Glioma-initiating cell elimination by metformin activation of FOXO3 via AMPK

Abstract

Control of the cancer stem/initiating cell population is considered key to realizing the long-term survival of glioblastoma patients. Recently, we demonstrated that FOXO3 activation is sufficient to induce differentiation of glioma-initiating cells having stem-like properties and inhibit their tumor-initiating potential. Here we identified metformin, an antidiabetic agent, as a therapeutic activator of FOXO3. Metformin activated FOXO3 and promoted differentiation of such stem-like glioma-initiating cells into nontumorigenic cells. Furthermore, metformin promoted FOXO3 activation and differentiation via AMP-activated protein kinase (AMPK) activation, which was sensitive to extracellular glucose availability. Importantly, transient, systemic administration of metformin depleted the self-renewing and tumor-initiating cell population within established tumors, inhibited tumor formation by stem-like glioma-initiating cells in the brain, and provided a substantial survival benefit. Our findings demonstrate that targeting glioma-initiating cells via the AMPK-FOXO3 axis is a viable therapeutic strategy against glioblastoma, with metformin being the most clinically relevant drug ever reported for targeting of glioma-initiating cells. Our results also establish a novel, direct link between glucose metabolism and cancer stem/initiating cells.

Figures

Figure 1.

Metformin inhibits self-renewal capacity, induces differentiation, and suppresses tumor-initiating potential of stem-like glioma-initiating cells. (A,B): The indicated stem-like glioma-initiating cells were cultured in the presence of either metformin (1 mM) or vehicle control for 3 days and then subjected to sphere formation assay in the absence of metformin. (A): The number of spheres formed (means ± SD from three independent experiments). (B): Representative photomicrographs of spheres formed by SJ28P3 cells. (C): The indicated cells cultured in the presence of either metformin (1 mM) or vehicle control for 3 days (or 6 days where indicated) were subjected to immunoblot analysis of neural stem cell/progenitor (Nestin, Musashi, and Bmi1) and differentiation (GFAP and βIII-tubulin) marker expression. (D,E): SJ28P3 cells cultured in the presence of either metformin (1 mM) or vehicle control for 3 or 6 days were subjected to immunofluorescence analysis for differentiation marker expression. (D): Representative fluorescence images of GFAP and βIII-tubulin expression (green). Nuclei were counterstained with Hoechst 33342 (blue). (E): The proportion of GFAP (left)- and βIII-tubulin (right)-positive cells was determined. The data represent means ± SD from three independent experiments. (F,G): SJ28P3 cells (1 × 104) cultured in the presence of either metformin (1 mM) or vehicle control for 3 days were implanted intracranially into the brain of nude mice. (F): Representative hematoxylin and eosin staining of brain sections from mice sacrificed 30 days after implantation. The lower panels show magnified views of the boxed areas in the corresponding upper panels. The arrowheads in the upper panels indicate tumor areas. (G): Kaplan-Meier plot showing survival of mice (five mice per group) after intracranial implantation. Scale bars = 200 μm (B), 100 μm (D), 2 mm ([F], top), and 200 μm ([F], bottom). *, p < .05. Abbreviations: Cont., control; GFAP, glial fibrillary acidic protein; Met, metformin.

Figure 2.

AMPK activation by metformin is sensitive to extracellular glucose concentration and is closely correlated with the effects of metformin on stem-like glioma-initiating cells. (A): SJ28P3 cells cultured in the presence of the indicated concentrations of metformin for 3 days under the conventional stem cell culture condition (glucose concentration, 26.2 mM) were subjected to immunoblot analysis of the indicated proteins. (B–H): SJ28P3 cells were cultured with or without metformin (1 mM) for 3 days at the indicated glucose concentrations. The cells were then subjected to immunoblot analysis of the whole (B,D) and fractionated (C) cell lysates, to immunofluorescence analysis (E, F), to observation under a phase contrast microscope (G), or to sphere formation assay (H). Note that the exposure time for each protein is different from that of the corresponding protein in Figure 1C. The data in (F) and (H) represent means ± SD from three independent experiments. Scale bars = 100 μm (E, F) and 50 μm (G). *, p < .05. Abbreviations: ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; GFAP, glial fibrillary acidic protein; n.s., not statistically significant; PARP, poly(ADP-ribose) polymerase.

Figure 3.

Pivotal role for the AMPK-FOXO3 axis in metformin-promoted differentiation of stem-like glioma-initiating cells. (A–D): SJ28P3 cells cultured at the indicated glucose concentrations were transfected with a control small interfering RNA (siRNA) (Cont.) or with siRNAs against AMPKα (AMPK) and treated, 10 hours after transfection, with or without metformin (1 mM) for 3 days. The cells were then subjected to immunoblot analysis of the whole (A) and fractionated (B) cell lysates or to immunofluorescence analysis (C,D). Representative fluorescence images (C) and the proportion of cells positive for the indicated markers (D) are shown. (E–H): SJ28P3 cells cultured at the indicated glucose concentrations were transfected with a control siRNA (Cont.) or with an siRNA against FOXO3 (F3) and treated, 10 hours after transfection, with or without metformin (1 mM) for 3 days. The cells were then subjected to immunoblot (E,H) or to immunofluorescence analysis (F,G). Representative fluorescence images (F) and the proportion of cells positive for the indicated markers (G) are shown. The data in (D) and (G) represent means ± SD from three independent experiments. Scale bars = 100 μm. *, p < .05. Abbreviations: ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; Cont., control; F3, FOXO3; GFAP, glial fibrillary acidic protein; PARP, poly(ADP-ribose) polymerase; siRNA, small interfering RNA.

Figure 4.

AMPK activation by AICAR is sufficient to activate FOXO3, inhibit self-renewal capacity, and induce differentiation of stem-like glioma-initiating cells. (A): SJ28P3 cells cultured in the presence or absence of AICAR (1 mM) for 3 days under the conventional stem cell culture condition (glucose concentration, 26.2 mM) were subjected to immunoblot analysis for the indicated protein expression. (B,C): SJ28P3 cells transfected with a control siRNA (Cont.) or with siRNAs against AMPKα (AMPK) were treated, 10 hours after transfection, with or without AICAR (1 mM) for 3 days. The cells were then subjected to immunoblot analyses of the fractionated (B) and whole (C) cell lysates. (D): SJ28P3 cells treated with or without AICAR (1 mM) in the presence or absence of compound C (10 μM) for 3 days were subjected to immunoblot analysis of the indicated proteins. (E–H): SJ28P3 cells cultured in the presence or absence of AICAR (1 mM) for 3 days were subjected to sphere formation assay (E,F), immunoblot analysis (G), and immunofluorescence staining (H). Representative photomicrographs of the spheres formed in the primary sphere formation assay (E) and the number of spheres formed (F) are shown. The data in (F) represent means ± SD from three independent experiments. Scale bars = 200 μm. *, p < .05. Abbreviations: ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AMPK, AMP-activated protein kinase; Cont., control; GFAP, glial fibrillary acidic protein; PARP, poly(ADP-ribose) polymerase; siRNA, small interfering RNA.

Figure 5.

Inhibition of tumor-initiating potential of stem-like glioma-initiating cells by metformin and AICAR as activators of AMP-activated protein kinase. (A,B): SJ28P3 (A) or #38 (B) cells (1 × 104) cultured at the indicted glucose concentrations and treated with or without metformin (1 mM) for 3 days were implanted orthotopically into the brain of nude mice. (C,D): SJ28P3 (C) or #38 (D) cells (1 × 104) cultured under the conventional stem cell culture condition (glucose concentration, 26.2 mM) and treated with or without AICAR (1 mM) for 3 days were implanted orthotopically into the brain of nude mice. Left: Representative hematoxylin and eosin staining of brain sections from mice sacrificed 30 days after implantation. Right: Kaplan-Meier plots showing survival of mice (five mice per group) after implantation. Scale bars = 2 mm. *, p < .05. Abbreviation: AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside.

Figure 6.

Transient, systemic administration of metformin depletes the self-renewing and tumor-initiating cell population from established tumors. (A,B): Nude mice implanted subcutaneously with SJ28P3 cells were, after tumor formation, randomized into control and metformin treatment groups and received intraperitoneal injection of vehicle control and metformin (500 mg/kg per day, once daily for 10 days), respectively. On the next day of the final drug treatment, the mice were sacrificed, and dissociated tumor cells (1 × 104 viable cells) were subjected to sphere formation assay (A,B). Representative phase-contrast micrographs of primary spheres (A) and the number of spheres formed (B) are shown. The data in (B) represent means ± SD from three independent cultures derived from three independent tumors. *, p < .05. (C–E): Alternatively, the dissociated tumor cells (1 × 106 viable cells per mouse) were transplanted subcutaneously into the right flank of nude mice (five mice per group). Tumor volume was measured at the indicated time points (C), and at the end of the observation period (18 days after transplantation), the secondary tumors derived from control-treated (tumors formed in five of five mice) and metformin-treated (tumors formed in four of five mice) primary tumors were excised, photographed (D) and evaluated for their weight (E). (F–H): Serial dilutions of the dissociated tumor cells (1 × 105, 1 × 104, or 1 × 103 viable cells) derived from control- and metformin-treated primary tumors were also transplanted intracranially into nude mice. (F): Representative hematoxylin and eosin staining of brain sections from mice receiving transplantation of cells (1 × 104) from primary tumors treated with metformin or vehicle control (sacrificed at 27 days after transplantation). (G): The survival of mice (five mice per group) receiving transplantation of control- or metformin-treated primary tumor cells (1 × 103) was evaluated by Kaplan-Meier analysis. (H): A table showing survival of mice at 160 days after intracranial transplantation of control- or metformin-treated primary tumor cells (upper rows) and the median survival time (lower rows). Scale bars = 200 μm (A) and 2 mm (F).

Figure 7.

Prevention of brain tumor formation by stem-like glioma-initiating cells implanted in the brain parenchyma via transient, systemic administration of metformin. (A–D): Nude mice implanted intracranially with SJ28P3 (A,B) or #38 (C,D) cells (1 × 104) underwent transient, systemic administration (intraperitoneal delivery, once daily for 5 or 10 consecutive days) of metformin (500 mg/kg per day) or vehicle control, which started on the next day of intracranial implantation. (A,C): Representative hematoxylin and eosin staining of brain sections from mice sacrificed at 25 days after implantation. (B,D): Survival of mice was evaluated by Kaplan-Meier analysis. Scale bars = 2 mm (A, C). *, p < .05; **, p < .01.