Molecular Mechanism Of RIPK1 Mediating Antitumor Immunity

BackgroundThe receptor-interacting protein kinase 1(RIPK1)regulates the cellular necroptosis process.According to existing research,RIPK1 performs a "dual role" in the regulation of cell death signals.On the one hand,RIPK1 forms a complex with cell inhibitor of apoptosis protein-1/2(cIAP 1/2)which mediates the activation of MAPK signaling pathway and NF-κB signaling pathway to promote cell survival.On the other hand,RIPK plays a key role in Tumor necrosis factor(TNF)family-mediated cellular necroptosis.Necroptosis and necroptosis are two different cell death signals.Normally,Fas-associated with death domain protein(FADD),caspase-8,FLICE-like inhibitory protein(FLIP)activates the classical necroptosis pathway,whereas in the absence of caspase-8,RIPK1 and RIPK3 form heterodimers and are involved in the formation of necrosome,when cell death signal will switch from apoptosis to necrosome.In this case,RIPK1 acts as a structural scaffold rather than exerting its kinase activity.Immunohistological analysis of a small number of samples from our preliminary work showed that RIPK1 was highly expressed in tissues that failed programmed cell death protein 1(PD-1)monoclonal antibody treatment.We used CRISPR-Cas9 genome editing technology to screen tumor cell genes for immunotherapeutic targets in an article that was published in Nature in 2007.In that article,the RIPK1 gene was identified as a possible sensitizer gene for PD-1 monoclonal antibody therapy.This therapy is used to treat various types of cancer.Studies that focus on inhibiting RIPK1 have garnered an increasing amount of attention in recent years due to its significance as an important inflammation-related target.On the other hand,there are not many research outcomes currently accessible on tumor immunity.Objectives1.To look into RIPK1’s fundamental roles in tumor cells(proliferation,invasive and migration).2.To interfere with tumor cell RIPK1 expression,and investigates the changes of immune microenvironment caused by RIPK1 in mouse transplanted tumors.3.To explore the mechanism of tumor cell RIPK1 in mediating anti-tumor immunity.4.To investigate the impact of RIPK1 expression on the effectiveness of PD-1 inhibitor therapy in non-small cell lung cancer tissues.MethodsPart Ⅰ.Expression and biological role of RIPK1 in tumor1.To examine the function and signaling mechanism of RIPK1 in lung squamous cell carcinoma and lung adenocarcinoma,as well as the expression of RIPK1 in lung squamous cell carcinoma and lung adenocarcinoma,using data from the Cancer Genome Atlas,a public database.2.MC38 cell lines knocked out by RIPK1 were constructed and plate clonal formation assay and CCK-8 assay were performed to evaluate the effects of RIPK1 on cell proliferation.3.The effects of RIPK1 on invasion and migration of MC38 cells were evaluated by transwell assay and caudal vein lung metastasis mouse model.Part Ⅱ Tumor cells RIPK1 affect the tumor immune microenvironment1.Construct subcutaneous tumor models using RIPK1 knockdown MC38 cells and control cells on BALB/c nude mouse and C57BL/6J black mouse skin to assess the effect of RIPK1 on T lymphocyte immunity2.Flow cytometry was used to analyze the effects of tumor RIPK1 deletion on the infiltration and activation status of CD8+T lymphocytomas and the polarization of TAMs in the immune microenvironment of C57BL/6J mouse subcutaneous transplantation tumors3.To examine the "abscopal effects" and "memory effects" of RIPK1-triggered immune responses,tumor cells were bilaterally implanted into C57BL/6J mice under the skin to generate "abscopal tumor" and "rechallenge tumor".Part Ⅲ Molecular mechanisms of tumor cell RIPK1 knockdown-mediated antitumor immunity1.Western Blot assay was used to analyze the phosphorylation levels of p65,IkB,RIPK3,MLKL,and cleaved protein levels of Caspase-8 and Caspase-1 in RIPK1 knockout cells and mouse transplanted tumor tissues;Enzyme-Linked Immunosorbnent Assay(ELISA)assay was used to analyze the levels of High Mobility Group Protein 1(HMGB1)and Adenosine Triphosphate(ATP).2.Co-Immunoprecipitation(Co-IP)was used to investigate the interactions of RIPK1,ZBP1(Z-DNA binding protein 1),and RIPK3.3.Utilizing flow cytometry to analyze the effect of RIPK1 on PD-Ll expression in tumor cells,and using the TCGA database to assess the association between RIPK1 expression and Tumor mutation burden(TMB).4.A multifactor assay,flow cytometry,real-time quantitative PCR and other experiments were used to investigate the changes in 40 cytokines and the polarization of tumor-associated macrophages in C57BL/6J mice subcutaneous tumors with RIPK1 knockdown in tumor cells.Part IV Correlation analysis of tumor RIPK1 expression and immunotherapy efficacy in patients with non-small cell lung cancer1.Using TCGA data,the effect of RIPK1 expression on overall survival in lung adenocarcinoma and lung squamous carcinoma was investigated.2.Immunohistochemistry was used to assess RIPK1 expression and CD8+T lymphocyte infiltration in 152 non-small cell lung canc.er tissues treated with immunotherapy.The relationship between RIPK1 expression and CD8+T lymphocyte count was investigated,as well as the effect of RIPK1 expression on immunotherapeutic efficacy.ResultsPart Ⅰ.Expression and biological role of RIPK1 in tumor1.A search of the TCGA database for RIPK1 revealed that it is a broadly expressed gene in both tumor and normal tissues,with significant changes in expression between tumor and normal tissues.RIPK1 is involved in gene transcriptional control,protein ubiquitination regulation and transcription factors,carcinogenesis,tumor immunity,and DNA damage response in lung squamous carcinoma and lung adenocarcinoma.2.The CCK-8 assay was performed after the construction of a stable knocked out RIPK1 MC38 cell line.The results showed that OD 450 in the knocked out RIPK1 group was significantly lower than that in the control group after 5 days of culture(p<0.0001).The results of plate cloning formation experiment showed that the size and number of cell clones formed by RIPK1-gRNA MC38 cells were significantly reduced compared with the control cells after 14 days of culture(P<0.05).3.The results of Transwell experiment showed that the number of MC38 cells with RIPK1 knocked out at 36h or 48h traversed the small compartment membrane was significantly lower than that in CRISPR-Ctr1 group(P<0.0001).Compared with CRISPR-Ctr1 MC38 cells,knockout RIPK1 significantly reduced the number and area of nodular metastases formed by MC38 cells in mouse lung.Part Ⅱ Tumor cells RIPK1 affect the tumor immune microenvironment1.BALB/c nude mouse and C57BL/6J black mouse were used to create subcutaneous tumor models.The growth curves of two types of mouse subcutaneous tumors showed that MC38 cells knocked out by RIPK1 could form tumors and grow stably under the skin of nude mouse.However,after many days of tumor growth,RIPK1 knockdown MC38 cells gradually decreased and eliminated in C57BL/6J black mouse(CRISPR-Ctr1 group versus RIPK1-gRNAlgroup,P<0.001).2.Flow cytometry and multicolor immunofluorescence analysis of immune infiltration and functional status of CD8+T lymphocytes in subcutaneous tumors of RIPK1-gRNA MC38 and CRISPR-Ctr1 MC38 groups of C57BL/6J black mouses.The results showed that the percentage of TNF α+IFN γ+CD8+T lymphocytes was significantly higher in the RIPK1 knockdown group than that in the RIPK1 non-knockdown group(P<0.05).The knockout of RIPK1 cells resulted in the increase of M1-type TAMs and the decrease of M2-type TAMs in mouse transplanted tumors.3.Experiments utilizing CD 8-neutralizing antibodies to deplete CD8+T lymphocytes revealed that CD8+T lymphocyte depletion effectively reversed the suppression of RIPK1-induced MC38 cell subcutaneous tumor development.An anti-PD-1 mouse subcutaneous tumor model was constructed using RIPK1 knockdown MC38 cells and B16-F10 cells,and the tumor growth curve indicated that interfering with RIPK1 expression considerably boosted the anti-tumor efficacy of the anti-PD-1 antibody.4.C57BL/6J mice were bilaterally injected with tumor cells beneath the skin to imitate "abscopal tumor" and "rechallenge tumor," and mouse growth curves revealed that RIPK1 activated local immune response may generate abscopal effects and memory effects.Part Ⅲ Molecular mechanism of tumor cell RIPK1 mediating antitumor immunity1.Changing the expression level of RIPK1 had no effect on the expression of PD-L1 in tumor cells;According to the analysis of the TCGA database,the expression level of RIPK1 was not correlated with TMB in 32 out of 33 cancer species.2.Westren Blot assay,real-time quantitative PCR assay and ELISA assay were used to detect the pro-survival and pro-death signals of RIPK1-knocked out cells and transplanted tumor tissues.The results showed that the p65 phosphorylation level of NF-κB decreased,and the expressions of downstream pro-survival genes Bcl-XL and Tnfaip3 were down-regulated.However,the increased phosphorylation of RIPK3 and MLKL resulted in the increased release of HMGBI and ATP,as well as the increased apoptotic proteins in the cleaved form of csapase-8 and csapase-1.3.CO-IP assay showed increased binding of ZBP1 to RIPK3 in subcutaneous tumor tissues with tumor cells devoid of RIPK1.4.Using the QAM-INF-1 multifactor assay kit from Guangzhou Riboao Company,the quantitative measurement of 40 immune-related cytokines was performed on the subcutaneous tumor tissues of the RIPK1 knockdown group and the control group.CCL2,CCL3,CCL5,CCL11,CCL12,CCL24,CXCL5,CXCL9,and ICAM-1 were raised in mouse transplanted tumors due to RIPK1 deletion(P<0.05),although IL-10 was decreased(P<0.05).Flow cytometry revealed that RIPK1 deletion resulted in TAM polarization to M1-type TAMs in mouse transplanted tumors.5.Real-time quantitative PCR analysis of cultured cells showed that RIPK1 knockdown inhibited the expression of CCL2 stimulated by TNFa;Depletion of CCL2 in mouse transplanted tumors using CCL2-neutralizing antibodies,and depletion of CCL2 partially reversed tumor growth inhibition in the RIPK1 knockdown group as shown by the mouse growth curve(RIPK1-gRNA group vs.RIPKl-gRNA+anti-CCL2 group,P<0.05).Flow cytometry showed that depletion of CCL2 led to decreased infiltration of CD206+M2 type TAMs in RIPK1 knockout group(RIPK1-gRNA group vs RIPK 1-gRNA+anti-CCL2 group,P<0.05),and the CD86+M1/CD206+M2 ratio was significantly increased(P<0.05).These results indicate that the absence of RIPK1 inhibits the secretion of CCL2 by tumor cells.Therefore,the increase of CCL2 in mouse subcutaneous grafts caused by the absence of RIPK1 is not the secretion of tumor cells.CCL2 can promote the polarization of TAMs to M2 TAMs,and also promote the infiltration of CD8+T lymphocytes.The increase of Ml-type TAM2 in transplanted tumors of RIPK1-deficient mouse may be related to other factors besides CCL2.Part Ⅳ Correlation analysis of tumor RIPK1 expression and immunotherapy efficacy in patients with non-small cell lung cancer1.Correlation analysis of the expression of RIPK1 in the TCGA database with the prognosis of lung adenocarcinoma and lung squamous cell carcinoma showed that there was no significant correlation between the expression of RIPK1 in lung adenocarcinoma and overall survival(P=0.16),but low expression of RIPK1 in lung squamous cell carcinoma showed a good overall survival trend,but no statistical difference(P=0.055).2.Immunohistochemical analysis of 152 cases of non-small cell lung cancer showed that RIPK1 expression was negatively correlated with CD8+T lymphocyte infiltration(r=-0.249,p=0.002).Kaplan-Meier survival curve showed that patients with low expression of RIPK1 had significantly better progression-free survival than those with high expression of RIPK1(HR=0.59,P=0.019).Conclusions1.RIPK1 is a gene commonly expressed in tumor and normal tissues.Its expression varies greatly in different tumor tissues and corresponding normal tissues,and its involved functions are also diverse.These characteristics indirectly verified RIPK1 as an upstream regulator of cell signal transduction.RIPK1 plays an important role in the proliferation,invasion and migration of MC38 cells.Whether this conclusion can be extended to other tumors needs to be further verified by other cell lines.2.Interfering with RIPK1 expression in tumor cells can alter the tumor immune milieu and improve the anti-tumor impact of anti-PD-1 antibodies;The local immune response caused by RIPK1 deletion can induce abscopal effects and memory effects.3.Interference with the expression of RIPK1 cells leads to the high expression of CCL2 and other cytokines in mouse subcutaneous graft tumors,which has an important effect on the polarization of tumor-related macrophages and the chemotaxis of T lymphocytes.4.The absence of RIPK1 cells led to the weakening of the pro-survival signal and the enhancement of the pro-death signal in the subcutaneous grafts of mouses,which promoted the occurrence of necrotic apoptosis and inflammatory apoptosis in the tumor tissues.5.The expression of RIPK1 in tumor tissues of non-small cell lung cancer patients was negatively correlated with CD8+ T lymphocyte infiltration.RIPK1 can predict the efficacy of immune checkpoint inhibitors in patients with non-small cell lung cancer.