Forced Induction Of Differentiation In Osteosarcoma Tumor Initiating Cells

Osteosarcoma (OS) is a highly malignant bone cancer that is defined histologically by the secretion of immature osteoid. The chemotherapeutic regimen has not changed in the last 40 years, and similarly the five-year survival rate has remained a dismal ∼60%, largely due to metastatic spread to the lungs. OS is believed to originate from osteogenic committed progenitors, involving disruption of extracellular matrix synthesis in favor of proliferation. In previous work, our lab developed a method to selectively identify Tumor Initiating Cells (TICs) in OS xenografts based on their ability to activate a transcriptional reporter comprised of a human OCT4 promoter linked to the coding sequence for Green Fluorescent Protein (GFP). Clonally derived, stably transfected OS Oct4/GFP+ cells are capable of initiating and maintaining the growth of heterogeneous tumors and driving disease progression. The loss of GFP expression and tumorigenic capacity occurs during tumor formation in response to cues within the tumor microenvironment. These conditions appear to reprogram the GFP+ cells, resulting in loss of reporter activity, reduced proliferation, and adoption of a specialized secretory phenotype as an adaptive survival mechanism: a phenotypic change similar to that seen in normal physiological differentiation. During skeletal formation, Bone Morphogenetic Proteins (BMP) play key roles in osteogenic maturation. Because the OS lineage of origin suggests an innate sensitivity to BMP proteins, we hypothesize that BMP stimulation will force the induction of differentiation in OS TICs and will impair the ability of these cells to initiate and maintain tumor growth. To test our hypothesis, we investigated the expression of BMP receptors in two primary OS cell lines, using Mesenchymal Stem Cells and Human Fetal Osteoblasts as positive controls. Expression of BMP Receptors II, 1A, and 1B was confirmed using flow cytometry indicating that the OS cells could potentially respond to BMP stimulation. The functionality of the BMP signaling pathway was subsequently investigated after stimulation with BMP heterodimers, BMP2/7 and BMP4/7. Immunoblotting showed OS TICs are capable of activating canonical and non-canonical BMP signaling pathways after BMP stimulation, suggesting a functional and intact signaling network potentially able to induce differentiation. However, only cells treated with BMP4/7 showed changes in cell cycle distribution as visualized by DAPI. Furthermore, BMP4/7-stimulated TICs showed reduced tumor formation in our xenograft mouse model Our data suggests an inhibition of OS tumorigenicity due to a BMP4/7 induced differentiation based response. Currently, we are investigating if forced expression of BMPs using an OS specific recombinant Adeno-Associated Virus can inhibit tumor growth in vivo. Such a differentiation-based therapy could become a more effective and safer alternative to cytotoxic chemotherapy. Citation Format: Margaret E. White, Emma Viktoria Marie Hyddmark, Ali Zarezadeh, Elham Nasri, Maria V. Guijarro, Padraic P. Levings, Glyn Palmer, Steven C. Ghivizzani, C. Parker Gibbs. Forced induction of differentiation in osteosarcoma tumor initiating cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1188.