title: Toll-Like Receptor 4 as a Novel Therapeutic Target in the Treatment of Malignant Mesothelioma
Creators: name: Coupland, Philippa Louise
Publisher: name: Griffith University
Year: 2017
Subjects: subject: Malignant mesothelioma
subject: Toll-Like Receptor 4

description: Background: Malignant mesothelioma is an aggressive and rare cancer. Mesothelioma affects the mesothelial cells of the pleural, pericardial and peritoneal cavities surrounding the lungs, heart and abdomen. Long latency periods, short patient survival times and limited treatment options associated with mesothelioma underlines the need to identify novel therapeutic drug targets. Numerous molecular mediators and pathways have been identified as potential therapeutic targets in malignant mesothelioma, with one being Toll-Like Receptor (TLR4). TLR4 is a protein that plays a significant role in the autocrine and paracrine feedback loop of High Mobility Group Box 1 (HMGB1) protein, which contributes to mesothelioma tumourogenesis and proliferation. Methods: Two main cell lines used in this study were the non-malignant cell line, MeT-5A, and malignant cell lines NCI-H28, IST-Mes2 and MM-Miller. Various small molecule-inhibiting drugs were used to investigate the role of TLR4 in these cell lines, including those that inhibit TLR4, and components downstream of the TLR4 signaling pathway. Short-term effects of these drugs were assessed by 3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)SO (MTT) assay, and longterm effects assessed using colony-forming assays (CFA). The mechanism of cell death following drug treatment was determined by Western blotting analysis. To determine the validity of hypotheses suggesting that TLR4 is up-regulated in malignant mesothelioma, Western blotting analysis for TLR4 was performed on all four cell lines. To examine the effect of TLR4 gene silencing on short-term cell viability, MeT-5A and NCI-H28 cell lines were treated with a small interfering ribonucleic acid (siRNA) targeting TLR4 before performing an MTT assay. Results: The TLR4 inhibitor, TAK-242, was the only drug that exhibited marginal reductions in cell viability in the NCI-H28 cell line compared to the MeT-5A cell line. IST-Mes2 and MM-Miller cell lines showed variable sensitivity to all molecular inhibitors. CFAs performed on MeT-5A and NCI-H28 cell lines using TAK-242 demonstrated that cell death continues to occur 10 days post-treatment, indicating that the drug exhibits long-term effectiveness on reducing cell viability. Western blot analysis showed induction of apoptosis in several cell lines following molecular inhibition. However this was not consistent between drug treatments and between different cell lines. TLR4 protein expression varied between all four cell lines as determined by Western blotting, with the malignant NCI-H28, IST-Mes2 and MMMiller cell lines expressing a 1.1-, 1.32-, and 1.35-fold increase in TLR4 protein expression compared to the non-malignant MeT-5A cell lines. Conclusion: The TLR4 inhibitor, TAK-242, was the only drug that showed marginal specificity for one of the malignant cell lines. Taken with data indicating that TLR4 protein expression is up-regulated in mesothelioma cell lines, TLR4 may play a role in mesothelioma, however it does not appear to be crucial to malignant mesothelioma cell survival. While various aspects of drug treatment and mesothelioma biology was investigated, future work need to focus on identifying other potential therapeutic targets in mesothelioma. The extent of the involvement of other molecules such as HMGB1, and further understanding of mesothelioma biology, is also warranted.