Transcri Tional Regulation Of Autophagy-Lysosomal Function In BRAF-driven Melanoma Progression And Chemoresistance

Autophagy can maintain homeostasis,and plays an important role in the cells growth and development.Autophagy is closely related to the pathogenesis and development of malignant melanoma.However,current research shows that the relationship between the development of melanoma and autophagy is contradictory.On the one hand,autophagy can inhibit the tumorigenesis driven by BRAFV600E.On the other hand,autophagy can promote the growth of melanoma.Yet,this paradox has not been resolved,and the molecular mechanism of autophagy interact with oncogenic signaling remains elusive.The BRAFV600E gene mutation is the most common genetic alteration of melanoma.Although the relationship between BRAFV600E and autophagy has been ambiguous,the autophagy process in melanoma patients who treated with BRAFV600E inhibitor(BRAFi)always has been induced.Possible mechanisms for BRAFi-induced autophagy in melanoma which current researched do not explain the intrinsic link between oncogenic BRAF signaling and autophagy very well.Therefore,the molecular mechanism of BRAFi-induced autophagy needs further investigation.In addition,autophagy is a key mechanism for the development of drug resistance in melanoma cells,and autophagy may act as a stress evasion mechanism leading to drug resistance.However,the specific mechanism of drug resistance is not clear.Therefore,this study aimed to investigate the specific molecular mechanism of BRAFi-activated autophagy-lysosome in BRAFV600E melanoma and its effect on the development,metastasis and drug resistance of oncogenic signaling and malignant tumors.First,by detecting autophagy regulation in melanoma,in BRAFV600E-melanoma cell lines and patient samples,it was found that autophagy is induced by BRAF inhibitor(BRAFi),as part of a transcriptional program coordinating lysosome biogenesis/function,mediated by the TFEB transcription factor which regulates transcription of the autophagy-lysosomal gene network.BRAFi no longer triggers autophagy-lysosomal activation even though the ER stress mechanism remains intact and activated when TFEB is silenced.Subsequently,we found that TFEB is phosphorylated and thus inactivated by BRAFV600E via its downstream ERK independently of mTORCl in the cells of BRAFV600E.BRAFV600E activates ERK,which promotes phosphorylation of TFEB S142,resulting in the inactivation of TFEB interacting with 14-3-3 and retention in the cytoplasm,which is independent of the phosphorylation status of TFEB S211.BRAFi disrupts TFEB phosphorylation,allowing its nuclear translocation,which is synergized by increased phosphorylation/inactivation of the ZKSCAN3 transcriptional repressor by JNK2/p38-MAPK.Secondly,our experiments demonstrated that BRAFi activates JNK2/p38 MAPK-mediated phosphorylation of ZKSCAN3,and phosphorylation of ZKSCAN3 translocates to the cytoplasm,thereby abolishing transcriptional repression and leading to increased transcription of autophagy-lysosomal function.This process is independent of mTORCl and ERK.We firstly demonstrate BRAFi disrupts TFEB phosphorylation,allowing its nuclear translocation,which is synergized by increased phosphorylation/inactivation of the ZKSCAN3 transcriptional repressor by JNK2/p38-MAPK,which affects the response of melanoma to carcinogenic stress.Finally,through animal experiments,it was found that Blockade of BRAFi-induced transcriptional activation of autophagy-lysosomal function in melanoma xenografts causes enhanced tumor progression,invasion,EMT-transdifferentiation,metastatic dissemination,and chemoresistance,affecting melanoma.Targeted therapy,which is associated with elevated TGF-? levels and enhanced TGF-? signaling.We demonstrated for the first time that TGF-? protein can interact with autophagosomes and eventually degrade in lysosomes by autophagy.We show that TFEB/ZKSACAN3-mediated autophagy-lysosomal activation plays an important role in inhibiting TGF-? signaling.Autophagy-lysosomal blockade results in abnormal activation of TGF-? signaling and induces EMT-TF expression and leads to EMT activation.Inhibition of TGF-? signaling inhibits tumor growth,restores tumor differentiation and drug responsiveness in melanoma cells.Inhibition of TGF-?signaling in a melanoma cell model can inhibit tumor growth,restore tumor differentiation,and alter the drug tolerance status of the tumor.Taken together,we have revealed for the first time that BRAFi-induced autophagy-lysosomal biogenesis is synergistically mediated through the autophagy-lysosomal transcription factor TFEB and the transcriptional repressor ZKSCAN3.We have demonstrated that BRAFV600E-TFEB/ZKSCAN3-autophagy-lysosomal signaling pathway is a key regulatory pathway for BRAFV600E-mediated TGF-? signaling,activation of EMT,and control of carcinogenesis,BRAFV600E mediates autophagy-lysosomal inactivation Lead to the development of tumors and increased metastasis and the development of resistance to BRAF,affecting the targeted treatment of melanoma.In this article,we explored the molecular mechanism of autophagy and its effects on disease progression and drug resistance by carcinogenic BRAFV600E and its inhibitors,revealing a kind of the "BRAF-TFEB-autophagy-lysosome" axis represents an intrinsic regulatory pathway in BRAF-mutant melanoma,coupling BRAF signaling with TGF-? signaling to drive tumor progression and chemoresistance.This reaserch may be very important to clinical tumor treatment,we believe that drugs which inhibit autophagy are not beneficial to the treatment of tumors,and it may be necessary to re-examine cancer treatment related autophagy in clinical treatment.