Glioblastoma (GBM) is an aggressive and fatal primary adult brain tumor. Even with surgery, chemotherapy with temozolomide (TMZ), and radiation, tumor re-growth and patient relapse are inevitable. Brain tumor initiating cells (BTICs), a rare subset of GBM cells with stem cell properties, were shown to be both chemo- and radio-resistant. We hypothesize that these treatment-resistant BTICs cause tumor relapse and a subset of neural stem cell genes regulate BTIC self-renewal, driving GBM recurrence.
Using patient-derived primary GBM samples, we designed an in vitro model of tumor recurrence by treating cells with TMZ and radiation. We also adapted the existing treatment protocol for adults with primary GBM for in vivo treatment of immunocompromised mice engrafted with GFP+ GBM cells. Post-chemoradiotherapy, GFP+ cells were recovered from mouse brains and profiled for self-renewal, proliferation and mRNA expression of important stem cell genes. Using in vitro and in vivo gain-of-function/loss-of-function experiments, we investigated the regulatory functions of Bmi1 in primary neural stem & progenitor cells (NSPCs) and GBM tumor formation. To understand the consequences of Bmi1 dysregulation on target gene expression, we performed global RNA-seq profiling on NSPCs and GBMs.
GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy, suggesting the presence of a treatment-refractory BTICs. GFP+ cells extracted from chemoradiotherapy treated human tumor xenografts showed increased self-renewal and elevated BTIC marker expression. Although treated mice responded to therapy with decreased tumor size, we observed tumor relapse post-chemoradiotherapy with increased Bmi1 protein expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis in xenografted mice, highlighting a critical role for Bmi1 in tumor initiation and maintenance. Conversely, over-expressing Bmi1 in NSPCs induced stem cell properties in vitro, but failed to initiate tumor formation in vivo. Using high-throughput sequencing data, we generated a map of signaling pathways dysregulated in GBM that may lead to tumor recurrence.
Our data confirms the existence of a rare treatment-refractory BTICs population that escapes therapy, and drives tumor relapse and recurrence with enhanced self-renewal capacity. Our human BTIC in vitro assays and human-mouse BTIC xenograft model provide fundamental tools to characterize the functional relevance and of key stem cell self-renewal genes in GBM recurrence.
Citation Format: Parvez Vora, Maleeha Qazi, Chitra Venugopal, Minomi Subapanditha, Sujeivan Mahendram, Chirayu Chokshi, Mohini Singh, David Bakhshinyan, Nicole McFarlane, Sheila Singh. Bmi1 identifies treatment-refractory stem cells in human glioblastoma. [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 2512.