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

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 WNT 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 Wnt pathway-targeted drugs in periostin overexpressing canine OS may be a potential therapeutic target.

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

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 IC50 values greater than 5 µM, but exhibited greater sensitivity to a paradox-breaking BRAF inhibitor (IC50: 0.2-1 µM). All TCC cell lines had IC50 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.

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

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.

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

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.

It's not always histiocytic sarcoma: Immunocytochemistry to identify two unusual tumors in a Bernese Mountain dog.

A 7-year-old female spayed Bernese Mountain dog was presented for evaluation of hematuria. Incidentally, a right stifle sarcoma was diagnosed via cytology, which raised concern for histiocytic sarcoma (given the patient's signalment) versus another joint-associated sarcoma. Histopathology and immunohistochemistry revealed a CD18-negative, non-histiocytic origin cell population. Findings were consistent with a joint-associated grade II soft tissue sarcoma (STS). The patient's hematuria was progressive over 5 months, and urinary bladder transitional cell carcinoma (TCC) was diagnosed via cystoscopy and histopathology. An enlarged right medial iliac lymph node was identified on routine restaging via abdominal ultrasound 3 months later. Cytology of the lymph node revealed a markedly pleomorphic cell population, again raising concern for histiocytic sarcoma (HS). Other differentials included an anaplastic metastatic population from the joint-associated STS or the TCC. Immunocytochemistry revealed a cytokeratin-positive, CD18-, CD204-, and vimentin-negative cell population, consistent with a carcinoma. DNA was extracted from cytology slides to sequence cells for BRAF mutation status. Sequencing revealed a homozygous V596E (transcript ENSCAFT00845055173.1) BRAF mutation, consistent with the known biology of TCC. In neither case was HS truly present in this patient, but immunocytochemistry provided information that helped to optimize the patient's chemotherapy recommendations.

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

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.

Integrated analysis of canine soft tissue sarcomas identifies recurrent mutations in TP53, KMT genes and PDGFB fusions.

Canine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features. Histologically, canine STS sub-types are difficult to discern at the light microscopic level due to their overlapping features. Thus, genomic, and transcriptomic profiling of canine STS may prove valuable in differentiating the diverse sub-types of mesenchymal neoplasms within this group. To this purpose we sought to characterize the transcript expression and genomic mutation profiles of canine STS. To delineate transcriptomic sub-types, hierarchical clustering was used to identify 4 groups with district expression profiles. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B ( PDGFB ) and collagen genes. Sensitivity to imatinib was evaluated in a canine STS cell line also bearing a PDGFB fusion. Using whole exome sequencing, recurrent driver variants were identified in the cancer genes KMT2D (21% of the samples) and TP53 (21%) along with copy number losses of RB1 and CDKN2A. Gene amplifications and resulting transcript increases were identified in genes on chromosomes 13, 14, and 36. A subset of STS was identified with high T-cell infiltration. This multi-omics approach has defined canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for therapy.

Integrated analysis of canine soft tissue sarcomas identifies recurrent mutations in TP53, KMT genes and PDGFB fusions.

Soft tissue sarcomas (STS) are a heterogenous group of mesenchymal tumors representing over 50 distinct types with overlapping histological features and non-specific anatomical locations. Currently, localized sarcomas are treated with surgery + / - radiation in both humans and dogs with few molecularly targeted therapeutic options. However, to improve precision-based cancer therapy through trials in pet dogs with naturally occurring STS tumors, knowledge of genomic profiling and molecular drivers in both species is essential. To this purpose, we sought to characterize the transcriptomic and genomic mutation profiles of canine STS subtypes (fibrosarcoma, undifferentiated pleomorphic sarcoma, and peripheral nerve sheath tumors), by leveraging RNAseq, whole exome sequencing, immunohistochemistry, and drug assays. The most common driver mutations were in cell cycle/DNA repair (31%, TP53-21%) and chromatin organization/binding (41%, KMT2D-21%) genes. Similar to a subset of human sarcomas, we identified fusion transcripts of platelet derived growth factor B and collagen genes that predict sensitivity to PDGFR inhibitors. Transcriptomic profiling grouped these canine STS tumors into 4 clusters, one PNST group (H1), and 3 FSA groups selectively enriched for extracellular matrix interactions and PDFGB fusions (H2), homeobox transcription factors (H3), and elevated T-cell infiltration (H4). This multi-omics approach provides insights into canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for chemo- and immuno-therapy.

Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation.

Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (Dm ) values ranging from 4.6-15.8 µM and 2.1-5.1 µM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with Dm values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.

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.

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

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.

Characterizing the molecular and immune landscape of canine bladder cancer.

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.

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

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.

Immune pathways and TP53 missense mutations are associated with longer survival in canine osteosarcoma.

Osteosarcoma affects about 2.8% of dogs with cancer, with a one-year survival rate of approximately 45%. The purpose of this study was to characterize mutation and expression profiles of osteosarcoma and its association with outcome in dogs. The number of somatic variants identified across 26 samples ranged from 145 to 2,697 with top recurrent mutations observed in TP53 and SETD2. Additionally, 47 cancer genes were identified with copy number variations. Missense TP53 mutation status and low pre-treatment blood monocyte counts were associated with a longer disease-free interval (DFI). Patients with longer DFI also showed increased transcript levels of anti-tumor immune response genes. Although, T-cell and myeloid cell quantifications were not significantly associated with outcome; immune related genes, PDL-1 and CD160, were correlated with T-cell abundance. Overall, the association of gene expression and mutation profiles to outcome provides insights into pathogenesis and therapeutic interventions in osteosarcoma patients.

Prospective clinical trial testing COXEN-based gene expression models of chemosensitivity in dogs with spontaneous osteosarcoma.

BACKGROUND: This study is a prospective clinical trial in dogs with osteosarcoma testing a gene expression model (GEM) predicting the chemosensitivity of tumors to carboplatin (CARBO) or doxorubicin (DOX) developed using the COXEN method. PATIENTS AND METHODS: Sixty dogs with appendicular osteosarcoma were enrolled in this trial. RNA isolation and gene expression profiling were conducted with 2 biopsies for 54/63 screened tumors, and with a single biopsy for 9 tumors. Resulting gene expression data were used for calculation of a COXEN score for CARBO and DOX based on a previous study showing the significance of this predictor on patient outcome utilizing retrospective data (BMC Bioinformatics 17:93). Dogs were assigned adjuvant CARBO, DOX or the combination based on the results of the COXEN score following surgical removal of the tumor via amputation and were monitored for disease progression by chest radiograph every 2 months. RESULTS: The COXEN predictor of chemosensitivity to CARBO or DOX was not a significant predictor of progression-free interval or overall survival for the trial participants. The calculation of DOX COXEN score using gene expression data from two independent biopsies of the same tumor were highly correlated (P < 0.0001), whereas the calculated CARBO COXEN score was not (P = 0.3039). CONCLUSION: The COXEN predictor of chemosensitivity to CARBO or DOX is not a significant predictor of outcome when utilized in this prospective study. This trial represents the first prospective trial of a GEM predictor of chemosensitivity and establishes pet dogs with cancer as viable surrogates for prospective trials of prognostic indicators.

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

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.

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

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.

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

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 (IC50 < 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.

ETS transcription factors in endocrine systems.

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.

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

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.