RESUMO
STING (Stimulator of Interferon Genes) is an endoplasmic reticulum-anchored adaptor of the innate immunity best known to trigger pro-inflammatory cytokine expression in response to pathogen infection. In cancer, this canonical pathway can be activated by intrinsic or drug-induced genomic instability, potentiating antitumor immune responses. Here we report that STING downregulation decreases cell survival and increases sensitivity to genotoxic treatment in a panel of breast cancer cell lines in a cell-autonomous manner. STING silencing impaired DNA Damage Response (53BP1) foci formation and increased DNA break accumulation. These newly identified properties were found to be independent of STING partner cGAS and of its canonical pro-inflammatory pathway. STING was shown to partially localize at the inner nuclear membrane in a variety of breast cancer cell models and clinical tumor samples. Interactomics analysis of nuclear STING identified several proteins of the DNA Damage Response, including the three proteins of the DNA-PK complex, further supporting a role of STING in the regulation of genomic stability. In breast and ovarian cancer patients that received adjuvant chemotherapy, high STING expression is associated with increased risk of relapse. In summary, this study highlights an alternative, non-canonical tumor-promoting role of STING that opposes its well-documented function in tumor immunosurveillance.
Assuntos
Neoplasias da Mama/prevenção & controle , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Proteínas de Membrana/metabolismo , Recidiva Local de Neoplasia/prevenção & controle , Nucleotidiltransferases/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Proteínas de Membrana/genética , Camundongos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Nucleotidiltransferases/genética , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Because of their favorable properties as macromolecular drugs, antibodies are a very successful therapeutic modality for interfering with disease-relevant targets in the extracellular space or at the cell membrane. However, a large number of diseases involve cytosolic targets and designing antibodies able to efficiently reach intracellular compartments would expand the antibody-tractable conditions. Here, we genetically fused cell penetrating peptides (CPPs) at various positions to an antibody targeting cancer cells, evaluated the developability features of the resulting antibody-peptide fusions and the ability of selected constructs to reach the cytosol. We first determined positions in the IgG structure that were permissive to CPP incorporation without destabilizing the antibody. Fusing CPPs to the C-terminus of the light chain and either before or after the hinge had the least effect on antibody developability features. These constructs were further evaluated for cell penetration efficiency. Two out of five tested CPPs significantly enhanced antibody penetration into the cytosol, in particular when fused before or after the hinge. Finally, we demonstrate that specific antibody binding to the cell surface target is necessary for efficient cell penetration of the CPP-antibody fusions. This study provides a solid basis for further exploration of therapeutic antibodies for intracellular targets.
Assuntos
Anticorpos/administração & dosagem , Peptídeos Penetradores de Células/administração & dosagem , Sistemas de Liberação de Medicamentos , Animais , Antígeno Carcinoembrionário/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Separação Celular , Citoplasma/metabolismo , Citosol/metabolismo , Espaço Extracelular , Citometria de Fluxo , Proteínas Ligadas por GPI/química , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Imunoglobulina G/administração & dosagem , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Proteínas Recombinantes de Fusão/administração & dosagem , Ressonância de Plasmônio de SuperfícieRESUMO
[This corrects the article DOI: 10.18632/oncotarget.12858.].
RESUMO
Activation of the IFN/STAT1 pathway is closely associated with drug response and recurrence of breast cancer treated by chemotherapy. The aim of the current study was to elucidate the molecular mechanisms involved upstream and downstream of this pathway in order to identify distinct entities that might be manipulated to improve treatment efficacy. Four breast cancer cell lines (T-47D, MCF7, MDA-MB-231 and HBCx-19 established from the eponymous PDX) were treated in vitro with mafosfamide, a DNA damage inducer. In two of these cell lines (MCF7 and HBCx-19), genotoxic treatment upregulated type I IFN expression leading to paracrine activation of IFN/STAT1 signaling pathway after 6-8 days. We show that STING, a well-characterized inducer of IFN in immune cells, is rapidly triggered in MCF7 cells under genotoxic stress and forms nuclear foci that co-localize with phosphorylated IRF-3 and γH2AX. STING silencing abrogated chemotherapy-induced type I IFN production and signaling and potentiated genotoxic treatment efficacy as it promoted cell death extent and delayed cell colony regrowth. Similar results were obtained after silencing PARP12, one selected gene of the IFN/STAT1 pathway fingerprint. In summary, this study provides the first demonstration of STING activation in breast cancer cells. Our data suggest that genotoxic-induced, STING-mediated type I IFN signaling is a cell-intrinsic mechanism of breast cancer cell survival and regrowth.
Assuntos
Neoplasias da Mama/genética , Ciclofosfamida/análogos & derivados , Proteínas de Membrana/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Ciclofosfamida/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferons/metabolismo , Células MCF-7 , Comunicação Parácrina/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
BACKGROUND: Oestrogen receptor-negative (ER-) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer. METHODS: Gene and protein expression profiles were analysed in a panel of ER- breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively. RESULTS: The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells. CONCLUSIONS: Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER- breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.
