Objectives:Intravesical Bacillus Calmette-Guérin(BCG)therapy is a gold standard for patients with high-risk non-muscle invasive bladder cancer(NMIBC).Although a long-lasting therapeutic response is observed in mo...Objectives:Intravesical Bacillus Calmette-Guérin(BCG)therapy is a gold standard for patients with high-risk non-muscle invasive bladder cancer(NMIBC).Although a long-lasting therapeutic response is observed in most patients,BCG failure occurs in 30%–50%of patients and a progression to muscle-invasive disease is found in 10%–15%.Therefore,predicting high-risk patients who might not benefit from BCG treatment is critical.The purpose of this study was to identify,whether the presence of specific oncogenic mutations might be indicative of BCG treatment response.Methods:Nineteen high-grade NMIBC patients who received intravesical BCG were retrospectively enrolled and divided into“responders”and“non-responders”groups.Tissue samples from transurethral resection of bladder cancer were performed before starting therapy and were examined using a multigene sequencing panel.Results:Mutations in TP53,FGFR3,PIK3CA,KRAS,CTNNB1,ALK and DDR2 genes were detected.TP53 and FGFR3 were found to be the most frequently mutated genes in our cohort(31.6%and 26.3%,respectively),followed by PIK3CA(15.8%).In the BCG-responsive patient group,90%of samples were found to have mutated genes,with almost 50%of them showing mutations in tyrosine kinase receptors and CTNNB1 genes.On the other hand,in the BCG-unresponsive group,we found mutations in 44.4%of samples,mainly in TP53 gene.Conclusions:Our findings suggest that a Next-Generation Sequencing(NGS)multigene panel is useful in predicting BCG response in patients with NMIBC.展开更多
Early colostrum feeding facilitates the passive transfer of immunoglobulin G(IgG),which contributes to the defensive establishment of neonates;however,the molecular mechanisms of IgG absorption in the small intestine ...Early colostrum feeding facilitates the passive transfer of immunoglobulin G(IgG),which contributes to the defensive establishment of neonates;however,the molecular mechanisms of IgG absorption in the small intestine of neonatal mammals remain largely unknown.In this study,a total of 16 neonatal goat kids with similar body weight(2.05±0.31 kg)were selected and randomly assigned to 1 of 2 feeding treatments:normal colostrum feeding(NCF,n=8)or delayed colostrum feeding(DCF,n=8).Multi-omics coupled with individual bioinformatics analyses were employed to obtain a comprehensive understanding of the molecular mechanisms of IgG absorption.Phenotypic analysis showed that the capacity of IgG absorption was largely affected(P<0.05)by colostrum feeding time in neonatal goat kids.Weighted gene co-expression network analysis generated 23 gene modules(gene module defined M1 to M23)and the M12 module was highly correlated(|r|>0.70 and adjusted P<0.01)with IgG absorption.Genes in M12 were involved in the endocytosis pathway,especially related to clathrin-mediated endocytosis and macropinocytosis.The differentially expressed genes(DEGs)enriched in the above-mentioned pathways regulated the clathrin synthesis(CLTC),the formation of clathrin-coated vesicles(ARPC1A),and the sorting and recycling endosomes(CAPZA2,KIAA0196,RAB10,RAB11A and VPS35)as well as the formation of macropinosomes(FGFR4 and RhoA)in micropinocytosis,which induced differences in serum IgG concentrations.Additionally,5 differentially expressed miRNAs(miR-2755-3p,miR-10400-5p,miR-71-5p,miR-2944-3p and miR-2411-3p)were predicted to regulate mRNA involved in clathrin-coated vesicles,Fc receptor for IgG(FcRn)-IgG sorting,and macropinosomes formation that may cause the difference in IgG absorption ability.This study provides new insights into the molecular mechanisms controlling IgG absorption of neonatal ruminants and reveals novel mRNA and miRNA markers involved in clathrin-mediated endocytosis and macropinocytosis which may provide the fundamental knowledge related to IgG absorption to support further study in other mammals.展开更多
Background:Small extracellular vesicles(sEVs)mediate intercellular commu-nication that contributes to hepatocellular carcinoma(HCC)progression via multifaceted pathways.The success of cell entry determines the effect ...Background:Small extracellular vesicles(sEVs)mediate intercellular commu-nication that contributes to hepatocellular carcinoma(HCC)progression via multifaceted pathways.The success of cell entry determines the effect of sEV on recipient cells.Here,we aimed to delineate the mechanisms underlying the uptake of sEV in HCC.Abbreviations:AF,Alexa Fluor;ANOVA,analysis of variance;ATP9A,ATPase Phospholipid Transporting 9A;BCECF-AM,2’,7’-bis-(2-barboxyethyl)-5-(and-6)-carboxyfluorescein,acetoxymethyl ester;BSA,bovine serum albumin;CCMR,Centre for Comparative Medicine Research;CRISPR,clustered regularly interspaced short palindromic repeats;CTL,ctrl;CXCR4,C-X-C Chemokine Receptor Type 4;DAPI,4′,6-diamidino-2-phenylindole;DFS,disease-free survival;DMEM,Dulbecco’s Modified Eagle Medium;DMSO,dimethyl sulfoxide;Dox,doxycycline;EEA1,early endosome antigen 1;EIPA,5-(N-ethyl-N-isopropyl)-amiloride;FBS,fetal bovine serum;FITC,fluorescein isothiocyanate;GAPDH,glyceraldehyde-3-phosphate dehydrogenase;GM130,Golgi matrix protein 130;HCC,hepatocellular carcinoma;HEPES,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;HPRT1,hypoxanthine phosphoribosyltransferase 1;H-score,histoscore;IAA,indole-3-acetic acid;KD,knockdown;KO,knockout;mAID,mini-auxin-inducible degron;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;NHE7,Na(+)/H(+)exchanger 7;ns,non-significant;OD,optical density;OS,overall survival;PBS,phosphate-buffered saline;PCR,polymerase chain reaction;pHe,endosomal pH;pHi,intracellular pH;PKH67,Paul Karl Horan 67;Rab21,Ras-associated binding protein 21;RIPA,radioimmunoprecipitation assay;SAM,synergistic activation mediator;SEMs,standard error of the means;sEVs,small extracellular vesicles;sgRNA,single-guide RNA;shRNA,short-hairpin RNA;SLC9,solute carrier gene 9;SLiCE,Seamless Ligation Cloning Extract;TCGA,The Cancer Genome Atlas;TCL,total cell lysates;TGN,trans-Golgi network;TMA,tissue microarray;TMR,tetramethyl rhodamine;TSG101,tumor susceptibility gene 101.Yue Yao and Yi Xu contributed equally to this work.Methods:Macropinocytosis was examined by the ability of cells to internalize dextran and sEV.Macropinocytosis was analyzed in Na(+)/H(+)exchanger 7(NHE7)-knockdown and-overexpressing cells.The properties of cells were stud-ied using functional assays.pH biosensor was used to evaluate the intracellular and endosomal pH.The expression of NHE7 in patients’liver tissues was exam-ined by immunofluorescent staining.Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7.Results:The data revealed that macropinocytosis controlled the internaliza-tion of sEVs and their oncogenic effect on recipient cells.It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non-metastatic HCC cells.Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride(EIPA)limited the entry of sEVs and compromised cell aggressiveness.Mechanistically,we delineated that high level of NHE7,a sodium-hydrogen exchanger,alkalized intracellular pH and acidized endosomal pH,leading to the maturation of macropinosomes.Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis.Clinically,NHE7 expression was upregulated and linked to dismal prognosis of HCC.Conclusions:This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells.Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.展开更多
Cancer is the second leading cause of death in the US.Current major treatments for cancer management include surgery,cytotoxic chemotherapy,targeted therapy,radiation therapy,endocrine therapy and immunotherapy.Despit...Cancer is the second leading cause of death in the US.Current major treatments for cancer management include surgery,cytotoxic chemotherapy,targeted therapy,radiation therapy,endocrine therapy and immunotherapy.Despite the endeavors and achievements made in treating cancers during the past decades,resistance to classical chemotherapeutic agents and/or novel targeted drugs continues to be a major problem in cancer therapies.Drug resistance,either existing before treatment(intrinsic)or generated after therapy(acquired),is responsible for most relapses of cancer,one of the major causes of death of the disease.Heterogeneity among patients and tumors,and the versatility of cancer to circumvent therapies make drug resistance more challenging to deal with.Better understanding the mechanisms of drug resistance is required to provide guidance to future cancer treatment and achieve better outcomes.In this review,intrinsic and acquired resistance will be discussed.In addition,new discoveries in mechanisms of drug resistance will be reviewed.Particularly,we will highlight roles of ATP in drug resistance by discussing recent findings of exceptionally high levels of intratumoral extracellular ATP as well as intracellular ATP internalized from extracellular environment.The complexity of drug resistance development suggests that combinational and personalized therapies,which should take ATP into consideration,might provide better strategies and improved efficacy for fighting drug resistance in cancer.展开更多
The cancer stem cell(CSC)state and epithelial-mesenchymal transition(EMT)activation are tightly interconnected.Cancer cells that acquire the EMT/CSC phenotype are equipped with adaptive metabolic changes to maintain l...The cancer stem cell(CSC)state and epithelial-mesenchymal transition(EMT)activation are tightly interconnected.Cancer cells that acquire the EMT/CSC phenotype are equipped with adaptive metabolic changes to maintain low reactive oxygen species levels and stemness,enhanced drug transporters,anti-apoptotic machinery and DNA repair system.Factors present in the tumor microenvironment such as hypoxia and the communication with non-cancer stromal cells also promote cancer cells to enter the EMT/CSC state and display related resistance.ATP,particularly the high levels of intratumoral extracellular ATP functioning through both signaling pathways and ATP internalization,induces and regulates EMT and CSC.The three of them work together to enhance drug resistance.New findings in each of these factors will help us explore deeper into mechanisms of drug resistance and suggest new resistance-associated markers and therapeutic targets.展开更多
文摘Objectives:Intravesical Bacillus Calmette-Guérin(BCG)therapy is a gold standard for patients with high-risk non-muscle invasive bladder cancer(NMIBC).Although a long-lasting therapeutic response is observed in most patients,BCG failure occurs in 30%–50%of patients and a progression to muscle-invasive disease is found in 10%–15%.Therefore,predicting high-risk patients who might not benefit from BCG treatment is critical.The purpose of this study was to identify,whether the presence of specific oncogenic mutations might be indicative of BCG treatment response.Methods:Nineteen high-grade NMIBC patients who received intravesical BCG were retrospectively enrolled and divided into“responders”and“non-responders”groups.Tissue samples from transurethral resection of bladder cancer were performed before starting therapy and were examined using a multigene sequencing panel.Results:Mutations in TP53,FGFR3,PIK3CA,KRAS,CTNNB1,ALK and DDR2 genes were detected.TP53 and FGFR3 were found to be the most frequently mutated genes in our cohort(31.6%and 26.3%,respectively),followed by PIK3CA(15.8%).In the BCG-responsive patient group,90%of samples were found to have mutated genes,with almost 50%of them showing mutations in tyrosine kinase receptors and CTNNB1 genes.On the other hand,in the BCG-unresponsive group,we found mutations in 44.4%of samples,mainly in TP53 gene.Conclusions:Our findings suggest that a Next-Generation Sequencing(NGS)multigene panel is useful in predicting BCG response in patients with NMIBC.
基金supported by the Chinese Academy of Sciences(Strategic Priority Research Program Grant No.XDA26050102)the National Natural Science Foundation of China(32072760)the Natural Science Foundation of Hunan Province of China(2022JJ10054).
文摘Early colostrum feeding facilitates the passive transfer of immunoglobulin G(IgG),which contributes to the defensive establishment of neonates;however,the molecular mechanisms of IgG absorption in the small intestine of neonatal mammals remain largely unknown.In this study,a total of 16 neonatal goat kids with similar body weight(2.05±0.31 kg)were selected and randomly assigned to 1 of 2 feeding treatments:normal colostrum feeding(NCF,n=8)or delayed colostrum feeding(DCF,n=8).Multi-omics coupled with individual bioinformatics analyses were employed to obtain a comprehensive understanding of the molecular mechanisms of IgG absorption.Phenotypic analysis showed that the capacity of IgG absorption was largely affected(P<0.05)by colostrum feeding time in neonatal goat kids.Weighted gene co-expression network analysis generated 23 gene modules(gene module defined M1 to M23)and the M12 module was highly correlated(|r|>0.70 and adjusted P<0.01)with IgG absorption.Genes in M12 were involved in the endocytosis pathway,especially related to clathrin-mediated endocytosis and macropinocytosis.The differentially expressed genes(DEGs)enriched in the above-mentioned pathways regulated the clathrin synthesis(CLTC),the formation of clathrin-coated vesicles(ARPC1A),and the sorting and recycling endosomes(CAPZA2,KIAA0196,RAB10,RAB11A and VPS35)as well as the formation of macropinosomes(FGFR4 and RhoA)in micropinocytosis,which induced differences in serum IgG concentrations.Additionally,5 differentially expressed miRNAs(miR-2755-3p,miR-10400-5p,miR-71-5p,miR-2944-3p and miR-2411-3p)were predicted to regulate mRNA involved in clathrin-coated vesicles,Fc receptor for IgG(FcRn)-IgG sorting,and macropinosomes formation that may cause the difference in IgG absorption ability.This study provides new insights into the molecular mechanisms controlling IgG absorption of neonatal ruminants and reveals novel mRNA and miRNA markers involved in clathrin-mediated endocytosis and macropinocytosis which may provide the fundamental knowledge related to IgG absorption to support further study in other mammals.
基金The work was funded by Research Grants Council General Research Fund(Grant No.17105322)Hong Kong Scholars Program(Grant No.XJ2020012 and 2020-036)+3 种基金University Research Committee Seed Fund for Basic Research(Grant No.202111159009)of The University of Hong KongMarshal Initiative Fund-ing of Harbin Medical University(Grant No.HMUMIF-22008)Open Funds of State Key Laboratory of Oncology in South China(Grant No.HN2023-02)Natural Sci-ence Foundation of Heilongjiang Province(Grant No.LH2023H043).
文摘Background:Small extracellular vesicles(sEVs)mediate intercellular commu-nication that contributes to hepatocellular carcinoma(HCC)progression via multifaceted pathways.The success of cell entry determines the effect of sEV on recipient cells.Here,we aimed to delineate the mechanisms underlying the uptake of sEV in HCC.Abbreviations:AF,Alexa Fluor;ANOVA,analysis of variance;ATP9A,ATPase Phospholipid Transporting 9A;BCECF-AM,2’,7’-bis-(2-barboxyethyl)-5-(and-6)-carboxyfluorescein,acetoxymethyl ester;BSA,bovine serum albumin;CCMR,Centre for Comparative Medicine Research;CRISPR,clustered regularly interspaced short palindromic repeats;CTL,ctrl;CXCR4,C-X-C Chemokine Receptor Type 4;DAPI,4′,6-diamidino-2-phenylindole;DFS,disease-free survival;DMEM,Dulbecco’s Modified Eagle Medium;DMSO,dimethyl sulfoxide;Dox,doxycycline;EEA1,early endosome antigen 1;EIPA,5-(N-ethyl-N-isopropyl)-amiloride;FBS,fetal bovine serum;FITC,fluorescein isothiocyanate;GAPDH,glyceraldehyde-3-phosphate dehydrogenase;GM130,Golgi matrix protein 130;HCC,hepatocellular carcinoma;HEPES,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;HPRT1,hypoxanthine phosphoribosyltransferase 1;H-score,histoscore;IAA,indole-3-acetic acid;KD,knockdown;KO,knockout;mAID,mini-auxin-inducible degron;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;NHE7,Na(+)/H(+)exchanger 7;ns,non-significant;OD,optical density;OS,overall survival;PBS,phosphate-buffered saline;PCR,polymerase chain reaction;pHe,endosomal pH;pHi,intracellular pH;PKH67,Paul Karl Horan 67;Rab21,Ras-associated binding protein 21;RIPA,radioimmunoprecipitation assay;SAM,synergistic activation mediator;SEMs,standard error of the means;sEVs,small extracellular vesicles;sgRNA,single-guide RNA;shRNA,short-hairpin RNA;SLC9,solute carrier gene 9;SLiCE,Seamless Ligation Cloning Extract;TCGA,The Cancer Genome Atlas;TCL,total cell lysates;TGN,trans-Golgi network;TMA,tissue microarray;TMR,tetramethyl rhodamine;TSG101,tumor susceptibility gene 101.Yue Yao and Yi Xu contributed equally to this work.Methods:Macropinocytosis was examined by the ability of cells to internalize dextran and sEV.Macropinocytosis was analyzed in Na(+)/H(+)exchanger 7(NHE7)-knockdown and-overexpressing cells.The properties of cells were stud-ied using functional assays.pH biosensor was used to evaluate the intracellular and endosomal pH.The expression of NHE7 in patients’liver tissues was exam-ined by immunofluorescent staining.Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7.Results:The data revealed that macropinocytosis controlled the internaliza-tion of sEVs and their oncogenic effect on recipient cells.It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non-metastatic HCC cells.Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride(EIPA)limited the entry of sEVs and compromised cell aggressiveness.Mechanistically,we delineated that high level of NHE7,a sodium-hydrogen exchanger,alkalized intracellular pH and acidized endosomal pH,leading to the maturation of macropinosomes.Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis.Clinically,NHE7 expression was upregulated and linked to dismal prognosis of HCC.Conclusions:This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells.Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.
文摘Cancer is the second leading cause of death in the US.Current major treatments for cancer management include surgery,cytotoxic chemotherapy,targeted therapy,radiation therapy,endocrine therapy and immunotherapy.Despite the endeavors and achievements made in treating cancers during the past decades,resistance to classical chemotherapeutic agents and/or novel targeted drugs continues to be a major problem in cancer therapies.Drug resistance,either existing before treatment(intrinsic)or generated after therapy(acquired),is responsible for most relapses of cancer,one of the major causes of death of the disease.Heterogeneity among patients and tumors,and the versatility of cancer to circumvent therapies make drug resistance more challenging to deal with.Better understanding the mechanisms of drug resistance is required to provide guidance to future cancer treatment and achieve better outcomes.In this review,intrinsic and acquired resistance will be discussed.In addition,new discoveries in mechanisms of drug resistance will be reviewed.Particularly,we will highlight roles of ATP in drug resistance by discussing recent findings of exceptionally high levels of intratumoral extracellular ATP as well as intracellular ATP internalized from extracellular environment.The complexity of drug resistance development suggests that combinational and personalized therapies,which should take ATP into consideration,might provide better strategies and improved efficacy for fighting drug resistance in cancer.
基金This work was supported in part by a NIH grant R15 CA242177-01 to Chen X.
文摘The cancer stem cell(CSC)state and epithelial-mesenchymal transition(EMT)activation are tightly interconnected.Cancer cells that acquire the EMT/CSC phenotype are equipped with adaptive metabolic changes to maintain low reactive oxygen species levels and stemness,enhanced drug transporters,anti-apoptotic machinery and DNA repair system.Factors present in the tumor microenvironment such as hypoxia and the communication with non-cancer stromal cells also promote cancer cells to enter the EMT/CSC state and display related resistance.ATP,particularly the high levels of intratumoral extracellular ATP functioning through both signaling pathways and ATP internalization,induces and regulates EMT and CSC.The three of them work together to enhance drug resistance.New findings in each of these factors will help us explore deeper into mechanisms of drug resistance and suggest new resistance-associated markers and therapeutic targets.
