The Research On The Mechanism Of TLR4 Promoting EGFR-target Therapy Resistance In Head And Neck Squamous Cell Carcinoma

Objective:Recent studies have confirmed that TOLL-like receptor 4(TLR4)is able to induce the release of angiogenic factors and immunosuppressive cytokines in tumor and protect tumor cells from apoptosis.However,the diagnostic,target-therapeutic and prognostic value of TLR4 in head and neck squamous cell carcinoma have not been widely studied.In our previous work,we found that the expression of TLR4 was tightly related with the poor therapeutic effect of EGFR-targeted drug in the pre-chemotherapeutic tumor specimens of randomized comparison clinical trial in our department,which indicated that TLR4 might be the key molecule related with EGFR-target drug resistance.Our research start with the uncovered phenomenon that expression of TLR4 is tightly related with EGFR-targeted drug resistance,further investigate the potential mechanism of high-expressed TLR4 promoting the EGFR-target drug resistance.It could help us to verify TLR4 as a predictive marker in current therapeutic strategy of EGFR monoclonal antibody(mAb),to explore the benefit situation in different populations towards theEGFR-target therapy,and to establish the personalized treatment plan and system.Methods: 1.We performed RT-PCR and immunohistochemical staining to detect the expression of TLR4 and EGFR,conducted correlational analysis of response rate and survival rate combined with EGFR-target therapy in 48 collected paraffin-embedded tissues of head and neck squamous cell carcinoma(HNSCC)and matched normal samples.2.CCK-8 and western blot were conducted to examine the resist effect of growth inhibition and changes of TLR4 signaling towards EGFR-target drugs(Cetuximab,Gefinitib)after TLR4 activation by LPS in HNSCC cells(SCC4,CAL27,SCC9).3.ELISA were performed to detect the changes of TLR4 signaling-related proinflammatory cytokines in HNSCC cells(SCC4,CAL27)pretreating Cetuximab or Gefitinib.4.Co-immunoprecipitation were using to examine the changes of related proteins between EGFR signaling and TLR4 signaling in HNSCC cells(SCC4,CAL27)pretreating Cetuximab or Gefitinib,and further investigate the cross talk mechanism between two signal pathway.5.TLR4 or MyD88 overexpressed SCC4 were injected into dorsal subcut is of NOD-SCID mice and Cetuximab or Gefitinib were peritoneal injected respectively,the tumor volume,western blot and RT-PCR were conducted to detect the carcinogenic cytokines expression to study the impact on drug resistance by overexpressed TLR4 or TLR4 downstream molecule MyD88.Results 1.TLR4 was highly expressed in 50% EGFR overexpressed in HNSCC biopsies,showed a positive correlation with high expression of EGFR,which was inversely proportional to the overall survival rate of patients.2.Growth inhibition pretreated with LPS was significant lower than control and NF-?B,MAPK signaling related proteins were significant upregulatedin SCC4,SCC9 and CAL27.3.Inhibition of EGFR by cetuximab or Gefitinib increased TLR4 induced TNF-?,PGE2 and NO production in CAL27 and SCC4.4.Inhibition of EGFR by cetuximab or Gefitinib,thetreated group showed cbl-bdecreased with MyD88 upregulated than control when coprecipitation cbl-b and MyD88 bySrcandthe treated group showed both cbl-band Src decreased with MyD88 upregulated when coprecipitation cbl-b and Src byMyD88,indicated that EGFR inhibition lead to decreased cbl-b-mediated degradation of MyD88 by attenuating Src activation.5.Tumor volumes of tumor-bearing nude mice overexpressed TLR4 or MyD88 were significant larger than control,increased mRNA expression of TNF-?,iNOS and COX2 were observed in the tumor,indicated drug resistance existed.Conclusions 1.Highly expressed TLR4 is tightly related with EGFR mAb resistance and TLR4 is the predictive biomarker for response to EGFR inhibition therapy.2.EGFR affects TLR4 by Src-cbl-b-MyD88,TLR4 acts on EGFR by NF-?B and MAPK pathway,there exists cross-talk between two signaling pathways.