Development of combined cellular and molecular target-directed therapies for glioblastoma

2014 Fiscal Year Annual Research Report

• Project/Area Number: 26893096
• Research Institution: Kanazawa University
• Principal Investigator: PYKO Ilya 金沢大学, がん進展制御研究所, 博士研究員 (00731853)
• Project Period (FY): 2014-08-29 – 2016-03-31
• Keywords: glioblastoma / temozolomide / mesenchymal stem cells / GSK 3β

Outline of Annual Research Achievements

Studies in vitro and in animal model were carried out complementary to explore whether mesenchymal stem cells (MSCs) transplantation benefits to treatment of glioblastoma (GBM) by enhancing anti-tumor effect in combination with GSK3β inhibitors and temozolomide (TMZ). Our experiments showed that transplantation of MSCs and GSK3β inhibition synergizes for treatment of experimental GBM and that MSCs co- cultured with GBM cells participate in regulation of GBM stemness phenotype similar to that observed under GSK3β inhibition. At present we examine the interaction between patient-derived GBM stem-like cells and adipose tissue-derived MSCs in the presence and absence of GSK3β inhibition and TMZ and its influence on GBM stemness phenotype. I found that the interaction of MSCs with GBM stem-like cells is enhanced by a combined treatment by GSK3β inhibitor. This observation encouraged us to investigate biological mechanisms by which MSCs regulate GBM stemness phenotype under the control by GSK3β starting from the screening for the phenomena of their interaction by confocal time-lapse microscopy.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

The following steps have been performed for the study. (1) preparation of 18 primary glioblastoma (GBM) stem-like cell cultures from patients with primary GBM; among which 14 with high O6-methylguanine DNA methyltransferase (MGMT) expression and resistant to temozolomide were used in experiments;
(2) 5 primary cultures of bone marrow mesenchymal stem cells (MSCs) and a primary neural stem cell (NSC) culture were isolated from C57BL mice;
(3) 7 primary MSC cultures were isolated from human adipose tissue samples with confirmed phenotype (CD14, CD29, CD31, CD34, CD44, CD45, CD90, CD105);
(4) I have investigated the effects of TMZ, GSK3β inhibition and co-culture of primary GBM stem-like cells and MSCs on GBM stemness phenotype by proliferation assay, time-lapse microscopy and by evaluation of MGMT promoter methylation status and gene expression;
(5) I have investigated the effects of GSK3β inhibition and intra-tumor transplantation of MSCs on neurological state, cognitive functions and survival of C57BL mice with GBM model. This model is generated by using GBM line EPNT-5(Institute for Cytology, Saint-Petersburg) it was induced in С57BL mouse by 7,12-dimethylbenzanthracene and adequately represents human GBM; I have investigated with fluorescent microscopy the effects on mice EPNT-5 GBM cells by co-culture with mice MSCs.

Strategy for Future Research Activity

For in vitro study, we isolate glioblastoma (GBM) stem-like cell culture from patients’ GBM tumors, mesenchymal stem cells (MSCs) from human adipose tissues and C57BL mice bone marrow, and primary neural stem cell cultures from C57BL mice. Molecular characteristics of these cells are examined to confirm the phenotypes of the respective cells. MSCs are labeled with a fluorescent linker and MSCs and GBM stem-like cells are transfected with GSK3β-specific short hairpin (sh)RNA expression vector. We then examine effects of MSCs, neural stem cells, GSK3β inhibition, TMZ and various combination of them against GBM stem-like cells. To address our working hypothesis of interaction between GBM stem-like cells and MSCs via GSK3β-mediated signaling, we investigate (a) c-Myc and O6-methylguanine DNA methyltransferase (MGMT) expression and MGMT promoter methylation; and (b) c-Myc and DNA (cytosine-5)-methyltransferase (DNMT) 3A binding to MGMT promoter according to our previous study (Carcinoenesis 2013).
For animal model study, we examine effects and underlying mechanism of intracranial transplantation of fluorescence-labeled MSCs, neural-transdifferentiated MSCs and neural stem cells against GBM in C57BL mice bearing mouse GBM. We observe survival as well as consequence of neurological state and cognitive functions in mice following transplantation of the respective cells.

Research Products

• [Journal Article] Glycogen Synthase Kinase 3β Sustains Invasion of Glioblastoma via the Focal Adhesion Kinase, Rac1, and c-Jun N-Terminal Kinase-Mediated Pathway (2015)
  • Author(s): Yuri Chikano, Takahiro Domoto, Takuya Furuta, Hemragul Sabit, Ayako Kitano-Tamura, Ilya V. Pyko, Takahisa Takino, Yoshimichi Sai, Yutaka Hayashi, Hiroshi Sato, Ken-ichi Miyamoto, Mitsutoshi Nakada, and Toshinari Minamoto
  • Journal Title: Mol Cancer Ther Volume: 14 Pages: 564-574
  • DOI: 10.1158/1535-7163.MCT-14-0479
  • Peer Reviewed
• [Presentation] Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O6-methylguanine DNA methyltransferase promoter methylation via c-Myc signaling (2015)
  • Author(s): Ilya V. Pyko, Mitsutoshi Nakada, Hemragul Sabit, Lei Teng, Natsuki Furuyama, Yutaka Hayashi, Kazuyuki Kawakami, Toshinari Minamoto, Аliaksandr S. Fedulau and Jun-ichiro Hamada
  • Organizer: Japan Cancer Association (JCA) Symposium
  • Place of Presentation: Kanazawa, Ishikawa, Japan
  • Year and Date: 2015-01-21 – 2015-01-22