| PurposeTo investigate the correlation of JAK2and CD95expression in colorectal cancertissues and cells; to investigate the impact of JAK2and CD95on cellproliferation, apoptosis, and epithelial-mesenchymal transition(EMT) incolorectal cancer cells, thus to confirm the relationship between JAK2-mediatedinflammatory pathway and immune function in colorectal cancer cells on bothcellular and molecular basis.Method1. Bioinformatic analysis1.1Using R2: Genomics Analysis and Visualization Platform, thecorrelation between the mRNA level of CD95and JAK2and survival in8separate colorectal cancer datasets were analyzed via Kaplan-Meier curve.1.2CD95associated proteins and their functions were analyzed usingSTRING online software. Overlap genes were identified using Gvenn toolanalysis and their correlation with CD95was ordered using online metasoftware (r value and p value).1.3CD95associated genes which were classified according to theirfunctions were showed in Gene heatmap form according to CD95expression inR2: Genomics Analysis and Visualization Platform.2. Preparation of colorectal cancer tissue slices and cell linesColorectal cancer tissue slices were obtained from resected colorectalcancer tissues during Oct.2011~May.2012in Gastrointestinal Surgery of University Medical Center Utrecht after formaldehyde fixation and paraffinembedding; cell lines were obtained from fresh cancer tissues after longtermexpansion.3. Correlation analysis between CD95and JAK2The expression level of CD95and JAK2in colorectal cancer (CRC) tissueswas identified by immunohistochemistry (IHC) staining, and expression level ofCD95and JAK2in CRC cell lines were identified by western blot. Thencorrelation between CD95and JAK2was analyzed.4. Inhibition of JAK2/STAT3in CRC cellsJAK2specific inhibitors AZD1480and CEP33779were added in differentdoses (0.5μM~5μM) to culturing system to block JAK2activation. Afterinhibition, JAK2, STAT3and phosphorylated STAT3were probed in westernblot, compared with DMSO treated as normal control and IL-6(10ng/ml，30min)treated as positive control.5. Clone formation assaySingle cell was prepared by disaggregation of CRC cell speroids andseeded in12-well plate in a density of1000cells/100μl Matrigel. AfterMatrigel coagulates, culture medium supplemented with or without JAK2inhibition was added2ml/well into each well and refreshed2days a time. Clonenumbers were counted after12days.6. Apoptosis test by flow cytometryPropidium Iodide (PI) staining was applied to the cells with or withoutJAK2inhibition to identify apoptosis.7. Immunofluorescence and Real time fluorescent quantitative PCR(RT-FQ-PCR)EMT associated proteins were tested via immunofluorescence afterJAK2/STAT3signal inhibition by AZD1480or CEP33779. EMT commonmarkers HIF-1, ZEB-1, ZEB-2, FABP, Snail-1, Snail-2and vimentin were quantified by RT-FQ-PCR.8. Cytokine detection under FasL stimulationIn culturing medium supplemented by FasL in a concentration of20ng/mltreated for12hours, cytokines in supernatant were detected after dialysis andconcentration.9. Constructing YFP-CD95or YFP expressing cell linesContruct pWPT-CD95-YFP and pWPT-YFP (as control) were prepared byJamila.293T cells were transfected with lentiviral packaging constructs andpWPT-CD95-YFP or pWPT-YFP with FuGene. Virus (cell supernatant) washarvested after24-48h and filtered through0.2um filter. CRC29and L145weredissociated6-16h before and transduced with the (concentrated orunconcentrated) virus supernatant for24h using plybrene. Cells were sorted byflowcytometry (BD Company) according to YFP expression. Thus, cellsexpressing CD95-YFP or YFP were sorted out.Results1. Correlation between CD95and JAK2in CRCs（1）Bioinformatical study shows high correlation between CD95, JAK2and prognosisIn8separate CRC cohorts including totally1293patients, CD95and JAK2are median-high expressed and no significant difference among these cohorts.The expression levels of CD95and JAK2are both correlated with patients’survival. High expression correlates with poor survival.（2）Fas and JAK2are co-expressed and functionally correlated in CRCSTRING analysis suggests that CD95is associated with immune response,antigen processing response and inflammatory response. Furthermore, Gvennanalysis shows that CD95associated genes are highly interconnected with JAK2and JAK2is the top in CD95associated genes. The Fas-associated genes are involved in inflammatory response, JAK2/STAT3singnal pathway,epithelial-mesenchymal transition (EMT). Immunohistochemistry for tissueslices of10colorectal cancers showed high expression of Fas accompanied byhigh expression of JAK2, and vice versa. Fas and JAK2are co-expressed andhave good correlation.（3）Fas and JAK2have correlation in CRC cell linesCRC cell lines that were originated from colon or rectum include CRC26,CRC29, CR9, CR16, CRC48, CRC47, while those resected from CRC livermetastasis include L145, L167, L169. With regard to JAK2expression level,CRC29, CR9, CR16are highest, followed by L169, CRC47, CRC48, L167,L145, CRC26. With regard to Fas expression level, the order from high to low isCRC29, L145, CRC47, CR16, CRC48, CRC26, L169, CR9, L167. Theexpression of JAK2and Fas has a certain correlation (r2=0.5087，p=0.0470),while better correlation is found in those cell lines originated from CRC(r2=0.7360，p=0.0289).2. The impact of JAK2on CRC cell proliferation, apoptosis and EMT（1）JAK2plays a positive role in CRC cells proliferationUnder JAK2inhibition, there is a significant reduction in number of tumorclones in a dose dependent manner, compared with DMSO treated group inMatrigel culturing system. Besides, the size of tumor clones under JAK2inhibition is much smaller than those under DMSO treatment.（2）JAK2activation benefits CRC cells proliferationCultured in Matrigel, CRC cells can have much higher proliferation onceappropriate dose of IL-6is added, compared with control group without IL-6.Concentration of IL-6doesn’t seem to effect proliferation.（3）JAK2inhibition doesn’t seem to induce apoptosisCultured in floating medium, DNA fragment was determined using propidium iodide (PI) staining after JAK2inhibition for2days. Compared withcontrol which was treated by equivalent dose of DMSO, there’s no significantapoptosis (p>0.05).（4）JAK2inhibition facilitates FasLinduced apoptosisCultured in floating medium, CRC cells were treated by FasL with orwithout JAK2inhibition (AZD1480and CEP33779). In FasL treated groupapoptosis is33.52%, and AZD1480followed by FasL goes up to41.20%,45.19%. In FasL treated group apoptosis is38.76%, and CEP33779followed byFasL goes up to62.34%,62.65%.（5）Level changes of EMT related proteins and transcriptors after JAK2inhibition.①Cultured in floating medium,2hours after CEP33779treatment to blockJAK2activation, Caveolin-1and E-cadherin are found to increase by westernblot, and co-focal microscopy detects increased expression signals of Caveolin-1and E-cadherin on cell-cell junctions.②Cultured in floating medium, JAK2was efficiently inhibited in CRC cells by CEP33779while in control group CRCcells were treated by same dose of DMSO. After2hours each group wastransferred into another culture system with constant IL-6supplement bydifferentiated CRC cells attached to the bottom.24hours after co-culture, JAK2inhibited CRC cells have less mRNA production for HIF-1, ZEB-1, ZEB-2,Snail-1，Snail-2，and higer vimentin expression.3. Specific inhibition of JAK2affects Fas isotype on CRC cellmembrane（1）FasLstimulates secretion of cytokines in CRC cells.Treated by a certain dose of FasL, CRC cells secret elevated amount ofcytokines including IL-8and IL-15R alpha, while JAK2inhibition facilatatesIL-8secretion. （2）Transfection of YFP-CD95and cell response to FasL and JAK2inhibitionCRC cell lines CRC29and L145were successfully transfected byCD95-YFP reporter gene vectors or YFP only. After these new cell linesCRC29-CD95/YFP, CRC29-YFP, L145-CD95/YFP, L145-CD95/YFP stablyproliferated, specific JAK2inhibition was introduced to culturing system, takingDMSO treatment as control. After1h,2h treatment, enhanced signals emittedfrom YFP-CD95were detected on cell membranes especially cell-cell junctions.And also, under JAK2inhibition, Fas trimmers on cell surface are obviouslyformed compared with control. However, in CEP33779followed by FasLtreated group, weak YFP-CD95signals on cell membranes were detected.Conclusion1. Meta-analysis of Kaplan Meier curve of multiple colorectal cancerdatasets suggests that high expression of CD95and JAK2in CRC tumor tissuescorrelate with poor survival.2. Expression of CD95and JAK2as well as CD95associated pathwaystudy demonstrate that CD95and JAK2are highly correlated in CRC tissuesmainly in tumor immunity, inflammatory response and chemokines,JAK2/STAT3signal transduction, EMT.3. In vitro study on CRC cell lines revealed a lower but necessary level ofJAK2/STAT3activation in the process of proliferation. Specific JAK2inhibitionimpairs colon formation capacity which suggest that JAK2may be essential fortumor growth; specific JAK2inhibition to some degree hindersepithelial-mesenchymal transition (EMT). Over-activation of JAK2/STAT3improves CRC cell proliferation capacity. JAK2inhibition alone doesn’t seem toinduce apoptosis but can facilitate FasL induced apoptosis.4. FasL can stimulate IL-8secretion which can be facilitated by CEP33779.As an important factor in the process of CRC metastasis and progression,the mechanism study of JAK2in CRC cells is expect to provide somepreliminary knowledge in CRC target therapy. In this research, we reveal theassociation of JAK2which is normally involved in inflammatory response andFasL normally involved in immune response, and their interconnectivity in CRCcell proliferation, EMT and also in Fas function, which is also the innovation ofthis study. We hope it will do some benefit in the development of CRC treatment.JAK2may stablize CD95on CRC cell membrane. |
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