The Regulation Of MiR-155/Wnt Signaling Pathway On IL-6-mediated Differentiation Block And Immunosuppressive Activity Of EMDSC In Breast Cancer

Objective:IL-6 is an important trigger for the expansion and recruitment of early-stage myeloid-derived suppressor cells（e MDSCs）,which are regarded to be major coordinators of the immunosuppressive tumor microenvironment.The purpose of this study was to explore the molecular events involved in the IL-6-mediated effects on mice e MDSC development.Methods:（1）IL-6 knockdown 4T1 breast cancer mice(4T1^IL-6low)models were constructed to explore the IL-6-mediated effect on MDSCs development,and a new e MDSCs subset was identified.The percentage of e MDSCs was detected by Flow Cytometry and immunohistochemistry.The expression of SOCS1-3 and JAK/STAT pathway were determined by q RT-PCR and Western Blot.Proliferation and apoptosis of T cells co-cultured with e MDSCs were detected by Brdu and Annexin V methods.（2）To confirm the effect of IL-6 on e MDSCs development in vitro,the BM cells isolated from BALB/c mice were co-cultured with 4T1 breast cancer cells to induce e MDSCs.And the IL-6R antibody was utilized to abolish the effect of IL-6.The percentage of e MDSCs was detected by Flow Cytometry.（3）To evaluate the efficacy of blocking IL-6/STAT3 pathway on e MDSCs development,IL-6R antibody and JSI-124 were administrated intraperitoneally,and the tumor growth and pulmonary metastatic nodules were monitored.（4）To explore the role of SOCS3 in e MDSCs development,conditional SOCS3 knockout mice(SOCS3^{Mye KO})with SOCS3deficiency specifically in the myeloid linage was constructed.The percentage of e MDSCs in spleen and bone marrow was detected by Flow Cytometry.（5）The e MDSCs separated from SOCS3^fl/fl(e MDSCs^fl/fl)and SOCS3^{Mye KO} mice(e MDSCs^SOCS3KO)were induced to differentiate into myeloid terminal mature cells using GM-CSF in vitro.The percentage of Gr-1 cells were detected by Flow Cytometry.The MPO,IL-1βand TNF-αexpression were determined by q RT-PCR.（6）The whole-genome RNA sequencing of e MDSCs^fl/fl,e MDSCs^SOCS3KO and CD11b⁺Gr-1⁺cells were carried out to enrich pathways involved in SOCS3 mediated e MDSCs development,the autophagy and Wnt pathway were filtered out.The activation of the AMPK,AKT/m TOR and Wnt pathways were determined by RT-PCR and Western Blot.The e MDSCs^fl/flwas treated with 3-MA to block autophagy,and e MDSCs^SOCS3KOwere treated with AZD5363 or Rapamycin to stimulate autophagy during GM-CSF induction.The e MDSCs^SOCS3KO was treated with Wnt antagonist to explore the effect of Wnt pathway in e MDSCs development.The percentage of e MDSCs was detected by Flow Cytometry.The LC3BII,Wnt3a,Wnt5a,Wnt10a,β-catenin,GSK-3β,p-GSK-3βin e MDSCs was detected by q RT-PCR and Western Blot.（7）Mi RNA microarray in e MDSCs^fl/fl,e MDSCs^SOCS3KO and CD11b⁺Gr-1⁺cells were performed to screen mi RNAs involved in SOCS3 mediated e MDSCs development,and mi R-155 was filtered out.The e MDSCs^fl/fl were transfected with mi R-155 mimics,the e MDSCs^SOCS3KO were transfected with mi R-155 inhibitor before GM-CSF induction.The percentage of e MDSCs was detected by Flow Cytometry.The autophagy activity was detected by Western Blot and cell immunofluorescence.（8）The Targetscan was utilized to predict the transcription factor regulated by mi R-155 which was further verified using luciferase reporter analysis.To confirm the role of C/EBPβin e MDSCs development,a lentiviral vector overexpressing C/EBPβwas transfected into e MDSCs^SOCS3KO before GM-CSF induction.The percentage of e MDSCs was detected by Flow Cytometry.And the C/EBPβexpression was determined by q RT-PCR and Western Blot.（9）To evaluate the efficacy of mi R-155-dependent autophagy block in breast cancer,the mi R-155 antagonist and Rapamycin were administrated.The tumor growth curve was plotted.The percentage of e MDSCs,CD3⁺T cells and proliferating CD3⁺T in tumors were detected by Flow Cytometry.Results:（1）IL-6 knockdown in 4T1 cells inhibited tumor growth and reduced metastatic nodules in the lungs in vivo.But no significant effect of IL-6 on the proliferation,apoptosis,migration,and invasion of cancer cells was observed in 4T1^IL-6low cells compared to 4T1^NC.（2）A newly defined subset of e MDSCs with phenotype of CD11b⁺Gr-1^-F4/80^-MHCII^-increased in 4T1 tumors compared to 4T1^IL-6low.The e MDSCs could differentiate into classical MDSCs after induction in vitro,and suppressed T cells more potently.（3）In IL-6R Ab and JSI-124 treated 4T1 bearing mice,the growth of tumors was repressed,the number and size of metastatic lung nodules decreased,and the percentage of e MDSCs in tumor decreased significantly.（4）The levels of p-JAK1,p-JAK2,p-TYK2,p-STAT1,and p-STAT3 increased in primary e MDSCs,which was decreased after reducing the tumor-derived IL-6.The SOCS1 and SOCS2 expression increased in e MDSCs,but the SOCS3 level decreased compared to normal control.after reducing the tumor-derived IL-6,SOCS1 decreased and SOCS3 increased,but SOCS2 showed no changes.（5）The e MDSC population in BM and spleen substantially increased in SOCS3^{Mye KO} mice compared to that in SOCS3^fl/fl mice,which showed similar T cell suppression efficiency as that of primary e MDSCs.And e MDSC^SOCS3KOfailed to differentiate into terminal myeloid cells in vitro.（6）The ratio of LC3BII/I expression in e MDSCs^fl/fl increased gradually,while no significant change occurred in e MDSCs^SOCS3KO after GM-CSF stimulation.3-MA treatment could block e MDSC^fl/fl differentiation,and Rapamycin rather than AZD5363could promote e MDSC^SOCS3KO differentiation.The levels of phosphorylated m TOR protein increased in e MDSCs^SOCS3KO compared to that in e MDSCs^fl/fl,while the level of phosphorylated AKT and AMPK showed no significant difference in e MDSCs^SOCS3KO.（7）Mi R-155 increased in e MDSCs^SOCS3KO,and mi R-155 inhibitor repressed the development of e MDSCs.Moreover,autophagy activity increased in mi R-155 inhibitor-transfected e MDSCs^SOCS3KO compared to that in NC-transfected e MDSCs^SOCS3KO.The AMPK and AKT showed no significant changes in either mi R-155 mimic-transfected e MDSCs^fl/flor mi R-155 inhibitor-transfected e MDSCs^SOCS3KO.But the mi R-155 mimics stimulated the phosphorylation of m TOR proteins in e MDSCs^fl/fl,and the mi R-155 inhibitor interfered with m TOR in e MDSCs^SOCS3KO.（8）The expression of Wnt3a increased dramatically in e MDSCs^SOCS3KO compared to e MDSCs^fl/fl,while Wnt5a and Wnt10a showed no significant changes.The phosphorylation levels of GSK-3βand the expression ofβ-catenin increased significantly in e MDSCs^SOCS3KO,while phosphorylated PKC and JNK showed no difference in e MDSCs^SOCS3KO.The e MDSCs decreased significantly after Wnt antagonist treatment compared to untreated group.Furthermore,Wnt agonist abolished the suppressive effect of mi R-155 inhibitor on e MDSCs.（9）The C/EBPβexpression decreased in e MDSCs^SOCS3KO.And the luciferase reporter gene assay confirmed a direct binding of mi R-155 to the 3’UTR region of C/EBPβ.Overexpressing C/EBPβdecreased e MDSCs significantly.The expression of Wnt3a andβ-catenin decreased,and the ratio of LC3BII/I increased in C/EBPβ-overexpressing e MDSCs ^SOCS3KO.（10）The mi R-155 inhibitor or rapamycin significantly inhibited the growth of tumors in vivo.The amount of e MDSCs decreased,total and proliferating T cells increased in primary tumor tissues after above treatment.Conclusion:（1）Tumor-derived IL-6 promoted the development of CD11b⁺Gr-1^-F4/80^-MHCII^-e MDSCs via activating the JAK/STAT3 pathway,which was caused by IL-6-related SOCS3 deficiency.（2）SCOS3 knockout specifically in myeloid cells promoted the development of e MDSCs,which was caused by autophagy suppression-related myeloid lineage differentiation block.（3）Mi R-155 increased upon SOCS3 deficiency,which consequently activated the Wnt/β-catenin pathway via targeting C/EBPβto inhibit autophagy.（4）SOCS3 deficiency resulted in mi R-155 upregulation via activating the noncanonical NF-κB pathway.（5）Both targeting the IL-6/STAT3pathway to reduced e MDSCs accumulation or administrating mi R-155 antagonist and Rapamycin to forcing e MDSCs differentiation via activating autophagy could suppressing tumor growth,which may be promising therapeutic strategies for breast cancer patients.