Kenichiro Matsuda

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reduced activation of p70S6K, and siRNA‐mediated knockdown of p70S6K resulted in the activation of both pathways, which no longer maintained the cross‐inhibitory relationship.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

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.

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

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.

An esophageal neuroendocrine tumor metastatic to the pineal region

With the aging of society, the number of patients with cancer has been increasing, and cases of metastatic brain tumors have also been increasing. Autopsy studies to date have reported metastatic brain lesions in 10–15 % of cancer patients. The clinical incidence of metastatic brain lesions has been also reported to be 6–10 %. Primary tumors with a high incidence in adults include lung cancer (small cell carcinoma, adenocarcinoma), breast cancer, renal cell carcinoma, colon cancer, and malignant melanoma. However, the primary tumor is unknown in about 10 % of patients with metastatic brain lesions [1–3]. The presence of metastatic brain lesions indicates an advanced stage (stage 4) of the primary cancer and a poor prognosis. However, various neurological symptoms due to an intracranial tumor or due to treatment itself can also often markedly decrease QOL. Research on the treatment of metastatic brain tumors is being conducted at many institutions, and it is important not to impair QOL. A multicenter collaborative study of metastatic brain tumors is now also being conducted in Japan. Most metastatic brain tumors are located in the cerebral hemispheres, but a few occur in the cerebellum or meninges. However, pineal region metastases are very rare; according to recent statistics, they account for only 0.3 % of all intracranial metastases [3]. Moreover, the pineal region is a site of predilection for primary tumors of the pineal region and germ cell tumors. Thus, when lesions are seen in the pineal region, these tumors have higher priority in the differential diagnosis. The patient in this case report had an esophageal neuroendocrine tumor (NET), which itself is rare, as a primary tumor. Therefore, this very rare case is reported.

A case of Langerhans cell histiocytosis of the skull in which preoperative methionine positron emission tomography was useful in comprehending the spreading of the lesion.

Background: The problem with treatments against skull lesions of Langerhans cell histiocytosis (LCH) is that invasions often reach the bones and dura mater, making it difficult to accurately comprehend the range thereof prior to surgery. We herein report that 11C-methionine positron emission tomography (PET) (Met-PET) carried out prior to surgery was useful in comprehending the spreading of the lesion. Case Description: A 20-year-old female presented with swelling and dull pain on the left side of the head. A slightly heterogeneously reinforced tumor was observed inside the bone defect in the gadolinium-enhanced T1-wighted image upon magnetic resonance imaging (MRI) and the dura mater contacting the tumor was observed with an enhancing effect. Accumulation was poor in the center of the tumor upon Met-PET, and accumulation with a well-defined border was observed in the border thereof as well as the area adjacent to the brain. Surgical resection was performed; the pathological diagnosis was LCH. An invasion of tumor cells was observed in the dura mater with accumulation observed according to Met-PET. Moreover, the accumulation of tumor cells was observed in the area observed with accumulation inside the bone; however, the center part with poor accumulation lacked tumor cells, with fibrous tissue accounting for most parts. Conclusion: Met-PET was believed to be helpful in comprehending the spreading of the tumor in the surroundings of the brain surface for skull lesions.

Opening the ventricle during surgery diminishes survival among patients with newly diagnosed glioblastoma treated with carmustine wafers: a multi-center retrospective study

Carmustine wafers (CW) were approved in Japan for newly diagnosed and recurrent malignant gliomas during 2013. The ventricle is often opened during surgery to achieve maximum resection. While not generally recommended in such situations, CW might be safely achieved by occluding an opened ventricle using gelform or collagen sheets. However, whether CW implantation actually confers a survival benefit for patients who undergo surgery with an open ventricle to treat glioblastoma remains unclear. Clinical, imaging, and survival data were collected in this multicenter retrospective study of 122 consecutive patients with newly diagnosed glioblastoma to determine adverse events and efficacy. Overall, 54 adverse events of all grades developed in 35 (28.6%) patients, with the most common being new seizures (16%). Adverse events did not significantly differ between patients with opened and closed ventricles during surgery. The 10- and 21.7-month, median, progression-free (PFS) and overall survival (OS), respectively did not significantly differ according to resection rates. However, median PFS and OS were significantly longer among patients with closed, than open ventricles (12.8 vs. 7.4 months; p = 0.0039 and 26.9 vs. 18.6 months; p = 0.011, respectively). Implanting CW into the resection cavity during concomitant radiochemotherapy with temozolomide seems to yield better survival rates without increased adverse events. Occlusion of the ventricular opening during surgery might be safe for CW implantation, but less so for treating patients with newly diagnosed glioblastoma.

Functional significance of the electrocorticographic auditory responses in the premotor cortex

Other than well-known motor activities in the precentral gyrus, functional magnetic resonance imaging (fMRI) studies have found that the ventral part of the precentral gyrus is activated in response to linguistic auditory stimuli. It has been proposed that the premotor cortex in the precentral gyrus is responsible for the comprehension of speech, but the precise function of this area is still debated because patients with frontal lesions that include the precentral gyrus do not exhibit disturbances in speech comprehension. We report on a patient who underwent resection of the tumor in the precentral gyrus with electrocorticographic recordings while she performed the verb generation task during awake brain craniotomy. Consistent with previous fMRI studies, high-gamma band auditory activity was observed in the precentral gyrus. Due to the location of the tumor, the patient underwent resection of the auditory responsive precentral area which resulted in the post-operative expression of a characteristic articulatory disturbance known as apraxia of speech (AOS). The language function of the patient was otherwise preserved and she exhibited intact comprehension of both spoken and written language. The present findings demonstrated that a lesion restricted to the ventral precentral gyrus is sufficient for the expression of AOS and suggest that the auditory-responsive area plays an important role in the execution of fluent speech rather than the comprehension of speech. These findings also confirm that the function of the premotor area is predominantly motor in nature and its sensory responses is more consistent with the “sensory theory of speech production,” in which it was proposed that sensory representations are used to guide motor-articulatory processes.