Mitochondrial Hsp90s suppress calcium-mediated stress signals propagating from mitochondria to the ER in cancer cells

Abstract

Background: Resistance to cell death in the presence of stressful stimuli is one of the hallmarks of cancer cells acquired during multistep tumorigenesis, and knowledge of the molecular mechanism of stress adaptation can be exploited to develop cancer-selective therapeutics. Mitochondria and the endoplasmic reticulum (ER) are physically interconnected organelles that can sense and exchange various stress signals. Although there have been many studies on stress propagation from the ER to mitochondria, reverse stress signals originating from mitochondria have not been well reported.

Methods: After inactivation of the proteins by pharmacologic and genetic methods, the signal pathways were analyzed by fluorescence microscopy, flow cytometry, MTT assay, and western blotting. A mouse xenograft model was used to examine synergistic anticancer activity and the action mechanism of drugs in vivo.

Results: We show in this study that mitochondrial heat shock protein 90 (Hsp90) suppresses mitochondria-initiated calcium-mediated stress signals propagating into the ER in cancer cells. Mitochondrial Hsp90 inhibition triggers the calcium signal by opening the mitochondrial permeability transition pore and, in turn, the ER ryanodine receptor, via calcium-induced calcium release. Subsequent depletion of ER calcium activates unfolded protein responses in the ER lumen, thereby increasing the expression of a pro-apoptotic transcription factor, CEBP homologous protein (CHOP). Combined treatment with the ER stressor thapsigargin and the mitochondrial Hsp90 inhibitor gamitrinib augmented interorganelle stress signaling by elevating CHOP expression, and showed synergistic cytotoxic activity exclusively in cancer cells in vitro and in vivo.

Conclusions: Collectively, mitochondrial Hsp90s confer cell death resistance to cancer cells by suppressing the mitochondria-initiated calcium-mediated interorganelle stress response.

Figure 1

Mitochondrial Hsp90s modulate the mitochondrial calcium store. (A) Time course of cytosolic calcium increase. The ratio of the emission fluorescence intensities at 340 and 380 nm excitation of Fura-2 labeled HeLa cells in calcium-free medium was measured after 30 μM gamitrinib treatment as described in Materials and Methods. (B) Increase of cytosolic calcium in 22Rv1 and MDA-MB-231 cells. Fura-2 fluorescence ratio after 30 μM gamitrinib (Gami) treatment for 1 hour was calculated. Data are the mean ± SEM of duplicated experiments and collected from 40 regions of interest (ROIs). (C) Mitochondrial membrane permeabilization. TMRM-loaded HeLa cells were imaged to measure mitochondrial membrane potential depolarization (ΔΨm) (left); alternatively, cytochrome c redistribution was analyzed (right) at the indicated times after 30 μM gamitrinib treatment as previously described [35]. White bar, 20 μm. (D) Caspase activation and cell death induction. After 30 μM gamitrinib treatment, HeLa cells were labeled with FITC-DEVD-fmk (left, DEVDase activity) or propidium iodide (right, PI staining) and analyzed by flow cytometry at the selected time points. (E) Cyclosporin A (CsA) blocks cytosolic calcium increase. Cytosolic calcium changes in Fura-2-labeled HeLa cells treated for 1 hour with 5 μM CsA and/or 30 μM gamitrinib were analyzed. Bar, 50 μm. (F) Summary of sequential events following mitochondrial Hsp90 inhibition. PTP opening is directly linked with the loss of ΔΨm and increase of cytosolic calcium. The calcium flux occurs prior to mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release. *, p < 0.0001.

Figure 2

Inhibition of mitochondrial Hsp90s depletes stored calcium in both mitochondria and the ER. (A) Mitochondrial calcium depletion. After 30 μM gamitrinib and 10 μM FCCP treatment, confocal FRET images of mtCameleon-expressing HeLa cells were reconstructed from their emission fluorescence ratios at 535/480 nm with excitation at 440 nm (left). FRET ratios at the indicated time intervals were averaged and plotted (right). (B) ER calcium depletion. FRET images of HeLa cells transiently expressing D1ER were acquired at the indicated time points after gamitrinib treatment (left) and analyzed to plot the FRET ratio (right). Selected ROIs are indicated as white circles. Bar, 10 μm. Data in (A) and (B) are mean ± SEM collected from 30 ROIs. R.U., relative units. (C) CHOP induction and eIF2α phosphorylation. HeLa cells were treated with 30 μM gamitrinib, 5 μM CsA, and 10 μM BAPTA as indicated and analyzed by western blotting. #, not significant; *, p < 0.001; **, p < 0.0001.

Figure 3

Ryanodine receptor (RyR)-mediated cytosolic calcium elevation. (A) IP₃R silencing. After IP₃R siRNA treatment, Fura-2 labeled HeLa cells were treated with 30 μM gamitrinib for 1 hour. The fluorescence ratio (340/380) was plotted. The data are mean ± SEM collected from 30 ROIs in two independent experiments. (B) IP₃R knockdown effect on CHOP expression. Control or IP₃R1 siRNA-transfected HeLa cells were incubated with or without 30 μM gamitrinib for 2 hours. Cell extracts were analyzed by western blotting. (C) RyR inhibitors compromise cytosolic calcium increase. Fura-2 labeled HeLa cells were treated with 30 μM gamitrinib for an hour in the presence or absence of 300 μM tetracaine, 100 μM ryanodine, and 5 μM CsA, and emission fluorescence intensity ratios (340/380 nm excitation) were measured. Data are mean ± SEM calculated from 40 ROIs in two independent experiments. (D) Fura-2 imaging and RyR2 silencing. Control or RyR2-#2 siRNA-treated HeLa cells were labeled with Fura-2 and imaged after 30 μM gamitrinib treatment for an hour (left). The fluorescence ratio (340/380) was plotted (middle). Knockdown efficiency of RyR2-#2 siRNA by western blotting (right). The data are mean ± SEM collected from 30 ROIs in two independent experiments. Bar, 50 μm. (E) Inhibition of CHOP induction by RyR inactivation. HeLa cells were treated with 30 μM gamitrinib in the presence or absence of 100 μM ryanodine. Cell extracts were analyzed by western blotting. (F) RyR2 silencing and CHOP expression. HeLa cells were treated with two different RyR2 siRNAs, incubated with 30 μM gamitrinib, for 2 hours and analyzed by western blotting. #, not significant; *, p < 0.0001.

Figure 4

Inhibition of mitochondrial Hsp90s sensitizes HeLa cells toward thapsigargin. (A) Cytoplasmic calcium and mitochondrial membrane potential by suboptimal dose of gamitrinib. Fluo-4 or TMRM/MitoTracker-labeled HeLa cells were incubated with 5 μM gamitrinib for 24 hours and analyzed by confocal microscope. Bar, 20 μm. (B) Combination effect in HeLa. HeLa cells were treated with various concentrations of Thap in the presence of 5 μM of either 17AAG or gamitrinib, and analyzed by MTT assay (left). Alternatively, HeLa cells were treated with 5 μM gamitrinib and/or 0.06 μM Thap for 24 hours and analyzed by the MTT assay. ***, p < 0.0001. (C) Combination effect in 22Rv1. 22Rv1 cells were treated with various concentrations of thapsigargin in the presence of 2.5 μM of either 17AAG or gamitrinib for 24 hours, and analyzed by the MTT assay (left). Alternatively, 22Rv1 cells were treated with 2.5 μM gamitrinib (Gami) and 0.06 μM Thap as indicated for 24 hours and analyzed by the MTT assay. **, p = 0.0006. (D) Combination treatment induces apoptosis. HeLa cells were treated with 10 μM gamitrinib and 0.5 μM Thap as indicated, labeled with FITC-DEVD-fmk and propidium iodide, and analyzed by flow cytometry. (B) and (C) represent mean ± SEM from three independent experiments.

Figure 5

Gamitrinib and Thap combination treatment elevates CHOP expression. (A) Synergistic increase in CHOP induction. 22Rv1 and H460 cells were treated with 5 μM gamitrinib and 0.06 μM Thap as indicated and analyzed by western blotting. (B) CHOP reporter assay. PC3 cells stably transfected with a CHOP::GFP reporter plasmid [50] were incubated with 2.5 μM gamitrinib and/or 0.02 μM Thap as indicated and analyzed as described in Materials and Methods (left). Cells with more than twice the background fluorescence intensity were considered as positive cells (right). Bar, 100 μm. Mean ± SEM. **, p = 0.0036. (C) Silencing CHOP expression. Control or CHOP siRNA-transfected HeLa cells were treated with 0.06 μM Thap and 5 μM gamitrinib for 24 hours, and analyzed by MTT assay. Knockdown efficiency analyzed by western blotting (bottom right). Mean ± SEM. *, p < 0.05. (D) Silencing RyR expression. Control or RyR siRNA-treated HeLa cells were incubated with 0.06 μM Thap and 5 μM gamitrinib for 24 hours, and analyzed by MTT assay. Knockdown efficiency analyzed by western blotting (bottom right). Mean ± SEM. *, p < 0.05. (E) Knockdown of RyR2 by siRNA. Control or RyR2 siRNA-transfected 22Rv1 cells were incubated with 2.5 μM gamitrinib and/or 0.06 μM Thap as indicated and analyzed by western blotting.

Figure 6

Synergistic cancer-specific cytotoxicity in vivo. (A) CHOP induction in astrocytes. Astrocytes treated with 30 μM gamitrinib for 2 hours were analyzed by western blotting. (B) Thap in combination with gamitrinib. Astrocytes were treated with various concentrations of Thap in the presence of 0, 2.5, 5, or 10 μM gamitrinib and the cell viability was analyzed by MTT assay. Data are from three independent experiments. (C) TRAP1 expression in astrocytes. TRAP1 and cyclophilin D (Cyp-D) expression in astrocytes isolated from mouse brain was compared with cancer cell lines by western blotting. (D) Tumor xenograft experiment. Subcutaneous 22Rv1 xenografts were established as described in Materials and Methods. At the end of the experiment, final tumor volumes were plotted (right). We used five mice per group and two tumors per animal. (E) Analysis of CHOP expression in liver and tumor. Liver and tumor samples from three randomly selected mice for each treatment (total 12 mice) were analyzed by western blotting (left). After normalization of CHOP band intensities with β-actin, relative CHOP intensities were calculated (right). (F) Schematic diagram of the mitochondria-initiated stress signal. Chemical inhibitors are indicated in red. (B), (D), and (E) are the mean ± SEM. ***, p = 0.0003; *, p = 0.039; #, p > 0.1.