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Targeting JNK for therapeutic depletion of stem-like glioblastoma cells.

Matsuda K, Sato A, Okada M, Shibuya K, Seino S, Suzuki K, Watanabe E, Narita Y, Shibui S, Kayama T, Kitanaka C - Sci Rep (2012)

Bottom Line: Control of the stem-like tumour cell population is considered key to realizing the long-term survival of patients with glioblastoma, one of the most devastating human malignancies.To date, possible therapeutic targets and targeting methods have been described, but none has yet proven to target stem-like glioblastoma cells in the brain to the extent necessary to provide a survival benefit.Here we show that targeting JNK in vivo, the activity of which is required for the maintenance of stem-like glioblastoma cells, via transient, systemic administration of a small-molecule JNK inhibitor depletes the self-renewing and tumour-initiating populations within established tumours, inhibits tumour formation by stem-like glioblastoma cells in the brain, and provide substantial survival benefit without evidence of adverse events.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cancer Science, Yamagata University School of Medicine, Yamagata, 990-9585, Japan.

ABSTRACT
Control of the stem-like tumour cell population is considered key to realizing the long-term survival of patients with glioblastoma, one of the most devastating human malignancies. To date, possible therapeutic targets and targeting methods have been described, but none has yet proven to target stem-like glioblastoma cells in the brain to the extent necessary to provide a survival benefit. Here we show that targeting JNK in vivo, the activity of which is required for the maintenance of stem-like glioblastoma cells, via transient, systemic administration of a small-molecule JNK inhibitor depletes the self-renewing and tumour-initiating populations within established tumours, inhibits tumour formation by stem-like glioblastoma cells in the brain, and provide substantial survival benefit without evidence of adverse events. Our findings not only implicate JNK in the maintenance of stem-like glioblastoma cells but also demonstrate that JNK is a viable, clinically relevant therapeutic target in the control of stem-like glioblastoma cells.

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JNK is required for the maintenance of self-renewal capacity of stem-like glioblastoma cells.(a) Stem-like glioblastoma cells cultured in the presence of SP600125 (40 μM, 7 days for TGS01; 20 μM, 3 days for GS-Y01) were subjected after washout of the inhibitor to serial (i.e., primary to tertiary) sphere formation assays in the absence of SP600125. Top, phase-contrast micrographs of the primary spheres (scale bars, 200 μm). Bottom, number of tumourspheres formed (mean ± s.d. from 3 experiments). *P < 0.05. (b–d) TGS01 and GS-Y01 cells were cultured in the presence of the indicated concentrations of SP600125 for 7 and 3 days, respectively. Cells were then subjected to immunoblot analysis for the expression of phospho-c-Jun (b) and stem cell and differentiation markers (c) and to immunofluorescence staining (green) of nestin, GFAP, and βIII-tubulin (d). In (d), nuclei were counterstained with Hoechst 33342 (blue), and the values in the graph represent mean ± s.d. from 3 experiments. Scale bars, 100 μm. *P < 0.05. (e–f) TGS01 and GS-Y01 cells were transiently transfected with siRNAs against JNK1 or JNK2, or with a control siRNA. Cells were then subjected to serial sphere formation assays (e, values represent mean ± s.d. from 3 experiments)) or to immunoblot analysis to examine the JNK signalling pathway (3 days after transfection) and stem cell/differentiation status (7 and 3 days after transfection for TGS01 and GS-Y01, respectively) (f). *P < 0.05.
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f2: JNK is required for the maintenance of self-renewal capacity of stem-like glioblastoma cells.(a) Stem-like glioblastoma cells cultured in the presence of SP600125 (40 μM, 7 days for TGS01; 20 μM, 3 days for GS-Y01) were subjected after washout of the inhibitor to serial (i.e., primary to tertiary) sphere formation assays in the absence of SP600125. Top, phase-contrast micrographs of the primary spheres (scale bars, 200 μm). Bottom, number of tumourspheres formed (mean ± s.d. from 3 experiments). *P < 0.05. (b–d) TGS01 and GS-Y01 cells were cultured in the presence of the indicated concentrations of SP600125 for 7 and 3 days, respectively. Cells were then subjected to immunoblot analysis for the expression of phospho-c-Jun (b) and stem cell and differentiation markers (c) and to immunofluorescence staining (green) of nestin, GFAP, and βIII-tubulin (d). In (d), nuclei were counterstained with Hoechst 33342 (blue), and the values in the graph represent mean ± s.d. from 3 experiments. Scale bars, 100 μm. *P < 0.05. (e–f) TGS01 and GS-Y01 cells were transiently transfected with siRNAs against JNK1 or JNK2, or with a control siRNA. Cells were then subjected to serial sphere formation assays (e, values represent mean ± s.d. from 3 experiments)) or to immunoblot analysis to examine the JNK signalling pathway (3 days after transfection) and stem cell/differentiation status (7 and 3 days after transfection for TGS01 and GS-Y01, respectively) (f). *P < 0.05.

Mentions: Prompted by observation of a uniform JNK pathway activation in self-renewing stem-like glioblastoma cells, we next investigated whether JNK is involved in the maintenance of the stem-like properties of self-renewing cells. We first tested the effect of SP600125, a “reversible”, ATP-competitive inhibitor of JNK17, on the ability of stem-like glioblastoma cells to self-renew themselves as tumourspheres at concentrations that inhibited c-Jun phosphorylation (and expression) (Fig. 2b) but not cellular viability (Supplementary Fig. 2). Whereas the cells pretreated with the control vehicle maintained the ability to form tumourspheres over serial passages, stem-like glioblastoma cells pretreated with SP600125 showed reduced ability to form tumourspheres even in the absence of the inhibitor (Fig. 2a), suggesting that transient JNK inhibition had deprived the cells of their self-renewing capacity. To determine whether such decreased tumoursphere formation truly reflects loss of stem-like properties, the expression of stem cell and differentiation markers was next examined. SP600125 treatment was found to cause decreased expression of stem cell markers such as Nestin, Sox2, and Musashi-1, accompanied by increased expression of the differentiation markers, glial fibrillary acidic protein (GFAP) and βIII-tubulin (Fig. 2c). These changes in marker expression level reflected the change in the ratio of undifferentiated to differentiated cell populations, as revealed by immunocytochemical analysis (Fig. 2d). The results of these pharmacological inhibitor assays were confirmed by subsequent knockdown experiments. Unexpectedly, we found that knockdown of either JNK1 or JNK2 in stem-like glioblastoma cells is sufficient to effectively inhibit the JNK pathway activity (Fig. 2f). This finding may be in line with a previous study using mouse embryonic fibroblasts for either JNK1 or JNK2, which found that both JNK1 and JNK2 are required for JNK pathway activation18. We therefore knocked down either JNK1 or JNK2 singly in the following experiments. The results indicate that, similar to those regarding SP600125, short-interfering RNA (siRNA)-mediated knockdown of JNK1 or JNK2 inhibits tumoursphere formation (Fig. 2e) and stem cell marker expression while inducing the expression of differentiation markers (Fig. 2f). Intriguingly, we found that expression of the FOXO1 transcription factor but not of FOXO3, which has previously been implicated in the differentiation of stem-like glioblastoma cells19, is upregulated accompanied by its nuclear translocation upon JNK inhibition in stem-like glioblastoma cells (Supplementary Figs. 3 and 4). We also found that FOXO1 knockdown inhibits commitment of stem-like glioblastoma cells to differentiation (Supplementary Fig. 3). These results suggest that prevention of FOXO1 activation is at least in part responsible for the JNK-mediated maintenance of stem-like glioblastoma cells. Collectively, the data suggest that continuous, uninterrupted activation of the JNK pathway is essential for preventing premature activation of the differentiation-inducing program, and therefore, for the maintenance of the self-renewal capacity of glioblastoma cells. Strikingly, such JNK dependence was confirmed in all 10 patient-derived stem-like glioblastoma cell lines tested in this study, which had been originally established in 3 independent institutions, as well as in the 2 stem-like cell lines established from conventional, serum-cultured glioblastoma cell lines (Supplementary Figs. 5–7). Furthermore, JNK was found to be required for tumoursphere formation and/or maintenance of the undifferentiated state in putative stem-like glioblastoma cells that ultimately failed to become established cell lines (Supplementary Fig. 8), in support of the idea that JNK dependence of self-renewal is not a unique feature of established cell lines. Thus, the critical role of JNK in the control of self-renewal and differentiation could be a cardinal feature shared by stem-like glioblastoma cells.


Targeting JNK for therapeutic depletion of stem-like glioblastoma cells.

Matsuda K, Sato A, Okada M, Shibuya K, Seino S, Suzuki K, Watanabe E, Narita Y, Shibui S, Kayama T, Kitanaka C - Sci Rep (2012)

JNK is required for the maintenance of self-renewal capacity of stem-like glioblastoma cells.(a) Stem-like glioblastoma cells cultured in the presence of SP600125 (40 μM, 7 days for TGS01; 20 μM, 3 days for GS-Y01) were subjected after washout of the inhibitor to serial (i.e., primary to tertiary) sphere formation assays in the absence of SP600125. Top, phase-contrast micrographs of the primary spheres (scale bars, 200 μm). Bottom, number of tumourspheres formed (mean ± s.d. from 3 experiments). *P < 0.05. (b–d) TGS01 and GS-Y01 cells were cultured in the presence of the indicated concentrations of SP600125 for 7 and 3 days, respectively. Cells were then subjected to immunoblot analysis for the expression of phospho-c-Jun (b) and stem cell and differentiation markers (c) and to immunofluorescence staining (green) of nestin, GFAP, and βIII-tubulin (d). In (d), nuclei were counterstained with Hoechst 33342 (blue), and the values in the graph represent mean ± s.d. from 3 experiments. Scale bars, 100 μm. *P < 0.05. (e–f) TGS01 and GS-Y01 cells were transiently transfected with siRNAs against JNK1 or JNK2, or with a control siRNA. Cells were then subjected to serial sphere formation assays (e, values represent mean ± s.d. from 3 experiments)) or to immunoblot analysis to examine the JNK signalling pathway (3 days after transfection) and stem cell/differentiation status (7 and 3 days after transfection for TGS01 and GS-Y01, respectively) (f). *P < 0.05.
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f2: JNK is required for the maintenance of self-renewal capacity of stem-like glioblastoma cells.(a) Stem-like glioblastoma cells cultured in the presence of SP600125 (40 μM, 7 days for TGS01; 20 μM, 3 days for GS-Y01) were subjected after washout of the inhibitor to serial (i.e., primary to tertiary) sphere formation assays in the absence of SP600125. Top, phase-contrast micrographs of the primary spheres (scale bars, 200 μm). Bottom, number of tumourspheres formed (mean ± s.d. from 3 experiments). *P < 0.05. (b–d) TGS01 and GS-Y01 cells were cultured in the presence of the indicated concentrations of SP600125 for 7 and 3 days, respectively. Cells were then subjected to immunoblot analysis for the expression of phospho-c-Jun (b) and stem cell and differentiation markers (c) and to immunofluorescence staining (green) of nestin, GFAP, and βIII-tubulin (d). In (d), nuclei were counterstained with Hoechst 33342 (blue), and the values in the graph represent mean ± s.d. from 3 experiments. Scale bars, 100 μm. *P < 0.05. (e–f) TGS01 and GS-Y01 cells were transiently transfected with siRNAs against JNK1 or JNK2, or with a control siRNA. Cells were then subjected to serial sphere formation assays (e, values represent mean ± s.d. from 3 experiments)) or to immunoblot analysis to examine the JNK signalling pathway (3 days after transfection) and stem cell/differentiation status (7 and 3 days after transfection for TGS01 and GS-Y01, respectively) (f). *P < 0.05.
Mentions: Prompted by observation of a uniform JNK pathway activation in self-renewing stem-like glioblastoma cells, we next investigated whether JNK is involved in the maintenance of the stem-like properties of self-renewing cells. We first tested the effect of SP600125, a “reversible”, ATP-competitive inhibitor of JNK17, on the ability of stem-like glioblastoma cells to self-renew themselves as tumourspheres at concentrations that inhibited c-Jun phosphorylation (and expression) (Fig. 2b) but not cellular viability (Supplementary Fig. 2). Whereas the cells pretreated with the control vehicle maintained the ability to form tumourspheres over serial passages, stem-like glioblastoma cells pretreated with SP600125 showed reduced ability to form tumourspheres even in the absence of the inhibitor (Fig. 2a), suggesting that transient JNK inhibition had deprived the cells of their self-renewing capacity. To determine whether such decreased tumoursphere formation truly reflects loss of stem-like properties, the expression of stem cell and differentiation markers was next examined. SP600125 treatment was found to cause decreased expression of stem cell markers such as Nestin, Sox2, and Musashi-1, accompanied by increased expression of the differentiation markers, glial fibrillary acidic protein (GFAP) and βIII-tubulin (Fig. 2c). These changes in marker expression level reflected the change in the ratio of undifferentiated to differentiated cell populations, as revealed by immunocytochemical analysis (Fig. 2d). The results of these pharmacological inhibitor assays were confirmed by subsequent knockdown experiments. Unexpectedly, we found that knockdown of either JNK1 or JNK2 in stem-like glioblastoma cells is sufficient to effectively inhibit the JNK pathway activity (Fig. 2f). This finding may be in line with a previous study using mouse embryonic fibroblasts for either JNK1 or JNK2, which found that both JNK1 and JNK2 are required for JNK pathway activation18. We therefore knocked down either JNK1 or JNK2 singly in the following experiments. The results indicate that, similar to those regarding SP600125, short-interfering RNA (siRNA)-mediated knockdown of JNK1 or JNK2 inhibits tumoursphere formation (Fig. 2e) and stem cell marker expression while inducing the expression of differentiation markers (Fig. 2f). Intriguingly, we found that expression of the FOXO1 transcription factor but not of FOXO3, which has previously been implicated in the differentiation of stem-like glioblastoma cells19, is upregulated accompanied by its nuclear translocation upon JNK inhibition in stem-like glioblastoma cells (Supplementary Figs. 3 and 4). We also found that FOXO1 knockdown inhibits commitment of stem-like glioblastoma cells to differentiation (Supplementary Fig. 3). These results suggest that prevention of FOXO1 activation is at least in part responsible for the JNK-mediated maintenance of stem-like glioblastoma cells. Collectively, the data suggest that continuous, uninterrupted activation of the JNK pathway is essential for preventing premature activation of the differentiation-inducing program, and therefore, for the maintenance of the self-renewal capacity of glioblastoma cells. Strikingly, such JNK dependence was confirmed in all 10 patient-derived stem-like glioblastoma cell lines tested in this study, which had been originally established in 3 independent institutions, as well as in the 2 stem-like cell lines established from conventional, serum-cultured glioblastoma cell lines (Supplementary Figs. 5–7). Furthermore, JNK was found to be required for tumoursphere formation and/or maintenance of the undifferentiated state in putative stem-like glioblastoma cells that ultimately failed to become established cell lines (Supplementary Fig. 8), in support of the idea that JNK dependence of self-renewal is not a unique feature of established cell lines. Thus, the critical role of JNK in the control of self-renewal and differentiation could be a cardinal feature shared by stem-like glioblastoma cells.

Bottom Line: Control of the stem-like tumour cell population is considered key to realizing the long-term survival of patients with glioblastoma, one of the most devastating human malignancies.To date, possible therapeutic targets and targeting methods have been described, but none has yet proven to target stem-like glioblastoma cells in the brain to the extent necessary to provide a survival benefit.Here we show that targeting JNK in vivo, the activity of which is required for the maintenance of stem-like glioblastoma cells, via transient, systemic administration of a small-molecule JNK inhibitor depletes the self-renewing and tumour-initiating populations within established tumours, inhibits tumour formation by stem-like glioblastoma cells in the brain, and provide substantial survival benefit without evidence of adverse events.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cancer Science, Yamagata University School of Medicine, Yamagata, 990-9585, Japan.

ABSTRACT
Control of the stem-like tumour cell population is considered key to realizing the long-term survival of patients with glioblastoma, one of the most devastating human malignancies. To date, possible therapeutic targets and targeting methods have been described, but none has yet proven to target stem-like glioblastoma cells in the brain to the extent necessary to provide a survival benefit. Here we show that targeting JNK in vivo, the activity of which is required for the maintenance of stem-like glioblastoma cells, via transient, systemic administration of a small-molecule JNK inhibitor depletes the self-renewing and tumour-initiating populations within established tumours, inhibits tumour formation by stem-like glioblastoma cells in the brain, and provide substantial survival benefit without evidence of adverse events. Our findings not only implicate JNK in the maintenance of stem-like glioblastoma cells but also demonstrate that JNK is a viable, clinically relevant therapeutic target in the control of stem-like glioblastoma cells.

Show MeSH
Related in: MedlinePlus