Late-stage epithelial ovarian cancer (EOC) involves the widespread dissemination of malignant disease throughout the peritoneal cavity, often accompanied by ascites. EOC metastasis relies on the formation of multicellular aggregates, called spheroids. Given that Liver Kinase B1 (LKB1) is required for EOC spheroid viability and LKB1 loss in EOC cells decreases tumor burden in mice, we investigated whether the LKB1 complex controls the invasive properties of human EOC spheroids. LKB1 signalling was antagonized through the CRISPR/Cas9 genetic knockout of LKB1 and/or the RNAi-dependent targeting of STE20-related kinase adaptor protein (STRAD, an LKB1 activator). EOC spheroids expressing nuclear GFP (green) or mKate2 (red) constructs were embedded in Matrigel for real-time live-cell invasion monitoring. Migration and invasion were also assessed in spheroid culture using Transwell chambers, spheroid reattachment, and mesothelial clearance assays. The loss of LKB1 and STRAD signalling decreased cell invasion through Matrigel and Transwell membranes, as well as mesothelial cell clearance. In the absence of LKB1, zymographic assays identified a loss of matrix metalloproteinase (MMP) activity, whereas spheroid reattachment assays found that coating plates with fibronectin restored their invasive potential. A three-dimensional EOC organoid model demonstrated that organoid area was greatly reduced by LKB1 loss. Overall, our data indicated that LKB1 and STRAD facilitated EOC metastasis by promoting MMP activity and fibronectin expression. Given that LKB1 and STRAD are crucial for EOC metastasis, targeting LKB1 and/or STRAD could disrupt the dissemination of EOC, making inhibitors of the LKB1 pathway an alternative therapeutic strategy for EOC patients.
晚期上皮性卵巢癌（EOC）表现为恶性病变在腹膜腔内广泛播散，常伴有腹水。EOC的转移依赖于称为球体的多细胞聚集体形成。鉴于肝激酶B1（LKB1）对EOC球体存活至关重要，且EOC细胞中LKB1缺失会降低小鼠肿瘤负荷，本研究探讨了LKB1复合物是否调控人EOC球体的侵袭特性。通过CRISPR/Cas9基因敲除LKB1和/或RNAi靶向STE20相关激酶衔接蛋白（STRAD，一种LKB1激活剂）来拮抗LKB1信号通路。将表达核定位GFP（绿色）或mKate2（红色）荧光蛋白的EOC球体包埋于基质胶中，进行实时活细胞侵袭监测。同时采用Transwell小室、球体再附着及间皮清除实验评估球体培养体系中的迁移和侵袭能力。LKB1与STRAD信号缺失显著降低了细胞通过基质胶和Transwell膜的侵袭能力，并减弱了间皮细胞清除作用。在LKB1缺失条件下，酶谱分析显示基质金属蛋白酶（MMP）活性丧失，而球体再附着实验发现纤连蛋白包被培养板可恢复其侵袭潜能。三维EOC类器官模型显示LKB1缺失会大幅缩小类器官面积。总体而言，我们的数据表明LKB1和STRAD通过促进MMP活性和纤连蛋白表达来驱动EOC转移。鉴于LKB1与STRAD对EOC转移至关重要，靶向LKB1和/或STRAD可阻断EOC播散，这使得LKB1通路抑制剂成为EOC患者的潜在治疗策略。
LKB1 and STRADα Promote Epithelial Ovarian Cancer Spheroid Cell Invasion
肿瘤(癌症)患者之家