Effect And Mechanism Of Rae1 On Tumor Development And CpG ODN Therapy In Mice

Rae1 is one of the members of the mouse NKG2 DLs family and is highly expressed on tumor cells,such as glioma cells.In addition to Rae1,mouse NKG2 DLs also include H60 and MULT1.NKG2 DLs are a class of proteins expressed on the surface of tumor cells that can be recognized by NKG2 D on the surface of NK cells and cytotoxic T cells,therefore activate NK cells or T cells to kill target cells.The sequence and affinity of variety NKG2 DLs expressed in human and mice tumor cells are totally different.And tumor cells generally express one or more kinds of,not all kinds of,NKG2 DLs,indicating that NKG2 DLs may regulate other’s expression and the regulatory role between NKG2 DLs may require further study.NKG2D/NKG2 DL signal has been considered to be crucial in tumor immune surveillance.The binding of NKG2 D and NKG2 DLs induces the activation of NK cells and cytotoxic T cells and the production of the proteins including TNF-α and IFN-γ,perforin and granzyme that could lysis and eliminate tumor cells.In recent studies,the NKG2DL-expressing tumors grew faster in the NKG2 D sufficient mice than that in the NKG2 D deficient mice with multiple types of tumors including diethylnitrosamine induced hepatocellular carcinoma,methylcholanthrene induced fibrosarcoma or TRAMP prostate cancer.However,the mechanism behind NKG2D/NKG2 DLs signaling to promote tumor development is unclear.Studies have shown that the activation of transcription factor E2F1 can induce the transcription of human and mouse NKG2 DLs genes and the expression of NKG2 DLs protein on the surface of tumor cells.m TOR and STAT3,which are closely related to tumorigenesis and development,can also activate E2F1,suggesting that NKG2 DLs may promote tumor development through m TOR and STAT3 signaling pathways.As an agonist of TLR9,Cp G ODN has been used in the research of vaccine adjuvant and tumor immunotherapy.The Cp G ODNs designed in our lab could enhance the immunogenicity of the vaccine and have a significant therapeutic effect on a variety of tumor-bearing mice.However,in the clinical study,the efficacy of Cp G ODN often shows huge differences due to different tumors.Studies have shown that TLR9 expressed by a variety of cells can upregulate the expression levels of NKG2 DLs on the cell surface,which suggests that NKG2DL-expressing tumor cells may be the cause of the effect of Cp G ODN tumor treatment.In order to explore the effects and possible mechanisms of NKG2 DLs on tumor development and tumor cells response,we took Rae1 as the main research object and established Rae1 wildtype or Rae1 defective tumor-bearing mouse models or in vitro cell models.The study explored the relationship between Rae1 and expression of other NKG2 DLs,the effect of NKG2D/NKG2 DL signal and m TOR and STAT3 signaling pathways on tumor development,and the relationship between treatment effect of Cp G ODN and expression of NKG2 DLs.The study reveals the new mechanism of NKG2 DLs promoting tumor development and affecting the treatment of Cp G ODN,which provides new ideas for immunotherapy targeting NKG2 DLs.The specific research contents are as the following:1.The effect of Rae1 on the surface NKG2 DL expression of tumor cellsIn order to investigate the regulation between NKG2 DLs on tumor cells,we established the orthotopic and ectopic glioma tumor models by intracranially or subcutaneously inoculating the Rae1+/+ GL261 cells or Rae1-/-GL261 cells into mice.Then we tested the expression of NKG2 DLs,including Rae1,H60 and MULT1,on the tumor cells,and the proportion of NKG2D+ immune cells in the inoculation sites.We used other tumor cells with or without Rae1 to verify the effect of Rae1 on NKG2 DL expression of tumor cells and NKG2 D expression of immune cells.The results are as follows:1.1 The effect of Rae1 on NKG2 DL expression of the mouse glioma cellsThe H60 and MULT1 expression levels of Rae1+/+ GL261 cells recovered from tumor tissues increased by dozens of times,but that of Rae1-/-GL261 cells recovered from tumor tissues increased by about 5 times.1.2 The relationship between Rae1 regulated NKG2 DL expression and immune cellsCompared with the Rae1-/-GL261 cells,the Rae1+/+ GL261 cells recruited more NKG2D+ immune cells in the tumor injected sites;PBMCs induced H60 and MULT1 expression on the Rae1+/+ GL261 cells,not Rae1-/-GL261 cells.1.3 The effect of Rae1 on the NKG2 DL expression on other tumor cellsAfter coculturing with PBMCs,the expression levels of H60 and MULT1 on LLC cells were upregulated,but the expression level of MULT1 on B16 cells and the expression levels of H60 and MULT1 on Pan02 cells were downregulated.The results suggest that Rae1 on the tumor cells mediates the upregulation of the expression of other NKG2 DLs induced by immune cells.2.function and mechanism of Rae1 on biological characteristics of tumor cellsTo investigate the effect of Rae1 on biological characteristics,including migration capacity and vitality,tumorigenicity of tumor cells,we detected the migration capacity and vitality of Rae1+/+ GL261 cells and Rae1-/-GL261 cells recovered from tumor tissues,and observed the survival rates and tumor size of the tumor-bearing mice.To explore the possible mechanism,we cocultured the Rae1-expressing Rae1+/+ GL261 cells,LLC cells,Rae1-/-GL261 cells,B16 cells and Pan02 cells with PBMCs,with NKG2 D blockade or NKG2 DL blockade.Then we detected the phosphorylation of m TOR and STAT3 in the above tumor cells using Western blotting.Then,the migration capacity and vitality of the GL261 cells treated with m TOR inhibitor and/or STAT3 inhibitor were tested using transwell assay and CCK8 assay.The results are as the following:2.1 The effect of Rae1 on the biological characteristics of glioma cells in vivoCompared with parental cells,the migration capacity of Rae1+/+ GL261 cells recovered from subcutaneous tumor tissues was increased and the cell vitality were decreased,whereas those of Rae1-/-GL261 cells recovered from subcutaneous tumor tissues were barely changed;compared with Rae1-/-GL261 cells,the Rae1+/+ GL261 cells formed tumor early and formed bigger tumor tissues.2.2 The effect of Rae1 on the related signaling pathways of glioma growth and biological characteristicsThe in vivo inoculation did not affect the MEK/ERK signaling pathway in GL261 cells;the STAT3 in Rae1+/+ GL261 cells and Rae1-/-GL261 cells recovered from tumor tissues was phosphorylated;the m TOR in Rae1+/+ GL261 cells,not Rae1-/-GL261 cells,recovered from subcutaneous tumor tissues were phosphorylated.2.3 The effect of Rae1/NKG2 D signal on the activation of m TOR and STAT3 signalingAfter cocultured with PBMCs,the extent of phosphorylated m TOR and STAT3 in Rae1+/+ GL261 cells,not Rae1-/-GL261 cells were increased;NKG2D or Rae1 blockade eliminated the PBMCs induced phosphorylation of m TOR and STAT3 in Rae1+/+ GL261 cells.2.4 The effect of m TOR and STAT3 activation on the migration and vitality of glioma cellsPBMCs increased the number of migrated cells and vitality of Rae1+/+ GL261 cells;m TOR and/or STAT3 inhibitors decreased the PBMCs increased migrated cells and vitality of Rae1+/+ GL261 cells;the PBMCs or inhibitors did not affect the migrated cells and vitality of Rae1-/-GL261 cells.2.5 The effect of Rae1/NKG2 D signal on the m TOR and STAT3 activation in other mouse tumor cellsNKG2D blockade decreased PBMCs induced phosphorylation of m TOR and STAT3 in LLC cells;PBMCs and NKG2 D blockade did not affect the phosphorylation of m TOR and STAT3 in B16 cells;PBMCs induced the expression of m TOR and the phosphorylation of STAT3 in Pan02 cells,which were not affected by NKG2 D blockade.The results suggest that Rae1/NKG2 D interaction between tumor cells and immune cells promotes the migration capacity and the in vivo growth by activating m TOR and STAT3.3.The impact of Rae1 on the antitumor effect of Cp G ODNTo investigate the effect of Rae1 on the anti-tumor effect of Cp G ODN,we designed and synthesized a Cp G ODN,named Cp G ODN5802,and tested its ability to activate NK cells in vitro and in vivo.The expression levels of NKG2 DLs and migration capacity of Rae1+/+ GL261 cells and Rae1-/-GL261 cells treated with Cp G ODN5802 were also tested using flow cytometry,scratch test and transwell assay.Then,we established mouse subcutaneous and intracranial tumor model of Rae1+/+ GL261 cells or Rae1-/-GL261 cells,the subcutaneous tumor model of LLC cells and B16 cells and treated the mice with Cp G ODN5802.The survival and tumor volume of the tumor model mice were monitored.The expression level of TLR9 in the tumor cells was tested using Western blotting.The results are as the following:3.1 Study on the immunostimulatory of Cp G ODNCp G ODN5802 increased the percentages of CD69-expressing NK cells in DLN cells,spleen cells and PBMCs in vivo,and increased that in spleen cells in vitro.3.2 The impact of Rae1 on the anti-glioma effect of CpG ODNCompared with the Rae1+/+ GL261 cells,the mice intracranially inoculated with Rae1-/-GL261 cells treated with Cp G ODN5802 lived longer,and the mice subcutaneously inoculated with Rae1-/-GL261 cells treated with Cp G ODN5802 developed smaller tumors.3.3 The effect of Rae1 on the NKG2 DL expression and biological characteristics of glioma cells regulated by Cp G ODNCp G ODN5802 did not affect the expression levels of NKG2 DLs on Rae1+/+ GL261 cells or Rae1-/-GL261 cells,and inhibited the motor capacity,migration capacity and vitality of Rae1+/+ GL261 cells and Rae1-/-GL261 cells.3.4 The effect of Rae1 on the Cp G ODN regulated TLR9 expression of glioma cellsAfter incubating with Cp G ODN5802,the expression level of TLR9 in Rae1+/+ GL261 cells and Rae1-/-GL261 cells was increased;TLR9 expression in Rae1+/+ GL261 cells treated with Cp G ODN5802 was decreased,whereas that in Rae1-/-GL261 cells treated with Cp G ODN5802 was increased.3.5 The effect of Rae1 on Cp G ODN regulated TLR9 expression and tumor development of other tumorsCompared with control group,the expression of TLR9 in LLC cells recovered tumor tissues was decreased,whereas that in B16 cells was increased;Cp G ODN5802 treatment prolonged the median survival of subcutaneous LLC cell model mice by 26% and prolonged that of subcutaneous B16 cell model mice by 100%.The results suggest that Rae1 inhibits the anti-tumor effect of Cp G ODN5802 though interfering Cp G ODN5802-induced the upregulation of TLR9 in tumor cells.In conclusion,our study demonstrated that Rae1 on tumor cells could regulate the expression of NKG2 DLs on tumor cells;the interaction of Rae1 and NKG2 D could activate the m TOR and STAT3 signaling pathway thereby promote tumor growth;Rae1 could also inhibit the antitumor effect of Cp G ODN by inhibiting the TLR9 expression on tumor cells.The findings might provide the insights for understanding the mechanism by which NKG2D/NKG2 DL signaling could promote tumor development,and provide a new strategy for tumor treatment of TLR9 agonists combined with NKG2 DL inhibitors.