The MDM2-p53 Axis Represents a Therapeutic Vulnerability Unique to Glioma Stem Cells.

The prevention of tumor recurrence by the successful targeting of glioma stem cells endowed with a tumor-initiating capacity is deemed the key to the long-term survival of glioblastoma patients. Glioma stem cells are characterized by their marked therapeutic resistance; however, recent evidence suggests that they have unique vulnerabilities that may be therapeutically targeted. We investigated MDM2 expression levels in glioma stem cells and their non-stem cell counterparts and the effects of the genetic and pharmacological inhibition of MDM2 on the viability of these cells as well as downstream molecular pathways. The results obtained showed that MDM2 expression was substantially higher in glioma stem cells than in their non-stem cell counterparts and also that the inhibition of MDM2, either genetically or pharmacologically, induced a more pronounced activation of the p53 pathway and apoptotic cell death in the former than in the latter. Specifically, the inhibition of MDM2 caused a p53-dependent increase in the expression of BAX and PUMA and a decrease in the expression of survivin, both of which significantly contributed to the apoptotic death of glioma stem cells. The present study identified the MDM2-p53 axis as a novel therapeutic vulnerability, or an Achilles' heel, which is unique to glioma stem cells. Our results, which suggest that non-stem, bulk tumor cells are less sensitive to MDM2 inhibitors, may help guide the selection of glioblastoma patients suitable for MDM2 inhibitor therapy.

CEP-1347 Boosts Chk2-Mediated p53 Activation by Ionizing Radiation to Inhibit the Growth of Malignant Brain Tumor Cells.

Radiation therapy continues to be the cornerstone treatment for malignant brain tumors, the majority of which express wild-type p53. Therefore, the identification of drugs that promote the ionizing radiation (IR)-induced activation of p53 is expected to increase the efficacy of radiation therapy for these tumors. The growth inhibitory effects of CEP-1347, a known inhibitor of MDM4 expression, on malignant brain tumor cell lines expressing wild-type p53 were examined, alone or in combination with IR, by dye exclusion and/or colony formation assays. The effects of CEP-1347 on the p53 pathway, alone or in combination with IR, were examined by RT-PCR and Western blot analyses. The combination of CEP-1347 and IR activated p53 in malignant brain tumor cells and inhibited their growth more effectively than either alone. Mechanistically, CEP-1347 and IR each reduced MDM4 expression, while their combination did not result in further decreases. CEP-1347 promoted IR-induced Chk2 phosphorylation and increased p53 expression in concert with IR in a Chk2-dependent manner. The present results show, for the first time, that CEP-1347 is capable of promoting Chk2-mediated p53 activation by IR in addition to inhibiting the expression of MDM4 and, thus, CEP-1347 has potential as a radiosensitizer for malignant brain tumors expressing wild-type p53.

Domatinostat Targets the FOXM1-Survivin Axis to Reduce the Viability of Ovarian Cancer Cells Alone and in Combination with Chemotherapeutic Agents.

The deregulation of the FOXM1 transcription factor is a key molecular alteration in ovarian cancer, contributing to the development and progression of ovarian cancer via activation of the target genes. As such, FOXM1 is a highly attractive therapeutic target in the treatment of ovarian cancer, but there has been no clinically tested FOXM1 inhibitor to date. We investigated in this study the effects of domatinostat, a class I-selective HDAC inhibitor currently in the clinical stage of development as a cancer therapeutic, on the expression of FOXM1 and viability of ovarian cancer cells. Cell viability, as well as protein and mRNA expression of FOXM1 and its transcriptional target survivin, was examined after domatinostat treatment of TOV21G and SKOV3 ovarian cancer cell lines in the absence or presence of cisplatin and paclitaxel. The effect of FOXM1 knockdown on survivin expression and those of genetic and pharmacological inhibition of survivin alone or in combination with the chemotherapeutic agents on cell viability were also examined. Domatinostat reduced the protein and mRNA expression of FOXM1 and survivin and also the viability of ovarian cancer cells alone and in combination with cisplatin or paclitaxel at clinically relevant concentrations. Knockdown experiments showed survivin expression was dependent on FOXM1 in ovarian cancer cells. Survivin inhibition was sufficient to reduce the viability of ovarian cancer cells alone and in combination with the chemotherapeutic agents. Our findings suggest that domatinostat, which effectively targets the FOXM1-survivin axis required for the viability of ovarian cancer cells, is a promising option for the treatment of ovarian cancer.

The Real-World status and risk factors for a poor prognosis in elderly patients with primary central nervous system malignant lymphomas: a multicenter, retrospective cohort study of the Tohoku Brain Tumor Study Group.

BACKGROUND: Elderly patients with primary central nervous system malignant lymphoma (EL-PCNSL) may not be given sufficient treatment due to their poor pre-treatment Karnofsky Performance Status (KPS) and comorbidities. Therefore, a retrospective, cohort study was performed to evaluate risk factors associated with a poor prognosis of EL-PCNSL in the Tohoku Brain Tumor Study Group. METHODS: Patients aged ≥ 71 years with PCNSL were enrolled from eight centers. Univariate analysis was performed with the log-rank test. A Cox proportional hazards model was used for multivariate analysis. RESULTS: Three of the total 142 cases received best supportive care (BSC). Treatment was given to 30 cases without a pathological diagnosis, 3 cases with cerebrospinal fluid (CSF) cytology, and 100 cases with a pathological diagnosis. After confirmation of no differences in progression-free survival (PFS) and overall survival (OS) between the group treated without pathology and the groups diagnosed by pathology or CSF cytology and between median age ≥ 76 years and < 76 years, a total of 133 patients were studied. The median pre-treatment KPS was 50%. Median PFS and median OS were 16 and 24 months, respectively. Risk factors associated with poor prognosis on Cox proportional hazards model analysis were pre-treatment cardiovascular disease and central nervous system disease comorbidities, post-treatment pneumonia and other infections, and the absence of radiotherapy or chemotherapy. CONCLUSIONS: Pre-treatment comorbidities and post-treatment complications would affect the prognosis. Radiation and chemotherapy were found to be effective, but no conclusions could be drawn regarding the appropriate content of chemotherapy and whether additional radiotherapy should be used.

Inhibition of the Phospholipase Cε-c-Jun N-Terminal Kinase Axis Suppresses Glioma Stem Cell Properties.

Glioma stem cells (GSCs), the cancer stem cells of glioblastoma multiforme (GBM), contribute to the malignancy of GBM due to their resistance to therapy and tumorigenic potential; therefore, the development of GSC-targeted therapies is urgently needed to improve the poor prognosis of GBM patients. The molecular mechanisms maintaining GSCs need to be elucidated in more detail for the development of GSC-targeted therapy. In comparison with patient-derived GSCs and their differentiated counterparts, we herein demonstrated for the first time that phospholipase C (PLC)ε was highly expressed in GSCs, in contrast to other PLC isoforms. A broad-spectrum PLC inhibitor suppressed the viability of GSCs, but not their stemness. Nevertheless, the knockdown of PLCε suppressed the survival of GSCs and induced cell death. The stem cell capacity of residual viable cells was also suppressed. Moreover, the survival of mice that were transplanted with PLCε knockdown-GSCs was longer than the control group. PLCε maintained the stemness of GSCs via the activation of JNK. The present study demonstrated for the first time that PLCε plays a critical role in maintaining the survival, stemness, and tumor initiation capacity of GSCs. Our study suggested that PLCε is a promising anti-GSC therapeutic target.

HDAC Class I Inhibitor Domatinostat Preferentially Targets Glioma Stem Cells over Their Differentiated Progeny.

Cancer stem cells (CSCs) are in general characterized by higher resistance to cell death and cancer therapies than non-stem differentiated cancer cells. However, we and others have recently revealed using glioma stem cells (GSCs) as a model that, unexpectedly, CSCs have specific vulnerabilities that make them more sensitive to certain drugs compared with their differentiated counterparts. We aimed in this study to discover novel drugs targeting such Achilles' heels of GSCs as anti-GSC drug candidates to be used for the treatment of glioblastoma, the most therapy-resistant form of brain tumors. Here we report that domatinostat (4SC-202), a class I HDAC inhibitor, is one such candidate. At concentrations where it showed no or minimal growth inhibitory effect on differentiated GSCs and normal cells, domatinostat effectively inhibited the growth of GSCs mainly by inducing apoptosis. Furthermore, GSCs that survived domatinostat treatment lost their self-renewal capacity. These results suggested that domatinostat is a unique drug that selectively eliminates GSCs not only physically by inducing cell death but also functionally by inhibiting their self-renewal. Our findings also imply that class I HDACs and/or LSD1, another target of domatinostat, may possibly have a specific role in the maintenance of GSCs and therefore could be an attractive target in the development of anti-GSC therapies.

Therapeutic targeting of pancreatic cancer stem cells by dexamethasone modulation of the MKP-1-JNK axis.

Postoperative recurrence from microscopic residual disease must be prevented to cure intractable cancers, including pancreatic cancer. Key to this goal is the elimination of cancer stem cells (CSCs) endowed with tumor-initiating capacity and drug resistance. However, current therapeutic strategies capable of accomplishing this are insufficient. Using in vitro models of CSCs and in vivo models of tumor initiation in which CSCs give rise to xenograft tumors, we show that dexamethasone induces expression of MKP-1, a MAPK phosphatase, via glucocorticoid receptor activation, thereby inactivating JNK, which is required for self-renewal and tumor initiation by pancreatic CSCs as well as for their expression of survivin, an anti-apoptotic protein implicated in multidrug resistance. We also demonstrate that systemic administration of clinically relevant doses of dexamethasone together with gemcitabine prevents tumor formation by CSCs in a pancreatic cancer xenograft model. Our study thus provides preclinical evidence for the efficacy of dexamethasone as an adjuvant therapy to prevent postoperative recurrence in patients with pancreatic cancer.

Targeting Folate Metabolism Is Selectively Cytotoxic to Glioma Stem Cells and Effectively Cooperates with Differentiation Therapy to Eliminate Tumor-Initiating Cells in Glioma Xenografts.

Glioblastoma (GBM) is one of the deadliest of all human cancers. Developing therapies targeting GBM cancer stem cells or glioma stem cells (GSCs), which are deemed responsible for the malignancy of GBM due to their therapy resistance and tumor-initiating capacity, is considered key to improving the dismal prognosis of GBM patients. In this study, we found that folate antagonists, such as methotrexate (MTX) and pemetrexed, are selectively cytotoxic to GSCs, but not to their differentiated counterparts, normal fibroblasts, or neural stem cells in vitro, and that the high sensitivity of GCSs to anti-folates may be due to the increased expression of RFC-1/SLC19A1, the reduced folate carrier that transports MTX into cells, in GSCs. Of note, in an in vivo serial transplantation model, MTX alone failed to exhibit anti-GSC effects but promoted the anti-GSC effects of CEP1347, an inducer of GSC differentiation. This suggests that folate metabolism, which plays an essential role specifically in GSCs, is a promising target of anti-GSC therapy, and that the combination of cytotoxic and differentiation therapies may be a novel and promising approach to effectively eliminate cancer stem cells.

Cytoplasmic localization of DGKζ exerts a protective effect against p53-mediated cytotoxicity.

The transcription factor p53 plays a crucial role in coordinating the cellular response to various stresses. Therefore, p53 protein levels and activity need to be kept under tight control. We report here that diacylglycerol kinase ζ (DGKζ) binds to p53 and modulates its function both in the cytoplasm and nucleus. DGKζ, a member of the DGK family that metabolizes a lipid second messenger diacylglycerol, localizes primarily to the nucleus in various cell types. Recently, reports have described that excitotoxic stress induces DGKζ nucleocytoplasmic translocation in hippocampal neurons. In the study reported here we found that cytoplasmic DGKζ attenuates p53-mediated cytotoxicity against doxorubicin-induced DNA damage by facilitating cytoplasmic anchoring and degradation of p53 through a ubiquitin-proteasome system. Concomitantly, decreased levels of nuclear DGKζ engender downregulation of p53 transcriptional activity. Consistent with these in vitro cellular experiments, DGKζ-deficient brain exhibits high levels of p53 protein after kainate-induced seizures and even under normal conditions. These findings provide novel insights into the regulation of p53 function and suggest that DGKζ serves as a sentinel to control p53 function both during normal homeostasis and in stress responses.

Treatment outcomes in glioblastoma patients aged 76 years or older: a multicenter retrospective cohort study.

Age is one of the most important prognostic factors in glioblastoma patients, but no standard treatment has been established for elderly patients with this condition. We therefore conducted a retrospective cohort study to evaluate treatment regimens and outcomes in elderly glioblastoma patients. The study population consisted of 79 glioblastoma patients aged ≥ 76 years (median age 78.0 years; 34 men and 45 women). The median preoperative Karnofsky performance status (KPS) score was 60. Surgical procedures were classified as biopsy (31 patients, 39.2 %), <95 % resection of the tumor (21 patients, 26.9 %), and ≥ 95 % resection of the tumor (26 patients, 33.3 %). Sixty-seven patients (81.0 %) received radiotherapy and 45 patients (57.0 %) received chemotherapy. The median overall progression-free survival time was 6.8 months, and the median overall survival time was 9.8 months. Patients aged ≥ 78 years were significantly less likely to receive radiotherapy (p = 0.004). Patients with a postoperative KPS score of ≥ 60 were significantly more likely to receive maintenance chemotherapy (p = 0.008). Multivariate analyses identified two independent prognostic factors: postoperative KPS score ≥ 60 (hazard ratio [HR] = 0.531, 95 % confidence interval [CI] 0.315-0.894, p = 0.017) and temozolomide therapy (HR = 0.442, 95 % CI 0.25-0.784, p < 0.001).The findings of this study suggest that postoperative KPS score is an important prognostic factor for glioblastoma patients aged ≥ 76 years, and that these patients may benefit from temozolomide therapy.

Melanocortin Receptor Agonist Bremelanotide Induces Cell Death and Growth Inhibition in Glioblastoma Cells via Suppression of Survivin Expression.

BACKGROUND/AIM: Glioblastoma is the most aggressive form of brain tumor and has a dismal prognosis; therefore, novel therapeutic approaches based on the mechanisms underlying its aggressive nature are urgently required. A growing body of evidence suggests that neurotransmitters play a key role in modulating the biology of glioblastoma; however, the role of melanocortins remains unclear. MATERIALS AND METHODS: The effects of bremelanotide, a melanocortin receptor agonist, alone or in combination with chemotherapeutic agents, on survivin expression and cell viability were investigated in human glioblastoma cell lines. RESULTS: Bremelanotide reduced survivin expression and induced cell death in glioblastoma cells at concentrations that were not toxic to normal human cells, and both of these effects were canceled in the presence of an antagonist of melanocortin receptors 3 and 4. Bremelanotide-induced cell death was prevented by the forced over-expression of survivin in glioblastoma cells, suggesting that bremelanotide induces glioblastoma cell death by inhibiting the expression of survivin. Bremelanotide also promoted cell death induced by chemotherapeutic agents, such as temozolomide and osimertinib. CONCLUSION: The present results identified melanocortin receptors 3 and 4 as novel and viable therapeutic targets for glioblastoma. Activation of these receptors by bremelanotide may inhibit the expression of survivin, thereby sensitizing glioblastoma cells to cell death.

Symptomatic recurrence of intracranial arterial dissections: follow-up study of 143 consecutive cases and pathological investigation.

BACKGROUND AND PURPOSE: The frequency and pattern of symptomatic recurrence of spontaneous intracranial arterial dissection (IAD) are unknown. METHODS: A follow-up study of 143 patients (85 men, 58 women; mean age, 50.7 [7-83] years) with spontaneous IADs at The University of Tokyo and affiliated hospitals from 1980 to 2000 was conducted. Tissue samples of IAD vessels obtained from 13 patients at various intervals from onset were also examined histologically. RESULTS: With a mean follow-up of 8.2 years, symptomatic recurrence occurred in 47 patients (33%). Of 37 cases initially presenting with hemorrhage, 35 developed hemorrhagic recurrence with a mean interval of 4.8 days, and 2 developed nonhemorrhagic recurrences after 21 and 85 months, respectively. Of 10 patients initially presenting with nonhemorrhagic symptoms, 1 developed hemorrhagic recurrence 4 days later, and 9 developed nonhemorrhagic recurrences with a mean interval of 8.6 months. Histopathologically, the affected vessels in the acute stage of hemorrhage (days 0-6) demonstrated insufficient granulation formation within the pseudolumen, followed by marked intimal thickening around the pseudolumen and recanalizing vessel formation in the late stage (>day 30). In the late stage of brain ischemia, subintimal and subadventitial hemorrhage accompanied with intimal thickening was observed. CONCLUSIONS: These data indicate that IAD is a disease carrying a relatively high risk of symptomatic recurrence, apparently occurring in 3 phases and patterns: early hemorrhagic recurrence, late nonhemorrhagic recurrence, and chronic fusiform aneurysm transformation. Knowledge of this triphasic recurrence and corresponding histopathological characteristics help determine the treatment and follow-up strategy for IAD patients.

Serum prolactin levels and prolactin mRNA expression in peripheral blood mononuclear cells in hepatitis C virus infection.

Prolactin is not only a pituitary hormone but an immunoregulatory hormone secreted from lymphocytes. Prolactin induction in relation to hepatitis C virus (HCV) infection has not been elucidated. The serum levels of prolactin were examined in 232 HCV-infected subjects positive for anti-HCV antibody and 65 healthy controls negative for it, who were recruited in the cohort study. The prolactin mRNAs were measured in peripheral blood mononuclear cells (PBMCs) of eleven healthy volunteers including five men and six women before and after stimulation by HCV in vitro. The serum level of prolactin and prolactin mRNA in PBMCs were measured by chemiluminescence immunoassay and real-time PCR, respectively. The serum levels of prolactin were significantly higher in the HCV-infected subjects (median: 7.5, IQR: 5.7-10.9 ng/ml) than in the controls (median: 5.6, IQR: 4.4-8.3 ng/ml) (P < 0.01). They were significantly higher in HCV-infected males (median: 8.0, IQR: 5.9-11.8 ng/ml) than in the controls (median: 4.8, IQR: 4.2-5.9 ng/ml) (P < 0.001), however, the difference was not significant between HCV-infected females (median: 7.3, IQR: 5.6-10.5 ng/ml) and the controls (median: 6.4, IQR: 5.3-9.8 ng/ml). The mRNA expression of prolactin was induced in PBMCs of all males, but it was induced in PBMCs of the two of six females examined in vitro. These results suggest that the serum level of prolactin is higher in HCV-infected males than in healthy males, and that HCV infection induces the mRNA expression of prolactin in PBMCs that is more apparent in male than in females.

Givinostat Inhibition of Sp1-dependent MGMT Expression Sensitizes Glioma Stem Cells to Temozolomide.

BACKGROUND/AIM: Givinostat is a pan-histone deacetylase (HDAC) inhibitor that has demonstrated excellent tolerability as well as efficacy in patients with polycythemia vera. Accumulating in vitro and in vivo evidence suggests givinostat is also promising as a therapeutic agent targeting glioma stem cells (GSCs), the cancer stem cells of glioblastoma (GBM) considered responsible for its intractable nature. However, it remains to be shown how givinostat impacts the therapeutic effects of temozolomide, a DNA-alkylating agent and the key component of GBM treatment given not only during postoperative radiotherapy but also thereafter as maintenance chemotherapy. MATERIALS AND METHODS: The effects of givinostat and knockdown of O6-methylguanine-DNA methyltransferase (MGMT) or Sp1 on the mRNA and protein expression of relevant genes in human GSC lines were examined by RT-PCR and western blot analyses. The dye exclusion method was used to evaluate cell viability. RESULTS: Givinostat enhanced the cytotoxic activity of temozolomide in GSC lines expressing MGMT, in which the MGMT expression was shown to contribute to their temozolomide resistance. Givinostat inhibited MGMT expression in GSCs and, in parallel, the expression of Sp1, a transcription factor involved in the control of MGMT promoter activity. Knockdown experiments demonstrated Sp1 expression was indeed required for MGMT expression in GSCs. CONCLUSION: Givinostat, in addition to its own cytotoxic activity, sensitizes GSCs to temozolomide by inhibiting Sp1-dependent MGMT expression in GSCs. Combining givinostat with temozolomide could therefore be a rational therapeutic strategy to effectively eliminate GSCs and thus help overcome the therapy resistance of GBM.

Antagonizing MDM2 Overexpression Induced by MDM4 Inhibitor CEP-1347 Effectively Reactivates Wild-Type p53 in Malignant Brain Tumor Cells.

The development of MDM4 inhibitors as an approach to reactivating p53 in human cancer is attracting increasing attention; however, whether they affect the function of MDM2 and how they interact with MDM2 inhibitors remain unknown. We addressed this question in the present study using CEP-1347, an inhibitor of MDM4 protein expression. The effects of CEP-1347, the genetic and/or pharmacological inhibition of MDM2, and their combination on the p53 pathway in malignant brain tumor cell lines expressing wild-type p53 were investigated by RT-PCR and Western blot analyses. The growth inhibitory effects of CEP-1347 alone or in combination with MDM2 on inhibition were examined by dye exclusion and/or colony formation assays. The treatment of malignant brain tumor cell lines with CEP-1347 markedly increased MDM2 protein expression, while blocking CEP-1347-induced MDM2 overexpression by genetic knockdown augmented the effects of CEP-1347 on the p53 pathway and cell growth. Blocking the MDM2-p53 interaction using the small molecule MDM2 inhibitor RG7112, but not MDM2 knockdown, reduced MDM4 expression. Consequently, RG7112 effectively cooperated with CEP-1347 to reduce MDM4 expression, activate the p53 pathway, and inhibit cell growth. The present results suggest the combination of CEP-1347-induced MDM2 overexpression with the selective inhibition of MDM2's interaction with p53, while preserving its ability to inhibit MDM4 expression, as a novel and rational strategy to effectively reactivate p53 in wild-type p53 cancer cells.

The Novel MDM4 Inhibitor CEP-1347 Activates the p53 Pathway and Blocks Malignant Meningioma Growth In Vitro and In Vivo.

A significant proportion of meningiomas are clinically aggressive, but there is currently no effective chemotherapy for meningiomas. An increasing number of studies have been conducted to develop targeted therapies, yet none have focused on the p53 pathway as a potential target. In this study, we aimed to determine the in vitro and in vivo effects of CEP-1347, a small-molecule inhibitor of MDM4 with known safety in humans. The effects of CEP-1347 and MDM4 knockdown on the p53 pathway in human meningioma cell lines with and without p53 mutation were examined by RT-PCR and Western blot analyses. The growth inhibitory effects of CEP-1347 were examined in vitro and in a mouse xenograft model of meningioma. In vitro, CEP-1347 at clinically relevant concentrations inhibited MDM4 expression, activated the p53 pathway in malignant meningioma cells with wild-type p53, and exhibited preferential growth inhibitory effects on cells expressing wild-type p53, which was mostly mimicked by MDM4 knockdown. CEP-1347 effectively inhibited the growth of malignant meningioma xenografts at a dose that was far lower than the maximum dose that could be safely given to humans. Our findings suggest targeting the p53 pathway with CEP-1347 represents a novel and viable approach to treating aggressive meningiomas.

CEP-1347 Dually Targets MDM4 and PKC to Activate p53 and Inhibit the Growth of Uveal Melanoma Cells.

Uveal melanoma (UM) is among the most common primary intraocular neoplasms in adults, with limited therapeutic options for advanced/metastatic disease. Since UM is characterized by infrequent p53 mutation coupled with the overexpression of MDM4, a major negative regulator of p53, we aimed to investigate in this study the effects on UM cells of CEP-1347, a novel MDM4 inhibitor with a known safety profile in humans. We also examined the impact of CEP-1347 on the protein kinase C (PKC) pathway, known to play a pivotal role in UM cell growth. High-grade UM cell lines were used to analyze the effects of genetic and pharmacological inhibition of MDM4 and PKC, respectively, as well as those of CEP-1347 treatment, on p53 expression and cell viability. The results showed that, at its clinically relevant concentrations, CEP-1347 reduced not only MDM4 expression but also PKC activity, activated the p53 pathway, and effectively inhibited the growth of UM cells. Importantly, whereas inhibition of either MDM4 expression or PKC activity alone failed to efficiently activate p53 and inhibit cell growth, inhibition of both resulted in effective activation of p53 and inhibition of cell growth. These data suggest that there exists a hitherto unrecognized interaction between MDM4 and PKC to inactivate the p53-dependent growth control in UM cells. CEP-1347, which dually targets MDM4 and PKC, could therefore be a promising therapeutic candidate in the treatment of UM.

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 O(6) -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.

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.

Gemcitabine Cooperates with Everolimus to Inhibit the Growth of and Sensitize Malignant Meningioma Cells to Apoptosis Induced by Navitoclax, an Inhibitor of Anti-Apoptotic BCL-2 Family Proteins.

Despite several clinical trials with encouraging findings, effective standard systemic therapies have yet to be established for malignant meningioma and the prognosis of these patients remains poor. Accumulating preclinical and clinical evidence suggests that gemcitabine is effective against malignant meningioma. To identify drugs with therapeutic effects that may be enhanced in combination with gemcitabine, we screened drugs that have been tested in preclinical and clinical trials for meningioma. In IOMM-Lee and HKBMM malignant meningioma cells, gemcitabine enhanced the growth inhibitory effects of the mTOR inhibitor everolimus, the clinical benefits of which have been demonstrated in patients with meningioma. The synergistic growth inhibitory effects of this combination were accompanied by cellular senescence characterized by an increase in senescence-associated ß-galactosidase activity. To enhance the effects of this combination, we screened senolytic drugs that selectively kill senescent cells, and found that navitoclax, an inhibitor of anti-apoptotic BCL-2 family proteins, effectively reduced the number of viable malignant meningioma cells in combination with everolimus and gemcitabine by inducing apoptotic cell death. The suppression of tumor growth in vivo by the combination of everolimus with gemcitabine was significantly stronger than that by either treatment alone. Moreover, navitoclax, in combination with everolimus and gemcitabine, significantly reduced tumor sizes with an increase in the number of cleaved caspase-3-positive apoptotic cells. The present results suggest that the addition of gemcitabine with or without navitoclax to everolimus is a promising strategy that warrants further evaluation in future clinical trials for malignant meningioma.