Kaori Sakurada

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activation of p70S6K, and siRNA‐mediated knockdown of p70S6K resulted in the activation of both pathways, which no longer maintained the cross‐inhibitory relationship.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

Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32.

AbstractBcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74–282)) and the conserved C-terminal region (p32 (1–221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.

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

Association of stem cell marker CD133 expression with dissemination of glioblastomas

Dissemination of glioblastoma was once considered rare but is now increasingly encountered with longer survival of glioblastoma patients. Despite the potential negative impact of dissemination on clinical outcome, however, molecular markers useful for prediction of dissemination risk still remains ill defined. We tested in this study for an association between the expression of stem cell marker CD133 and the risk of dissemination in 26 cases of glioblastoma (16 with dissemination and 10 without dissemination). The protein expression of CD133 was examined by western blot analysis of tumor specimens, and the CD133 expression levels were quantified by densitometry and normalized to β-actin. The results indicated that CD133 expression levels are significantly higher in glioblastomas with dissemination (mean 10.3, range 0.20-27.8) than in those without (mean 1.18, range 0.07-3.58). The results suggest that CD133 could be a molecular predictor of glioblastoma dissemination, and also give rise to an intriguing idea that CD133-positive cancer stem cells may be implicated in the initiation of disseminated lesions.

Surgical resection of tumors located in subcortex of language area.

SummaryObject. Although functional mapping facilitates the planning of surgery in and around eloquent areas, the resection of tumors adjacent to language areas remains challenging. In this report, we took notice that the language areas (Broca’s and Wernicke’s) present at the perisylvian fissure. We posit that if there is non-essential language area on the inner surface of the Sylvian fissure, safe tumor resection may be possible even if the tumor is located under the language cortex. Methods. The study population consisted of 5 patients with intrinsic brain tumors (frontal glioma, n = 3; temporal cavernous angioma, n = 1; primary malignant central nervous system lymphoma, n = 1) located in the perisylvian subcortex, in the language-dominant hemisphere. All patients underwent awake surgery and we performed intra-operative bipolar cortical functional language mapping. When the tumor was located under the language area, the Sylvian fissure was opened and the inner surface of the opercular cortex was exposed with the patient asleep, and additional functional mapping of that cortex was performed. This enabled us to remove the tumor from the non-functioning cortex.In our series, 4 of 5 patients had not language function on the inner surface of the operculum. Only one patient, a 52-year-old man with frontal glioblastoma (Case 3) had language function on the inner surface of the frontal operculum. Conclusion. We suggest that even perisylvian tumors located in the subcortex of the language area may be resectable via the nonfunctioning intrasylvian cortex by a transopercular approach without resultant language dysfunction.

Nestin expression in central nervous system germ cell tumors

Central nervous system (CNS) germ cell tumors constitute a unique class of rare tumors that mainly affect children and adolescents. These tumors are believed to originate from displaced primordial germ cells. Recently, results of treatment of germ cell tumors have improved with use of radiotherapy and combination chemotherapy. However, some tumors have proven refractory to intensive treatment with surgery, radiation, and combination chemotherapy. Nestin is an intermediate filament protein expressed in undifferentiated cells during CNS development and in CNS tumors and is used as a marker of immature elements of tumors, including brain tumor stem cells. In this study, we examined for the first time nestin expression in 19 CNS germ cell tumors (nine pure germinomas, five germinomas with syncytiotrophoblastic giant cells, one yolk sac tumor, one choriocarcinoma, one embryonal carcinoma, and two mature teratomas). Nestin was expressed in 14 cases but was not expressed in three pure germinomas and two mature teratomas. Clinically, nestin-negative tumors did not exhibit dissemination, while all tumors that exhibited dissemination also strongly expressed nestin protein. These findings suggest that the detection of nestin expression could be useful in the management of CNS germ cell tumors, as an auxiliary predictor of dissemination and/or progression.

A rare case of extraventricular neurocytoma

We report an unusual case of extraventricular (cerebral) neurocytoma with a long, 25-year history, and which appeared to transform to neuroblastoma. In 1978, an 18-year-old woman was treated for right frontal oligodendroglioma. Eighteen years later (in 1996), recurrence of tumor in the fourth ventricle was noted and was treated with gamma-knife radiotherapy. The tumor shrunk transiently, but 7 years later (in 2004), MRI study demonstrated a second recurrence and ventricular dissemination. Partial removal was performed, and histological examination revealed that tumor cells had round or oval nuclei with halos. Frequent mitoses and vascular proliferation were observed. The MIB-1 LI was 80%. Despite postoperative whole-brain radiotherapy to a total dose of 30 Gy, the tumor progressed, and she died at 4 months after the second surgery.

Two cases of atypical central neurocytomas

Central neurocytomas (CNs) are usually considered benign; however, some subsets of these tumors with histologic atypia and elevated proliferation potential have been reported to have a poor outcome. It has been proposed that those CNs exhibiting a MIB-1 labeling index (LI) greater than 2% and/or vascular proliferation be defined as atypical central neurocytoma (ACN). Two cases of ACN are reported here. Case 1 was a 13-year-old male who complained of headache. An MRI showed an enhancement of the tumor in the third ventricle and left lateral ventricle. The tumor was completely removed transcortically by a left frontotemporal craniotomy. The tumor showed anaplastic features, including microvascular proliferation, mitosis, and necrosis. The MIB-1 LI was 7.8%. The patient received 50 Gy of focal irradiation. After 16 months of follow-up, no recurrence was observed. Case 2 was a 17-year-old female who presented with headache. An MRI showed an enhancement of the tumor in the right lateral ventricle. To preserve the internal cerebral vein, the tumor was partially removed by the transcingulate approach after a bifrontal craniotomy. Histologically, the tumor displayed the typical features of a CN. The MIB-1 LI was 7.0%. Complete remission was achieved through 50 Gy of focal irradiation. After 15 months of follow-up, there was no recurrence. The differences in the histopathological findings in these two cases indicate the presence of a variety of histopathological features among ACNs.