CD24-dependent MAPK Pathway Activation Is Required For Colorectal Cancer Cell Proliferation

ObjectiveCD24 is a glycosylphosphatidylinositol(GPI)-anchored membrane protein with several potential O-or N-linked glycosylation sites.CD24 was initially described to be up-regulated in breast cancer and later confirmed to be overexpressed in human cancers such as prostate cancer and cholangiocarcinoma,suggesting that this gene may play a key role in tumorigenesis.Its expression is usually associated with poor prognosis.In colorectal cancer(CRC),CD24 was commonly up-regulated and cytoplasmic CD24 expression remained a significant independent prognostic marker. Down-regulation of CD24 expression induced growth inhibition in CRC,and this effect was augmented in combination with classic chemotherapies.Taken together, CD24 does play an important role in tumor development and progression.However, the underlying mechanisms of how CD24 contributes to the progression of malignant tumor remain largely unclear.In our present study,we determined the role of CD24 in the proliferation of CRC cells and attempted to elucidate the potential mechanism in this process.Materials and methodsReagents and meterials SW1116,SW480,SW620,HCT8,LoVo and Colo205 colorectal cell lines, MEK1/2 inhibitor U0126,p38 MAPK inhibitor SB203580,G418,ERK1/2(137F5) antibody,p38 MAPK antibody,SAPK/JNK antibody,phospho-SAPK/JNK (Thr183/Tyr185) antibody and phospho-p38 MAPK antibody,CD24(C-20) polyclonal antibody,Raf-1(C-12) antibody,p-Raf-1(Ser 338) antibody,p-ERK(E-4) antibody,formalin-fixed and paraffin-embedded tissue samples from 106 primary human CRCs.Construction of the CD24 expression plasmid and stable transfectionA full-length human CD24 cDNA corresponding to bases 111 to 353 of Genbank accession(no.NM₀13230) was amplified by PCR from normal intestinal cDNA library.The primer pairs for CD24 were 5'-TAGGTACCACTATGGGCAGAGCAA TGG-3'(F) and 5'-CCGGAATTCCGTTAAGAGTAGAGATGC-3'(R).The PCR product was cloned into the pcDNA3.1(+) vector(Invitrogen) to create the pcDNA3.1(+)-CD24 plasmid.The orientation of the insert and the sequence of cDNA were verified by sequencing.Cultured SW480 cells were transfected with the recombinant plasmids and the empty vector using the lipofectamine 2000 reagent (Invitrogen,Life Technologies) according to the manufacturer's instructions. Transfectants were selected in medium containing 600μg/ml G418.After selection and isolation of stably transfected clones,the clones were analyzed for CD24 expression using RT-PCR and flow cytometry.Reverse transcription-polymerase chain reaction(RT-PCR)Total RNA was extracted from cells using Trizol(Invitrogen).RNA samples(3μg) were subjected to reverse transcription using a RevertAid^TM First Strand cDNA Synthesis Kit(Fermentas).The PCR was initiated by 5 min incubation at 94℃,ended after a 10 min extension at 72℃,36 cycles for denaturation at 94℃for 30s, annealing at 55℃,and extension 72℃for 1 min using PCR kit(SBS Genetech Co. Ltd.,Beijing,China). Flow cytometryCells were harvested and resuspended in PBS/5%FBS in a density of 2×10⁵/ml. CD24 monoclonal antibodies(Ab-1,Neomarker) were applied at 5μg/ml(1:100). After incubation for 60 min on ice,the cells were washed with PBS/5%FBS twice and incubated for a further 40 min with fluorescein isothiocyanate(FITC)-conjugated secondary antibody on ice.The cells were washed with PBS/5%FBS twice again and analyzed by a Flow Cytometer.As negative controls,cells were stained with both isotype-matched control antibodies.Western blottingCells lysed with ice-cold lysis buffer and PMSF.Protein samples(30μg) were separated by 12%acrylamide gel using a Bio-Rad Mini-Proteanâ…¢system(100V for 3h).Proteins were transferred to PVDF membranes in 2.0mA/cm² for 2h in transfer buffer.Following transferring,the membranes were blocked for 1 h at room temperature with 5%skimmed milk powder in 0.05%TBS-T.Blots were then incubated at room temperature with primary antibodies in 2%BSA dissolved in TBS-T(1:500 dilution).Primary antibodies were removed and the blots were extensively washed with TBS-T for three times.Blots were then incubated for 1 h at room temperature with the proper horseradish peroxidase(HRP)-conjugated secondary antibodies(1:5000 dilution).Then,blots were extensively washed as above and detected by using the Enhanced ECL System.Actin was also detected as internal control.All experiments were repeated twice and blots were analyzed using quantity one 1-D analysis software(Bio-Rad).Proliferation assaysCell growth was evaluated using a WST-8 assay(Beyotime Institute Biotech, Shanghai,China).The cells were seeded into the 96-well culture plates at a density of 2000 cells,and the medium was replaced with 100μl of fresh medium and 10μl of WST-8 reagent in each well at 24,48,72 and 96h,respetively.After the plate was returned to incubator for 1-2h,the absorbency of A450 was measured using an enzyme-linked immunosorbent assay plate reader.Immunohistochemical analysisRepresentive 4μM sections were prepared,deparaffinized,and rehydrated in xylene, graded alcohol,and water.Antigen retrieval was enhanced by microwaving the slides in 0.01M citrate buffer(pH 6.0) for a total of 10min.Endogenous peroxidase activity was quenched by treatment with 3%hydrogen peroxide for 30 min,followed by incubation with goat or rabbit serum for 30min.The primary antibodies were applied and incubated overnight at 4℃.Subsequent steps utilized the UltraSensitive^TM SP kit (Maxin-bio,Fuzhou,China) according to the manufacture's instructions.Peroxidase activity was visualized with diaminobenzidine and slides were counterstained with haematoxylin,dehydrated and mounted.For negative controls,blocking solution was added instead of the primary antibody.All slides were assessed by two pathologists independently and blinded to the case.Statistical analysis.Data were compiled with the software package SPSS 13.0.Factorial analysis ANOVA was employed to determine whether the results of proliferation assays had statistical significance.The non-parametric Kruskal-Wallis or Mann-Whitney tests were applied to test the significance of differences among groups of clinicopathological parameters,whereas correlations between expression of proteins were evaluated by the Spearman rank-order correlation coefficient.The level of significance was defined as P＜0.05.ResultExpression of CD24 was cell type-dependent in colorectal cancer cell linesFlow cytometry was used to detect CD24 expression at protein level in the colorectal cancer cell lines.Of six cell lines,SW1116 maintained moderate expression of CD24,SW620 and Colo205 contained higher expression,but expression was scarcely observed in SW480,HCT8,and LoVo cell lines.To investigate CD24 mRNA at the level of transcription,a standard RT-PCR experiment described in methods was performed.The result was consistent with Flow cytometric analysis.These results showed Expression of CD24 was cell type-dependent in colorectal cancer cell lines.Overexpression of CD24 induced proliferation in vitroCD24 recombinant plasmid,pcDNA3.1(+)-CD24,was constructed using expression vector pcDNA3.1(+),and was then stably transfected into the SW480 cells. Two clones,named as SW480^CD24+1 and SW480^CD24+4,exhibited dramatically increased expression of CD24 at mRNA transcripts level compared with the parental and vector control cells.Expression of CD24 at protein level in the clones was confirmed by Flow cytometric analysis.The growth ability of SW480^CD24+1 cells in vitro was assessed by WST-8 assay,and the result showed that these cells proliferated more quickly than SW480^vec cells,which were transfected with the vector alone,and the parental SW480 cells.Overexpression of CD24 induced activation of ERK1/2,Raf-1 and p38 MAPK,but it had no effect on JNK1/2To elucidate the potiential mechanisms of CD24-induced proliferation,we examined the activation of MAPK pathways.The rusults showed that ERK1/2 and p38 MAPK were activated in the SW480^CD24+1 and SW480^CD24+4 cells compared with SW480^vec cells and the parental SW480 cells.However,no activation of JNK1/2 was observed.To determine the role of CD24 in Raf-ERK pathway,we further detected the activity of Raf-1,the upstream ERK1/2 activator,and our data showed that Raf-1 protein levels and kinase activity were elevated after the SW480 cells overexpressed with CD24. Suppressing the activation of ERK1/2 and p38 MAPK abrogated CD24-induced proliferation in the SW480^CD24+1 cellsTo further evaluated the contribution of the ERK1/2 and p38 MAPK in the CD24-induced proliferation,we inhibited their activations using specific pharmacological inhibitors.The starved SW480^CD24+1 cells were treated either with inhibitor of the upstream ERK1/2 activator MEK1/2,U0126,or the p38 MAPK inhibitor SB203580.DMSO was used as control.The cells were treated separately with 5,10 and 20μM inhibitors and observed at 24 and 72h after treatment followed by WST-8 assay for cell viability.Meantime,parallel cultures were analyzed for total phosphor-tyrosine content by western blotting.Our data showed that the activation of ERK1/2 and p38 MAPK were inhibited,and there were significant decreases of cell viability in the cells treated with different concentration U0126 and SB203580 after 72h.Cytoplasmic CD24 expression increased with tumor stage and gradeCD24 showed a membranous and a cytoplasmic immunostainings in colorectal tissue.For cytoplasmic CD24 staining,89.6%cases showed positive and 45.3%cases were moderately or strongly positive,while its expression in adjacent normal mucosa was either absent or barely detectable on cell membrane.There was a significantly positive correlation between cytoplasmic CD24 expression and tumor stage and grade, while no correlation was observed between cytoplasmic CD24 expression and age, gender,localization,and tumor grade.For CD24 membranous staining,only 34.9% was classified as positive.CD24 membranous expression showed a significantly negative correlation with tumor grade,but no association with other clinicopathologic parameters.Activations of the ERK1/2 and p38 MAPK strongly correlated with cytoplasmic CD24 expression in human CRC tissue To investigate the correlations among ERK1/2,p38 MAPK and CD24 in vivo, immmunohistochemical analysis was performed.Immunostaining of p-ERK1/2 was cytoplasmic,while p-p38 MAPK revealed strong cytoplasmic and partly nuclear staining.Cytoplasmic expression of p-ERK1/2 and p-p38 MAPK was observed in 84.0%and in 78.3%cases,respectively.In adjacent normal mucosa,the staining of both molecules was weak or absent.Cytoplasmic expression of both molecules had no significant correlation with tumor stage and grade.Nuclear p-p38 MAPK staining were classified as 24.5%,which had no significant correlation and trend with any clinicopathological parameters.Expression of p-ERK1/2 correlated with cytoplasmic p-p38 MAPK expression in CRCs(r=0.780,P＜0.001).More importantly,our data showed that strong correlations were found between cytoplasmic CD24 expression and immounostainings of ERK1/2 or p38 MAPK in CRCs(r=0.571,P＜0.001; r=0.528,P＜0.001;respectiveely),suggesting that activations of the ERK1/2 and p38 MAPK strongly correlated with cytoplasmic CD24 expression in human CRC tissue.ConclusionIn the present study,we evaluated the role of CD24 in the proliferation of CRC cells and the potential mechanisms.Our data showed that increased CD24 expression was cell type-dependent and occurred in 89.6%of human CRCs,and cytoplasmic CD24 expression increased with tumor stage and grade.CD24 could induced proliferation of CRC cells in vitro.In addition,we showed,for the first time, CD24-induced proliferation was dependent on activation of the ERK1/2 and p38 MAPK in CRC.Moreover,activation of ERK1/2 and p38 MAPK also appears to be strongly correlated with CD24 expression in CRC tissue.Our results suggest that CD24 plays a key role in the proliferation of CRC.Furthermore,CD24-dependent MAPK pathway activation is required for CRC cell proliferation.Our finding provided a novel interpretation for the mechanism of CD24-induced proliferation in CRC.The linkage of CD24 and MAPK pathway may unravel a novel mechanism in regulation of proliferation of CRCs.