C-MYC Target Gene Network in the Ewing’s Sarcoma Cancer Stem Cell
Tumors are not homogenous in nature, but are heterogeneous populations of cells. One population, the cancer stem cell (CSC), is thought to be responsible for the initiation and progression of the disease as well as contain inherent properties that make it more resistant to modern therapies. One of the key regulatory genes in these cells may be the oncogenic transcription factor c-Myc. We propose to study the regulatory and functional role c-Myc has in the Ewing’s sarcoma cancer stem cells (ESSCs) to increase our understanding of the genetic and molecular landscape of these cells. These studies can enhance our insights into the mechanisms of tumor initiation, therapeutic resistance, reoccurrence and metastasis. Aim1. Role of c-Myc and determination of its transcriptional network in ESSCs. Aim1a. Determination of the c-Myc specific target genes in Ewing’s sarcoma through Chromatin Immunoprecipitation and sequencing (ChIP-Seq) studies. We will perform a global investigation into the Ewing’s sarcoma c-Myc target gene network using Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) technology. This will provide insights into direct c-Myc targets within Ewing’s. Aim1b. c-Myc dependence and transcriptional alterations in ESSCs. We intend to utilize the high Aldehyde Dehydrogenase (ALDH1) activity within Ewing’s cell lines to isolate the CSC population. Illumina TruSeq preparation and HiSeq PE100 sequencing on the GAIIx instrumentation for full sequencing of the mRNAs isolated from the control and shMyc ESSCs will provide insights into the genetic landscape of the cancer stem cells. Aim2. Functional significance of the c-Myc transcriptional target gene network and pathways in ESSCs. We will use si/shRNA and overexpression lentiviral technology to genetically alter specific c-Myc target genes in ESSCs. When feasible, we will assess small molecule inhibitors on pertinent genetic pathways, such as the Wnt signaling pathway. We will perform in vitro and in vivo assays to assess alterations on ESSC self-renewal, proliferation and tumorigenicity.