Abstract B68: Differential gene expression associated with temozolomide resistance development in glioblastoma xenografts.
Authors: Youland Ryan S. , Kitange Gaspar J. , Mladek Ann C. , Carlson Brett L. , Schroeder Mark A. , Sarkaria Jann N. 
Published: June 2012

Purpose/Background: The first-line chemotherapeutic temozolomide (TMZ) does not offer any therapeutic benefit in some cases of glioblastoma (GBM) and emergence of resistance leads to tumor progression in almost all patients. While the expression of O-6-methylguanine-DNA-methyltransferase (MGMT) correlates well with TMZ resistance, its expression varies among GBM tumors. The goal of this study is to identify additional genes with potential roles in TMZ resistance.

Materials and Methods: Five primary GBM xenografts were generated from patient samples, and subsequent secondary models for resistance were developed by subjecting tumor-bearing mice to escalating doses of TMZ. Triplicate samples from parental and TMZ-resistant xenografts were analyzed for gene expression using the Illumina BeadChip microarray platform. The top 100 differentially expressed genes for each line were considered candidates. Genes were selected for qRT-PCR validation after ontological and pathway analysis. A lentivirus-mediated shRNA knockdown was performed to validate the role of several candidate genes in TMZ resistance in U87 and primary GBM12 cell lines. Knockdown was confirmed with qRT-PCR and western blots. TMZ responsiveness was assessed using CyQUANT and Neurosphere assays.

Results: Upregulated MGMT expression was found in 3 of the 5 xenografts (GBM12TMZ, GBM14TMZ and GBM28TMZ) and was mechanistically linked with TMZ resistance in these lines using O6-benzylguanine to reverse the resistance phenotype. Differential expression of other DNA damage response genes included CCND2, CDKN1A, GADD45B, UBB as well as apoptosis regulators NFKB2 and BIRC3. In lines with unchanged MGMT expression (GBM22TMZ and GBM39TMZ), changes in anti-apoptosis genes NFKB2 and MAP3K1, transcription factor SRF and signal transduction gene PRKCDBP were validated by qRT-PCR. In GBM12TMZ, in addition to MGMT upregulation, expression of Ubiquitin B (UBB) was found to be the most significantly down-regulated gene both on microarray (Log2FC = −4.55, Fold change = 0.043) and qRT-PCR (Log2RQ =-4.04, RQ = 0.061). To test a possible role of UBB in TMZ resistance, knockdown was achieved in U87 (knockdown efficiency=91.2%) and in primary xenograft line GBM12 (knockdown efficiency=84.4%). The CyQUANT assay for U87UBB-KD and U87NT treated with escalating doses of TMZ revealed no significant difference in cell survival (10 M TMZ relative survival= 89.7% for U87UBB-KD and 79.2% for U87NT; p-value=0.53). Similarly, a neurosphere assay for GBM12UBB-KD and GBM12NT also yielded no significant difference in cell survival (10 M TMZ relative survival= 25.13% for GBM12UBB-KD and 23.35% for GBM12NT; p-value=0.83).

Conclusions: We have used a TMZ resistance xenograft model to identify candidate TMZ resistance genes. The current data provides evidence on the role of MGMT in the development of acquired TMZ resistance while ruling out UBB as potential TMZ resistance gene in GBM. Additional studies are underway to assess the functional significance of other candidate genes, particularly those with differential expression in the setting of constant MGMT expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B68.