Introduction: Epithelial ovarian cancer (EOC) is usually diagnosed late in tumour progression and despite initial success of surgery and chemotherapy, patients often relapse with chemo-resistance. Late-stage EOC involves widespread dissemination of malignant disease throughout the peritoneal cavity, oftentimes accompanied by ascites fluid. EOC metastasis relies on the formation of multicellular aggregates called spheroids. Given our findings that Liver kinase B1 (LKB1) is required for EOC spheroid viability and LKB1 loss in EOC cells decreases tumour burden in mice, herein we investigated whether the LKB1 complex controls invasive properties of human EOC spheroids.
Methods: LKB1 signalling was antagonized through CRISPR/Cas9 genetic knockout of LKB1 and RNAi-dependent targeting of STE20-related kinase adaptor protein (STRAD; an LKB1 activator). Migration and invasion were assessed in spheroid culture via Transwell chambers, spheroid reattachment assays, and mesothelial clearance assays. EOC spheroids expressing nuclear GFP or mKate2 constructs were generated using Ultra-Low Attachment culture plates and embedded in Matrigel where an IncuCyte S3 real-time live-cell imager monitored invasion.
Results: The loss of LKB1 and STRAD signalling decreased mesothelial clearance as well as cell invasion through Matrigel and across Transwell membranes. A three-dimensional EOC organoid model demonstrated that organoid area was reduced by LKB1 knockout. 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.
Conclusions: LKB1 and STRAD facilitate EOC metastasis through MMP activity and fibronectin expression. Given that LKB1 and STRAD are crucial to EOC metastasis, targeting LKB1 could disrupt the dissemination of EOC making LKB1-specific inhibitors an alternative therapeutic strategy for EOC patients.