Abstract 2432: Epigenetic reprogramming of osteosarcoma tumor initiating cells alters histologic and metastatic phenotype
Authors: Hyddmark Emma V. , Levings Padraic , White Margaret , Guijarro Maria , Nasri Elham , Zarezadeh Ali , Palmer Glyn , Ghivizzani Steve , Gibbs Charles P. 
Published: July 2016

Abstract
Osteosarcoma (OS) is a pediatric cancer with 40% mortality despite aggressive treatments. Major treatment challenges are that some tumors do not respond to therapies or develop resistance over time, processes partially attributed to cellular heterogeneity within a tumor. We have previously demonstrated such heterogeneity using a reporter consisting of an Oct4 promoter driving expression GFP. Cells capable of activating the reporter, the GFP+ cells, are 100-fold more tumorigenic than the GFP- cells. Pure populations of GFP+ cells injected into mice produces heterogeneous tumors, indicating that the GFP- cells arise from the GFP+ cells. The two populations display distinct gene expression profiles, which are reproducible between tumors and representative of global changes. These characteristics suggest that the emergence of a GFP- cell population is due to epigenetic changes. The objective of this study was to investigate to what extent tumorigenic potential in OS is governed at the epigenetic level and can be manipulated exogenously by agents that modify or re-pattern the epigenome.
To test this, sorted GFP+ OS cells were treated with the histone deacetylase inhibitor Trichostatin A (TSA) in vitro at a dose that increased histone acetylation without inducing cell death. Although TSA treatment showed promising antitumoral effects in vitro by inhibiting proliferation and inducing cell cycle arrest, however, global interrogation of the gene expression changes induced by TSA treatment revealed an activation of many cancer associated pathways. In fact, TSA treated Oct4-GFP+ cells injected into NSG mice generated tumors that displayed enhanced metastatic dissemination, a reduced GFP+ population, and histologic changes. Differential expression analyses between the GFP+ cells isolated from TSA tumors or tumors generated from DMSO treated cells showed that the new phenotype was associated with enrichment for cell cycle pathways and a decrease in extracellular matrix pathways. This was confirmed at the protein level and with proliferation assays. As rapidly proliferating cells are generally more sensitive to chemotherapy, we investigated the sensitivity of the GFP+ cells from TSA and Control tumors to Doxorubicin (Dox). Cells isolated from TSA tumors were significantly more receptive to Dox-induced cell death.
Our results suggest that OS malignancy is partially governed at the epigenetic level and that it is possible to reduce intratumoral heterogeneity. A reduction in intraturmoral heterogeneity can be exploited in cases of tumors that are refractory to treatment or have acquired epigenetic resistance. Intermittent treatment with epigenetic modifiers could potentially sensitize the tumors to the other treatments, as have been demonstrated for other cancer types.
Citation Format: Emma V. Hyddmark, Padraic Levings, Margaret White, Maria Guijarro, Elham Nasri, Ali Zarezadeh, Glyn Palmer, Steve Ghivizzani, Charles P. Gibbs. Epigenetic reprogramming of osteosarcoma tumor initiating cells alters histologic and metastatic phenotype. [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 2432.