Meecham Amelia, Cutmore Lauren C, Protopapa Pantelitsa, Rigby Lauren G, Marshall John F
Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom.
University of California, San Diego, San Diego, CA, United States.
Front Cell Dev Biol. 2022 Aug 24;10:920303. doi: 10.3389/fcell.2022.920303. eCollection 2022.
The integrin αvβ6 is expressed at low levels in most normal healthy tissue but is very often upregulated in a disease context including cancer and fibrosis. Integrins use endocytosis and trafficking as a means of regulating their surface expression and thus their functions, however little is known of how this process is regulated in the context of αvβ6. As αvβ6 is a major target for the development of therapeutics in cancer and fibrosis, understanding these dynamics is critical in the development of αvβ6-targeted therapies. Following development of a flow cytometry-based assay to measure ligand (A20FMDV2 or LAP)-bound αvβ6 endocytosis, an siRNA screen was performed to identify which genes were responsible for internalising αvβ6. These data identified 15 genes (DNM2, CBLB, DNM3, CBL, EEA1, CLTC, ARFGAP3, CAV1, CYTH2, CAV3, CAV2, IQSEC1, AP2M1, TSG101) which significantly decreased endocytosis, predominantly within dynamin-dependent pathways. Inhibition of these dynamin-dependent pathways significantly reduced αvβ6-dependent migration (αvβ6-specific migration was 547 ± 128 under control conditions, reduced to 225 ± 73 with clathrin inhibition, and 280 ± 51 with caveolin inhibition). Colocalization studies of αvβ6 with endosome markers revealed that up to 6 h post-internalisation of ligand, αvβ6 remains in Rab11-positive endosomes in a perinuclear location, with no evidence of αvβ6 degradation up to 48 h post exposure to A20FMDV2. Additionally, 60% of ligand-bound αvβ6 was recycled back to the surface by 6 h. With studies ongoing using conjugated A20FMDV2 to therapeutically target αvβ6 in cancer and fibrosis, these data have important implications. Binding of A20FMDV2 seemingly removes much of the αvβ6 from the cell membrane, and upon its recycling, a large fraction appears to still be in the ligand-bound state. While these results are observed with A20FMDV2, these data will be of value in the design of αvβ6-specific therapeutics and potentially the types of therapeutic load.
整合素αvβ6在大多数正常健康组织中低水平表达，但在包括癌症和纤维化在内的疾病背景下常常上调。整合素利用内吞作用和运输作为调节其表面表达从而调控其功能的一种方式，然而对于在αvβ6背景下该过程如何被调控却知之甚少。由于αvβ6是癌症和纤维化治疗药物开发的主要靶点，了解这些动态变化对于开发靶向αvβ6的疗法至关重要。在开发出一种基于流式细胞术的检测方法以测量与配体（A20FMDV2或LAP）结合的αvβ6内吞作用后，进行了一项小干扰RNA筛选以确定哪些基因负责αvβ6的内化。这些数据鉴定出15个基因（DNM2、CBLB、DNM3、CBL、EEA1、CLTC、ARFGAP3、CAV1、CYTH2、CAV3、CAV2、IQSEC1、AP2M1、TSG101），它们显著降低内吞作用，主要在依赖发动蛋白的途径中。抑制这些依赖发动蛋白的途径显著降低了αvβ6依赖的迁移（在对照条件下αvβ6特异性迁移为547±128，用网格蛋白抑制时降至225±73，用小窝蛋白抑制时降至280±51）。αvβ6与内体标记物的共定位研究表明，在配体内化后长达6小时，αvβ6仍位于核周位置的Rab11阳性内体中，在暴露于A20FMDV2后长达48小时没有αvβ6降解的证据。此外，60%与配体结合的αvβ6在6小时内被循环回到表面。随着使用偶联的A20FMDV2在癌症和纤维化中靶向治疗αvβ6的研究不断进行，这些数据具有重要意义。A20FMDV2的结合似乎从细胞膜上去除了大部分αvβ6，并且在其循环后，很大一部分似乎仍处于与配体结合的状态。虽然这些结果是在A20FMDV2上观察到的数据，但这些数据对于设计αvβ6特异性疗法以及潜在的治疗载荷类型将具有价值。
