Abstract 2596: Toll like receptor 4 as a potential DNA repair modulator in U87MG-GBM cells

Abstract

Abstract Toll like receptor 4 (TLR4) has a dual role in cellular fate, depending on the stimuli and microenvironment context. Its activation may promotes cell survival and proliferative phenotype via MyD88 with NFkB activation, or leads to cell death via TRIF. Glioblastoma (GBM), grade IV astrocytoma, is the most common and aggressive primary malignancy in the brain. Surgical tumor cytoreduction followed by radiotherapy and adjunct chemotherapy with Temozolomide (TMZ) have been considered the standard of care for GBM. And, in spite of several combinatory rescue therapeutic trials, the median overall survival of GBM patients continue to be approximately 15 months. In this context, new therapeutic strategies are urgent, and immune modulation has demonstrated optimistic results in several tumor types. Therefore, the aim of this study was to analyze the role of TLR4 in human astrocytomas. First, we evaluated TLR4 expression in 140 human astrocytoma of different grades of malignancy, by qRT-PCR. TLR4 molecular signaling pathways were investigated through LPS stimuli in U87MG-GBM cell line. In astrocytoma samples, TLR4 was up-regulated when compared to brain non-neoplastic samples (p<0.05, Kruskall Wallis and Dunn tests), with increased expression in Mesenchymal compare to Classical and Proneural subtypes. U87MG cells stimulation with LPS (10ug/ml) lead to NF-kB (p65) nuclear translocation after 12hr through western blot and immunofluorescence, which was unexpected as such translocation through the MyD88/TRAF6 canonical pathway occurs within 30min to 2hr after LPS stimulation. TRIF, IL1B, RIPK1/3 expressions were increased after LPS stimulation, suggesting an activation of inflammasome and ripoptosome pathways. Moreover, the combined LPS treatment with TMZ showed a higher apoptotic rate after 48hr when compared to TMZ alone. However, the transcriptomic analysis by RNASeq of U87MG cells after LPS stimulation showed an upregulation of DNA repair genes, particularly MBD4 and PLAUR. Both genes are upregulated in mesenchymal GBM subtype in the TCGA RNASeq dataset, and they present correlated expression with TLR4 (r=0.406, p=0.004 and r=0.338, p=0.019 by Spearman's test). Our results suggest that the anti-tumoral effect by TLR4 pathway activation by enhancement of tumor apoptosis might be more efficient with combined inhibition of DNA repair cascade. In fact, OSI-296 and BKM-120 have shown to decrease MBD4 expression, and increased overall survival was observed in a phase II clinical trial for solid tumor. Citation Format: Isabele F. Moretti, Antonio Lerario, Sueli M. Oba-Shinjo, Suely K. Marie, Marina Trombetta Lima. Toll like receptor 4 as a potential DNA repair modulator in U87MG-GBM cells [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 2596.