Glioblastoma Metastasis Research Articles

Differentiation of Glioblastoma from Brain Metastasis: Qualitative and Quantitative Analysis Using Arterial Spin Labeling MR Imaging.

PurposeTo evaluate the diagnostic performance of cerebral blood flow (CBF) by using arterial spin labeling (ASL) perfusion magnetic resonance (MR) imaging to differentiate glioblastoma (GBM) from brain metastasis.Materials and MethodsThe institutional review board of our hospital approved this retrospective study. The study population consisted of 128 consecutive patients who underwent surgical resection and were diagnosed as either GBM (n = 89) or brain metastasis (n = 39). All participants underwent preoperative MR imaging including ASL. For qualitative analysis, the tumors were visually graded into five categories based on ASL-CBF maps by two blinded reviewers. For quantitative analysis, the reviewers drew regions of interest (ROIs) on ASL-CBF maps upon the most hyperperfused portion within the tumor and upon peritumoral T2 hyperintensity area. Signal intensities of intratumoral and peritumoral ROIs for each subject were normalized by dividing the values by those of contralateral normal gray matter (nCBFintratumoral and nCBFperitumoral, respectively). Visual grading scales and quantitative parameters between GBM and brain metastasis were compared. In addition, the area under the receiver-operating characteristic curve was used to evaluate the diagnostic performance of ASL-driven CBF to differentiate GBM from brain metastasis.ResultsFor qualitative analysis, GBM group showed significantly higher grade compared to metastasis group (p = 0.001). For quantitative analysis, both nCBFintratumoral and nCBFperitumoral in GBM were significantly higher than those in metastasis (both p < 0.001). The areas under the curve were 0.677, 0.714, and 0.835 for visual grading, nCBFintratumoral, and nCBFperitumoral, respectively (all p < 0.001).ConclusionASL perfusion MR imaging can aid in the differentiation of GBM from brain metastasis.

CSN6 controls the proliferation and metastasis of glioblastoma by CHIP-mediated degradation of EGFR.

CSN6, a critical subunit of the constitutive photomorphogenesis 9 (COP9) signalosome (CSN), has received attention as a regulator of the degradation of cancer-related proteins such as p53, c-myc and c-Jun, through the ubiquitin-proteasome system, suggesting its importance in cancerogenesis. However, the biological functions and molecular mechanisms of CSN6 in glioblastoma (GBM) remain poorly understood. Here, we report that GBM tumors overexpressed CSN6 compared with normal brain tissues and that CSN6 promoted GBM cell proliferation, migration, invasion and tumorigenesis. Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, was used to reveal that the proliferative and metastatic effects of CSN6 on GBM cells were EGFR dependent. We also found that CSN6 positively regulated EGFR stability via reduced levels of EGFR ubiquitination, thereby elevating steady expression of EGFR. In addition, this study is the first description of a novel role for the CSN6-interacting E3 ligase, CHIP (carboxyl terminus of heat-shock protein 70-interacting protein), regulating EGFR ubiquitination in cancer cells. We showed that CSN6 associated with CHIP and led to CHIP destabilization by increasing CHIP self-ubiquitination. Moreover, CSN6 decreased CHIP expression and increased EGFR expression in the tumor samples. Deregulation of this axis promoted GBM cell's proliferation and metastasis. Thus, our study provides insights into the applicability of using the CSN6-CHIP-EGFR axis as a potential therapeutic target in cancer.

Extramedullary skeletal muscle metastasis of glioblastoma: A case report and literature review ⁎

Abstract Objective The aim of the study was to explore the clinicopathologic, immunophenotypic, and diagnostic features of extramedullary metastases of glioblastoma. Methods One case of extramedullary skeletal muscle metastasis of glioblastoma was studied, including the clinical, histological, and immunohistochemical features. Results A 24-year-old man underwent surgical resection for glioblastoma (WHO grade IV) in the left temporal parietal region followed by radiotherapy and temozolomide therapy. One year and nine months later, he developed an extramedullary skeletal muscle metastasis in L4, and the histology was remarkably different from that of the primary glioblastoma specimen. The immunohistochemical analysis also showed changes. In the metastasis, the small cells were negative for GFAP; weakly positive for S-100; and positive for nestin, NSE, and CD56, with 60% of cells positive for p53 and 40% positive for Ki-67. The giant cells showed strong positivity for GFAP and S-100, and weak expression of p53, Ki-67, nestin, NSE, and CD56. The primary glioblastoma specimen showed strong positivity for GFAP and S-100 and was negative for NSE, nestin, and CD56, with around 25% of the tumor cells positive for p53 and a Ki-67 labeling index of 20%. Conclusion Extraneural metastasis (ENM) is a rare complication of glial tumors and glioma stem cells may be related to the metastasis. Since extraneural metastasis may occur in patients without central nervous symptoms, any unusual signs during the follow-up of patients diagnosed with glioblastoma should not be underestimated.

Molecular determinants of transforming growth factor beta-1 action on human glioblastoma cells

Background. Increased expression of transforming growth factor beta-1 (TGF-β1) in malignant brain tumors promotes cancer cells survival enhancing their growth, migration, invasion, angiogenesis, immune system suppression. Objective is to study molecular mechanisms of TGF-β1 action on U87 human glioblastoma cells by means of proteomic high-resolution massspectrometry. Results. We have identified intracell signal pathways responsible for TGF-β1 involvement in malignant gliomas oncogenesis including differential expressed proteins of tight cell junctions, focal adhesion, histone deacetylases, heat shock, S100 family. Conclusions. Important patterns are determined that could be used for the development of new approaches for detection of glioblastoma metastasis candidate markers and potential therapy targets of this decease.

Anti-miR delivery strategies to bypass the blood-brain barrier in glioblastoma therapy

Small non-coding RNAs called miRNAs are key regulators in various biological processes, including tumor initiation, propagation, and metastasis in glioblastoma as well as other cancers. Recent studies have shown the potential for oncogenic miRNAs as therapeutic targets in glioblastoma. However, the application of antisense oligomers, or anti-miRs, to the brain is limited due to the blood-brain barrier (BBB), when administered in the traditional systemic manner. To induce a therapeutic effect in glioblastoma, anti-miR therapy requires a robust and effective delivery system to overcome this obstacle. To bypass the BBB, different delivery administration methods for anti-miRs were evaluated. Stereotaxic surgery was performed to administer anti-Let-7 through intratumoral (ITu), intrathecal (ITh), and intraventricular (ICV) routes, and each method's efficacy was determined by changes in the expression of anti-Let-7 target genes as well as by immunohistochemical analysis. ITu administration of anti-miRs led to a high rate of anti-miR delivery to tumors in the brain by both bolus and continuous administration. In addition, ICV administration, compared with ITu administration, showed a greater distribution of the miR across entire brain tissues. This study suggests that local administration methods are a promising strategy for anti-miR treatment and may overcome current limitations in the treatment of glioblastoma in preclinical animal models.

Targeting ROR1 inhibits the self‐renewal and invasive ability of glioblastoma stem cells

Glioblastoma is the most malignant of brain tumours and is difficult to cure because of interruption of drug delivery by the blood-brain barrier system, its high metastatic capacity and the existence of cancer stem cells (CSCs). Although CSCs are present as a small population in malignant tumours, CSCs have been studied as they are responsible for causing recurrence, metastasis and resistance to chemotherapy and radiotherapy for cancer. CSCs have self-renewal characteristics like normal stem cells. The aim of this study was to investigate whether receptor tyrosine kinase-like orphan receptor 1 (ROR1) is involved in stem cell maintenance and malignant properties in human glioblastoma. Knockdown of ROR1 caused reduction of stemness and sphere formation capacity. Moreover, down-regulation of ROR1 suppressed the expression of epithelial-mesenchymal transition-related genes and the tumour migratory and invasive abilities. The results of this study indicate that targeting ROR1 can induce differentiation of CSCs and inhibit metastasis in glioblastoma. In addition, ROR1 may be used as a potential marker for glioblastoma stem cells as well as a potential target for glioblastoma stem cell therapy.

EGFRvIII/integrin β3 interaction in hypoxic and vitronectinenriching microenvironment promote GBM progression and metastasis.

Glioblastoma (GBM) is one of the most lethal brain tumors with a short survival time. EGFR amplification and mutation is the most significant genetic signature in GBM. About half of the GBMs with EGFR amplification express a constitutively autophosphorylated variant of EGFR, known as EGFRvIII. Our in vitro data demonstrated further enhanced EGFRvIII activity and tumor cell invasion in the tumor microenvironment of hypoxia plus extracellular matrix (ECM) vitronectin, in which EGFRvIII and integrin β3 tended to form complexes. The treatment with ITGB3 siRNA or the integrin antagonist cilengetide preferentially interrupted the EGFRvIII/integrin β3 complex, effectively reduced tumor cell invasion and activation of downstream signaling effectors. Cilengitide is recently failed in Phase III CENTRIC trial in unselected patients with GBM. However, we found that cilengitide demonstrated efficacious tumor regression via inhibition of tumor growth and angiogenesis in EGFRvIII orthotopic xenografts. Bioinformatics analysis emphasized key roles of integrin β3, hypoxia and vitronectin and their strong correlations with EGFRvIII expression in malignant glioma patient samples in vivo. In conclusion, we demonstrate that EGFRvIII/integrin β3 complexes promote GBM progression and metastasis in the environment of hypoxia and vitronectin-enrichment, and cilengitide may serve as a promising therapeutics for EGFRvIII-positive GBMs.

SRPX2 Enhances the Epithelial-Mesenchymal Transition and Temozolomide Resistance in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and most aggressive central nervous system tumor in adults. Due to GBM cell invasiveness and resistance to chemotherapy, current medical interventions are not satisfactory, and the prognosis for GBM is poor. It is necessary to investigate the underlying mechanism of GBM metastasis and drug resistance so that more effective treatments can be developed for GBM patients. sushi repeat-containing protein, X-linked 2 (SRPX2) is a prognostic biomarker in many different cancer cell lines and is associated with poor prognosis in cancer patients. SRPX2 overexpression promotes interactions between tumor and endothelial cells, leading to tumor progression and metastasis. We hypothesize that SRPX2 also contributes to GBM chemotherapy resistance and metastasis. Our results revealed that GBM tumor samples from 42 patients expressed higher levels of SRPX2 than the control normal brain tissue samples. High-SRPX2 expression levels are correlated with poor prognosis in those patients, as well as resistance to temozolomide in cultured GBM cells. Up-regulating SRPX2 expression in cultured GBM cell lines facilitated invasiveness and migration of GBM cells, while down-regulating SRPX2 through RNA interference was inhibitory. These results suggest that SRPX2 plays an important role in GBM metastasis. Epithelial to mesenchymal transition (EMT) is one of the processes that facilitate GBM metastasis and resistance to chemotherapy. EMT marker expression was decreased in SRPX2 down-regulated GBM cells, and MAPK signaling pathway marker expression was also decreased when SRPX2 is knocked down in GBM-cultured cells. Blocking the MAPK signaling pathway inhibited GBM metastasis but did not inhibit cell invasion and migration in SRPX2 down-regulated cells. Our results indicate that SRPX2 facilitates GBM metastasis by enhancing the EMT process via the MAPK signaling pathway.

MicroRNA-663 inhibits the proliferation, migration and invasion of glioblastoma cells via targeting TGF-β1.

Cell migration and invasion are key processes involved during tumor metastasis. Recently, microRNAs (miRs) have been demonstrated to play important roles in the regulation of cancer metastasis. However, the underlying mechanisms remain unknown. Here, we aimed to investigate the exact role of miR-663 in the metastasis of glioblastoma as well as the underlying mechanisms. By performing quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, we demonstrated that miR-663 was significantly downregulated in glioblastoma tissues (n=25), when compared to that in normal brain tissues (n=15). In addition, its expression levels were also reduced in human glioblastoma cell lines, A172 and U87. Furthermore, restoration of miR-663 expression led to a significant decrease in the cell proliferation, migration and invasion of human glioblastoma A172 and U87 cells. We further identified TGF-β1 as a direct target of miR-663, and found that the expression of TGF-β1 was negatively mediated by miR-663 at the post-transcriptional level in glioblastoma cells. Moreover, overexpression of TGF-β1 significantly reversed the inhibitory effects of miR-663 upregulation on the proliferation, migration and invasion in A172 and U87 cells. In addition, our data suggest that MMP2 and E-cadherin, a key factor in epithelial-mesenchymal transition (EMT), are involved in the miR-633/TGF-β1-mediated metastasis of glioblastoma. In summary, miR-663 plays an inhibitory role in the regulation of proliferation, migration and invasion of glioblastoma cells, partly at least, via direct mediation of TGF-β1 as well as downstream MMP2 and E-cadherin. Therefore, we suggest that miR-663 is a potential candidate for the prevention of glioblastoma metastasis.

Intramedullary spinal glioblastoma metastasis from anaplastic astrocytoma of cerebellum: A case report and review of the literature

Cerebellar anaplastic astrocytoma is infrequently encountered even nowadays, and drop metastasis with progression into spinal glioblastoma is not reported in the English literature. We report a case of cerebellar anaplastic astrocytoma receiving operation and subsequent concurrent chemoradiotherapy. One year later, progressive weakness of both lower limbs and unsteady gait occurred. Spine magnetic resonance imaging showed cervical and thoracic spine intramedullary tumor. We then performed laminectomy and tumor biopsy. The histopathological report demonstrated primary spinal cord glioblastoma multiforme with positive glial fibrillary acidic protein, high MIB-1 labeling index and negative staining of isocitrate dehydrogenase-1 mutation. After reviewing the English literature to date, most metastatic spinal glioblastoma resulted from previous intracranial glioblastoma, and there are few studies mentioning spinal glioblastoma originating from intracranial low-grade gliomas. Over time, improvement in local control of the primary tumor has raised patient risk of the possibility of spinal metastasis, and clinical physicians should be aware of this aspect so that quicker diagnosis and management will be accomplished, even in patients with lower grade of intracranial gliomas.

Downregulation of solute carriers of glutamate in gliosomes and synaptosomes may explain local brain metastasis in anaplastic glioblastoma.

Advanced grades of glioblastoma are highly aggressive, especially in terms of multisite spread within the brain or even to distant sites at the spinal cord. In advanced grades of glioblastoma, glutamate and glutamine are reported to be increased in concentration in the extracellular fluid. It has been reported that glutamate acts as an extracellular signaling molecule for facilitating local spread of advanced grades of glioblastoma. In the present study, we aimed to examine whether glutamate uptake mechanisms is impaired in advanced glioblastoma. The possible downregulated mechanisms of glutamate uptake would facilitate persistence of glutamate in the extracellular environment, rather than intracellular uptake. We obtained biobanked human specimens of glioblastoma and tested expression of proteins belonging to the solute carrier families of proteins that are known to function as membrane-located excitatory amino acid like glutamate transporters. The present study provides preliminary evidence of the downregulation of membrane expression of excitatory amino acid transporters solute carrier family 1 member 3 (SLC1A3) and its palmitoylated form in gliosomes, as well as SLC1A2 in the glio-synaptosomes. Compounds like riluzole used in the treatment of amyotrophic lateral sclerosis and the antibiotic ceftriaxone have the potential to facilitate glutamate uptake. These medications may be examined as adjunct chemotherapy in the massively aggressive tumor glioblastoma multiforme.

Extracranial metastasis of gliobastoma: Three illustrative cases and current review of the molecular pathology and management strategies

Glioblastoma (GBM) is the most common and the most malignant primary brain tumor in adults, accounting for ~12-15% of all intracranial neoplasms. Despite advances in surgical, medical and radiation therapies, the mortality of GBM remains high, with a median survival ranging between 40 and 70 weeks. Similar to other primary brain tumors, the extracranial metastasis of GBM is extremely rare, occurring in <2% of patients. To demonstrate the clinical characteristics of this rare tumor, we herein present three cases of extracranial GBM metastasis: One to the lungs, which represents the longest reported survival of lung metastases from GBM to date; the second to the soft tissue of the posterior neck; and the third to the lumbar intradural space. Unlike tumors elsewhere, there are unique barriers in the brain that prevent the hematogenous and lymphatic spread of intracranial tumors, such as the dura mater and the thickened basement membrane of the blood vessels. In addition, central nervous system tumor cells lack extracellular matrix proteins required to invade surrounding connective tissue, a prerequisite for tumor dissemination. In this study, we aimed to investigate the different possible mechanisms underlying the extracranial metastasis of GBM and determine the biomolecular and genetic characteristics differentiating GBMs that metastasize from those that do not. We also reviewed the role of systemic chemotherapy and bevacizumab in the treatment of disseminated GBMs. Early identification and differentiation of these tumors may enable patients to benefit from surgical resection, radiation and combination chemotherapy prior to developing other comorbidities from metastatic disease, which may translate into prolonged survival with an acceptable quality of life.

Antiangiogenic Therapies and Extracranial Metastasis in Glioblastoma: A Case Report and Review of the Literature.

We present a case report of a patient with glioblastoma multiforme (GBM) complicated by extracranial metastasis (ECM) whose survival of nearly four years surpassed the anticipated life expectancy given numerous negative prognostic factors including EGFRvIII-mutation, unmethylated MGMT promoter status, and ECM. Interestingly, while this patient suffered from locally aggressive disease with multiple intracranial recurrences, the proximal cause of death was progressive extracranial disease and complications related to pulmonary metastases. Herein, we review potential mechanisms of ECM with an emphasis upon glioblastoma molecular and genetic profiles and the potential implications of targeted agents such as bevacizumab.

PM-02 * DIACYLGLYCEROL KINASE ALPHA INHIBITION PROLONGS SURVIVAL OF MICE WITH PRIMARY AND METASTATIC BRAIN TUMORS

Diacylglycerol kinases (DGKs) are a family of highly conserved enzymes that are promiscuously responsive to growth factors and other mitogenic signaling cascades. DGKs are activated by binding of Ca2+ and phosphoserine, and catalyze conversion of diacylglycerol (DAG) to phosphatidic acid (PA). PA has multiple effects on downstream cellular signaling, while activated DGKs can associate with scaffolding proteins, or undergo degradation or translocation to the nucleus through atypical localization signals and thereby promote cell division and survival. A repurposed small-molecule inhibitor of DGKα activity is shown here to antagonize tumor growth in xenografts of primary and metastatic brain tumors and syngeneic mouse tumor grafts. Based on recent prior research and in this work in xenograft models, DGKα inhibition may arrest or kill tumors through cancer addiction to DGKα activity. Here, in syngeneic tumor models of primary and metastatic brain tumors, an additional anti-tumor effect of DGKα inhibition via up-regulation of normal immune function is possibly present. This work uses two syngeneic in vivo tumor models to compare DGKα inhibition with the monoclonal antibody immunotherapy anti-CTLA-4, and also tests the effect of combination of the two treatments on survival, tumor size, and immunologic activity. The anti-tumor activity of the DGKα inhibitor and the contribution of other DGK family members is shown using cell-based toxicity and flow cytometry assays. Direct interaction between the inhibitor and DGKα is shown with biochemical reconstitution of inhibitor-enzyme interactions These early results suggest that DGKα inhibition may be a promising strategy for patients with glioblastoma and melanoma brain metastasis.

MiRNA-181c inhibits EGFR-signaling-dependent MMP9 activation via suppressing Akt phosphorylation in glioblastoma.

As the most aggressive malignant primary human brain tumor, glioblastoma is noted with extremely poor patient survival. Previous studies have demonstrated that expression of matrix metalloproteinase-9 (MMP9) in glioblastoma cells is critical for cancer metastasis. However, the molecular signaling pathways that control MMP9 activation remain undefined. Here, we reported a strong negative correlation of microRNA (miRNA)-181c levels with either MMP9 levels or activation of epidermal growth factor receptor (EGFR) signaling in glioblastoma patients. EGF-induced activation of EGFR in a human glioblastoma line, A-172 cells, increased MMP9 expression through activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway, without affecting expression of miRNA-181c. On the other hand, overexpression of miRNA-181c in A-172 cells inhibited MMP9 expression by inhibiting Akt phosphorylation, but not phosphorylation of EGFR receptor. Taken together, these findings suggest that EGFR signaling activates downstream PI3K/Akt to increase MMP9 expression in glioblastoma, while phosphorylation of Akt is a control point by miRNA-181c. Our work thus provides new insights into the molecular basis underlying the metastasis of glioblastoma.

Inhibition of FoxO1 nuclear exclusion prevents metastasis of glioblastoma.

Glioblastoma is the most aggressive malignant primary brain tumor in humans, with extremely poor patient survival. Although previous studies have demonstrated that expression of matrix metalloproteinase-9 (MMP9) in glioblastoma promotes cancer metastasis, the upstream molecular signaling cascades that control activation of MMP9 remain largely unknown. Here, we used a human glioblastoma line, A-172, to examine molecular signaling to activate MMP9. We found that epidermal growth factor (EGF)-induced activation of epidermal growth factor receptor (EGFR) in A-172 cells activated MMP9, resulting in an increase in cancer invasiveness. A specific inhibitor for EGFR efficiently blocked EGF-induced activation of MMP9 and then cancer invasiveness. Moreover, an inhibitor for phosphatidylinositol 3-kinase (PI-3 K)/protein kinase B (Akt) significantly inhibited the EGF-induced activation of MMP9. Furthermore, nuclear exclusion of a major Akt downstream target, Forkhead box protein O1 (FoxO1), was induced by Akt activation, which could be inhibited by either an EGFR inhibitor or by PI-3 K/Akt inhibitor. An expression of a constitutive nuclear form of FoxO1 significantly inhibited MMP9 activation induced by EGF. Taken together, these findings suggest that EGF/EGFR signaling activates downstream PI-3 K/Akt to induce FoxO1 nuclear exclusion, which activates MMP9 to promote glioblastoma invasiveness. Thus, FoxO1 appears to be a novel therapeutic target for inhibiting metastasis of glioblastoma.

Disrupting the CXCL12/CXCR4 axis disturbs the characteristics of glioblastoma stem-like cells of rat RG2 glioblastoma

BackgroundGlioblastoma stem-like cells (GSC) have been shown to promote tumor growth, tumor-associated neovascularization, therapeutic resistance, and metastasis. CXCR4 receptors have been found involved in the proliferation, metastasis, angiogenesis, and drug-resistant characteristics of glioblastoma. However, the role of CXCR4 in modulating the stem-like cell properties of rat glioblastoma remains ambiguous.MethodsTo explore the role of the CXCL12/CXCR4 axis in maintaining rat GSC properties, we disrupted the CXCR4 signaling by using small hairpin interfering RNA (shRNA). To investigate the role of the CXCL12/CXCR4 axis in maintaining rat GSC properties, we used a spheroid formation assay to assess the stem cell self-renewal properties. A western blot analysis and PCR arrays were used to examine the genes involved in proliferation, self-renewal, and cancer drug resistance. Finally, DNA content and flow cytometry, an immunohistochemical analysis, and methylcellulose colony formation, in vitro invasive and intracranial injection xenograft assays were employed to examine the disruptive effect of CXCR4 on the characteristics of GSCs of the RG2 cell line.ResultsDisrupting CXCR4 inhibited the proliferation of RG2 cells both in vitro and in vivo. The spheroid formation assay indicated that CXCR4 was vital for the self-renewal of RG2 GSCs. Disrupting the CXCL12/CXCR4 pathway also reduced the expression of GSC cell markers, including Nestin, ABCG2, and musashi (Msi), and the expression of genes involved in regulating stem cell properties, including Oct4, Nanog, maternal embryonic leucine zipper kinase (MELK), MGMT, VEGF, MMP2, and MMP9.ConclusionThe chemokine receptor CXCR4 is crucial for maintaining the self-renewal, proliferation, therapeutic resistance, and angiogenesis of GSCs of rat RG2 glioblastoma.

Abstract 5343: MicroRNAs regulate epithelial-mesenchymal transition in human glioma cells through modulation of the miRNA-VHL- β-catenin axis.

Abstract Background. Glioblastoma (GBM) is the most common malignant primary brain tumor in adults and is a leading cause of cancer-related death due to its invasive nature. Despite advances in radiation and chemotherapy following surgical resection of the tumor, the prognosis of GBM remains poor with an average survival time of less than one year. Complex biological processes are involved in the establishment of invasion and metastasis of GBM and epithelial- mesenchymal transition (EMT) has been reported to play crucial role. The EMT converts epithelial cells into mesenchymal cells, a normal embryological process that frequently get activated during cancer invasion and metastasis. While increasing evidence indicates that the deregulation of miRNAs and beta-catenin signal pathway may play a critical role in EMT in a wide range of human cancers, there is no direct evidence showing that this pathway actually determines the EMT regulated by miRNAs in GBM. Methods. The expression of miRNAs (miR-21, miR-23b, and miR-566) in 48 clinical samples and five glioma cell lines with different grades was examined by FISH and qRT-PCR. TOP/FOP flash luciferase assays and transwell test were employed to identify the effect of the above miRNAs on the Wnt/β-catenin pathway activity and invasive activity of the glioma cells. Luciferase assay and western blot analysis were used to examine that VHL is a direct target of the above miRNAs. We further investigated the role of miRNAs on EMT by Western blot analysis. Results. We identified that expression of all of the above miRNAs are elevated in both glioma tissue samples and glioma cells by qRT-PCR and FISH analyses. Knock-down each of the above miRNAs could suppress invasion of glioma cells in vitro. Luciferase assay and western blot analysis revealed that VHL is a direct target of miR-23b. As such, restored expression of VHL inhibited glioma cell invasion. Mechanistic investigation revealed that miRNAs deletion suppressed β-catenin/Tcf-4 transcription activity in a VHL-dependent manner. Furthermore, expression of VHL was inversely correlated with miRNAs in glioblastoma samples. Conclusion. Our results indicate that down-regulation each of miRNAs (miR-21, miR-23b, and miR-566) inhibits EMT and invasion of glioma by expression of VHL. Genetic restoration of VHL bypassed the effects of miRNA overexpression, suppressing the transcriptional activities of β-catenin/Tcf-4 pathways. Based upon these observations, we conclude a role for the these miRNAs, upstream regulators of beta-catenin signal pathways, represent therapeutic targets to inhibit tumor metastasis in glioma cells. Citation Format: Lei Han, Luyue Chen, Kailiang Zhang, Zhendong Shi, Lingchao Chen, Peiyu Pu, Chunsheng Kang. MicroRNAs regulate epithelial-mesenchymal transition in human glioma cells through modulation of the miRNA-VHL- β-catenin axis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5343. doi:10.1158/1538-7445.AM2013-5343

Cytological diagnosis of metastatic glioblastoma in the pleural effusion of a lung transplant patient

The extracranial metastasis of glioblastoma is a rare event. We report the case of a patient who developed metastatic glioblastoma in pleural effusion 15 months after lung transplant, with emphasis on differential diagnosis based on cytological material. In our case, tumor cells had pleomorphic nuclei, prominent nucleoli, and fine vesicular chromatin. Some were arranged in a poorly formed pseudo-glandular architecture, mimicking a poorly differentiated adenocarcinoma. The cytological diagnosis of metastatic glioblastoma is difficult and depends critically on clinical history and suspicion, particularly in the transplant setting. Review of the literature indicates that transmission/metastasis of intracranial malignancy occurs rarely following organ transplantation, with some debate on the suitability for transplant of organs from affected donors. Although the situation is uncommon, this report of the cytological findings of extracranial glioblastoma may extend our current knowledge and provide additional differential diagnostic information for this entity.

Abstract B83: Multikinase inhibitors for antiangiogenesis and tumor metastasis in zebrafish model

Abstract Tumor metastasis is a central issue in the treatment of malignancies, and metastases rather than the primary tumor contributes to about 90% of cancer-related deaths. Current therapies for solid tumors focus on preventing the development of blood vessels supplying nutrients to the tumor; however, metastasis to distant organs leads to relapse and patient mortality and resistance to treatment often results in disease progression. The host microenvironment is a critical regulator of cancer progression because the normal vascular endothelium, unlike the tumor cells, does not carry mutations, which potentially facilitates a clinical response to anti-angiogenesis therapeutic agents. Macrophages and neutrophils play a critical role in this process since macrophage plasticity results in the expression of genes which can switch the macrophage ‘type’ to one which enhances tumor metastasis. Thus, it is beneficial to target immune cells and change their gene expression profile such that they become beneficial to the host and prevent metastasis. The vascular endothelial growth factor (VEGF) pathway is central to regulating angiogenesis and metastasis. The VEGF blocking antibody, bevacizumab and small molecules that inhibit receptor tyrosine kinases including the VEGF receptor, sunitinib and sorafenib, have prolonged lives of cancer patients in the clinic; however, their long term effects in patients is unknown. Based on preclinical mouse model studies indicating that the “anti-angiogenesis” agent sunitinib increased tumor metastases, the anti-angiogenesis approach in cancer has acquired a controversial “paradigm” that anti-angiogenesis therapy can lead to harmful effects on tumor growth and metastasis. This highlights the urgency of unraveling the molecular and cellular mechanism of action of anti-angiogenesis agents on tumor growth and metastasis. Zebrafish/tumor xenograft model is a live, complex, 3-dimensional tumor xenograft animal model allows the continuous delivery of tumor angiogenic factors and includes the participation of host stroma and vasculature in the cellular and molecular events contributing to tumor invasion and metastasis; thus mimicking the host disease. The zebrafish model facilitates the high-throughput manipulation of the host environment using morpholino knockdown of genes and small molecule inhibitors to block tumor angiogenesis. Zebrafish neoplasms induced by carcinogens or oncogenes recapitulate the human disease at the histological, molecular and pathological levels. Moreover, the similarity in gene-expression profile was consistent between zebrafish and human cancer, supporting its use as a model for human tumors. The zebrafish xenograft does not need treatment with immunosuppressive drugs prior to the xenograft, the neo-vascularization can be seen within 24 hours and metastasis can be detected within 6 days of tumor cell transplantation. Recently it was also demonstrated that neutrophils mediated enhanced metastasis in zebrafish/breast cancer xenograft after treatment with sunitinib and macrophages changed their phenotype in response to treatment. Here we determined if specific VEGF inhibitors block tumor angiogenesis without induction of tumor metastasis. Sunitinib is an “anti-angiogenesis” agent acting as a multikinase inhibitor that selectively targets the VEGF receptors but also inhibits PDGFR and KIT. Although sunitinib effectively inhibits neovascularization, it also increased local invasion and distant metastasis in pancreatic neuroendocrine carcinoma and glioblastoma in a mouse xenograft model. In addition, accelerated metastasis of breast cancer and melanoma was observed after short-term treatments with sunitinib, even though it exhibited a potent inhibition of tumor angiogenesis. As with the mouse studies, our zebrafish/tumor xenograft studies demonstrated that sunitinib efficiently suppresses angiogenesis and promotes tumor metastasis. Subsequently, we identified another small molecule multikinase inhibitor, which, like sunitinib, selectively blocks VEGFRs (as well as inhibits PDGFR and KIT), but produces an opposite outcome with respect to metastasis. In our zebrafish/tumor model transplanted with human prostate, lung or pancreatic cancer cells, this newly identified multikinase inhibitor efficiently blocks angiogenesis and effectively suppresses tumor metastasis. This new data promises to shift the current anti-angiogenesis “paradigm” that all anti-angiogenesis agents promote tumor metastasis. Therefore, we argue that different small molecule inhibitors of angiogenesis can successfully target angiogenesis and block metastasis at the same time. Potential stromal-epithelial interactions essential for cancer progression and metastasis through these inhibitors include pathways regulating angiogenesis, hypoxia, inflammation and metastasis. Our data suggest that the mechanism by which two similar receptor tyrosine kinase inhibitors produce the same effect on anti-angiogenesis but have very different outcomes in tumor metastasis by modulating these pathways. Citation Format: Karine F. Ferri-Lagneau, Jamil Haider, TinChung leung. Multikinase inhibitors for antiangiogenesis and tumor metastasis in zebrafish model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B83.