EGFR-mediated Signaling Pathway Influences The Sensitivity Of Oral Squamous Cell Carcinoma To JQ1

Inhibiting BRD4 has emerged as a promising anticancer strategy,and inhibitors such as JQ1 can suppress cell growth in oral squamous cell carcinoma(OSCC).However,the mechanism through which JQ1 exerts its anticancer activity has not been reported.Moreover,JQ1 does not markedly inhibit proliferation and increase apoptosis in OSCC when used as a monotherapy.Herein,we explore the mechanism of JQ1 in OSCC and probe ways to increase its therapeutic potential.In this study,we used two cell lines,Ca127 and Scc25.We found that BRD4 was highly expressed in OSCC tissues when compared with adjacent non-tumor tissues,and JQ1 worked through the EGFR-mediated signaling pathway in tumor cells.Furthermore,we demonstrated that JQ1 induced an increased treatment effect in vitro and in vivo when combined with a PI3K inhibitor.Interestingly,subsequent mechanistic analyses indicated that further suppressing EGFR and BRD4 expression was instrumental to this functional synergism.Moreover,we found that upregulating EGFR expression by EGF stimulation protected cells treated with JQ1 from apoptosis,while knockdown of EGFR before addition of JQ1 successfully mimicked the combination treatment results.In summary,our findings revealed that JQ1 can act by inhibiting the EGFR-mediated signaling pathway,and EGFR expression influences the sensitivity of OSCC to JQ1.Regarding clinical use,this study demonstrates that BRD4 is a novel therapeutic target and EGFR can be used as a biomarker to identify the most appropriate anti-BRD4 treatment strategy in OSCC.Chapter 1 BRD4 is overexpressed of OSCCObjective:To investigate the expression of BRD4 in 30 cases OSCC patients and further verifying BRD4 can be used as a therapeutic target for oral squamous cell carcinoma.Methods:The expressions of BRD4 in OSCC tissues obtained from 30 patients and were detected by Immunohistochemistry.Results:We found that the expression of BRD4 in OSCC tissues was significantly greater than in the corresponding adjacent normal tissues.Conclusion:The study verified that BRD4 was overexpressed in OSCC.Chapter2 BRD4 inhibitor can act by EGFR/PTEN/PI3K/AKT pathwayObjective:The effect of JQ1 on EGFR/PTEN/PI3K/AKT signaling pathway was detected of oral squamous cell carcinoma.Methods:We analyzed the ability of JQ1 to alter EGFR and the downstream pathway expression using western blotting and RT-PCR.We treated tumor cells with JQ1 at a concentration of 0.2,1,and 5?M for 24 h.Results:This experiment verified that JQ1 treatment markedly influenced levels of EGFR and the downstream pathway(*P<0.05)(**P<0.01).Thus,we concluded that JQ1 can work through inhibition of the EGFR-mediated signaling pathway in OSCC.Conclusion:BRD4 inhibitor can act by EGFR/PTEN/PI3K/AKT pathwayChapter3 The addition of a PI3K inhibitor increases sensitivity to JQ1Objective:To investigate the synergistic effect of JQ1 and a PI3K inhibitor in OSCC.Methods:To test this hypothesis,we treated Cal27 and Scc25 cells with a PI3K inhibitor,JQ1,or the combination and analyzed them using CCK8 and Western blot assays.Next,we evaluated the effect of using JQ1 and a PI3K inhibitor in nude mice.Results:Our results showed that the combination of JQ1 and BKM120 led to a significant decrease in tumor cell viability compared with the use of individual inhibitors alone(*P<0.05).Western blot assay results showed that the cleaved caspase-3 was markedly upregulated after combination treatment for 24 h,compared with JQ1 or BKM120 when either was used alone.The combination of JQ1 and BKM120 significantly inhibited tumor growth compared with JQ1 or BKM120 treatment alone,which was only moderately inhibited(**P<0.01).Body weights of mice at the time of sacrifice were similar to those of control mice,suggesting minimal adverse effects of the combined treatment.Conclusion:These observations suggested a broadly functional interaction between JQ1 and the PI3K inhibitor in OSCC,particularly regarding cell proliferation and apoptosis.Chapter 4 The additional treatment effect was due to further inhibiting EGFR and BRD4 expression in OSCC.Objective:To understand the mechanism of synergism treatment.Methods:Ca127 and Scc25 cells were treated with JQ1,BKM120,or the combination.Subsequently,we analyzed relevant protein and mRNA levels using western blotting and RT-PCR.Results:BRD4 and EGFR were markedly downregulated following combination treatment with JQ1 and BKM120Conclusion:Collectively,our results demonstrated that the additional treatment effect was due to further inhibiting EGFR and BRD4 expression in OSCC.Chapter5 The antitumor activity of JQ1 in OSCC was dependent upon EGFR expression.Objective:To verify that EGFR counteracts the effect of JQ1 treatment in OSCC.Methods:We used 1Ong/ml EGF to stimulate EGFR expression or siEGFR to knockdown EGFR.Subsequently,we used the CCK8 assay to investigate proliferation after treatment for 1-4 d and measured apoptosis using flow cytometry after 24 h.We next evaluated whether EGFR activation modulated BRD4 expression in JQ1-treated tumor cells using RT-PCR and western blotting.Results:The data indicated that treatment with JQ1,BKM120,and EGF protein,increased proliferation compared with dual drug treatment(**P<0.01).Flow cytometry verified that adding EGF led to a decreased percentage of apoptotic Ca127 cells compared with when JQ1 was used alone.After 24 h treatment,western blot assays consistently demonstrated that BRD4 was activated by EGF and that the combination of JQ1 and EGF protein increased BRD4 expression in Ca127 and Scc25 cells compared with JQ1 alone.Moreover,treatment with JQ1,BKM120,and EGF led to a significant increase in BRD4 levels compared with treatment with JQI and BKM120.The RT-PCR results were consistent with the western blot data(**P<0.01).The results showed that knockdown of EGFR strongly decreased both BRD4 mRNA and protein levels compared with JQ1 treatment alone(**P<0.01).Conclusion:Collectively,these results demonstrated that the antitumor activity of JQ1 in OSCC was dependent upon EGFR expression.