Shinichiro Wakisaka

Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR

Neuropoletic cytokines such as ciliary neurotrophic factor (CNTF) can activate multiple signaling pathways in parallel, including those involving Janus kinase (JAK)-signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamydn (mTOR)-p70 S6 kinase . Crosstalk occurs between these pathways, because studies have shown that STAT3 requires phosphorylation on tyrosine and serine residues by independent protein kinase activities for maximal activation of target gene transcription. Members of the JAK/Tyk family of tyrosine kinases mediate phosphorylation of STAT3 at Tyr705 during CNTF signaling; however, the kinase responsible for phosphorylation at STAT3 Tyr727 appears to depend on both the extracellular stimulus and the cellular context. Here we investigate the kinase activity responsible for phosphorylation of STAT3 on Ser727 in CNTF-stimulated neuroblastoma cells. We found that CNTF-induced phosphorylation of Ser727 was inhibited by the mTOR inhibitor rapamycin, but not by inhibitors of MAPK and protein kinase C (PKC) activation. A STAT3 peptide was efficiently phosphorylated on Ser727 in a CNTF-dependent manner by mTOR, but not by a kinase-inactive mTOR mutant or by p70 S6 kinase. In agreement with these biochemical studies, rapamycin treatment of cells transfected with a STAT-responsive promoter reporter decreased activation of the reporter to the same degree as a STAT3 Ser727Ala mutant The ability of mTOR to contribute to activation of STAT3 extends the function of mTOR in mammalian cells to include transcriptional regulation.

Glioma cell extracellular matrix metalloproteinase inducer (EMMPRIN) (CD147) stimulates production of membrane-type matrix metalloproteinases and activated gelatinase A in co-cultures with brain-derived fibroblasts

Extracellular matrix metalloproteinase inducer (EMMPRIN) also called CD147, basigin or M6 in the human is a member of the immunoglobulin superfamily that is enriched on the surface of tumor cells and stimulates adjacent stromal cells to produce several matrix metalloproteinases (MMPs). In this study, we have demonstrated that coculturing of EMMPRIN-expressing human glioblastoma multiforme cells (U251) with brain-derived human fibroblasts not only stimulates production, but also activation of pro-gelatinase A (proMMP-2), an enzyme that is enriched in malignant gliomas and most likely crucial to tumor progression. Production of membrane types 1 and 2-MMPs (MT1-MMP and MT2-MMP), which are activators of proMMP-2, was also stimulated in these cocultures. Stimulation of MMP-2, MT1-MMP and MT2-MMP production was inhibited by anti-EMMPRIN monoclonal antibody in a dose-dependent manner. Thus, we have shown, for the first time, that EMMPRIN causes increased expression of MT1-MMP and MT2-MMP, as well as increased production and activation of MMP-2.

Expression of emmprin (CD147), a cell surface inducer of matrix metalloproteinases, in normal human brain and gliomas

EMMPRIN (extracellular matrix metalloproteinase inducer), also called CD147, basigin or M6 in the human, is a member of the immunoglobulin superfamily that is present on the surface of tumor cells and stimulates adjacent fibroblasts to produce matrix metalloproteinases (MMPs). In our study, we investigated expression of EMMPRIN in human normal brain and gliomas, since mouse basigin and chicken HT7, the species homologues of human EMMPRIN, are associated with neuronal interactions and normal blood‐brain barrier function, respectively. EMMPRIN expression was detected in all samples of non‐neoplastic brain and glioma tissues examined. However, expression levels of EMMPRIN mRNA and protein were significantly higher in gliomas than in non‐neoplastic brain. Moreover, levels of mRNA expression and immunohistochemical staining correlated with tumor progression in gliomas: They were highest in the most malignant form of glioma, glioblastoma multiforme, followed by anaplastic astrocytoma and then low‐grade astrocytoma. Also, immunolocalization revealed quite different distributions in non‐neoplastic brain and glioma: EMMPRIN was demonstrated only in vascular endothelium in non‐neoplastic regions of the brain, whereas it was present in tumor cells but not in proliferating blood vessels in malignant gliomas. These data indicate that an MMP inducer molecule EMMPRIN is differently expressed in human normal brain and gliomas and could be associated with astrocytoma progression. Possible mechanisms whereby glioma cell EMMPRIN could influence tumor progression will be discussed. Int. J. Cancer 88:21–27, 2000.

Expression of c‐Met correlates with grade of malignancy in human astrocytic tumours: an immunohistochemical study

Recent studies suggest the involvement of hepatocyte growth factor/scatter factor (HGF/SF) in glioma cell invasion and tumour progression. We investigated the distribution and rate of tumour cells that express c‐Met protein, which is the cell‐surface receptor for HGF/SF, in astrocytic tumours. The type of cells that express c‐Met in tumour tissues was also identified.

Prediction of hemorrhagic complications after thrombolytic therapy for middle cerebral artery occlusion: value of pre- and post-therapeutic computed tomographic findings and angiographic occlusive site.

OBJECTIVE To evaluate the usefulness of pre- and post-therapeutic computed tomographic (CT) findings in predicting hemorrhagic complications, we retrospectively examined 35 patients treated with intra-arterial thrombolytic therapy for middle cerebral artery (MCA) occlusion. METHODS The presence or absence of early CT findings (loss of the insular ribbon, obscuration of the lentiform nucleus, and cortical effacement) and the presence and location of extravasation of contrast medium were evaluated on pre- and post-therapeutic CT scans, respectively. According to the angiographic occlusive site, the patients were classified into the following three groups: Group 1 (n = 13), MCA trunk occlusion involved lenticulostriate arteries; Group 2 (n = 11), occlusion of the MCA trunk without involvement of the lenticulostriate arteries; Group 3 (n = 11), occlusion of a branch of the MCA. Hemorrhagic complications (hemorrhagic transformation and/or massive brain swelling) were evaluated by reviewing CT scans obtained 3 to 14 days after thrombolytic therapy. RESULTS No patient without extravasation (n = 17) showed hemorrhagic complications, and extravasation is the most useful finding in predicting hemorrhagic complications. There was significant correlation between extravasation and hemorrhagic complications (P < 0.01). In Groups 1 and 2, there was also significant correlation between early CT findings and hemorrhagic complications (P < 0.01), indicating that early CT findings are also useful in predicting hemorrhagic complications. In Group 1, 10 of 13 (76.9%) patients had both early CT findings and extravasation, and 6 of these 10 patients had hemorrhagic complications with clinical deterioration, suggesting the difficulty of thrombolytic therapy in this group. On the contrary, in Group 2, 8 of 11 (72.7%) patients had neither early CT findings nor extravasation and none of these 8 patients had hemorrhagic complications. In Group 3, however, early CT findings and extravasation had no correlation. Because the affected area was small in this group, it was difficult to evaluate cortical effacement. Although negative early CT findings did not always mean absence of extravasation and hemorrhagic complications in this group, the patients with hemorrhagic complications did not clinically deteriorate because of the small affected area. CONCLUSION Hemorrhagic complications could be predicted by evaluation of angiographic occlusive site and pre- and post-therapeutic CT findings.

Comparative analysis of expression of hepatocyte growth factor and its receptor, c-met, in gliomas, meningiomas and schwannomas in humans

Expression of hepatocyte growth factor (HGF) and c-met, a proto-oncogene that encodes a receptor for HGF, was examined in 45 cases of human primary intracranial tumors by means of RT-PCR. In gliomas, HGF and c-met mRNAs were preferentially expressed in high-grade tumors. Co-expression of both genes was observed in glioblastomas (6/15) and in one anaplastic astrocytoma (1/5) but not in low-grade astrocytomas (0/3). By contrast, the c-met gene was consistently expressed in meningiomas (12/14) and schwannomas (8/8). The presence of c-Met protein was confirmed in the tumor cells of glioblastoma, meningioma and schwannoma by immunohistochemical staining. Moreover, all of the schwannoma cases co-expressed the HGF gene. These observations suggest that HGF/c-met expression is somehow related to the disease progression in gliomas, whereas c-Met protein might have an important fundamental biological role in meningioma and schwannoma. Moreover, since all of the schwannoma cases concomitantly expressed the ligand (HGF) and the receptor (c-met) genes, HGF may act in an autocrine fashion in schwannoma.

Time sequential single photon emission computed tomography studies in brain tumour using thallium-201

Time sequential single photon emission computed tomography (SPECT) studies using thallium-201 were performed in 25 patients with brain tumours to evaluate the kinetics of thallium in the tumour and the biological malignancy grade preoperatively. After acquisition and reconstruction of SPECT data from 1 min post injection to 48 h (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 15–20 min, followed by 4–6, 24 and 48 h), the thallium uptake ratio in the tumour versus the homologous contralateral area of the brain was calculated and compared with findings of X-ray CT, magnetic resonance imaging, cerebral angiography and histological investigations. Early uptake of thallium in tumours was related to tumour vascularity and the disruption of the blood-brain barrier. High and rapid uptake and slow reduction of thallium indicated a hypervascular malignant tumour; however, high and rapid uptake but rapid reduction of thallium indicated a hypervascular benign tumour, such as meningioma. Hypovascular and benign tumours tended to show low uptake and slow reduction of thallium. Long-lasting retention or uptake of thallium indicates tumour malignancy.

Reduced expression of hepatocyte growth factor activator inhibitor type‐2/placental bikunin (HAI‐2/PB) in human glioblastomas: Implication for anti‐invasive role of HAI‐2/PB in glioblastoma cells

Hepatocyte growth factor activator inhibitor type‐2/placental bikunin (HAI‐2/PB) is a serine proteinase inhibitor that contains 2 Kunitz‐domains and a presumed transmembrane domain. It has broad inhibitory spectra against various serine proteinases showing potent inhibitory activities not only to hepatocyte growth factor activator but also to plasmin, trypsin and kallikreins. In this study, we investigated the expression of HAI‐2/PB in human gliomas in vivo and the effects of HAI‐2/PB on the fibrinolytic and invasive capabilities of human glioblastoma cells in vitro. With RNA blot analysis, HAI‐2/PB mRNA was expressed in normal brain and in low‐grade astrocytomas, but was hardly detectable in anaplastic astrocytomas and glioblastomas, indicating that its expression levels were inversely correlated with the histological grade of human gliomas. To further explore the possible role of HAI‐2/PB in glioma progression, cultured human glioblastoma cell lines (U251 and YKG‐1) were transiently transfected with an expression vector harboring human HAI‐2/PB cDNA. Subsequent analysis indicated that the expression of HAI‐2/PB suppressed the fibrinolytic activities of both glioblastoma cell lines. Moreover, HAI‐2/PB inhibited Matrigel invasion of U251 and YKG‐1 cells by 30% and 64%, respectively. This anti‐invasive effect appeared to be mediated primarily by the inhibitory activity of HAI‐2/PB against the serine proteinase‐dependent matrix degradation. These findings suggest that the reduced expression of HAI‐2/PB is possibly involved in the progression of human gliomas.

Intracranial and Orbital Metastasis of Hepatocellular Carcinoma: Report of Two Cases

Two cases of rare intracranial and orbital metastasis of hepatocellular carcinoma are presented. A 61-year-old woman was found to have a metastatic tumor in the right temporo-occipital lobe 1 year after undergoing treatment for a primary hepatoma. An osteolytic tumor was removed from the left orbit of a 58-year-old man and the primary tumor, a hepatoma, was discovered postoperatively. The intracranial and orbital tumors were verified to be hepatocellular carcinoma. Both patients died within 1 year of surgery. The relevant literature is briefly reviewed.

Role of hepatocyte growth factor activator (HGF activator) in invasive growth of human glioblastoma cells in vivo

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that is involved in invasive growth of tumor cells via its receptor MET, a protein product of c‐met proto‐oncogene. HGF activator (HGFA) is a serine proteinase responsible for the activation of proform of HGF/SF (proHGF/SF). In our study, we examined the effects of engineered expression of HGFA on 2 human glioblastoma cell lines (YKG‐1 and U251). Both cells expressed MET, while only YKG‐1 expressed endogenous proHGF/SF. Enhanced MET phosphorylation and increased migratory activity were induced by the expression of HGFA in YKG‐1 cells in vitro in the presence of thrombin, which is a known activator of proHGFA. In contrast, MET phosphorylation was consistently observed in U251 that lacked endogenous HGF/SF, suggesting ligand‐independent activation of MET in this cell line. Consequently, the expression of HGFA in U251 did not enhance the MET phosphorylation and following cellular response even with the thrombin treatment. However, addition of exogenous proHGF/SF resulted in enhanced migratory activity of HGFA‐expressing U251 cells in the presence of thrombin in vitro. The engineered HGFA expression resulted in significantly enhanced tumor growth with increased vascular density in vivo when YKG‐1 cells were implanted in nude mouse brain. This effect was not observed in U251 lacking endogenous proHGF/SF. These results indicate the possible existence of multiple mechanisms of MET activation in glioblastomas and that the activation system of proHGF/SF is important in progression of glioblastomas that express endogenous proHGF/SF and require ligand‐dependent MET activation.