Pharmacologic screens reveal metformin that suppresses GRP78-dependent autophagy to enhance the anti-myeloma effect of bortezomib

Abstract

Although the therapeutic benefit of proteasome inhibition in multiple myeloma remains unchallenged, drug resistance inevitably emerges through mechanisms that remain elusive. Bortezomib provokes unwanted protein accumulation and the endoplasmic reticulum stress to activate the unfolded protein response (UPR) and autophagy as compensatory mechanisms that restore protein homeostasis. High-throughput screens to detect pharmacologics that modulated autophagy to enhance the anti-myeloma effect of bortezomib revealed metformin, a widely used antidiabetic agent with proven efficacy and limited adverse effects. Metformin co-treatment with bortezomib suppressed induction of the critical UPR effector glucose-regulated protein 78 (GRP78) to impair autophagosome formation and enhance apoptosis. Gene expression profiling of newly diagnosed myeloma patient tumors further correlated the hyperexpression of GRP78-encoding HSPA5 with reduced clinical response to bortezomib. The effect of bortezomib was enhanced with metformin co-treatment using myeloma patient tumor cells and the chemoresistant, stem cell-like side population that may contribute to disease recurrence. The relevance of the findings was confirmed in vivo as shown by metformin co-treatment with bortezomib that delayed the growth of myeloma xenotransplants. Taken together, our results suggest that metformin suppresses GRP78, a key driver of bortezomib-induced autophagy, and support the pharmacologic repositioning of metformin to enhance the anti-myeloma benefit of bortezomib.

Figures

Figure 1

Effect of proteasome inhibitors on myeloma SP and MP viability, proliferation and induction of apoptosis. (a) Representative flow cytometric dot plots of SP analysis. Dot plots show RPMI8226 cells incubated in Hoechst 33342 alone (left), Hoechst 33342 accumulation in the presence of 100 μM verapamil (middle) and Hoechst 33342 accumulation in the presence of 50 μM reserpine (right). Abscissa is Hoechst red fluorescence intensity and ordinate is Hoechst blue fluorescence intensity with the gate representing the SP fraction. (b) Effect of bortezomib, carfilzomib or ixazomib at indicated concentrations on SP and MP viability. SP and MP were isolated from RPMI8226 cells and viability determined using the XTT assay. Error bars represent s.d. values determined from triplicate measurements. (c) Effect of bortezomib, carfilzomib and ixazomib on SP or MP cells isolated from MM patients. Cells were incubated with drugs as indicated and viability determined using the XTT assay. Error bars represent s.d. values determined from triplicate measurements. (d) Effect of bortezomib on SP and MP cell proliferation. Cells were incubated with bortezomib at indicated concentrations for 8 h and BrdU then added for 2 h. BrdU incorporation was determined relative to untreated MP cells. Error bars represent s.d. values determined from triplicate measurements. (e) Effect of bortezomib on the generation of annexin-positive SP and MP cells. Cells were incubated with bortezomib as indicated for 24 h and the percentage of annexin-positive cells determined relative to untreated SP or MP cells. Shown is the percentage increase in annexin-V-positive cells as determined by flow cytometry. Error bars represent s.d. values from triplicate measurements.

Figure 2

Effect of metformin co-treatment with bortezomib on myeloma SP and MP cell viability, proliferation and induction of apoptosis. (a) Dose-dependent effect of bortezomib alone or combined with metformin (500 μM) on the viability of SP and MP isolated from RPMI8226 cells. SP and MP cells were incubated with bortezomib alone or combined with metformin (500 μM). Cell viability was determined using the XTT assay. Error bars represent s.d. values determined from triplicate measurements. (b) Dose-dependent effect of bortezomib added at the indicated concentrations alone or combined with metformin (500 μM) on myeloma patient's SP and MP cell viability. (c) Dose-dependent effect of bortezomib added at indicated concentrations on colony formation. Values were normalized relative to the number of colonies formed from untreated SP. (d) Dose-dependent effect of bortezomib added at indicated concentrations alone or combined with metformin on colony formation. Values were normalized relative to the number of colonies detected with untreated SP cells. (e) Effect of bortezomib on SP and MP cell proliferation. Cells were incubated with bortezomib for 8 h at indicated concentrations and BrdU then added for 2 h. BrdU incorporation was determined relative to untreated MP cells. Error bars represent s.d. values determined from triplicate measurements. (f) Effect of bortezomib on the percentage of annexin-positive SP and MP cells. Cells were incubated with bortezomib at indicated concentrations for 24 h. The relative percentage increase in annexin-positive cells was determined relative to untreated SP or MP cells. Shown is the percentage increase in annexin-positive cells determined by flow cytometry. Error bars represent s.d. values determined from triplicate measurements.

Figure 3

Effect of metformin on GRP78 and bortezomib-induced autophagy. (a) Western blot to determine the effect of bortezomib treatment on GRP78 levels in RPMI8226 cell lysates. Cells were treated with bortezomib at indicated concentrations for 24 h, lysates prepared and probed using a GRP78-specific antibody. (b) Western blot to determine the effect of metformin co-treatment with bortezomib on GRP78 levels in RPMI8226 cells. Cells were treated with bortezomib and metformin at indicated concentrations for 24 h; lysates prepared and probed using an antibody specific to GRP78. (c) Western blot to determine the effect of metformin co-treatment with bortezomib on GRP78 levels in SP and MP cells isolated from RPMI8226. Cells were treated with bortezomib and metformin at indicated concentrations for 24 h; lysates prepared and probed using an antibody specific to GRP78. (d) Effect of metformin co-treatment with bortezomib on the conversion of LC3B-I to LC3B-II in RPMI8226 cells. Cells were treated with bortezomib (5 nM), metformin (500 μM) for 24 h. Bafilomycin A1 (100 nM), a specific inhibitor of the vacuolar type H+-ATPase was then added for the final 4 h of incubation to inhibit autophagy flux and to prevent autophagosome recycling and lead to LC3B-II accumulation. Lysates were prepared and probed by western blot to detect the relative levels of LC3B-I and LC3B-II. (e) Effect of bortezomib (5 nM) and metformin (500 μM) alone and combined on the levels of GRP78 and autophagosomes in RPMI8226 cells. Cells were treated with drugs for 16 h and GRP78 was then visualized by immunohistochemistry staining and confocal microscopy. Autophagosomes were detected using the cytologic identification (cyto-ID) autophagy detection kit (Enzo Life Sciences, Farmingdale, NY, USA) and visualized using a Zeiss LSM710 confocal microscope (Zeiss Microscopy, Thornwood, NY, USA). Settings for fluorescein isothiocyanate detection were EX488nm and EM550nm. (f) Effect of HSPA5-specific shRNA on autophagosome formation. RPMI8226 cells were transfected with control or HSPA5-specific shRNA and transfectants treated as indicated. Autophagosomes were detected by dye-based staining and confocal microscopy. (g) Autophagosomes were detected by the dye-based method and the relative amount of green fluorescence was determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Statistically significant differences in fluorescence are indicated by *. (h) Effect of bortezomib (5 nM) and metformin (500 μM) alone and combined on autophagosome formation in SP cells. SP were treated with drugs as indicated for 16 h and autophagosome formation visualized by dye-based staining and confocal microscopy. Shown are representative images observed in multiple experiments.

Figure 4

Effect of bortezomib and metformin on tumor volume (TV) and overall survival. MM.1S cells (3 × 106/injection) were subcutaneously placed in the flanks of 5-week-old nude NCr nu/nu female mice. Mice were then randomly distributed into four groups (10/group) and after the formation of palpable tumors treatment was commenced. Groups received intravenous injection of either vehicle (phosphate-buffered saline containing 10% dimethyl sulfoxide (DMSO)) or bortezomib (0.5 mg/kg in phosphate-buffered saline, 10% DMSO) administered with or without metformin (600 μg/ml diluted in drinking water that contained 5% dextrose). Metformin was administered throughout treatment and vehicle or bortezomib administered on days 1–4. TVmeasurements were made using a vernier caliper and calculated using the formula: TV=0.5(a × b2) where a is the long diameter and b the short diameter. Mice were euthanized when tumors reached 2 cm3, became ulcerated or elicited neurologic or musculoskeletal complications that limited mobility and feeding. TV was evaluated from the first day of treatment. Shown is the average of replicate measurements. (b) Kaplan–Meier survival curves after treated with either vehicle, metformin, bortezomib or both.