RESUMO
In many solid tumors including triple-negative breast cancer (TNBC), upregulation of the interleukin-4 receptor (IL-4R) has been shown to promote cancer cell proliferation, apoptotic resistance, metastatic potential, and a Th2 response in the tumor microenvironment (TME). Since immunosuppressive cells in the TME and spleen including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) also express the IL-4R, we hypothesized that selective depletion of IL-4R-bearing cells in TNBC would result in the direct killing of tumor cells and the depletion of immunosuppressive cells and lead to an enhanced antitumor response. To selectively target IL-4R+ cells, we employed DABIL-4, a fusion protein toxin consisting of the catalytic and translocation domains of diphtheria toxin fused to murine IL-4. As anticipated, DABIL-4 has potent cytotoxic activity against TNBC cells both in vitro and in vivo. We demonstrate in the murine 4T1 TNBC model that DABIL-4 significantly reduces tumor growth, splenomegaly, and lung metastases. Importantly, we also show that the administration of DABIL-4 results in the selective depletion of MDSCs, TAMs, and regulatory T cells in treated mice, with a concomitant increase in IFN-γ+ CD8 effector T cells in the TME. Since the 4T1 antitumor activity of DABIL-4 was largely diminished in IL-4R knockout mice, we postulate that DABIL-4 functions primarily as an immunotherapeutic by the depletion of MDSCs, TAMs, and regulatory T cells. NanoString analysis of control and treated tumors confirmed and extended these observations by showing a marked decline of mRNA transcripts that are associated with tumorigenesis and metastasis. In conclusion, we demonstrate that DABIL-4 targeting of both tumor and immunosuppressive host cells likely represents a novel and effective treatment strategy for 4T1 TNBC and warrants further study.
Assuntos
Adenocarcinoma/tratamento farmacológico , Células Supressoras Mieloides/efeitos dos fármacos , Proteínas Recombinantes de Fusão/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Contagem de Células , Linhagem Celular Tumoral , Feminino , Humanos , Interleucina-4/química , Interleucina-4/uso terapêutico , Subunidade alfa de Receptor de Interleucina-4/química , Subunidade alfa de Receptor de Interleucina-4/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Terapia de Alvo Molecular , Células Supressoras Mieloides/patologia , Proteínas Recombinantes de Fusão/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.
Assuntos
Receptores de Citocinas/metabolismo , Fenômenos Biofísicos , Linhagem Celular , Cristalografia por Raios X , Endocitose , Variação Genética , Células HeLa , Humanos , Interleucina-13/química , Interleucina-13/genética , Interleucina-13/metabolismo , Subunidade alfa1 de Receptor de Interleucina-13/química , Subunidade alfa1 de Receptor de Interleucina-13/genética , Subunidade alfa1 de Receptor de Interleucina-13/metabolismo , Subunidade alfa de Receptor de Interleucina-4/química , Subunidade alfa de Receptor de Interleucina-4/genética , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Ligantes , Modelos Biológicos , Modelos Moleculares , Fosforilação , Ligação Proteica , Engenharia de Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Receptores de Citocinas/química , Receptores de Citocinas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT6/metabolismo , Transdução de SinaisRESUMO
Interleukin-4 (IL-4) and Interleukin-13 (IL-13), key cytokines in the pathogenesis of allergic inflammatory disease, mediate their effects via a receptor composed of IL-13Rα1 and IL-4Rα. A third (decoy) receptor called IL-13Rα2 regulates interleukin signaling through this receptor complex. We employed a variety of biophysical and cell-based techniques to decipher the role of this decoy receptor in mediating IL-4 signaling though the IL-4Rα-IL-13Rα1 receptor complex. Surface plasmon resonance (SPR) analysis showed that IL-13Rα2 does not bind IL-4, and does not affect binding of IL-4 to IL-4Rα. These results indicate that the extracellular domains of IL-4Rα and IL-13Rα2 are not involved in the regulation of IL-4 signaling by IL-13Rα2. We next used a two-hybrid system to show that the cytoplasmic domains of IL-4Rα and IL-13Rα2 interact, and that the secondary structure of the IL-13Rα2 intracellular domain is critical for this interaction. The cellular relevance of this interaction was next investigated. BEAS-2B bronchial epithelial cells that stably express full length IL-13Rα2, or IL-13Rα2 lacking its cytoplasmic domain, were established. Over expression of IL-13Rα2 attenuated IL-4 and IL-13 mediated STAT6 phosphorylation. IL-13Rα2 lacking its cytoplasmic domain continued to attenuate IL-13-mediated signaling, but had no effect on IL-4-mediated STAT6 signaling. Our results suggest that the physical interaction between the cytoplasmic domains of IL-13Rα2 and IL-4Rα regulates IL-4 signaling through the IL-4Rα-IL-13Rα1 receptor complex.
Assuntos
Subunidade alfa2 de Receptor de Interleucina-13/química , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Subunidade alfa de Receptor de Interleucina-4/química , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Sítios de Ligação , Linhagem Celular , Citocinas/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-4/metabolismo , Estrutura Secundária de Proteína , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Ressonância de Plasmônio de SuperfícieRESUMO
Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Ralpha receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.