Transcriptomic analysis of STUB1 knockdown in HEK293 cells under normal and heat-shock conditions
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE310700 | GEO | 2025/11/27
REPOSITORIES: GEO
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Project description:Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNg) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNg response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models – with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNg sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.
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Project description:Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNg) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNg response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models - with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNg sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.
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Project description:Transcriptomic analysis of STUB1 knockdown in HEK293 cells under normal and heat-shock conditions
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Project description:Chronic hypoxia induces pulmonary vascular remodeling and pulmonary hypertension (PH). While it is established that transcription factors, hypoxia-inducible factors (HIF-1α/HIF-2α) activate gene programs that drive hypoxia-induced PH, the mechanism of HIF-1/2 activation is less clear. Here, we report that carboxylterminus of Hsp70-interacting protein (CHIP or Stub1) modulates HIF-1α and HIF-2α transcription rather than reducing their stability. Knocking-down Stub1 reduced hypoxic activation of HIF-1α mRNA, protein, and activity while enhancing hypoxic induction of HIF-2α mRNA, protein, and target genes in pulmonary vascular cells. Mechanistically, CBP/p300-mediated acetylation of lysine (K287) inactivates the ubiquitin ligase activity of Stub1 and triggers its translocation from the cytoplasm into the nucleus. There, it recognizes the HIF promoter and hypoxia response elements (HREs) in target genes. Expression of Stub1-K287Q mutant (mimicking acetylation) enhanced hypoxia-induced HIF-1α expression, while acetyl-deficient Stub1-K287R mutant had the opposite effect on HIF-α but enhanced hypoxia-induced HIF-2α transcriptional activity. Endothelial-Stub1 transgenic mice tolerated chronic hypoxia better, had less pulmonary vascular remodeling, reduced pulmonary vascular resistance, and greater cardioprotection. Thus, Stub1 nuclear translocation enhances hypoxic induction of HIF-1α activity while suppressing deleterious effects of HIF-2α. These observations indicate that nuclear-Stub1 synergizes with HIF-1α to promote transcriptional responses and antagonizes HIF2α-driven PH in chronic hypoxia.
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Project description:The cytokine IFNγ differentially impacts on tumors upon immune checkpoint blockade (ICB). Despite our understanding of downstream signaling events, less is known about 36 regulation of its receptor (IFNγ-R1). With an unbiased genome-wide CRISPR/Cas9 screen for critical regulators of IFNγ-R1 cell surface abundance, we identified STUB1 as an E3 ubiquitin ligase for IFNγ-R1 in complex with its signal-relaying kinase JAK1. STUB1 mediates ubiquitination-dependent proteasomal degradation of IFNγ-R1/JAK1 complex through IFNγ-R1K285 and JAK1K249. Conversely, STUB1 inactivation amplifies IFNγ signaling, sensitizing tumor cells to cytotoxic T cells in vitro. This was corroborated by an anticorrelation between STUB1 expression and IFNγ response in ICB-treated patients. Consistent with the context-dependent effects of IFNγ in vivo, anti-PD-1 response was increased in heterogenous tumors comprising both wildtype and STUB1-deficient cells but not full STUB1 knockout tumors. These results uncover STUB1 as a critical regulator of IFNγ-R1, and highlight the context-dependency of STUB1-regulated IFNγ signaling for ICB outcome.
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