Histone deacetylase activity mediates acquired resistance towards structurally diverse HSP90 inhibitors

Abstract

Heat shock protein 90 (HSP90) regulates multiple signalling pathways critical for tumour growth. As such, HSP90 inhibitors have been shown to act as effective anticancer agents in preclinical studies but, for a number of reasons, the same effect has not been observed in the clinical trials to date. One potential reason for this may be the presence of de novo or acquired resistance within the tumours. To investigate mechanisms of resistance, we generated resistant cell lines through gradual dose escalation of the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The resultant resistant cell lines maintained their respective levels of resistance (7-240×) in the absence of 17-AAG and were also cross-resistant with other benzoquinone ansamycin HSP90 inhibitors. Expression of members of the histone deacetylase family (HDAC 1, 5, 6) was altered in the resistant cells. To determine whether HDAC activity contributed to resistance, pan-HDAC inhibitors (TSA and LBH589) and the class II HDAC-specific inhibitor SNDX275 were found to resensitize resistant cells towards 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin. Most significantly, resistant cells were also identified as cross-resistant towards structurally distinct HSP90 inhibitors such as radicicol and the second-generation HSP90 inhibitors CCT018159, VER50589 and AUY922. HDAC inhibition also resensitized resistant cells towards these classes of HSP90 inhibitors. In conclusion, we report that prolonged 17-AAG treatment results in acquired resistance of cancer cells towards not just 17-AAG but also to a spectrum of structurally distinct HSP90 inhibitors. This acquired resistance can be inhibited using clinically relevant HDAC inhibitors. This work supports the potential benefit of using HSP90 and HDAC inhibitors in combination within the clinical setting.

Keywords: 17-AAG; HSP90; acquired resistance; cancer; histone deacetylases.

Figure 1

Acquired resistance of MDA‐435 and MDA‐231 cells towards 17‐AAG and 17‐DMAG. Dose–response curves and resistance index (RI = IC ₅₀ ratio relative to parental cell line) for MDA‐435‐ and MDA‐435R‐resistant cells treated for 3 days with 17‐AAG (A) and 17‐DMAG (C). MDA‐231‐ and MDA‐231R‐resistant cells treated for 3 days with 17‐AAG (B) and 17‐DMAG (D). Growth curves, representative of four independent experiments; bars, SD. Columns, mean of four independent experiments; bars, SD. *P < 0.05, **P < 0.01, ***P < 0.001 using Student's t‐test.

Figure 2

Cross‐resistance of MDA‐435R and MDA‐231R cells towards geldanamycin. Dose–response growth and resistance index of MDA‐435R (A) and MDA‐231R (B) treated with geldanamycin determined by SRB assay after 72 h. Curves, representative of three independent experiments; bars, SD. Columns, mean of at least two independent experiments; bars, SD. *P < 0.05, **P < 0.01 and ***P < 0.001 using Student's t‐test.

Figure 3

Inhibition of P‐gp by verapamil does not sensitize resistant cells towards 17‐AAG. Dose–response growth and resistance index of MDA‐435R (A) and MDA‐231R (B) treated with 17‐AAG alone or 17‐AAG with 25 mm of verapamil were determined by SRB assay after three days. Curves, representative of at least two independent experiments; bars, SD. Columns, mean of at least two independent experiments; bars, SD. *P < 0.05, **P < 0.01 and ***P < 0.001 using Student's t‐test.

Figure 4

Molecular effects of HSP90 inhibition by 17‐AAG on HSP90 client proteins in parental and resistant cell lines. MDA‐435 parental and resistant MDA‐435R cells treated with 0.1 μm of 17‐AAG (~ 5 × 17‐AAG IC ₅₀ concentrations of the parental cell line) (A) and with 30 μm of 17‐AAG (~ 5 × 17‐AAG IC ₅₀ concentrations of the resistant cell line) (B). MDA‐231 parental and resistant MDA‐231R cells treated with 5.0 μm of 17‐AAG (~ 5 × 17‐AAG IC ₅₀ concentrations of the parental cell line) (C) and with 50 μm of 17‐AAG (~ 5 × 17‐AAG IC ₅₀ concentrations of the resistant cell line) (D). Total cell lysates were collected at the indicated time‐points and analysed by western blotting.

Figure 5

Altered NQO1 levels, HDAC family member expression and altered acetylation status in 17‐AAG‐resistant cell lines. Analysis of parental and resistant cell lines demonstrated altered expression levels of a number of molecules. Semiquantitative PCR demonstrated that the expression levels of NQO1 in resistant MDA‐435 cells were decreased when compared with parental cells, while no alteration was noted between MDA‐231 and MDA‐231R cell lines (A). Western blot analysis of parental and resistant MDA‐231 total cell lysates examining levels of HDAC family members in the presence and absence of 17‐AAG for a period of 24 h (B). Analysis of acetylated HSP90 by immunoprecipitation of HSP90 and western blot analysis with antiacetylated lysine antibody of total cell lysates of parental and resistant MDA‐231 cells treated with and without 17‐AAG demonstrated increased acetylated HSP90 (C). Analysis of acetylation of Grp94 (D) and Trap1 (E) by immunoprecipitation and western blot analysis of MDA‐231 and MDA‐231R total cell lysates demonstrated no alteration in acetylation status. Acetylated lysine residue was detected by western blotting. Western blot analysis of acetylated histone 3 in parental and resistant MDA‐231 cells treated with and without 17‐AAG demonstrated decreased nuclear acetylation (F).

Figure 6

Inhibition of HDACs resensitizes MDA‐231R‐resistant cells towards 17‐AAG and 17‐DMAG. Dose response and resistance index (RI) of MDA‐231 and MDA‐231R cells treated with the pan‐HDAC inhibitor LBH589 (10 nm) in combination with 17‐AAG (A) or 17‐DMAG (B) for a period of 72 h. MDA‐231 and MDA‐231R cells treated with 17‐AAG (C) in combination with the more specific HDAC inhibitor SNDX275 (1 μm) that targets class I HDACs. Growth curves, representative of four independent experiments; bars, SD. Columns, mean of four independent experiments; bars, SD. *P < 0.05, **P < 0.001 using Student's t‐test.

Figure 7

Inhibition of HDACs resensitizes MDA‐231R‐resistant cells towards structurally unrelated to the BAs and next‐generation HSP90 inhibitors. Dose response and resistance index of cells treated with radicicol (A) or with CCT018159 (B) in combination with LBH589 (10 nm) for a period of 72 h. Growth curves, representative of four independent experiments; bars, SD. Columns, mean of four independent experiments; bars, SD. *P < 0.05, **P < 0.01 using Student's t‐test.