A Novel Function of Molecular Chaperone HSP70: SUPPRESSION OF ONCOGENIC FOXM1 AFTER PROTEOTOXIC STRESS

Abstract

The oncogenic transcription factor FOXM1 is overexpressed in the majority of human cancers, and it is a potential target for anticancer therapy. We identified proteasome inhibitors as the first type of drugs that target FOXM1 in cancer cells. Here we found that HSP90 inhibitor PF-4942847 and heat shock also suppress FOXM1. The common effector, which was induced after treatment with proteasome and HSP90 inhibitors or heat shock, was the molecular chaperone HSP70. We show that HSP70 binds to FOXM1 following proteotoxic stress and that HSP70 inhibits FOXM1 DNA-binding ability. Inhibition of FOXM1 transcriptional autoregulation by HSP70 leads to the suppression of FOXM1 protein expression. In addition, HSP70 suppression elevates FOXM1 expression, and simultaneous inhibition of FOXM1 and HSP70 increases the sensitivity of human cancer cells to anticancer drug-induced apoptosis. Overall, we determined the unique and novel mechanism of FOXM1 suppression by proteasome inhibitors.

Keywords: 70-kD heat shock protein (Hsp70); FOXM1; anticancer drug; cell death; gene expression; protein-protein interaction.

FIGURE 1.

Treatment with proteasome and HSP90 inhibitors or heat shock leads to the down-regulation of FOXM1 and the up-regulation of HSP70. A–C, RPMI 8266 multiple myeloma and MDA-MB-231 breast cancer cells were treated with the proteasome inhibitors bortezomib (Bor) or thiostrepton (Thio) as indicated. Twenty-four hours after treatment, cells were harvested, and immunoblotting was carried out with antibodies against FOXM1 and HSP70. β-Actin was used as the loading control. D and E, MIA PaCa-2 pancreatic and MDA-MB-231 breast cancer cells were treated with the indicated concentrations of the HSP90 inhibitor PF-4942847. Cells were harvested 24 h after treatment, and the protein levels of FOXM1 and HSP70 were assessed by immunoblotting. β-Actin was used as the loading control. F, MDA-MB-231 breast cancer cells were subjected to heat shock at 42 °C for the indicated time points. Following treatment, cells were harvested, and immunoblotting was performed for FOXM1, HSP70, and β-actin as the loading control.

FIGURE 2.

Inhibition of HSP70 restores the protein expression of FOXM1. A–D, DU145 and MDA-MB-231 cells were treated with thiostrepton (Thio) in combination with the HSP70 inhibitor 9AA. MDA-MB-231 and U2OS cells were treated with the HSP90 inhibitor PF-4942847 along with the HSP70 inhibitor 9AA or quercetin, respectively. Twenty-four hours after treatment, cell lysates were immunoblotted for FOXM1, HSP70, and β-actin as the loading control. E–G, MIA PaCa-2 and MDA-MB-231 cells were transfected with control and HSP70 siRNAs. Forty-eight hours after transfection, cells were treated with the indicated concentrations of thiostrepton or the HSP90 inhibitor PF-4942847. Immunoblot analysis was performed for FOXM1, HSP70, and β-actin as the loading control.

FIGURE 3.

Simultaneous suppression of FOXM1 and HSP70 sensitizes human cancer cells to programmed cell death. A–C, transient FOXM1 knockdown cells and their control counterparts were treated with thiostrepton (Thio) in the presence or absence of the HSP70 inhibitor 9AA. Twenty-four hours after treatment, cell lysates were immunoblotted for FOXM1, HSP70, cleaved caspase-3, and β-actin as the loading control. D, MDA-MB-231 control and transient FOXM1 knockdown breast cancer cells were treated as indicated for 24 h. Cell death was determined by flow cytometry after propidium iodide staining. The graph shows quantification as the percentage of propidium iodide-positive sub-G₁ cells compared with propidium iodide-positive sub-G₁ control cells ± S.D. of a representative triplicate experiment. ***, p < 0.0001.

FIGURE 4.

HSP70 binds to FOXM1 and negatively regulates FOXM1 by interfering with the DNA-binding ability of FOXM1. A and B, C3 and C3-luc cells were induced with doxycycline (Doxy) and treated with thiostrepton (Thio) for 24 h or subjected to heat shock at 42 °C overnight. After treatment, the cells were lysed and immunoprecipitated with the FOXM1 antibody or control rabbit IgG. Immunoblotting (IB) was performed with anti-FOXM1 and HSP70 antibodies. C, the C3 cell line was transiently transfected with the control enhanced GFP and the HSP70 expression plasmids. Doxycycline was added to the culture medium 48 h after transfection for an additional 24 h. Cells were harvested, and immunoblotting was performed with antibodies against FOXM1 and HSP70. β-actin was used as the loading control. D, the C3 cell line was transiently transfected with the control enhanced GFP (EGFP) and the HSP70 expression plasmids. Doxycycline was added to the culture medium 48 h after transfection for an additional 24 h. Cells were processed for the ChIP experiments as described under “Experimental Procedures.” The graph shows the mean ± S.E. of three independent ChIP experiments. *, p < 0.05. E, during proteotoxic stress, HSP70 expression increases, and it binds to FOXM1 and inhibits FOXM1 binding to its own regulatory elements, disrupting the autoregulation of FOXM1, which leads to the down-regulation of both FOXM1 mRNA and protein expression. Consequently, HSP70 acts as a negative regulator of FOXM1 after proteotoxic stress.