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HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma-induced apoptosis.

ABSTRACT: The success of cancer immunotherapy is limited to a subset of patients, highlighting the need to identify the processes by which tumors evade immunity. Using CRISPR/Cas9 screening, we reveal that melanoma cells lacking HOIP, the catalytic subunit of LUBAC, are highly susceptible to both NK and CD8⁺ T-cell-mediated killing. We demonstrate that HOIP-deficient tumor cells exhibit increased sensitivity to the combined effect of the inflammatory cytokines, TNF and IFN-γ, released by NK and CD8⁺ T cells upon target recognition. Both genetic deletion and pharmacological inhibition of HOIP augment tumor cell sensitivity to combined TNF and IFN-γ. Together, we unveil a protective regulatory axis, involving HOIP, which limits a transcription-dependent form of cell death that engages both intrinsic and extrinsic apoptotic machinery upon exposure to TNF and IFN-γ. Our findings highlight HOIP inhibition as a potential strategy to harness and enhance the killing capacity of TNF and IFN-γ during immunotherapy.

SUBMITTER: Freeman AJ

PROVIDER: S-EPMC8567220 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma-induced apoptosis.

Freeman Andrew J AJ Vervoort Stephin J SJ Michie Jessica J Ramsbottom Kelly M KM Silke John J Kearney Conor J CJ Oliaro Jane J

EMBO reports 20210901 11

The success of cancer immunotherapy is limited to a subset of patients, highlighting the need to identify the processes by which tumors evade immunity. Using CRISPR/Cas9 screening, we reveal that melanoma cells lacking HOIP, the catalytic subunit of LUBAC, are highly susceptible to both NK and CD8<sup>+</sup> T-cell-mediated killing. We demonstrate that HOIP-deficient tumor cells exhibit increased sensitivity to the combined effect of the inflammatory cytokines, TNF and IFN-γ, released by NK and ...[more]

PMID: 34467615

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Project description:The objective of this study was to examine the combined effect of Interferon-gamma (IFN-γ) and Tumor Necrosis factor-alpha (TNF-α) on cytotoxicity and expression of prostate apoptosis response-4 (Par-4) and Par-4 interacting proteins B-cell lymphoma (Bcl-2), nuclear factor kappa-light-chain-enhancer of activated B cells/p65 subunit (NF-κB/p65), Ak mouse strain thymoma (Akt) in human neuroblastoma (NB) cells. Materials and methods included human neuroblastoma cell lines-SK-N-MC, SK-N-SH, and SH-SY5Y, which were treated with IFN-γ and TNF-α individually, or in combination, and were assessed for viability by tetrazolium (MTT) assay. Apoptosis was monitored by hypodiploid population (by flow cytometry), DNA fragmentation, Poly (ADP-ribose) polymerase (PARP) cleavage, and caspase-8 activity. Transcript level of Par-4 was measured by RT-PCR. Protein levels of Par-4 and suppressor of cytokine signaling 3 (SOCS-3) were assessed by immunoblotting. Cellular localization of Par-4 and p65 was examined by immunofluorescence. Unbiased transcript analysis for IFN-γ, TNF-α, and Par-4 were analyzed from three independent clinical datasets from neuroblastoma patients. In terms of results, SK-N-MC cells treated with a combination of, but not individually with, IFN-γ and TNF-α induced apoptosis characterized by hypodiploidy, DNA fragmentation, PARP cleavage, and increased caspase-8 activity. Apoptosis was associated with up-regulation of Par-4 mRNA and protein expression. Immunofluorescence studies revealed that Par-4 was localized exclusively in cytoplasm in SK-N-MC cells cultured for 24 h. but showed nuclear localization at 48 h. Treatment with IFN-γ and TNF-α together enhanced the intensity of nuclear Par-4. In gene expression, data from human neuroblastoma patients, levels of IFN-γ, and TNF-α have strong synergy with Par-4 expression and provide good survival advantage. The findings also demonstrated that apoptosis was associated with reduced level of pro-survival proteins-Bcl-2 and Akt and NF-κB/p65. Furthermore, the apoptotic effect induced by IFN-γ-induced Signal Transducer and Activator of Transcription-1(STAT-1), and could be due to down-regulation of suppressor of cytokine signaling-3 (SOCS3). The study concludes that a combinatorial approach using IFN-γ and TNF-α can be explored to maximize the effect in chemotherapy in neuroblastoma, and implies a role for Par-4 in the process.

Dataset Information

HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma-induced apoptosis.

Publications

HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma-induced apoptosis.

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