Cellular Interactions with Thrombospondin

细胞与血小板反应蛋白的相互作用

基本信息

批准号：
10262702
负责人：
david d roberts
金额：
$ 155.14万
依托单位：
DIVISION OF CLINICAL SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10262702
关键词：
Acetylation Acute Angiogenesis Inhibition Anthracycline Antibodies Antigens Apoptosis Autophagocytosis Binding Blocking Antibodies Blood Platelets Blood Pressure Blood flow Bone Marrow C57BL/6 Mouse CD3 Antigens CD47 gene CD8-Positive T-Lymphocytes CD80 gene CD86 gene CTLA4 gene Cell Count Cell Proliferation Cell Surface Receptors Cell Survival Cell physiology Cells Cyclic GMP Cyclic GMP-Dependent Protein Kinases Cytotoxic Chemotherapy DNA Damage Data Effectiveness Etoposide Exhibits Extracellular Matrix Family suidae Gene Expression Gene Expression Profiling Genes Genetic Transcription Genotoxic Stress Glycoproteins Granzyme Growth Hemostatic function Histone Deacetylase Histone H3 Human Immunity Immunocompetent Immunologic Surveillance Impairment In Vitro Interferon Type II Ionizing radiation Irradiated tumor Ischemia Jurkat Cells KDR gene Kinetics Knockout Mice Ligands Maintenance Malignant Neoplasms Mediating Melanoma Cell Metabolism Methylation Mitochondria Mus Myocardium NOS3 gene Natural Increases Natural Killer Cells Nitric Oxide Non-Malignant Null Lymphocytes PC3 cell line Pathway interactions Patients Phagocytes Phenotype Play Promoter Regions Protons Radiation Injuries Radiation Protection Radiation Tolerance Radiation therapy Rattus Reactive Oxygen Species Receptor Cell Receptor Signaling Recovery Regulation Reperfusion Injury Reporting Resistance Role Signal Pathway Signal Transduction Signal Transduction Pathway Smooth Muscle Myocytes Soluble Guanylate Cyclase Stress Stromal Cells T-Cell Receptor T-Lymphocyte The Cancer Genome Atlas Therapeutic Therapeutic antibodies Thrombospondin 1 Thrombospondins Tissue Survival Tissues Topoisomerase Inhibitors Transgenic Mice Translations Tumor Immunity Tumor-infiltrating immune cells Vascular Smooth Muscle Wild Type Mouse adaptive immune response angiogenesis anti-cancer anti-tumor immune response base c-myc Genes cancer cell cancer immunotherapy cancer stem cell chemotherapy cytotoxic drug sensitivity exhaustion gene discovery high-throughput drug screening histone methylation immune cell checkpoints immune checkpoint improved ischemic injury knock-down mRNA Expression macrophage malignant breast neoplasm melanoma mouse model neoplastic cell novel promoter prostate cancer cell protein expression radiation resistance receptor recruit response selective expression self-renewal soft tissue stem cells targeted treatment therapeutic target tissue culture tissue stress transcription factor transcriptome tumor tumor microenvironment tumor progression tumor xenograft

项目摘要

Elevated CD47 expression in some cancers is associated with decreased survival and limited clearance by phagocytes expressing the CD47 counterreceptor SIRPalpha. In contrast, elevated CD47 mRNA expression in human melanomas was associated with improved survival. Gene-expression data were analyzed to determine a potential mechanism for this apparent protective function and suggested that high CD47 expression increases recruitment of natural killer (NK) cells into the tumor microenvironment. The CD47 ligand thrombospondin-1 inhibited NK cell proliferation and CD69 expression in vitro Cd47-/- NK cells correspondingly displayed augmented effector phenotypes, indicating an inhibitory function of CD47 on NK cells. Treating human NK cells with a CD47 antibody that blocks thrombospondin-1 binding abrogated its inhibitory effect on NK cell proliferation. Similarly, treating wild-type mice with a CD47 antibody that blocks thrombospondin-1 binding delayed B16 melanoma growth, associating with increased NK cell recruitment and increased granzyme B and interferon-gamma levels in intratumoral NK but not CD8+ T cells. However, B16 melanomas grew faster in Cd47-/- than in wild-type mice. Melanoma-bearing Cd47-/- mice exhibited decreased splenic NK cell numbers, with impaired effector protein expression and elevated exhaustion markers. Proapoptotic gene expression in Cd47-/- NK cells was associated with stress-mediated increases in mitochondrial proton leak, reactive oxygen species, and apoptosis. Global gene-expression profiling in NK cells from tumor-bearing mice identified CD47-dependent transcriptional responses that regulate systemic NK activation and exhaustion. Therefore, CD47 positively and negatively regulates NK cell function, and therapeutic antibodies that block inhibitory CD47 signaling can enhance NK immune surveillance of melanomas. Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translation-blocking morpholino alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 morpholino resulted in the greatest increase in intratumoral granzyme B, interferon-gamma, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors. Knockdown or gene disruption of CD47 protects non-malignant cells from genotoxic stress caused by ionizing radiation or cytotoxic chemotherapy but sensitizes tumors in an immune competent host to genotoxic stress. The selective radioprotection of non-malignant cells is mediated in part by enhanced autophagy and protection of anabolic metabolism pathways, but differential H2AX activation kinetics suggested that the DNA damage response is also CD47-dependent. A high throughput screen of drug sensitivities indicated that CD47 expression selectively sensitizes Jurkat T cells to inhibitors of topoisomerases, which are known targets of Schlafen-11 (SLFN11). CD47 mRNA expression positively correlated with schlafen-11 mRNA expression in a subset of human cancers but not the corresponding non-malignant tissues. CD47 mRNA expression was also negatively correlated with SLFN11 promoter methylation in some cancers. CD47 knockdown, gene disruption, or treatment with a CD47 function-blocking antibody decreased SLFN11 expression in Jurkat cells. The CD47 signaling ligand thrombospondin-1 also suppressed schlafen-11 expression in wild type but not CD47-deficient T cells. Re-expressing SLFN11 restored radiosensitivity to a CD47-deficient Jurkat cells. Disruption of CD47 in PC3 prostate cancer cells similarly decreased schlafen-11 expression and was associated with a CD47-dependent decrease in acetylation and increased methylation of histone H3 in the SLFN11 promoter region. The ability of histone deacetylase or topoisomerase inhibitors to induce SLFN11 expression in PC3 cells was lost when CD47 was targeted in these cells. Disrupting CD47 in PC3 cells increased resistance to etoposide but, in contrast to Jurkat cells, not to ionizing radiation. These data identify CD47 as a context-dependent regulator of SLFN11 expression and suggest an approach to improve radiotherapy and chemotherapy responses by combining with CD47-targeted therapeutics.

某些癌症中的CD47表达升高与表达CD47反受体sirpalpha的吞噬细胞的存活率降低和有限的清除有关。相反，人黑色素瘤中CD47 mRNA表达升高与提高的生存有关。分析了基因表达数据，以确定这种明显的保护功能的潜在机制，并表明高CD47表达会增加自然杀伤（NK）细胞的募集到肿瘤微环境中。 CD47配体血小板素-1在体外CD47 - / - NK细胞中抑制了NK细胞增殖和CD69表达，相应地显示出增强的效应子表型，表明CD47对NK细胞的抑制作用。用CD47抗体处理人NK细胞，该抗体阻断血小板素-1结合，废除了其对NK细胞增殖的抑制作用。同样，用CD47抗体处理野生型小鼠，该抗体阻断了血小板蛋白-1结合的延迟B16黑色素瘤的生长，与NK细胞募集的增加，颗粒酶B和肿瘤内NK中的干扰素 - γ水平增加，但不是CD8+ T细胞。但是，在CD47 - / - 中B16黑色素瘤的生长速度要比野生型小鼠快。含有黑色素瘤的CD47 - / - 小鼠表现出脾NK细胞数量降低，效应蛋白表达受损和衰竭标记升高。 CD47 - / - NK细胞中的凋亡基因表达与应激介导的线粒体质子泄漏，活性氧和凋亡的增加有关。含有肿瘤小鼠的NK细胞中的全球基因表达分析确定了调节全身NK激活和精疲力尽的CD47依赖性转录反应。因此，CD47对NK细胞功能进行积极和负面的调节，并且阻断抑制性CD47信号传导的治疗抗体可以增强黑色素瘤的NK免疫监测。针对T细胞免疫检查点细胞毒性T淋巴细胞抗原-4（CTLA4）的抗体增强了放射疗法对黑色素瘤患者的有效性，但许多人仍然具有抗性。为了进一步提高响应率，我们探索了将抗CTLA4阻滞与CD47的反义抑制（一种限制T细胞的抑制受体的反义抑制）的结合，该抑制受体限制了T细胞受体信号传导和杀死受照射靶细胞的杀伤。来自癌症基因组地图集的人类黑色素瘤数据揭示了CD47 mRNA表达与T细胞调节剂的表达之间的正相关，包括CTLA4及其反受体CD80和CD86。单独使用翻译障碍的形态抑制了CD47对人T细胞对人T细胞的抑制作用，或与抗CTLA4相结合，增强了抗原依赖性的抗原依赖性杀伤辐照性黑素瘤细胞。相应地，使用CD47和CTLA4的联合封闭的局部辐照的B16F10黑色素瘤的治疗显着提高了小鼠相对于任何一种治疗的生存率。单独或与抗CTLA4结合使用CD47 m增加了辐照肿瘤中CD3+ T细胞的浸润。抗CTLA4还增加了CD3+和CD8+ T细胞浸润以及非辐照肿瘤中NK细胞的标记。抗CTLA4与CD47变形杆结合，导致肿瘤内颗粒状B，Interferon-Gamma和NK细胞标记mRNA表达的增加最大。这些数据表明，将CTLA4和CD47封锁结合起来可以通过增强辐照肿瘤中的适应性T-和NK细胞免疫来提供生存益处。 CD47的敲低或基因破坏可保护非恶性细胞免受因电离辐射或细胞毒性化学疗法而引起的遗传毒性应激，但会使免疫胜任宿主中的肿瘤敏感到遗传毒性应激。非恶性细胞的选择性辐射保护部分是通过增强的自噬和对合成代谢代谢途径的保护的部分介导的，但是差异H2AX激活动力学表明，DNA损伤响应也依赖于CD47。药物敏感性的高吞吐量表明，CD47表达选择性地将Jurkat T细胞敏感到拓扑异构酶的抑制剂，这是Schlafen-11的已知靶标（SLFN11）。 CD47 mRNA表达与人类癌的子集中的schlafen-11 mRNA表达正相关，而与相应的非恶性组织无关。在某些癌症中，CD47 mRNA表达也与SLFN11启动子甲基化负相关。 CD47敲低，基因破坏或用CD47功能阻断抗体治疗降低了Jurkat细胞中SLFN11的表达。 CD47信号配体血小板素-1还抑制了野生型中的schlafen-11表达，但不抑制CD47缺陷型T细胞。重新表达SLFN11将放射敏性恢复到CD47缺陷的Jurkat细胞。 PC3前列腺癌细胞中CD47的破坏类似地降低了Schlafen-11表达，并且与SLFN11启动子区域中CD47依赖性降低和组蛋白H3的甲基化增加有关。当这些细胞中CD47靶向时，组蛋白脱乙酰基酶或拓扑异构酶抑制剂在PC3细胞中诱导SLFN11表达的能力失去了能力。破坏PC3细胞中的CD47增加了对依托泊苷的抗性，但与Jurkat细胞相比而不是电离辐射。这些数据将CD47识别为SLFN11表达的上下文依赖性调节剂，并提出了一种通过与CD47靶向的治疗剂结合的方法来改善放射疗法和化学疗法反应的方法。