Toll-like receptor -3 is a nucleic acid-sensing innate immune receptor that detects RNA released from narcotic cells in the tumor microenvironment. TLR3 is expressed on the endosomal compartments but the surface expression of TLR3 has also been reported in various cancers, including breast cancer from our group (Bandopadhya et al., 2015). TLR3 exclusively uses TRIF dependent pathway to mediate its effect on cellular apoptosis and hence used in different clinical trials. But, the outcomes of clinical trials using TLR3 agonists are not always favorable. In the present work, we have addressed the involvement of canonical MyD88 mediated pathway through cell surface to explain the anti-apoptotic effect.
Human breast cancer cells MDA-MB-231, MCF-7 and T47D were challenged with TLR3 ligand Poly (I:C) and MyD88 inhibitor (ST2825). Cell proliferation, BrdU incorporation assay, ELISA, Confocal Microscopy, Western blotting, Co-Immunoprecipitation, and immunohistochemistry were carried out to investigate the signaling cascade.
Poly (I:C) treatment enhances the cell proliferation, DNA content and cell viability while MyD88 inhibitor reversed the effect of Poly (I:C). The expression level of secretary IL-6 as well as nuclear localization of NF-kB (p65) was significantly increased with Poly (I:C) addition but the level was decreased with the addition of MyD88 inhibitor. The expression level of P-IRAK1, P-TAK1, IRAK1, TAK1, TAB1, TRAF-6, and Cyclin D1 had significantly increased with Poly (I:C) addition but the level was decreased with the addition of MyD88 inhibitor. The formation of TRAF-6, TAK1 and TAB1 complex also showed the same pattern.
The mechanistic process includes surface ligand bound TLR3 induces MyD88 dimerization, which promotes the early phase nuclear localization of p65, to up-regulate IL-6 production and finally promote Cyclin-D1 production. Elevated Cyclin-D1 can justify cellular proliferation than apoptosis which is independent of established TRIF meditated TLR3 signaling promoting apoptosis. Thus, this is the first time reporting the surface TLR3 might use MyD88 mediated canonical pathway. Thus our work can help to TLR3 ligand mediated immunotherapy that got set back earlier clinical trial.
Anupam Basu, PhD Molecular Biology and Human Genetics Laboratory Professor Department of Zoology the University of Burdwan Purbo Bardhaman - 713104.
Science and Engineering Research Board, Department of Science & Technology, Government of India.
All authors have declared no conflicts of interest.