Aldehyde dehydrogenase 1A1--a new mediator of resistance to temozolomide in glioblastoma

Abstract

Implementation of chemotherapy with the drug temozolomide increased the overall survival of patients with glioblastoma multiforme (GBM; WHO grade IV), in particular when the O(6)-methylguanine DNA methyltransferase (MGMT) promoter is epigenetically silenced. Nevertheless, the prognosis remains poor, and relapse in GBM occurs regularly. This clinical behavior seems to be due to the existence of a therapy-resistant subpopulation of cells that induce tumor regrowth. The objective of this work was to analyze the role of aldehyde dehydrogenase (ALDH) 1A1 in mediating temozolomide resistance and its value as a predictor of clinical outcome in GBM patients. Nine GBM cell lines were treated with temozolomide alone or in combination with 4-diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH1A1, or with ALDH1A1 short hairpin (sh)RNA. ALDH1A1 expression and MGMT status of 70 primary GBM patients were correlated with median survival. ALDH1A1 overexpression predicted temozolomide resistance in vitro. Sensitivity of ALDH1A1 positive/MGMT-positive cells to temozolomide could be restored by inhibition of ALDH1A1 by DEAB or by knockdown with shRNA, as indicated by increased cytotoxicity, reduced clonogenicity, and accumulation in the G2/M cell-cycle phase. The prognosis of patients with a high level of ALDH1A1 expression was poor compared with that of patients with low levels (P < .0001). ALDH1A1 is a new mediator for resistance of GBM to temozolomide and a reliable predictor of clinical outcome and may serve as a potential target to improve treatment of human GBM.

Fig. 1.

ALDH1A1 expression, MGMT status, and response to treatment with TMZ. (A) Western blot analysis showed a high level of ALDH1A1 in LN18, T30, T39, T40, T98G, and R28 cells. The cell lines LN229, G139, and T16 were negative for ALDH1A1. MGMT protein expression was detected in LN18, T98G, T39, T40, and R28 cells. (B) No relevant MGMT promoter methylation was found in LN18, T98G, G139, T39, T40, or R28 (MGMT⁺). The MGMT promoter of T16, T30, and LN229 was methylated to 60%, 30%, and 100%, respectively (MGMT^–).The MGMT promoter of G139 cells was completely unmethylated, but no expression of MGMT protein was detected (MGMT^–*). (C) Response of GBM cell lines to treatment with TMZ for 7 days was assessed by metabolic MTT assay.

Fig. 2.

Concomitant treatment with TMZ and DEAB reduced the clonogenic capacity of GBM cell lines. (A) Colony formation assay demonstrates that the clonogenic capacity of LN18 exposed to 200 µM TMZ in combination with 100 µM DEAB for 10 days was significantly attenuated. (B) TMZ in combination with ALDH1A1 inhibition impaired sphere formation. Neurospheres of LN18, T98G, and R28 cells decreased in size and number after TMZ and DEAB administration; **P < .01, ***P < .001; mean values are shown above the box plots. Scale bar = 100 µM.

Fig. 3.

Knockdown or inhibition of ALDH1A1 restored sensitivity to TMZ. (A) Downregulation of ALDH1A1 at the protein level was verified by Western blot analysis. MGMT protein expression was stable upon shRNA transduction. (B) Metabolic MTT assay revealed an increased cytotoxicity of TMZ when combined with ALDH1A1 inhibition or knockdown. LN18 and T98G shALDH1A1 cells were sensitive to TMZ compared with WT and mock transfected control cells, especially when medium doses of TMZ were applied. (C) Treatment of LN18 shALDH1A1 cells with 200 µM TMZ for 7 days led to accumulation in G2/M. (D) MGMT protein expression was not affected by treatment with TMZ, DEAB, or TMZ + DEAB for 7 days. (E) Colony formation assay showed a significant reduction of the clonogenic capacity of LN18 WT cells treated twice with TMZ + DEAB (striped bar) or LN18 shALDH1A1 cells treated twice with TMZ alone (black bar).

Fig. 4.

ALDH1A1^– GBM cell lines showed prolonged G2/M arrest following treatment with TMZ. (A) Representative flow cytometry histograms of LN18 cells after treatment with 500 µM TMZ, 300 µM DEAB, or a combination of both for 48 h; y–axes: DNA content, x-axes: FL3-H. (B) Column graph shows the percentage of cells in G2/M after treatment with 500 µM (LN18, T98G) or 200 µM TMZ (LN229, T40, R28) and 300 µM DEAB for 48 h or 72 h in DMEM + FBS. R28 cells were also analyzed after treatment with 200 µM TMZ and 100 µM DEAB in neurobasal medium (R28, B27); mean values of 3 independent experiments are shown; *P < .05, **P < .01, ***P < .001. (C) Western blot analysis of LNZ308 WT and ALDH1A1 transfected (ALDH1A1) cells. No MGMT protein expression was detected. (D) Cell-cycle analysis of ALDH1A1^–/MGMT^– (LNZ308 WT, LN229) and ALDH1A1⁺/MGMT^– cells (LNZ308 ALDH1A1) following exposure to 50–500 µM TMZ for 48 h.

Fig. 5.

ALDH1A1 expression was found to correlate with an adverse prognosis in human GBM. (A) The percentage of ALDH1A1⁺ cells in human GBM tissue was analyzed by IHC staining. FFPE sections from primary tumors with a low percentage of ALDH1A1⁺ cells (≤10%) (A) and with a high level of ALDH1A1 expression (>10%) (B) are shown; tissue section from relapsed GBM demonstrated a very high percentage of ALDH1A1⁺ cells (> 40%) (C). (B–E) Kaplan–Meier plot of patient median survival; patients with tumors with a high level of ALDH1A1 expression (blue curve) had significantly shorter median survival than did patients with low-level tumors (green curve) (P < .0001). Poor clinical outcome of patients with ALDH1A1-high tumors was found in the group of patients with and without MGMT promoter methylation (P = .005 and P = .0004). (F) The median percentage of ALDH1A1⁺ cells in relapsed GBM was significantly higher than in the respective primary tumors (P = .002).