Key Laboratory of Human Functional Genomics of Jiangsu Province, National Health Commission Key Laboratory of Antibody Techniques, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, Jiangsu, China.
Bio-medical Center, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China.
Hepatology. 2019 Oct;70(4):1231-1245. doi: 10.1002/hep.30646. Epub 2019 May 24.
Wnt signaling is one of the key regulators of hepatocellular carcinoma (HCC) tumor progression. In addition to the classical receptor frizzled (FZD), various coreceptors including heparan sulfate proteoglycans (HSPGs) are involved in Wnt activation. Glypican-3 (GPC3) is an HSPG that is overexpressed in HCC and functions as a Wnt coreceptor that modulates HCC cell proliferation. These features make GPC3 an attractive target for liver cancer therapy. However, the precise interaction of GPC3 and Wnt and how GPC3, Wnt, and FZD cooperate with each other are poorly understood. In this study, we established a structural model of GPC3 containing a putative FZD-like cysteine-rich domain at its N-terminal lobe. We found that F41 and its surrounding residues in GPC3 formed a Wnt-binding groove that interacted with the middle region located between the lipid thumb domain and the index finger domain of Wnt3a. Mutating residues in this groove significantly inhibited Wnt3a binding, β-catenin activation, and the transcriptional activation of Wnt-dependent genes. In contrast with the heparan sulfate chains, the Wnt-binding groove that we identified in the protein core of GPC3 seemed to promote Wnt signaling in conditions when FZD was not abundant. Specifically, blocking this domain using an antibody inhibited Wnt activation. In HCC cells, mutating residue F41 on GPC3 inhibited activation of β-catenin in vitro and reduced xenograft tumor growth in nude mice compared with cells expressing wild-type GPC3. Conclusion: Our investigation demonstrates a detailed interaction of GPC3 and Wnt3a, reveals the precise mechanism of GPC3 acting as a Wnt coreceptor, and provides a potential target site on GPC3 for Wnt blocking and HCC therapy.
Wnt 信号通路是调控肝细胞癌（HCC）肿瘤进展的关键调节因子之一。除了经典的受体卷曲蛋白（FZD）外，各种核心受体，包括硫酸乙酰肝素蛋白聚糖（HSPGs），也参与了 Wnt 的激活。磷脂酰肌醇聚糖-3（GPC3）是一种 HSPG，在 HCC 中过度表达，作为一种 Wnt 核心受体，调节 HCC 细胞增殖。这些特征使 GPC3 成为肝癌治疗的一个有吸引力的靶点。然而，GPC3 与 Wnt 的精确相互作用以及 GPC3、Wnt 和 FZD 如何相互合作的机制尚不清楚。在本研究中，我们建立了一个包含 GPC3 假定的 FZD 样富含半胱氨酸结构域的结构模型。我们发现，GPC3 中的 F41 及其周围残基形成了一个 Wnt 结合沟，与 Wnt3a 的脂质指状结构域和食指结构域之间的中间区域相互作用。突变该沟中的残基会显著抑制 Wnt3a 的结合、β-连环蛋白的激活以及 Wnt 依赖性基因的转录激活。与肝素硫酸链不同，我们在 GPC3 蛋白核心中鉴定的 Wnt 结合沟似乎在 FZD 不丰富的情况下促进了 Wnt 信号通路。具体来说，使用抗体阻断该结构域会抑制 Wnt 的激活。在 HCC 细胞中，与表达野生型 GPC3 的细胞相比，突变 GPC3 上的残基 F41 会抑制β-连环蛋白的体外激活，并减少裸鼠异种移植肿瘤的生长。结论：我们的研究表明了 GPC3 与 Wnt3a 的详细相互作用，揭示了 GPC3 作为 Wnt 核心受体的精确作用机制，并为 GPC3 提供了一个用于阻断 Wnt 和 HCC 治疗的潜在靶点。
