Chung Joon-Yong, Lee Woonghee, Lee Olivia W, Ylaya Kris, Nambiar Divya, Sheehan-Klenk Julia, Fayn Stanley, Hewitt Stephen M, Choyke Peter L, Escorcia Freddy E
Molecular Imaging Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health Bethesda, MD 20892, USA.
Laboratory of Genetic Susceptibility, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health Bethesda, MD 20892, USA.
Am J Cancer Res. 2024 Jul 15;14(7):3348-3371. doi: 10.62347/TTNY4279. eCollection 2024.
Glypican-3 (GPC3) is overexpressed in hepatocellular carcinomas and hepatoblastomas and represents an important therapeutic target but the biologic importance of GPC3 in liver cancer is unclear. To date, there are limited data characterizing the biological implications of GPC3 knockout (KO) in liver cancers that intrinsically express this target. Here, we report on the development and characterization of GPC3-KO liver cancer cell lines and compare to them to parental lines. GPC3-KO variants were established in HepG2 and Hep3B liver cancer cell lines using a lentivirus-mediated CRISPR/Cas9 system. We assessed the effects of GPC3 deficiency on oncogenic properties and in murine xenograft models. Downstream cellular signaling pathway changes induced by GPC3 deficiency were examined by RNAseq and western blot. To confirm the usefulness of the models for GPC3-targeted drug development, we evaluated the target engagement of a GPC3-selective antibody, GC33, conjugated to the positron-emitting zirconium-89 (Zr) in subcutaneous murine xenografts of wild type (WT) and KO liver cancer cell lines. Deletion of GPC3 significantly reduced liver cancer cell proliferation, migration, and invasion compared to the parental cell lines. Additionally, the tumor growth of GPC3-KO liver cancer xenografts was significantly slower compared with control xenografts. RNA sequencing analysis also showed GPC3-KO resulted in a reduction in the expression of genes associated with cell cycle regulation, invasion, and migration. Specifically, we observed the downregulation of components in the AKT/NFκB/WNT signaling pathways and of molecules related to cell cycle regulation with GPC3-KO. In contrast, pMAPK/ERK1/2 was upregulated, suggesting an adaptive compensatory response. KO lines demonstrated increased sensitivity to ERK (GDC09994), while AKT (MK2206) inhibition was more effective in WT lines. Using antibody-based positron emission tomography (immunoPET) imaging, we confirmed that Zr-GC33 accumulated exclusively in GPC3-expression xenografts but not in GPC3-KO xenografts with high tumor uptake and tumor-to-liver signal ratio. We show that GPC3-KO liver cancer cell lines exhibit decreased tumorigenicity and altered signaling pathways, including upregulated pMAPK/ERK1/2, compared to parental lines. Furthermore, we successfully distinguished between GPC3+ and GPC3- tumors using the GPC3-targeted immunoPET imaging agent, demonstrating the potential utility of these cell lines in facilitating GPC3-selective drug development.
磷脂酰肌醇蛋白聚糖-3（GPC3）在肝细胞癌和肝母细胞瘤中过表达，是一个重要的治疗靶点，但GPC3在肝癌中的生物学重要性尚不清楚。迄今为止，关于GPC3基因敲除（KO）在天然表达该靶点的肝癌中的生物学意义的数据有限。在此，我们报告GPC3基因敲除肝癌细胞系的建立和特征，并将它们与亲代细胞系进行比较。使用慢病毒介导的CRISPR/Cas9系统在HepG2和Hep3B肝癌细胞系中建立了GPC3基因敲除变体。我们评估了GPC3缺失对致癌特性的影响，并在小鼠异种移植模型中进行了研究。通过RNA测序和蛋白质免疫印迹检测了GPC3缺失诱导的下游细胞信号通路变化。为了确认这些模型对GPC3靶向药物开发的有用性，我们在野生型（WT）和基因敲除肝癌细胞系的皮下小鼠异种移植物中评估了与正电子发射锆-89（Zr）偶联的GPC3选择性抗体GC33的靶点结合情况。与亲代细胞系相比，GPC3的缺失显著降低了肝癌细胞的增殖、迁移和侵袭能力。此外，GPC3基因敲除肝癌异种移植物的肿瘤生长明显慢于对照异种移植物。RNA测序分析还表明，GPC3基因敲除导致与细胞周期调控、侵袭和迁移相关的基因表达减少。具体而言，我们观察到GPC3基因敲除后AKT/NFκB/WNT信号通路中的成分以及与细胞周期调控相关的分子下调。相反，pMAPK/ERK1/2上调，提示存在适应性代偿反应。基因敲除细胞系对ERK（GDC09994）的敏感性增加，而AKT（MK2206）抑制在野生型细胞系中更有效。使用基于抗体的正电子发射断层扫描（免疫PET）成像，我们证实Zr-GC33仅在表达GPC3的异种移植物中积聚，而不在GPC3基因敲除的异种移植物中积聚，且肿瘤摄取和肿瘤与肝脏的信号比很高。我们表明，与亲代细胞系相比，GPC3基因敲除肝癌细胞系的致瘤性降低，信号通路改变，包括pMAPK/ERK1/2上调。此外，我们使用GPC3靶向免疫PET成像剂成功区分了GPC3阳性和GPC3阴性肿瘤，证明了这些细胞系在促进GPC3选择性药物开发方面的潜在效用。
