Tatsuya Takezaki

Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Recent findings have shown that malignant tumors contain cancer-initiating cells (CIC), which self-renew and are tumorigenic. However, CICs have not been characterized properly due to lack of specific markers. We recently established a mouse glioma cell line, NSCL61, by overexpressing an oncogenic HRas(L61) in p53-deficient neural stem cells. Using limiting dilution assays, we show that only 2 of 24 NSCL61 clones retained their tumorigenicity in vivo, although the others also expressed oncogenic HRas(L61) and could proliferate in culture. A comparison of the gene expression profiles of tumorigenic and nontumorigenic clones showed that the tumorigenic clones had lost Sox11 expression. We show that overexpression of sox11 prevented tumorigenesis of NSCL61s by inducing their neuronal differentiation accompanied with decreased levels of plagl1. We also show that overexpression of plagl1 abolished neuronal commitment of nontumorigenic cells and induced them to become tumorigenic. Moreover, we show that human glioma-initiating cells lost sox11 expression, and overexpression of sox11 prevented their tumorigenesis in vivo. Together with the clinical evidence showing that downregulation of sox11 mRNA correlates with a significant decrease in survival, these findings suggest that Sox11 prevents gliomagenesis by blocking the expression of oncogenic plagl1.

Essential role of the Hedgehog signaling pathway in human glioma‐initiating cells

Recent findings have demonstrated that malignant tumors, including glioblastoma multiforme, contain cancer‐initiating cells (also known as cancer stem cells), which self‐renew and are malignant, with features of tissue‐specific stem cells. As these cells are resistant to irradiation and anti‐cancer drugs, it is important to characterize them and find targeting therapies. In this study, we established two primary human glioma cell lines from anaplastic oligodendroglioma and glioblastoma multiforme. These lines were enriched in glioma‐initiating cells, as just 10 cells formed malignant glioma when injected into mouse brain. We used these cell lines to examine the roles of the Notch, Hedgehog and Wnt signaling pathways, which are involved in stem‐cell maintenance and tumorigenesis, to determine which of these pathways are crucial to glioma‐initiating cells and their regulation. Here we show that the Hedgehog pathway is indispensable for glioma‐initiating cell proliferation and tumorigenesis; the Hedgehog signaling inhibitors prevented glioma‐initiating cell proliferation, while signaling inhibitors for Notch or Wnt did not. Overexpression of Gli2ΔC, a C‐terminal‐truncated form of Gli2 that antagonizes Gli transcription factor functions, blocked glioma‐initiating cell proliferation in culture and tumorigenesis in vivo. Knockdown of the Gli downstream factor Cdc2 also prevented glioma‐initiating cell proliferation. Taken together, these results show that the Hedgehog→ Gli→ Cdc2 signaling cascade plays a role in the proliferation and malignancy of glioma‐initiating cells. (Cancer Sci 2011; 102: 1306–1312)

TGF-β Mediates Homing of Bone Marrow-Derived Human Mesenchymal Stem Cells to Glioma Stem Cells

Although studies have suggested that bone marrow human mesenchymal stem cells (BM-hMSC) may be used as delivery vehicles for cancer therapy, it remains unclear whether BM-hMSCs are capable of targeting cancer stem cells, including glioma stem cells (GSC), which are the tumor-initiating cells responsible for treatment failures. Using standard glioma models, we identify TGF-β as a tumor factor that attracts BM-hMSCs via TGF-β receptors (TGFβR) on BM-hMSCs. Using human and rat GSCs, we then show for the first time that intravascularly administered BM-hMSCs home to GSC-xenografts that express TGF-β. In therapeutic studies, we show that BM-hMSCs carrying the oncolytic adenovirus Delta-24-RGD prolonged the survival of TGF-β-secreting GSC xenografts and that the efficacy of this strategy can be abrogated by inhibition of TGFβR on BM-hMSCs. These findings reveal the TGF-β/TGFβR axis as a mediator of the tropism of BM-hMSCs for GSCs and suggest that TGF-β predicts patients in whom BM-hMSC delivery will be effective.

Mesenchymal Stem Cells Isolated From Human Gliomas Increase Proliferation and Maintain Stemness of Glioma Stem Cells Through the IL-6/gp130/STAT3 Pathway.

Although mesenchymal stem cells (MSCs) have been implicated as stromal components of several cancers, their ultimate contribution to tumorigenesis and their potential to drive cancer stem cells, particularly in the unique microenvironment of human brain tumors, remain largely undefined. Consequently, using established criteria, we isolated glioma‐associated‐human MSCs (GA‐hMSCs) from fresh human glioma surgical specimens for the first time. We show that these GA‐hMSCs are nontumorigenic stromal cells that are phenotypically similar to prototypical bone marrow‐MSCs. Low‐passage genomic sequencing analyses comparing GA‐hMSCs with matched tumor‐initiating glioma stem cells (GSCs) suggest that most GA‐hMSCs (60%) are normal cells recruited to the tumor (group 1 GA‐hMSCs), although, rarely (10%), GA‐hMSCs may differentiate directly from GSCs (group 2 GA‐hMSCs) or display genetic patterns intermediate between these groups (group 3 GA‐hMSCs). Importantly, GA‐hMSCs increase proliferation and self‐renewal of GSCs in vitro and enhance GSC tumorigenicity and mesenchymal features in vivo, confirming their functional significance within the GSC niche. These effects are mediated by GA‐hMSC‐secreted interleukin‐6, which activates STAT3 in GSCs. Our results establish GA‐hMSCs as a potentially new stromal component of gliomas that drives the aggressiveness of GSCs, and point to GA‐hMSCs as a novel therapeutic target within gliomas. Stem Cells 2015;33:2400–2415

Brain tumor stem cells as research and treatment targets

Glioblastoma multiforme (GBM) is one of the most malignant forms of human cancer. Despite intensive treatment, the mean survival of GBM patients remains about 1 year. Recent cancer studies revealed that cancer tissues are pathologically heterogeneous and only a small population of cells has the specific ability to reinitiate cancer. This small cell population is called cancer stem cells (CSCs); in brain tumors these are known as brain tumor stem cells (BTSCs). The identification of BTSCs yielded new insights into chemo-and radioresistance, by which BTSCs can survive selectively and initiate recurrence. Research focused on BTSCs as treatment targets may contribute to the discovery of new therapeutic strategies.

Ceacam1L modulates STAT3 signaling to control the proliferation of glioblastoma-initiating cells

Glioblastoma-initiating cells (GIC) are a tumorigenic cell subpopulation resistant to radiotherapy and chemotherapy, and are a likely source of recurrence. However, the basis through which GICs are maintained has yet to be elucidated in detail. We herein demonstrated that the carcinoembryonic antigen-related cell adhesion molecule Ceacam1L acts as a crucial factor in GIC maintenance and tumorigenesis by activating c-Src/STAT3 signaling. Furthermore, we showed that monomers of the cytoplasmic domain of Ceacam1L bound to c-Src and STAT3 and induced their phosphorylation, whereas oligomerization of this domain ablated this function. Our results suggest that Ceacam1L-dependent adhesion between GIC and surrounding cells play an essential role in GIC maintenance and proliferation, as mediated by signals transmitted by monomeric forms of the Ceacam1L cytoplasmic domain.

Glioma Initiating Cells Form a Differentiation Niche Via the Induction of Extracellular Matrices and Integrin αV

Glioma initiating cells (GICs) are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanism of GIC maintenance/differentiation, we established GIC clones having the potential to differentiate into malignant gliomas, and subjected to DNA microarray/iTRAQ based integrated proteomics. 21,857 mRNAs and 8,471 proteins were identified and integrated into a gene/protein expression analysis chart. Gene Ontology analysis revealed that the expression of cell adhesion molecules, including integrin subfamilies, such as α2 and αV, and extracellular matrices (ECMs), such as collagen IV (COL4), laminin α2 (LAMA2), and fibronectin 1 (FN), was significantly upregulated during serum-induced GIC differentiation. This differentiation process, accompanied by the upregulation of MAPK as well as glioma specific proteins in GICs, was dramatically accelerated in these ECM (especially FN)-coated dishes. Integrin αV blocking antibody and RGD peptide significantly suppressed early events in GIC differentiation, suggesting that the coupling of ECMs to integrin αV is necessary for GIC differentiation. In addition, the expression of integrin αV and its strong ligand FN was prominently increased in glioblastomas developed from mouse intracranial GIC xenografts. Interestingly, during the initial phase of GIC differentiation, the RGD treatment significantly inhibited GIC proliferation and raised their sensitivity against anti-cancer drug temozolomide (TMZ). We also found that combination treatments of TMZ and RGD inhibit glioma progression and lead the longer survival of mouse intracranial GIC xenograft model. These results indicate that GICs induce/secrete ECMs to develop microenvironments with serum factors, namely differentiation niches that further stimulate GIC differentiation and proliferation via the integrin recognition motif RGD. A combination of RGD treatment with TMZ could have the higher inhibitory potential against the glioma recurrence that may be regulated by the GICs in the differentiation niche. This study provides a new perspective for developing therapeutic strategies against the early onset of GIC-associated glioma.

Antitumor effect of fibrin glue containing temozolomide against malignant glioma.

Temozolomide (TMZ), used to treat glioblastoma and malignant glioma, induces autophagy, apoptosis and senescence in cancer cells. We investigated fibrin glue (FG) as a drug delivery system for the local administration of high‐concentration TMZ aimed at preventing glioma recurrence. Our high‐power liquid chromatography studies indicated that FG containing TMZ (TMZ‐FG) manifested a sustained drug release potential. We prepared a subcutaneous tumor model by injecting groups of mice with three malignant glioma cell lines and examined the antitumor effect of TMZ‐FG. We estimated the tumor volume and performed immunostaining and immunoblotting using antibodies to Ki‐67, cleaved caspase 3, LC3 and p16. When FG sheets containing TMZ (TMZ‐FGS) were inserted beneath the tumors, their growth was significantly suppressed. In mice treated with peroral TMZ plus TMZ‐FGS the tumors tended to be smaller than in mice whose tumors were treated with TMZ‐FGS or peroral TMZ alone. The TMZ‐FGS induced autophagy, apoptosis and senescence in subcutaneous glioma tumor cells. To assess the safety of TMZ‐FG for normal brain, we placed it directly on the brain of living mice and stained tissue sections obtained in the acute and chronic phase immunohistochemically. In both phases, TMZ‐FG failed to severely damage normal brain tissue. TMZ‐FG may represent a safe new drug delivery system with sustained drug release potential to treat malignant glioma.

New Treatment Strategies to Eradicate Cancer Stem Cells and Niches in Glioblastoma

Glioblastoma multiforme (GBM) harbors are not only rapidly dividing cells but also small populations of slowly dividing and dormant cells with tumorigenesity, self-renewal, and multi-lineage differentiation capabilities. Known as glioblastoma stem cells (GSCs), they are resistant to conventional chemo- and radiotherapy and may be a causative factor in recurrence. The treatment outcome in patients with GBM remains unsatisfactory and their mean survival time has not improved sufficiently. We studied clinical evidence and basic research findings to assess the possibility of new treatment strategies that target GSCs and their specific microenvironments (GBM niches) and raise the possibility of adding new treatments to eradicate GSCs and GBM niches.

Characteristics of brain metastases from esophageal carcinoma

Background: Esophageal carcinoma (EC) is a major malignancy with a poor prognosis. Although esophageal cancers rarely metastasize to the brain, the number of patients diagnosed with brain metastases (BM) from EC is steadily increasing. Therefore, the risk factors for BM from EC should be known. Here we reviewed our experiences and the previous literature regarding BM from EC. Methods: Between 2000 and 2013, we retrospectively reviewed the clinical features and neurological findings of 19 patients diagnosed with and treated for BM from EC to determine the clinical risk factors and features. Results: In all patients, the lesions were partially or completed located in the thoracic esophagus, and the average size of the EC lesion at diagnosis was 5.8 ± 2.9 cm, which was smaller than the previously reported size of EC lesions accompanied by BM. Patients without lung metastases were more common than those with lung metastases. The lesions in the 13 patients included squamous cell carcinoma (SqCC) in 9 (69.2%) and small cell carcinoma (SmCC) in 3 (23.0%). Six patients were not examined. Although there was no trend toward a higher incidence of BM in patients with adenocarcinoma and SqCC, this trend was observed in patients with SmCC. Excluding a single patient with SmCC, all patients had beyond stage III disease at EC diagnosis. Conclusions: Our study suggests that BM can occur in patients with EC lesions smaller than those previously reported; moreover, SmCC may be a risk factor for BM from EC.