Abstract B19: RB function as a central component of osteosarcoma behavior: A comparative assessment in dogs and humans
Authors: Scott Milcah C. , Sarver Aaron L. , Phan Frances , Gupta Rachit , Thayanithy Venugopal , Subramanian Subbaya , Modiano Jaime F. 
Published: May 2015

Abstract
Introduction: There remain major gaps in our understanding of the pathogenesis of osteosarcoma (OS). This diagnosis represents a group of highly heterogeneous tumors, and an unresolved problem in their management is the lack of robust prognostic and predictive factors. Neither topography nor morphology, nor other conventional laboratory and ancillary tests can distinguish between highly aggressive and more indolent types of OS. Spontaneous OS is commonly diagnosed in dogs, and we showed recently that human and canine OS achieve similar phenotypes by altering convergent molecular pathways. Specifically canine and human OS can be stratified into two prognostically significant subgroups with inversely correlated expression of two gene clusters. This pattern was revealed only by the use of canine samples that also eliminated stromal signatures, as it was masked in studies that used human tumors or that employed different analyses. These two prognostic groups have significantly different behavior defined by survival, as well as by their ability to engraft and metastasize in mice. Equally important, there is exquisite overlap with a predictive signature called CINSARC (Complexity Index in SARComa) of soft tissue sarcomas, suggesting that OS can be used as proof of concept to support the canine model more generally for studies of sarcoma pathogenesis and therapy.
Experimental Procedures: We used bioinformatics and genetic complementation, respectively, to identify and validate driver events that are responsible for the biological behavior of canine and human OS. The MEME Suite and Ingenuity Pathway Analysis were used for de novo motif discovery and to identify transcription factors with significant target enrichment in the prognostic signature. Immunoblotting, dual luciferase reporter assays, mutagenesis assays, and qRT-PCR were used to confirmed differential expression and activity of RB protein in cell lines derived from aggressive and indolent OS. Co-immunoprecipitation and ChIP were used to characterize complexes associated with endogenous or ectopic RB. Gene expression microarrays and bioinformatics were used to assess changes in genome wide gene expression after restoration of RB in aggressive OS.
New Unpublished Data: The promoter sequences of all of the genes in the prognostic signature contained variants of RB-E2F-DP-1 binding sites. The gene signature also was significantly enriched for targets regulated by the RB and E2F families. RB expression was not the sole determinant of RB function, but genetic restoration of functional RB reversed the phenotype of each aggressive cell line analyzed to one resembling indolent tumors or normal osteoblasts, and it inevitably led to cell death. In contrast, indolent tumors that retained endogenous RB function were highly tolerant of RB addition, showing no (or minimal) phenotypic changes upon expression of ectopic RB. Somewhat surprisingly, restoration of RB also corrected other gene defects associated with the aggressive OS phenotype, such as activity of TP53 pathway.
Conclusions: Taken together, our results suggest that RB inactivation is not necessary for the development of OS, but the integrity of the RB pathway is an important factor that contributes to behavior, clinical progression, and outcome for dogs and humans with OS. Our ongoing work seeks to develop pharmacological agents that mimic RB-dependent gene expression and chromatin organization that can be used to improve outcomes for patients with the most aggressive forms of OS.
Citation Format: Milcah C. Scott, Aaron L. Sarver, Frances Phan, Rachit Gupta, Venugopal Thayanithy, Subbaya Subramanian, Jaime F. Modiano. RB function as a central component of osteosarcoma behavior: A comparative assessment in dogs and humans. [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 B19.