Segovia, José

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.