Masaki Morishige

GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion

Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.

Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities

Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA‐mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1‐mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.

The EGFR‐GEP100‐Arf6‐AMAP1 Signaling Pathway Specific to Breast Cancer Invasion and Metastasis†

Tumors are tissue‐specific diseases, and their mechanisms of invasion and metastasis are highly diverse. In breast cancer, biomarkers that specifically correlate with the invasive phenotypes have not been clearly identified. A small GTPase Arf6 primarily regulates recycling of plasma membrane components. We have shown that Arf6 and its effector AMAP1 (DDEF1, DEF1, ASAP1 and centaurin β4) are abnormally overexpressed in some breast cancers and used for their invasion and metastasis. Overexpression of these proteins is independent of the transcriptional upregulation of their genes, and occurs only in highly malignant breast cancer cells. We recently identified GEP100 (BRAG2) to be responsible for the Arf6 activation to induce invasion and metastasis, by directly binding to ligand‐activated epidermal growth factor receptor (EGFR). A series of our studies revealed that for activation of the invasion pathway of EGFR, it is prerequisite that Arf6 and AMAP1 both are highly overexpressed, and that EGFR is activated by ligands. Pathological analyses indicate that a significant large population of human ductal cancers may utilize the EGFR‐GEP100‐Arf6‐AMAP1 pathway for their malignancy. Microenvironments have been highly implicated in the malignancy of mammary tumors. Our results reveal an aspect of the precise molecular mechanisms of some breast cancers, in which full invasiveness is not acquired just by intracellular alterations of cancer cells, but extracellular factors from microenvironments may also be necessary. Possible translation of our knowledge to cancer therapeutics will also be discussed.

GEP100-Arf6-AMAP1-Cortactin Pathway Frequently Used in Cancer Invasion Is Activated by VEGFR2 to Promote Angiogenesis

Angiogenesis and cancer invasiveness greatly contribute to cancer malignancy. Arf6 and its effector, AMAP1, are frequently overexpressed in breast cancer, and constitute a central pathway to induce the invasion and metastasis. In this pathway, Arf6 is activated by EGFR via GEP100. Arf6 is highly expressed also in human umbilical vein endothelial cells (HUVECs) and is implicated in angiogenesis. Here, we found that HUVECs also highly express AMAP1, and that vascular endothelial growth factor receptor-2 (VEGFR2) recruits GEP100 to activate Arf6. AMAP1 functions by binding to cortactin in cancer invasion and metastasis. We demonstrate that the same GEP100-Arf6-AMAP1-cortactin pathway is essential for angiogenesis activities, including cell migration and tubular formation, as well as for the enhancement of cell permeability and VE-cadherin endocytosis of VEGF-stimulated HUVECs. Components of this pathway are highly expressed in pathologic angiogenesis, and blocking of this pathway effectively inhibits VEGF- or tumor-induced angiogenesis and choroidal neovascularization. The GEP100-Arf6-AMAP1-cortactin pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion and metastasis, and provides their new therapeutic targets.

The association between high VEGF levels and multiple probable punctuate cavernous malformations.

Cerebral cavernous malformations (CCMs) are congenital abnormalities of the cerebral vessels. The de novo development of new lesions in this disease has been reported. However, the underlying mechanism of progressive CCMs in such patients remains unclear. This report documents two cases of multiple probable CCMs that showed a progressive behaviour. The plasma levels of vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1) were measured using an enzyme-linked immunosorbent assay (ELISA). The concentration of both VEGF and TGF-β1 in plasma was increased in these patients. A relationship was observed between high concentrations of growth factors and progressive CCMs. Even though a causal linkage between these conditions cannot be confirmed, a continuous high VEGF level in plasma could be a possible clinical indicator for subsequent intracerebral haemorrhages in the CCM patients.

The EGFR-GEP100-Arf6 pathway in breast cancer: Full invasiveness is not from the inside

Arf6 and its effector AMAP1 are overexpressed in malignant breast cancer cells, and are involved in their invasion and metastasis. We recently revealed that GEP100, a guanine nucleotide exchanging factor, is responsible for the activation of Arf6 which induces invasion and metastasis. GEP100 associated directly with ligand-activated epidermal growth factor receptor (EGFR) to be activated. Disruption of E-cadherin-mediated cell-cell adhesion is one of the major steps involved in acquisition of invasive phenotypes of most carcinomas. The EGFR-GEP100-Arf6 pathway not only activated matrix invasion activity but also perturbed E-cadherin function. GEP100 was found to be expressed in more than 80% of invasive ductal carcinomas. However, 60% of ductal carcinomas in situ were also positive for GEP100, in which GEP100 was preferentially coexpressed with EGFR in their malignant cases. Microenvionments have been highly implicated in the development of tumor malignancy. Our results reveal an aspect of the precise molecular mechanism of cancer invasion and metastasis, in which full invasiveness is not acquired just by alterations of cancer cells themselves, but their microenvironments may also play pivotal roles.

Spontaneous chronic head and spinal subdural haematoma

SummarySpinal chronic subdural haematomas (SSDH) are extremely rare. We report a case of a SSDH combined with intracranial SDH. After tapping the SSDH in addition to the evacuation of SDH, the clinical symptoms dramatically improved. SSDH are considered to have an uncertain prognosis; however, good results can be obtained with an early diagnosis and prompt treatment.

Primary central nervous system lymphoma with cortical laminar hemorrhage.

Here we report a case of primary central nervous system (CNS) lymphoma with cortical laminar hemorrhage. The present case showed an acute onset of focal neurologic signs and bilateral cortical lesions surrounded by peripheral white matter edema on magnetic resonance imaging. A part of the left frontal cortical lesion was hyperintense on T1-weighted images and hypointense on T2-weighted and T2-weighted gradient-echo images, suggesting subacute laminar hemorrhage. The patient was initially diagnosed with multiple hemorrhagic infarctions, but a biopsy specimen revealed diffuse large B-cell lymphoma with hemosiderin deposits. Immunohistochemical studies revealed that the tumor cell cytoplasm and membrane stained positively for anti-vascular endothelial growth factor antibody. The present case reconfirms the danger of making a specific lymphoma diagnosis based on magnetic resonance imaging findings alone and that histopathologic examination following brain biopsy is necessary for a correct diagnosis. Vascular endothelial growth factor expression might be associated with the intratumoral hemorrhage.

Cognitive alteration after carotid revascularization is correlated with cortical GABAB-ergic modulations

The present study evaluates the hypotheses that a GABAergic mechanism underlies neurobehavioral sequelae of carotid stenosis and that it can be reversed by carotid revascularization. We used the Rivermead Behavioural Memory Test (RBMT), short interval intracortical inhibition (SICI), intracortical facilitation (ICF), long interval intracortical inhibition (LICI), and cortical silent period (CSP) to evaluate cognitive function and cerebral cortical excitability in 16 carotid artery stenosis patients with cognitive impairment before carotid arterial stenting (CAS) and 1 month later. We compared the pre- and post-CAS results and those of 16 healthy controls. CSP was prolonged in patients compared with controls (195.8±18ms vs. 157.8±13.9ms; p<0.0001, unpaired t-test). Patients tended to a have high resting motor threshold and less pronounced SICI and ICF than controls, but differences were not significant. Decreased RBMT score was correlated with hyperperfusion and CSP increase after CAS. RBMT score increase was correlated with CSP normalization. LICI showed positive correlation with CSP. CSP may provide a means of probing the integrity of GABA(B)-ergic networks in an ischemic human brain. CSP and LICI are potential tools to explore neuronal function for improvement as well as impairment after carotid revascularization.

Superficial Temporal Artery-To-Middle Cerebral Artery Anastomosis with Encephalo-Duro-Myo-Synangiosis as a Modified Operative Procedure for Moyamoya Disease

BACKGROUND Various types of revascularization surgery have been performed for moyamoya disease. Although the efficacies of these operations are well recognized, the optimal operative procedure remains undecided. In this report, we describe our modified surgical revascularization procedure for moyamoya disease and retrospectively analyze the results of such surgeries on six sides in six adult patients. METHODS Our operative procedure, combining direct and indirect bypasses, is a superficial temporal artery to middle cerebral artery anastomosis with encephalo-duro-myo-synangiosis. The encephalo-duro-myo-synangiosis is an indirect bypass combining the encephalo-duro- and encephalo-myo-synangioses. This operative procedure has been used routinely in adult patients since 2002. RESULTS Perioperative complications were noted in one of the six operations. This complication was transient and no attributive lesions were detected on CT or MRI. Revascularization was seen in cerebral blood flow studies in all patients, and the clinical outcomes were excellent or good. Effective neovascularization through the grafts was observed in all patients in follow-up angiographies. CONCLUSIONS This operative procedure provides needed revascularization and prevents ischemic deficits. This modified procedure is useful for responding to subsequent additional ischemia in the area of the anterior cerebral artery and should be considered one of the optimal procedures for treating moyamoya disease.