RIP3 promotes colitis-associated colorectal cancer by controlling tumor cell proliferation and CXCL1-induced immune suppression.

Rationale: Necroptosis is a programmed form of non-apoptotic cell death that requires receptor-interacting protein 3 (RIP3). RIP3 has been shown to be relevant in multiple tumor types and has differential impact on tumor progression. We investigated whether RIP3 is involved in the progression of colitis-associated cancer (CAC) in mice. Methods: Tissues from colorectal cancer patients were examined for RIP3 expression. CAC was induced using azoxymethane (AOM) injection followed by dextran sodium sulfate (DSS) treatment in RIP3-deficient or wild-type mice. Colon tissues were collected and analyzed by Western blotting and gene expression profile analyses. Immune cell infiltration and CXCL1 expression were examined by flow cytometry and Real-time PCR, respectively. Results: RIP3 expression was upregulated in mouse CAC and human colon cancer. RIP3-deficient mice showed significantly attenuated colitis-associated tumorigenesis. Bone marrow transplantation experiments suggested that RIP3's function in hematopoietic cells primarily contributes to the phenotype. RIP3 supported epithelial proliferation and tumor growth via JNK signaling but had no effect on apoptosis. RIP3 deletion increased T cell accumulation and reduced infiltration by immunosuppressive subsets of myeloid cells during acute colitis and CAC. The immune-suppressive tumor microenvironment was dependent on RIP3-induced expression of the chemokine attractant CXCL1, and administration of recombinant CXCL1 during CAC restored tumorigenesis in Rip3-/- mice. Conclusion: Our results reveal an unexpected function of RIP3 in enhancing the proliferation of premalignant intestinal epithelial cells (IECs) and promoting myeloid cell-induced adaptive immune suppression. These two distinct mechanisms of RIP3-induced JNK and CXCL1 signalling contribute to CAC progression.

Receptor-Interacting Protein Kinase 3 Deficiency Recruits Myeloid-Derived Suppressor Cells to Hepatocellular Carcinoma Through the Chemokine (C-X-C Motif) Ligand 1-Chemokine (C-X-C Motif) Receptor 2 Axis.

Receptor-interacting protein kinase 3 (RIP3) is the core regulator that switches cell death from apoptosis to necrosis. However, its role in tumor immunity is unknown. In this study, decreased RIP3 expression was observed in patients with hepatocellular carcinoma (HCC), which correlates with myeloid-derived suppressor cell (MDSC) accumulation. Moreover, RIP3 is a prognosis factor for patients with HCC. We further found that RIP3 knockdown results in an increase of MDSCs and a decrease of interferon gamma-positive (IFN-γ+ ) cluster of differentiation 8-positive (CD8+ ) tumor-infiltrating lymphocytes (IFN-γ+ CD8+ T cells) in hepatoma tissues, thus promoting immune escape and HCC growth in immunocompetent mice. By phosphorylating P65Ser536 and promoting phosphorylated P65Ser536 nuclear translocation, RIP3 knockdown increases the expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in HCC cells. RIP3 knockdown induces MDSC recruitment through the CXCL1-chemokine (C-X-C motif) receptor 2 (CXCR2) axis. Furthermore, a CXCR2 antagonist substantially suppresses MDSC chemotaxis and HCC growth in RIP3 knockout mice. Conclusion: RIP3 deficiency is an essential factor directing MDSC homing to HCC and promoting CXCL1/CXCR2-induced MDSC chemotaxis to facilitate HCC immune escape and HCC progression; blocking the CXCL1-CXCR2 chemokine axis may provide an immunological therapeutic approach to suppress progression of RIP3 deficiency HCC.

Alterations of the Immunologic Co-Stimulator B7 and TNFR Families Correlate with Hepatocellular Carcinoma Prognosis and Metastasis by Inactivating STAT3.

Blockade of the immunosuppressive checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA4) or programmed death 1 (PD-1) and its cognate ligand, programmed death 1 ligand (PD-L1), has altered the landscape of anti-tumor immunotherapy. B7 family and tumor necrosis factor receptor (TNFR) superfamily play a crucial role in T cell activation, tolerance, and anergy through co-stimulatory and inhibitory signal transduction. Investigating the immune molecular landscapes of the B7 and TNFR families is critical in defining the promising responsive candidates. Herein, we performed comprehensive alteration analysis of the B7 and TNFR family genes across six hepatocellular carcinoma (HCC) datasets with over 1000 patients using cBioPortal TCGA data. About 16% of patients had both B7 and TNFR gene alterations. TNFR gene amplifications were relatively more common (1.73⁻8.82%) than B7 gene amplifications (1.61⁻2.94%). Analysis of 371 sequenced samples revealed that all genes were upregulated: B7 and TNFR mRNA were upregulated in 23% of cases (86/371) and 28% of cases (105/371), respectively. Promoter methylation analysis indicated an epigenetic basis for B7 and TNFR gene regulation. The mRNA levels of B7 and TNFR genes were inversely correlated with promoter methylation status. B7-H6 expression was significantly associated with worse overall survival, and B7-H6 mRNA was increased gradually in cases with gene copy number alterations. B7-H6 overexpression was associated with aggressive clinicopathologic features and poor prognosis in HCC. Downregulation of B7-H6 in HCC cells significantly inhibited cell adhesion, proliferation, migration, and invasion. Knockdown of B7-H6 in HCC cells inhibited tumor growth and metastasis in vivo. B7-H6 promoted HCC metastasis via induction of MMP-9 expression and STAT3 activation. B7-H6 and STAT3 performed functional overlapping roles on enhancing the MMP-9 promoter activity in HCC cells. These results suggest that alterations of the immunologic co-stimulator B7 and TNFR families correlate with HCC metastasis and prognosis, and especially B7-H6 plays a critical role in promoting metastasis of HCC.