Decabromodiphenyl Ether Induces The Tumorigenesis And Progression Of Papillary Thyroid Carcinoma And Drug Resistance Of Dabrafenib

Part Ⅰ Long-term exposure to decabromodiphenyl ether promotes the tumorigenesis and progression of papillary thyroid carcinoma by inhibiting TRβBackground:Thyroid cancer is one of the most common endocrine cancers,of which papillary thyroid carcinoma(PTC)accounts for about 70-90%of all cases.With the great progress in industrial production,it has been observed that the incidence of thyroid cancer has the same increasing trend as the concentrations of decabromodiphenyl ether(BDE209)detected in the environment.BDE209 is similar to thyroid hormones(THs)in structure and can bind to thyroid receptors(TRs),which belongs to endocrine disrupting chemicals(EDCs).In this study,in vivo and in vitro assays were used to comprehensively analyze the chronic toxic effect of long-term exposure to environmental concentration of BDE209 on thyroid tissues,and to explore whether it is potential role in inducing the tumorigenesis and progression of PTC by inhibiting TRβ.Methods:Human normal thyroid follicular cell line and PTC cell lines with different genetic backgrounds,were exposed to environmentally concentrations of BDE209 for 27 days.1.MTS,EdU,and colony formation assays were used to detect cell proliferation.Wound healing assay and transwell assay were used to detect the migration and invasion ability of cancer cell line.2.The thyroid tissues of BDE209 exposed mice were stained with HE,and the xenograft tumors were stained with Ki67 immunohistochemistry.3.RT-qPCR and Western blot were used to detect the expression of TRβ at the transcriptional and translational levels,and luciferase reporter assay was used to detect the changes in the transcriptional function of TRβ.4.THRB expression levels were analyzed using gene expression omnibus(GEO)data.Human normal thyroid follicular epithelial cells exposed to long-term environmental concentrations of BDE209 were sent for RNA high-throughput sequencing(RNA-seq)analysis,and gene enrichment analysis was performed on the returned data.Results:1.BDE209 has biphasic effects on the proliferation of thyroid cells.BDE209 at an environmentally relevant dose(0.5-5 μM)significantly promoted the proliferation of Nthy ori 3-1 cells(P<0.05),while higher concentrations of BDE209 inhibited cell proliferation in a concentration-dependent manner.On the basis of these results,the environmental exposure concentrations with a proliferation effect(1 and 2.5 μM)were initially determined and used in subsequent experiments.2.Hormesis effect of long-term exposure BDE209 on thyroid cells proliferation,cell cycle,invasion and migration.Long-term exposure of BDE209 to thyroid cell lines could promote cell proliferation and tumor cell migration and invasion in a concentration-and time-dependent manner.And BDE209 exposure could increase the proportion of S and G2/M phase in the cell cycle.The results of PTC cell lines showed that the proliferative effect of BDE209 on TPC-1 cell line was higher than that on BCPAP cell line,but lower than that on normal thyroid follicular cell line.3.BDE209 promotes the proliferation of thyroid follicular cells and the BCPAP cells in BALB/c female mouse xenograft models.Thyroid tissues of the control group and the BDE209 exposure group were stained with HE,and the xenograft tumors were stained with Ki67.The results showed that BDE209 could promote follicular hyperplasia of thyroid tissues and accelerate the growth of PTC xenografts.4.Long-term exposure to BDE209 promotes tumorigenesis by inhibiting IRβ expression and function.Compared with the control group and DMSO group,the environmental concentration BDE209 decreased the TRβ mRNA level and the protein expression of TRP in a dose-dependent manner.The results of luciferase reporter assay showed that BDE209 could reduce the transcriptional activity of TR,and the reduction was more significant when BDE209 was used in combination with triiodothyronine.5.Inverse association between expression of TRB and thyroid cancer.GEO database analysis showed that THRB expression in thyroid tumor samples was significantly lower than that in corresponding normal thyroid samples.On this basis,GSEA analysis of the data returned by RNA-seq showed that genes related to thyroid hormone response pathway(P=0.0)and thyroid cancer(P=0.001)were significantly enriched in the BDE209 exposure group.Conclusions:These results suggest that BDE209 may promote the tumorigenesis and progression of papillary thyroid carcinoma through inhibiting the transcription function and protein expression of TRβ.Part Ⅱ Decabromodiphenyl ether induces dabrafenib resistance in papillary thyroid carcinoma by activating EGFR-CRAF-MAPK pathwayBackground:Papillary thyroid carcinoma is low malignant thyroid cancer,and comprehensive treatment including surgery and postoperative hormone suppression therapy is adopted.However,the prognosis of aggressive subtypes of differentiated thyroid cancer,highgrade follicular carcinoma,advanced recurrent or metastatic thyroid cancer and iodine refractory thyroid cancer is still a difficult problem in clinical treatment.BRAFV600E mutation is the most common oncogenic initiator of PTC,which leads to continuous phosphorylation of downstream targets of MAPK signaling pathway.This makes the activation of MAPK cascade the key to the occurrence of thyroid cancer,and it is also the target pathway for clinical precise targeted therapy.However,dabrafenib resistance rapidly occur after single-dose use,which has limitation for clinical use,but the mechanism of dabrafenib resistance is unclear.This study is the first to explore the effect of BDE209 on dabrafenib from the perspective of environmental pollutants,and to explore the correlation between the potential mechanism and EGFR-CRAFMAPK signaling pathway.Methods:1.Western blot and IF assays were used to detect the phosphorylation level of MAPK signaling pathway after BDE209 exposure.2.BCPAP cell lines with BRAFV600E mutation sensitive to dabrafenib were selected to detect the dabrafenib resistance index IC50 after BDE209 exposure.BCPAP cells were divided into four groups:control group,BDE209 treatment group,dabrafenib treatment group and BDE209+dabrafenib combination group.3.Flow cytometry was used to detect the level of apoptosis and cell cycle in different groups.Combined MTS,EdU and colony formation assays were used to verify the inhibitory effect of BDE209 on dabrafenib drug sensitivity.4.RNA-seq of long-term exposure to environmental concentration BDE209,and the returned data were analyzed by KEGG enrichment analysis using DAVID website,and then the results were verified by GSEA enrichment analysis.5.The phosphorylation levels of EGFR and CRAF were detected by Western blot.6.After interfering EGFR protein expression,the cell proliferation level of BDE209 treatment group was detected by rescue experiment.Results:1.Environmental concentrations BDE209 activates the MAPK pathway.Western blot and IF experiments showed that the phosphorylation levels of MEK and ERK1/2 proteins were significantly increased in the BDE209 group in a dose-dependent manner,suggesting that BDE209 may promote the tumorigenesis of PTC by activating the MAPK signaling pathway.2.BDE209 induces drug resistance of dabrafenib in BRAFV600E mutation PTC cells.IC50 of dabrafenib was significantly increased by the use of environmental doses BDE209.Compared with dabrafenib treatment group,BDE209 treatment significantly reduced the level of apoptosis of BCPAP cells.Dabrafenib significantly inhibited the proliferation of BCPAP cells and the proportion of BCPAP cells in S and G2/M phases.However,dabrafenib combined with BDE209 could significantly reverse the inhibitory effects.3.BDE209 induces dabrafenib resistance by activating the MAPK pathway.Dabrafenib treatment significantly reduced the phosphorylation levels of MEK and ERK1/2 proteins in BCPAP cells.Compared with dabrafenib treatment group,the phosphorylation level of MAPK pathway was significantly increased in dabrafenib+ BDE209 treatment group.These results suggest that dabrafenib can specifically target PTC cells with BRAFV600E mutation,but BDE209 can inhibit its efficacy of cells to dabrafenib.4.Environmental concentrations BDE209 activates the EGFR pathway.KEGG and GSEA enrichment analysis of differential genes showed that,EGFR tyrosine kinase activation pathway was significantly enriched in the BDE209 exposure group.Western blot was used to verify the expression and phosphorylation levels of EGFR and CRAF.The results showed that the activation levels of EGFR and CRAF were significantly increased in Nthy ori 3-1 and PTC cell lines.BDE209 has a regulatory effect on EGFR signaling pathway.5.BDE209 induces dabrafenib resistance through the EGFR-CRAF-MAPK pathway.Rescue assay confirmed that BDE209 or EGF alone promoted cell proliferation in BAPAP cells,which was significantly reduced after EGFR knockdown.Western blot showed that the phosphorylation of EGFR-CRAF-MAPK pathway increased after BDE209 exposure.Dabrafenib alone significantly inhibited MAPK activation,but had little effect on EGFR and CRAF protein phosphorylation.However,MAPK was reactivated when BDE209 and dabrafenib were used in combination.Conclusions:These results suggest that environmental concentrations BDE209 induces dabrafenib resistance in PTC with BRAFV600E mutation through EGFR-CRAF bypass to regulate MAPK reactivation.