Adrienne Weeks

ECT2 and RASAL2 Mediate Mesenchymal-Amoeboid Transition In Human Astrocytoma Cells

Malignant astrocytomas are highly invasive brain tumors. The Rho family of cytoskeletal GTPases are key regulators of astrocytoma migration and invasion; expression of the guanine nucleotide exchange factor ECT2 is elevated in primary astrocytomas and predicts both survival and malignancy. Mice bearing orthotopically implanted astrocytoma cells with diminished ECT2 levels following ECT2 knockdown exhibit longer survival. Although ECT2 is normally expressed in the nucleus, we show that ECT2 is aberrantly localized to the cytoplasm in both astrocytoma cell lines and primary human astrocytomas, and colocalizes with RAC1 and CDC42 at the leading edge of migrating astrocytoma cells. Inhibition of ECT2 expression by RNA interference resulted in decreased RAC1 and CDC42 activity, but no change in RHO activity, suggesting that ECT2 is capable of activating these pro-migratory Rho family members. ECT2 overexpression in astrocytoma cells resulted in a transition to an amoeboid phenotype that was abolished with the ROCK inhibitor, Y-27632. Cytoplasmic fractionation of astrocytoma cells followed by ECT2 immunoprecipitation and mass spectrometry were used to identify protein-binding partners that modulate the activity of ECT2 toward RAC1 and RHO/ROCK. We identified RASAL2 as an ECT2-interacting protein that regulates RHO activity in astrocytoma cells. RASAL2 knockdown leads to a conversion to an amoeboid phenotype. Our studies reveal that ECT2 has a novel role in mesenchymal-amoeboid transition in human astrocytoma cells.

Role of the Cofilin Activity Cycle in Astrocytoma Migration and Invasion

The cofilin pathway plays a central role in the regulation of actin polymerization and the formation of cell membrane protrusions that are essential for cell migration. Overexpression of cofilin has been linked to the aggressiveness of a variety of different cancers. In these cancers, the phosphorylation of cofilin at Ser3 is a key regulatory mechanism modulating cofilin activity. The activation status of cofilin has been directly linked to tumor invasion. Accordingly, in this study, we examined the expression of cofilin and its activation status in astrocytoma cell lines and astrocytic tumors. We show that cofilin expression was increased and correlated with increasing grade malignant astrocytoma. In addition, both cofilin and LIMK had elevated expression in astrocytoma cell lines. Knockdown of cofilin by siRNA altered astrocytoma cell morphology and inhibited astrocytoma migration and invasion. Conversely, overexpression of a cofilin phosphorylation mutant in an in vivo intracranial xenograft model resulted in a more highly invasive phenotype than those xenographs expressing wild-type cofilin. Animals harboring astrocytomas stably expressing the cofilin phosphorylation mutant (cofilin-S3A) demonstrated marked local invasiveness and spread across the corpus callosum to the contralateral hemisphere in all animals. Taken together, these data indicate that the cofilin activity pathway may represent a novel therapeutic target to diminish the invasion of these highly malignant tumors.

Overexpression of CD99 Increases the Migration and Invasiveness of Human Malignant Glioma Cells.

The malignant glioma is the most common primary human brain tumor, and its migration and invasiveness away from the primary tumor mass are considered a leading cause of tumor recurrence and treatment failure. Recently, gene expression profiling revealed that the transmembrane glycoprotein CD99 is more highly expressed in malignant glioma than in normal brain. Although its function is not completely understood, CD99 is implicated in cell adhesion and migration in a variety of different cell types. CD99 has wild-type and splice variant isoforms. Previous studies have shown that wild-type CD99 may be an oncosuppressor in some tumors, distinct from the role of the splice variant isoform. In this study, our data reveal that only wild-type CD99 is expressed in human glioma cells and tissues. Using a tissue microarray, we validated that gliomas demonstrate higher expression of CD99 compared with nonneoplastic brain. To assess the role of CD99 in glioma migration and invasion, we inhibited CD99 expression by siRNA and demonstrated decreased glioma migration and invasion. In contrast, when CD99 was overexpressed in glioma cells, we observed enhancement of cell migration and invasiveness. An orthotopic brain tumor model demonstrates that CD99 overexpression significantly increases invasiveness and decreases survival rate. Interestingly, Rac activity was decreased and Rho activity was increased in CD99 overexpressing glioma cells, and the proportion of amoeboid cells to mesenchymal cells was significantly increased. Taken together, our findings suggest that CD99 may play an important role in the migration and invasion of human gliomas independent of Akt, ERK, or JNK signaling pathways. Moreover, CD99 might be involved in amoeboid-mesenchymal transition in glioma migration. CD99 may be an important future target to inhibit migration and invasion, especially in CD99-expressing gliomas.

The evolution and application of techniques in molecular biology to human brain tumors: a 25 year perspective

Since the establishment of the AANS/CNS Section on Tumors in 1984, neurosurgeons have been actively involved in basic science research of human brain tumors that has moved the field forward considerably. Here, we chronicle the major advances that have been made with respect to our understanding of the concepts guiding the biology of human malignant brain tumors. Numerous technical advances in science, such as the development of gene transfer techniques, the polymerase chain reaction, the discovery of oncogenes and tumor suppressor genes, and the refinement of approaches to cancer cytogenetics have enabled researchers to identify many of the non-random genetic alterations associated with brain tumor growth, invasion, immunology, angiogenesis and apoptosis. These data led to some astounding progress, for example with the use of gene therapy, whereby in the 1990s several human clinical trials were conducted for patients with brain tumors. More recently, the human genome project has been completed providing a blueprint for the human species. What has followed are exciting new techniques in molecular biology such as transcriptional profiling, single nucleotide polymorphism (SNP)-arrays, array comparative genomic hybridization (array-CGH), microRNA profiling, and detection of epigenetic silencing of tumor suppressor genes. The cancer genome is now being sequenced at break neck speed using advanced DNA sequencing techniques. We are on the threshold of cataloguing the major genetic alterations observed in all human brain tumors. What will follow is modeling of these genetic alterations in systems that will allow for the development of novel pharmacotherapeutics and translational research therapies.

Molecular Biology of Human Brain Tumors

The Central Brain Tumor Registry of the USA estimates the annual incidence of primary brain and central nervous system (CNS) tumors at 7.3/100,000 for malignant and 13.3/100,000 for nonmalignant tumors. A true comparison of incidence numbers across different time periods and countries is difficult given the inconsistency of data collection methods and the diversity in diagnostic criteria. Available diagnosis rates for malignant gliomas spanning the last three decades do not allow the determination of an increase or decline of age adapted incidence (Ohgaki and Kleihues, Acta Neuropathol 109:93–108, 2005; Ostrom et al., Neuro Oncol 16:896–913, 2014), despite remarkable changes of environment and lifestyle in industrial nations.

Epithelial Cell Transforming 2 and Aurora Kinase B Modulate Formation of Stress Granule–Containing Transcripts from Diverse Cellular Pathways in Astrocytoma Cells

Stress granules are small RNA-protein granules that modify the translational landscape during cellular stress to promote survival. The RhoGTPase RhoA is implicated in the formation of RNA stress granules. Our data demonstrate that the cytokinetic proteins epithelial cell transforming 2 and Aurora kinase B (AurkB) are localized to stress granules in human astrocytoma cells. AurkB and its downstream target histone-3 are phosphorylated during arsenite-induced stress. Chemical (AZD1152-HQPA) and siRNA inhibition of AurkB results in fewer and smaller stress granules when analyzed using high-throughput fluorescent-based cellomics assays. RNA immunoprecipitation with the known stress granule aggregates TIAR and G3BP1 was performed on astrocytoma cells, and subsequent analysis revealed that astrocytoma stress granules harbor unique mRNAs for various cellular pathways, including cellular migration, metabolism, translation, and transcriptional regulation. Human astrocytoma cell stress granules contain mRNAs that are known to be involved in glioma signaling and the mammalian target of rapamycin pathway. These data provide evidence that RNA stress granules are a novel form of epigenetic regulation in astrocytoma cells, which may be targetable by chemical inhibitors and enhance astrocytoma susceptibility to conventional therapy, such as radiation and chemotherapy.

Abstract 3847: Cytoplasmic ECT2: Implications for invasion and migration and RAS-RAC cross-talk in glioma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Gliomas are a highly malignant primary brain tumour characterized by their adept ability to invade normal brain parenchyma. We have previously identified the cytoplasmic and normally nuclear sequestered pro-cytokinetic, small cytoskeletal GTPase ECT2 to be involved in the invasive process of malignant glioma. ECT2 shows dysregulated spatial regulation in malignant gliomas with increased cytoplasmic expression, in particular at the leading edge of invading primary glioma cells. This aberrant ECT2 plays a role in RAC1 and CDC42 activation, as shRNA mediated loss of ECT2 leads to no effect on cell cycle, but a less invasive phenotype and diminished RAC1 and CDC42 levels. Using immunoprecipitation and mass spectrometry we identified the novel RAS-GAP, RASAL2 as a significant cytoplasmic interactor with ECT2 in gliomas. We show this interaction represents a mechanism by which RAS (which is aberrantly activated in gliomas) can cross-talk with the RAC/RHO pathway and coordinate growth and invasion of gliomas. Over-expression of ECT2 leads to mesenchymal-amoeboid transition (MAT) in glioma cells. Cells expressing GFP-ECT2 show hyperactive cortical dynamics with the formation of membrane blebs, similar to an amoeboid phenotype. ECT2 localized to the cortical blebs during retraction in a similar manner to active RHOA in amoeboid cells. It is known that MAT relies on an antagonistic relationship between RHOA (pro-amoeboid) and RAC1 (promesenchymal). We tested whether our phenotype was mediated by RHOA or RAC1 by administration of the RHOA downstream inhibitor of ROCK, Y27632 and RAC1 [NSC23766][1]. RHOA pathway inhibition by Y27632 at 25uM was able to reverse this hypercortical activity and resulted in formation of lamellipodia, as seen with RAC1 activation. [NSC23766][1] was unable to abolish the amoeboid phenotype in concentrations of up to 100uM. A proportion of GFP-ECT2 amoeboid cells were capable of migrating at velocities of up to 10 um/sec. Interestingly, although loss of ECT2 has no phenotypic changes in cells plated in a 2D context, loss of ECT2 by shRNA in cells plated within a collagen matrix show marked reduction in amoeboid phenotype. The ability of glioma cells to undergo MAT is a relatively new concept, however well documented in other cancer subtypes such as melanoma. The molecular switches involved in MAT remain elusive, however our data reveals a potential role for ECT2 in migratory phenotype switching. Importantly, when a stably expressing ECT2 shRNA glioma line is orthotopically injected into mouse brains, these ECT2 knockdown xenografts exhibit enhanced survival and diminished tumour engraftment as compared to controls. Taken together, this suggests that ECT2 is a viable target for malignant gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3847. doi:10.1158/1538-7445.AM2011-3847 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=NSC23766&atom=%2Fcanres%2F71%2F8_Supplement%2F3847.atom