Sunitinib Treatment Promotes Metastasis Of Drug-resistant Renal Cell Carcinoma VIA TFE3 Signaling Pathway

Research backgroundRenal cell carcinoma is one of the most aggressive malignant tumors in human urinary system,accounting for 2-3%of adult malignant tumors.According to the global cancer watch,there will be about 400000 new cases and 175000 deaths in 2018.Renal cell carcinoma(RCC)is resistant to chemoradiotherapy.Surgical resection is still the only effective treatment for early non metastatic RCC.About 33%of the patients had metastatic lesions at the initial diagnosis,and 30%of the patients eventually developed metastatic renal cell carcinoma(mRCC)after surgery.Targeted therapy can significantly improve the prognosis of patients with metastatic renal cell carcinoma.Sunitinib is a tyrosine kinase inhibitor(TKI)targeting vascular endothelial growth factor receptor(VEGFR).Compared with interferon ?,sunitinib can significantly prolong progression free survival(PFS),but not overall survival(OS).At present,sunitinib is the first-line treatment for patients with mRCC.However,in key phase ? trials,7%of patients developed disease progression at the first assessment,and almost all patients eventually developed progressive disease.Although the academic community has been exploring the prognostic factors of MRCC patients receiving targeted therapy and proposed several drug resistance mechanisms,the mechanism of sunitinib sensitivity or secondary drug resistance has not been determined.Therefore,we urgently need to better understand the pathogenesis and drug resistance mechanism of renal cell carcinoma,so as to provide more effective treatment strategies for patients with metastatic renal cell carcinoma.The MIT TFE family consists of four members:TFEB,TFE3,tfec and MITF.As elicitors of lysosome synthesis and energy regulators of cells,these factors play an important role in intracellular homeostasis and autophagy.The subcellular localization of MIT TFE family is mainly on lysosomal surface,cytoplasm and nucleus,and it can sense the energy fluctuation and various forms of cell pressure in cells,and shuttle between these organelles under the mechanism of phosphorylation.The main kinases responsible for the phosphorylation of MiT TFE protein include mTOR,ERK and GSK3.In addition,calcineurin regulates lysosomal calcium release through dephosphorylation of MiT TFE protein.The factors of MiT TFE family were initially described as oncogenes.The increased expression and activity of TFEB,TFE3 and TFE3 are associated with more than 20 kinds of cancers,and have a direct effect on the proliferation and migration of these cancer cells.TFEB and TFE3 can directly regulate the expression of PD-L1 in renal cell carcinoma,and are closely related to drug resistance and immune escape.MiT TFE family not only plays an important role in the progression of RCC,but also plays an important role in the chemotherapy resistance of RCC tumor.Autophagy in a broad sense is also called macroautophagy.Its main function is responsible for the degradation and circulation of intracellular macromolecules and organelles to maintain the homeostasis of intracellular environment.Stress including endoplasmic reticulum stress is the main inducer of autophagy.Endoplasmic reticulum autophagy(ER-phagy),as a recognized selective autophagy mechanism that controls the quality and morphology of endoplasmic reticulum,is an autophagy that specifically degrades endoplasmic reticulum to balance the expansion of endoplasmic reticulum during the reaction of unfolded proteins on endoplasmic reticulum.At present,it is recognized that specific autophagy receptors are the main regulatory factors of selective autophagy,and the research of endoplasmic reticulum autophagy mainly focuses on the discovery and functional exploration of specific receptors.The function of ER-phagy,especially in the occurrence and development of cancer,is rarely studied.Whether ER-phagy is involved in anticancer response remains unclear.Part?TFE3 is the core factor that mediates the metastasis of renal cell carcinoma after sunitinib resistanceObjectiveOur team treated a 21-year-old female patient with mRCC.The patient received sunitinib for 3 months.After imaging showed that the tumor volume was significantly reduced,the patient received tumor reduction surgery for primary renal cell carcinoma,and then received maintenance sunitinib treatment.Three years later,the patient received neck new RCC metastasis resection.Our team objectively believed that the primary tumor specimen obtained from 3 years ago was sunitinib sensitive renal cell carcinoma tissue,and the new metastasis lesion removed from 3 years later was RCC tissue that resistance to sunitinib.We sequenced the two-stage lesions of this case by proteomics.and carried out some in vitro simulation experiments according to the sequencing results.The purpose is to explore the molecular mechanism of the clinical process of RCC from sunitinib sensitive to sunitinib resistant.Method1.One patient with advanced metastatic renal cell carcinoma was enrolled.Proteomic analysis was performed on the renal cell carcinoma tissues in the sunitinib sensitive stage and the metastatic tissues after sunitinib resistance,and the changes of molecular pathways in these two states were determined by enrichment analysis.2.We infected the RCC cell line 786O with lentivirus carrying TFE3 gene plasmid for constructing a cell line stably expressing TFE3 gene.The subcutaneous tumorigenesis of two groups of nude mice was carried out with this new cell line.When the tumor was implanted successfully,one group was treated with sunitinib,and the other group was treated with DMSO repeatedly.The two groups of tumors were induced for three generations at the same time.Finally,the sunitinib resistant 786O/OE-SR cell line(sunitinib induced group)and sunitinib sensitive 786O/OE cell line(DMSO induced group)were constructed.3.The subcellular localization of TFE3 before and after sunitinib resistance was verified by IHC and Western blotting in the case of before and after sunitinib.4.Q-PCR and Western blotting were used to verify the changes of TFE3 downstream factor expression under sunitinib treatment.5.Transwell test and scratch test were used to verify the invasion and migration ability of renal cancer cells before and after sunitinib resistance in the condation of TFE3 silence.Result1.Our team treated a patient with mRCC diagnosed by enhanced CT.After 3 months of sunitinib treatment,the tumor diameter was reduced from 15cm to 10cm,and then the left RCC received resection.After 3 years' sunitinib maintenance treatment,CT showed that this case suffered new metastasis of RCC in the left cervical region with resection of metastatic lesion was performed.We believe that the left renal tumor specimens obtained 3 years ago are sensitive to sunitinib,and the metastatic specimens obtained 3 years later are resistant to sunitinib.2.TMT proteomics was used to analyze sunitinib sensitive samples and sunitinib resistant samples.Two parts of each lesion were randomly selected for resection.Thermographic analysis showed that the expression of specific cluster proteins was only up-regulated or down regulated in sunitinib resistant group compared with other groups.Compared with sunitinib sensitive tumor samples,lysosomal proteins were significantly up-regulated in metastatic samples,and the corresponding down regulated proteins were mainly enriched in extracellular matrix(ECM)proteins3.Through scanning electron microscope we found that the number of lysosomes in drug-resistant lesions was significantly more than that in sunitinib sensitive RCC tissues.IHC staining showed that TFE3 was strongly expressed in the nucleus of drug-resistant metastatic foci,while TFE3 was diffusely expressed in the cytoplasm of sunitinib sensitive RCC tissues.4.We constructed 786O/OE cell line and 786O/OE-SR cell line in vivo to verify the subsequent experiments and recorded the weight,volume and physical diagram of all tumors during the process.After silencing the TFE3 of 786O/OE-SR cells by lentivirus,the tumor formation experiment in nude mice was carried out with sunitinib treatment,which showed that TFE3 knocked down could reverse 786O/OE-SR cell' resistance ability to sunitinib.The results of immunohistochemistry showed that compared with 786O/OE cells,a large number of TFE3 nucleation appeared in 786O/OE-SR cells,which was consistent with the results of clinical specimens.5.WB assay showed that the subcellular localization of TFE3 in 786O/OE-SR cells was dynamic.When sunitinib was given,TFE3 would enter the nucleus in large quantities,and when sunitinib was withdrawn,TFE3 would return to the cytoplasm.Western blotting showed that with the increase of sunitinib concentration,the expression of downstream proteins of TFE3,CTSA,CTSB,FTH1 and E-syt1 increased.6.In vitro invasion and migration experiments showed that with the increase of sunitinib concentration,the invasion and migration ability of 786O/OE-SR cells were also enhanced.Lentivirus sh-TFE3 silencing TFE3 could reverse the enhanced effect of sunitinib on the invasion and migration ability of 786O/OE-SR cells.ConclusionThrough in vivo experiments,we confirmed our previous conjecture:the high expression of TFE3 is the key factor to induce the sunitinib resistance of RCC.once RCC obtains resistance to sunitinib,sunitinib maintenance treatment will continuously stimulate TFE3 to enter the nucleus,thus activating the expression of lysosomal pathway.As a result,a large number of lysosomal exocytosis will degrade the extracellular matrix so as to promote the metastasis of RCC.Part ? TFE3 promotes the metastasis of RCC by promoting endoplasmic reticulum autophagy and lysosome exocytosisObjectiveIn the first part of this study,when we observed the lysosomes of clinical specimens by electron microscope,we unexpectedly found that a large number of ER fragments appeared in the drug-resistant RCC tissue,accompanied by autophagosomes phagocytizing ER fragments,which is what we call ER-phagy.It has never been reported that ER fragmentation occur in the contact area between lysosome and ER.How does TFE3 regulate massive ER-phagy?What is the relationship between ER-phagy and drug resistance metastasis of RCC?In order to clarify the above problems,we used the drug-resistant cell lines constructed from the first part of this study to carry out a series of simulation and exploration experiments.Method1.Sunitinib resistant RCC tissues from the first part of this study were observed by scanning electron microscope.2.The correlation between TFE3 and Esyt1 was analyzed by TCGA and GEO database.The sequence ratio of promoter region and double luciferase reporter gene experiment proved the regulation principle of TFE3 on Esyt1.3.We proved the activation of Esyt1 on ER-phagy by our own designed CCER experiment,EATR experiment and WB experiment.4.Co-IP and confocal microscopy were used to detect the binding of Estl and syt7 domains.All the proteins that bind to Syt7 protein in 786o/oe-sr cells were extracted by CoIP experiment,and their functions were classified and annotated.5.The changes of ER-phagy,lysosome were observed by scanning electron microscope after 786O/OE-SR was transferred into Syt7 plasmid and empty plasmid.After 786O/OE-SR cells were transfected with Syt7 and its mutants,ER tracker staining was used to observe the morphological changes of ER.The changes of colocalization between ER protein Esytl and lysosomal cathepin B(CTSB)were observed by confocal microscopy after silencing Syt7 by si-syt7.786O/OE-SR cells were transfected with Syt7 plasmid and then CTSB was specifically inhibited by CA-074Me.Er tracker staining was used to observe the morphological changes of ER.6.CCK-8 assay was used to detect the role of C2 domain of syt7 in sunitinib resistance.Transwell assay was used to detect whether sunitinib could promote the invasion and metastasis of 786O/OE-SR cells by silencing syt7.Four weeks old female nude mice were randomly divided into four groups,and each group was injected with the following:DMSO+sh NC treated 786O/OE-SR cells,DMSO+sh-syt7 treated 786O/OE-SR cells,sunitinib+sh NC treated 786O/OE-SRcells,sunitinib +sh-syt7 treated 786O/OE-SR cells.Three weeks later,the lungs of nude mice in each group were stained with HE and the number of tumor nodules was counted.7.After the cytosolic Ca2+was increased by ionomycin treatment,the exocytosis of lysosome mediated by syt7 was observed by confocal microscopy.Flow cytometry was used to compare the changes of intracellular calcium concentration under the action of ER-phagy receptor FAM134b and CA-074Me.After lentivirus knockout of Esyt-1 in 786O/OE-SR cells,confocal microscopy was used to observe whether sunitinib could still stimulate Syt7 mediated lysosomal exocytosis.Result1.TFE3,as an upstream transcription factor,can directly bind to the promoter region of Esyt1,thus initiating the expression of Esyt1 gene.2.As a chaperone of FAM134B,Esyt1 regulates ER-phagy before FAM134B.3.Syt7 mediates the organelle contact between lysosome and ER by binding its C2 domain to the C2 domain of Esyt1.4.After exogenous overexpression of syt7,a large number of contact sites were formed between lysosome and endoplasmic reticulum,at which ER fragmentation and e ER-phagy occurred.5.In 786O/OE-SR cells,the regulation of ER fragmentation by Syt7 depends on the local digestion of ER by lysosomal enzymes mediated by Esyt1/Syt7 heterodimer.6.Overexpression of Syt7 can enhance the resistance of 786O cells to sunitinib,and silencing syt7 can reverse the resistance of 786O/OE-SRcells to sunitinib.Transwell and lung metastasis experiments in nude mice showed that silencing syt7 could reverse the enhancement of sunitinib on the metastatic ability of 786O/OE-SR cells.7.WB and flow cytometry showed that Esyt1/Syt7 mediated ER membrance was the key factor to increase intracellular calcium concentration.Confocal microscopy showed that intracellular Ca2+stimulated the exocytosis of lysosomes mediated by the C2 domain of syt7.When Esyt1 was knocked out,sunitinib could not stimulate the lysosomal exocytosis induced by C2 domain of Syt7.ConclusionWhen renal cell carcinoma is resistant to sunitinib,continuous sunitinib treatment can stimulate TFE3 to enter the nucleus and directly promote the expression of endoplasmic reticulum protein Esyt-1.The heterodimer formed by Esyt-1 and Syt7 directly mediates the contact site formation between lysosome and ER.Lysosome directly releases cathepsin through the gap of lysosome membrane to degrade the ER in the contact site area,thus directly mediating the key step of ER-phagy,which is named ER fragmentation.A large number of ER degradation will promote the release of calcium ions into the cytoplasm,which directly mediates the exocytosis of lysosomes by stimulating the C2 domain of Syt7,and ultimately mediates the metastasis of drug-resistant RCC.