RNA-seq after CD95 gene knockout in 4T1 cells
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ABSTRACT:
PROVIDER: PRJNA647117 | ENA |
REPOSITORIES: ENA
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Project description:We examine the differential RNA expression after knocking out CD95/Fas gene in 4T1 cells
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Project description:Novel therapies targeting cancer stem cells (CSCs), which play critical roles in chemo- and radio-resistance, metastasis, and possibly resistance against cancer immunotherapy including granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccines, may provide beneficial clinical outcomes. Here, we used syngeneic immunocompetent mice that allowed precise evaluation of the immunogenicity of the side population (SP) isolated from 4T1 murine breast carcinoma (4T1-SP) cells as putative CSCs. 4T1-SP cells showed various stem cell properties including high capacities for colony formation and tumorigenicity as well as high expression of phosphorylated signal transducer and activator of transcription-3 and vascular endothelial growth factor that are inductive of immune tolerance. Despite these progressive malignant characteristics of 4T1-SP cells, subcutaneous injection of non-transmissible Sendai virus-mediated GM-CSF gene-transduced 4T1-SP (4T1-SP/GM) cells remarkably impaired their tumorigenicity compared with that of the controls. This impairment of tumorigenicity was partially dependent on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Notably, therapeutic vaccinations using irradiated 4T1-SP/GM cells markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with that of the controls including irradiated 4T1-non-SP/GM cells. Tumor suppression was accompanied by robust accumulation of mature dendritic cells at vaccination sites and systemic Th1-based cellular immunity. Moreover, vaccinations comprising primary 4T1-SP cells isolated from transplanted 4T1-SP tumors elicited antitumor effects. cDNA microarray analysis showed that 4T1-SP cells predominantly expressed genes of cancer-related antigens including cancer/testis antigens. Collectively, we demonstrate that SP cell-based vaccinations induce effective antitumor immunity that may improve the efficacy of SP cell-based immunotherapy. Gene expression profiles were compared between sorted 4T1-SP and 4T1-NSP cells.
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Project description:CD95L is expressed by tumor-infiltrating lymphocytes to eliminate CD95-expressing tumor cells and thereby CD95 loss by tumor cells is often considered as a consequence of an immunoediting process. Nonetheless CD95 expression is maintained in most triple negative breast cancers (TNBCs), and we recently reported that CD95 loss in TNBC cells triggers the induction of a pro-inflammatory program promoting the recruitment of cytotoxic NK and CD8+ T-cells and impairing tumor growth. Using a comprehensive proteomic approach, we have identified two yet unknown CD95 interaction partners, Kip1 ubiquitination-promoting complex protein 2 (KPC2) and p65. KPC2 contributes to the partial degradation of p105 (NFκB1) and the subsequent generation of p50 homodimers, which transcriptionally represses pro-inflammatory NF-κB-driven gene expression. Mechanistically, KPC2 directly interacts with the C-terminal region of CD95 and links the receptor to RelA (p65) and KPC1, the catalytic subunit of the KPC complex that acts as E3 ubiquitin-protein ligase promoting the partial degradation of p105 into p50. Loss of CD95 in TNBC cells releases KPC2, limiting the formation of the NF-κB inhibitory homodimer complex (p50/p50), promoting NF-κB activation and the production of pro-inflammatory cytokines including CSF1, CSF2, CXCL1 and IL1 members, known to promote recruitment and differentiation of certain adaptive and innate immune effector cells.
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