REGγ Ablation Reverses Differentiation Of Anaplastic Thyroid Carcinoma And Accentuates Radio-therapeutic Response By Inhibiting TGF-β 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 REGγ,a proteasome activator,is highly expressed in human ATC tissues and cells,suggesting that REGγ is a factor that promotes ATC formation.Subsequently,PCR,Western Blot and IF experiments confirmed that REGγ 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,REGγ knockdown significantly restored the expression of thyroid-specific genes.Cell iodine absorption experiments showed that REGγ 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 REGγ deletion,regardless of the xenograft tumor model established by SW1736 cells or K18 cells.These results indicate that REGγ 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 REGγ can activate Smad3 in SW1736 and K18 cells,to promote the p-Smad3 and Smad4 into nuclear,resulting in the activation of the TGF-β signaling pathway,inhibiting the expression of thyroid-specific genes.We also found that REGγ is inversely related to the protein level of Smad7,while mRNA levels are not affected.CHX experiments showed that REGγ regulates the stability of Smad7 in SW1736 and K18 cells.Importantly,we found that the REGγ(N151Y)mutant lost its effect on the stability of Smad7.In vitro degradation experiments further confirmed that REGγ can directly promote the degradation of Smad7.Therefore,these results indicate that Smad7,an antagonist of the TGF-β signaling pathway,is a novel target protein of the REGγ-proteasome degradation system,REGγ directly binds to Smad7 and promotes its degradation.With gain-and loss-of-function studies,we demonstratethat Smad7 is a key mediator for the REGγ function in ATC cell dedifferentiation and metastasis.we performed bioinformatics analysis and IHC staining and quantitative analysis.The results also showed that REGγ was negatively correlated with Smad7 and positively correlated with NIS and Pax8.It is proved that REGγ 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 REGγ activates TGF-β 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,REGγ may serve as a novel therapeutic target for patients with poor prognosis of ATC.Repression of REGγ 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.