Regulation and function of exosomal cargo loading in cancer progression

胞外货物负荷在肿瘤进展中的调节和作用 外切小体是50-150 nm的内小体来源的胞外囊泡，由大多数细胞分泌。这个 肿瘤细胞分泌的外切体含有高水平的致癌和免疫抑制蛋白 并在肿瘤进展、免疫抑制和治疗耐药中发挥重要作用。 外体货物装载是一个高度调控的过程，而ESCRT(内体分选复合体所需的 (运输)被认为在这一过程中起着关键作用。ESCRT通过促进膜起作用 内陷和囊泡的形成，识别和连接货物到膜上，并促进 将含有货物的囊泡释放到内体的管腔中。尽管多个ESCRT 已有报道具有致癌或抑瘤作用的成分，系统研究 ESCRT组分在肿瘤来源的外体中的作用仍然缺乏。我的长期研究目标是 了解外体货物装载的规则和特定外体分选对 肿瘤发生学。His结构域蛋白酪氨酸磷酸酶(HD-PTP)是ESCRT相关的接头蛋白 其推测的磷酸酶活性由PTPN23基因编码，位于人类染色体3p21.3， 该基因在肿瘤中经常被缺失。作为一种肿瘤抑制基因，频繁的PTPN23半合子缺失， 已经在癌症患者中检测到突变和HD-PTP表达减少。然而，这些机制 对于HD-PTP的肿瘤抑制作用，人们知之甚少。我的初步研究表明HD的丧失- PTP显著增加三阴性乳腺癌(TNBC)细胞胞外PD-L1水平，并提示 HD-PTP缺失的促肿瘤作用是通过胞外体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，其缺失通过增加胞外体促进肿瘤进展 为了检验这一假设，我将首先检验HD-PTP对外体PD-L1的影响是否 由其ESCRT功能或磷酸酶活性或两者共同介导。我还将进一步调查是否 外源PD-L1水平升高诱导HD-PTP缺失对小鼠的促瘤作用 模特们。接下来，我将研究依赖HD-PTP对胞外体和细胞PD-L1的调节机制 表情。为此，我将首先研究HD-PTP丢失对定位、细胞内运输的影响 以及PD-L1的翻译后修饰。将进行CRISPR/Cas9筛查以寻找新的途径 这介导了HD-PTP对细胞表面PD-L1的影响。最后，我将进行外体蛋白质组学研究 目的：系统探讨ESCRT机械调节失调对胞外体含量变化和功能的影响。 外切体在肿瘤进展、免疫反应和治疗耐药中的作用。在……里面 综上所述，这项研究将揭示ESCRT在肿瘤进展中的新功能，使其具有潜在的作用 外切体生物发生和货物装载的治疗靶向。

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.