MEK2 is a prognostic marker and potential chemo-sensitizing target for glioma patients undergoing temozolomide treatment

Abstract

Although temozolomide (TMZ) is the first-line chemotherapeutic agent for glioblastoma, it is often non-curative due to drug resistance. To overcome the resistance of glioblastoma cells to TMZ, it is imperative to identify prognostic markers for outcome prediction and to develop chemo-sensitizing agents. Here, the gene expression profiles of TMZ-resistant and TMZ-sensitive samples were compared by microarray analysis, and mitogen-activated protein kinase kinase 2 (MEK2) was upregulated specifically in resistant glioma cells but not in sensitive tumor cells or non-tumor tissues. Moreover, a comprehensive analysis of patient data revealed that the increased level of MEK2 expression correlated well with the advancement of glioma grade and worse prognosis in response to TMZ treatment. Furthermore, reducing the level of MEK2 in U251 glioma cell lines or xenografted glioma models through shRNA-mediated gene knockdown inhibited cell proliferation and enhanced the sensitivity of cells toward TMZ treatment. Further analysis of tumor samples from glioma patients by real-time PCR indicated that an increased MEK2 expression level was closely associated with the activation of many drug resistance genes. Finally, these resistance genes were downregulated after MEK2 was silenced in vitro, suggesting that the mechanism of MEK2-induced chemo-resistance could be mediated by the transcriptional activation of these resistance genes. Collectively, our data indicated that the expression level of MEK2 could serve as a prognostic marker for glioma chemotherapy and that MEK2 antagonists can be used as chemo-sensitizers to enhance the treatment efficacy of TMZ.

Figure 1

MEK2 expression in glioma tissues. (a) The hierarchical cluster of differentially expressed probes in three temozolomide-sensitive (TS) and seven temozolomide-resistant (TR) patients based on cDNA microarray analysis (left). The detailed MEK2 probe (MAPK2K) expression pattern was enlarged (right). (b) The statistical results of MEK2 mRNA expression measured by real-time PCR in the normal brain (Normal, n = 8) and glioma tissues (Tumor, n = 26). **P < 0.01 vs. Normal. (c) The representative results and statistical analysis of MEK2 protein expression measured by immunoblot in the normal brain (Normal, n = 8) and glioma tissues (Tumor, n = 26). **P < 0.01 vs. Normal. (d) The representative images of MEK2 immunostaining in normal brain and glioma tissues (×400). All results are expressed as the mean ± SD from at least three independent experiments.

Figure 2

The association of MEK2 expression and clinical outcome in glioma. (a) The representative images of MEK2 immunostaining in the low-grade (left, a case of WHO-I grade) and high-grade (right, a case of WHO-IV grade) glioma tissues (×400). (b) The statistical results of MEK2 mRNA expression measured by real-time PCR in normal brain (Normal, n = 8), low-grade glioma tissues (Low Grade, n = 13) and high-grade glioma tissues (High Grade, n = 13). **P < 0.01 vs. Normal, #P < 0.01 vs. Low Grade. (c) The statistical results of MEK2 protein expression measured by immunoblot in normal brain (Normal, n = 8), low-grade glioma tissues (Low Grade, n = 13) and high-grade glioma tissues (High Grade, n = 13). **P < 0.01 vs. Normal, #P < 0.01 vs. Low Grade. (d) Kaplan–Meier curve of overall survival in 87 Grade IV patients with low (blue curve) and high (green curve) MEK2 expression. (e) Kaplan–Meier curve of overall survival in 50 Grade IV patients receiving temozolomide treatment with low (blue curve) and high (green curve) MEK2 expression. All results are expressed as the mean ± SD from at least three independent experiments.

Figure 3

The function of MEK2 in glioma cell growth. (a) The immunoblotting results of protein lysates extracted from U251 and U87 cells with MEK2 silenced (si-MEK2) or its control group (si-ctl). (b) The growth curve of U251 cells with MEK2 silenced (si-MEK2) or its control group (si-ctl) measured by MTT assay. **P < 0.01 vs. si-ctl at the same time point. (c) The growth curve of U87 cells with MEK2 silenced (si-MEK2) and its control group (si-ctl) measured by MTT assay. **P < 0.01 vs. si-ctl at the same time point. (d) The apoptotic rates of U251 and U87 cells with MEK2 silenced (si-MEK2) or its control group (si-ctl) measured by Annexin V staining. (e) The cell cycle profile of U87 cells with MEK2 silenced (si-MEK2) and its control group (si-ctl). (f) The percentages of cells at G1, S, and G2/M phases in U251 and U87 cells with MEK2 silenced (si-MEK2) or its control group (si-ctl). All results are expressed as the mean ± SD from at least three independent experiments.

Figure 4

The effect of MEK2 expression on the temozolomide sensitivity of glioma cells. The representative plots (a) and statistical results (b) of the apoptotic rates of U251 and U87 cells with MEK2 silenced (si-MEK2) or its control group (si-ctl) treated with 100 μM temozolomide (TMZ) for 48 hrs as measured by Annexin V staining. **P < 0.01, *P < 0.05 vs si-ctl. (c) The inhibition curve of U251 with MEK2 silenced (si-MEK2) or its control group (si-ctl) treated with 1, 10, 25, 50, 100, 200, 400, or 800 μM TMZ and measured by MTT assay. The representative plots (d) and statistical results of the apoptotic rates (e) of U251 and U87 cells with MEK2 silenced (si-MEK2) or its control group (si-ctl) treated with 100 μM TMZ and MEK inhibitors (GSK: GSK1120212 at 10 nM, PD: PD184352 at 50 nM) for 48 hrs as measured by Annexin V staining. **P < 0.01, *P < 0.05 vs. DMSO + TMZ group; ##P < 0.01 vs. DMSO within each treatment. All results are expressed as the mean ± SD from at least three independent experiments.

Figure 5

The downstream genes of MEK2 in regulation of temozolomide sensitivity. ABCB1, ABCC1, ABCC2, ABCG2, BCL2, or MGMT mRNA expression levels in U251 (a) and U87 (b) cells with MEK2 silenced (si-MEK2) or its control group (si-ctl) as measured by real-time PCR.**P < 0.01 vs. si-ctl. The correlation coefficients of MEK2 mRNA expression and ABCG2 (c) or MGMT (d) mRNA expression in 34 human tissues (including normal brain and glioma tissues) as measured by real-time PCR. All results are expressed as the mean ± SD from at least three independent experiments.

Figure 6

Effect of the Inhibition of MEK2 on chemotherapy sensitivity to TMZ in vivo. (a) Nude mice were intracranially transplanted with 5 × 10⁵ glioma cells infected with Si-MEK2 or Si-ctl (control). The survival curve was plotted by Kaplan–Meier analysis. Mice-bearing gliomas from Si-MEK2-infected U87 cells treated with TMZ survived longer than those in the control group (n = 8 per group). Each curve was compared using the two-sided log-rank test. P < 0.001, Si-MEK2 vs. Si-ctl. The number of mice at risk at each time point is also shown. (b) Representative tumor images were obtained by MRI 1 or 3 weeks after glioma transplantation. (c) Tumor size was quantified and analyzed. (d) In the subcutaneous model, the tumor volumes in Si-MEK2- and Si-ctl-infected groups treated with TMZ (n = 8 per group) were determined every 5 days for 30 days after implantation of U87 cells. The means and upper 95% confidence intervals are shown, Si-MEK2 vs. Si-ctl. P values were calculated using the two-sided student's t-test. (E) The volume of xenografted tumors on day 30 of implantation. Error bars represent the interquartile range. *P = 0.013, Si-ctl vs. Si-MEK2, P values were calculated using the two-sided student's t-test.