Glioblastomas (GBM) are the most malignant and common intrinsic brain tumor. Despite aggressive treatment the disease is uniformly fatal and patients survive on average less than a year. Progress against this lethal disease requires the identification of specific molecular targets and tools for rapid screening of therapeutic agents that inhibit invasive glioma stem cell (GSC) function. We first identified the role of TWIST1 in GBM invasion and demonstrated that TWIST1 knockdown inhibits human glioma stem cell activity in in vitro assays. Stable knockdown of TWIST1 expression in human GSCs or mouse-transformed neural stem and progenitor cells demonstrated significant inhibition of tumor growth in vivo. Our results show the critical role of TWIST1 in GSC tumorigenicity, suggesting that inhibition of TWIST1 may have therapeutic significance. However, the generation of TWIST1 inhibitors has to date proven nearly impossible because 1) transcription factors are difficult to target;2) there is very limited knowledge of the TWIST1 pathway;and, 3) reliable reporters of TWIST1 activity do not exist. The innovation of our approach is to develop a """"""""workaround"""""""" to this roadblock. This feasibility study is ideally suited for the R21 mechanism, as it will investigate the TWIST1 pathway in GSCs and will generate important new research tools to facilitate developing TWIST1 inhibitors in GSCs. Using a combined analysis of gene expression microarrays of GSCs with TWIST1 knockdown referenced to CHIP-sequencing to define direct TWIST1 targets, pathway analysis will identify genes/networks regulated through direct and indirect interactions with TWIST1. Bioinformatic analysis of the resulting data will lead to identification of candidate transcriptional regulatory motifs associated with TWST1 activity. These motifs will then be used to generate reporter constructs that inform on regulation of TWIST1 pathway activity in GSCs. Dual reporters that reflect TWIST1 regulation through activation or repression of genes are expected to increase the sensitivity and specificity of this approach in subsequent high-throughput screening for TW pathway inhibitors. Because TWIST1 is involved in the progression of gliomas and in the invasion and metastasis of a large variety of malignant tumors, the generation of TWIST1 reporters to screen for TWIST1 inhibitors is expected to have broad impact on the field of clinical oncology.
We recently found that stable inhibition of TWIST1 expression in primary glioma stem cells (GSCs) isolated from patients dramatically reduced stem cell activity and tumorigenicity. We propose now to generate TWIST1 reporters suitable for application in High-throughput screening for TWIST1 inhibitors targeting specifically cancer stem cell activity. We will use an innovative approach that combines microarrays profiling of our human primary GSCs with and without TWIST1 inhibition, CHIP-sequencing and interrogation of transcriptional factor network using bioinformatics tools followed by reporters construction and validation.
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