Katsuyoshi Miyashita

Inhibition of GSK‐3β activity attenuates proliferation of human colon cancer cells in rodents

The authors’ recent discovery that glycogen synthase kinase‐3β (GSK‐3β) participates in colon cancer cells’ survival and proliferation prompted us to investigate whether GSK‐3β inhibition alters proliferation of colon cancer cells in vivo. Groups of four or five athymic mice (Balb/c, nu/nu) with subcutaneous xenografts of SW480 human colon cancer cells were treated with dimethyl sulfoxide (DMSO) or different doses (1, 2 and 5 mg/kg body weight) of either small‐molecule GSK‐3β inhibitor (SB‐216763 and AR‐A014418) by intraperitoneal injection three times per week for 5 weeks. Compared with DMSO (a diluent of the GSK‐3β inhibitors) as a control, either GSK‐3β inhibitor significantly inhibited proliferation of cancer cell xenografts in the rodents in a dose‐dependent manner. Histochemical and immunohistochemical analysis of tumor xenografts demonstrated a significant, dose‐dependent decrease in fractions of proliferating cells and an increase in the incidence of apoptosis of cancer cells in mice treated with either GSK‐3β inhibitor. No adverse events or effects were observed in the rodents during the course of treatment, except for rare lethal accidents due to intraperitoneal injection. Morphological examination showed no apparent pathologic changes in major organs including the lungs, liver, pancreas, kidneys, spleen and large bowel of rodents treated with DMSO and the GSK‐3β inhibitors. The results indicate that the GSK‐3β inhibitors would be a novel class of therapeutic agent for colon cancer. (Cancer Sci 2007; 98: 1388–1393)

Potential Therapeutic Effect of Glycogen Synthase Kinase 3β Inhibition against Human Glioblastoma

Purpose: Glioblastoma represents the malignant brain tumor that is most refractory to treatment and in which the identification of molecular target(s) is urgently required. We investigated the expression, activity, and putative pathologic role of glycogen synthase kinase 3β (GSK3β), an emerging therapeutic target for neurodegenerative diseases, in human glioblastoma. Experimental Design: The active fraction of GSK3β that is phosphorylated at the tyrosine 216 residue (pGSK3βY216) was identified in glioblastoma cell lines. GSK3β activity for phosphorylating its substrate was detected in these cells by nonradioisotopic in vitro kinase assay. Results: Higher expression levels of GSK3β and pGSK3βY216 were frequently detected in glioblastomas compared with nonneoplastic brain tissues. Inhibition of GSK3β activity by escalating doses of a small-molecule inhibitor (AR-A014418) or inhibition of its expression by RNA interference induced the apoptosis and attenuated the survival and proliferation of glioblastoma cells in vitro. Inhibition of GSK3β was associated with increased expression of p53 and p21 in glioblastoma cells with wild-type p53 and with decreased Rb phosphorylation and expression of cyclin-dependent kinase 6 in all glioblastoma cell lines. Administration of AR-A014418 at a low dose significantly sensitized glioblastoma cells to temozolomide and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea, chemotherapeutic agents used in the clinical setting, as well as to ionizing radiation. Conclusion: These results indicate that GSK3β exerts a pathologic role by promoting the survival and proliferation of glioblastoma cells and by protecting them from apoptosis via the inactivation of p53- and/or Rb-mediated pathways. Consequently, we propose that GSK3β provides a potential therapeutic target in glioblastoma.

Deregulated GSK3β Sustains Gastrointestinal Cancer Cells Survival by Modulating Human Telomerase Reverse Transcriptase and Telomerase

Purpose: Glycogen synthase kinase-3 (GSK3) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3 in gastrointestinal, pancreatic, and liver cancers. Experimental Design: Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3 expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3 activity by in vitro kinase assay. The effects of small-molecule GSK3 inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3 inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells. Results: Cancer cell lines and most cancer tissues showed increased GSK3 expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3 resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3 inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity. Conclusion: The results indicate that deregulated GSK3 sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase. (Clin Cancer Res 2009;15(22):68109)

An Emerging Strategy for Cancer Treatment Targeting Aberrant Glycogen Synthase Kinase 3β

Improvement in the outcome of cancer patients who are refractory to currently available treatments relies on the development of target-directed therapies. One group of molecular targets with potential clinical relevance is a set of protein tyrosine kinases encoded mostly by proto-oncogenes and that are frequently deregulated in cancer. Glycogen synthase kinase 3beta (GSK3beta), a serine/threonine protein kinase, has emerged as a therapeutic target for common chronic diseases including type 2 diabetes mellitus, neurodegenerative disorders, inflammation and osteoporosis. This is based on its currently known functions and primary pathologic causalities. GSK3beta has well characterized roles in the regulation of gene transcription and in oncogenic signaling. We have shown that deregulated GSK3beta promotes gastrointestinal, pancreatic and liver cancers and glioblastomas. Furthermore, we have demonstrated that inhibition of GSK3beta attenuates cell survival and proliferation, induces cell senescence and apoptosis and sensitizes tumor cells to chemotherapeutic agents and ionizing radiation. This has led us to propose GSK3beta as a potential therapeutic target in cancer. The anti-tumor effects of GSK3beta inhibition are mediated by changes in the expression and phosphorylation of molecules critical to the regulation of cell cycling, proliferation and apoptosis and underlie the pathological role for GSK3beta in cancer. Investigation of the mechanisms responsible for deregulation of GSK3beta and the consequent downstream pathologic effects in cancer cells has shed light on the molecular pathways leading to tumorigenesis. This will allow exploration of novel therapeutic strategies for cancer that target aberrant GSK3beta.

Glycogen synthase kinase-3β is a pivotal mediator of cancer invasion and resistance to therapy.

Tumor cell invasion and resistance to therapy are the most intractable biological characteristics of cancer and, therefore, the most challenging for current cancer research and treatment paradigms. Refractory cancers, including pancreatic cancer and glioblastoma, show an inextricable association between the highly invasive behavior of tumor cells and their resistance to chemotherapy, radiotherapy and targeted therapies. These aggressive properties of cancer share distinct cellular pathways that are connected to each other by several molecular hubs. There is increasing evidence to show that glycogen synthase kinase (GSK)‐3β is aberrantly activated in various cancer types and this has emerged as a potential therapeutic target. In many but not all cancer types, aberrant GSK3β sustains the survival, immortalization, proliferation and invasion of tumor cells, while also rendering them insensitive or resistant to chemotherapeutic agents and radiation. Here we review studies that describe associations between therapeutic stimuli/resistance and the induction of pro‐invasive phenotypes in various cancer types. Such cancers are largely responsive to treatment that targets GSK3β. This review focuses on the role of GSK3β as a molecular hub that connects pathways responsible for tumor invasion and resistance to therapy, thus highlighting its potential as a major cancer therapeutic target. We also discuss the putative involvement of GSK3β in determining tumor cell stemness that underpins both tumor invasion and therapy resistance, leading to intractable and refractory cancer with dismal patient outcomes.

Detection of Active Fraction of Glycogen Synthase Kinase 3β in Cancer Cells by Nonradioisotopic in vitro Kinase Assay

Glycogen synthase kinase 3β (GSK3β) is a well-known marker and potential therapeutic target in non-insulin-dependent diabetes mellitus and Alzheimer’s disease. Our recent demonstration that GSK3β has a previously unrecognized role in colorectal cancer facilitates the development of a nonradioisotopic in vitro kinase assay (NRIKA) for detecting GSK3β activity in gastrointestinal cancer cells. The NRIKA uses a sequential combination of immunoprecipitations to isolate GSK3β in sample cells’ lysates, and an in vitro kinase reaction that uses recombinant β-catenin protein (substrate) and nonradioisotopic ATP, followed by immunoblotting to detect β-catenin phosphorylated in serine 33, 37 and/or threonine 41 residues. The NRIKA detected higher expression of active GSK3β in stomach, colon, pancreas and liver cancer cell lines than in human embryonic kidney cells (HEK293) considered nonneoplastic. Inhibition of cancer cell-derived GSK3β activity by GSK3β inhibitors (SB-216763, AR-A014418) was detected by the NRIKA. GSK3β inhibition attenuated survival and proliferation and induced apoptosis in all types of cancer cells but not in HEK293. These findings supported the idea that the pathologic roles of GSK3β are definite and common in various types of cancer. The NRIKA provides a basis for evolving a high-throughput tool for testing substances for GSK3β inhibition, and for screening and identifying novel GSK3β inhibitors with a view to discovering drugs for treatment of cancer as well as non-insulin-dependent diabetes mellitus and Alzheimer’s disease.

Chronic spatial working memory deficit associated with the superior longitudinal fasciculus: a study using voxel-based lesion-symptom mapping and intraoperative direct stimulation in right prefrontal glioma surgery

OBJECTIVE Although the right prefrontal region is regarded as a silent area, chronic deficits of the executive function, including working memory (WM), could occur after resection of a right prefrontal glioma. This may be overlooked by postoperative standard examinations, and the disabilities could affect the patients professional life. The right prefrontal region is a part of the frontoparietal network and is subserved by the superior longitudinal fasciculus (SLF); however, the role of the SLF in spatial WM is unclear. This study investigated a persistent spatial WM deficit in patients who underwent right prefrontal glioma resection, and evaluated the relationship between the spatial WM deficit and the SLF. METHODS Spatial WM was examined in 24 patients who underwent prefrontal glioma resection (right, n = 14; left, n = 10) and in 14 healthy volunteers using a spatial 2-back task during the long-term postoperative period. The neural correlates of spatial WM were evaluated using lesion mapping and voxel-based lesion-symptom mapping. In addition, the spatial 2-back task was performed during surgery under direct subcortical electrical stimulation in 2 patients with right prefrontal gliomas. RESULTS Patients with a right prefrontal lesion had a significant chronic spatial WM deficit. Voxel-based lesion-symptom mapping analysis revealed a significant correlation between spatial WM deficit and the region that overlapped the first and second segments of the SLF (SLF I and SLF II). Two patients underwent awake surgery and had difficulties providing the correct responses in the spatial 2-back task with direct subcortical electrical stimulation on the SLF I, which was preserved and confirmed by postoperative diffusion tensor imaging tractography. These patients exhibited no spatial WM deficits during the postoperative immediate and long-term periods. CONCLUSIONS Spatial WM deficits may persist in patients who undergo resection of the tumor located in the right prefrontal brain parenchyma. Injury to the dorsal frontoparietal subcortical white matter pathway, i.e., the SLF I or SLF I and II, could play a causal role in this chronic deficit. A persistent spatial WM deficit, without motor and language deficits, could affect the professional life of the patient. In such cases, awake surgery would be useful to detect the spatial WM network with appropriate task during tumor exploration.

Biological basis and clinical study of glycogen synthase kinase- 3β-targeted therapy by drug repositioning for glioblastoma

Background Glycogen synthase kinase (GSK)-3β has emerged as an appealing therapeutic target for glioblastoma (GBM). Here, we investigated the therapeutic effect of the current approved drugs against GBM via inhibition of GSK3β activity both, in experimental setting and in a clinical study for recurrent GBM patients by repositioning existent drugs in combination with temozolomide (TMZ). Materials and Methods Progression-free and overall survival rates were compared between patients with low or high expression of active GSK3β in the primary tumor. GBM cells and a mouse model were examined for the effects of GSK3β-inhibitory drugs, cimetidine, lithium, olanzapine, and valproate. The safety and efficacy of the cocktail of these drugs (CLOVA cocktail) in combination with TMZ were tested in the mouse model and in a clinical study for recurrent GBM patients. Results Activation of GSK3β in the tumor inversely correlated with patient survival as an independent prognostic factor. CLOVA cocktail significantly inhibited cell invasion and proliferation. The patients treated with CLOVA cocktail in combination with TMZ showed increased survival compared to the control group treated with TMZ alone. Conclusions Repositioning of the GSK3β-inhibitory drugs improved the prognosis of refractory GBM patients with active GSK3β in tumors. Combination of CLOVA cocktail and TMZ is a promising approach for recurrent GBM.

Direct evidence for the causal role of the left supplementary motor area in working memory: A preliminary study

Working memory is defined as active short-term memory nvolved in purpose-oriented tasks [1]. It plays an essential role n activities of daily living and social life, such as conversation, eading, and calculation. Previous functional magnetic resonance maging (MRI) studies have reported activation in some brain egionsduringa language-relatedN-back task that evaluatedworkng memory; these regions include the dorsolateral prefrontal ortex, anterior cingulate cortex, Broca’s area, supramarginal gyrus, uperior parietal lobule, and supplementary motor area (SMA) [2]. lthough several studies involving human brain mapping have ndicated that short-term memory could be subserved by several rain regions, including the perisylvian cortex, superior frontal yrus, and fornix, there is no direct evidence that the SMA plays a ole in working memory in the human brain [3–6]. Here, we report hat the left SMA plays a role in verbal working memory and the sefulness of the intraoperative 2-back task for evaluatingworking emory during awake surgery.

Intraoperative Motor Symptoms during Brain Tumor Resection in the Supplementary Motor Area (SMA) without Positive Mapping during Awake Surgery

Awake surgery could be a useful modality for lesions locating in close proximity to the eloquent areas including primary motor cortex and pyramidal tract. In case with supplementary motor area (SMA) lesion, we often encounter with intraoperative motor symptoms during awake surgery even in area without positive mapping. Although the usual recovery of the SMA syndrome has been well documented, rare cases with permanent deficits could be encountered in the clinical setting. It has been difficult to evaluate during surgery whether the intraoperative motor symptoms lead to postoperative permanent deficits. The purpose of this study was to demonstrate the intraoperative motor symptoms could be reversible, further to provide useful information for making decision to continue surgical procedure of tumor resection. Eight consecutive patients (from July 2012 to June 2014, six men and two women, aged 33–63 years) with neoplastic lesions around the SMA underwent an awake surgery. Using a retrospective analysis of intraoperative video records, intraoperative motor symptoms during tumor resection were investigated. In continuous functional monitoring during resection of SMA tumor under awake conditions, the following motor symptoms were observed during resection of the region without positive mapping: delayed motor weakness, delay of movement initiation, slowness of movement, difficulty in dual task response, and coordination disturbance. In seven patients hemiparesis observed immediately after surgery recovered to preoperative level within 6 weeks. During awake surgery for SMA tumors, the above-mentioned motor symptoms could occur in area without positive mapping and might be predictors for reversible SMA syndrome.