Osteosarcoma (OS) is a malignant bone tumor that afflicts over 10,000 dogs. Most dogs and approximately 30-40% of children with OS succumb to metastatic disease. We identified elevated insulin¬-like growth factor 2 mRNA binding protein 1 (IGF2BP1) as one of the biomarkers of poor prognosis in canine OS. IGF2BP1 is an oncofetal protein that regulates mRNA subcellular localization, nuclear export, stability, and translation. IGF2BP1 controls the expression of oncogene targets and correlates with poor outcome in a variety of human cancers.
Using microarray analysis, we identified elevated insulin-like growth factor II mRNA binding protein 1 (IGF2BP1) expression as a biomarker of poor prognosis in canine osteosarcoma. Also, our preliminary data show that IGF2BP1 knockdown (shRNA) in a human OS cell line increased sensitivity to doxorubicin by ≥ tenfold compared to control. Significant reductions in cellular migration, invasion, proliferation, and tumor growth in nude mice were also observed (p < 0.05). The current research explores mechanisms for increased IGF2BP1 expression in panels of human and canine osteosarcoma cell lines. Gene amplification, hypomethylation, increased transcription, and alterations in microRNA (miRNA) regulation directly or through 3’UTR shortening have all been hypothesized by many studies as mechanisms to increase IGF2BP1 expression in cancer. We evaluated the expression and alternative polyadenylation of IGF2BP1 using RT-qPCR and western blot analysis in human and dog osteosarcoma cell lines. We assessed transcriptional activation of IGF2BP1 using luciferase reporters containing promoter sequences from the human and canine IGF2BP1 genes. To detect genomic amplification and methylation, we used qPCR to assess gene copy numbers and treatment with the DNA methylase inhibitor, 5-Azacytidine, to explore activation of gene expression through hypomethylation.
Using qPCR analysis, we observed genomic amplification in 35% of canine tumors and cell lines and correlated amplification with IGF2BP1 transcript expression (p = 0.0006, Pearson r = 0.88). We observed no genomic amplification in human cell lines. Significant loss of 3’UTR regulatory sequences was found in 20% of canine cell lines (p < 0.05). The promoter analysis showed that most regulatory elements were located within ~580bp from the translational start site in both species. Using pathway-focused luciferase reporter assays, we identified activation of the following factors: MYC, NF-Kappa B, AP-1, and TCF4: β-catenin. Thus, our data show that multiple mechanisms can contribute to elevated IGF2BP1 expression, and these results can be used to develop new treatment strategies that target elevated IGF2BP1 or regulatory mechanisms.
Using the McKinley canine OS cell line, we generated and validated stable overexpression of IGF2BP1 (IGF2BP1-pLVX-Puro, Clontech). The stable OS cell line pool and individual clones with a corresponding empty vector control were analyzed and tested for migration, invasion, proliferation, and resistance to standard chemotherapeutic agents. We analyzed migration and invasion using a scratch wound assay and measured cellular proliferation as a surface confluence for 90 hours on an IncuCyte Zoom. We also assessed the clones’ sensitivity to doxorubicin over 48 hours using a bioreductive resazurin-based fluorometric assay. We assessed changes in transcript expression in response to IGF2BP1 from isolated total RNA analyzed on Affymetrix Canine 1.0ST microarrays (University of Colorado Cancer Center Genomic and Microarray Shared Resource).
The overexpressing IGF2BP1 clones had increased resistance to doxorubicin compared to the control, and the IC50 levels correlated with IGF2BP1 mRNA levels (p < 0.05, r2 = 0.89). For cellular proliferation, we found that only the IGF2BP1-expressing pool, that represents random insertion of the plasmid without selecting isolated clones, exhibited a significantly higher rate of proliferation relative to the empty vector control (p < 0.05). However, one of the highest expressing IGF2BP1 isolated clones had significantly greater cellular mobility and invasion than this pool, and both the pool and isolated clone had significantly higher rates of migration and invasion that cells transfected with the empty plasmid (p < 0.05).
Microarray analysis of control and over¬expressing cells was used to detect global changes in gene expression and to identify potential targets of IGF2BP1. Differentially expressed genes were cross ¬referenced to the RNA¬ Binding Protein Immunoprecipitation database, published by Conway et al. (2016) using human stem cells, to identify direct mRNA targets bound by IGF2BP1. We identified 162 genes that were differentially expressed between control and overexpressing cells (FC ≥2, FDR< 0.05), and 13 of those genes have been previously reported to bind IGF2BP1 directly. Pathway analysis of these 13 genes identified enrichment for genes involved in the regulation of cell adhesion, migration, and the extracellular matrix. Altered expression and IGF2BP1 binding of a subset of these transcripts were confirmed using RNA immunoprecipitation and RT-qPCR. Our data suggest that IGF2BP1 plays a significant role in human and canine osteosarcoma. This study revealed the functional relevance of IGF2BP1 and identified it as a biomarker for aggressiveness in osteosarcoma. With this knowledge, new treatment strategies can be developed that target IGF2BP1 or it is signaling pathways for osteosarcoma, or any cancer that expresses high levels of IGF2BP1. This treatment may have a high impact on the cell’s ability to metastasize.
|Commitee:||Wilusz, Jeffrey, Argueso, Lucas, Stargell, Laurie|
|School:||Colorado State University|
|Department:||Cell and Molecular Biology (Graduate Degree Program)|
|School Location:||United States -- Colorado|
|Source:||DAI-B 81/3(E), Dissertation Abstracts International|
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