Atsushi Sato

Crosstalk Between the PI3K/mTOR and MEK/ERK Pathways Involved in the Maintenance of Self-Renewal and Tumorigenicity of Glioblastoma Stem-Like Cells

The molecular signaling pathways orchestrating the biology of cancer stem‐like cells (CSLCs), including glioblastoma, remain to be elucidated. We investigated in this study the role of the MEK/extracellular signal‐regulated kinase (ERK) pathway in the control of self‐renewal and tumorigenicity of glioblastoma CSLCs, particularly in relation to the PI3K/mTOR (mammalian target of rapamycin) pathway. Targeted inactivation of MEK alone using pharmacological inhibitors or siRNAs resulted in reduced sphere formation of both cell line‐ and patient‐derived glioblastoma CSLCs, accompanied by their differentiation into neuronal and glial lineages. Interestingly, this effect of MEK inactivation was apparently augmented in the presence of NVP‐BEZ235, a dual inhibitor of PI3K and mTOR. As a potential explanation for this observed synergy, we found that inactivation of either the MEK/ERK or PI3K/mTOR pathway triggered activation of the other, suggesting that there may be mutually inhibitory crosstalk between these two pathways. Significantly, inactivation of either pathway led to the reduced activation of p70S6K, and siRNA‐mediated knockdown of p70S6K resulted in the activation of both pathways, which no longer maintained the cross‐inhibitory relationship. Finally, combinational blockade of both pathways in glioblastoma CSLCs suppressed their tumorigenicity, whether transplanted subcutaneously or intracranially, more efficiently than blockade of either alone. Our findings suggest that there is p70S6K‐mediated, cross‐inhibitory regulation between the MEK/ERK and PI3K/mTOR pathways, in which each contribute to the maintenance of the self‐renewal and tumorigenic capacity of glioblastoma CSLCs. Thus, combinational disruption of these pathways would be a rational and effective strategy in the treatment of glioblastoma. STEM CELLS 2010;28:1930–1939

Glioma-Initiating Cell Elimination by Metformin Activation of FOXO3 via AMPK

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.

Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells

Glioblastoma, the most intractable cerebral tumor, is highly lethal. Recent studies suggest that cancer stem-like cells (CSLCs) have the capacity to repopulate tumors and mediate radio- and chemoresistance, implying that future therapies may need to turn from the elimination of rapidly dividing, but differentiated, tumor cells to specifically targeting the minority of tumor cells that repopulate the tumor. However, the mechanism by which glioblastoma CSLCs maintain their immature stem-like state or, alternatively, become committed to differentiation is poorly understood. Here, we show that the inactivation of mammalian target of rapamycin (mTor) by the mTor inhibitor rapamycin or knockdown of mTor reduced sphere formation and the expression of neural stem cell (NSC)/progenitor markers in CSLCs of the A172 glioblastoma cell line. Interestingly, combination treatment with rapamycin and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, not only reduced the expression of NSC/progenitor markers more efficiently than single-agent treatment, but also increased the expression of βIII-tubulin, a neuronal differentiation marker. Consistent with these results, a dual PI3K/mTor inhibitor, NVP-BEZ235, elicited a prodifferentiation effect on A172 CSLCs. Moreover, A172 CSLCs, which were induced to undergo differentiation by pretreatment with NVP-BEZ235, exhibited a significant decrease in their tumorigenicity when transplanted either subcutaneously or intracranially. Importantly, similar results were obtained when patient-derived glioblastoma CSLCs were used. These findings suggest that the PI3K/mTor signaling pathway is critical for the maintenance of glioblastoma CSLC properties, and targeting both mTor and PI3K of CSLCs may be an effective therapeutic strategy in glioblastoma.

MEK‐ERK Signaling Dictates DNA‐Repair Gene MGMT Expression and Temozolomide Resistance of Stem‐Like Glioblastoma Cells via the MDM2‐p53 Axis

Overcoming the resistance of glioblastoma cells against temozolomide, the first‐line chemotherapeutic agent of choice for newly diagnosed glioblastoma, is a major therapeutic challenge in the management of this deadly brain tumor. The gene encoding O6‐methylguanine DNA methyltransferase (MGMT), which removes the methyl group attached by temozolomide, is often silenced by promoter methylation in glioblastoma but is nevertheless expressed in a significant fraction of cases and is therefore regarded as one of the most clinically relevant mechanisms of resistance against temozolomide. However, to date, signaling pathways regulating MGMT in MGMT‐expressing glioblastoma cells have been poorly delineated. Here in this study, we provide lines of evidence that the mitogen‐activated protein/extracellular signal‐regulated kinase kinase (MEK)–extracellular signal‐regulated kinase (ERK)‐‐murine double minute 2 (MDM2)‐p53 pathway plays a critical role in the regulation of MGMT expression, using stem‐like glioblastoma cells directly derived from patient tumor samples and maintained in the absence of serum, which not only possess stem‐like properties but are also known to phenocopy the characteristics of the original tumors from which they are derived. We show that, in stem‐like glioblastoma cells, MEK inhibition reduced MDM2 expression and that inhibition of either MEK or MDM2 resulted in p53 activation accompanied by p53‐dependent downregulation of MGMT expression. MEK inhibition rendered otherwise resistant stem‐like glioblastoma cells sensitive to temozolomide, and combination of MEK inhibitor and temozolomide treatments effectively deprived stem‐like glioblastoma cells of their tumorigenic potential. Our findings suggest that targeting of the MEK‐ERK‐MDM2‐p53 pathway in combination with temozolomide could be a novel and promising therapeutic strategy in the treatment of glioblastoma. STEM CELLS 2011;29:1942–1951.

Pivotal role for ROS activation of p38 MAPK in the control of differentiation and tumor-initiating capacity of glioma-initiating cells

Reactive oxygen species (ROS) are involved in various aspects of cancer cell biology, yet their role in cancer stem cells (CSCs) has been poorly understood. In particular, it still remains unclear whether and how ROS control the self-renewal/differentiation process and the tumor-initiating capacity of CSCs. Here we show that ROS-mediated activation of p38 MAPK plays a pivotal role in the control of differentiation and tumor-initiating capacity of glioma-initiating cells (GICs) derived from human glioblastomas. Mechanistically, ROS triggered p38-dependent Bmi1 protein degradation and FoxO3 activation in GICs, which were shown to be responsible for the loss of their self-renewal capacity and differentiation, respectively. Thus, the results suggest that Bmi1 and FoxO3 govern distinct phases of transition from undifferentiated to fully differentiated cells. Furthermore, we also demonstrate in this study that oxidative stress deprives GICs of their tumor-initiating capacity through the activation of the ROS-p38 axis. As such, this is the first study to the best of our knowledge to delineate how ROS control self-renewal/differentiation and the tumor-initiating capacity of stem-like cancer cells. This study also suggests that targeting of the ROS-p38 axis could be a novel approach in the development of therapeutic strategies against gliomas, represented by glioblastoma.

Regulation of neural stem/progenitor cell maintenance by PI3K and mTOR

Control of stem cell state and differentiation of neural stem/progenitor cells is essential for proper development of the nervous system. EGF and FGF2 play important roles in the control of neural stem/progenitor cells, but the underlying mechanism still remains unclear. Here we show, using in vitro primary cultures of mouse neural stem/progenitor cells, that both PI3K and mTOR are activated by EGF/FGF2 but that inhibiting the activation of either PI3K or mTOR alone results in only reduced proliferation of neural stem/progenitor cells without affecting their stem cell state, namely, the capacity to self-renew. However, significantly, concurrent inhibition of PI3K and mTOR promoted exit from the stem cell state together with astrocytic differentiation of neural stem/progenitor cells. These findings suggest that PI3K and mTOR are involved in the EGF/FGF2-mediated maintenance of neural stem/progenitor cells and that they may act in parallel and independent pathways, complementing and backing up each other to maintain the stem cell state.

FoxO3a functions as a key integrator of cellular signals that control glioblastoma stem-like cell differentiation and tumorigenicity.

Glioblastoma is one of the most aggressive types of human cancer, with invariable and fatal recurrence even after multimodal intervention, for which cancer stem‐like cells (CSLCs) are now being held responsible. Our recent findings indicated that combinational inhibition of phosphoinositide‐3‐kinase/Akt/mammalian target of rapamycin (mTOR) and mitogen‐activated protein/extracellular signal‐regulated kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) pathways effectively promotes the commitment of glioblastoma CSLCs to differentiation and thereby suppresses their tumorigenicity. However, the mechanism by which these two signaling pathways are coordinated to regulate differentiation and tumorigenicity remains unknown. Here, we identified FoxO3a, a common phosphorylation target for Akt and ERK, as a key transcription factor that integrates the signals from these pathways. Combinational blockade of both the pathways caused nuclear accumulation and activation of FoxO3a more efficiently than blockade of either alone, and promoted differentiation of glioblastoma CSLCs in a FoxO3a expression‐dependent manner. Furthermore, the expression of a constitutively active FoxO3a mutant lacking phosphorylation sites for both Akt and ERK was sufficient to induce differentiation and reduce tumorigenicity of glioblastoma CSLCs. These findings suggest that FoxO3a may play a pivotal role in the control of differentiation and tumorigenicity of glioblastoma CSLCs by the PI3K/Akt/mTOR and MEK/ERK signaling pathways, and also imply that developing methods targeting effective FoxO3a activation could be a potential approach to the treatment of glioblastoma. STEM CELLS 2011;29:1327–1337

Resveratrol promotes proteasome-dependent degradation of Nanog via p53 activation and induces differentiation of glioma stem cells.

Glioblastoma is the most common and aggressive primary brain tumor. Glioma stem cells (GSCs) are relatively resistant to chemo-radiotherapy and are responsible for tumor progression and the recurrence of glioblastomas after conventional therapy. Thus, the control of the GSC population is considered key to realizing long-term survival of glioblastoma patients. Here, we identified that resveratrol significantly reduced the self-renewal and tumor-initiating capacity of patient-derived GSCs. Furthermore, resveratrol promoted Nanog suppression via proteasomal degradation, which was inhibited by MG132, a proteasome inhibitor. p53 activation is an important factor in Nanog suppression and treatment with resveratrol was also found to activate the p53/p21 pathway. Importantly, inhibition of Nanog by siRNA provoked inhibitory effects on both the self-renewal and tumor-forming capacity of GSCs. Our findings indicate that Nanog is an essential factor for the retention of stemness and may contribute to the resveratrol-induced differentiation of GSCs. Our results also suggest that targeting GSCs via the p53-Nanog axis, with resveratrol for instance, could be a therapeutic strategy against glioblastoma.

Reduction of cadmium uptake in spinach (Spinacia oleracea L.) by soil amendment with animal waste compost.

A field experiment was conducted to evaluate the efficacy of animal waste compost (AWC) in reducing Cd uptake by spinach (Spinacia oleracea L.). Spinach was grown in a field that had been treated by having cattle, swine, or poultry waste compost incorporated into the soil before each crop throughout 4 years of rotational vegetable production. Cadmium concentration was 34-38% lower in spinach harvested from the AWC-treated soils than in the chemical fertilizer-treated soil. Although the repeated application of swine and poultry compost caused significant P accumulation in the cropped soils, that of cattle compost did not. These results indicate that cattle compost with high affinity for Cd and low P content should be the preferred soil amendment when used to reduce Cd uptake by spinach.

Targeting JNK for therapeutic depletion of stem-like glioblastoma cells

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.