The GAL-8/EGFR axis in the antitumoral effects of D-Propranolol on T98G glioblastoma cells

Abstract

Glioblastoma (GB) is the most severe type of brain neoplasia, as patients present a lifespan of roughly 12 months. Current treatments only partially diminish GB aggressiveness and do not improve lifespan significantly. The high migratory activity invading secondary regions of the brain is one of the most difficult of its malignant properties to counteract. New therapeutic alternatives are urgently needed. A potential approach to reduce GB malignancy is to simultaneously interfere with the function of different proteins involved in GB pathogenicity. In this thesis we addressed the epidermal growth factor receptor (EGFR) and Galectin8 (Gal-8). EGFR is the most commonly altered gene in GB, found overexpressed in 40% to 50% of cases and proposed as a marker of poor prognosis (Li, Liang, Song, Xiang, & Liu, 2018). EGFR variant III (EGFRvIII), a deletion mutant that lacks most of the extracellular domain of the receptor precluding its binding to ligand is one of the most aggressive oncoproteins. EGFR belongs to the ErbB tyrosine kinase family receptor that regulates signaling pathways involved in a variety of cellular processes, such as cell growth, viability, and migration (Ciardiello & Tortora, 2008). Drugs that counteract the activity of EGFR and are currently used in other cancers have little effectiveness in GB. Our laboratory has shown that the secondary amine D-Propranolol can be used to interfere with EGFR function inducing alterations in its endocytic trafficking behavior that result in its intracellular accumulation. This effect associates with effects against proliferation and viability on different cancer cells and involves a little-known signaling pathway downstream phosphatidic acid (PA). D-Propranolol induces rapid increments of PA, which activates type 4 phosphodiesterases leading to decreased cAMP levels and PKA activity. Direct inhibitors of PKA activity mimic the endocytic effect of D-Propranolol on the EGFR. As a secondary amine, D-Propranolol may in principle induce lysosomal damage, a condition actively studied as a potential route for the elimination of cancer cells. Damaged endolysosomes are detected by a control system in the 2 cytosolic compartment constituted by Galectins, which are carbohydrates recognizing proteins, and in this context, Galectin-3 (Gal-3), Galectin-9 (Gal-9) and Galectin-8 (Gal-8), which play different and complementary functions in autophagic removing or repairing of these organelles (Jia et al., 2020). In particular, when endolysosomal membrane present leakages Gal-8 interacts with the lysosome amino acid transporter SLC38A9 and the Ragulator/Rag complex resulting in displacement and inactivation of mTORC1 (Jia et al., 2018). These events serve as a protection mechanism for reestablishing lysosomal function and cell homeostasis. To this date, these intracellular protecting properties of Gal-8 have been investigated in processes of bacterial and viral infections, and tau protein dispersal, remaining unknown in cancer. Gal-8 is secreted by unconventional mechanisms and its extracellular functions have been associated with multiple pro-tumoral features. Results from our laboratory have described that Gal-8 overexpression in Madin-Darbey Canine Kidney cells (MDCK), a non-cancerous cell type, increases cell proliferation and promotes formation of tumors in xenografts experiments (Oyanadel et al., 2018), while Gal-8 silencing increases apoptosis by 15% in U87 GB cells (Metz et al., 2016). Soluble Gal-8 in the extracellular media induces the migration of U87 GB cells in Transwell assays (Metz et al., 2016). Furthermore, GB cells have been described to manifest aggressive invasion to healthy brain tissue, which leads to recurrence after surgical procedures of tumor removal (Chintala et al., 1999). Preliminary results in collaboration with the laboratory of Dr. Patricia Burgos have shown that D-Propranolol induces Gal-3 recruitment to lysosome-like compartments in HeLa cells, suggesting a potential lysosomal damaging effect of this drug that might contribute to its deleterious effects in cancer cells. We propose the following hypothesis: D-Propranolol decreases viable cell number and invasion of T98G glioblastoma cells interfering with the function of EGFR and Gal-8 . The general objective is to determine whether D-Propranolol is an effective drug against glioblastoma cells that express malignancy factors such as EGFR and Gal-8. The specific objectives are: 1) To evaluate the effect of 3 D-Propranolol on the viability of T98G cells, 2) To evaluate the effect of DPropranolol in lysosomes in T98G cells, 3) To evaluate the impact of Gal-8 expression on T98G cell viability, EGFR signaling and invasion in response to DPropranolol. Methodology: We used human glioblastoma T98G cells to assess the effects of D-Propranolol on EGFR endocytosis, using indirect immunofluorescence (IFI), immunoblot, and flow cytometry. Viable cell number was evaluated with trypan blue, and proliferation was assessed with EdU thymidine analogue. Lysosomal damage was evaluated by IFI of Gal-3 and Gal8, as well with transduced Gal-8-ZsGreen in T98G cells, Lysotracker red and Magic red. Gal-8 silencing was achieved by transduction with lentiviral particles containing sh-Gal-8. EGFR and ERK phosphorylation were evaluated by immunoblot. Invasive migration was evaluated by an inverted invasive migration assay.