Hypoxia regulates Toll-like receptor-9 expression and invasive function in human brain cancer cells in vitro.

Jouko Sandholm,Kevin W Harris,David Graves,Joonas H Kauppila,Johanna Tuomela,Katri S Selander

doi:10.3892/ol.2014.2095

Jouko Sandholm, Kevin W Harris + Show 4 more

Open Access

https://doi.org/10.3892/ol.2014.2095

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Abstract

Toll-like receptor-9 (TLR9) is a cellular DNA sensor of the innate immune system. TLR9 is widely expressed in a number of tumors, including brain cancer; however, little is known regarding its regulation and involvement in cancer pathophysiology. The present study demonstrated that hypoxia upregulates and downregulates TLR9 expression in human brain cancer cells in vitro, in a cell-specific manner. In addition, hypoxia-induced TLR9 upregulation was associated with hypoxia-induced invasion; however, such invasion was not detected in cells where hypoxia had suppressed TLR9 expression. Furthermore, suppression of TLR9 expression through TLR9 siRNA resulted in an upregulation of matrix metalloproteinase (MMP)-2, -9 and -13 and tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) mRNA, and a decreased invasion of cells in normoxia, in a cell-specific manner. In cells where hypoxia induced TLR9 expression, TLR9 expression and invasion were reduced by TLR9 siRNA. The decreased invasion observed in hypoxia was associated with the decreased expression of the MMPs and a concomitant increase in TIMP-3 expression. In conclusion, hypoxia regulates the invasion of brain cancer cells in vitro in a TLR9-dependent manner, which is considered to be associated with a complex expression pattern of TLR9-regulated mediators and inhibitors of invasion.

Highlights

The Toll‐like receptors (TLRs) are key regulators of the innate immune system and function as pattern recognition proteins that detect microbe‐ and host‐derived endogenous molecular patterns [1,2]
The present study demonstrated that the surrounding oxygen level has an important effect on Toll‐like receptor‐9 (TLR9) expression and function in human brain cancer cells in vitro
Our studies further suggest that TLR9 expression is significantly associated with the invasive machinery in brain cancer cells and may mediate hypoxia‐induced invasion in brain cancer

Summary

Introduction

The Toll‐like receptors (TLRs) are key regulators of the innate immune system and function as pattern recognition proteins that detect microbe‐ and host‐derived endogenous molecular patterns [1,2]. Ligand binding to TLRs activates transcription factors, most notably NF-κB and the eventual outcome of TLR activation in cells of the immune system is an innate immune reaction, which is characterized by increased cytokine and interleukin production [2] This inflammatory reaction results in the activation of the adaptive immune system and thereby, elimination of the invading pathogens and infected cells [7]. Synthetic TLR9‐ligands (CpG‐sequence containing oligonucleotides) have been demonstrated to stimulate the in vitro invasion of TLR9‐expressing cancer cells, including astrocytoma and glioblastoma cells [14,16]. This increased invasion is mediated via the downregulation of the tissue inhibitor of matrix metalloproteinases‐3 (TIMP‐3) and the upregulation of matrix metalloproteinase‐13 (MMP‐13) activity [15,16]. Synthetic TLR9 ligands have been investigated in preclinical models of brain cancer immunotherapy; CpG‐sequence containing oligonucleotides were shown to induce apoptosis in brain cancer cells in vitro and in vivo and, may provide long‐term antitumor immunity against gliomas [22,23]

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