a tool for evaluation of periostin expression in dogs and suggest that investigation of pathway-targeted drugs in periostin overexpressing canine OS may be a potential therapeutic target.
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http://dx.doi.org/10.1177/0300985821996671DOI Listing
March 2021

A systematic analysis of genomics-based modeling approaches for prediction of drug response to cytotoxic chemotherapies.

BMC Med Genomics 2019 06 17;12(1):87. Epub 2019 Jun 17.

School of Biomedical Engineering, Colorado State University, Fort Collins, 80523, CO, USA.

Background: The availability and generation of large amounts of genomic data has led to the development of a new paradigm in cancer treatment emphasizing a precision approach at the molecular and genomic level. Statistical modeling techniques aimed at leveraging broad scale in vitro, in vivo, and clinical data for precision drug treatment has become an active area of research. As a rapidly developing discipline at the crossroads of medicine, computer science, and mathematics, techniques ranging from accepted to those on the cutting edge of artificial intelligence have been utilized. Given the diversity and complexity of these techniques a systematic understanding of fundamental modeling principles is essential to contextualize influential factors to better understand results and develop new approaches.

Methods: Using data available from the Genomics of Drug Sensitivity in Cancer (GDSC) and the NCI60 we explore principle components regression, linear and non-linear support vector regression, and artificial neural networks in combination with different implementations of correlation based feature selection (CBF) on the prediction of drug response for several cytotoxic chemotherapeutic agents.

Results: Our results indicate that the regression method and features used have marginal effects on Spearman correlation between the predicted and measured values as well as prediction error. Detailed analysis of these results reveal that the bulk relationship between tissue of origin and drug response is a major driving factor in model performance.

Conclusion: These results display one of the challenges in building predictive models for drug response in pan-cancer models. Mainly, that bulk genotypic traits where the signal to noise ratio is high is the dominant behavior captured in these models. This suggests that improved techniques of feature selection that can discriminate individual cell response from histotype response will yield more successful pan-cancer models.
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http://dx.doi.org/10.1186/s12920-019-0519-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580596PMC
June 2019

Identifying Candidate Druggable Targets in Canine Cancer Cell Lines Using Whole-Exome Sequencing.

Mol Cancer Ther 2019 08 7;18(8):1460-1471. Epub 2019 Jun 7.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado.

Cancer cell culture has been a backbone in cancer research, in which analysis of human cell line mutational profiles often correlates with oncogene addiction and drug sensitivity. We have conducted whole-exome sequence analyses on 33 canine cancer cell lines from 10 cancer types to identify somatic variants that contribute to pathogenesis and therapeutic sensitivity. A total of 66,344 somatic variants were identified. Mutational load ranged from 15.79 to 129.37 per Mb, and 13.2% of variants were located in protein-coding regions (PCR) of 5,085 genes. PCR somatic variants were identified in 232 genes listed in the Cancer Gene Census (COSMIC). Cross-referencing variants with human driving mutations on cBioPortal identified 61 variants as candidate cancer drivers in 30 cell lines. The most frequently mutated cancer driver was TP53 (15 mutations in 12 cell lines). No drivers were identified in three cell lines. We identified 501 non-COSMIC genes with PCR variants that functionally annotate with COSMIC genes. These genes frequently mapped to the KEGG MAPK and PI3K-AKT pathways. We evaluated the cell lines for ERK1/2 and AKT(S473) phosphorylation and sensitivity to the MEK1/2 inhibitor, trametinib. Twelve of the 33 cell lines were trametinib-sensitive (IC < 32 nmol/L), all 12 exhibited constitutive or serum-activated ERK1/2 phosphorylation, and 8 carried MAPK pathway cancer driver variants: NF1(2), BRAF(3), N/KRAS(3). This functionally annotated database of canine cell line variants will inform hypothesis-driven preclinical research to support the use of companion animals in clinical trials to test novel combination therapies.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-1346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679748PMC
August 2019

Identifying the ErbB/MAPK Signaling Cascade as a Therapeutic Target in Canine Bladder Cancer.

Mol Pharmacol 2019 07 2;96(1):36-46. Epub 2019 May 2.

Flint Animal Cancer Center, Department of Clinical Sciences (K.E.C., B.G.H., D.L.G., D.L.D.), and Cell and Molecular Biology Graduate Program (K.E.C., D.L.G., D.L.D.), Colorado State University, Fort Collins, Colorado; and University of Colorado Cancer Center, Aurora, Colorado (D.L.G., D.L.D.)

Transitional cell carcinoma (TCC) of the bladder comprises 2% of diagnosed canine cancers. TCC tumors are generally inoperable and unresponsive to traditional chemotherapy, indicating a need for more effective therapies. BRAF, a kinase in the mitogen-activated protein kinase (MAPK) pathway, is mutated in 70% of canine TCCs. In this study, we use BRAF mutant and wild-type TCC cell lines to characterize the role of BRAF mutations in TCC pathogenesis and assess the efficacy of inhibition of the MAPK pathway alone and in combination with other gene targets as a treatment for canine TCC. Analysis of MAPK target gene expression and assessment of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation following serum starvation indicated constitutive MAPK activity in all TCC cell lines. BRAF mutant TCC cell lines were insensitive to the BRAF inhibitor vemurafenib, with IC values greater than 5 M, but exhibited greater sensitivity to a paradox-breaking BRAF inhibitor (IC: 0.2-1 M). All TCC cell lines had IC values less than 7 nM to the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor trametinib independent of their BRAF mutation status. ERK1/2 phosphorylation decreased after 6-hour treatments with MAPK inhibitors, but rebounded by 24 hours, suggesting the presence of resistance mechanisms. Microarray analysis identified elevated expression of the ErbB family of receptors and ligands in TCC cell lines. The pan-ErbB inhibitor sapitinib synergized with BRAF inhibition in BRAF mutant Bliley TCC cells and synergized with MEK1/2 inhibition in Bliley and BRAF wild-type Kinsey cells. These findings suggest the potential for combined MAPK and ErbB receptor inhibition as a therapy for canine TCC. SIGNIFICANCE STATEMENT: The results of this study (1) identify a novel combination strategy for canine bladder cancer treatment: targeting the ErbB/MAPK signaling cascade and (2) establish the utility of canine bladder cancer as a naturally-occurring model for human MAPK-driven cancers.
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http://dx.doi.org/10.1124/mol.119.115808DOI Listing
July 2019

Canine sarcomas as a surrogate for the human disease.

Pharmacol Ther 2018 08 9;188:80-96. Epub 2018 Mar 9.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; Flint Animal Cancer Center, Colorado State University, Fort Collins, CO 80523, USA; University of Colorado Cancer Center, Anschutz Medical Campus, Aurora, CO 80045, USA.

Pet dogs are becoming increasingly recognized as a population with the potential to inform medical research through their treatment for a variety of maladies by veterinary health professionals. This is the basis of the One Health initiative, supporting the idea of collaboration between human and animal health researchers and clinicians to study spontaneous disease processes and treatment in animals to inform human health. Cancer is a major health burden in pet dogs, accounting for approximately 30% of deaths across breeds. As such, pet dogs with cancer are becoming increasingly recognized as a resource for studying the pharmacology and therapeutic potential of anticancer drugs and therapies under development. This was recently highlighted by a National Academy of Medicine Workshop on Comparative Oncology that took place in mid-2015 (http://www.nap.edu/21830). One component of cancer burden in dogs is their significantly higher incidence of sarcomas as compared to humans. This increased incidence led to canine osteosarcoma being an important component in the development of surgical approaches for osteosarcoma in children. Included in this review of sarcomas in dogs is a description of the incidence, pathology, molecular characteristics and previous translational therapeutic studies associated with these tumors. An understanding of the patho-physiological and molecular characteristics of these naturally occurring canine sarcomas holds great promise for effective incorporation into drug development schemas, for evaluation of target modulation or other pharmacodynamic measures associated with therapeutic response. These data could serve to supplement other preclinical data and bolster clinical investigations in tumor types for which there is a paucity of human patients for clinical trials.
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http://dx.doi.org/10.1016/j.pharmthera.2018.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432917PMC
August 2018

Intra- and interspecies gene expression models for predicting drug response in canine osteosarcoma.

BMC Bioinformatics 2016 Feb 19;17:93. Epub 2016 Feb 19.

Cell and Molecular Biology Program, Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA.

Background: Genomics-based predictors of drug response have the potential to improve outcomes associated with cancer therapy. Osteosarcoma (OS), the most common primary bone cancer in dogs, is commonly treated with adjuvant doxorubicin or carboplatin following amputation of the affected limb. We evaluated the use of gene-expression based models built in an intra- or interspecies manner to predict chemosensitivity and treatment outcome in canine OS. Models were built and evaluated using microarray gene expression and drug sensitivity data from human and canine cancer cell lines, and canine OS tumor datasets. The "COXEN" method was utilized to filter gene signatures between human and dog datasets based on strong co-expression patterns. Models were built using linear discriminant analysis via the misclassification penalized posterior algorithm.

Results: The best doxorubicin model involved genes identified in human lines that were co-expressed and trained on canine OS tumor data, which accurately predicted clinical outcome in 73 % of dogs (p = 0.0262, binomial). The best carboplatin model utilized canine lines for gene identification and model training, with canine OS tumor data for co-expression. Dogs whose treatment matched our predictions had significantly better clinical outcomes than those that didn't (p = 0.0006, Log Rank), and this predictor significantly associated with longer disease free intervals in a Cox multivariate analysis (hazard ratio = 0.3102, p = 0.0124).

Conclusions: Our data show that intra- and interspecies gene expression models can successfully predict response in canine OS, which may improve outcome in dogs and serve as pre-clinical validation for similar methods in human cancer research.
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http://dx.doi.org/10.1186/s12859-016-0942-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759767PMC
February 2016

DOG1 is a sensitive and specific immunohistochemical marker for diagnosis of canine gastrointestinal stromal tumors.

J Vet Diagn Invest 2015 May 10;27(3):268-77. Epub 2015 Apr 10.

Veterinary Diagnostic Laboratories (Bass, Ehrhart, Powers), Colorado State University, Fort Collins, COCell and Molecular Biology Graduate Program (Dailey, Ehrhart, Duval), Colorado State University, Fort Collins, CODepartments of Microbiology, Immunology and Pathology (Ehrhart, Powers), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, COClinical Sciences (Duval), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, COFlint Animal Cancer Center (Dailey, Ehrhart, Duval), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO.

Gastrointestinal stromal tumors (GISTs) and leiomyosarcomas are histologically similar primary neoplasms commonly occurring in the gastrointestinal tract of dogs and humans. Immunohistochemical staining (IHC) is needed to differentiate between these 2 entities and positive reactivity for KIT (cluster of differentiation [CD]117) is regarded as the gold standard for diagnosis of canine GIST. Studies estimate 5-10% of human GISTs stain negative or only weakly positive for KIT and have identified DOG1 (discovered on gastrointestinal stromal tumors protein 1) as a highly sensitive and specific marker for human GISTs. The purpose of this study was to evaluate immunoreactivity of a commercially available DOG1 antibody for use in diagnosis of canine GISTs. Fifty-five primary mesenchymal gastrointestinal tumors with histologic features consistent with GIST or leiomyosarcoma were evaluated via IHC for KIT, DOG1, and desmin. A subset of tumors was additionally evaluated for reactivity for smooth muscle actin (SMA). Thirty-three tumors (60%) were diagnosed as GIST based on positive immunoreactivity for KIT or DOG1 regardless of reactivity for desmin or SMA. Most GISTs (32/33, 97.0%) had similar staining for both KIT and DOG1. DOG1 expression was identified in 2 tumors (1 study tumor and 1 additional tumor) negative for KIT and desmin that had histologic features consistent with KIT-negative, platelet-derived growth factor receptor-alpha (PDGFRA)-mutant human GISTs. Our results suggest that DOG1 has improved specificity and sensitivity to that of KIT for differentiating between canine GISTs and leiomyosarcomas. Inclusion of both DOG1 and KIT IHC in diagnostic panels will improve the accuracy of canine GIST diagnosis.
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http://dx.doi.org/10.1177/1040638715578878DOI Listing
May 2015

Development of an in vitro model of acquired resistance to toceranib phosphate (Palladia®) in canine mast cell tumor.

BMC Vet Res 2014 May 6;10:105. Epub 2014 May 6.

Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO, USA.

Background: Mast cell tumors (MCTs) are the most common skin tumors in dogs and exhibit variable biologic behavior. Mutations in the c-kit proto-oncogene are associated with the tumorigenesis of MCTs, resulting in growth factor-independent and constitutive phosphorylation of the KIT receptor tyrosine kinase (RTK). Toceranib (TOC) phosphate (Palladia®) is a KIT RTK inhibitor that has biological activity against MCTs. Despite these benefits, patients ultimately develop resistance to TOC. Therefore, there is a need to identify distinguishing clinical and molecular features of resistance in this population.

Results: The canine C2 mastocytoma cell line contains an activating mutation in c-kit. Three TOC-resistant C2 sublines (TR1, TR2, TR3) were established over seven months by growing cells in increasing concentrations of TOC. TOC inhibited KIT phosphorylation and cell proliferation in a dose-dependent manner in the treatment-naïve, parental C2 line (IC50 < 10 nM). In contrast, the three sublines were resistant to growth inhibition by TOC (IC50 > 1,000 nM) and phosphorylation of the KIT receptor was less inhibited compared to the TOC-sensitive C2 cells. Interestingly, sensitivity to three structurally distinct KIT RTK inhibitors was variable among the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents vinblastine and lomustine. Sequencing of c-kit revealed secondary mutations in the juxtamembrane and tyrosine kinase domains of the resistant sublines. These included point mutations in TR1 (Q574R, M835T), TR2 (K724R), and TR3 (K580R, R584G, A620S). Additionally, chronic TOC exposure resulted in c-kit mRNA and KIT protein overexpression in the TOC-resistant sublines compared to the parental line. C2, TR1, TR2, and TR3 cells demonstrated minimal P-glycoprotein (P-gp) activity and no functional P-gp.

Conclusions: This study demonstrates the development of an in vitro model of acquired resistance to targeted therapy in canine MCTs harboring a c-kit-activating mutation. This model may be used to investigate the molecular basis of and strategies to overcome TOC resistance.
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http://dx.doi.org/10.1186/1746-6148-10-105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049511PMC
May 2014

Transcription factor Ets1 cooperates with estrogen receptor α to stimulate estradiol-dependent growth in breast cancer cells and tumors.

PLoS One 2013 9;8(7):e68815. Epub 2013 Jul 9.

Flint Animal Cancer Center, Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado, United States of America.

The purpose of this study was to explore the role of transcription factor Ets1 in estrogen receptor α (ERα)-positive breast cancer progression. We expressed human Ets1 or empty vector in four human ERα-positive breast cancer cell lines and observed increased colony formation. Further examination of cellular responses in stable Ets1-expressing MCF7 clones displayed increased proliferation, migration, and invasion. Ets1-expressing MCF7 tumors grown in the mammary fat pads of nude mice exhibited increased rates of tumor growth (7.36±2.47 mm(3)/day) compared to control MCF7 tumors (2.52±1.70 mm(3)/day), but maintained their dependence on estradiol for tumor growth. Proliferation marker Ki-67 staining was not different between control and Ets1-expressing tumors, but Ets1-expressing tumors exhibited large necrotic centers and elevated apoptotic staining. Ets1 was shown to cooperate with ERα and the p160 nuclear receptor coactivator (NCOA/SRC) family to increase activation of a consensus estrogen response element luciferase reporter construct. Ets1-expressing MCF7 cells also exhibited elevated expression of the ERα target genes, progesterone receptor and trefoil factor 1. Using GST-pulldown assays, Ets1 formed stable complexes containing both ERα and p160 nuclear receptor coactivators. Taken together, these data suggest that the Ets1-dependent estradiol sensitization of breast cancer cells and tumors may be partially due to the ability of Ets1 to cooperate with ERα and nuclear receptor coactivators to stimulate transcriptional activity of estrogen-dependent genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068815PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706316PMC
February 2014

HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors.

BMC Vet Res 2013 Jul 1;9:130. Epub 2013 Jul 1.

Background: Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression.

Results: Reverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes. Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI.

Conclusions: Our findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.
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http://dx.doi.org/10.1186/1746-6148-9-130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701487PMC
July 2013

Polymerase chain reaction-based species verification and microsatellite analysis for canine cell line validation.

J Vet Diagn Invest 2011 Jul 13;23(4):780-5. Epub 2011 Jun 13.

Animal Cancer Center, Department of Clinical Sciences, Colorado State University, 300 West Drake Road, Fort Collins, Colorado 80523, USA.

Cell line cross-contamination as well as genetic drift during passaging have been acknowledged as widespread problems since the 1960s. Improper cell line identification can invalidate results and, if not discovered, pollute the scientific community's body of knowledge with regard to cancer cell lines, their gene expression, and their drug susceptibilities. Despite the obvious need, validation of cell line identity is not yet widely required, and the problem persists. A highly sensitive polymerase chain reaction (PCR)-based approach and short tandem repeat (STR) profiling were used to examine the prevalence of inter- and intraspecies cell line contamination in a veterinary research setting. First, 60 cell lines from 6 laboratories were tested with multiplex species-specific PCR capable of identifying 6 commonly used species. Of these, 3 were determined to be misidentified by species. Second, to identify intraspecies contamination among canine cancer cell lines, 29 canine lines from 3 different laboratories were analyzed with STR fingerprinting. Using this methodology, 3 canine cell lines were determined to be misidentified or cross-contaminated by other canine cell lines. Finally, genetic drift was observed within 1 cell line obtained from different laboratories. These findings emphasize the importance of cell line validation as a critical component of "good cell culture practice." A database of the STR profiles obtained in the current study has been established for future comparison and validation of canine cell lines by investigators at Colorado State University and other institutions.
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http://dx.doi.org/10.1177/1040638711408064DOI Listing
July 2011

Characterization of canine osteosarcoma by array comparative genomic hybridization and RT-qPCR: signatures of genomic imbalance in canine osteosarcoma parallel the human counterpart.

Genes Chromosomes Cancer 2011 Nov 11;50(11):859-74. Epub 2011 Aug 11.

Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, USA.

Osteosarcoma (OS) is the most commonly diagnosed malignant bone tumor in humans and dogs, characterized in both species by extremely complex karyotypes exhibiting high frequencies of genomic imbalance. Evaluation of genomic signatures in human OS using array comparative genomic hybridization (aCGH) has assisted in uncovering genetic mechanisms that result in disease phenotype. Previous low-resolution (10-20 Mb) aCGH analysis of canine OS identified a wide range of recurrent DNA copy number aberrations, indicating extensive genomic instability. In this study, we profiled 123 canine OS tumors by 1 Mb-resolution aCGH to generate a dataset for direct comparison with current data for human OS, concluding that several high frequency aberrations in canine and human OS are orthologous. To ensure complete coverage of gene annotation, we identified the human refseq genes that map to these orthologous aberrant dog regions and found several candidate genes warranting evaluation for OS involvement. Specifically, subsequenct FISH and qRT-PCR analysis of RUNX2, TUSC3, and PTEN indicated that expression levels correlated with genomic copy number status, showcasing RUNX2 as an OS associated gene and TUSC3 as a possible tumor suppressor candidate. Together these data demonstrate the ability of genomic comparative oncology to identify genetic abberations which may be important for OS progression. Large scale screening of genomic imbalance in canine OS further validates the use of the dog as a suitable model for human cancers, supporting the idea that dysregulation discovered in canine cancers will provide an avenue for complementary study in human counterparts.
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http://dx.doi.org/10.1002/gcc.20908DOI Listing
November 2011

PRKA/AMPK: integrating energy status with fertility in pituitary gonadotrophs.

Authors:
Dawn L Duval

Biol Reprod 2011 Feb 17;84(2):205-6. Epub 2010 Nov 17.

Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado, USA.

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http://dx.doi.org/10.1095/biolreprod.110.089722DOI Listing
February 2011

Expression profiling in canine osteosarcoma: identification of biomarkers and pathways associated with outcome.

BMC Cancer 2010 Sep 22;10:506. Epub 2010 Sep 22.

Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado, USA.

Background: Osteosarcoma (OSA) spontaneously arises in the appendicular skeleton of large breed dogs and shares many physiological and molecular biological characteristics with human OSA. The standard treatment for OSA in both species is amputation or limb-sparing surgery, followed by chemotherapy. Unfortunately, OSA is an aggressive cancer with a high metastatic rate. Characterization of OSA with regard to its metastatic potential and chemotherapeutic resistance will improve both prognostic capabilities and treatment modalities.

Methods: We analyzed archived primary OSA tissue from dogs treated with limb amputation followed by doxorubicin or platinum-based drug chemotherapy. Samples were selected from two groups: dogs with disease free intervals (DFI) of less than 100 days (n = 8) and greater than 300 days (n = 7). Gene expression was assessed with Affymetrix Canine 2.0 microarrays and analyzed with a two-tailed t-test. A subset of genes was confirmed using qRT-PCR and used in classification analysis to predict prognosis. Systems-based gene ontology analysis was conducted on genes selected using a standard J5 metric. The genes identified using this approach were converted to their human homologues and assigned to functional pathways using the GeneGo MetaCore platform.

Results: Potential biomarkers were identified using gene expression microarray analysis and 11 differentially expressed (p < 0.05) genes were validated with qRT-PCR (n = 10/group). Statistical classification models using the qRT-PCR profiles predicted patient outcomes with 100% accuracy in the training set and up to 90% accuracy upon stratified cross validation. Pathway analysis revealed alterations in pathways associated with oxidative phosphorylation, hedgehog and parathyroid hormone signaling, cAMP/Protein Kinase A (PKA) signaling, immune responses, cytoskeletal remodeling and focal adhesion.

Conclusions: This profiling study has identified potential new biomarkers to predict patient outcome in OSA and new pathways that may be targeted for therapeutic intervention.
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http://dx.doi.org/10.1186/1471-2407-10-506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2955038PMC
September 2010

An orthotopic, postsurgical model of luciferase transfected murine osteosarcoma with spontaneous metastasis.

Clin Exp Metastasis 2010 Mar 7;27(3):151-60. Epub 2010 Mar 7.

Animal Cancer Center, Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523, USA.

Osteosarcoma (OS) is the most common bone tumor in humans. Newer, more clinically relevant models of OS are required to investigate novel therapeutics. The ability to study spontaneous micrometastases in the absence of a primary tumor is important since this is the manner in which most patients are treated clinically. Therefore, we have developed a novel model of murine OS using the DLM8 cell line, which is syngeneic to C3H mice. We have engineered these cells to express firefly luciferase so the development of metastases can be followed serially and non-invasively. These cells form osteolytic/osteoproductive lesions and metastasize spontaneously after orthotopic implantation in the proximal tibia, and the development of soft-tissue metastasis can be followed serially by luciferase expression following amputation. We have demonstrated a significant prolongation of disease-free and overall survival in the surgical adjuvant setting following treatment with doxorubicin or carboplatin, drugs which form the mainstay of treatment for human OS. In conclusion, we have developed a novel surgical adjuvant model of metastatic OS in immunocompetent mice that closely recapitulates the clinical situation, allowing the evaluation of novel therapeutics in the context of minimal residual disease.
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http://dx.doi.org/10.1007/s10585-010-9318-zDOI Listing
March 2010

A Pit-1 threonine 220 phosphomimic reduces binding to monomeric DNA sites to inhibit Ras and estrogen stimulation of the prolactin gene promoter.

Mol Endocrinol 2010 Jan 3;24(1):91-103. Epub 2009 Nov 3.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Anschutz Medical Center, Aurora, Colorado 80045, USA.

Pit-1 is a POU-homeodomain transcription factor that dictates the ontogeny of pituitary somatotrophs, lactotrophs, and thyrotrophs through regulation of their respective protein hormone genes: GH, prolactin (PRL), and TSHbeta. Although Pit-1 threonine 220 (T220) and serine 115 are protein kinase phospho-acceptor sites, the transcriptional role of Pit-1 phosphorylation remains unclear. In the rat PRL promoter (rPRL), Ras-stimulated transcription is mediated by binding of Ets-1 and Pit-1 at a composite site (FPIV). Ets-1 and Pit-1 physically interact, and Pit-1 T220 is a major Ets-1 contact point. T220 was mutated to aspartic acid (D, to mimic phosphorylation) or a neutral alanine (A), and DNA binding and transcriptional activity were tested. The Pit-1 T220D mutation reduced binding at monomeric Pit-1 sites (FPIV, PRL-1d), but not dimeric Pit-1 sites (FPI). Pit-1 T220A bound all sites with wild-type (WT) affinity. In transfections of HeLa cells, each Pit-1 mutant transcriptionally activated the -425rPRL promoter and cooperated with Ets-1 to WT levels. In contrast, Pit-1-mediated Ras activation of the -425 rPRL promoter was significantly inhibited by T220D. Finally, Pit-1 synergistic activation of the 2500-bp rPRL promoter with estrogen receptor was reduced by T220D compared with T220A and WT Pit-1. Thus, phosphorylation of Pit-1 T220 reduces binding to monomeric sites blunting Ras and estrogen/estrogen receptor stimulation of the rPRL gene promoter. Consequently, T220 phosphorylation of Pit-1 by protein kinase A, protein kinase C, or cell cycle-dependent kinases appears to serve as a regulatory switch, inhibiting Ras and estrogen/estrogen receptor regulatory pathways, while enhancing the cAMP/protein kinase A response, thus allowing a more precise integration of pituitary responses to distinct signaling stimuli.
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http://dx.doi.org/10.1210/me.2009-0279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2802898PMC
January 2010

The 26-amino acid beta-motif of the Pit-1beta transcription factor is a dominant and independent repressor domain.

Mol Endocrinol 2009 Sep 25;23(9):1371-84. Epub 2009 Jun 25.

University of Colorado Denver, Anschutz Medical Center, P.O. Box 6511, Mail Stop 8106, Aurora, Colorado 80045, USA.

The POU-homeodomain transcription factor Pit-1 governs the pituitary cell-specific expression of Pit-1, GH, prolactin (PRL), and TSHbeta genes. Alternative splicing generates Pit-1beta, which contains a 26-amino acid beta-domain inserted at amino acid 48, in the middle of the Pit-1 transcription activation domain (TAD). Pit-1beta represses GH, PRL, and TSHbeta promoters in a pituitary-specific manner, because Pit-1beta activates these same promoters in HeLa nonpituitary cells. Here we comprehensively analyze the role of beta-domain sequence, position, and context, to elucidate the mechanism of beta-dependent repression. Repositioning the beta-motif to the Pit-1 amino terminus, hinge, linker, and carboxyl terminus did not affect its ability to repress basal rat (r) PRL promoter activity in GH4 pituitary cells, but all lost the ability to repress Ras-induced rPRL promoter activity. To determine whether beta-domain repression is independent of Pit-1 protein and DNA binding sites, we generated Gal4-Pit-1TAD, Gal4-Pit-1betaTAD, and Gal4-beta-domain fusions and demonstrated that the beta-motif is sufficient to actively repress VP16-mediated transcription of a heterologous promoter. Moreover, beta-domain point mutants had the same effect whether fused to Gal4 or within the context of intact Pit-1beta. Surprisingly, Gal4-beta repression lost histone deacetylase sensitivity and pituitary specificity. Taken together, these results reveal that the beta-motif is a context-independent, modular, transferable, and dominant repressor domain, yet the beta-domain repressor activity within Pit-1beta contains cell type, promoter, and Pit-1 protein context dependence.
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http://dx.doi.org/10.1210/me.2008-0137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737555PMC
September 2009

ETS transcription factors in endocrine systems.

Trends Endocrinol Metab 2007 May-Jun;18(4):150-8. Epub 2007 Mar 26.

University of Colorado HSC, P. O. Box 6511, Mail Stop 8106, Aurora, CO 80045, USA.

E26 transformation-specific (ETS) transcription factors have become increasingly recognized as key regulators of differentiation, hormone responses and tumorigenesis in endocrine organs and target tissues. The ETS family is highly diverse, consisting of both transcription activators and repressors that mediate growth factor signaling and regulate gene expression through combinatorial interactions with multiple protein partners on composite DNA elements. ETS proteins have a role in the endocrine system in establishing pituitary-specific gene expression, mammary gland development and cancers of the breast, prostate and reproductive organs.
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http://dx.doi.org/10.1016/j.tem.2007.03.002DOI Listing
June 2007

Differential utilization of transcription activation subdomains by distinct coactivators regulates Pit-1 basal and Ras responsiveness.

Mol Endocrinol 2007 Jan 4;21(1):172-85. Epub 2006 Oct 4.

Department of Medicine, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA.

The POU-homeodomain transcription factor Pit-1 governs ontogeny and cell-specific gene expression of pituitary lactotropes, somatotropes, and thyrotropes. The splice isoform, Pit-1beta, inserts a 26-amino acid (AA) repressor at AA48 in the Pit-1 transcription activation domain (TAD). The Pit-1 TAD contains a basal regulatory subregion, R1 (AA1-45), and a basal and Ras-responsive region, R2 (AA46-80). To precisely map these activities, we generated GAL4-Pit-1/Pit-1betaTAD fusions and, in full-length HA-Pit-1, a series of substitution mutants of R2. Analysis in GH4 cells identified an activation domain at AA50-70, followed by an overlapping, dual-function, Ras-responsive-inhibitory domain, located from AA60-80. In contrast, GAL4-Pit-1betaTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the beta-domain, we inserted the beta-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1beta, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, beta-domain insertion at AA61 and 71 resulted in greater repression of Ras responsiveness, defining a critical R2 TAD spanning AA61-71 of Pit-1. Furthermore, Ras activation is augmented by steroid receptor coactivator 1, whereas cAMP response element binding protein-binding protein is not a Ras mediator in this system. In summary, the Pit-1/Pit-1beta TADs are composed of multiple, modular, and transferable subdomains, including a regulatory R1 domain, a basal activation region, a selective inhibitory-Ras-responsive segment, and a beta-specific repressor domain. These data provide novel insights into the mechanisms by which the Pit-1 TAD integrates DNA binding, protein partner interactions, and distinct signaling pathways to fine-tune Pit-1 activity.
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http://dx.doi.org/10.1210/me.2006-0245DOI Listing
January 2007

The gonadotropin releasing hormone (GnRH) receptor activating sequence (GRAS) is a composite regulatory element that interacts with multiple classes of transcription factors including Smads, AP-1 and a forkhead DNA binding protein.

Mol Cell Endocrinol 2003 Aug;206(1-2):93-111

Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA.

Activin responsiveness of the murine GnRH receptor gene promoter is mediated at a regulatory element we termed the GnRH receptor activating sequence (GRAS). Here, we have sought to define the complex of transcription factors that interact at this element. Consistent with activin regulation at GRAS, gel shift analyses and yeast one-hybrid assays reveal Smad4 interaction at the 5' end of GRAS. While overexpression of Smad3 activates a GRAS reporter, Smad3 binding at GRAS was not detectable. A functional interaction of Smad3 at GRAS was, however, detectable in yeast expressing Smad4. Thus, Smad3 interaction at GRAS appears to be dependent on the presence of Smad4. Mutations located at the 3' end of GRAS do not affect Smad binding but eliminate functional activity. Thus, Smad binding alone cannot account for the functional attributes of GRAS. Consistent with this notion, we find that AP-1 binding is immediately juxtaposed to and, in fact, partially overlaps the Smad binding site. Finally, a recently identified member of the forkhead family of transcription factors, FoxL2, is also capable of interacting at GRAS. Furthermore, FoxL2 activation at GRAS is lost with mutation of either the 5' Smad binding site or a putative forkhead binding site located at the 3' end of the element. We suggest that GRAS is a composite regulatory element whose functional activity is dependent on the organization of a multi-protein complex consisting of Smads, AP-1 and a member of the forkhead family of DNA binding proteins.
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http://dx.doi.org/10.1016/s0303-7207(03)00235-1DOI Listing
August 2003

Ras signaling and transcriptional synergy at a flexible Ets-1/Pit-1 composite DNA element is defined by the assembly of selective activation domains.

J Biol Chem 2003 Oct 5;278(41):39684-96. Epub 2003 Aug 5.

Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

Pit-1 and Ets-1 binding to a composite element synergistically activates and targets Ras-mitogen-activated protein kinase signaling to the rat prolactin promoter. These transcriptional responses appear to depend on three molecular features: organization of the Ets-1/Pit-1 composite element, physical interaction of these two factors via the Pit-1 homeodomain (amino acids 199-291) and the Ets-1 regulatory III domain (amino acids 190-257), and assembly of their transcriptional activation domains (TADs). Here we show that the organization of the Ets-1/Pit-1 composite element tolerates significant flexibility with regard to Ras stimulation and synergy. Specifically, the putative monomeric Pit-1 binding site can be substituted with bona fide binding sites for either a Pit-1 monomer or dimer, and these sites tolerated a separation of 28 bp. Additionally, we show that the physical interaction of Ets-1 and Pit-1 is not required for Ras responsiveness or synergy because block mutations of the Pit-1 interaction surface in Ets-1, which reduced Ets-1/Pit-1 binding in vitro, did not significantly affect Ets-1 stimulation of Ras responsiveness or synergy. We also show differential use of distinct TAD subtypes and Pit-1 TAD subregions to mediate either synergy or Ras responsiveness. Specifically, TADs from Gal4, VP16, or Ets-2 regulatory III domain linked to Ets-1 DNA binding domain constructs restored synergy to these TAD/Ets-1 DNA binding domain fusions. Conversely, deletion of the defined Pit-1 TAD (amino acids 2-80) retained synergy, but not Ras responsiveness. Consequently, we further defined the Pit-1 amino-terminal TAD into region 1 (R1, amino acids 2-45) and region 2 (R2, amino acids 46-80). R1 appears to regulate basal and synergistic responses, whereas the Ras response was mapped to R2. In summary, Ras responsiveness and Pit-1/Ets-1 synergy are mediated through the assembly of distinct TADs at a flexible composite element, indicating that different mechanisms underlie these two transcriptional responses and that the Pit-1 R2 subregion represents a novel, tissue-specific Ras-responsive TAD.
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http://dx.doi.org/10.1074/jbc.M302433200DOI Listing
October 2003

Structural characterization of the PIT-1/ETS-1 interaction: PIT-1 phosphorylation regulates PIT-1/ETS-1 binding.

Proc Natl Acad Sci U S A 2002 Oct 19;99(20):12657-62. Epub 2002 Sep 19.

Department of Physiological Chemistry, University Medical Center Utrecht and Center for Biomedical Genetics, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands.

The POU-domain transcription factor Pit-1 and Ets-1, a member of the ETS family of transcription factors, can associate in solution and synergistically activate the prolactin promoter by binding to a composite response element in the prolactin promoter. We mapped the minimal region of Ets-1 required for the interaction with the Pit-1 POU-homeodomain. Here, we describe a detailed NMR study of the interaction between the POU-homeodomain of Pit-1 and the minimal interacting region of Ets-1. By using heteronuclear single quantum coherence titration experiments, we were able to map exact residues on the POU-homeodomain that are involved in the interaction with this minimal Ets-1 interaction domain. By using our NMR data, we generated point mutants in the POU-homeodomain and tested their effect on the interaction with Ets-1. Our results show that phosphorylation of Pit-1 can regulate the interaction with Ets-1.
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http://dx.doi.org/10.1073/pnas.192693499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC130516PMC
October 2002