Modulation in the expression levels and protein localization of cell volume and actin cytoskeleton associated proteins by the Slit/ Robo signaling pathway in Glioblastoma multiforme.

Honorable Mention Winner Glioblastoma multiforme (GBM) is the most aggressive and malignant primary brain tumor in adults1. Even after surgical resection, chemotherapy, and radiation, the average patient survival rate is 14 months2. Recent studies indicate that GBM tumors that contact the lateral ventricles within the subventricular zone (SVZ) tend to migrate and recur at distal locations after surgical resection3. One important signaling pathway in Neural Progenitor Cells (NPCs) migration from the SVZ to the olfactory bulbs is the Slit/Robo signaling pathway4. Slit2 proteins are produced by the choroid plexus and create a gradient across ependymal cells. Slit2 proteins then interact with the Roundabout (Robo) receptor on NPCs and acts as a chemo-repelling agent5. This Slit2/ Robo1 signaling pathway is a possible mechanism that contributes to GBM migration from the SVZ to distal locations. The intracellular changes when the Slit/Robo pathway is activated includes changes in cell volume regulation and actin cytoskeleton dynamics6. We then examined how the Slit2/ Robo1 signaling pathway causes changes in the expression of cell volume regulatory genes (AQP4, KCC1, NKCC1, NCC), actin cytoskeleton genes regulatory genes (p-ERM, p-Cofilin), and the localization of actin-associated proteins (FAK, Paxillin, N-WASP, Vinculin). It was found that the Slit/Robo signaling pathway increases the expression levels of genes that contribute to cell volume regulation in GBM cells, and the Slit/Robo signaling pathway increases the phosphorylation of p-Cofilin and p-ERM. This indicates that the Slit2/ Robo1 pathway participates in the regulation of cell volume and actin cytoskeleton dynamics in GBM cells

Determining glioblastoma proteome changes in response to lateral ventricle neural stem cells

Glioblastoma (GBM) is the most common and malignant primary tumor in adults. When GBM tumors are located close to the lateral ventricle they display a more aggressive recurrence pattern and negatively impact patient survival. These findings suggest the involvement of the subventricular zone neurogenic niche in GBM malignancy. To define the inter-cellular communication between neural stem cells and GBM cells, we optimized a tool to determine cell-specific proteomic changes of GBM cells in response to neural stem cell proximity. We cloned the mutated methionyl-tRNA synthetase (MetRS) gene into the lentiviral plasmid MetRS puro. MetRS allows for incorporation of azide-tagged methionine analog azidonorleucine (ANL) into newly formed proteins, effectively labeling proteins synthesized by expressing cells. We utilized the pLKO.1 vector backbone allowing puromycin resistance as a selection method. The MetRS L274 modification was confirmed, as only MetRS-transduced cells of both commercial HEK and primary GBM1A cell lines selectively incorporated ANL. Following verification, we successfully packaged the plasmid into a lentivirus. We transduced primary human fetal neural stem cell (hfNSC) and GBM lines and selected the MetRS-expressing cells by puromycin exposure. After 96 hours, wild type (WT) cells died while successfully transduced cells exhibited resistance and the ANL-compatible MetRS enzyme. Co-cultures consisting of MetRS-transduced GBM and WT hfNSCs were used to simulate a similar environment of glioblastoma neighboring lateral ventricles. Proteome Profiler results showed a significant downregulation of an angiogenesis inhibitor and upregulation of malignancy promoting proteins in GBM1A. Going forward, this analysis method will be used for cell-specific proteomics in vivo

Molecular cloning of the gene promoter encoding the human CaVγ2/Stargazin divergent transcript (CACNG2-DT): characterization and regulation by the cAMP-PKA/CREB signaling pathway

CaVγ2 (Stargazin or TARPγ2) is a protein expressed in various types of neurons whose function was initially associated with a decrease in the functional expression of voltage-gated presynaptic Ca2+ channels (CaV) and which is now known to promote the trafficking of the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) towards the cell membrane. Alterations in CaVγ2 expression has been associated with several neurological disorders, such as absence epilepsy. However, its regulation at the transcriptional level has not been intensively addressed. It has been reported that the promoter of the Cacng2 gene, encoding the rat CaVγ2, is bidirectional and regulates the transcription of a long non-coding RNA (lncRNA) in the antisense direction. Here, we investigate the proximal promoter region of the human CACNG2 gene in the antisense direction and show that this region includes two functional cAMP response elements that regulate the expression of a lncRNA called CACNG2-DT. The activity of these sites is significantly enhanced by forskolin, an adenylate cyclase activator, and inhibited by H89, a protein kinase A (PKA) antagonist. Therefore, this regulatory mechanism implies the activation of G protein-coupled receptors and downstream phosphorylation. Interestingly, we also found that the expression of CACNG2-DT may increase the levels of the CaVγ2 subunit. Together, these data provide novel information on the organization of the human CACNG2-DT gene promoter, describe modulatory domains and mechanisms that can mediate various regulatory inputs, and provide initial information on the molecular mechanisms that regulate the functional expression of the CaVγ2 protein

Phospho-Tau Protein Expression in the Cell Cycle of SH-SY5Y Neuroblastoma Cells : A Morphological Study

ACKNOWLEDGMENTS Authors want to express their gratitude to Dr. P. Davies (Albert Einstein College of Medicine, Bronx, NY, USA); Lester I. Binder † (North Western, Chicago, IL, USA) for the generous gift of mAbs (TG-3, Alz-50) and (Tau-1, Tau-5, Tau-7), respectively; Tec. Amparo Viramontes Pintos for the handling of the brain tissue; M. en C. Samadhi Moreno-Campuzano for her technical assistance; M en C.J. Iván Gálvan for his support in confocal microscopy, and the confocal microscopy unit of Laboratorio Nacional de Servicios Experimentales (LaNSE), CINVESTAV. We also want to express our gratitude to the Mexican families who donated brains of loved ones affected with Alzheimer’s disease, and made our research possible. This work is dedicated to the memory of Professor Dr. José Raúl Mena López †. This work was financially supported by CONACyT grants, No. 142293 (to R.M.), 266492 (I.V-F), 239516 (J.S) and the Mancera-Reséndiz family.Peer reviewedPostprin

Editorial: Neural Stem Cells of the Subventricular Zone: From Neurogenesis to Glioblastoma Origin

EM is funded by VI Plan Propio de Investigación (Universidad de Sevilla) Grant number 2020/0000081. NZ and HG-C are funded by the NIH-NINDS K01NS110930-03 and the Neuro oncology convergence. CC is funded by the Integrated Territorial Investment Operational Programme of the European Commission and by the Department of Department of Health and Families (Consejerıa de Salud y Familias) of the Regional Government of Andalusia. Project reference: ITI-0042-2019: ITI Cadiz 2019Ye

Circulatory shear stress induces molecular changes and side population enrichment in primary tumor-derived lung cancer cells with higher metastatic potential

Abstract Cancer is a leading cause of death and disease worldwide. However, while the survival for patients with primary cancers is improving, the ability to prevent metastatic cancer has not. Once patients develop metastases, their prognosis is dismal. A critical step in metastasis is the transit of cancer cells in the circulatory system. In this hostile microenvironment, variations in pressure and flow can change cellular behavior. However, the effects that circulation has on cancer cells and the metastatic process remain unclear. To further understand this process, we engineered a closed-loop fluidic system to analyze molecular changes induced by variations in flow rate and pressure on primary tumor-derived lung adenocarcinoma cells. We found that cancer cells overexpress epithelial-to-mesenchymal transition markers TWIST1 and SNAI2, as well as stem-like marker CD44 (but not CD133, SOX2 and/or NANOG). Moreover, these cells display a fourfold increased percentage of side population cells and have an increased propensity for migration. In vivo, surviving circulatory cells lead to decreased survival in rodents. These results suggest that cancer cells that express a specific circulatory transition phenotype and are enriched in side population cells are able to survive prolonged circulatory stress and lead to increased metastatic disease and shorter survival

Glioblastoma disrupts the ependymal wall and extracellular matrix structures of the subventricular zone

Abstract Background Glioblastoma (GBM) is the most aggressive and common type of primary brain tumor in adults. Tumor location plays a role in patient prognosis, with tumors proximal to the lateral ventricles (LVs) presenting with worse overall survival, increased expression of stem cell genes, and increased incidence of distal tumor recurrence. This may be due in part to interaction of GBM with factors of the subventricular zone (SVZ), including those contained within the cerebrospinal fluid (CSF). However, direct interaction of GBM tumors with CSF has not been proved and would be hindered in the presence of an intact ependymal cell layer. Methods Here, we investigate the ependymal cell barrier and its derived extracellular matrix (ECM) fractones in the vicinity of a GBM tumor. Patient-derived GBM cells were orthotopically implanted into immunosuppressed athymic mice in locations distal and proximal to the LV. A PBS vehicle injection in the proximal location was included as a control. At four weeks post-xenograft, brain tissue was examined for alterations in ependymal cell health via immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. Results We identified local invading GBM cells within the LV wall and increased influx of CSF into the LV-proximal GBM tumor bulk compared to controls. In addition to the physical disruption of the ependymal cell barrier, we also identified increased signs of compromised ependymal cell health in LV-proximal tumor-bearing mice. These signs include increased accumulation of lipid droplets, decreased cilia length and number, and decreased expression of cell channel proteins. We additionally identified elevated numbers of small fractones in the SVZ within this group, suggesting increased indirect CSF-contained molecule signaling to tumor cells. Conclusions Our data is the first to show that LV-proximal GBMs physically disrupt the ependymal cell barrier in animal models, resulting in disruptions in ependymal cell biology and increased CSF interaction with the tumor bulk. These findings point to ependymal cell health and CSF-contained molecules as potential axes for therapeutic targeting in the treatment of GBM