REG? Ablation Reverses Differentiation Of Anaplastic Thyroid Carcinoma And Accentuates Radio-therapeutic Response By Inhibiting TGF-? Pathway

Anaplastic thyroid cancer(ATC)is the most aggressive human thyroid malignancy derived from thyroid follicular cells,Tumor cells do not retain any biological characteristics of the original follicular cells,characterized by dedifferentiation and resistance to radioiodine therapy,poor prognosis,and short survival.To date,the underlying mechanisms regulating ATC dedifferentiation are largely unknown.Here,we show that REG?,a proteasome activator,is highly expressed in human ATC tissues and cells,suggesting that REG? is a factor that promotes ATC formation.Subsequently,PCR,Western Blot and IF experiments confirmed that REG? can significantly inhibit the expression of thyroid-specific genes(NIS,Pax8,TTF1,Tg,TSHR and TPO)in ATC(SW1736 and K18)cells,affecting the differentiation status of ATC cells.In contrast,REG? knockdown significantly restored the expression of thyroid-specific genes.Cell iodine absorption experiments showed that REG? gene silencing significantly increased the radioactive iodine uptake of SW1736 and K18 cells.In animal studies,we established a mouse model of ATC xenograft tumor and conducted a therapeutic experiment on radioactive iodine 131.The results showed that the therapeutic effect of radioactive iodine 131 was significantly improved after REG? deletion,regardless of the xenograft tumor model established by SW1736 cells or K18 cells.These results indicate that REG? is an important and new regulatory factor involved in the differentiation status of anaplastic thyroid cancer cells.Mechanistically,IF,Western Blot and Q-PCR experiments confirmed that REG? can activate Smad3 in SW1736 and K18 cells,to promote the p-Smad3 and Smad4 into nuclear,resulting in the activation of the TGF-? signaling pathway,inhibiting the expression of thyroid-specific genes.We also found that REG? is inversely related to the protein level of Smad7,while mRNA levels are not affected.CHX experiments showed that REG? regulates the stability of Smad7 in SW1736 and K18 cells.Importantly,we found that the REG?(N151Y)mutant lost its effect on the stability of Smad7.In vitro degradation experiments further confirmed that REG? can directly promote the degradation of Smad7.Therefore,these results indicate that Smad7,an antagonist of the TGF-? signaling pathway,is a novel target protein of the REG?-proteasome degradation system,REG? directly binds to Smad7 and promotes its degradation.With gain-and loss-of-function studies,we demonstratethat Smad7 is a key mediator for the REG? function in ATC cell dedifferentiation and metastasis.we performed bioinformatics analysis and IHC staining and quantitative analysis.The results also showed that REG? was negatively correlated with Smad7 and positively correlated with NIS and Pax8.It is proved that REG? also affects the expression of thyroid-specific genes by regulating Smad7 in human ATC cells,which has very important clinical significance.This study demonstrates for the first time that REG? activates TGF-? signaling pathway by degrading Smad7 in ATC cells,thereby inhibiting the expression of thyroid-specific genes,promoting the dedifferentiation of thyroid cancer cells,and accelerating the malignant development of anaplastic thyroid cancer.Therefore,REG? may serve as a novel therapeutic target for patients with poor prognosis of ATC.Repression of REG? is expected to enable ATC to be treated with radioactive iodine.It also provides new ideas and theoretical basis for drug treatment of undifferentiated thyroid cancer.