Abstract B103: The impact of ULK1 deficiency on autophagy, cell viability, and tumor progression in experimental models of ovarian cancer metastasis
Authors: Jack Webb, Bipradeb Singha, Matthew Borrelli, Lauren Viola, Adrian Buensuceso, Yudith Ramos-Valdes, Trevor Shepherd
Journal: Cancer Research (2024)

Abstract Introduction: Epithelial ovarian cancer (EOC) is a primary cause of mortality from gynaecologic cancers. The late-stage diagnosis and absence of effective strategies for treating chemo-resistant disease are significant issues. Mechanisms ensuring EOC cells' survival during metastasis and after treatment may support the onset of resistant disease. EOC's spread involves direct dissemination into the peritoneal cavity, often leading to multicellular clusters, or spheroids. Our work has shown autophagy as a cell survival mechanism in EOC spheroids, with ULK1 (unc-51-like kinase-1) playing an essential role. We've developed ULK1 knockout EOC cells to further investigate its function in tumor growth and metastasis. Methods: We utilized CRISPR/Cas9 to eliminate ULK1 expression in EOC cell lines OVCAR8 and HEYA8 and control cell line FT190. To verify ULK1 loss and evaluate autophagy markers LC3 and p62, we used Western blotting in adherent and spheroid culture conditions. Autophagic flux was observed through fluorescence microscopy and monomeric mCherry protein expression with the mCherry-eGFP-LC3 reporter. We determined cell growth and viability via NucLight GFP labelled cells' fluorescence microscopy, Trypan Blue exclusion counting, Cell Titer-Glo and Caspase-Glo assays. To measure sensitivity to carboplatin and paclitaxel, Alamarblue assays were employed. We used cells expressing luc2tdTomato for bioluminescent imaging to evaluate tumor progression in xenografts. Results: ULK1 loss impairs autophagy activation in EOC-ULK1KO spheroids, visible by loss of LC3 processing, p62 accumulation, and monomeric mCherry expression attenuation (OVCAR8). However, FT190 spheroids maintain autophagy activation despite ULK1 loss, possibly due to compensatory ULK2 activity. OVCAR8, HEYA8, and FT190-ULK1KO spheroids exhibited decreased cell viability and spheroid integrity reduction. Notably, OVCAR8-ULK1KO spheroids are substantially more sensitive to carboplatin treatment, while HEYA8-ULK1KO spheroids appear resistant to both carboplatin and paclitaxel. There were no differences in EOC spheroid growth due to ULK1 absence. OVCAR8-ULK1KO cells reduced the tumor burden in mice at all time points, with significant reductions at late-stage disease. In contrast, HEYA8-ULK1KO cells significantly reduced the tumor burden during the early disease progression stages. Discussion: ULK1 appears to be essential for EOC spheroid formation and cell survival in suspension, probably through its regulation of autophagy, but may not be as significant in precursor fallopian tube secretory epithelial cells. Unexpectedly, ULK1 deficiency doesn't increase EOC cell sensitivity to standard-of-care chemotherapy, potentially indicating that chemotherapy affects autophagy via drug-specific action mechanisms. However, ULK1 seems to play a crucial role in the early dissemination of tumor cells and late-stage disease progression. We are currently expanding our investigation into HGSOC spheroids to identify altered signaling pathways, novel ULK1 targets, and synergistic treatments. Citation Format: Jack Webb, Bipradeb Singha, Matthew Borrelli, Lauren Viola, Adrian Buensuceso, Yudith Ramos-Valdes, Trevor Shepherd. The impact of ULK1 deficiency on autophagy, cell viability, and tumor progression in experimental models of ovarian cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B103.