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Integrated pathways associated with metastasis and chemoresistance in canine osteosarcoma




Dailey, Deanna D., author
Duval, Dawn, advisor
Bouma, Jerry, committee member
Ehrhart, EJ, committee member
Page, Rod, committee member

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Osteosarcoma (OS) is the most common canine primary bone tumor affecting 10,000 dogs every year. This aggressive cancer is characterized by both a high rate of metastasis and chemotherapeutic resistance. It is estimated up to 80% of patients carry silent metastases at the time of diagnosis, and most will progress despite removal of the primary tumor and chemotherapy. Canine OS is strikingly similar to the disease in humans following a similar clinical course and sharing genetic and molecular aberrations. Thus the canine disease has gained recognition as a relevant spontaneous tumor model for human OS. Unfortunately, survival rates for both species have plateaued with no significant gains made in the last 20-30 years. New treatment strategies are needed and will likely consist of combined therapies including conventional chemotherapy drugs along with targeted and immune modulating agents. The success of clinical trials to evaluate these novel therapies will rely on improved understanding of molecular pathways contributing to progression and chemotherapy resistance of OS. Further, molecular characterization of OS will provide biomarkers essential for prognosis, treatment planning and patient monitoring. Gene expression profiling of canine tumors from poor responders compared to tumors from good responders implicated pathways critical to normal bone development including hedgehog and Wnt/β-catenin. During bone development, there is significant crosstalk between these pathways and the Notch signaling pathway, a third developmental pathway associated with growth and survival in a variety of human cancers. We performed pathway focused gene expression studies using canine and human OS cells, canine OS tumors and normal bone samples to evaluate two Notch receptors and two downstream effectors. We identified expression changes consistent with Notch activation in OS compared to normal bone. We further determined that, while expression of three Notch associated genes remained elevated in tumors from the poor responders, expression of hairy/enhancer of split 1 (HES1) was significantly lower in tumors from poor responders than in tumors from good responders. Survival analyses based on immunoreactivity for HES1 in fixed tissues from an independent tumor set confirmed the association between low HES1 expression and poor outcome. To further explore the Notch pathway in OS and elucidate potential mechanisms underlying the disruption of Notch/HES1 signaling in the most aggressive tumors, we performed miRNA expression profiling of canine tumors. Our goals included identification of miRNA signatures associated with patient outcome in OS as well as integration of miRNA and gene expression data for additional pathway-focused explorations. Further, we endeavored to find miRNA biomarkers in serum of OS patients with prognostic potential. We successfully identified a tumor-based three-miRNA signature and a serum-based two-miRNA signature that separated patients into distinct outcome groups with good accuracy. In silico miRNA-mRNA interaction analyses of dysregulated miRNAs and Notch-associated genes in tumors compared to normal bone revealed nearly 20 interactions, validated experimentally in other systems, potentially associated with OS. Interaction and pathway analysis of aberrant miRNA and gene expression in tumors from poor responders vs. good responders identified insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), an oncogene of interest in OS, as a common target of seven down-regulated miRNAs. Finally, these analyses suggested interactions with the tumor microenvironment are important to the progression of OS. We expanded our miRNA expression profiling to include microarray analysis of 29 canine cancer cell lines. This allowed us to utilize drug sensitivity data from in vitro assays, where cells were treated with either doxorubicin or carboplatin, to identify "drug-resistance-related" miRNAs associated with outcome in canine OS tumors. We identified an additional miRNA from this cell-based approach, which participated in a tumor-based four-miRNA predictive signature. In silico miRNA-gene regulatory pathway analyses of outcome associated miRNAs and dysregulated genes from predictive doxorubicin and carboplatin models, developed using the COXEN algorithm, implicated the Notch pathway as contributing to doxorubicin resistance. Finally, pathway analyses of the top five miRNAs associated with progression of OS and chemoresistance: let-7b, miR-98, miR-130a, miR-181b and miR-223 implicated the PI3K/AKT pathway in progression of OS. Taken together, the studies described herein, provide an integrated picture of Notch signaling in OS elaborating candidate miRNA-gene interactions associated with development and progression of OS and resistance to doxorubicin. Further, these studies have revealed key miRNA-mRNA interactions that implicate other targetable pathways and thus, may serve as biomarkers for patient stratification, enhancing efforts towards integration of individualized targeted therapies in OS. Finally, we have identified miRNA-based prognostic signatures measurable from OS tumors or patient serum, which laid the groundwork for development of a clinically useful prognostic screen for OS.


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gene expression


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