Abstract
Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with an estimated 5-year survival of less 10%. The current standard of care involves maximal tumor resection followed by radiation and chemotherapy with the alkylating agent temozolomide (TMZ). Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) gene predicts response to TMZ therapy, but does not explain all of the heterogeneity in responses observed in the clinic. The establishment of additional molecular biomarkers, is therefore of significant interest.
The aim of the present study was to analyze the impact of endogenous EGFRvIII expression on sensitivity of cell cultures and tumor xenografts to TMZ and to investigate the mechanisms underlying differential responses.
Approximately 20–30% of GBM show expression of the epidermal growth factor receptor (EGFR) variant III (EGFRvIII), which is associated with EGFR gene amplification. EGFRvIII lacks the exons 2–7, leading to a constitutively activated receptor. Most mechanistic studies have been performed using transfected cell lines, therefore artificially overexpressing EGFRvIII. We have recently reported the establishment of an isogenic cell system with either a low (EGFRvIII-) or a high (EGFRvIII+) fraction of cells with endogenous EGFRvIII expression. Using isogenic pairs of MGMT methylated DKMG and BS153 cells we observed that expression of EGFRvIII was associated with a significant increase in TMZ sensitivity in both cell cultures and orthotopic xenografts in mice. Knockdown of EGFRvIII restored TMZ resistance confirming that EGFRvIII expression is responsible for increased TMZ sensitivity. Since both cell lines do not express MGMT, TMZ sensitivity is predominantly determined by functional mismatch repair (MMR). In line with this, we found that the expression of EGFRvIII was associated with upregulated expression of MMR proteins in both cultured cells and in BS153 tumor xenografts. Furthermore, even a modest decrease in MSH6 and MLH1 expression using siRNA had a profound effect on the response of EGFRvIII expressing cells to TMZ, clearly demonstrating that TMZ sensitivity in EGFRvIII expressing cells is determined by MMR protein expression.
In summary, we have clearly shown that MGMT methylated GBM with endogenous EGFRvIII expression are significantly more sensitive to TMZ than their isogenic EGFRvIII negative counterparts. Overall, our data highlight a new mechanism, which mediates TMZ sensitivity and suggest that EGFRvIII expression could be a predictive marker for initial therapeutic response to TMZ treatment in MGMT methylated patients.