Van Impe Katrien, Bethuyne Jonas, Cool Steven, Impens Francis, Ruano-Gallego David, De Wever Olivier, Vanloo Berlinda, Van Troys Marleen, Lambein Kathleen, Boucherie Ciska, Martens Evelien, Zwaenepoel Olivier, Hassanzadeh-Ghassabeh Gholamreza, Vandekerckhove Joël, Gevaert Kris, Fernández Luis Ángel, Sanders Niek N, Gettemans Jan
Breast Cancer Res. 2013 Dec 13;15(6):R116. doi: 10.1186/bcr3585.
Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit.
We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading.
With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS).
CapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.
肌动蛋白细胞骨架的异常周转与癌细胞的迁移和侵袭密切相关。然而，相关证据往往只是间接的，而且由于缺乏像大多数传统药物那样针对蛋白质生物学特性而非相应基因的抑制剂，对基因产物治疗意义的可靠评估受到了影响。蛋白质组学研究表明，肌动蛋白细胞骨架的组成成分CapG在乳腺癌中过表达。间接证据表明CapG参与肿瘤细胞的扩散和转移。在本研究中，我们在乳腺癌转移模型中使用源自骆驼的CapG单域抗体或纳米抗体，以探讨抑制CapG活性是否具有治疗价值。
我们制备了针对人CapG的单域抗体（纳米抗体），并在乳腺癌细胞经慢病毒转导后将其用作胞内抗体（免疫调节）。对纳米抗体进行功能表征，以确定CapG的哪些生化特性受到干扰。使用裸鼠原位和尾静脉体内转移模型评估癌细胞的扩散情况。
通过G-肌动蛋白和F-肌动蛋白结合试验，我们鉴定出一种与CapG第一个结构域具有纳摩尔亲和力的CapG纳米抗体。因此，CapG与肌动蛋白单体或肌动蛋白丝的相互作用被阻断。细胞内定位实验表明，该纳米抗体在细胞质环境中与CapG相互作用。纳米抗体在乳腺癌细胞中的表达抑制了细胞迁移和基质胶侵袭。值得注意的是，在免疫缺陷小鼠的原位异种移植和尾静脉转移模型中，该纳米抗体阻止了肺转移瘤的形成。我们表明，利用携带III型蛋白分泌系统（T3SS）的细菌可将CapG纳米抗体递送至癌细胞中。
抑制CapG可显著降低乳腺癌转移。基于纳米抗体的方法为评估药物靶点的治疗价值提供了一条捷径。纳米抗体-CapG界面的图谱绘制可能为合理设计药理化合物提供一个平台。
