A Study On The Role Of CD74 In Migration Of Breast Cancer Cells

BackgroundCD74,also named Ii.is a multifunctional cytokine.As a invariant chain of MHC class ?,CD74 involves in MHC class ? mediated antigen presentation process.MHC class ? correctly folded in endoplasmic reticulum and transfered out of endoplasmic reticulum are both regulated by CD74,CD74 also participates in MHC class ? transfering from golgi body to antigen processing compartments.MIF is a ligand of CD74,MIF interacts with CD74 and induces production of proinflammatory cytokines,inhibition of apoptosis and increase of cell survival.In addition.CD74 was also associated with many human diseases,including autoimmune disease(such as systemic lupus erythematosus),atherosclerosis,alzheimer's disease and cancer,Studies have indicated that CD74 was overexpressed in a variety of tumors,including breast cancer.Furthermore,clear cell renal cell carcinoma,gastric cancer and pancreatic cancer cell invasion are associated with CD74.Recent studies indicate that CD74 is overexpressed in breast cancer and has a linear relationship with lymph node metastasis and triple negative breast cancer.The molecular mechanism is still unknown.CD44 is the receptor of hyaluronic acid.CD44 interacts with receptor tyrosine kinase directly or through adapter protein when hyaluronic acid binds to CD44.Downstream signal pathways,such as the PI3K-AKT signal pathway and MAPK cascade,are activated by the interaction of CD44 and receptor tyrosine kinase.Activated signal pathways mediate cell survival,proliferation,apoptosis or drug resistance of tumor cells.CD44 also interacts with the Ras superfamily of small GTPases through adapter protein and activates RHOA or RAC,RHO GTPases involve in cytoskeleton assembly and rearrangement.In addition,CD44 is associated with characteristics of tumor stem cells.CD44+CD24-subgroup separated from breast cancer cells shows characteristics of tumor stem cells,EMT related TWIST and SNAIL are upregulated in the subgroup.RHO GTPases are members of Ras superfamily of small GTPases.and it is divided into 8 superfamilies based on the amino acid sequence similarity.RHOA,RACl and CDC42 are studied extensively.Myosin light chain is phosphorylated by RHOA through downstream effector ROCK.Phosphorylation of myosin light chain induced myosin contraction.Besides,CFL is phosphorylated by RHOA-ROCK pathway induced LIMK phosphorylation and rearrangement of the actin cytoskeleton is regulated by CFL.PAK is a effector of RACl and CDC42,PAK also phosphorylates LIMK and effects actin cytoskeleton stability.RAC1 regulates ARP2/3 through WAVE and CDC42 regulates ARP2/3 through WASP,both of the RAC 1 and CDC42 pathways mediate actin cytoskeleton assembly and rearrangement.Studies indicate that RHO GTPases are overexpressed in a variety of tumors and involve in invasion and metastasis.CFL is an actin-binding protein.It has a deactivated form with Ser3 phosphorylation on N-terminal and an activated form without Ser3 phosphorylation.Activated CFL tends to bind with GDP · F-actin,then the F-actin is severed and depolymerized.CFL severing produces G-actin and free barbed ends,GDP bound G-actin is exchanged for GTP bound form and added to the elongation terminal,hence,CFL mediates actin polymerization.Furthermore,free barbed ends are used for actin nucleation,elongation and actin branching.All together,CFL regulates actin polymerization and depolymerization.Our research sets to confirm whether CD74 regulates invasion and metastasis through effecting cytoskeleton related proteins in breast cancer cells.Methods1.Immunohistochemical detected CD74 distribution in BIDC,the association between CD74 and breast cancer was assessed by analysis of clinical data.2.Cell invasion changes induced by CD74 inhibition are detected by matrigel invasion assay.Cell migration changes induced by CD74 inhibition are detected by wound-healing scratch assay.3.F-actin is stained with TRITC-phalloidin,CD74 effects on protrusions formation is visualized by confocal laser scanning microscope.4.Cytoskeletal regulatory protein changes induced by CD74 inhibition or overexpression are detected by western blot.5.Immunofluorescence and co-immunoprecipitation are used to assay the interaction between CD74 and CD44.6.CD74 or/and CD44 is inhibited,western blot detects CFL1 phosphorylation.7.CD74 or/and CD44 is inhibited.TRITC-phalloidin stains F-actin.CD74 or/and CD44 effects on protrusions formation is visualized by confocal laser scanning microscope.8.CD44 is suppressed under the CD74 overexpressed condition,western blot detects the regulatory relationship between CD74 and CD44.9.MG 132 is added under the CD74 inhibited condition,western blot assesses CD74 effects on stability of CD44.10.MIF is suppressed by specific siRNA or inhibitor,western blot detects CFL1 phosphorylation.11.Co-immunoprecipitation shows CD74 and CD44 interaction is attenuated by MIF overexpression.12.Breast cancer cells transfects with RHO GTPases related plasmids.CD74 mediates CFL1 phosphorylation through RHO GTPases is detected by western blot.13.CD74 regulates cell migration through RHOA is detected by wound-healing scratch assay.14.Lentiviral packaging system is used for preparation of virus,then virus infection and stable expression cell lines are screened.15.CD74 effects on tumorigenesis and metastasis in vivo are assessed by xenograft studies.Results1.In BIDC tissues,CD74 was overexpressed on membrane and in cytoplasm.CD74 was associated with breast cancer,clinical stages and lymph node metastasis.2.MDA-MB-231 cells were transfected with CD74 shRNA contained plasmid,invasion assay suggested MDA-MB-231 cell invasion ability was inhibited by CD74 down regulation.3.CD74 expression was inhibited by CD74 shRNA contained plasmid in MDA-MB-231 cells,wound-healing scratch assay indicated MDA-MB-231 cell migration was inhibited by CD74 down regulation.4.CD74 was inhibited by CD74 shRNA contained plasmid in MDA-MB-231 cells(or T47D cells).F-actin was stained with TRITC-phalloidin,confocal laser scanning microscope visualized cell protrusions formation.Results showed protrusions formation was suppressed by CD74 inhibition.5.CD74 was overexpressed in MCF-7 cells,F-actin was stained with TRITC-phalloidin,confocal laser scanning microscope visualized cell protrusions formation.Results indicated overexpression of CD74 increased protrusions formation.6.Cytoskeleton rearrangement related ROCK1 and CFL1 phosphorylation level were decreased in CD74 shRNA transfected MDA-MB-231 cells.Consistent results were obtained in T47D cells.7.Cytoskeleton rearrangement related ROCK1 and CFL1 phosphorylation level were increased in CD74 overexpressed MCF-7 cells.Similar results were gained in HCC1806 cells.8.CD74 was stained with AlexaFluor 568,CD44 was stained with AlexaFluor 488.immunofluorescence result suggested CD74 colocalized with CD44 in MDA-MB-231 cells and T47D cells.9.MDA-MB-231 cells(or T47D cells)were transfected with pcDNA3.1-Flag-CD74 plasmid,co-immunoprecipitation result showed CD74 interacted with CD44.10.CD44 in MDA-MB-231 cells was used for endogenous co-immunoprecipitation,result also suggested CD74 interacted with CD44.11.CD44 was silenced with CD44 specific siRNA in MDA-MB-231 cells or T47D cells.Western blot showed down regulation of CD74,ROCK1 and p-CFL1 protein levels.12.pLT-shCD74#1 or pLT-shCD74#2 plasmid was used to inhibit CD74 expression,CD44 was suppressed by specific siRNA.Western blot assay indicated CFL1(?)phosphorylation level was inhibited by CD74 or/and CD44 silence.Consistent results were obtained in MDA-MB-231 cells and T47D cells.13.CD74 was suppressed by pLT-shCD74#1 or pLT-shCD74#2 plasmid.CD44 was inhibited with CD44 specific siRNA.F-actin was stained with TRITC-phalloidin,confocal laser scanning microscope visualized cell protrusions formation.Results showed protrusions formation was suppressed by CD74 or/and CD44 silence.Consistent results were obtained in MDA-MB-231 cells and T47D cells.14.MDA-MB-231 cells(T47D cells)were transfected with CD44 specific siRNA or/and pcDNA3.1-CD74.western blot showed inhibition of CD44 suppressed p-CFL1 upregulation induced by CD74 overexpression.15.CD74 was inhibited by CD74 shRNA contained plasmid in MDA-MB-231 cells,western blot assay suggested MG 132 restrained CD44 reduction.16.MIF was inhibited by MIF specific siRNA or specific inhibitor,or MIF was overexpressed in breast cells,western blot results suggested CFL1 phosphorylation level was negatively correlated with MIF expression.17.HEK 293FT cells were co-transfected with pcDNA3.1-Flag-CD74 and pcDNA3.1-MIF,cell lysate was immunoprecipitated with anti-Flag antibody,co-immunoprecipitation result indicated decreased interaction between CD74 and CD44 under MIF overexpression condition.18.CD74 overexpression increased CFL1 phosphorylation was inhibited by RHO GDI or RHOA N19 in MCF-7 cells.CD74 suppression decreased CFL1 phosphorylation was rescued by RHOA L63 in MDA-MB-231 cells.19.CD74 was silenced in MDA-MB-231 cells and RHOA L63 plasmid was transfected,wound-healing scratch assay showed CD74 down regulation decreased cell migration was rescued by RHOA L63.MCF-7 cells were co-transfected with CD74 and RHOA N19,wound-healing scratch assay and immunofluorescence result suggested CD74 overexpression increased cell migration and protrusions formation were inhibited by RHOA N19.20.Three groups of nude mice were injected with MDA-MB-231 pLT-shCTRL,MDA-MB-231 pLT-shCD74#1 or MDA-MB-231 pLT-shCD74#2 cells.respectively.Abdominal subcutaneous injection result showed inhibition of CD74 suppressed tumorigenesis.A case of liver metastasis was found in MDA-MB-231 pLT-shCTRL cell injected group.21.Three groups of nude mice were injected with MDA-MB-231 pLT-shCTRL,MDA-MB-231 pLT-shCD74#1 or MDA-MB-231 pLT-shCD74#2 cells,respectively.Tail vein injection result showed two cases of lung metastasis were found in MDA-MB-231 pLT-shCTRL cell injected group.Conclusion1.CD74 was associated with breast cancer,clinical stages and lymph node metastasis.2.Inhibition of CD74 restrains migration and invasion in breast cancer cells.3.CD74 expression is positively correlated with protrusions formation.4.CFL1 phosphorylation level is positively correlated with CD74 expression5.CD74 colocalizes with CD44 in breast cancer cells.6.CD74 and CD44 co-regulate CFL1 phosphorylation and protrusions formation,CD74 is a upstream regulator of CD44.7.CD74 may increase CD44 stability by suppressing CD44 degradation through proteasome pathway.8.CD74 and CD44 interaction is attenuated by MIF overexpression,the interaction between CD74 and MIF inhibits CD74 regulated CFL1 phosphorylation.9.CD74 regulates CFLI phosphorylation and protrusions formation through RHOA in breast cancer cells.10.Inhibition of CD74 suppresses breast cancer tumorigenesis.11.Inhibition of CD74 suppresses breast cancer metastasis.Taken together,our results show that CD74 is associated with breast cancer cell migration,invasion and protrusions formation.In breast cancer cells,CD74 interacts with CD44 and enhances tumorigenesis and metastasis via RHOA-mediated CFL1 phosphorylation.In addition,our results indicate that CD74 may increase CD44 stability by suppressing CD44 degradation through proteasome pathway;CD74 regulates CFLI phosphorylation through CD44 may be suppressed by MIF overexpression.