Loss of ULK1 impairs autophagy activation, spheroid viability, and tumor progression in epithelial ovarian cancer

Abstract

Abstract Introduction: Epithelial ovarian cancer (EOC) remains a leading cause of gynecological cancer-related deaths due its late diagnosis and the absence of effective treatments for chemo-resistant disease. EOC metastasizes through peritoneal dissemination, often forming multicellular spheroids, in which autophagy—a cell survival mechanism—is induced, requiring ULK1 (Unc-51-like kinase 1) activity. Our study aims to further understand the role of ULK1 in EOC tumor growth and metastasis. Methods: Using CRISPR/Cas9 technology, we ablated the ULK1 gene in EOC cell lines OVCAR8 and HEYA8, and the fallopian tube derived FT190 control line. Western blotting confirmed ULK1 loss and key autophagy markers. Autophagic flux was assessed using fluorescence microscopy and cell viability by Trypan Blue, Cell Titer-Glo, and Caspase-Glo assays. We tested sensitivity to carboplatin and paclitaxel treatments in cell culture, while bioluminescent imaging monitored tumor progression of xenograftsULK1KO. Immunohistochemistry (IHC) was performed to assess Ki67 for cell proliferation and cleaved caspase-3 for apoptosis. Results: Our results show that ULK1 loss leads to impaired autophagy in EOC spheroids, with reduced LC3 processing and elevated p62 levels. Intriguingly, FT190 cells maintained autophagy which correlated with elevated ULK2 expression. All cell lines lacking ULK1KO had reduced spheroid cell viability and spheroid integrity. Surprisingly, ULK1 loss led to differential sensitivity to chemotherapy agents carboplatin and paclitaxel between OVCAR8 and HEYA8 cells. ULK1 deficiency reduced tumor burden in xenografted mice, although differences were observed in tumor growth rate and extent of metastasis between OVCAR8 and HEYA8 cells. Furthermore, Ki67 and cleaved caspase-3 staining revealed reduced cell proliferation and increased apoptosis respectively in tumors derived from ULK1KO cells. Conclusions: ULK1 is required for EOC spheroid formation and cell survival while in suspension likely through its regulation of autophagy, but it may have a lesser role for autophagy regulation in precursor cells. ULK1 deficiency does not increase EOC cell sensitivity to standard-of-care chemotherapy, possibly indicating that other therapeutic strategies would be needed to synergize with autophagy inhibition for EOC treatment.. Altogether, ULK1 may have a multifaceted role in EOC beyond autophagy regulation, by contributing to early dissemination of as spheroids and establishment of secondary tumors