Abstract 3765: High-throughput RNAi screening identifies genes controlling glioblastoma stem cell migration and survival.

Abstract

Abstract Glioblastoma is incurable. Glioblastoma stem cells (GSC) became a model for explaining tumor recurrence due to their tumorigenic capacity, migratory nature, and radio-chemoresistant phenotype. To prevent tumor recurrence, a strategy targeting of GSC must be identified and added into the treatment. To identify vital genes that promote GSC migration and sustain self-renewal and anti-apoptotic features we have established tumorigenic CD133+ GSC lines (n=3 patients), which are capable of clonal self-renewal and asymmetric division for producing fast-growing CD133- daughter cells that form tumor spheres. CD133+ GSC can migrate outward from primary spheres and form spheres again, which provide an visible, functional model for identifying essential genes of CD133+GSC via loss-of-function RNA interference (RNAi) screen. To identify genes and pathways that confer the migration nature and super longevity of GSC, we constructed a siRNA library consisting of siRNA clones targeting 2079 genes overexpressed in tumorigenic CD133+ GSC when compared to non-tumorigenic, autologous tumor cell lines, normal brain tissue, human embryonic stem cells, normal neural stem cells, fetal neural progenitor cells and glioblastoma tumors, respectively, as determined by the comparative expression microarray analysis (E/B>2X, E-B>100, p value<0.05). An automated high-throughput siRNA screen was performed in 384-well plates using a reverse transfection procedures and the functional consequence were scored in 3 categories: score 1- degree of cell migration and size of tumor spheres were slightly decreased, score 2- cell migration was interrupted, and score 3-cell apoptosis was detected thereby no tumor spheres were formed. The siRNA targeting endothelin 3 served as positive siRNA control, which suppress GSC migration and induction of GSC apoptosis. Our primary screen has identified 193 genes whose inhibition by RNAi had led to impaired cell migration while knockdown of 128 genes led to the cell apoptosis and loss of self-repopulating capacity. Overlapped gene functions in regulation of both GSC migration and survival were detected, and genes which have never been reported to be associated with cell migration and cell survival were identified. Genes that maintain GSC survival and self-renewal capacity (score 3) mainly include those with roles in stemness maintenance, tumor suppressor/antigrowth/antidifferentiation, DNA repair, cell cycle checkpoint, While genes involved in GSC migration mainly include those with a role in cell adhesion, cell spreading, chemotaxis, axon guidance, neural crest cell migration. A high confidence gene list will be presented in the meeting. The development of an innovative therapy targeting essential genes of GSC that promote cell migration and/or cell survival could attenuate the infiltrating nature of GBM tumor, prolong treatment sensitivity, and prevent tumor recurrence. Citation Format: Yue Liu, Yibei Zhang, Jonathan L. Tso, Jimmy C. Menjivar, Jane Y. Tian, Linda Liau, William McBride, Cho-Lea Tso. High-throughput RNAi screening identifies genes controlling glioblastoma stem cell migration and survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3765. doi:10.1158/1538-7445.AM2013-3765