Hemragul Sabit

Serine/Threonine Kinase MLK4 Determines Mesenchymal Identity in Glioma Stem Cells in an NF-κB-dependent Manner

Activation of nuclear factor κB (NF-κB) induces mesenchymal (MES) transdifferentiation and radioresistance in glioma stem cells (GSCs), but molecular mechanisms for NF-κB activation in GSCs are currently unknown. Here, we report that mixed lineage kinase 4 (MLK4) is overexpressed in MES but not proneural (PN) GSCs. Silencing MLK4 suppresses self-renewal, motility, tumorigenesis, and radioresistance of MES GSCs via a loss of the MES signature. MLK4 binds and phosphorylates the NF-κB regulator IKKα, leading to activation of NF-κB signaling in GSCs. MLK4 expression is inversely correlated with patient prognosis in MES, but not PN high-grade gliomas. Collectively, our results uncover MLK4 as an upstream regulator of NF-κB signaling and a potential molecular target for the MES subtype of glioblastomas.

Enhanced expression of basement-membrane-type heparan sulfate proteoglycan in tumor fibro-myxoid stroma of intrahepatic cholangiocarcinoma

To investigate the molecular mechanism for enhanced fibrous stroma formation in intrahepatic cholangiocarcinoma (ICC), we surveyed the expression pattern of basement‐membrane‐type heparan sulfate proteoglycan (HSPG; also known as perlecan) at the core protein and the mRNA level in ICC as well as in other liver neoplasms and reactive hepatic diseases. Immunohistochemistry of paraffin‐embedded liver sections with hyaluronidase pretreatment showed that HSPG was present in small amounts in normal liver around the bile ducts and the blood vessels within the portal area. There was no evident expression within the hepatic lobules. Intense immunoexpression of HSPG was seen in the tumor‐specific fibro‐myxoid stroma of ICC and metastatic liver cancer originating from the colon. However, tumor‐specific stroma of hepatocellular carcinomas showed little or no expression of HSPG. At the mRNA level, signals for HSPG were found in tumor cells of cholangiocarcinoma and metastatic colonic carcinomas, and in myofibroblasts in the tumor fibro‐myxoid‐specific stroma. From immunoprecipitation and reverse transcription–polymerase chain reaction (RT–PCR) analyses, a cultured human intrahepatic cholangiocarcinoma cell line (CCKS1), was found to express high levels of HSPG core protein and mRNA. These findings suggest that biliary and metastatic colon carcinoma cells as well as stromal myofibroblasts have a potential for HSPG production. In order to investigate the growth, invasion and metastatic ability of ICC, further study of the ‘self‐made’ stromal component of ICC may provide a new approach.

Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O6-methylguanine DNA methyltransferase promoter methylation via c-Myc signaling

Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase involved in human cancers including glioblastoma. We have previously demonstrated that GSK3β inhibition enhances temozolomide effect in glioma cells. In this report, we investigated the molecular mechanisms of sensitization of glioblastoma cells to temozolomide by GSK3β inhibition, focusing on O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing. Glioblastoma tissues from patients treated with the GSK3β-inhibiting drugs were subjected to immunohistochemistry and methylation-specific PCR assay. Human glioblastoma cell lines T98G, U138, U251 and U87 were treated with a small-molecule GSK3β inhibitor, AR-A014418 or GSK3β-specific small interfering RNA. The combined effect of temozolomide and AR-A014418 on cell proliferation was determined by AlamarBlue assay and an isobologram method. MGMT promoter methylation was estimated by methylation-specific PCR and MethyLight assay. MGMT gene expression was evaluated by real-time quantitative reverse transcriptase-PCR. c-Myc and DNA (cytosine-5)-methyltransferase 3A binding to the MGMT promoter was estimated by chromatin immunoprecipitation assay. GSK3β inhibition decreased phosphorylation of glycogen synthase and reduced MGMT expression and increased MGMT promoter methylation in clinical tumors. In glioblastoma cell lines, GSK3β inhibition decreased cell viability, enhanced temozolomide effect and downregulated MGMT expression with relevant changes in the methylation levels of the MGMT promoter. Here, we showed for the first time that c-Myc binds to the MGMT promoter with consequent recruitment of DNA (cytosine-5)-methyltransferase 3A, regulating the levels of MGMT promoter methylation. The results of this study suggest that GSK3β inhibition enhances temozolomide effect by silencing MGMT expression via c-Myc-mediated promoter methylation.

miR-150-5p and miR-133a suppress glioma cell proliferation and migration through targeting membrane-type-1 matrix metalloproteinase.

Gliomas are the most frequent primary tumors of the brain, and there is no successful treatment for highly malignant gliomas. MicroRNAs (miRNAs) are involved in a variety of biological processes. Recent studies showed that miR-150-5p and miR-133a are downregulated in various human malignancies, and one of target mRNAs was shown to be membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA. However, their detailed role in the processes of cancer remains to be determined. Here we found that miR-150-5p and miR-133a expression was significantly downregulated in glioma tissues compared with normal tissues, and that MT1-MMP expression was inversely upregulated in glioma tissues. Knockdown of MT1-MMP by specific siRNAs in U87 and U251 glioma cells induced suppression of cell proliferation and invasion/migration. Transfection of miR-150-5p or miR-133a mimics into glioma cell lines reduced MT1-MMP expression and MMP-2 activation by these cells, and cell proliferation and invasion/migration were also suppressed by it. Co-transfection of specific inhibitor oligo DNA for miR-150-5p or miR-133a abrogated miR-150-5p or miR-133a mimics actions, respectively. These results suggest that miR-150-5p and miR-133a may suppress malignancy of gliomas by targeting MT1-MMP, and could be used as an anti-metastatic therapy for glioma patients.

Combination therapy using Notch and Akt inhibitors is effective for suppressing invasion but not proliferation in glioma cells

Molecular targeted therapy can potentially provide more effective treatment for patients with high-grade gliomas. Notch and Akt are notable target molecules as they play important roles in a variety of cellular processes, such as regeneration, differentiation, proliferation, migration, and invasion. Here, we assessed the therapeutic possibility of inhibiting Notch and Akt in gliomas using the clinically available, selective small molecule inhibitors MRK003 and MK-2206. We evaluated their efficacy individually and as a combination therapy in U251 and U87 glioma cell lines. We confirmed that MK-2206 effectively inhibits Akt phosphorylation in a dose-dependent manner, whereas MRK003 inhibits Notch signaling and Akt phosphorylation. Both MRK003 and MK-2206 significantly inhibited cell growth, migration, and invasion in a dose-dependent manner. Akt dephosphorylation was enhanced by combination therapy with MRK003 and MK-2206. However, the effect of combination treatment did not exceed that of MK-2206 monotherapy in proliferation assay. Inhibition of invasion, further enhanced by combination therapy, correlated with increased Akt inactivation. In summary, combination therapy with MRK003 and MK-2206 may be effective for inhibiting invasion but not proliferation.

Characterizing invading glioma cells based on IDH1-R132H and Ki-67 immunofluorescence

Abstract Glioma, the most common primary brain tumor, is characterized by proliferative-invasive growth. However, the detailed biological characteristics of invading glioma cells remain to be elucidated. A monoclonal antibody (clone HMab-1) that specifically and sensitively recognizes the isocitrate dehydrogenase-1 (IDH1) protein carrying the R132H mutation can identify invading glioma cells by immunostaining. To investigate the degree of invasion in gliomas of distinct grades and the proliferative capacity of the invading cells, immunofluorescent staining was conducted using antibodies against IDH1-R132H and Ki-67 on 11 surgical and autopsy specimens of the tumor core and the invading area. Higher numbers of IDH1-R132H-positive cells in the invading area correlated with a higher tumor grade. Double staining for IDH1-R132H and Ki-67 demonstrated that most invading cells that expressed IDH1-R132H were not stained by the Ki-67 antibody, and the ratio of Ki-67-positive cells among IDH1-R132H-positive cells was significantly lower in the invasion area than in the tumor core in all grades of glioma. These data suggest that higher grade gliomas have a greater invasive potential and that invading cells possess low proliferative capacity.

Glycogen Synthase Kinase 3β Sustains Invasion of Glioblastoma via the Focal Adhesion Kinase, Rac1, and c-Jun N-Terminal Kinase-Mediated Pathway

The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3β (GSK3β), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3β compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3β also contributes to the highly invasive nature of glioblastoma. The effects of GSK3β inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3β attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3β as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance. Mol Cancer Ther; 14(2); 564–74. ©2014 AACR.

Recurrent anaplastic meningioma treated by sunitinib based on results from quantitative proteomics

Anaplastic meningiomas are aggressive tumours that account for 1% to 3% of all meningiomas [1]. They tend to recur even after complete surgical resection and subsequent radiation therapy. The prognosis for anaplastic meningiomas is poor, with a median survival of less than 2 years [2]. However, none of the chemotherapeutic drugs have shown any convincing effect on anaplastic meningiomas [3]. Currently, novel therapeutic drugs that act on growth factor receptors are being manufactured and tested for anaplastic meningiomas [3]. In this report, we present the case of a patient with intractable anaplastic meningioma that recurred repeatedly in a short period. Our proteomics analysis showed that platelet-derived growth factor receptor (PDGFR) b was highly expressed in the tumour. Immunohistochemistry confirmed expression and activation of PDGFRb in the surgical specimen. According to our results, sunitinib (Sutent, Pfizer Inc., New York , NY, USA), a rationally designed small-molecule inhibitor that blocks PDGFRb signalling, was administered [4]. To the best of our knowledge, this is the first case where a tailored chemotherapy has been administered for anaplastic meningioma on the basis of proteomics results. A 62-year-old woman was admitted to our hospital (Kanazawa University Hospital) for the treatment of sphenoid ridge meningioma. The detailed clinical course was reported by Kawahara et al. as the extremely aggressive anaplastic meningioma [5]. Briefly, she was diagnosed with anaplastic meningioma and underwent surgery three times and radiotherapy twice for the repeated recurrences. However, the tumour showed local recurrence and cerebrospinal fluid dissemination 4 months after the first surgery. We analysed the tumour samples resected during the first surgery with quantitative target absolute proteomics, as described previously [6]. The research protocols for the present study were approved by the ethical committees of the Graduate School of Pharmaceutical Sciences, Tohoku University and Graduate School of Medical Science, Kanazawa University Hospital. The plasma membrane fractions of the brain samples were processed using differential centrifugation with a sucrose density gradient and were digested by trypsin followed by reduction and alkylation. The tryptic digests were acidified with formic acid for analysis with the high-performance liquid chromatography (HPLC) system (Agilent1200 system, Agilent, Santa Clara, CA, USA), which was connected to an electrospray ionization-triple quadrupole mass spectrometer (API5000, ABSCIEX, Foster City, CA, USA). HPLC was performed with C18 capillary columns (ZORBAX SBC18 0.5 mm inner diameter 150 mm, 5 mm particles; Agilent). The mass spectrometer was set up to run a multichannel reaction monitoring for 11 target molecules that have available molecular-targeted drugs (Table 1). Linear gradients of 1–50% acetonitrile in 0.1% formic acid were applied to elute the peptides at a flow rate of 50 ml/min for 50 min. The protein expression levels were determined as described previously [7]. Peptide sequences and selected ions for quantification of each membrane protein are shown in Table S1. As shown in Table 1, PDGFRb, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), CD33 and CD37 were detected in the plasma membrane fraction of the tumour tissues, and PDGFRb exhibited the greatest protein expression levels. Moreover, PDGFRb protein level in this case was higher than the levels obtained from glioblastomas (mean SD, 0.270 0.731 fmol/mg protein: n = 21), whereas PDGFRb is undetectable in normal brain (n = 1). Immunohistochemistry was performed to validate the proteomics data. Briefly, paraffin-embedded tissue blocks were sectioned (6-mm thick) on to slides and then deparaffinized. Sections were immunostained using the ABC Elite Kit (Funakoshi, Tokyo, Japan) with anti-PDGFRb monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA) and phosphorylated PDGFRb (Tyr857) polyclonal antibody (Santa Cruz Biotech., Santa Cruz, CA, USA). Sections were quenched with 3% hydrogen peroxide in methanol for 20 min, microwaved for 15 min in citrate buffer, blocked by incubation in 0.3% H2O2 solution in methanol for 20 min, and blocked for 20 min with diluted

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.

Activation of the receptor tyrosine kinase AXL regulates the immune microenvironment in glioblastoma

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1-AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors.Significance: These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. Cancer Res; 78(11); 3002-13. ©2018 AACR.