Late p65 nuclear translocation in glioblastoma cells indicates non-canonical TLR4 signaling and activation of DNA repair genes

Isabele F Moretti,Paula R Sola,Suely K N Marie,Sueli M Oba-Shinjo,Marina Trombetta-Lima,Roseli Da Silva Soares,Antonio M Lerario

doi:10.1038/s41598-020-79356-1

Isabele F Moretti, Paula R Sola + Show 5 more

Open Access

https://doi.org/10.1038/s41598-020-79356-1

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Abstract

Glioblastoma (GBM) is the most aggressive brain primary malignancy. Toll-like receptor 4 (TLR4) has a dual role in cell fate, promoting cell survival or death depending on the context. Here, we analyzed TLR4 expression in different grades of astrocytoma, and observed increased expression in tumors, mainly in GBM, compared to non-neoplastic brain tissue. TLR4 role was investigated in U87MG, a GBM mesenchymal subtype cell line, upon LPS stimulation. p65 nuclear translocation was observed in late phase, suggesting TLR4-non-canonical pathway activation. In fact, components of ripoptosome and inflammasome cascades were upregulated and they were significantly correlated in GBMs of the TCGA-RNASeq dataset. Moreover, an increased apoptotic rate was observed when the GBM-derived U87MG cells were co-treated with LPS and Temozolomide (TMZ) in comparison to TMZ alone. Increased TLR4 immunostaining was detected in nuclei of U87MG cells 12 h after LPS treatment, concomitant to activation of DNA repair genes. Time-dependent increased RAD51, FEN1 and UNG expression levels were confirmed after LPS stimulation, which may contribute to tumor cell fitness. Moreover, the combined treatment with the RAD51 inhibitor, Amuvatinib in combination with, TMZ after LPS stimulation reduced tumor cell viability more than with each treatment alone. In conclusion, our results suggest that stimulation of TLR4 combined with pharmacological inhibition of the DNA repair pathway may be an alternative treatment for GBM patients.

Highlights

Glioblastoma (GBM) is the most aggressive brain primary malignancy
We first recapitulated Toll-like receptor 4 (TLR4) expression in our cohort of 140 human astrocytoma of different grades of malignancy (26 astrocytoma grade II (AGII), 18 astrocytoma grade III (AGIII), and 96 GBM compared to 22 non-neoplastic [NN] brain tissue), and we analyzed TLR4 signaling pathways
TLR4 expression was significantly higher in AGII, AGIII, and GBM when compared to NN (p < 0.05, Kruskal– Wallis and Dunn tests)

Summary

Introduction

Glioblastoma (GBM) is the most aggressive brain primary malignancy. Toll-like receptor 4 (TLR4) has a dual role in cell fate, promoting cell survival or death depending on the context. The non-canonical pathway consists of the TLR4 internalization to the endosome compartment by a phosphoinositide 3-kinase (PI3K)-dependent mechanism[13] This process results in the activation of TRIF, which promotes an anti-inflammatory response by inducing the expression of interferon type I, interferon regulatory factor 3 and 7 (IRF3/7), and IL-102,14. TRIF may trigger a cell death pathway by interacting with receptor interacting protein kinase 1 and 3 (RIPK1, RIPK3), and the fas adaptor death domain (FADD), which in turn activate caspase 8 (CASP8), leading to apoptosis. Inflammatory cells in a solid tumor microenvironment may exhibit a pro- or anti-inflammatory profile depending on the s timuli[21] Inflammatory receptors such as TLR4 are expressed in tumor cells, adding another layer of complexity that results from potentially distinct effects of TLR4 downstream signaling in each cell c ompartment[22]. Among the GBM molecular subtypes, the mesenchymal (MES) subtype, which harbors neurofibromin 1 (NF1) and RB transcription corepressor 1 (RB1) mutations, presents the poorest outcome, compared to the proneural (PN) subtype harboring somatic mutations in tumor protein p53 (TP53), platelet-derived growth factor receptor A (PDGFRA), and isocitrate dehydrogenase 1 (IDH1), and to the classical subtype with epidermal growth factor receptor (EGFR) mutations[25,26]

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