ALCAM-mediated cell adhesion and extracellular vesicle biogenesis in bladder cancer

膀胱癌中 ALCAM 介导的细胞粘附和细胞外囊泡生物发生

• 批准号: 10204708
• 负责人: Ariana Kathryn von Lersner
• 金额: $ 3.08万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10204708
• 关键词: 3-Dimensional; ALCAM gene; Activated-Leukocyte Cell Adhesion Molecule; Adaptor Signaling Protein; Adhesions; Adhesives; Affect; Alternative Splicing; Binding; Biogenesis; Biological; Biology; Birds; Bladder; Bladder papilloma; Cancer Patient; Cancer Relapse; Carcinoma; Cell Adhesion; Cell Communication; Cell Line; Cell membrane; Cells; Chemotaxis; Cytometry; Cytoplasmic Tail; DLG4 gene; Data; Detection; Development; Disease; Disease Progression; Electron Microscopy; Embryo; Environment; Event; Goals; Immunoblotting; Immunoglobulin Domain; Immunoglobulins; In Vitro; Intercellular Junctions; Intervention; Laboratories; Length; Leukocyte Adhesion Molecules; Link; Lipid Bilayers; Liquid substance; Literature; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane; Modeling; Molecular; Monitor; Multivesicular Body; Neoplasm Metastasis; Pathologic; Pathway interactions; Physiology; Plasma; Production; Property; Protein Analysis; Protein Isoforms; Proteins; Publishing; Regulation; Research; Risk; Scaffolding Protein; Site; Techniques; Testing; Tight Junctions; Tumor Cell Migration; Tumor-Derived; Urine; Vesicle; Western Blotting; Work; base; cancer cell; cell motility; clinically relevant; exosome; experimental study; extracellular; extracellular vesicles; in vivo Model; insight; liquid biopsy; member; microvesicles; migration; molecular marker; mortality; nanoparticle; neoplastic cell; novel; particle; prevent; response; synergism; targeted treatment; treatment response; treatment strategy; tumor progression

PROJECT SUMMARY Extracellular vesicles (EVs) are a heterogenous group of lipid bilayer enclosed particles secreted from cells and are found in most, if not all, biological fluids. The term EV encompasses a wide range of vesicle classes, including exosomes (50-150 nm) and microvesicles (150 nm - 1000+ µm), derived from the endosomal pathway after fusion of multivesicular bodies with the plasma membrane or from direct budding from the plasma membrane, respectively. EVs are further characterized by membrane and luminal cargoes that give rise to distinct functional properties. An emerging body of literature suggests that EVs are major contributors in cell-cell communication in normal physiology and pathological events, such as cancer. EVs have been shown to affect cancer cell chemotaxis and metastatic organotropism. Notably, a specific class of EV is dependent upon syntenin-1, an adaptor protein that contains two PSD95/Dlg/zonula occludens 1 (PDZ) domains, for biogenesis and cargo loading through the endosomal pathway. This suggests that upstream effectors which bind to syntenin-1 could modulate its availability, and in turn, regulate exosome biogenesis and cargo loading. Activated Leukocyte Cell Adhesion Molecule (ALCAM) is an adhesion protein in the immunoglobulin superfamily found at sites of cell-cell junctions. ALCAM is a dynamic regulator of cell adhesion through differential shedding of its ectodomain. Our laboratory demonstrated that the full-length ALCAM Isoform (Iso1) resists shedding, promotes adhesion and limits metastasis. Conversely, the alternatively spliced isoform 2 (Iso2) is susceptible to shedding, facilitates motility and metastasis. ALCAM shedding and elevated expression of ALCAM Iso2 correlates with bladder cancer (BCa) disease progression. ALCAM can be linked to EV biogenesis through its association with the cytoplasmic scaffolding protein syntenin-1. Sequestering syntenin-1 via the cytoplasmic tail of ALCAM abrogates motility and metastasis, further underscoring the functional relevance of the regulatory mechanism. Additional preliminary studies revealed that the expression of Iso1 suppresses large EV biogenesis while Iso2-facilitates the biogenesis of large EVs. Based on these findings we hypothesize that ALCAM shedding controls syntenin-dependent EV biogenesis in tumor cells. In addition, we hypothesize that ALCAM-mediated adhesion can control the pro-migratory function of EVs by regulating their cargo. To test this hypothesis, I will utilize cell lines expressing different isoforms of ALCAM and BCa patient-derived plasma to determine the mechanism in which ALCAM-mediated cell adhesion affects EV biology. Experiments proposed in Aim 1 will investigate how modulation of a cells' adhesive state through ALCAM expression alters EV biogenesis and function. EV biogenesis will be assessed through complementary techniques of nanoparticle tracking analysis, western blotting, and microflow cytometry. EV function will be determined with the avian embryo model of metastasis and a 3D organotypic bladder model. Aim 2 will characterize the changes in EV cargos upon ALCAM shedding and disease progression in BCa through palmitoylated-mass spectrometry. Collectively, these data will define a relationship between cell adhesion EV biology, both mechanistically and functionally. Additionally, these findings will identify informative molecular markers to aid in the monitoring of disease state through a non-invasive means.

项目摘要 细胞外囊泡（EV）是从细胞分泌的一组异质的脂质双层封闭颗粒， 存在于大多数生物体液中术语EV包括广泛的囊泡类别，包括外来体 （50-150 nm）和微泡（150 nm - 1000+ µm），来源于多泡融合后的内体途径 体与质膜或从质膜直接出芽。EV更进一步 其特征在于产生不同功能性质的膜和腔货物。一个新兴的 文献表明EV是正常生理和病理事件中细胞-细胞通讯的主要贡献者， 例如癌症。EV已显示影响癌细胞趋化性和转移性器官向性。值得注意的是， 一类EV依赖于syntenin-1，syntenin-1是一种含有两个PSD 95/Dlg/闭合小带1（PDZ）的衔接蛋白， 结构域，用于通过内体途径的生物发生和货物装载。这表明，上游效应， 与syntenin-1结合可以调节其可用性，并进而调节外泌体生物发生和货物装载。激活 白细胞粘附分子（ALCAM）是免疫球蛋白超家族中的一种粘附蛋白，发现于 细胞间连接ALCAM是通过其胞外域的差异脱落的细胞粘附的动态调节剂。我们 一个实验室证明，全长ALCAM亚型（Iso 1）抵抗脱落，促进粘附和限制 转移相反，选择性剪接同种型2（Iso 2）易于脱落，促进运动性， 转移ALCAM脱落和ALCAM Iso 2表达升高与膀胱癌（BCa）疾病相关 进展ALCAM可以通过与细胞质支架蛋白的结合与EV的生物发生联系起来 合成蛋白-1。通过ALCAM的胞质尾区隔离syntenin-1消除了运动性和转移， 强调监管机制的功能相关性。进一步的初步研究表明， Iso 1的表达抑制大EV的生物发生，而Iso 2-促进大EV的生物发生。基于这些发现 我们假设ALCAM脱落控制肿瘤细胞中的突触蛋白依赖性EV生物发生。另外我们 假设ALCAM介导的粘附可以通过调节EV的货物来控制EV的促迁移功能。到 为了验证这一假设，我将利用表达ALCAM和BCa患者来源血浆的不同亚型的细胞系， 确定ALCAM介导的细胞粘附影响EV生物学的机制。目标1中提出的实验 将研究通过ALCAM表达调节细胞粘附状态如何改变EV生物发生和功能。 EV生物发生将通过纳米颗粒跟踪分析、蛋白质印迹和免疫印迹等补充技术进行评估。 微量流式细胞术EV功能将用转移的禽胚胎模型和3D器官型 膀胱模型目的2将描述BCa中ALCAM脱落和疾病进展时EV货物的变化 通过棕榈酰化-质谱法。总的来说，这些数据将定义细胞粘附EV 生物学，无论是机械上还是功能上。此外，这些发现将确定信息分子标记， 有助于通过非侵入性手段监测疾病状态。