Differentiation Therapy Of Thyroid Cancer Induced By Histone Epigenetic Modification Combined With MAPK Inhibition

Background:Aberrant activity of mitogen-activated protein kinase（MAPK）signaling pathway induced by BRAF^V600E mutation down-regulates the expression of iodide-metabolizing genes,which is one of the reasons resulting in radioiodine refractory differentiation thyroid cancer（RR-DTC）.Therefore,MAPK inhibitors（MAPKi,including BRAF inhibitors and MEK inhibitors）have become an important research direction in differentiation therapy of RR-DTC.Although in vitro studies including our research group on differentiation therapy induced by MAPK in RR-DTC have achieved promising efficacy,the clinical efficacy urgently needs to be improved.This may be due to insufficient studies on the mechanism of dedifferentiation and insufficient targeting intervention,which makes it difficult for the existed therapy strategies to promote differentiation in a sustained and efficient manner,and subsequently,it is impossible to ensure that the tumor can achieved the lethal internal irradiation dose in ¹³¹I therapy.Previous studies have confirmed that aberrant activation of MAPK pathway can affect gene expression by regulating modifications on histone acetylation and methylation.We aim to enhance the differentiation of RR-DTC and radioiodine uptake by increasing expression of Nis in tumor cells through histone modification enzyme inhibitors combined with MAPKi,and explore the underlying mechanism.Methods:BRAF^V600E mutant cell lines（BCPAP and K1）and BRAF wild-type cell lines（BHP 2-7 or TPC-1）were treated with histone deacetylase inhibitor（HDACi,panobinostat）or histone methyltransferase inhibitor（HMTi,tazemetostat）alone,or combined with BRAF inhibitor（dabrafenib）or MEK inhibitor（selumetinib）,respectively.Subsequently,Chromatin immunoprecipitation（ChIP）,quantitative real time polymerase chain reaction（qRT-PCR）,Western Blotting,immunofluorescence,radioiodine uptake assay,and in vitro clonogenic assay were performed to verify the effect of combination therapy on tumor differentiation status.Results:We found that HDACi alone increased the acetylation level of histone at Nis promoter irrespective of BRAF status.Although MAPKi（dabrafenib or selumetinib）improved the acetylation level of histone at Nis promoter in BRAF^V600Emutation cells,they did not affect in BRAF wild-type cell.HDACi combined with MAPKi only displayed a more robust improvement of the acetylation level of histone at Nis promoter in BRAF^V600Emutation cells.We also found that HMTi mildly reduced the level of global histone H3lysine 27 trimethylation（H3K27me3）in all the PTC cells.MAPKi only reduced the global H3K27me3 level in BRAF^V600E mutation cell lines,and H3K27me3 was further decreased in BRAF^V600E mutation cells when cells were treated with dual inhibition with HMTi and MAPKi compared with MAPKi or HMTi treatment alone.Besides,HMTi slightly increased the expression of iodide-metabolizing genes ignoring BRAF status.Likewise,MEK inhibitor also mildly increased the expression of iodide-metabolizing genes,radioiodine uptake and the toxic effect of radioiodine on tumor cells in both BRAF^V600E mutant cell lines and BRAF wild-type cell line.However,BRAF inhibitor displayed the above effects only in BRAF^V600E mutant cell lines.In BRAF^V600E mutation cell lines,combined treatment with HMTi and MAPKi significantly improved iodide-metabolizing genes,radioiodine uptake and the toxic effect of radioiodine on tumor cells when compared with that in single treatment groups.Furthermore,thyroid stimulating hormone（TSH）alone can not improve the above effects in tumor cells,however,when tumor cells treated with these inhibitors,additional TSH can further improve the differentiation effect induced by these treatments in tumor cells.Conclusion:Changes in histone epigenetic modifications such as acetylation and methylation induced by aberrant activity of MAPK via BRAF^V600E mutation result in tumor dedifferentiation.The differentiation efficacy of HDACi on RR-DTC enhanced by MAPKi is mainly due to promoting histone acetylation level at Nis gene promoter,which has BRAF^V600E dependencies.Also,we found that HMTi enhanced thyroid cancer cells differentiation by reducing global H3K27me3 irrespective BRAF status for the first time.MAPKi promoted thyroid cancer cell differentiation exclusively in BRAF^V600E mutation cell lines by reducing global H3K27me3.Compared with single treatment groups,combined HMTi with MAPKi significantly enhanced differentiation status of tumor cells via decreasing global H3K27me3,which also had BRAF^V600E dependencies,besides,additional TSH can further enhance the differentiation efficacy of combination therapies.Significance:We aim at the clinical dilemma and explore a novel therapeutic strategy to enhance the differentiation efficacy of single treatment,providing a new theoretical basis for targeted therapy in RR-DTC patients.Also,we found that reduction of global H3K27me3 could induce the tumor cells differentiation for the first time,suggesting that epigenetics plays a vital role in the process of tumor genesis,development and dedifferentiation,which indicates a new horizon for the targeted differentiation therapy for RR-DTC.