Regulation and function of exosomal cargo loading in cancer progression

外泌体负载在癌症进展中的调节和功能

• 批准号: 10381669
• 负责人: Sheng Sun
• 金额: $ 17.82万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10381669
• 关键词: 3p21; Adaptor Signaling Protein; Affect; Binding; Biogenesis; Breast Cancer Cell; Breast Cancer Patient; CRISPR screen; CRISPR/Cas technology; Cancer Patient; Cell surface; Cells; Complex; Data; Deletion Mutation; Down-Regulation; Drug resistance; Endosomes; Environment; Epidermal Growth Factor Receptor; Foundations; Genes; Goals; Human Chromosomes; Immune response; Immunofluorescence Immunologic; Immunosuppression; Immunotherapeutic agent; Inbred BALB C Mice; Link; Longitudinal Studies; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Mus; NF-kappa B; Neoplasm Metastasis; Oncogenic; Pathway interactions; Phosphoric Monoester Hydrolases; Play; Post-Translational Protein Processing; Process; Protein Tyrosine Phosphatase; Proteins; Proteomics; Regulation; Reporting; Research; Role; Signal Transduction; Sorting - Cell Movement; Survival Rate; Tertiary Protein Structure; Testing; Therapeutic immunosuppression; Tumor Immunity; Tumor Suppressor Proteins; Tumor-Derived; Ubiquitination; Vesicle; Work; breast cancer progression; cancer cell; endosome lumen; exosome; extracellular vesicles; gain of function; genome wide screen; glycosylation; in vivo; intercellular communication; kinase inhibitor; knock-down; live cell imaging; mouse model; mutant; neoplastic cell; novel; overexpression; particle; programmed cell death ligand 1; protein complex; protein degradation; protein function; src-Family Kinases; stable cell line; therapeutic target; therapy resistant; trafficking; triple-negative invasive breast carcinoma; tumor; tumor growth; tumor progression; tumorigenesis; vesicular release

Regulation and function of exosomal cargo loading in cancer progression Exosomes are 50-150 nm, endosome-derived, extracellular vesicles that are secreted by most cells. The exosomes secreted by tumor cells contain increased levels of proteins with oncogenic and immunosuppressive functions and play important roles in tumor progression, immunosuppression and therapeutic resistance. Exosomal cargo loading is a highly regulated process, and ESCRTs (Endosomal Sorting Complex Required for Transport) are believed to be critically involved in this process. ESCRTs function by promoting membrane invagination and vesicle formation, recognizing and linking cargoes to the membrane and facilitating the release of the vesicles containing the cargoes into the lumen of endosomes. Although multiple ESCRT components have been reported to have oncogenic or tumor suppressor function, systematic studies of the roles of ESCRT components in tumor-derived exosomes are still lacking. My long-term research goal is to understand the regulation of exosomal cargo loading and the contribution of specific exosomal sorting to tumorigenesis. HD-PTP (His Domain Protein Tyrosine Phosphatase) is an ESCRT associated adaptor protein with putative phosphatase activity encoded by the PTPN23 gene, located on human chromosome 3p21.3, which is frequently deleted in tumors. As a tumor suppressor, frequent PTPN23 hemizygous deletions, mutations, and reduced HD-PTP expression have been detected in cancer patients. However, the mechanisms for tumor suppressor function of HD-PTP are poorly understood. My preliminary studies show that loss of HD- PTP significantly increases exosomal PD-L1 levels in triple negative breast cancer (TNBC) cells and suggest that the tumor promoting effects of HD-PTP loss is mediated by exosomal PD-L1. Thus, I hypothesize that HD- PTP functions as a tumor suppressor, loss of which promotes tumor progression through increasing exosomal PD-L1 level.To test the hypothesis, I will first examine whether the effect of HD-PTP on exosomal PD-L1 is mediated by its ESCRT function or phosphatase activity or both. I will also further investigate whether increased exosomal PD-L1 level induces the tumor promoting effect of HD-PTP loss that I observe in mouse models. Next, I will study the mechanisms for HD-PTP-dependent regulation of exosomal and cellular PD-L1 expression. For this purpose, I will first examine the effects HD-PTP loss on localization, intracellular trafficking and posttranslational modifications of PD-L1. CRISPR/Cas9 screen will be performed to look for novel pathway that mediates the effect of HD-PTP on cell surface PD-L1. Finally, I will perform exosomal proteomics studies to systematically explore the effects of dysregulation of ESCRT machinery on exosomal content change and the exosome-mediated function in tumor progression, immune response and therapeutic resistance. In summary, this research will unveil the novel functions of ESCRTs in tumor progression, enabling potential therapeutics targeting of exosome biogenesis and cargo loading.

外泌体货物装载在癌症进展中的调节和功能 外泌体是由大多数细胞分泌的50-150 nm、内体衍生的细胞外囊泡。的 肿瘤细胞分泌的外来体含有增加水平的具有致癌和免疫抑制作用的蛋白质， 在肿瘤进展、免疫抑制和治疗抗性中发挥重要作用。 外泌体货物装载是一个高度调节的过程，ESCRT（内体分选复合物， 运输）被认为是关键参与这一进程。ESCRTs通过促进膜 内陷和囊泡形成，识别和连接货物的膜，并促进 释放含有货物的囊泡进入内体的内腔。虽然多个ESCRT 已报道组分具有致癌或肿瘤抑制功能，对这些组分的系统研究表明， ESCRT组分在肿瘤来源的外来体中的作用仍然缺乏。我的长期研究目标是 了解外泌体货物装载的调节和特定外泌体分选对 肿瘤发生HD-PTP（His Domain Protein Tyrosine Phosphatase）是一种与ESCRT相关的衔接蛋白 具有由位于人染色体3p21.3上的PTPN 23基因编码的推定磷酸酶活性， 在肿瘤中经常缺失。作为一种肿瘤抑制因子，频繁的PTPN 23半合子缺失， 在癌症患者中检测到HD-PTP突变和HD-PTP表达降低。然而，机制 对HD-PTP的肿瘤抑制功能知之甚少。我的初步研究表明，HD的损失- PTP显著增加三阴性乳腺癌（TNBC）细胞中的外泌体PD-L1水平，并提示 HD-PTP丢失的肿瘤促进作用是由外泌体PD-L1介导的。因此，我假设HD- PTP作为一种肿瘤抑制因子发挥作用，其缺失通过增加外泌体表达促进肿瘤进展。 为了验证这一假设，我将首先检查HD-PTP对外泌体PD-L1的影响是否与HD-PTP的作用有关。 通过其ESCRT功能或磷酸酶活性或两者介导。我还将进一步调查 增加的外泌体PD-L1水平诱导了我在小鼠中观察到的HD-PTP损失的肿瘤促进作用 模型接下来，我将研究HD-PTP依赖性调节外泌体和细胞PD-L1的机制， 表情为此，我将首先检查HD-PTP丢失对定位、细胞内运输和细胞内转运的影响。 和PD-L1的翻译后修饰。将进行CRISPR/Cas9筛选以寻找新的途径 其介导HD-PTP对细胞表面PD-L1的作用。最后，我将进行外泌体蛋白质组学研究 系统地探讨ESCRT机制失调对外泌体含量变化的影响， 外泌体介导的肿瘤进展、免疫应答和治疗抗性中的功能。在 总之，这项研究将揭示ESCRT在肿瘤进展中的新功能， 靶向外泌体生物发生和货物装载的治疗剂。

项目成果

Parallel Genomic Alterations of Antigen and Payload Targets Mediate Polyclonal Acquired Clinical Resistance to Sacituzumab Govitecan in Triple-Negative Breast Cancer.

抗原和有效载荷靶标的平行基因组改变介导多克隆获得对三阴性乳腺癌的sacituzumab govitecan的临床抗性。

• DOI: 10.1158/2159-8290.cd-21-0702
• 发表时间: 2021-10
• 期刊: Cancer discovery
• 影响因子: 28.2
• 作者: Coates JT;Sun S;Leshchiner I;Thimmiah N;Martin EE;McLoughlin D;Danysh BP;Slowik K;Jacobs RA;Rhrissorrakrai K;Utro F;Levovitz C;Denault E;Walmsley CS;Kambadakone A;Stone JR;Isakoff SJ;Parida L;Juric D;Getz G;Bardia A;Ellisen LW
• 通讯作者: Ellisen LW