Zarco, Natanael

Abstract Background Glioblastomas (GBMs) are the main primary brain tumors in adults with almost 100% recurrence rate. Patients with lateral ventricle proximal GBMs (LV-GBMs) exhibit worse survival compared to distal locations for unknown reasons. One hypothesis is the proximity of these tumors to the cerebrospinal fluid (CSF) and its chemical cues that can regulate cellular phenotype. We therefore investigated the role of CSF on GBM gene expression and the role of a CSF-induced gene, SERPINA3, in GBM malignancy in vitro and in vivo. Methods We utilized human CSF and GBM brain tumor-initiating cells (BTICs). We determined the impact of SERPINA3 expression in glioma patients using The Cancer Genome Atlas (TCGA) database. SERPINA3 expression changes were evaluated at mRNA and protein levels. The effects of knockdown (KD) and overexpression (OE) of SERPINA3 on cell migration, viability and cell proliferation were evaluated. Stem cell characteristics on KD cells were evaluated by differentiation and colony formation experiments. Tumor growth was studied by intracranial and flank injections. Results GBM-CSF increased BTIC migration accompanied by upregulation of the SERPINA3 gene. In patient samples and TCGA data, we observed SERPINA3 to correlate directly with brain tumor grade and indirectly with GBM patient survival. SERPINA3 KD induced a decrease in cell proliferation, migration, invasion, and stem cell characteristics, while SERPINA3 OE increased cell migration. In vivo, SERPINA3 KD BTICs showed increased survival in a murine model. Conclusions SERPINA3 plays a key role in GBM malignancy and its inhibition results in a better outcome using GBM preclinical models.

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with a median survival of 14 months despite multimodal treatment. Patients with GBM tumors exhibit a post-resection recurrence rate of almost 100%. The tumor recurrence and treatment resistance properties of GBM reside in a stem-like cell population called Brain Tumor Initiating Cells (BTIC). Among primary GBMs, periventricular tumors exhibit worse survival than tumors distal to the lateral ventricles (LV), the cause for this worse outcome is not known. A potential explanation is the proximity of these tumors to the cerebrospinal fluid (CSF) and the neurogenic niche in the sub-ventricular zone (SVZ). Chemotactic signals present in the cerebrospinal fluid (CSF) regulate neural progenitor cells migratory response and differentiation profile. Our preliminary data show that cancer-derived CSF induces an increase in the migration and proliferation of GBM cells when compared to non-cancer CSF. We evaluated the transcriptome of human GBM cells in response to cancer and non-cancer CSF and identified alpha-1-antichymotrypsin (SERPINA3) as one of the genes that show higher over expression upon cancer CSF-stimulation. SERPINA3 is a circulating anti-protease that is produced and released in different tissues such as liver, prostate, lungs, endometrium and brain that is over-expressed in multiple cancers. We hypothesize that the overexpression of SERPINA3 in response to CSF is a contributing factor to the increase malignancy of periventricular GBM tumors. We tested this hypothesis in silico, in vitro and in vivo. Using TCGA and Rembrandt databases we observed a higher expression of SERPINA3 in brain tumor tissue compared to controls with no differences among GBM subtypes. In GBM patients, expression of SERPINA3 negatively affects survival expectancy. In vitro, Using intraoperative samples, we confirmed that SERPINA3 is overexpressed in brain cancer tissue by western-blot and qRT-PCR. Interestingly, we observed a higher nuclear localization of SERPINA3 in cancer tissue and cells. To determine if SERPINA3 has an effect on the malignancy of GBM cells, we silenced its expression using shRNA. SERPINA3 silencing induced a decrease in cell proliferation (p<0.05), as well as migration (p<0.05), and invasion (p<0.05). Remarkably CD133 expression and stem cell markers were decreased when SERPINA3 is silenced. In contrast, the addition of recombinant SERPINA3 induced an increase in cell migration (p<0.05) of GBM cells. Moreover, cancer CSF is no longer able to induce an increase in GBM cell migration in SERPINA3-KD cells (p<0.05). In vivo, mice orthotopically-injected with SERPINA3-KD cells showed a prolonged overall survival (p<0.05). In summary, our results support the hypothesis that SERPINA3 plays a key role in GBM malignancy in response to CSF and its inhibition results in a better outcome using GBM preclinical models. Targeting the CSF-GBM interaction is a potential therapeutic approach that remains to be explored. Citation Format: Montserrat A. Lara Velazquez, Natanael Zarco, Anna Carrano, Jordan Phillips, Paula Schiapparelli, Emily S. Norton, Stephanie Jeanneret, Teresita Corona, Jose Segovia, Kaisorn Chaichana, Yan Asmann, Alfredo Quinones Hinojosa, Hugo Guerrero-Cazares. Cerebrospinal fluid-responsive factor SERPINA3 induces an increase in the malignancy of glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3780.

Abstract Glioblastoma (GBM), also known as grade IV astrocytoma, is the most common and devastating primary cancer of the central nervous system in adults. Despite the most advanced therapeutic strategies combining surgery, chemotherapy, and radiation, survival expectancy of GBM patients averages 14 months, with a recurrence rate of almost 100%. Remarkably, tumor location has great implications in the prognosis of GBM. Approximately 50-60% of GBM tumors infiltrates or contacts the subventricular zone (SVZ). These patients have significantly worse outcomes, in terms of median overall survival, time to progression and reoccurrence. The reason for the worse prognosis of SVZ-infiltrating tumors is unknown. The SVZ is the largest neurogenic niche in the adult brain, here the differentiation and migration of SVZ cells is regulated by direct contact with the cerebrospinal fluid (CSF). We suggest that CSF contribute to enhance GBM malignant characteristics in SVZ-infiltrating tumors. This study evaluated the impact of CSF exposure on GBM cells proliferation, migration in vitro and in vivo, as well as changes in gene expression profile. We demonstrated that CSF affects the malignancy of GBM cells by increasing their ability to proliferate and migrate. GBM cells stimulated with cancer CSF, non-cancer CSF, or control conditions at different time points (24-72 hrs) were evaluated for viability (by Alamar Blue assay) and proliferation rate (Ki67+ immunostaining), as well as for migratory capabilities (by means of transwell assay and time-lapse microscopy). We recapitulated these CSF effects in vivo using an in vivo model in which both female and male nude mice were implanted with GBM cells in suspension with human cancer or non-cancer CSF in an hydrogel as vehicle (n=8 mice per group). Tumor growth was followed via IVIS imaging and then studied by measuring tumor area on H&E slides and immunohistochemistry staining for Ki67, 21 days after tumor implantation. Animals receiving cancer CSF presented with greater tumor size and Ki67 proliferation index. To further dissect the mechanisms behind these observations, we performed a transcriptome analysis of CSF-treated GBM cells. At the molecular level we observed that cancer CSF induced changes in pathways regulating apoptosis, survival, integrins signaling, angiogenesis, glucose metabolism and response to inflammatory stimuli, all of which might be responsible for the increase in GBM malignancy. Taken together, this study provides direct evidence that CSF is an important player in determining tumor growth and invasion through the activation of complex gene expression patterns characteristic of a malignant phenotype. Understanding the interaction of brain tumors and CSF may lead to new therapeutic strategies that will target the CSF components in GBM patients. Citation Format: Anna Carrano, Jordan Phillipps, Montserrat Lara-Velazquez, Natanael Zarco, Paola Suarez-Meade, Kaisorn Chaichana, Alfredo Quinones-Hinojosa, Yan Asmann, Hugo Guerrero-Cázares. The role of human cerebrospinal fluid in glioblastoma malignancy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1574.

Voltage-gated Ca2+(CaV) channels are expressed in endocrine cells where they contribute to hormone secretion. Diverse chemical messengers, including epidermal growth factor (EGF), are known to affect the expression of CaVchannels. Previous studies have shown that EGF increases Ca2+currents in GH3 pituitary cells by increasing the number of high voltage-activated (HVA) CaVchannels at the cell membrane, which results in enhanced prolactin (PRL) secretion. However, little is known regarding the mechanisms underlying this regulation. Here, we show that EGF actually increases the expression of the CaVα2δ-1 subunit, a key molecular component of HVA channels. The analysis of the gene promoter encoding CaVα2δ-1 ( CACNA2D1) revealed binding sites for transcription factors activated by the Ras/Raf/MEK/ERK signaling cascade. Chromatin immunoprecipitation and site-directed mutagenesis showed that ELK-1 is crucial for the transcriptional regulation of CACNA2D1 in response to EGF. Furthermore, we found that EGF increases the membrane expression of CaVα2δ-1 and that ELK-1 overexpression increases HVA current density, whereas ELK-1 knockdown decreases the functional expression of the channels. Hormone release assays revealed that CaVα2δ-1 overexpression increases PRL secretion. These results suggest a mechanism for how EGF, by activating the Ras/Raf/MEK/ERK/ELK-1 pathway, may influence the expression of HVA channels and the secretory behavior of pituitary cells.

Abstract Glioblastoma (GBM) is the most aggressive of all the brain tumors with a median patient survival less than 15 months. Despite of surgical resection, radiotherapy, and chemotherapy, recurrence rate is almost 100%. A great percentage of GBM tumors (~60%) infiltrate and contact the ventricular-subventricular zone (V-SVZ). Interestingly, these tumors are the most aggressive, and invariably lead to higher distal recurrence rates, shorter time to tumor progression, and lower overall survival of the patient. The reason for this role of V-SVZ-proximity on the outcome of GBM patients is unknown. We suggest that a potential explanation is the interaction of GBM with the V-SVZ. This region is the largest neurogenic niche in the adult brain where neural stem cells (NSCs) give rise to newborn neuroblasts that migrate toward the olfactory bulb. In GBM there is a cell subpopulation called brain tumor stem cells (BTSCs) with NSCs-like characteristics, but with added potential for tumor initiation, recurrence and invasiveness. Tumor microenvironment plays an important role in migration and invasion process. In the present work, we used the total exosome isolation kit to purify Extracellular Vesicles (EVs) from human primary cultures of BTSCs. We determined that BTSCs-derived EVs contain specific information that is transfer to primary cultures of human Neural Progenitors Cells (NPCs) modulating their proliferation rate, cell viability, and migration. In addition, we identify that NPCs taken up BTSCs-derived EVs and significantly increase the expression levels of stemness-related genes such as Nestin, Nanog, and Sox2, suggesting that a phenotypic transdifferentiation is being carry out. These results support our hypothesis that GBM modulate the tumor microenvironment close to the V-SVZ by releasing EVs that target cellular components in this region and promote their phenotypic transformation, highlighting that NPCs biology changes in the context of tumor environment.