Sun Hanxue, Rana Sanyukta, Wang Zhe, Zhao Kun, Schnölzer Martina, Provaznik Jan, Hackert Thilo, Lv Qingjie, Zöller Margot
Tumor Cell Biology, University Hospital of Surgery, Heidelberg, Germany.
Functional Proteome Analysis, German Cancer Research Center, Heidelberg, Germany.
J Oncol. 2019 Aug 7;2019:3516973. doi: 10.1155/2019/3516973. eCollection 2019.
Pancreatic cancer-initiating cells (PaCIC) express CD44v6 and Tspan8. A knockdown (kd) of these markers hinders the metastatic capacity, which can be rescued, if the cells are exposed to CIC-exosomes (TEX). Additional evidence that CD44v6 regulates Tspan8 expression prompted us to explore the impact of these PaCIC markers on nonmetastatic PaCa and PaCIC-TEX. We performed proteome, miRNA, and mRNA deep sequencing analyses on wild-type, CD44v6kd, and Tspan8kd human PaCIC and TEX. Database comparative analyses were controlled by qRT-PCR, Western blot, flow cytometry, and confocal microscopy. Transcriptome analysis of CD44 versus CD44v6 coimmunoprecipitating proteins in cells and TEX revealed that Tspan8, several signal-transducing molecules including RTK, EMT-related transcription factors, and proteins engaged in mRNA processing selectively associate with CD44v6 and that the membrane-attached CD44 intracytoplasmic tail supports Tspan8 and NOTCH transcription. Deep sequencing uncovered a CD44v6 contribution to miRNA processing. Due to the association of CD44v6 with Tspan8 in internalization prone tetraspanin-enriched membrane domains (TEM) and the engagement of Tspan8 in exosome biogenesis, most CD44v6-dependent changes were transferred into TEX such that the input of CD44v6 to TEX activities becomes largely waved in both a CD44v6kd and a Tspan8kd. Few differences between CD44v6kd- and Tspan8kd-TEX rely on CD44v6 being also recovered in non-TEM derived TEX, highlighting distinct TEX delivery from individual cells that jointly account for TEX-promoted target modulation. This leads us to propose a model in which CD44v6 strongly supports tumor progression by cooperating with signaling molecules, altering transcription of key molecules, and through its association with the mRNA processing machinery. The association of CD44v6 with Tspan8, which plays a crucial role in vesicle biogenesis, promotes metastases by transferring CD44v6 activities into TEM and TEM-independently derived TEX. Further investigations of the lead position of CD44v6 in shifting metastasis-promoting activities into CIC-TEX may offer a means of targeting TEX-CD44v6 in therapeutic applications.
胰腺癌起始细胞（PaCIC）表达CD44v6和四跨膜蛋白8（Tspan8）。敲低这些标志物会阻碍其转移能力，但如果将细胞暴露于癌症起始细胞外泌体（TEX），这种能力可以得到恢复。CD44v6调节Tspan8表达的更多证据促使我们探究这些PaCIC标志物对非转移性胰腺癌（PaCa）和PaCIC-TEX的影响。我们对野生型、CD44v6敲低型和Tspan8敲低型的人PaCIC及TEX进行了蛋白质组、微小RNA（miRNA）和信使核糖核酸（mRNA）深度测序分析。数据库比较分析通过定量逆转录聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法、流式细胞术和共聚焦显微镜进行控制。对细胞和TEX中CD44与CD44v6共免疫沉淀蛋白的转录组分析表明，Tspan8、包括受体酪氨酸激酶（RTK）在内的几种信号转导分子、上皮-间质转化（EMT）相关转录因子以及参与mRNA加工的蛋白质选择性地与CD44v6结合，并且膜附着的CD44胞质尾支持Tspan8和Notch转录。深度测序揭示了CD44v6对miRNA加工的作用。由于CD44v6在易于内化的富含四跨膜蛋白的膜结构域（TEM）中与Tspan8结合，且Tspan8参与外泌体生物发生，大多数依赖CD44v6的变化被转移到TEX中，使得CD44v6对TEX活性的影响在CD44v6敲低型和Tspan8敲低型中都大幅减弱。CD44v6敲低型TEX和Tspan8敲低型TEX之间的差异很少，这是因为在非TEM来源的TEX中也能检测到CD44v6，这突出了来自单个细胞的不同TEX传递方式，它们共同导致TEX促进的靶标调节。这使我们提出一个模型，即CD44v6通过与信号分子合作、改变关键分子的转录以及与mRNA加工机制结合，有力地支持肿瘤进展。CD44v6与Tspan8的结合在囊泡生物发生中起关键作用，通过将CD44v6的活性转移到TEM和非TEM来源的TEX中促进转移。进一步研究CD44v6在将促进转移的活性转移到CIC-TEX中的主导地位，可能为在治疗应用中靶向TEX-CD44v6提供一种方法。
