Defining the Significance of Insulin‐like Growth Factor 2 mRNA‐Binding Protein 1 (IGF2BP1) Expression in both Human and Canine Osteosarcoma

Abstract

Osteosarcoma is a malignant bone tumor that afflicts over 10,000 dogs and 450 adolescents yearly. Most dogs and approximately 25% of children succumb to metastatic disease. We identified elevated insulin‐like growth factor II mRNA binding protein 1 (IGF2BP1) as a biomarker of poor prognosis in canine osteosarcoma. IGF2BP1 is an oncofetal protein that regulates mRNA subcellular localization, nuclear export, stability and translation. This empowers IGF2BP1 to control expression of oncogene targets that contribute to cellular proliferation and resistance to chemotherapy. IGF2BP1 expression correlates with poor outcome in a variety of human cancers. We over‐expressed IGF2BP1 in a canine osteosarcoma cell line and used shRNA knockdown constructs to decrease expression in a human osteosarcoma cell line. IGF2BP1 over‐expressing clones show a 1.67 fold increase in doxorubicin resistance compared to control and IGF2BP1 expression correlates to IC50 values (r2 = 0.89). IGF2BP1 knockdown increased sensitivity to doxorubicin by ≥ 10 fold compared to control and significant reductions in cellular migration, invasion, proliferation, and tumor growth in nude mice were also observed (p < 0.05). These results support the hypothesis that elevated expression of IGF2BP1 may contribute to tumor progression and inadequate therapeutic response. Microarray analysis of control and IGF2BP1 over‐expressing cells was used to detect global changes in gene expression and to identify potential targets for IGF2BP1. Differentially expressed genes were cross‐referenced to the RNA‐Binding Protein Immunoprecipitation database to identify direct mRNA targets bound by IGF2BP1 in osteosarcoma. We identified 162 genes that were differentially expressed (FC ≥2, FDR< 0.05). Pathway analysis identified enrichment for genes involved in regulation of cell adhesion, migration, and extracellular matrix. We identified 13 differentially expressed genes that directly bind IGF2BP1 and are involved in cell adhesion and protein kinase activity. We investigated mechanisms that contribute to increased IGF2BP1 expression in panels of human and canine osteosarcoma cell lines and tumors. Using qPCR analysis, we observed genomic amplification in 35% of canine tumors and cell lines and correlated amplification with IGF2BP1 transcript expression (p < 0.05, r2 = 0.8). Genomic amplification was not observed in human cell lines. Significant loss of 3’UTR regulatory sequences was observed in 20% of canine cell lines (p<0.05). IGF2BP1 promoter analysis indicated that most regulatory elements were located within 580 bp of the start site of translation in both species including transcriptional response elements for Myc and β‐catenin. Overall, our data suggests that increased IGF2BP1 expression contributes to the development and progression of human and canine osteosarcoma. Multiple mechanisms contribute to elevated IGF2BP1 expression including gene amplification and altered mRNA expression and stability. These results can be used to develop new treatment strategies that target elevated IGF2BP1.Support or Funding InformationMorris Animal Foundation, CVMBS Research Council, Cancer League of ColoradoThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.