分子生物学知识拓展 (15).pdf

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1、Crosstalk between colon cells and macrophages increases ST6GALNAC1 and MUC1-sTn expression in ulcerative colitis and colitisassociated colon cancerMichael Kvorjak1,Yasmine Ahmed2,Michelle L.Miller1,Raahul Sriram1,Claudia Coronello3,Jana G.Hashash4,Douglas J.Hartman5,Cheryl A.Telmer6,Natasa Miskov-Zi

2、vanov2,Olivera J.Finn1,Sandra Cascio1,3,71Department of Immunology,University of Pittsburgh,Pittsburgh,PA,USA,152612Department of Electrical and Computer Engineering,University of Pittsburgh,PA USA,152133Fondazione Ri.Med,via Bandiera 11,Palermo,Italy,901334Department of Gastroenterology,University

3、of Pittsburgh Medical Center,PA,USA,152135Department of Pathology University of Pittsburgh Medical Center,PA,USA,152136Molecular Biosensor and Imaging Center,Carnegie Mellon University,Pittsburgh,PA,USA,152137Department of Obstetrics,Gynecology,&Reproductive Sciences,University of Pittsburgh,Pittsbu

4、rgh,PA,USA,15213AbstractPatients with ulcerative colitis(UC)have an increased risk of developing colitis-associated colon cancer(CACC).Changes in glycosylation of the oncoprotein MUC1 commonly occur in chronic inflammation,including UC,and this abnormally glycosylated MUC1 promotes cancer developmen

5、t and progression.It is not known what causes changes in glycosylation of MUC1.Gene expression profiling of myeloid cells in inflamed and malignant colon tissues showed increased expression levels of inflammatory macrophage-associated cytokines compared to normal tissues.We analyzed the involvement

6、of macrophage-associated cytokines in the induction of aberrant MUC1 glycoforms.A co-culture system was used to examine the effects of M1 and M2 macrophages on glycosylation-related enzymes in colon cancer cells.M2-like macrophages induced the expression of the glycosyltransferase ST6GALNAC1,an enzy

7、me that adds sialic acid to O-linked GalNAc residues,promoting the formation of tumor-associated sialyl-Tn(sTn)O-glycans.Immunostaining of UC and CACC tissue samples confirmed the elevated number of M2-like macrophages as well as high expression of ST6GALNAC1 and the altered MUC1-sTn glycoform on co

8、lon cells.Cytokine arrays and blocking antibody experiments indicated that the macrophage-dependent ST6GALNAC1 activation was mediated by IL-13 and CCL17.We demonstrated that IL-13 promoted phosphorylation of STAT6 to activate transcription of ST6GALNAC1.A computational model of signaling pathways w

9、as assembled and used to test Corresponding Authors:Sandra Cascio,phone:412-641-1801,sac131pitt.edu.Conflict of Interest:The authors declare no potential conflicts of interest.HHS Public AccessAuthor manuscriptCancer Immunol Res.Author manuscript;available in PMC 2020 August 01.Published in final ed

10、ited form as:Cancer Immunol Res.2020 February;8(2):167178.doi:10.1158/2326-6066.CIR-19-0514.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptIL-13 inhibition as a possible therapy.Our findings revealed a novel cellular cross-talk between colon cells and macrophages within the infl

11、amed and malignant colon that contributes to the pathogenesis of UC and CACC.KeywordsIL-13;CCL17;ST6GALNAC1;MUC1-sTn;MacrophagesIntroductionApproximately 1.4 and 2.2 million people are affected with inflammatory bowel diseases(IBD)in the US and Europe,respectively(1,2).IBD,including ulcerative colit

12、is(UC)and Crohns disease(CD),are chronic intestinal inflammation disorders that affect the gastrointestinal tract;namely the colon in UC and anywhere in the gastrointestinal tract in CD.Patients with UC often present with bloody diarrhea while those with CD tend to have weight loss and abdominal pai

13、n.Histologically,there is a disruption of crypt architecture and inflammatory cell infiltration(3).Patients with UC and CD colitis have an increased risk of developing colitis-associated colon cancer(CACC).This risk is proportional to the duration and extent of disease,with a cumulative incidence as

14、 high as 30%in individuals with longstanding UC and widespread colonic involvement(4).During tumor initiation and progression,epithelial cells acquire new capabilities that allow them to become tumorigenic and ultimately malignant.Glycosylation changes are one of the most common post-translational m

15、odifications that occur during malignant transformation(5).Among all glycoproteins,mucin-type O-glycans are frequently altered during UC and progression to colon cancer(6-8).The mucin MUC1,is expressed at low levels on the apical surface of normal epithelial cells and is heavily glycosylated in the

16、tandem repeat region of the extracellular domain rich in proline,serine,and threonine residues(9,10).During colonic malignant transformation,MUC1 is over-expressed and it loses its apical polarity and displays an altered glycosylation profile,becoming predominantly hypoglycosylated(6-8).Aberrant and

17、 tumor-associated mucin glycoforms expose long stretches of naked peptide backbone decorated with sialyl-LewisX(SLeX),prematurely terminated monosaccharides(Tn antigens)or disaccharides(T antigens)and their sialylated forms sTn and sT to the immune system(9).We demonstrated that the presence of huma

18、n MUC1 exacerbates chronic inflammation and induces tumorigenesis in the azoxymethane/dextran sodium sulfate(AOM/DSS)mouse model of CACC(11).Chronic inflammation promotes the expression of aberrant MUC1 glycosylation in colon epithelial cells whereas no or very low expression of hypoglycosylated MUC

19、1 is detected in colon tissues of healthy mice(11).Altered O-glycans on mucins can result either from mutations in the cell chaperone COSMC or changes in ST6GALNAC1,enzymes involved in the biosynthesis of Tn/sTn glycans(12).An inflammatory microenvironment can also induce changes in the glycan compo

20、sition of cells via modulation of glycosyltransferases(13,14).Tumor-associated macrophages(TAMs),by secreting inflammatory cytokines and chemokines,play a key role in tumor initiation,promotion,invasion and metastasis.During Kvorjak et al.Page 2Cancer Immunol Res.Author manuscript;available in PMC 2

21、020 August 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptintestinal inflammation,such as UC,macrophages acquire a typical inflammatory phenotype,present antigens,undergo phagocytic activities and secrete inflammatory cytokines and chemokines thus contributing to UC pathogene

22、sis and progression to CACC(15-17).Differentiated macrophages are classified into two subpopulations:the classically activated macrophages(M1 phenotype)and the alternatively activated macrophages(M2 phenotype)(18).In vitro studies have shown that upon stimulation with inflammatory factors,interferon

23、-(IFN-)and lipopolysaccharides(LPSs),macrophages polarize to a M1 state and secrete pro-inflammatory cytokines IL-6,IL-1,and TNF-.By contrast,in response to anti-inflammatory signals IL-4 and IL-13,macrophages polarize to a M2 state,secrete tumor promoting cytokines such as arginase(Arg)-1,and expre

24、ss the mannose receptor(MR),IL-10,and Fizz1(18).Within tumors,both pro-and anti-inflammatory signals are simultaneously present,resulting in a more complex spectrum of macrophage polarization states(19).Here,we showed that markers associated with both types of macrophages were highly expressed in UC

25、 and CACC tissues.The expression of TAM markers,glycosylation-associated enzymes and tumor-associated MUC1 glycoforms were assessed in human tissues and in a co-culture model system.Computational modeling was carried out to evaluate the involvement of macrophage-induced cytokines in aberrant glycosy

26、lation,in particular in the regulation of glycosyltransferase ST6GALNAC1.A novel regulatory mechanism was discovered involving macrophage-derived CCL17 and macrophage-enhanced IL-13 in the induction of ST6GALNAC1 expression in colon cancer cells.Chromatin immunoprecipitation assays of human UC and C

27、ACC samples indicated that IL-13 via STAT6 directly promoted the transcriptional activity of the ST6GALNAC1 gene.Thus,increased expression of ST6GALNAC1 resulted in the production of the MUC1-sTn glycoform,which is associated with colonic inflammation and cancer.A computational model demonstrated th

28、e signaling,cross-talk and dynamics involved in regulating gene expression,and identified a potential therapeutic intervention.Material and MethodsCell culture and Reagents.SW480(ATCC CCL-228)and HT-29(ATCC HTB-38)cell lines were purchased from American Type Culture Collection in 2016 and frozen upo

29、n initial expansion(5 passages).Cells were cultured for a maximum of 15 passages in RPMI-1640(Royal Park Memorial Institute Medium,Cellgro,Mediatech,Inc.)medium supplemented with 10%heat-inactivated fetal bovine serum,100 units/mL penicillin,100 g/mL streptomycin and 2 mmol/L L-glutamine.Both cell l

30、ines were regularly tested for Mycoplasma contamination.Cells were not reauthenticated.For specific experiments IL-13 and CCL17(R&D Systems)were used at 20,50 100 ng/mL for 24h.IL-13 neutralizing antibody(JES10-5A2,Thermo Scientific)and CCL17(AF364,R&D Systems)were used at 1:200 dilution.Kvorjak et

31、al.Page 3Cancer Immunol Res.Author manuscript;available in PMC 2020 August 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptPatients and tissue samples.Archived paraffin sections of colonic biopsies of UC patients in remission(non-inflamed),with active disease,and those with co

32、litis associated colon cancer were selected and collected in the Department of Gastroenterology,University of Pittsburgh.The study was approved by the institutional review board of the University of Pittsburgh(PRO16090194).Chart review to identify patients who had biopsy samples during colonoscopy a

33、nd/or surgery was performed.In order to perform IHC staining,a waiver of consent and HIPAA was requested.Fresh colon tissues were obtained under the approved IRB PRO19070174.Patients had previously provided a signed informed consent at the time their tissue was collected.Tissues were processed and s

34、tored in under standard operating procedures of the Pittsburgh Biospecimen Core.Plasmids and Transfection.The cDNA for ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1(ST6GALNAC1)was subcloned into the pcDNA3 plasmid vector(Invitrogen)(20).Empty vector was used as the negative control.Trans

35、fections were performed with Lipofectamine 3000(cat.L3000008,Thermo Fisher Scientific,)according to manufacturers instruction.Antibodies.The following primary antibodies were used:anti-p65(sc-8008),anti-pp65(sc-166748)anti-IB(sc-1643),anti-MUC1 VU-4H5(sc-7313,Santa Cruz),anti-ST6GALNAC1(PA5-31200 an

36、d 15363-1-AP),anti-IL-13(AHC0132),anti-actin(MA5-11869,Thermo Fisher Scientific),anti-MUC1 5E5(TAB-418MZ,Creative Biolabs),anti-CD163(NBP2-36494,Novus Biologicals),anti-CCL17(ab182793),anti-sialyl Tn,clone sTn 219(ab115957),anti-CD68(ab955),anti-CD86(ab53004,Abcam),anti-AKT(C67E7,#4691),anti-p-AKT(D

37、9E,#4060),p-STAT1(D4A7,#7649),p-STAT3(D3A7,#9145),p-STAT6(D8S9Y,#56554,Cell Signaling).Western Blotting.Total proteins from human colon tissues and cancer cell lines were extracted using RIPA buffer(150 mM NaCl,0.5 sodium deoxycholate,0.1%SDS,1%NP-40 and 50 mM Tris-HCl)with commercial protease inhib

38、itors(Complete Protease Inhibitor Cocktail,Roche)and phosphatase inhibitors(Phosphatase Inhibitor Cocktail II,Sigma-Aldrich).Proteins were separated by SDS-PAGE and transferred to PVDF membranes.Blots were incubated with the primary antibodies indicated above.Immunoblots were developed with m-IgG BP

39、-HRP(sc-516102)or mouse anti-rabbit IgG-HRP(sc-2357,Santa Cruz)and chemiluminescence reagents(SuperSignal West Pico Substrate,cat.#34580,Pierce).Intensity of signals was determined by densitometric scanning(Kodak,Image Station 4000MM).Densitometry of western immunoblotting results was performed usin

40、g Image J software.Kvorjak et al.Page 4Cancer Immunol Res.Author manuscript;available in PMC 2020 August 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptELISA.IL-13 concentrations in the conditioned medium of HT-29 or SW480 cancer cells co-cultured with M1 or M2 macrophages we

41、re measured using a human ELISA kit(DY213,BD Biosciences)following manufacturers protocol.Mono-cultures of HT-29 or SW480,M1 and M2 cells were used as control.IL-13 concentrations detected were within the range of the standard curve.All points were done in triplicate,and the experiments were repeate

42、d three times.Samples were read in a microplate reader(Infinite 200 Pro,Tekan).Immunofluorescence confocal microscopy.Cultured cells were fixed in 4%paraformaldehyde for 20 min and permeabilized in 0.5%Triton-X100 for 20 min.The fixed tissues or cells were incubated with primary Abs for 1h at RT fol

43、lowed by secondary anti-mouse Alexa-488 or Cy3 antibody(Invitrogen Life Technologies)for 1h at RT.Abs were diluted in 1%bovine serum albumin(BSA).Nuclei were stained with mounting medium containing DAPI(VectorLab).Confocal images were captured on an Olympus Fluoview 1000 confocal microscope.Immunohi

44、stochemistry(IHC).Slides were deparaffinized by baking overnight at 59C.Endogenous peroxidase activity was eliminated by treatment with 30%H2O2 for 15 min at room temperature.Antigen retrieval was performed by microwave heating in 0.1%citrate buffer for 10 min at 850V.Nonspecific binding sites were

45、blocked with 2%BSA.Reaction with anti-CD163,anti-CD68,anti-CD86,anti-sTn,anti-ST6GALNAC1(listed above)was for 16h at 4C.Staining was performed by the avidin-biotin-peroxidase complex method with a commercial kit(PK4000,Vectastain ABC HRP kit;Vector Laboratories)according to the manufacturers protoco

46、l.Positive signals were visualized by a DAB Substrate Kit(cat.#550880,BD Pharmingen)according to the manufacturers protocol.The total inflammation score for each sample was determined as previously described 8.For double staining IHC,ImmPRESS duet staining HRP/AP polymer kits,anti-rabbit IgG-brown a

47、nd anti-mouse IgG red was used(MP-7714,Vector Laboratories)according the manufacturers protocol.Histology sections were observed using an Olympus BX40 microscope.Images were acquired using a Leica DFC420 camera and Leica Application Suite version 2.7.1 R1.Migration and invasion assay.Migration studi

48、es were conducted using 24-well Transwells,8 m pore size(Costar Transwell;Corning Inc.).In each well,4 X 104 HT-29 or SW480 cells were plated into the upper chamber in serum free medium whereas 700 l of 5%FBS containing medium was placed in the lower chamber.After 16 h,cells remaining in the upper c

49、hamber were removed using cotton swabs.Cells adhering to the lower surface were fixed with 4%paraformaldehyde for 20 min and stained with Christal Violet 1%.Cells were then counted in 5 different fields using a microscope at 40X magnification.Peripheral blood monocyte isolation and macrophage differ

50、entiation.Peripheral blood mononuclear cells(PBMCs)were freshly isolated by density gradient centrifugation using Ficoll Paque Plus(Sigma-Aldrich)for 50 min at 400g.At least 20 de-Kvorjak et al.Page 5Cancer Immunol Res.Author manuscript;available in PMC 2020 August 01.Author ManuscriptAuthor Manuscr