ULK1 is required for spheroid cell viability during metastatic transit in epithelial ovarian cancer
Jack D Webb1,2, Adrian Buensuceso1, Emily Tomas1,2, Matthew J Borrelli1,2, Lauren Viola1, Owen Hovey3, Yudith Ramos Valdes1, Bipradeb Singha 1,2, Shawn S-C Li3, Trevor G Shepherd1,2,4,5.
1The Mary & John Knight Translational Ovarian Cancer Research Unit, London Regional Cancer Program, London, ON, Canada; 2Anatomy & Cell Biology, The University of Western Ontario, London, ON, Canada; 3Biochemistry, The University of Western Ontario, London, ON, Canada; 4Oncology, The University of Western Ontario, London, ON, Canada; 5Obstetrics & Gynaecology, The University of Western Ontario, London, ON, Canada
Introduction: Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer, attributed to its late-stage detection and development of chemotherapy resistance. EOC metastasis involves the spread of cancer cells into the peritoneal cavity forming multicellular clusters, or spheroids. Our research has identified autophagy, mediated by ULK1 (unc-51-like kinase-1), as a crucial survival mechanism in these spheroids. Herein, we have generated new ULK1 knockout cells to further study its requirement during tumour growth and metastasis.
Methods: CRISPR/Cas9 technology facilitated ULK1 gene ablation in EOC cell lines OVCAR8, HEYA8, and the FT190 control line, while autophagy markers were verified by western blot. Cell viability was assessed by Trypan Blue and Caspase-Glo assays, bioluminescent imaging monitored tumour progression in xenografts, and ex vivo assessments of tumour and ascites specimens. Ascites-derived spheroid invasion was determined using the mesothelial clearance assay. Phospho-proteomic mass spectrometry and bioinformatics analyses were performed on OVCAR8 spheroids and validated by western blot.
Results: ULK1 knockout disrupted autophagy in EOC spheroids, as shown by altered LC3 processing, p62 accumulation, and reduced Beclin-1 phosphorylation. ULK1 loss decreased spheroid viability and increased apoptosis in vitro, and significantly decreased tumour burden and metastatic potential in vivo. ULK1 deletion hindered organoid growth yet did not impact spheroid invasiveness. Further analysis of altered signaling in spheroids revealed significant changes in MAPK and PI3K-AKT-mTOR pathways due to ULK1 loss.
Conclusion: Our findings reveal that ULK1 is pivotal for autophagy induction, spheroid viability, and ultimate metastatic progression in vivo. However, additional autophagy-independent ULK1 mechanisms may be implicated in EOC progression, too. We hope to discover new ULK1 signaling effects through future interrogation of the phospho-proteomic data.
We acknowledge funding support from the Cancer Research Society to TGS, and London Health Sciences Foundation through donations to the Mary & John Knight Translational Ovarian Cancer Research Unit.. We are grateful to Ronny Drapkin for providing the FT190 cell line. We are also very grateful to the many donors to the Mary and John Knight Translational Ovarian Cancer Research Unit through the London Health Sciences Foundation for additional infrastructure funding, including the Leica DMI 400B inverted microscope, Bio-Rad Chemidoc, IncuCyte S3, and IVIS Lumina imaging system used in this study..