Abstract A23: Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) drives growth, metastasis and chemoresistance in human and canine osteosarcoma
Authors: Pfaff Liza E. , Kalet Brian T. , Dailey Deanna D. , Duval Dawn L. 
Published: May 2015

Osteosarcoma (OSA) is the most common bone tumor in children. Dose intensive therapies have resulted in survival rates of 75%, however for patients with metastasis at diagnosis, the survival rate is only 20%, indicating the need for improved therapeutic strategies for metastatic osteosarcoma. Unfortunately, the availability of human OSA samples for study is extremely limited; however, over 10,000 canine patients spontaneously develop OSA annually and canine tumors share common histological features, genetic mutations and gene expression profiles with human OSA. To identify factors that contribute to metastasis and chemotherapeutic resistance of OSA, we assessed the gene expression signatures of normal bone and groups of primary canine OSA tumors surgically resected from dogs with short (&lt;100 days) and long (&gt;300 day) disease free intervals (DFI) following standard treatment. Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) was identified as a negative prognostic indicator in this canine OSA study. IGF2BP1 is an oncofetal protein that binds multiple RNA targets to regulate their nuclear transport, stability, translation and subcellular localization. We have further investigated the role of IGF2BP1 in the development and metastatic progression of both human and canine osteosarcoma.

IGF2BP1 transcripts were virtually undetectable on microarray analysis of normal bone and up-regulated greater-than 65-fold in both canine tumor groups relative to normal bone. RT-qPCR analysis validated these findings demonstrating a stair-step pattern of expression in which normal bone had virtually no IGF2BP1 expression, and IGF2BP1 expression was elevated 132-fold and 915-fold compared to normal bone in the long and short DFI tumor groups, respectively. Array CGH analysis revealed no genomic amplification of the IGF2BP1 locus, but 3′ UTR truncation may contribute to increased IGF2BP1 mRNA half-life. Similarly, human osteosarcoma cell lines, on average, expressed 14-fold higher levels of IGF2BP1 transcripts compared to human osteoblasts. Further, in the MG63.2 cell line, a metastatic variant of the MG63 human OSA cell line, we measured elevated mRNA transcripts (five-fold, p=0.0368) and protein levels (seven-fold), implicating IGF2BP1 in metastasis of osteosarcoma. IGF2BP1 knockdown in shRNA-expressing MG63.2 clones resulted in a three-fold (p&lt;0.001) decrease in cellular proliferation compared to cells stably expressing a non-targeted shRNA construct. IGF2BP1 knockdown also reduced cell migration and invasion using Boyden chamber-based assays (p&lt;0.05). MG63.2 cells from two independent IGF2BP1 knockdown shRNA constructs grown as subcutaneous tumors in nude mice (n=5/group) exhibited significantly delayed tumor appearance and reduced volume compared to scrambled controls (p&lt;0.001). Finally, IGF2BP1 stable knockdown cell lines were up to 10-fold (p&lt;0.05) more sensitive to doxorubicin compared to the control knockdown cell line. The sensitivity to doxorubicin correlated directly with IGF2BP1 knockdown (p = 0.0049, r2 = 0.9901). Overall, these data implicate IGF2BP1 gene expression in the development, metastatic progression, and chemoresistance of human and canine osteosarcoma. Further, these studies provide evidence that spontaneous canine OSA represents a valuable model for both target discovery and the development of novel therapeutic strategies for the treatment of metastatic OSA.

Citation Format: Liza E. Pfaff, Brian T. Kalet, Deanna D. Dailey, Dawn L. Duval. Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) drives growth, metastasis and chemoresistance in human and canine osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A23.