Acquired resistance to 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) in glioblastoma cells

Abstract

Heat shock protein 90 (HSP90) inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), which is currently in phase II/phase III clinical trials, are promising new anticancer agents. Here, we explored acquired resistance to HSP90 inhibitors in glioblastoma (GB), a primary brain tumor with poor prognosis. GB cells were exposed continuously to increased 17-AAG concentrations. Four 17-AAG-resistant GB cell lines were generated. High-resistance levels with resistance indices (RI = resistant line IC(50)/parental line IC(50)) of 20 to 137 were obtained rapidly (2-8 weeks). After cessation of 17-AAG exposure, RI decreased and then stabilized. Cross-resistance was found with other ansamycin benzoquinones but not with the structurally unrelated HSP90 inhibitors, radicicol, the purine BIIB021, and the resorcinylic pyrazole/isoxazole amide compounds VER-49009, VER-50589, and NVP-AUY922. An inverse correlation between NAD(P)H/quinone oxidoreductase 1 (NQO1) expression/activity and 17-AAG IC(50) was observed in the resistant lines. The NQO1 inhibitor ES936 abrogated the differential effects of 17-AAG sensitivity between the parental and resistant lines. NQO1 mRNA levels and NQO1 DNA polymorphism analysis indicated different underlying mechanisms: reduced expression and selection of the inactive NQO1*2 polymorphism. Decreased NQO1 expression was also observed in a melanoma line with acquired resistance to 17-AAG. No resistance was generated with VER-50589 and NVP-AUY922. In conclusion, low NQO1 activity is a likely mechanism of acquired resistance to 17-AAG in GB, melanoma, and, possibly, other tumor types. Such resistance can be overcome with novel HSP90 inhibitors.

Figure 1. Acquired in vitro resistance to 17-AAG in GB cell lines

Resistance index (RI = IC₅₀ ratio of resistant / parental line) of (A) SF268-RA12, (B) U87MG-RA6, (C) SF188-RA6, and (D) KNS42-RA4 resistant lines. Resistant cell lines were treated with 17-AAG continuously (drug on) or without drug pressure (drug off), as described in the Material and Methods section. The IC₅₀ values of 17-AAG in the parental and resistant cell lines are presented. Values are mean ± SD of at least 5 independent experiments.

Figure 2. Molecular signature of HSP90 inhibition by 17-AAG in SF268 parental and resistant lines

Western blot of cell lysates from (A) SF268 parental line and (B) SF268-RA6 resistant line treated with 5×IC₅₀ concentrations of 17-AAG of the parental line (60 nM; top) and 5×IC₅₀ of the resistant line (600 nM; bottom). Cells were harvested at 0, 8, 16, 24, 48, and 72 hours post-treatment. GAPDH was used as loading control.

Figure 3. Cross-resistance of 17-AAG resistant GB cell lines with other HSP90 inhibitors and chemotherapeutic agents

(A) SF268-RA12, (B) U87MG-RA6, (C) SF188-RA6, and (D) KNS42-RA4 resistant lines. Values (mean ± SD, n=3) are shown as resistance index (RI = IC₅₀ resistant/IC₅₀ parent line). Significant differences (t student test, P value < 0.05) between parental and resistant lines are indicated by *.

Figure 4. Expression of NQO1, heat shock and DNA repair proteins in the parental and 17-AAG-resistant GB cell lines

Western blots were performed with lysates from (A) SF268 parental and SF268-RA12 resistant line. (B) U87MG parental and U87MG-RA6 resistant line. (C) SF188 parental and SF188-RA6 resistant line. (D) KNS42 parental and KNS42-RA4 resistant line. Three samples from parental and resistant lines were harvested at 80% confluence. The resistant cells were analyzed after continuous 17-AAG pressure (+) and after cessation of 17-AAG pressure (−), as described in the Material and Methods section. The human colon cancer cell line BE vector control (BEneg) which carries the inactivating NQO1*2 polymorphism was used as the negative control (16, 25). NQO1 positive controls were the isogenic counterpart transfected with NQO1 BE-F397 clone 2 (BE2) and the naturally high NQO1-expressing human colon cancer cell line HT29 (16). The human colon cancer cell line HT29 was used as a positive control for MGMT (47), MLH1 and MSH2 (48). The human colon cancer cell line HCT116 was used as a negative control for MLH1 (48) and a positive control for HSP27 (49). The parental human ovarian cancer cell line CH1 and the doxorubicin-resistant cell line CH1DoxR were negative and positive controls for PgP expression (50), which was not detected in any of the GB lines (data not shown).

Figure 5. Inverse correlation between 17-AAG sensitivity and NQO1 enzyme activity in the parental and 17-AAG-resistant GB cell lines

The relationship between NQO1 activity and in vitro cellular sensitivity was evaluated in (A) SF268 parental and SF268-RA12 resistant line, (B) U87MG parental and U87MG-RA6 resistant line, (C) SF188 parental and SF188-RA6 resistant line, (D) KNS42 parental and KNS42-RA4 resistant line, where ■ represents the parental line, ● the resistant line under continuous drug pressure and △ the resistant line without drug pressure. The non-parametric Spearman test coefficient (r) and P value are shown. The negative correlation coefficient demonstrated an inverse correlation between NQO1 activity and 17-AAG IC₅₀. P values < 0.05 were considered significant.