PRMT5 Methylating SMAD4 Activates TGF-β Signaling And Promotes Colorectal Cancer Metastasis

Background & Objective: Colorectal cancer is a serious disease that endangers human life and health.Metastasis of CRC often leads to poor prognosis.Interference of TGF-βsignaling can lead to a variety of diseases,including cancer,and plays a critical role in the occurrence and development of CRC.When the classical TGF-β signaling is activated,the SMAD family proteins SMAD2,SMAD3 and SMAD4 form complexes,which enter the nucleus and initiate the transcription of multiple downstream genes,including EMT related genes SNAIL and SLUG.This is one of the key mechanisms by which TGF-β signaling promotes the invasion and migration of CRC.As the core component of SMAD complex,the function of SMAD4 is precisely regulated.Mutations and PTMs of SMAD molecules can lead to dysregulation of TGF-β signaling.This study aims to identify the methylation of SMAD4,and confirm the type of methylation.To identify the key enzyme mediating the modification,and investigate its role and mechanism in the formation of SMAD complexes,TGF-β signaling activation and EMT of tumor cells.And to explore the significance of detection and intervention of SMAD4 methylation level in the diagnosis and treatment of CRC.Methods: The methylation sites and binding proteins of SMAD4 were identified by protein mass spectrometry.Protein immunoprecipitation was used to verify the mass spectrometry results and screen the methyltransferase mediating the methylation of the corresponding site,and confirm the type of methylation.The effect of Protein arginine methyltransferase 5(PRMT5)on SMAD4 was demonstrated by in vitro methylation assay.The effects of SMAD4 R361 methylation on the formation and nuclear import of SMAD complexes were identified by quantitative protein immunoprecipitation and immunofluorescence.In vitro and in vivo models were established to study the effect of PRMT5 and SMAD4 R361 methylation on liver metastasis of CRC and to explore the effect of specific inhibition of PRMT5 on liver metastasis of CRC.SMAD4 R361 symmetric demethylation(SDMA)specific antibody was developed.Immunohistochemical(IHC)staining and scoring were performed on CRC tissue microarray to explore the relationship between the methylation level at SMAD4 R361 and tumor prognosis.Results: Protein mass spectrometry,immunoprecipitation and the in vitro methylation assay showed that SMAD4 could be symmetrical dimethylated(SDMA)at R361 site,which was mediated by PRMT5.Protein immunoprecipitate,Western blot and q PCR experiments showed that PRMT5 triggered SMAD4 R361 methylation under TGF-β1 treatment,promoting SMAD4 binding with SMAD2/3 and increasing SMAD complexes nuclear import,enhancing the expression of EMT-related molecules downstream of TGF-βsignaling pathway.Trans-well assay and animal model of CRC liver metastasis suggested that PRMT5 could affect invasion and migration of CRC in vitro and in vivo through SMAD4 R361-dependent manner.PRMT5 specific inhibitor GSK3326595 showed better metastasis inhibition in SMAD4 wild-type CRC cell lines.IHC staining of tumor samples from colorectal cancer patients showed that SMAD4 R361 SDMA level was correlated with PRMT5 level in tumor tissue.IHC staining and scoring of tissue microarray from 80 colorectal cancer patients showed that SMAD4 R361 SDMA level was positively associated with the risk of tumor metastasis and the number of metastatic lymph nodes,and negatively associated with overall survival in patients who did not receive postoperative chemotherapy.Conclusions: PRMT5 interacts with SMAD4 under the induction of TGF-β1,triggering symmetrical dimethylation at SMAD4 R361 and inducing SMAD complexes formation and nuclear import,increasing the expression of EMT-related genes,and thus promoting the invasion and migration of CRC.In CRC patients,high expression of PRMT5 or high level of SMAD4 R361 SDMA suggests a higher risk of metastasis and a poorer prognosis.In SMAD4 wild-type colorectal cancer,GSK3326595 specific inhibition of PRMT5 inhibited tumor metastasis.SMAD4 R361 symmetry dimethylation is a potential indicator to predict the prognosis of CRC and the efficacy of targeting PRMT5 therapy.Part Ⅰ: PRMT5 mediates SMAD4 R361 SDMA to activate TGF-β signaling pathwayObjective: To identify the methylation site and binding protein of SMAD4.To identify the methylation type of R361 site and the key enzyme mediating the modification.To clarify the role and mechanism of the methylation of SMAD4 R361 site in the formation and nuclear import of SMAD complexes.Methods: SMAD4 and its binding protein were enriched by protein immunoprecipitation,and the methylation sites and binding protein were identified by protein mass spectrometry.Quantitative protein co-immunoprecipitation was used to determine the type of SMAD4 R361 methylation.The key enzyme for SMAD4 R361 methylation was identified through methyltransferase plasmid overexpression or inhibitors.The co-localization of PRMT5 and SMAD4 in HEK293 T and human CRC cell lines SW48 and Lo Vo was observed by immunofluorescence and confocal scanning microscopy.The effect of PRMT5 on SDMA of SMAD4 R361 was confirmed by in vitro methylation assay.Using public databases to analyze the conserved status of SMAD4 R361 among species,and analyze the mutation in tumors.Gene set enrichment analysis(GSEA)was used to analyze the correlation between PRMT5 expression and TGF-β signaling pathway in SMAD4 wild-type and mutant CRC.SMAD4 R361 K mutant cell line,PRMT5 overexpression plasmid and PRMT5 specific inhibitor were used to detect the effect and mechanism of PRMT5 on the binding of SMAD4 to SMAD2/3 under TGF-β1 treatment by quantitative protein immunoprecipitation.Immunofluorescence,nuclear-cytoplasmic protein separation and Western blot assay were used to detect the effect of PRMT5 on SMAD complexes’ nuclear import under TGF-β1 treatment.Results: Protein mass spectrometry analysis showed that SMAD4 was methylated at R361 and PRMT5 could bind to SMAD4.Analysis of data from public databases showed that SMAD4 R361 was highly conserved among species,but had a high mutation frequency in CRC.GSEA analysis showed that the expression of PRMT5 was significantly correlated with TGF-β signaling pathway in CRC,but not in CRC patients with SMAD4 R361 mutation.Laser confocal scanning microscopy showed that PRMT5 and SMAD4 colocalized in cells,and the degree of colocalization increased under the effect of TGF-β1.Quantitative protein immunoprecipitation assay showed that GSK3326595 treatment or PRMT5 overexpression could affect the binding of SMAD4 to SMAD2/3 induced by TGF-β1 in SMAD4 wild-type cell lines,but the effect was not obvious in SMAD4 R361 K mutant.Immunofluorescence and nuclear-cytoplasmic protein isolation showed that intervention with PRMT5 affected TGF-β1-induced SMAD complexes nuclear import in SMAD4 wildtype cell lines,but this effect was not evident in SMAD4 R361 K mutant.Conclusions: PRMT5 interacts with SMAD4 under the induction of TGF-β1,triggering symmetrical dimethylation at SMAD4 R361 and inducing SMAD complexes formation and nuclear import.High expression of PRMT5 increased TGF-β signaling activity in a SMAD4 R361 dependent manner.Part Ⅱ: SMAD4 R361 SDMA promotes invasion and migration of CRCObjective: To explore the role and mechanism of PRMT5 methylation of SMAD4 in TGF-β-mediated EMT,and to clarify the correlation between PRMT5 expression and SMAD4 R361 SDMA,and invasion and migration ability of CRC.To investigate the effect of PRMT5 intervention on invasion and migration of SMAD4 wild type and R361 mutated CRC.Methods: Overexpressing PRMT5 in human CRC cell lines SW48 and Lo Vo or treating them with GSK3326595.The effect of PRMT5 on the expression of EMT-related molecules induced by TGF-β1 was detected by q PCR and Western blot.The SMAD4 R361 K mutant SW48 and Lo Vo were constructed using CRISPR-Cas9 system.The effects of PRMT5 on the expression of EMT-related molecules induced by TGF-β1 in wild-type and mutant SMAD4 cell lines were detected by q PCR and Western blot.Trans-well assay was conducted to investigate the effect of PRMT5 intervention on the invasion and migration of wild-type and SMAD4 R361 K mutant CRC in vitro.Tumor liver metastasis animal model was established to study the effect of PRMT5 intervention on the invasion and migration of wild-type and SMAD4 R361 K mutant CRC in vivo.Results: SMAD4 R361 K mutation or treatment with GSK3326595 reduced the expression of EMT-related genes induced by TGF-β1 in SW48 and Lo Vo cell lines.Overexpression of PRMT5 enhanced the expression of EMT-related genes induced by TGF-β1 in wild-type SW48 and Lo Vo cell lines,but not in SMAD4 R361 K mutant.Trans-well assay showed that overexpression of PRMT5 significantly enhanced the invasion and migration induced by TGF-β1 in wild-type SW48 and Lo Vo cell lines,but the effect was weaker in SMAD4 R361 K mutant.In the tumor liver metastasis animal model,overexpression of PRMT5 significantly enhanced the liver metastasis of SMAD4 wild-type cell lines,but the effect of overexpression of PRMT5 was not so obvious in R361 K mutant.Knockdown of PRMT5 or GSK3326595 significantly inhibited the liver metastasis of SMAD4 wild-type tumors,but in SMAD4 R361 K mutant tumors,the effect of GSK3326595 was weaker.Conclusions: PRMT5 enhances TGF-β signaling induced EMT in a SMAD4 R361 dependent manner and promotes invasion and migration of CRC in vivo and in vitro.Targeting PRMT5 can inhibit the invasion and metastasis of CRC in vivo and in vitro,and its inhibitory effect is better in SMAD4 wild-type tumors,which is potential for the treatment of metastatic CRC.Part Ⅲ: Targeting SMAD4 R361 SDMA in the diagnosis and treatment of CRCObjective: To clarify the correlation between PRMT5 expression level and SMAD4 R361 SDMA level in CRC patients,and to analyze the correlation between SMAD4 R361 SDMA and tumor metastasis and prognosis in CRC patients.To explore the possibility of SMAD4 R361 SDMA as a prognostic marker for CRC.Methods: IHC staining of clinical samples from the interface between tumor and paracancer tissues of CRC patients was performed.The relationship between PRMT5 expression and SMAD4 R361 SDMA in clinical samples and differences between tumor and paracancer tissues was verified.The SMAD4 R361 symmetric dimethylated specific antibody developed in this study was used to perform IHC staining of a tissue microarray of 80 CRC patients,and the IHC score of the tumor tissues was calculated.The relationship between the IHC score and the situation of tumor metastasis,the number of metastatic lymph nodes and OS was analyzed.Results: IHC staining of 6 CRC patients showed that IHC staining of PRMT5 and SMAD4 R361 symmetrical dimethylation was significantly up-regulated in tumor tissues compared to para cancer.Excluding non-tumor cases in the tissue microarray,6 of the 63 samples were unstained(not included in follow-up statistics).The IHC score in tumor lesions was higher than that in para-cancer tissue.Patients with lymph nodes or distant metastasis had higher IHC scores in tumor tissue.SMAD4 R361 SDMA level was positively correlated with the number of metastatic lymph nodes and was negatively correlated with overall survival in patients who did not receive postoperative chemotherapy.Conclusions: The SDMA levels of PRMT5 and SMAD4 R361 are higher in tumors than in para-cancer tissue.High levels of SMAD4 R361 SDMA predict greater risks of metastasis and poor prognosis and are potential to be used as a prognostic marker in CRC.