Recent molecular studies reveal global chromatin disorganization in glioblastoma (GBM), but how that affects individual gene loci to drive tumor growth and migration in vivo is still poorly understood. Previously, we have shown remarkable similarities in chromatin remodeling between germinal matrix stem/progenitors (NSPCs) and gliomas at the EGFR promoter, and implicated several epigenetic modifiers for the transcriptional activation of this oncogenic locus (Erfani et al, 2015). We now expand our study focus to investigate more systematically and in functionally distinct populations how developmentally regulated transcriptional networks become pathologically co-opted in gliomas to maintain tumorigenic properties of cell migration and cell proliferation. To this end, we have developed a simple EGF ligand-binding strategy to prospectively isolate populations with stem cell properties from fresh human samples and patient-derived glioma xenografts (PDX), in which the migratory human GBM cells can be regionally separated from those at the tumor core (Tome- Garcia et al, Stem Cell Rep, accepted in final revisions). Full transcriptome and chromatin accessibility analyses in these freshly sorted human GBM stem cell populations revealed distinct regulatory regions related to cell migration vs. self-renewal, the latter also present in NSPCs. We thus hypothesized that tumor properties of cell growth and migration in GBM are regulated through unique transcription factors (TFs) at regions of open chromatin, some of which are co-opted from a normal neural developmental state. To test this hypothesis, here we intend to profile the transcriptome (by RNA-seq) and associated open chromatin landscape (by ATAC-seq) in phenotypically defined migratory vs. core-proliferative human GBM populations, isolated from PDX gliomas, and compare them to our previously generated data in developing NSPCs. Differential analysis of RNAseq and ATACseq data in infiltrative vs. tumor-core GBM cells will allow us to define the transcriptional pathways and chromatin accessibility regulatory regions driving adaptations for a migratory GBM cell fate in vivo, while comparative analysis with similar data in NSPCs will elucidate how developmental pathways are aberrantly co- opted and re-activated in GBM. Combined ATACseq/RNAseq data analysis will provide a blueprint for specific TF binding occupancy at gene loci activated in infiltrative tumor cells, generating a candidate list of TF regulators for cell migration in GBM, which we will confirm biochemically. Defining the specific epigenetic landscape and associated transcriptional phenotype in neoplastic and progenitor populations derived from fresh human and PDX samples will advance deeply our understanding of the mechanisms maintaining plasticity in GBM stem cells and the specific molecular adaptations in a subset of these cells towards a migratory cell fate. By depositing our data in an open access platform, we will maximize data dissemination and facilitate further investigation into the role of cell migration in GBM, hoping to uncover new avenues for therapy aimed at targeting infiltrative tumor cells that currently evade surgical resection.
This proposal lays forward an innovative, translational methodology for the direct isolation of migratory and core-proliferative populations of glioblastoma cells from fresh human samples and mouse xenografts, addressing current challenges of tumor heterogeneity and immortalized cell culture artifact. Using this approach, the study aims to define the epigenetic landscape and transcriptional networks that drive tumorigenic properties of proliferation and migration in human glioblastoma cells, and to distinguish those that are co-opted from normal neural stem cell development.
|Tome-Garcia, Jessica; Erfani, Parsa; Nudelman, German et al. (2018) Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma. Nat Commun 9:4020
|Tome-Garcia, Jessica; Doetsch, Fiona; Tsankova, Nadejda M (2017) FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand. Bio Protoc 7: