Dataset Information

Calpain-dependent clearance of the autophagy protein p62/SQSTM1 is a contributor to ?PK oncolytic activity in melanoma.

ABSTRACT: Oncolytic virotherapy is a promising strategy for reducing tumor burden through selective virus replication in rapidly proliferating cells. However, the lysis of slowly replicating cancer stem cells (CSCs), which maintain neoplastic clonality, is relatively modest and the potential contribution of programmed cell death pathways to oncolytic activity is still poorly understood. We show that the oncolytic virus ?PK lyses CSC-enriched breast cancer and melanoma 3D spheroid cultures at low titers (0.1 pfu/cell) without resistance development and it inhibits the 3D growth potential (spheroids and agarose colonies) of melanoma and breast cancer cells. ?PK induces calpain activation in both melanoma and breast cancer 3D cultures as determined by the loss of the p28 regulatory subunit, and 3D growth is restored by treatment with the calpain inhibitor PD150606. In melanoma, ?PK infection also induces light chain 3 (LC3)-II accumulation and p62/SQSTM1 clearance, both markers of autophagy, and 3D growth is restored by treatment with the autophagy inhibitor chloroquine (CQ). However, expression of the autophagy-required protein Atg5 is not altered and CQ does not restore p62/SQSTM1 expression, suggesting that the CQ effect may be autophagy-independent. PD150606 restores expression of p62/SQSTM1 in ?PK-infected melanoma cultures, suggesting that calpain activation induces anti-tumor activity through p62/SQSTM1 clearance.

SUBMITTER: Colunga A

PROVIDER: S-EPMC3975656 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Calpain-dependent clearance of the autophagy protein p62/SQSTM1 is a contributor to ΔPK oncolytic activity in melanoma.

Colunga A A Bollino D D Schech A A Aurelian L L

Gene therapy 20140220 4

Oncolytic virotherapy is a promising strategy for reducing tumor burden through selective virus replication in rapidly proliferating cells. However, the lysis of slowly replicating cancer stem cells (CSCs), which maintain neoplastic clonality, is relatively modest and the potential contribution of programmed cell death pathways to oncolytic activity is still poorly understood. We show that the oncolytic virus ΔPK lyses CSC-enriched breast cancer and melanoma 3D spheroid cultures at low titers (0 ...[more]

PMID: 24553345

Similar Datasets

Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus devastating the global swine industry. DEAD-box helicases (DDXs) are a family of ATP-dependent RNA helicases that are predominantly implicated in modulating cellular RNA metabolism. Meanwhile, a growing number of studies have suggested that some DDXs are associated with innate immunity and virus infection, so they are considered potential antiviral targets. Herein, we screened 40 DDXs and found that ectopic expression of DDX10 exhibited a significant anti-PRRSV effect, while DDX10 knockdown promoted PRRSV proliferation. Further analysis revealed that DDX10 positively regulates type I interferon production, which may contribute to its anti-PRRSV effect. Interestingly, PRRSV infection promoted DDX10 translocation from the nucleus to the cytoplasm for macroautophagic/autophagic degradation to block the antiviral effect of DDX10. By screening PRRSV-encoded proteins, we found that the viral envelope (E) protein interacted with DDX10. In line with the autophagic degradation of DDX10 during PRRSV infection, E protein could induce autophagy and reduce DDX10 expression in wild-type cells, but not in ATG5 or ATG7 knockout (KO) cells. When further screening the cargo receptors for autophagic degradation, we found that SQSTM1/p62 (sequestosome 1) interacted with both DDX10 and E protein, and E protein-mediated DDX10 degradation was almost entirely blocked in SQSTM1 KO cells, demonstrating that E protein degrades DDX10 by promoting SQSTM1-mediated selective autophagy. Our study reveals a novel mechanism by which PRRSV escapes host antiviral innate immunity through selective autophagy, providing a new target for developing anti-PRRSV drugs.Abbreviations: ACTB: actin beta; ATG: autophagy related; co-IP: co-immunoprecipitation; CQ: chloroquine; DDX10: DEAD-box helicase 10; E: envelope; EGFP: enhanced green fluorescent protein; hpi: hours post infection; hpt: hours post transfection; IFA: indirect immunofluorescence assay; IFN-I: type I IFN; IFNB/IFN-β: interferon beta; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; mAb: monoclonal antibody; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NFKB/NF-κB: nuclear factor kappa B; OPTN: optineurin; ORF: open reading frame; PRRSV: porcine reproductive and respiratory syndrome virus; SeV: sendai virus; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID50: 50% tissue culture infective dose; WT: wild type.

Project description:The microtubule (MT) is a highly dynamic polymer that functions in various cellular processes through MT hyperacetylation. Thus, many viruses have evolved mechanisms to hijack the MT network of the cytoskeleton to allow intracellular replication of viral genomic material. Coronavirus non-structural protein 8 (nsp8), a component of the viral replication transcriptional complex, is essential for viral survival. Here, we found that nsp8 of porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with a zoonotic potential, inhibits interferon (IFN)-β production by targeting melanoma differentiation gene 5 (MDA5), the main pattern recognition receptor for coronaviruses in the cytoplasm. Mechanistically, PDCoV nsp8 interacted with MDA5 and induced autophagy to degrade MDA5 in wild-type cells, but not in autophagy-related (ATG)5 or ATG7 knockout cells. Further screening for autophagic degradation receptors revealed that nsp8 interacts with sequestosome 1/p62 and promotes p62-mediated selective autophagy to degrade MDA5. Importantly, PDCoV nsp8 induced hyperacetylation of MTs, which in turn triggered selective autophagic degradation of MDA5 and subsequent inhibition of IFN-β production. Overall, our study uncovers a novel mechanism employed by PDCoV nsp8 to evade host innate immune defenses. These findings offer new insights into the interplay among viruses, IFNs, and MTs, providing a promising target to develop anti-viral drugs against PDCoV.IMPORTANCECoronavirus nsp8, a component of the viral replication transcriptional complex, is well conserved and plays a crucial role in viral replication. Exploration of the role mechanism of nsp8 is conducive to the understanding of viral pathogenesis and development of anti-viral strategies against coronavirus. Here, we found that nsp8 of PDCoV, an emerging enteropathogenic coronavirus with a zoonotic potential, is an interferon antagonist. Further studies showed that PDCoV nsp8 interacted with MDA5 and sequestosome 1/p62, promoting p62-mediated selective autophagy to degrade MDA5. We further found that PDCoV nsp8 could induce hyperacetylation of MT, therefore triggering selective autophagic degradation of MDA5 and inhibiting IFN-β production. These findings reveal a novel immune evasion strategy used by PDCoV nsp8 and provide insights into potential therapeutic interventions.

Dataset Information

Calpain-dependent clearance of the autophagy protein p62/SQSTM1 is a contributor to ?PK oncolytic activity in melanoma.

Publications

Calpain-dependent clearance of the autophagy protein p62/SQSTM1 is a contributor to ΔPK oncolytic activity in melanoma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets