The Influence Of TLR3,7 And 9 Signaling On Tumor Progression Of Oral Squamous Cell Carcinoma

BACKGROUND:Oral squamous cell carcinoma(OSCC)is associated with five years survival rate of around 50%and traditional treatment has gained very limited improvement of OSCC survival rate over the last 30 years,new targets for OSCC treatment are needed.Toll-like receptors(TLRs)are a family of transmembrane receptors that recognize conserved patterns of microbial structures.TLRs were once believed to be expressed only on immune cells.Accumulating evidences indicated that they were also expressed on various cancer and cancer cell lines and have been reported to play an important role in tumor development.But their roles on tumor development are contradictory.As TLRs agonists might contribute quite differently to the development of various kinds of tumors,and the role of TLRs(especially nucleic acids detecting TLRs:TLR3,7 and 9)on OSCC is still not clear,we investigated in this study to find out whether OSCC express functional TLR3,7 and 9 and the effects of TLRs activation on OSCC development.METHODS:TLR3,7 and 9 expression profile in primary OSCC tissue and two OSCC cell lines(SCC4 and CAL27)were examined by immunofluorescence.TLRs mRNA and protein expression in these two cell lines were analyzed by Q-PCR and Western blot.Poly(I:C),imiquimod-R837 and ODN M362 were used to active TLR3,7 and 9 in OSCC.Then cell viability,apoptosis and migration were investigated by CCK-8 test,flow cytometry analysis(Annexin V-PI double staining)and transwell system respectively.Caspase-3 cleavage was evaluated to further confirm cell apoptosis.A pan-caspase inhibitor(Z-VAD-FMK)and a caspase-3 inhibitor(Ac-DEVD-CHO)were used to testify the involvement of caspase signaling in poly(I:C)-induced OSCC apoptosis.The mRNA expression of various cytokines was evaluated by Q-PCR.The changes of TLR3,7 and 9 mRNA and protein expression were also tested by Q-PCR and Western blot.As a marker of NF-?B activation,NF-?B nuclear translocation was investigated by immunofluorescence.An endosomal acidification antagonist chloroquine and NF-?B inhibitor BAY 11-7082 were used to further confirm the importance of TLR3 and NF-?B signaling.RESULTS:TLR3,7 and 9 were expressed by both primary OSCC tissue and OSCC cell lines.SCC4 and CAL27 showed strong expression of TLR3 and weak expression of TLR7 and TLR9.As TLR3 agonist,poly(I:C)stimulated robust responses in OSCC.It decreased cell viability by suppressing cell proliferation and inducing apoptosis and decreased cell migration.Poly(I:C)-TLR3-induced OSCC cell apoptosis was caspase-3-dependent.Poly(I:C)also provoked NF-?B nuclear translocation and stimulated several proinflammatory cytokines in OSCC including IL-6,IL-8,TNF-a,VEGF,IL-1? and MCP-1 but decreased TGF-? production.It also up-regulated NF-KB-dependent TLR3 and 7 expression and induced TLR9 expression changes.Although poly(I:C)can activate other receptors than TLR3(like PKR,RIG-1,MDA5),all those effect were testified to be mainly TLR3 related.In contrast to poly(I:C),imiquimod-R837 and ODN M362,function via weakly expressed TLR7 and TLR9,failed to influence OSCC cell proliferation,apoptosis and migration.But just like poly(I:C),Imiquimod-R837 and ODN M362 induced NF-?B nuclear translocation and regulated some cytokines expression,as well as changed TLR3,7 and 9 expression,only to a much more limited extent.CONCLUSIONS:The present study demonstrated strong expression of functional active TLR3 and weak TLR7 and TLR9 in OSCC cell lines(SCC4 and CAL27).TLR3 activation directly caused cell apoptosis and decreased cell migration in OSCC,suggesting that TLR3 might impact OSCC development and should be considered as an important target in future immunotherapy for OSCC.Exposure of OSCC to poly(I:C),imiquimod-R837 and ODN M362 elicited TLR3,7 and 9-mediated proinflammatory phenotypic changes,demonstrated by NF-?B dependent up-regulation of proinflammatory cytokines and chemokines.This response could potentially be triggered by viral infection of cells within OSCC.Changed production of various proinflammatory markers modulated tumor microenvironment and could further affect OSCC development by regulating local immune responses,as well as tumor cell growth and angiogenic potential.Our study also demonstrates for the first time the existence of an intricate regulatory network of cross-talk of the TLR3,TLR7 and TLR9 pathways in OSCC.The identified positive and negative amplification loops for the different TLR agonists indicate that TLRs expression in OSCC is highly dynamic.This may in turn changed the response of OSCC to a given condition and TLRs involvement in OSCC progression.Understand the intricate TLRs regulatory network in OSCC will open the possibility to modulate the response through a targeted ligand-driven intervention.