Shizuka Seino

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.

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.

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.

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.

JNK contributes to temozolomide resistance of stem-like glioblastoma cells via regulation of MGMT expression

While elimination of the cancer stem cell population is increasingly recognized as a key to successful treatment of cancer, the high resistance of cancer stem cells to conventional chemoradiotherapy remains a therapeutic challenge. O6-methylguanine DNA methyltransferase (MGMT), which is frequently expressed in cancer stem cells of glioblastoma, has been implicated in their resistance to temozolomide, the first-line chemotherapeutic agent against newly diagnosed glioblastoma. However, much remains unknown about the molecular regulation that underlies MGMT expression and temozolomide resistance of glioblastoma cancer stem cells. Here, we identified JNK as a novel player in the control of MGMT expression and temozolomide resistance of glioblastoma cancer stem cells. We showed that inhibition of JNK, either pharmacologically or by RNA interference, in stem-like glioblastoma cells derived directly from glioblastoma tissues reduces their MGMT expression and temozolomide resistance. Importantly, sensitization of stem-like glioblastoma cells to temozolomide by JNK inhibition was dependent on MGMT expression, implying that JNK controls temozolomide resistance of stem-like glioblastoma cells through MGMT expression. Our findings suggest that concurrent use of JNK inhibitors with temozolomide may be a rational therapeutic approach to effectively target the cancer stem cell population in the treatment of glioblastoma.

Specific role of JNK in the maintenance of the tumor-initiating capacity of A549 human non-small cell lung cancer cells

Deregulation of c-Jun NH2-terminal kinase (JNK) signaling is now increasingly reported in a variety of human malignancies. Non-small cell lung cancer (NSCLC) is among such human malignancies with aberrant JNK activation; yet the exact role(s) of JNK deregulation in NSCLC biology, in particular in vivo, remains unclear. Here, we demonstrated a specific role of JNK in the control of the tumor-initiating capacity of A549 cells derived from human lung adenocarcinoma, a major subtype of NSCLC. Despite its potent inhibitory activity on A549 cell growth in vitro, SP600125, a reversible JNK inhibitor, failed to inhibit the growth of pre-established A549 xenografts in vivo when systemically administered. Nevertheless, the same SP600125 treatment caused a marked reduction in the tumor-initiating population within the A549 tumors, suggesting that JNK may be specifically required in vivo for the maintenance of the tumor-initiating population of tumor cells rather than for proliferation and survival of the entire cell population. Furthermore, A549 cells either pre-treated with SP600125 or transiently transfected with siRNAs against the JNK genes in vitro showed substantially reduced ability to initiate tumor formation upon implantation into nude mice, implying that the cell intrinsic JNK activity of A549 cells is essential for the maintenance of their tumor-initiating capacity. To our knowledge, this is the first demonstration that JNK is involved in the control of the tumor-initiating capacity of NSCLC cells. Our findings also give rise to an intriguing possibility that therapies targeting JNK could contribute to prevention of relapse and/or metastasis of NSCLC through elimination of tumor-initiating cells.

Licochalcone A specifically induces cell death in glioma stem cells via mitochondrial dysfunction

Glioblastoma multiforme is the most malignant primary intrinsic brain tumor. Glioma stem cells (GSCs) are associated with chemoradiotherapy resistance and the recurrence of glioblastomas after conventional therapy. The targeting of GSCs is potentially an effective treatment for the long‐term survival of glioblastoma patients. Licochalcone A, a natural chalconoid from licorice root, exerts anticancer effects; however, its effect on GSCs remains unknown. We found that Licochalcone A induced massive caspase‐dependent death in GSCs but not in differentiated GSCs nor normal somatic and neural stem cells. Prior to cell death, Licochalcone A caused mitochondrial fragmentation and reduced the membrane potential and ATP production in GSCs. Thus, Licochalcone A induces mitochondrial dysfunction and shows promise as an anticancer stem cell drug.

Involvement of GLUT1-mediated glucose transport and metabolism in gefitinib resistance of non-small-cell lung cancer cells

Use of epidermal growth factor receptor (EGFR) inhibitors represented by gefitinib and erlotinib has become the standard of treatment for non-small-cell lung cancers (NSCLCs) with activating EGFR mutations. However, the majority of NSCLCs, which overexpress EGFR without such mutations, are resistant to EGFR inhibitors, and the mechanism(s) behind such primary resistance of NSCLCs without activating EGFR mutations to EGFR inhibitors still remains poorly understood. Here in this study, we show that glucose metabolism mediated by GLUT1, a facilitative glucose transporter, is involved in gefitinib resistance of NSCLC cells. We found that GLUT1 expression and glucose uptake were increased in resistant NSCLC cells after gefitinib treatment and that genetic as well as pharmacological inhibition of GLUT1 sensitized not only NSCLC cells with primary resistance but also those with acquired resistance to gefitinib. In vivo, the combination of systemic gefitinib and a GLUT1 inhibitor, both of which failed to inhibit tumor growth when administered alone, significantly inhibited the growth of xenograft tumors formed by the implantation of NSCLC cells with wild-type EGFR (wt-EGFR). Since our data indicated that GLUT1 was similarly involved in erlotinib resistance, our findings suggest that the activity of GLUT1-mediated glucose metabolism could be a critical determinant for the sensitivity of NSCLC cells to EGFR inhibitors and that concurrent GLUT1 inhibition may therefore be a mechanism-based approach to treating NSCLCs resistant to EGFR inhibitors, including those with wt-EGFR.