ULK1 promotes metastatic progression in epithelial ovarian cancer

ULK1 promotes metastatic progression in epithelial ovarian cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article ULK1 promotes metastatic progression in epithelial ovarian cancer Trevor Shepherd, Jack Webb, Adrian Buensuceso, Emily Tomas, Matthew Borrelli, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6148090/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2026 Read the published version in Oncogene → Version 1 posted 10 You are reading this latest preprint version Abstract Epithelial ovarian cancer (EOC) is a leading cause of gynecological cancer mortality, driven largely by late diagnosis and chemo-resistant disease. While autophagy plays a critical role in the survival of EOC spheroids during metastasis, the role of ULK1, a key regulator of autophagy, in EOC progression remains unclear. To investigate this, we utilized CRISPR/Cas9 technology to delete ULK1 in EOC cell lines OVCAR8 and HEYA8, and the immortalized fallopian tube epithelial cell line FT190. Immunoblotting confirmed ULK1 loss and its associated autophagy disruption in EOC spheroids, evidenced by reduced Beclin-1 phosphorylation, impaired LC3 processing, and p62 accumulation. Culture-based assays revealed that ULK1 knockout decreased EOC spheroid cell viability due to increased apoptosis and, notably, impaired matrix-bound organoid growth, offering new insights into the potential role of ULK1 in affecting EOC tumor growth and spread. These findings were further demonstrated by in vivo xenograft models, in which ULK1 loss significantly reduced tumor burden and metastatic potential. The potential for ULK1 requirement in metastatic properties was supported by diminished invasive capacity of ULK1 knockout spheroid cells in mesothelial clearance assays. To investigate the mechanisms by which ULK1 contributes EOC tumor progression and metastasis, we conducted proteomic analyses of OVCAR8 spheroids, which revealed that ULK1 loss disrupted critical signaling pathways, including MEK-MAPK, PI3K-AKT-mTOR, and apoptosis regulation. Although ULK1 knockout failed to synergize with standard-of-care chemotherapeutics, it significantly enhanced sensitivity to MEK and mTOR inhibition, revealing potential therapeutic combinations to target autophagy via ULK1 and MAPK and PI3K-AKT-mTOR pathway vulnerabilities in EOC. Overall, this study highlights ULK1 as a critical regulator of multiple steps of EOC growth and metastasis, underscoring its potential as a novel therapeutic target in advanced ovarian cancer. Biological sciences/Cancer/Gynaecological cancer/Ovarian cancer Biological sciences/Cell biology/Mechanisms of disease Autophagy ovarian cancer metastasis CRISPR/Cas9 spheroids organoids xenografts mass spectrometry proteome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 BACKGROUND Epithelial ovarian cancer (EOC) has the fifth-highest death-to-incidence ratio for all cancers in women and is the leading cause of death from gynecologic cancers due to its late-stage diagnosis and lack of effective strategies for treating chemoresistant disease ( 1 ). Patients with EOC are typically treated with aggressive surgical debulking and cytotoxic carboplatin/paclitaxel combination chemotherapy; however, nearly 80% of these patients relapse within five years ( 2 ). Investigations into the mechanisms that support cell survival during metastasis and regrowth of refractory EOC cells after treatment are of critical importance and are an area of active research. EOC spreads by tumor cells disseminating directly into the peritoneal space, often suspended in ascites, and then attaches to the serosal surfaces of the abdominal cavity to form secondary deposits ( 3 , 4 ). Clusters of metastatic EOC cells known as spheroids accumulate in the malignant fluid of patients with advanced disease ( 5 ). Spheroids are known to promote metastasis with increased cell survival in the face of chemotherapy and possess enhanced adhesive and invasive capabilities ( 6 , 7 ). Additionally, our lab and others have clearly demonstrated that spheroid cells undergo numerous phenotypic changes, including cellular quiescence ( 8 ), epithelial-mesenchymal transition ( 9 ), activated stress metabolism ( 10 , 11 ), and autophagy ( 12 ), all of which contribute to the tumor cell dormancy phenotype of residual disease, and that may drive chemo-resistant recurrence. Autophagy is an evolutionarily conserved and tightly controlled metabolic degradation process in which proteins and organelles are broken down in the lysosomes ( 13 ). This degradative process yields metabolic substrates from lysosomal activity thereby providing vital nutrients for essential cellular functions during nutrient scarcity or energy stress ( 14 ). Autophagy typically operates at basal levels to fulfill fundamental homeostatic functions but can quickly escalate under stress ( 15 ). Autophagy induction is controlled by the Unc-51-like kinase (ULK1) complex, consisting of ULK1/2, autophagy-related gene 13 (ATG13), and focal adhesion kinase-interacting protein (FIP200) ( 16 ). ULK1 is a serine-threonine kinase that responds to upstream signals of nutrient and energy availability to trigger autophagy through the initiation of phagophore formation. Under nutrient-abundant conditions, mechanistic target of rapamycin complex 1 (mTORC1) phosphorylates ULK1, thereby inhibiting its activity and initiating autophagy. Conversely, the absence of nutrients leads to mTORC1 deactivation, while AMP-activated protein kinase (AMPK) phosphorylates to activate ULK1 ( 17 ). Thus, ULK1 and autophagy are considered an essential regulatory hub to control energy supplies in an equilibrium with cellular demands. The role of ULK1 in autophagy initiation has been reviewed in ( 18 ), and its involvement in tumorigenesis—promoting tumor growth, invasiveness, and survival—has been documented across various cancer types ( 19 – 23 ). However, few studies have investigated the role of ULK1-mediated autophagy in EOC with a specific focus on tumor growth and metastatic progression. We have previously demonstrated that AMPK activation ( 24 ) and AKT-mTORC1 downregulation ( 8 ) work in coordination to induce autophagy in high-grade serous ovarian cancer (HGSOC) spheroids. We also showed that EOC spheroids had increased ULK1 expression, which parallels autophagy induction, and that transient knockdown and inhibitor treatments in vitro blocked autophagy and reduced cell viability ( 25 ). Building on our prior work on autophagy in EOC spheroids and utilizing CRISPR/Cas9 technology for ULK1 ablation, we aimed to elucidate ULK1's role in tumorigenesis by examining its contributions to spheroid survival, metastatic dissemination, and tumor growth to establish its potential as a therapeutic target in EOC metastasis. METHODS Cultured cell lines The cell lines OVCAR8, OVCAR8- ULK1 KO, HEYA8, and HEYA8- ULK1 KO were grown in RPMI-1640 medium (Wisent), whereas FT190 and FT190- ULK1 KO were grown in DMEM/F12 medium (Life Technologies). All growth media were supplemented with 10% fetal bovine serum. OVCAR8 and HEYA8 cells were procured from the American Type Culture Collection. Adherent cells were sustained on tissue culture-treated polystyrene (Sarstedt, Newton, NC, USA) and spheroids were maintained in Ultra-Low Attachment (ULA) cluster plates (Corning, NY, USA). The immortalized human fallopian tube secretory epithelial cell line FT190 ( 26 ) was generously provided by R. Drapkin from the University of Pennsylvania, Philadelphia, PA, USA. The human lung mesothelial ZT-GFP cell line ( 6 ) was generously provided by Marcin Iwanicki from the Stevens Institute of Technology All cell lines were authenticated through short tandem repeat analysis by the Center for Applied Genomics (The Hospital for Sick Children, Toronto, ON, Canada) and routinely examined them for mycoplasma using a Universal Mycoplasma Detection Kit (30-1012K; ATCC). Generation of ULK1 KO cell lines CRISPR/Cas9 (sc-400516-KO-2 Lot# C3016, Santa Cruz Biotechnology) was used to ablate ULK1 in OVCAR8, HEYA8, and FT190 cells. Briefly, cells were seeded at 1-1.5x10 5 per well in a 6-well plate and transfected the following day. Cells were trypsinized four days post-transfection and sorted using fluorescence-activated cell sorting (FACS) in 96-well plates. The clones were left to grow for a minimum of two weeks, after which colony formation was observed. Colonies were harvested and plated into 6-well plates and then 10 cm plates upon reaching confluency. Cells were harvested for protein lysates, screened for ULK1 loss via western blotting and passaged for continued culture and subsequent clone pooling. Generation of Nuclight GFP cell lines Cells were transduced with Incucyte® Nuclight Green Lentivirus (EF1a, Puro) (Sartorius, #4624) following the manufacturer's instructions. After transduction, cells were cultured in complete media supplemented with puromycin (1µg/mL; BioShop, #5E38885) to select successfully transduced cells. Transduced and puromycin-selected cells were subjected to FACS to isolate green fluorescent protein (GFP)-positive cells. The isolated GFP + cells were expanded and used for further analysis and experimentation. Generation of mCherry-eGFP-LC3 cell lines Parental and ULK1 KO cells were transfected with mCherry-eGFP-LC3B autophagy reporter plasmid [pBABE-puro mCherry-EGFP-LC3B was a gift from Jayanta Debnath (Addgene plasmid # 22418; http://n2t.net/addgene:22418;RRID:Addgene_22418 ] ( 27 ). After transfection, cells were grown in complete media containing G-418 (400µg/mL, Wisent, #450-130-QL) for two weeks to select those with successful reporter plasmid integration. Following the selection phase, the cells were cultured in complete medium without G-418 for another four weeks, allowing for growth and recovery. Cells were sorted using FACS to identify double-positive cells (GFP+, mCherry+). Generation of luc2tdTomato cell lines Cells were transduced with pCDH-EF1-Luc2-P2A-tdTomato, according to the manufacturer’s instructions [pCDH-EF1-Luc2-P2A-tdTomato was a gift from Kazuhiro Oka (Addgene plasmid # 72486; http://n2t.net/addgene:72486;RRID:Addgene_72486 )]. After transduction, the cells were subjected to FACS to isolate the tdTomato + cells. The cells were cultured in a complete medium for growth and recovery. The cells were then subjected to another round of FACS to gate and select for populations of cells with similar tdTomato expression intensities. Cell populations were seeded in a serial dilution for bioluminescent imaging (BLI) analysis using D-luciferin and the IVIS Lumina S5 system (PerkinElmer) to determine optimal populations (i.e., similar reporter gene expression among lines) to use for subsequent in vivo xenograft assays. Antibodies and reagents Antibodies against ULK1 (#8054S), p62 (#5114S), LC3B (#2775S), Beclin1 S30 (#5410S), Beclin1 (#3738S), AKT S473 (#9271), AKT (#9272S), MEK1/2 S217/221 (#9154S), MEK (#8727), ERK1/2 Thr202/Tyr204 (#9101), ERK1/2 (#9102), cleaved-PARP (#9541S), P70S6K Thr389 (#9234S), P70S6K (#2708S), p38 MAPK Thr180/Y182 (#4511S), 4EBP1 T37/46 (#2855S), 4EBP1 (#9452S) were purchased from Cell Signaling Technology. Anti-ULK2 antibody (AB97695), ATG16L1 (AB187671), ATG16L1 S30 (AB19016), and mCherry (AB167453; 1:500) were purchased from Abcam. Anti-actin antibody (A2066; 1:25000) was purchased from Millipore. Antibodies against tubulin (T5168; 1:40000) and vinculin (V9264; 1:25000) were purchased from Sigma-Aldrich. Horseradish peroxidase (HRP)-conjugated antibodies against mouse IgG (NA931; 1:10000) and rabbit IgG (NA934; 1:10000) were purchased from GE Healthcare. Antibodies were diluted in tris-buffered saline-Tween 20 containing either 5% bovine serum albumin or non-fat milk 1:1000 or as stated otherwise. Adenovirus expressing green fluorescent protein (Ad-GFP) was a kind gift from Dr. B. C. Vanderhyden (Ottawa Health Research Institute) and prepared as described previously ( 10 ). Rat-tail collagen was purchased from Gibco (#963791) and Matrigel was purchased from Corning (CLS356231). Paclitaxel was purchased from Cayman Chemical Company (#10461) and stored at -20°C as 5 mM in DMSO stocks. Carboplatin was received from the London Regional Cancer Program and stored at 4°C as 27 mM in saline stocks. Olaparib (#HY-10162) and ralimetinib (#HK-13241) were purchased from MedChemExpress, trametinib (#7709) was purchased from Tocris Bioscience, and AKT inhibitor VIII (Akti-1/2) was purchased from EMD/Calbiochem (#12408). Immunoblot analysis The Bio-Rad Mini-PROTEAN II Electrophoresis System was used for immunoblotting following the manufacturer's guidelines, utilizing in-house prepared gels (30% acrylamide/bis solution 37.5:1, catalog number 1610158; Bio-Rad). Densitometric analysis was conducted using Image Lab 6.05 software suite (Bio-Rad). Preparation of wholecell lysates For assessment of all proteins, 4-, 8-, 24-, 48, 72-hour whole-cell lysates were generated from adherent cells cultured at a density of 0.75-1× 10 6 cells in 10 mL medium (10 cm dish), or spheroid cells cultured at a density of 1–3× 10 6 cells in 15 mL medium (35 mm ULA well). Cell seeding numbers were chosen to obtain acceptable protein yields for each cell line. Wholecell lysates. Adherent cells cultivated on tissue culture-treated plates or dishes were collected by removing the medium, washing twice with cold PBS, and scraping into a modified radioimmunoprecipitation (RIPA) buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1.5 mM MgCl 2 , 1 mM EGTA, 1% Triton X-100, 0.1% SDS, 1 mM Na 3 VO 4 , 10 mM NaF, 1 mM PMSF, 1 × SIGMA FAST protease inhibitor cocktail (cat. S8820; Sigma), 10 mM beta-glycerophosphate). Spheroids (minimum of 1.5 × 10 6 cells per sample) were collected by transferring the cell suspension into an ice-cold conical tube, followed by centrifugation using a swinging bucket rotor (800 × g at 4°C for 4 min) to form a pellet. The medium was aspirated, and the pellet was resuspended in cold PBS (at least 10 mL of cold PBS. This process was repeated by resuspending the pellet in cold PBS, followed by centrifugation and aspiration of the PBS. The resulting cell pellets were lysed using modified RIPA buffer, vortexed, exposed to one freeze-thaw cycle, and clarified by centrifugation (maximum × g at 4°C for 20 min). Spheroid viability assays Bulk spheroids . Cells were placed in 24-well ultra-low attachment (ULA) cluster plates at a density of 0.5-1 × 10 5 cells per well in 1 mL of medium. The spheroids were then collected into chilled microcentrifuge tubes and centrifuged at 500×g for 3 min to form pellets. After aspirating the medium, the pellets were washed once with 500 µL of PBS, centrifuged once more, and resuspended in 50–250 µL of trypsin/EDTA. The suspension was incubated at 37°C with gentle agitation every 10 min until no aggregates were visible (10–30 minutes). Trypsin was inactivated by adding an equal volume of FBS, followed by the addition of Trypan Blue dye (Gibco™ 15250061) at 1:1 ratio and gentle mixing via pipetting. Cell counting was performed using the TC20 Automated Cell Counter (Bio-Rad Laboratories). Individual spheroids. Cells were seeded in a 96-well round-bottom ULA plate at a density of 2000 cells per well in 100 µL of medium. For alamarBlue assays, cells were incubated at a final dilution of 1:10 (alamarBlue to media) for 4, 24, or 48 h and fluorescence was measured using a Agilent Biotek Synergy H1 plate reader. For CellTiter-Glo (Promega, G7572) and Caspase-Glo 3/7 (Promega, G8092) assays, 100 µL of reagent was added to each well, and the plate was frozen at -80°C. After 24 h, the plates were thawed at room temperature in the dark for 60 min on a plate rocker. The contents of wells were transferred to individual wells of a 96-well opaque white plate, and luminescence was measured on the Agilent Biotek Synergy H1 plate reader. Cell Proliferation Cells expressing Nuclight GFP were placed in 96-well standard-well ultra-low attachment (ULA) cluster plates at a density of 0.5-1 × 10 5 cells per well in 200 µL of medium. Fluorescent images were captured at 4 h intervals in the Incucyte® S3 System (Sartorius). Growth curves and doubling times were calculated using GraphPad Prism 10. Adherent and spheroid doubling time calculations were performed using green and green mean intensity features of the Incucyte® S3 system, respectively. Organoids Cells were seeded at a density of 5000 cells/well as droplets in 50µL of Cultrex Basement Membrane Extract (BME) PathClear Type 2 (Cedarlane, Burlington, ON, Canada) on a 24-well standard tissue culture plate (Corning). Droplets were overlaid with an EOC organoid specific media containing Advanced DMEM/F-12 (Invitrogen) supplemented with B-27™ (Invitrogen), Forskolin (Sigma), GlutaMAX™ (Invitrogen), HEPES (Wisent), Human EGF (Peprotech Inc.), Human FGF-10 (Peprotech Inc.), Nicotinamide (Sigma), N-Acetyl-L-cysteine (Sigma), Recombinant Human Noggin (R&D Systems) and ROCK-inhibitor (Y27632 dihydrochloride, Sigma). Brightfield images were captured every 12 h with the Incucyte® S3 System. The total organoid area (µm²) and number of organoids per well were quantified using the Organoid Analysis Software via the Incucyte® S3 System. Scratch Wound Closure Migration Assay Confluent cell monolayers were scratched with a pipette tip and immediately imaged (0 h time point). Images were acquired up to 36 h post-scratch and ImageJ ( 28 ) was used to measure scratch width and calculate scratch area. Mesothelial Clearance Assay ZT-GFP mesothelial cell line with tdTomato-expressing spheroids. Human ZT-GFP mesothelial cells (1-1.5 × 10 5 cells in 1mL) were seeded into each well of a 24-well tissue culture plate and incubated for 24 h. An empty well containing 1mL of media served as a control. Cells expressing luc2tdTomato (2000 cells per well) were seeded into 96-well ULA plates and incubated for 24 h then individual spheroids were transferred using a P200 and placed onto mesothelial cell monolayers or control wells, with at least 5 spheroids per well. Green and red fluorescent images were captured 24 h later using a Leica DMI4000B inverted microscope and spheroid displacement was quantified using Fiji (Fiji is just ImageJ). Primary human mesothelial cells with mouse ascites-derived spheroids. Prior to cell culture, collagen (50µg) was dissolved in 70% ethanol and added to each well of a 24-well tissue culture plate and incubated at room temperature for 2 h. The wells were then aspirated, washed with PBS, and re-aspirated before adding mesothelial cells. To generate fluorescing primary mesothelial cells, 3.5µL of recombinant human Ad5-green fluorescent protein (Ad-GFP) vector stock per 100,000 cells was added to the cell suspension, which was then seeded to the collagen layer at a density of 1-1.5 × 10 5 cells and incubated for 24 h. Mouse xenograft ascites-derived spheroids were generated by seeding cells into 96-well ULA plates at 2000 cells per well in 100µL and incubated for 24 h. The following day, mesothelial cells were washed twice with PBS to remove Ad-GFP-containing media, and 1mL of fresh media was added to each well, including control wells without mesothelial cells. Spheroids were transferred to a 24-well plate at 5 per well using a P200. Green and red fluorescent images were captured 24 h later and spheroid displacement was quantified as described above. Xenotransplantation assays NOD/SCID female mice (8–10 weeks old; Charles River Laboratories) were injected intraperitoneally with luc2tdTomato cells with the following cell numbers in 150 µL PBS: OVCAR8/OVCAR8- ULK1 KO, 2 × 10 6 ; HeyA8/HeyA8- ULK1 KO, 1 × 10 6 ). For survival analyses, mice were monitored daily after intraperitoneal injection and euthanized using established criteria for humane endpoints (lethargy, hunched posture, impaired breathing, weight loss, and excessive ascites) as per protocol guidelines. Mice received weekly injections of D-luciferin (Perkin Elmer, #122799) at 75 mg/kg in 100µL PBS to monitor tumor progression via BLI using the IVIS Lumina S5 system (PerkinElmer). Tumor locations and evidence of ascites for each mouse was assessed and recorded at necropsy. The mice were provided chow (Envigo, #2919) and water ad libitum throughout the study. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Western Ontario (London, Ontario, Canada) and were performed in accordance with approved guidelines. Immunohistochemistry Sectioning and staining of tumor specimens were performed by the Molecular Pathology Core Facility at Robarts Research Institute (London, Ontario, Canada). Images of stained tumor sections were captured using an Aperio ScanScope slide scanner (Leica). IHC analysis was performed using the Fiji distribution in ImageJ software ( 29 ). Ki67-positive nuclei were masked using the Trainable Weka Segmentation plugin ( 30 ), and masked regions were counted using a minimum particle area of 120 pixels. Cleaved caspase-3 staining in xenograft tumor sections was evaluated using the “IHC Profiler” plugin for ImageJ, as described previously ( 31 ). Positive caspase-3 staining represents the combined “high-positive” and “positive” scores as defined by IHC profiler. Carboplatin and Paclitaxel Dose-Response Curves To determine carboplatin and paclitaxel half-maximal inhibitory concentration (IC 50 ) values, 2000 cells in 100 µL media were seeded in a standard 96-well plate for adherent culture, allowed to attach for 24 h, then treated with carboplatin or paclitaxel over a 12-point concentration gradient. After 72 h of treatment, cell viability was determined using the alamarBlue Cell Viability Reagent (Invitrogen CAT# DAL 1025) according to the manufacturer’s instructions. To determine carboplatin and paclitaxel IC 50 values of spheroids, 2000 cells in 100 µL media were seeded in a 96-well ULA plate. After 72 h, cells were treated individually over a 12-point concentration gradient for an additional 72 h. Following treatment, viability was determined by alamarBlue viability assay. Viability was assessed at 4 h and 48 h post alamarBlue incubation for carboplatin and paclitaxel, respectively, and IC 50 values were calculated using GraphPad Prism 10. Proteomic Mass Spectrometry Protein extraction and mass spectrometer analysis were performed on OVCAR8 wild-type and OVCAR8- ULK1 KO 24-hour spheroids. Spheroids were lysed using three 5-second pulse rounds of sonication at 35% amplitude lysed OVCAR8 wild-type and OVCAR8 ULK1 KO spheroids at 24 h post seeding in lysis buffer containing 200 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS; pH 8.6), 6 M guanidine, 1 mM PMSF, 100 mM NaF, and phosphatase inhibitor cocktail (2 mM NaF, 2 mM imidazole, 1.15 mM Na 2 MoO 4 , 1 mM Na 4 P 2 O 7 , 4 mM Na 2 C 4 H 4 O 6 , 2 mM Na 3 VO 4 , and 1 mM β-glycerophosphate). Lysates were incubated in the dark with 5 mM Tris (2-carboxyethyl) phosphine and 15 mM indole-3-acetic acid for 30 and 45 minutes, respectively, and quenched with 5 mM dithiothreitol. Sera-Mag™ SpeedBeads (GE Healthcare, Little Chalfont, UK; 65152105050250) were added to the lysates, followed by equal volumes of 100% ethanol. The resulting mixture was incubated on a shaker for 10 minutes. The supernatant was removed from the mixture and the beads were washed and resuspended in 50 mM EPPS buffer (pH 8.5). After beads and EPPS buffer were subjected to a 2-hour LysC digestion at 1 mAu per 100 µg of protein, trypsin was added at a 1:50 ratio for overnight digestion. The beads were washed with water and 30% acetonitrile the following day to elute the peptides, which were stored at − 80°C. For mass spectrometry, the peptides were analyzed using a Q Exactive Plus mass spectrometer coupled with an EASYLCn-1000 system (Thermo Fisher Scientific). The peptides were loaded onto an Easy-LCn-1000 and separated on an EASY-Spray 75µm x 500mm at 45°C using an ES803A analytical column (Thermo Fisher Scientific) at a flow rate of 300 nl/min. Raw mass spectrometry data were processed using FragPipe (version 20.0) and Rstudio with the Tidyverse R package for data manipulation, mice R package for imputing missing data, and LIMMA R package for differential expression analysis. Pathway analysis was performed using Kegg( 32 ) ( http://bioinformatics.sdstate.edu/go/ ) and Reactome databases( 1 ) ( https://reactome.org ). Spheroid Drug Treatments and Reattachment Cells were placed in 24-well ULA cluster plates at a density of 5 × 10 4 cells per well in 1 mL of medium. After 24 h, spheroids were treated with individually with carboplatin (100 µM), paclitaxel (50 nM), olaparib (20 µM), AKTi 1/2 (5 µM), trametinib (10 nM), or ralimetinib (15 µM). Spheroid cell viability was performed using Trypan Blue Exclusion assay as described above at 96 h for all drug treatments, except for Olaparib which was performed at 192 h. For spheroid reattachment assays, inhibitor-treated spheroids were reattached to standard tissue culture plates for 48 h and viability was assessed using alamarBlue at 1:10 dilution in media. Fluorescence was measured using the Agilent Biotek Synergy H1 plate reader. Statistical analysis Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software) and the details for specific statistical tests are described in each figure legend. RESULTS Differential requirement for ULK1 between EOC and noncancer precursor spheroids Autophagy induction is controlled by the ULK complex, notably by ULK1 kinase activity ( 16 ). Our previous studies showed that EOC spheroids exhibit elevated ULK1 expression, which is associated with increased autophagy activation, and that targeted ULK1 knockdown or inhibition effectively disrupted autophagy activation ( 25 ). To further elucidate the role of ULK1 in autophagy and tumorigenesis, we ablated ULK1 in OVCAR8 and HEYA8 EOC cells using CRISPR/Cas9 and pooled multiple independent clones to generate a population of ULK1 knockout ( ULK1 KO) cells. To validate the effect of ULK1 loss on autophagy, we examined proteins involved in the autophagic pathway. Upon examining parental OVCAR8 and HEYA8 spheroids, we observed increased ULK1 expression, whereas ULK1 KO cells exhibited a complete absence of ULK1 (Fig. 1 A). In both OVCAR8 and HEYA8 ULK1 KO day 3 spheroids, we detected a significant increase in p62 expression and a substantial reduction in LC3II:I compared to their parental cell lines (Fig. 1 A and B). This is important, as p62 accumulation serves as an indicator of autophagy inhibition, whereas its decrease suggests autophagy induction ( 32 ). Additionally, the LC3II:I ratio, derived from the processing of LC3I to LC3II, a marker of autophagosome membranes, reflects the activation of autophagy ( 33 ). We observed a significant decrease in the LC3II:I ratio observed as early as 4 and 24 h within OVCAR8 and HEYA8 ULK1 KO spheroids, respectively (Supplementary Fig. 1A and B). Interestingly, ULK1 loss resulted in an elevation of basal LC3I (Fig. 1 A and B) and a significant reduction in p-ATG16L1 (S278) (Supplementary Fig. 2A), supporting the established role of ULK1 in the early processing of LC3 ( 34 ). Additionally, we verified the abrogation of ULK1 activity through loss p-Beclin-1 (S30) levels (Fig. 1 A and B), as this is a known substrate for ULK1 activity ( 35 ). High-grade serous ovarian cancer (HGSOC) is the most prevalent EOC histotype that arises from the preneoplastic lesions in the secretory epithelium of the distal fallopian tube ( 37 ). Therefore, we ablated ULK1 in FT190 cells, an immortalized human fallopian tube secretory epithelial cell line ( 26 ). Although ULK1 loss resulted in reduced phosphorylated Beclin-1 (S30) levels as seen in EOC cells, no significant differences in the LC3II:I ratio were observed between FT190 parental and FT190 ULK1 KO spheroids (Fig. 1 A and B; Supplemental Fig. 1C), suggesting that ULK1 activity is not essential for autophagy activation in these precursor cells. To address the potential compensation due to ULK1 loss in FT190 cells, we investigated ULK2 protein expression, a homolog of ULK1 that is believed to be redundant in autophagy activation ( 38 ). No differences in ULK2 expression were observed among ULK1 KO EOC lines, however, a significant increase was observed in FT190 ULK1 KO adherent cells (Fig. 1 B). These results suggest that ULK1 is vital for autophagy activation in EOC spheroids, whereas its role may be redundant in HGSOC precursor cells. Cell viability is significantly impaired in EOC ULK1 KO spheroids Spheroids enhance EOC cell viability during metastasis by protecting from anoikis and chemotherapy-induced damage ( 39 ). To understand whether ULK1 and autophagy activities contribute to this property, ULK1 KO cells were grown in suspension culture to assay for differences in spheroid morphology, density, integrity, and viable cell number. We found that HEYA8 ULK1 KO spheroids showed obvious differences in morphology with decreased density and cell number, and impaired integrity compared to parental spheroids, whereas OVCAR8 ULK1 KO spheroids retained overall morphology but displayed reduced viable cell number also (Supplementary Fig. 3A). A significant reduction in viable cells was observed by Trypan Blue exclusion cell counting in both OVCAR8 and HEYA8 ULK1 KO spheroids on days 7 and 10 (Fig. 2 A). Although all EOC spheroids grew in cell number up to day 3, growth plateaued in OVCAR8 spheroids after this time; HEYA8 parental spheroids continued to expand in cell number, yet ULK1 KO spheroids failed to do so (Fig. 2 A). Interestingly, loss of ULK1 in FT190 spheroids resulted in a further reduction in viable cells as compared with parental spheroids (Fig. 2 A). EOC parental and ULK1 KO cells expressing nuclear-localized GFP were generated to facilitate fluorescence imaging as an indirect indicator of spheroid growth. However, no differences in growth rate were observed by fluorescence imaging between EOC parental and ULK1 KO spheroids (Supplementary Fig. 3B). To determine whether reduced viable cell number in ULK1 KO spheroids occurs via cytostasis or cell death, we assessed markers for proliferation and apoptosis. Since EOC spheroids display features of quiescent cells compared to adherent cells ( 8 ), we evaluated the expression of tumor suppressor proteins p21 and p27, which are established markers of tumor cell dormancy ( 40 ). In line with this, we have shown previously that cellular quiescence in EOC spheroids is associated with increased p27 ( 8 ). Herein, we observed significant reductions in p21 and p27 expression in HEYA8 ULK1 KO spheroids, yet in contrast these markers exhibited significant increases in OVCAR8 ULK1 KO spheroids (Fig. 2 B). We measured apoptosis activity in spheroids at multiple time points using the Caspase 3/7 Glo assay. We observed a significant surge in apoptosis within 24–48 h of spheroid formation, with elevated activity persisting for up to 72 h in HEYA8, OVCAR8 and FT190 spheroids due to ULK1 ablation (Fig. 2 C). These findings demonstrate that the primary mechanism whereby ULK1 loss impairs spheroid cell viability is through apoptosis induction. ULK1 is required for EOC tumor growth and spread in xenograft models Given our findings that ULK1 is critical for autophagy activation and spheroid viability, we sought to investigate its role in EOC tumor formation and metastasis directly, areas that have not been explored previously. To initiate these studies in cell culture, we grew parental and ULK1 KO cells as matrix-embedded organoids for up to 18 days and assessed expansion over time. Although total organoid number was similar between parental and ULK1 KO lines, there was a significant reduction organoid size over time due to ULK1 loss (Fig. 3 A and B; Supplementary Fig. 4A). To investigate ULK1 function in spheroid attachment, invasion and migration, we used the mesothelial clearance assay, an experimental model that mimics the early steps of EOC metastasis ( 41 ). We transferred pre-formed EOC spheroids expressing tdTomato onto either standard tissue culture plastic or GFP-expressing ZT human mesothelial cells. This allowed us to separately quantify spheroid attachment and dispersion from mesothelial cell displacement properties. OVCAR8 ULK1 KO cells displayed significantly reduced ability to displace mesothelial cells (Fig. 3 C) and disperse on tissue culture plastic (Supplementary Fig. 5B), whereas the displacement capacity of HEYA8 ULK1 KO cells was impaired in the presence of mesothelial cells only (Fig. 3 C; Supplementary Fig. 4B). These findings of altered cell motility properties were recapitulated using a scratch wound closure assay, where ULK1 loss significantly decreased wound closure rate in OVCAR8 cells but not in HEYA8 cells (Supplementary Fig. 4C). These cell culture-based results suggest ULK1 may have additional functions to promote EOC spheroid cell invasiveness and metastatic capacity in vivo. To model EOC metastasis, whereby malignant cells from the primary tumor are shed directly into the peritoneal cavity ( 2 , 3 ), we injected luciferase/tdTomato-expressing cells intraperitoneally into female NOD/SCID mice and monitored tumor progression over time via BLI (Fig. 4 A and D). Mice injected with OVCAR8 ULK1 KO cells showed reduced tumor burden at all time points, with significant decreases observed during the mid-to-late stage of disease progression (Fig. 4 B). In contrast, HEYA8 ULK1 KO cells had a significant decrease only at very early stages of disease progression (Fig. 4 E), which was lost at later time points. At experimental endpoint, EOC cells with ULK1 loss resulted in fewer tumor lesions observed at several metastatic sites, with notable decreases in ascites formation and omental metastasis (Fig. 4 C and F), two canonical features of metastatic EOC. Despite reduced tumor growth and metastatic spread, no significant differences were observed in survival rates (Supplementary Fig. 5A). In addition, no differences in either Ki67- or Caspase-3-positive IHC staining on tumor samples were seen (Supplementary Fig. 5C and D). Taken together, our findings suggest that ULK1 impacts EOC progression by affecting intrinsic tumor cell growth, and spheroid adhesion and invasion at later steps of metastasis. ULK1 knockout does not synergize with standard-of-care treatment Elevated autophagy levels have been linked to poor prognostic outcomes in cancer patients due to cytotoxic drug resistance ( 42 ). Since we observed reduced tumor burden and metastasis due to ULK1 ablation in injected EOC cells, yet this did not alter overall survival, we tested whether ULK1 loss and autophagy disruption would sensitize EOC spheroids to chemotherapy. Using our in vitro spheroid model system, we assessed cell viability by treating spheroids with either carboplatin or paclitaxel as single agents. Carboplatin treatment resulted in a significant increase in viable cells in OVCAR8 ULK1 KO spheroids and no difference in HEYA8 ULK1 KO spheroids. No significant differences in viability were observed between parental and ULK1 KO spheroids under paclitaxel treatment (Fig. 5 A and B). We also treated spheroids with Olaparib, a poly ADP ribose polymerase (PARP) inhibitor used as maintenance therapy in select HGSOC patients ( 43 ). Both parental and ULK1 KO spheroids showed significant sensitivity to Olaparib, yet ULK1 loss did not alter this effect (Fig. 5 C). These results suggest that while ULK1 knockout reduces spheroid cell viability, its combination with standard-of-care therapies elicited no further improvement, underscoring our subsequent studies to identify alternative strategies to improve efficacy. ULK1 loss disrupts key cell survival pathways in EOC spheroids In addition to its well-established role as a primary regulator of autophagy, ULK1 plays critical roles in energy metabolism, mitochondrial homeostasis, and vesicular trafficking ( 44 ). However, its non-canonical functions in EOC remain largely unexplored yet may be implicated in driving EOC progression and thus serve as new therapeutic targets to improve the limited efficacy of standard chemotherapies as seen in our findings. To this end, we performed proteomic mass spectrometry and bioinformatic analyses on OVCAR8 and OVCAR8 ULK1 KO spheroids to identify potential ULK1-regulated pathways in our experimental system. Through KEGG and Reactome pathway analyses of our resultant dataset (Fig. 6 A), we found significant changes biological pathways related to cell survival, including apoptosis, and PI3K-AKT-mTOR and MAPK signaling that were shared between both analyses. Since we had already observed enhanced apoptosis in ULK1KO spheroids (Fig. 2 C), we sought to validate members of PI3K-AKT-mTOR and MAPK signaling pathways, given their critical roles in regulating tumor progression ( 45 , 46 ). We observed significant reductions in p-MEK (S217/221) and p-p38 (Thr180/Tyr182) levels, while no changes in downstream p-ERK (Thr202/Tyr204) were detected (Fig. 6 B and C; Supplemental Fig. 6A). This trend extended to the PI3K-AKT-mTOR pathway, with a universal decrease in p-AKT (S473) in ULK1 KO spheroids. Further downstream analysis of AKT revealed contrasting effects, with increased P70S6K phosphorylation (T389) yet decreased 4EBP1 phosphorylation (Thr37/46) in ULK1 KO spheroids (Fig. 6 B and C; Supplementary Fig. 6B). Female mice xenografted with OVCAR8 and OVCAR- ULK1 KO cells developed malignant ascites with reduced prevalence due to ULK1 loss (Fig. 4 C). These ascites samples were returned to cell culture to study whether their inherent pathobiology had changed during disease progression in mice. OVCAR8 ULK1KO spheroids from ascites-derived lines remained autophagy deficient, as evidenced by a decreased LC3II:I ratio, increased p62, suppressed ULK1 activity with reduced p-Beclin-1 S30, and lower ULK2 expression. Phosphorylated-MEK1/2 was reduced, which was consistent with original OVCAR8- ULK1 KO spheroid cells. However, the changes in p-AKT (S473), p-P70S6K (T389), and p-p38 MAPK seen in pre-injection OVCAR8 ULK1 KO spheroids were not observed in ascites-derived lines, although total AKT protein decreased and total P70S6K increased in ascites-derived lines (Supplementary Fig. 7A and B). Despite the observed changes in MEK-MAPK and PI3K-AKT-mTOR signaling proteins, ascites-derived OVCAR8 ULK1 KO spheroids retained impaired metastatic potential in mesothelial clearance and reattachment assays, indicating persistent functional defects post-xenografting (Supplementary Fig. 7C and D). Collectively, these observations suggest that ULK1 disruption leads to reprogramming of key signaling pathways known to impact tumor progression and cancer cell survival. ULK1 ablation enhances efficacy of MEK and mTOR inhibition To explore novel synergistic treatment strategies in the context of ULK1 loss, we targeted the dysregulated PI3K-AKT-mTOR and MEK-MAPK pathways in ULK1 KO spheroids using specific inhibitors. Treatment with AKTi-1/2 (AKT inhibitor) and ralimetinib (p38 inhibitor) resulted in significantly increased cell viability in OVCAR8 ULK1 KO spheroids, while no differences were observed in HEYA8 ULK1 KO spheroids (Fig. 7 A). However, treatment with either trametinib (MEK inhibitor) or AZD-8055 (mTORC1/2 inhibitor) resulted in significantly reduced cell viability in ULK1 KO spheroids (Fig. 7 A), indicating that ULK1 loss may sensitize EOC spheroid cells to MEK and mTOR inhibition. We conducted spheroid reattachment assays to evaluate the effect of these inhibitors on this key step in the metastatic process ( 47 ). Untreated OVCAR8 ULK1 KO spheroids had significantly reduced reattachment, which was further decreased due to trametinib and AZD-8055 treatment (Fig. 7 B). So too did treatment with trametinib and AZD-8055 significantly reduce HEYA8 ULK1 KO spheroid reattachment (Fig. 7 B). Our findings demonstrate PI3K-AKT-mTOR and MEK-MAPK signaling pathways contribute to EOC spheroid viability and metastatic properties and may represent important therapeutic targets particularly when combined with ULK1 ablation and autophagy blockade. DISCUSSION Epithelial ovarian cancer is a highly lethal gynecologic cancer characterized by late-stage diagnosis, high relapse rates, and the formation of chemo-resistant spheroids that contribute to peritoneal metastasis. To the best of our knowledge, this is the first study to elucidate the role of ULK1 function using the combination of in vitro , ex vivo , and in vivo models of EOC metastasis. Our findings revealed that beyond its role in regulating autophagy, ULK1 deficiency significantly impacted tumor progression, leading to reduced spheroid viability, diminished invasive capacity, and impaired organoid growth. Additionally, our tumor xenograft models demonstrated that ULK1 loss significantly decreases tumor growth and spread, highlighting its critical role in supporting key processes in EOC metastasis and tumor development. Our study is underscored by our proteomic mass spectrometry analysis, which revealed dysregulated mTOR-PI3K-AKT and MAPK signaling and increased sensitivity to MEK and mTOR inhibition in ULK1 KO spheroids. The findings provide new insights into potential autophagy-independent functions of ULK1 and highlight novel therapeutic strategies in EOC. As we expected, ULK1 loss ablated LC3II:I processing and elevated p62 levels in EOC spheroids. Although our previous research suggests that Beclin-1 is not required for autophagy activation in EOC spheroids ( 49 ), the absence of p-Beclin-1 (S30) in ULK1 KO cells highlights its utility as a specific biomarker for ULK1 activity in EOC, particularly if assessing on-target activity of ULK1 inhibitors. Interestingly, ULK1 loss did not impair autophagy activation in the FT190 HGSOC precursor cell line. Instead, there was a substantial increase in ULK2 protein expression in FT190 ULK1 KO cells suggesting a compensatory mechanism for autophagy activation. Consistent with our findings, a previous study reported that ULK1 inhibition did not alter ULK2 expression in HGSOC cells ( 50 ). Additionally, ULK2 has been shown to compensate for ULK1 loss in mouse embryonic fibroblasts, but not in cerebellar granule neurons ( 51 ). Our observations suggest a unique requirement for ULK1 to activate autophagy in malignant EOC cells. EOC metastasis occurs by direct dissemination of tumor cells into the peritoneal cavity, where spheroid clusters suspended in ascites promote secondary tumor formation through enhanced survival, adhesion, and invasiveness. Using spheroid models that mimic these unique metastasis mechanisms, our results highlight ULK1's pivotal role in EOC progression. ULK1 loss significantly impaired viability, increased apoptosis, and reduced invasive capacity of EOC spheroids. These reductions in spheroid cell viability appear to be driven by increased apoptosis rather than altered cell growth. Anoikis, a programmed cell death triggered by the loss of cell attachment to the extracellular matrix, serves as a critical barrier to metastasis ( 52 ). Spheroids, however, resist anoikis by activating autophagy ( 52 ) and PI3K-AKT-mTOR and MAPK signaling to promote apoptosis resistance ( 53 ). Given our findings of decreased PI3K-AKT-mTOR and MAPK signaling in ULK1 KO spheroids and the role of autophagy as a defense mechanism preceding apoptosis ( 54 ), the disruption of these survival pathways likely compromises the ability of spheroids to resist anoikis, leading to increased apoptosis and reduced viability. In addition to reduced tumor cell dissemination, we observed fewer metastases and reduced ascites formation. We initially speculated that this reduction in secondary tumors was primarily due to ULK1’s role in regulating cell viability in suspension rather than directly on secondary growth or altered invasive capacities. However, we observed significant reductions in mesothelium invasion of spheroids lacking ULK1. Our findings corroborate a previous study demonstrating that inhibiting autophagy restricts the invasiveness of ovarian cancer cells via the negative regulation of p62 on ERK1/2 activity for invadopodium formation ( 55 ). In addition, ULK1 loss significantly impaired the growth of EOC cells in organoid culture, highlighting potential ULK1 function in tumor development. Patient-derived organoids (PDOs) represent another three-dimensional culture system similar to spheroids where PDOs serve to replicate the structural and functional characteristics of the original tissue, providing an accurate ex vivo model for studying tumor growth ( 56 ). Taken together, these data suggest that metastatic cells reaching secondary sites may exhibit compromised invasiveness and impaired re-initiation of tumor growth. In xenograft models, ULK1 knockout reduced tumor burden and limited metastatic spread, demonstrating its importance in systemic disease progression. Similarly, evidence from both in vitro and in vivo investigations indicate that ULK1 depletion significantly reduces pancreatic and hepatocellular carcinoma growth ( 57 , 58 ). However, ULK1’s role in cancer development might be context-specific among different malignancies. For example, in breast cancer models, ULK1 loss has been associated with an increased likelihood of osseous metastasis ( 59 ). In contrast, EOC rarely metastasizes to distant sites such as the lungs, skin, bones, or brain via hematogenous dissemination highlighting the underlying mechanisms driving EOC metastasis are biologically different from many other carcinomas ( 60 ). Despite significant reductions in tumor burden, mice injected with ULK1 knockout cells did not exhibit improved survival. These mice developed distended abdomens, jaundice, and significant weight loss, ultimately requiring sacrifice in accordance with approved guidelines. This underscores the lethality of EOC, as tumor cells, even with ULK1 loss, can still disseminate to vital abdominal organs, driving disease progression and mortality. We reported that autophagy levels and ULK1 mRNA overexpression are correlated with poor survival outcomes in advanced-stage ovarian cancer ( 25 ), making it a promising therapeutic target. While combining ULK1 inhibition with standard-of-care chemotherapeutics might seem promising to enhance anti-tumor effects, the results are not uniformly positive. Previous studies have suggested that ULK1 loss can enhance chemotherapy sensitivity in OVCAR8 cells ( 61 ); however, our findings, along with unpublished in vitro data (Johnston & Shepherd, in preparation), indicate that ULK1 inhibition may instead reduce the efficacy of standard first-line chemotherapeutics used in EOC. Previous studies in gastric cancer have shown that elevated p62 expression activates the transcription of Chemokine C-C motif ligand 2 (CCL2), a cytokine associated with drug resistance and contributes to cisplatin resistance ( 62 ). Perhaps the significant increase in p62 expression resulting from ULK1 loss decreased chemotherapeutic efficacy in our system as well. While ULK1 is widely recognized as a critical regulator of autophagy, its autophagy-independent functions, especially in the context of EOC, have been less studied. As such, we performed protein mass spectrometry on OVCAR8 and OVCAR8 ULK1 KO spheroids, which verified our findings of increased apoptosis and revealed significant alterations in critical signaling pathways, including PI3K- AKT- mTOR and MAPK signaling. The PI3K-AKT-mTOR pathway is a hallmark cancer promoter that governs essential processes such as cell growth, motility, survival, and metabolism ( 63 ). Similarly, MAPK pathways play a central role in regulating fundamental processes such as cell proliferation, differentiation, and stress responses ( 64 ). We demonstrated a universal decrease in p-MEK (S217/22), p-p38 (T180/Thr182), and p-AKT (S473), and an increase in p-p70S6K (T389) in EOC spheroids lacking ULK1. This aligns with previous studies implicating MAPK signaling in ovarian cancer, where it has been shown to regulate autophagy and inhibit apoptosis ( 65 ), and promote invasion and proliferation through combined AKT and MAPK signaling activities ( 66 ). Therefore, we were intrigued to assess the therapeutic potential of targeting these pathways in our ULK1 deficient system. While AKT and p38 inhibition significantly decreased EOC spheroid viability, ULK1 loss did not increase drug sensitivity and, in some cases, exhibited potential antagonistic effects. However, ULK1 loss significantly enhanced the sensitivity of EOC spheroids to MEK inhibition via trametinib and mTORC1/2 inhibition via AZD-8055. Many preclinical and clinical studies have explored targeting the PI3K-AKT-mTOR pathway, including the use of mTOR inhibitors, in EOC ( 67 ). However, the clinical application of AZD-8055 is limited by its pharmacokinetics, inadequate intratumoral concentrations, and dose-limiting toxicities ( 68 ). Its successor, AZD-2014 (vistusertib), initially showed reduced liver toxicity ( 68 ), but recent studies, such as those in meningiomas, reported poor tolerance, with most participants discontinuing the trial ( 69 ). Interestingly, combination therapy with vistusertib and anastrozole in advanced hormone receptor-positive endometrial cancer demonstrated manageable adverse events and improved overall response rates and progression-free survival (NCT02730923) ( 70 ). Trametinib, represents a new standard-of-care option for relapsed or persistent low-grade serous ovarian cancer ( 71 ), and has shown success in a patient with recurrent HGSOC who had several lines of prior therapy ( 72 ). These findings suggest that blocking autophagy via ULK1 inhibition combined with MEK inhibition via trametinib or mTORC1/2 inhibition could be effective in EOC. It would be worthwhile to investigate the efficacy of ULK1 inhibitors combined with these targeted agents in PDOs to reveal therapeutic potential ( 73 ). Furthermore, our findings suggest that by targeting ULK1 and its autophagy-dependent and -independent pathways would offer a promising new therapeutic strategy to disrupt EOC metastatic progression. CONCLUSION Our comprehensive analysis underscores ULK1's multifaceted role in EOC, where its influence extends beyond autophagy regulation to impact key cell survival pathways, particularly apoptosis and MAPK and PI3K-AKT-mTOR signaling networks. Although ULK1 loss did not enhance the efficacy of standard-of-care chemotherapeutics, it significantly sensitized EOC spheroids to MEK and mTORC1/2 inhibition. Ultimately, our research establishes that targeting ULK1 could offer a promising strategy for controlling tumor growth and reducing metastasis in EOC, providing a new avenue for therapeutic intervention aimed at improving patient outcomes in ovarian cancer. Abbreviations EOC Epithelial ovarian cancer ULK1 Unc51-like kinase ATG13 Autophagy-related gene 13 FIP200 Focal adhesion kinase interacting protein 200 mTOR Mammalian target of rapamycin AMPK AMP-activated protein kinase PARP Poly (ADP-ribose) polymerase MAPK1/2 Mitogen-activated protein kinase 1/2 MEK1/2 Mitogen-activated protein kinase kinase 1/2 ERK1/2 Extracellular signal-regulated kinase 1/2 HGSOC High-grade serous ovarian carcinoma PDO Patient-derived organoid FACS Fluorescence-activated cell sorting GFP Green fluorescent protein ULA Ultra-low attachment IHC Immunohistochemistry Declarations Ethics approval and consent to participate All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Western Ontario (London, Ontario, Canada) and were performed in accordance with approved guidelines. Consent for publication Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Competing interests The authors declare that they have no competing interests. Funding We acknowledge the funding support from the Cancer Research Society to TGS and the London Health Sciences Foundation through donations to the Mary and John Knight Translational Ovarian Cancer Research Unit. JDW was supported by an Obstetrics & Gynecology Graduate Scholarship from the Department of Obstetrics and Gynecology at the Western University and a Queen Elizabeth II Graduate Scholarship in Science and Technology (Ontario Government). Authors' contributions JDW and TGS conceptualized and designed the study. JDW, ET, LV, YRV, and AB acquired data. TGS supervised and obtained funding for this study. BS, MB, and YRV provided additional resources. 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Trametinib response in heavily pretreated high-grade ovarian cancer: One step towards precision medicine. Gynecol Oncol Rep. 2020;32. Chan WS, Mo X, Ip PPC, Tse KY. Patient-derived organoid culture in epithelial ovarian cancers—Techniques, applications, and future perspectives. Vol. 12, Cancer Medicine. John Wiley and Sons Inc; 2023. p. 19714–31. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files JDWSupplementalFigureLegends.docx Supplemental Figure Legends SupplementalFigure1.png Supplemental Figure 1 SupplementalFigure2.png Supplemental Figure 2 SupplementalFigure3.png Supplemental Figure 3 SupplementalFigure4.png Supplemental Figure 4 SupplementalFigure5.png Supplemental Figure 5 SupplementalFigure6.png Supplemental Figure 6 SupplementalFigure7.png Supplemental Figure 7 Cite Share Download PDF Status: Published Journal Publication published 05 Mar, 2026 Read the published version in Oncogene → Version 1 posted Editorial decision: revise 31 Jul, 2025 Review # 3 received at journal 27 Jul, 2025 Reviewer # 3 agreed at journal 14 Jul, 2025 Review # 2 received at journal 18 Apr, 2025 Reviewer # 2 agreed at journal 10 Apr, 2025 Reviewer # 1 agreed at journal 30 Mar, 2025 Reviewers invited by journal 28 Mar, 2025 Submission checks completed at journal 04 Mar, 2025 Editor assigned by journal 03 Mar, 2025 First submitted to journal 03 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6148090","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":435247488,"identity":"834ebce5-b675-4512-bb8f-162a5c0be620","order_by":0,"name":"Trevor Shepherd","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYFACHgYGxgYGOX4SNEC0GEs2kKol0eAAsVrs+c8efFy4wybB+EaO4QOGGjsibJHISzaeeSYtz+xGjrEBw7FkYrTwmEnzth0uNruRu02CsYGZCC38Z8x/87b9T9w8A6ylnggtDDlmzLxtBxI3SIC1HCZCC9AL0jPbko0lzrz/bJBw7DhhLez9Zww/F7bZyfG3pyU++FBTTVgLCCC8nECcBmQto2AUjIJRMAqwAQCasjUlrHkyJQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1853-8066","institution":"University of Western Ontario","correspondingAuthor":true,"prefix":"","firstName":"Trevor","middleName":"","lastName":"Shepherd","suffix":""},{"id":435247489,"identity":"9f945e95-eee5-40d4-94ac-87b447455424","order_by":1,"name":"Jack Webb","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jack","middleName":"","lastName":"Webb","suffix":""},{"id":435247490,"identity":"72208742-7b06-49bb-8fce-f3d5a522b367","order_by":2,"name":"Adrian Buensuceso","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Adrian","middleName":"","lastName":"Buensuceso","suffix":""},{"id":435247491,"identity":"d22e431b-ffe4-4c67-9530-6c637e1440c4","order_by":3,"name":"Emily Tomas","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"","lastName":"Tomas","suffix":""},{"id":435247492,"identity":"430bf6ca-778f-4297-99b7-6f37ac750176","order_by":4,"name":"Matthew Borrelli","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Borrelli","suffix":""},{"id":435247493,"identity":"12dff94c-0b8a-470d-af8b-eaf903cc30a5","order_by":5,"name":"Lauren Viola","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lauren","middleName":"","lastName":"Viola","suffix":""},{"id":435247494,"identity":"ef3d4ec6-a5f5-40e0-9bd5-ce50ca48781e","order_by":6,"name":"Owen Hovey","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Owen","middleName":"","lastName":"Hovey","suffix":""},{"id":435247495,"identity":"4dd5279d-2593-40b7-9943-46c7ca26a064","order_by":7,"name":"Yudith Ramos Valdes","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yudith","middleName":"Ramos","lastName":"Valdes","suffix":""},{"id":435247496,"identity":"29fdb003-033a-4548-943b-5f9afb737937","order_by":8,"name":"Bipradeb Singha","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bipradeb","middleName":"","lastName":"Singha","suffix":""},{"id":435247497,"identity":"cffb56bd-5b39-476e-aa49-f6d4a63d5929","order_by":9,"name":"Shawn Li","email":"","orcid":"https://orcid.org/0000-0003-3610-9035","institution":"Western University","correspondingAuthor":false,"prefix":"","firstName":"Shawn","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-03-03 16:52:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6148090/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6148090/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41388-026-03702-2","type":"published","date":"2026-03-05T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80725957,"identity":"abb51519-af08-4fed-bea5-1030f642e437","added_by":"auto","created_at":"2025-04-16 11:45:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1115567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential requirement for ULK1 between EOC and noncancer precursor spheroids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) OVCAR8 and HEYA8 EOC cells, and FT190 precursor cells with or without intact ULK1 were seeded as adherent and spheroid cultures. Protein lysates were harvested 72 h after seeding for western blot analysis of protein markers of autophagy and ULK1 activity as indicated. B) Densitometric analysis of autophagy markers and ULK1 activity in OVCAR8, HEYA8, and FT190 cells relative to their expression in parental cells in adherent conditions. Data are displayed as mean ± SEM; Two-way ANOVA followed by Šidák’s multiple comparisons test, *P\u0026lt;0.01, **P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/3a1364cbfc0dae015420588b.png"},{"id":80724469,"identity":"67b9085c-ecfd-4130-b7f9-7dbff01cfa2b","added_by":"auto","created_at":"2025-04-16 11:37:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":526395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViability is significantly impaired in EOC \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eULK1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eKO spheroids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) The number of viable OVCAR8, HEYA8, and FT190 cells were counted by Trypan Blue Exclusion Assay. Data displayed as mean ± SEM; Two-way ANOVA followed by Šidák’s multiple comparisons test, *P\u0026lt;0.01, **P\u0026lt;0.001, ****P\u0026lt;0.0001. B) OVCAR8 and HEYA8 parental and \u003cem\u003eULK1\u003c/em\u003eKO cells were seeded for spheroid culture and protein lysates were harvested at 72 h for western blot analysis. Densitometric analysis of p27 and p21 in OVCAR8 and HEYA8 cells relative to expression in parental spheroid conditions. Data displayed as mean ± SEM; Student’s\u003cem\u003e t\u003c/em\u003e-test, *P\u0026lt;0.05, **P\u0026lt;0.001. C) Apoptosis activity was measured using Caspase-Glo 3/7 luminescence assays. Cells were seeded at 2000 cells per well in a 96-well ULA plate. Data reflects luminescence relative to parental spheroids at each time point. Data displayed as mean ± SEM; Student’s \u003cem\u003et\u003c/em\u003e-test, *P\u0026lt;0.05, **P\u0026lt;0.001, ***P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/23f1fe44b24b253f9963eac4.png"},{"id":80726975,"identity":"452f364c-6c65-4b7b-8e70-d0f8bc42b91b","added_by":"auto","created_at":"2025-04-16 11:53:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2994293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eULK1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e loss impairs EOC organoid growth and spheroid invasion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) OVCAR8 and HEYA8 parental and \u003cem\u003eULK1\u003c/em\u003eKO cells were grown as organoids and images were captured for up to 18 days using the IncuCyte S3 Live-Cell Analysis System. Scale bar represents 800𝜇m and 200𝜇m, respectively. B) Organoid area was quantified using the IncuCyte S3 Live-Cell Analysis System. Data reflects the total organoid area and displayed as mean ± SEM. Two-way ANOVA followed by Šidák’s multiple comparisons test; *P\u0026lt;0.01, **P\u0026lt;0.001. C) The mesothelial clearance assay was utilized to evaluate EOC spheroid invasion. Spheroids expressing tdTomato were seeded onto ZT-GFP cells and imaged 24 h later. Displacement was quantified using Fiji. Data reflects area of ZT cells displaced normalized to the original spheroid size and displayed as mean ± SEM. Scale bar represents 300𝜇m.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/5c3e88bed59324519ff6a0d5.png"},{"id":80724475,"identity":"9042676d-116f-4e83-9d71-e56049f3305c","added_by":"auto","created_at":"2025-04-16 11:37:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1981290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eULK1 is required for EOC tumour growth and spread in xenograft models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Representative bioluminescent images of mice injected i.p. with OVCAR8 parental and \u003cem\u003eULK1\u003c/em\u003eKO cells. The BLI signal for each image is normalized independently based on the optimal imaging parameters for that specific mouse, including binning, radiance, and exposure values. Scale bar indicates luminescence intensity, with red representing higher luminescence values to blue representing lower luminescence values. B) Total flux (photons/sec) was used as a measure of tumor burden as assessed weekly via bioluminescence imaging. Data are displayed as mean ± SEM; Student’s \u003cem\u003et\u003c/em\u003e-test, *P\u0026lt;0.05, **P\u0026lt;0.01. C) Petal plots representing the proportion of mice displaying tumors at peritoneal sites or presence of ascites, as indicated. Radial gridlines represent 10% gradations (OVCAR8, n=6; OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO, n=7). D) Representative bioluminescent images of mice injected i.p. with HEYA8 parental and \u003cem\u003eULK1\u003c/em\u003eKO cells. E) Total flux (photons/sec) was as a measure of tumor burden assessed weekly via bioluminescence imaging. Data are displayed as mean ± SEM; Student’s \u003cem\u003et\u003c/em\u003e-test, *P\u0026lt;0.05. F) Petal plots representing the proportion of mice displaying tumors at peritoneal sites or presence of ascites, as indicated. Radial gridlines represent 10% gradations (HEYA8, n=7; HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO, n=6).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/dbec1378bc1a583616e5fe96.png"},{"id":80727415,"identity":"d57becb1-45fe-4738-87f0-46d2045733d5","added_by":"auto","created_at":"2025-04-16 12:01:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":175540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eULK1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e loss does not synergize with standard-of-care therapeutics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpheroids of each indicated cell line were cultured for 24 h before treating with either A) carboplatin (100 µM) or B) paclitaxel (50 nM) for 72 h, or C) olaparib (20 µM) for 168 h. Viable cell number was measured by Trypan Blue Exclusion Assay. Data displayed as mean ± SEM; Two-way ANOVA followed by Šidák’s multiple comparisons test, *P\u0026lt;0.01, **P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/1b70436a87037cec05857fd7.png"},{"id":80724478,"identity":"2e57c5b1-0981-498f-b056-5604d7fe34ff","added_by":"auto","created_at":"2025-04-16 11:37:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":909839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eULK1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e loss disrupts cell survival pathways in EOC spheroids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Differentially expressed proteins identified by label-free proteomics of OVCAR8 parental and \u003cem\u003eULK1\u003c/em\u003eKO spheroids were applied to KEGG and Reactome pathway enrichment analysis using STRING and Cytoscape applications. Proteins increased or decreased in OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids versus parental controls identified major pathways with the respective gene counts and false discovery rates as indicated. B) OVCAR8 and HEYA8 parental and \u003cem\u003eULK1\u003c/em\u003eKO cells were seeded into adherent and spheroid cultures. Protein lysates were harvested 72 h after seeding for western blot analysis of MEK-MAPK and PI3K- AKT-mTOR signaling pathways. C) Densitometric analysis of western blots was performed relative to parental adherent conditions. Phosphorylated proteins were normalized to their respective total protein levels (except p38 Thr180/Y182 that was normalized to vinculin). Data are displayed as mean ± SEM; Two-way ANOVA followed by Šidák’s multiple comparisons test; *P\u0026lt;0.01, **P\u0026lt;0.001, ***P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/0c5d575e37ca7e4224deba17.png"},{"id":80725960,"identity":"7b59c39b-dae1-460c-83ad-1010fcb3a7c7","added_by":"auto","created_at":"2025-04-16 11:45:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":446080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eULK1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ablation synergizes with MEK and mTORC1/2 inhibition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Spheroids were cultured for 24 h before treating with either AKTi-1/2 (5 µM), trametinib (10 nM), ralimetinib (15 µM) or AZD-8055 (10 nM) for 72 h before performing Trypan Blue Exclusion Assay. Data displayed as mean ± SEM; Two-way ANOVA followed by Šidák’s multiple comparisons test, *P\u0026lt;0.01, **P\u0026lt;0.001, ****P\u0026lt;0.0001. B) Inhibitor-treated spheroids were transferred to standard tissue-culture treated plates and cell viability was assessed at 48 h post-reattachment by alamarBlue assay. Data displayed as mean ± SEM; Two-way ANOVA followed by Šidák’s multiple comparisons test, *P\u0026lt;0.01, **P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/b7925f18c6105f9a53e51f72.png"},{"id":104410613,"identity":"b8d501a8-d0e1-470e-adcf-423590172fd1","added_by":"auto","created_at":"2026-03-11 12:53:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9717120,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/8223f03b-ff6c-4028-898a-f8131118a42b.pdf"},{"id":80724468,"identity":"331f1c79-7f6e-4797-9b9d-382f92e5bdf5","added_by":"auto","created_at":"2025-04-16 11:37:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22800,"visible":true,"origin":"","legend":"Supplemental Figure Legends","description":"","filename":"JDWSupplementalFigureLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/787871b2bda3700841548ff2.docx"},{"id":80724470,"identity":"b0a1d0c6-144e-4b14-8d02-be8547fbf560","added_by":"auto","created_at":"2025-04-16 11:37:51","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1671360,"visible":true,"origin":"","legend":"Supplemental Figure 1","description":"","filename":"SupplementalFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/1235f0b247e961e5c5ec3985.png"},{"id":80724477,"identity":"dc18c249-1455-4f94-ba99-430aabe424fc","added_by":"auto","created_at":"2025-04-16 11:37:52","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":176468,"visible":true,"origin":"","legend":"Supplemental Figure 2","description":"","filename":"SupplementalFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/3fe6d756728a5880cda00749.png"},{"id":80725964,"identity":"f690fa68-9274-44f9-a2ba-d02301a122a4","added_by":"auto","created_at":"2025-04-16 11:45:52","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":941702,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure 3\u003c/p\u003e","description":"","filename":"SupplementalFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/288c91742a3ef6e3a54c9c5e.png"},{"id":80725966,"identity":"8e1d89c3-cc71-4a06-9e0e-245e81b2843a","added_by":"auto","created_at":"2025-04-16 11:45:52","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":792255,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure 4\u003c/p\u003e","description":"","filename":"SupplementalFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/ab93520cf5d52e65905d2ca1.png"},{"id":80724490,"identity":"c36d3fd4-69c2-4a6d-b7ac-6900d9e93490","added_by":"auto","created_at":"2025-04-16 11:37:52","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":6653157,"visible":true,"origin":"","legend":"Supplemental Figure 5","description":"","filename":"SupplementalFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/3d0ca731e0d88e28f3b326fa.png"},{"id":80724487,"identity":"c9dda5dd-9c58-4a99-afc5-5ece8ffb4529","added_by":"auto","created_at":"2025-04-16 11:37:52","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":553385,"visible":true,"origin":"","legend":"Supplemental Figure 6","description":"","filename":"SupplementalFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/916b3ec8abe817b0d005fd90.png"},{"id":80724491,"identity":"36ed6350-72e3-4cdd-8352-0976ce839e46","added_by":"auto","created_at":"2025-04-16 11:37:52","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":2304202,"visible":true,"origin":"","legend":"Supplemental Figure 7","description":"","filename":"SupplementalFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6148090/v1/2a949ecb5e694383d167d3c9.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"ULK1 promotes metastatic progression in epithelial ovarian cancer","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eEpithelial ovarian cancer (EOC) has the fifth-highest death-to-incidence ratio for all cancers in women and is the leading cause of death from gynecologic cancers due to its late-stage diagnosis and lack of effective strategies for treating chemoresistant disease (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Patients with EOC are typically treated with aggressive surgical debulking and cytotoxic carboplatin/paclitaxel combination chemotherapy; however, nearly 80% of these patients relapse within five years (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Investigations into the mechanisms that support cell survival during metastasis and regrowth of refractory EOC cells after treatment are of critical importance and are an area of active research. EOC spreads by tumor cells disseminating directly into the peritoneal space, often suspended in ascites, and then attaches to the serosal surfaces of the abdominal cavity to form secondary deposits (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Clusters of metastatic EOC cells known as spheroids accumulate in the malignant fluid of patients with advanced disease (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Spheroids are known to promote metastasis with increased cell survival in the face of chemotherapy and possess enhanced adhesive and invasive capabilities (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Additionally, our lab and others have clearly demonstrated that spheroid cells undergo numerous phenotypic changes, including cellular quiescence (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), epithelial-mesenchymal transition (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), activated stress metabolism (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), and autophagy (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), all of which contribute to the tumor cell dormancy phenotype of residual disease, and that may drive chemo-resistant recurrence.\u003c/p\u003e \u003cp\u003eAutophagy is an evolutionarily conserved and tightly controlled metabolic degradation process in which proteins and organelles are broken down in the lysosomes (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This degradative process yields metabolic substrates from lysosomal activity thereby providing vital nutrients for essential cellular functions during nutrient scarcity or energy stress (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Autophagy typically operates at basal levels to fulfill fundamental homeostatic functions but can quickly escalate under stress (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Autophagy induction is controlled by the Unc-51-like kinase (ULK1) complex, consisting of ULK1/2, autophagy-related gene 13 (ATG13), and focal adhesion kinase-interacting protein (FIP200) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). ULK1 is a serine-threonine kinase that responds to upstream signals of nutrient and energy availability to trigger autophagy through the initiation of phagophore formation. Under nutrient-abundant conditions, mechanistic target of rapamycin complex 1 (mTORC1) phosphorylates ULK1, thereby inhibiting its activity and initiating autophagy. Conversely, the absence of nutrients leads to mTORC1 deactivation, while AMP-activated protein kinase (AMPK) phosphorylates to activate ULK1 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Thus, ULK1 and autophagy are considered an essential regulatory hub to control energy supplies in an equilibrium with cellular demands.\u003c/p\u003e \u003cp\u003eThe role of ULK1 in autophagy initiation has been reviewed in (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), and its involvement in tumorigenesis\u0026mdash;promoting tumor growth, invasiveness, and survival\u0026mdash;has been documented across various cancer types (\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, few studies have investigated the role of ULK1-mediated autophagy in EOC with a specific focus on tumor growth and metastatic progression. We have previously demonstrated that AMPK activation (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and AKT-mTORC1 downregulation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) work in coordination to induce autophagy in high-grade serous ovarian cancer (HGSOC) spheroids. We also showed that EOC spheroids had increased ULK1 expression, which parallels autophagy induction, and that transient knockdown and inhibitor treatments \u003cem\u003ein vitro\u003c/em\u003e blocked autophagy and reduced cell viability (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Building on our prior work on autophagy in EOC spheroids and utilizing CRISPR/Cas9 technology for \u003cem\u003eULK1\u003c/em\u003e ablation, we aimed to elucidate ULK1's role in tumorigenesis by examining its contributions to spheroid survival, metastatic dissemination, and tumor growth to establish its potential as a therapeutic target in EOC metastasis.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCultured cell lines\u003c/h2\u003e \u003cp\u003eThe cell lines OVCAR8, OVCAR8-\u003cem\u003eULK1\u003c/em\u003eKO, HEYA8, and HEYA8-\u003cem\u003eULK1\u003c/em\u003eKO were grown in RPMI-1640 medium (Wisent), whereas FT190 and FT190-\u003cem\u003eULK1\u003c/em\u003eKO were grown in DMEM/F12 medium (Life Technologies). All growth media were supplemented with 10% fetal bovine serum. OVCAR8 and HEYA8 cells were procured from the American Type Culture Collection. Adherent cells were sustained on tissue culture-treated polystyrene (Sarstedt, Newton, NC, USA) and spheroids were maintained in Ultra-Low Attachment (ULA) cluster plates (Corning, NY, USA). The immortalized human fallopian tube secretory epithelial cell line FT190 (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) was generously provided by R. Drapkin from the University of Pennsylvania, Philadelphia, PA, USA. The human lung mesothelial ZT-GFP cell line (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) was generously provided by Marcin Iwanicki from the Stevens Institute of Technology All cell lines were authenticated through short tandem repeat analysis by the Center for Applied Genomics (The Hospital for Sick Children, Toronto, ON, Canada) and routinely examined them for mycoplasma using a Universal Mycoplasma Detection Kit (30-1012K; ATCC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of\u003c/b\u003e \u003cb\u003eULK1\u003c/b\u003e\u003cb\u003eKO cell lines\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCRISPR/Cas9 (sc-400516-KO-2 Lot# C3016, Santa Cruz Biotechnology) was used to ablate \u003cem\u003eULK1\u003c/em\u003e in OVCAR8, HEYA8, and FT190 cells. Briefly, cells were seeded at 1-1.5x10\u003csup\u003e5\u003c/sup\u003e per well in a 6-well plate and transfected the following day. Cells were trypsinized four days post-transfection and sorted using fluorescence-activated cell sorting (FACS) in 96-well plates. The clones were left to grow for a minimum of two weeks, after which colony formation was observed. Colonies were harvested and plated into 6-well plates and then 10 cm plates upon reaching confluency. Cells were harvested for protein lysates, screened for \u003cem\u003eULK1\u003c/em\u003e loss via western blotting and passaged for continued culture and subsequent clone pooling.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneration of Nuclight GFP cell lines\u003c/h3\u003e\n\u003cp\u003eCells were transduced with Incucyte\u0026reg; Nuclight Green Lentivirus (EF1a, Puro) (Sartorius, #4624) following the manufacturer's instructions. After transduction, cells were cultured in complete media supplemented with puromycin (1\u0026micro;g/mL; BioShop, #5E38885) to select successfully transduced cells. Transduced and puromycin-selected cells were subjected to FACS to isolate green fluorescent protein (GFP)-positive cells. The isolated GFP\u0026thinsp;+\u0026thinsp;cells were expanded and used for further analysis and experimentation.\u003c/p\u003e\n\u003ch3\u003eGeneration of mCherry-eGFP-LC3 cell lines\u003c/h3\u003e\n\u003cp\u003eParental and \u003cem\u003eULK1\u003c/em\u003eKO cells were transfected with mCherry-eGFP-LC3B autophagy reporter plasmid [pBABE-puro mCherry-EGFP-LC3B was a gift from Jayanta Debnath (Addgene plasmid # 22418; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:22418;RRID:Addgene_22418\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:22418;RRID:Addgene_22418\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e] (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). After transfection, cells were grown in complete media containing G-418 (400\u0026micro;g/mL, Wisent, #450-130-QL) for two weeks to select those with successful reporter plasmid integration. Following the selection phase, the cells were cultured in complete medium without G-418 for another four weeks, allowing for growth and recovery. Cells were sorted using FACS to identify double-positive cells (GFP+, mCherry+).\u003c/p\u003e\n\u003ch3\u003eGeneration of luc2tdTomato cell lines\u003c/h3\u003e\n\u003cp\u003eCells were transduced with pCDH-EF1-Luc2-P2A-tdTomato, according to the manufacturer\u0026rsquo;s instructions [pCDH-EF1-Luc2-P2A-tdTomato was a gift from Kazuhiro Oka (Addgene plasmid # 72486; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:72486;RRID:Addgene_72486\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:72486;RRID:Addgene_72486\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)]. After transduction, the cells were subjected to FACS to isolate the tdTomato\u0026thinsp;+\u0026thinsp;cells. The cells were cultured in a complete medium for growth and recovery. The cells were then subjected to another round of FACS to gate and select for populations of cells with similar tdTomato expression intensities. Cell populations were seeded in a serial dilution for bioluminescent imaging (BLI) analysis using D-luciferin and the IVIS Lumina S5 system (PerkinElmer) to determine optimal populations (i.e., similar reporter gene expression among lines) to use for subsequent \u003cem\u003ein vivo\u003c/em\u003e xenograft assays.\u003c/p\u003e\n\u003ch3\u003eAntibodies and reagents\u003c/h3\u003e\n\u003cp\u003eAntibodies against ULK1 (#8054S), p62 (#5114S), LC3B (#2775S), Beclin1 S30 (#5410S), Beclin1 (#3738S), AKT S473 (#9271), AKT (#9272S), MEK1/2 S217/221 (#9154S), MEK (#8727), ERK1/2 Thr202/Tyr204 (#9101), ERK1/2 (#9102), cleaved-PARP (#9541S), P70S6K Thr389 (#9234S), P70S6K (#2708S), p38 MAPK Thr180/Y182 (#4511S), 4EBP1 T37/46 (#2855S), 4EBP1 (#9452S) were purchased from Cell Signaling Technology. Anti-ULK2 antibody (AB97695), ATG16L1 (AB187671), ATG16L1 S30 (AB19016), and mCherry (AB167453; 1:500) were purchased from Abcam. Anti-actin antibody (A2066; 1:25000) was purchased from Millipore. Antibodies against tubulin (T5168; 1:40000) and vinculin (V9264; 1:25000) were purchased from Sigma-Aldrich. Horseradish peroxidase (HRP)-conjugated antibodies against mouse IgG (NA931; 1:10000) and rabbit IgG (NA934; 1:10000) were purchased from GE Healthcare. Antibodies were diluted in tris-buffered saline-Tween 20 containing either 5% bovine serum albumin or non-fat milk 1:1000 or as stated otherwise. Adenovirus expressing green fluorescent protein (Ad-GFP) was a kind gift from Dr. B. C. Vanderhyden (Ottawa Health Research Institute) and prepared as described previously (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Rat-tail collagen was purchased from Gibco (#963791) and Matrigel was purchased from Corning (CLS356231). Paclitaxel was purchased from Cayman Chemical Company (#10461) and stored at -20\u0026deg;C as 5 mM in DMSO stocks. Carboplatin was received from the London Regional Cancer Program and stored at 4\u0026deg;C as 27 mM in saline stocks. Olaparib (#HY-10162) and ralimetinib (#HK-13241) were purchased from MedChemExpress, trametinib (#7709) was purchased from Tocris Bioscience, and AKT inhibitor VIII (Akti-1/2) was purchased from EMD/Calbiochem (#12408).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot analysis\u003c/h2\u003e \u003cp\u003eThe Bio-Rad Mini-PROTEAN II Electrophoresis System was used for immunoblotting following the manufacturer's guidelines, utilizing in-house prepared gels (30% acrylamide/bis solution 37.5:1, catalog number 1610158; Bio-Rad). Densitometric analysis was conducted using Image Lab 6.05 software suite (Bio-Rad).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of wholecell lysates\u003c/h3\u003e\n\u003cp\u003eFor assessment of all proteins, 4-, 8-, 24-, 48, 72-hour whole-cell lysates were generated from adherent cells cultured at a density of 0.75-1\u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in 10 mL medium (10 cm dish), or spheroid cells cultured at a density of 1\u0026ndash;3\u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in 15 mL medium (35 mm ULA well). Cell seeding numbers were chosen to obtain acceptable protein yields for each cell line.\u003c/p\u003e \u003cp\u003e\u003cem\u003eWholecell lysates.\u003c/em\u003e Adherent cells cultivated on tissue culture-treated plates or dishes were collected by removing the medium, washing twice with cold PBS, and scraping into a modified radioimmunoprecipitation (RIPA) buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM EGTA, 1% Triton X-100, 0.1% SDS, 1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 10 mM NaF, 1 mM PMSF, 1 \u0026times; SIGMA\u003cem\u003eFAST\u003c/em\u003e protease inhibitor cocktail (cat. S8820; Sigma), 10 mM beta-glycerophosphate). Spheroids (minimum of 1.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per sample) were collected by transferring the cell suspension into an ice-cold conical tube, followed by centrifugation using a swinging bucket rotor (800 \u0026times; g at 4\u0026deg;C for 4 min) to form a pellet. The medium was aspirated, and the pellet was resuspended in cold PBS (at least 10 mL of cold PBS. This process was repeated by resuspending the pellet in cold PBS, followed by centrifugation and aspiration of the PBS. The resulting cell pellets were lysed using modified RIPA buffer, vortexed, exposed to one freeze-thaw cycle, and clarified by centrifugation (maximum \u0026times; g at 4\u0026deg;C for 20 min).\u003c/p\u003e\n\u003ch3\u003eSpheroid viability assays\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eBulk spheroids\u003c/em\u003e. Cells were placed in 24-well ultra-low attachment (ULA) cluster plates at a density of 0.5-1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well in 1 mL of medium. The spheroids were then collected into chilled microcentrifuge tubes and centrifuged at 500\u0026times;g for 3 min to form pellets. After aspirating the medium, the pellets were washed once with 500 \u0026micro;L of PBS, centrifuged once more, and resuspended in 50\u0026ndash;250 \u0026micro;L of trypsin/EDTA. The suspension was incubated at 37\u0026deg;C with gentle agitation every 10 min until no aggregates were visible (10\u0026ndash;30 minutes). Trypsin was inactivated by adding an equal volume of FBS, followed by the addition of Trypan Blue dye (Gibco\u0026trade; 15250061) at 1:1 ratio and gentle mixing via pipetting. Cell counting was performed using the TC20 Automated Cell Counter (Bio-Rad Laboratories).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIndividual spheroids.\u003c/em\u003e Cells were seeded in a 96-well round-bottom ULA plate at a density of 2000 cells per well in 100 \u0026micro;L of medium. For alamarBlue assays, cells were incubated at a final dilution of 1:10 (alamarBlue to media) for 4, 24, or 48 h and fluorescence was measured using a Agilent Biotek Synergy H1 plate reader. For CellTiter-Glo (Promega, G7572) and Caspase-Glo 3/7 (Promega, G8092) assays, 100 \u0026micro;L of reagent was added to each well, and the plate was frozen at -80\u0026deg;C. After 24 h, the plates were thawed at room temperature in the dark for 60 min on a plate rocker. The contents of wells were transferred to individual wells of a 96-well opaque white plate, and luminescence was measured on the Agilent Biotek Synergy H1 plate reader.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell Proliferation\u003c/h2\u003e \u003cp\u003eCells expressing Nuclight GFP were placed in 96-well standard-well ultra-low attachment (ULA) cluster plates at a density of 0.5-1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well in 200 \u0026micro;L of medium. Fluorescent images were captured at 4 h intervals in the Incucyte\u0026reg; S3 System (Sartorius). Growth curves and doubling times were calculated using GraphPad Prism 10. Adherent and spheroid doubling time calculations were performed using green and green mean intensity features of the Incucyte\u0026reg; S3 system, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOrganoids\u003c/h2\u003e \u003cp\u003eCells were seeded at a density of 5000 cells/well as droplets in 50\u0026micro;L of Cultrex Basement Membrane Extract (BME) PathClear Type 2 (Cedarlane, Burlington, ON, Canada) on a 24-well standard tissue culture plate (Corning). Droplets were overlaid with an EOC organoid specific media containing Advanced DMEM/F-12 (Invitrogen) supplemented with B-27\u0026trade; (Invitrogen), Forskolin (Sigma), GlutaMAX\u0026trade; (Invitrogen), HEPES (Wisent), Human EGF (Peprotech Inc.), Human FGF-10 (Peprotech Inc.), Nicotinamide (Sigma), N-Acetyl-L-cysteine (Sigma), Recombinant Human Noggin (R\u0026amp;D Systems) and ROCK-inhibitor (Y27632 dihydrochloride, Sigma). Brightfield images were captured every 12 h with the Incucyte\u0026reg; S3 System. The total organoid area (\u0026micro;m\u0026sup2;) and number of organoids per well were quantified using the Organoid Analysis Software via the Incucyte\u0026reg; S3 System.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eScratch Wound Closure Migration Assay\u003c/h2\u003e \u003cp\u003eConfluent cell monolayers were scratched with a pipette tip and immediately imaged (0 h time point). Images were acquired up to 36 h post-scratch and ImageJ (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) was used to measure scratch width and calculate scratch area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMesothelial Clearance Assay\u003c/h2\u003e \u003cp\u003e \u003cem\u003eZT-GFP mesothelial cell line with tdTomato-expressing spheroids.\u003c/em\u003e Human ZT-GFP mesothelial cells (1-1.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells in 1mL) were seeded into each well of a 24-well tissue culture plate and incubated for 24 h. An empty well containing 1mL of media served as a control. Cells expressing luc2tdTomato (2000 cells per well) were seeded into 96-well ULA plates and incubated for 24 h then individual spheroids were transferred using a P200 and placed onto mesothelial cell monolayers or control wells, with at least 5 spheroids per well. Green and red fluorescent images were captured 24 h later using a Leica DMI4000B inverted microscope and spheroid displacement was quantified using Fiji (Fiji is just ImageJ).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePrimary human mesothelial cells with mouse ascites-derived spheroids.\u003c/em\u003e Prior to cell culture, collagen (50\u0026micro;g) was dissolved in 70% ethanol and added to each well of a 24-well tissue culture plate and incubated at room temperature for 2 h. The wells were then aspirated, washed with PBS, and re-aspirated before adding mesothelial cells. To generate fluorescing primary mesothelial cells, 3.5\u0026micro;L of recombinant human Ad5-green fluorescent protein (Ad-GFP) vector stock per 100,000 cells was added to the cell suspension, which was then seeded to the collagen layer at a density of 1-1.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells and incubated for 24 h. Mouse xenograft ascites-derived spheroids were generated by seeding cells into 96-well ULA plates at 2000 cells per well in 100\u0026micro;L and incubated for 24 h. The following day, mesothelial cells were washed twice with PBS to remove Ad-GFP-containing media, and 1mL of fresh media was added to each well, including control wells without mesothelial cells. Spheroids were transferred to a 24-well plate at 5 per well using a P200. Green and red fluorescent images were captured 24 h later and spheroid displacement was quantified as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eXenotransplantation assays\u003c/h2\u003e \u003cp\u003eNOD/SCID female mice (8\u0026ndash;10 weeks old; Charles River Laboratories) were injected intraperitoneally with luc2tdTomato cells with the following cell numbers in 150 \u0026micro;L PBS: OVCAR8/OVCAR8-\u003cem\u003eULK1\u003c/em\u003eKO, 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e; HeyA8/HeyA8-\u003cem\u003eULK1\u003c/em\u003eKO, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e). For survival analyses, mice were monitored daily after intraperitoneal injection and euthanized using established criteria for humane endpoints (lethargy, hunched posture, impaired breathing, weight loss, and excessive ascites) as per protocol guidelines. Mice received weekly injections of D-luciferin (Perkin Elmer, #122799) at 75 mg/kg in 100\u0026micro;L PBS to monitor tumor progression via BLI using the IVIS Lumina S5 system (PerkinElmer). Tumor locations and evidence of ascites for each mouse was assessed and recorded at necropsy. The mice were provided chow (Envigo, #2919) and water \u003cem\u003ead libitum\u003c/em\u003e throughout the study. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Western Ontario (London, Ontario, Canada) and were performed in accordance with approved guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eSectioning and staining of tumor specimens were performed by the Molecular Pathology Core Facility at Robarts Research Institute (London, Ontario, Canada). Images of stained tumor sections were captured using an Aperio ScanScope slide scanner (Leica). IHC analysis was performed using the Fiji distribution in ImageJ software (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Ki67-positive nuclei were masked using the Trainable Weka Segmentation plugin (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and masked regions were counted using a minimum particle area of 120 pixels. Cleaved caspase-3 staining in xenograft tumor sections was evaluated using the \u0026ldquo;IHC Profiler\u0026rdquo; plugin for ImageJ, as described previously (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Positive caspase-3 staining represents the combined \u0026ldquo;high-positive\u0026rdquo; and \u0026ldquo;positive\u0026rdquo; scores as defined by IHC profiler.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCarboplatin and Paclitaxel Dose-Response Curves\u003c/h2\u003e \u003cp\u003eTo determine carboplatin and paclitaxel half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values, 2000 cells in 100 \u0026micro;L media were seeded in a standard 96-well plate for adherent culture, allowed to attach for 24 h, then treated with carboplatin or paclitaxel over a 12-point concentration gradient. After 72 h of treatment, cell viability was determined using the alamarBlue Cell Viability Reagent (Invitrogen CAT# DAL 1025) according to the manufacturer\u0026rsquo;s instructions. To determine carboplatin and paclitaxel IC\u003csub\u003e50\u003c/sub\u003e values of spheroids, 2000 cells in 100 \u0026micro;L media were seeded in a 96-well ULA plate. After 72 h, cells were treated individually over a 12-point concentration gradient for an additional 72 h. Following treatment, viability was determined by alamarBlue viability assay. Viability was assessed at 4 h and 48 h post alamarBlue incubation for carboplatin and paclitaxel, respectively, and IC\u003csub\u003e50\u003c/sub\u003e values were calculated using GraphPad Prism 10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProteomic Mass Spectrometry\u003c/h2\u003e \u003cp\u003eProtein extraction and mass spectrometer analysis were performed on OVCAR8 wild-type and OVCAR8-\u003cem\u003eULK1\u003c/em\u003eKO 24-hour spheroids. Spheroids were lysed using three 5-second pulse rounds of sonication at 35% amplitude lysed OVCAR8 wild-type and OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids at 24 h post seeding in lysis buffer containing 200 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS; pH 8.6), 6 M guanidine, 1 mM PMSF, 100 mM NaF, and phosphatase inhibitor cocktail (2 mM NaF, 2 mM imidazole, 1.15 mM Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e, 1 mM Na\u003csub\u003e4\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, 4 mM Na\u003csub\u003e2\u003c/sub\u003eC\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e, 2 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, and 1 mM β-glycerophosphate). Lysates were incubated in the dark with 5 mM Tris (2-carboxyethyl) phosphine and 15 mM indole-3-acetic acid for 30 and 45 minutes, respectively, and quenched with 5 mM dithiothreitol. Sera-Mag\u0026trade; SpeedBeads (GE Healthcare, Little Chalfont, UK; 65152105050250) were added to the lysates, followed by equal volumes of 100% ethanol. The resulting mixture was incubated on a shaker for 10 minutes. The supernatant was removed from the mixture and the beads were washed and resuspended in 50 mM EPPS buffer (pH 8.5). After beads and EPPS buffer were subjected to a 2-hour LysC digestion at 1 mAu per 100 \u0026micro;g of protein, trypsin was added at a 1:50 ratio for overnight digestion. The beads were washed with water and 30% acetonitrile the following day to elute the peptides, which were stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003cp\u003eFor mass spectrometry, the peptides were analyzed using a Q Exactive Plus mass spectrometer coupled with an EASYLCn-1000 system (Thermo Fisher Scientific). The peptides were loaded onto an Easy-LCn-1000 and separated on an EASY-Spray 75\u0026micro;m x 500mm at 45\u0026deg;C using an ES803A analytical column (Thermo Fisher Scientific) at a flow rate of 300 nl/min. Raw mass spectrometry data were processed using FragPipe (version 20.0) and Rstudio with the Tidyverse R package for data manipulation, mice R package for imputing missing data, and LIMMA R package for differential expression analysis. Pathway analysis was performed using Kegg(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/go/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/go/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Reactome databases(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://reactome.org\u003c/span\u003e\u003cspan address=\"https://reactome.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSpheroid Drug Treatments and Reattachment\u003c/h2\u003e \u003cp\u003eCells were placed in 24-well ULA cluster plates at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in 1 mL of medium. After 24 h, spheroids were treated with individually with carboplatin (100 \u0026micro;M), paclitaxel (50 nM), olaparib (20 \u0026micro;M), AKTi 1/2 (5 \u0026micro;M), trametinib (10 nM), or ralimetinib (15 \u0026micro;M). Spheroid cell viability was performed using Trypan Blue Exclusion assay as described above at 96 h for all drug treatments, except for Olaparib which was performed at 192 h. For spheroid reattachment assays, inhibitor-treated spheroids were reattached to standard tissue culture plates for 48 h and viability was assessed using alamarBlue at 1:10 dilution in media. Fluorescence was measured using the Agilent Biotek Synergy H1 plate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 10 (GraphPad Software) and the details for specific statistical tests are described in each figure legend.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDifferential requirement for ULK1 between EOC and noncancer precursor spheroids\u003c/h2\u003e \u003cp\u003eAutophagy induction is controlled by the ULK complex, notably by ULK1 kinase activity (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Our previous studies showed that EOC spheroids exhibit elevated ULK1 expression, which is associated with increased autophagy activation, and that targeted \u003cem\u003eULK1\u003c/em\u003e knockdown or inhibition effectively disrupted autophagy activation (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). To further elucidate the role of ULK1 in autophagy and tumorigenesis, we ablated \u003cem\u003eULK1\u003c/em\u003e in OVCAR8 and HEYA8 EOC cells using CRISPR/Cas9 and pooled multiple independent clones to generate a population of \u003cem\u003eULK1\u003c/em\u003e knockout (\u003cem\u003eULK1\u003c/em\u003eKO) cells. To validate the effect of \u003cem\u003eULK1\u003c/em\u003e loss on autophagy, we examined proteins involved in the autophagic pathway. Upon examining parental OVCAR8 and HEYA8 spheroids, we observed increased ULK1 expression, whereas \u003cem\u003eULK1\u003c/em\u003eKO cells exhibited a complete absence of ULK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In both OVCAR8 and HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO day 3 spheroids, we detected a significant increase in p62 expression and a substantial reduction in LC3II:I compared to their parental cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). This is important, as p62 accumulation serves as an indicator of autophagy inhibition, whereas its decrease suggests autophagy induction (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Additionally, the LC3II:I ratio, derived from the processing of LC3I to LC3II, a marker of autophagosome membranes, reflects the activation of autophagy (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). We observed a significant decrease in the LC3II:I ratio observed as early as 4 and 24 h within OVCAR8 and HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids, respectively (Supplementary Fig.\u0026nbsp;1A and B). Interestingly, \u003cem\u003eULK1\u003c/em\u003e loss resulted in an elevation of basal LC3I (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B) and a significant reduction in p-ATG16L1 (S278) (Supplementary Fig.\u0026nbsp;2A), supporting the established role of ULK1 in the early processing of LC3 (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Additionally, we verified the abrogation of ULK1 activity through loss p-Beclin-1 (S30) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B), as this is a known substrate for ULK1 activity (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHigh-grade serous ovarian cancer (HGSOC) is the most prevalent EOC histotype that arises from the preneoplastic lesions in the secretory epithelium of the distal fallopian tube (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Therefore, we ablated \u003cem\u003eULK1\u003c/em\u003e in FT190 cells, an immortalized human fallopian tube secretory epithelial cell line (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Although \u003cem\u003eULK1\u003c/em\u003e loss resulted in reduced phosphorylated Beclin-1 (S30) levels as seen in EOC cells, no significant differences in the LC3II:I ratio were observed between FT190 parental and FT190 \u003cem\u003eULK1\u003c/em\u003eKO spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B; Supplemental Fig.\u0026nbsp;1C), suggesting that ULK1 activity is not essential for autophagy activation in these precursor cells. To address the potential compensation due to \u003cem\u003eULK1\u003c/em\u003e loss in FT190 cells, we investigated ULK2 protein expression, a homolog of ULK1 that is believed to be redundant in autophagy activation (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). No differences in ULK2 expression were observed among \u003cem\u003eULK1\u003c/em\u003eKO EOC lines, however, a significant increase was observed in FT190 \u003cem\u003eULK1\u003c/em\u003eKO adherent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These results suggest that ULK1 is vital for autophagy activation in EOC spheroids, whereas its role may be redundant in HGSOC precursor cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability is significantly impaired in EOC\u003c/b\u003e \u003cb\u003eULK1\u003c/b\u003e\u003cb\u003eKO spheroids\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSpheroids enhance EOC cell viability during metastasis by protecting from anoikis and chemotherapy-induced damage (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). To understand whether ULK1 and autophagy activities contribute to this property, \u003cem\u003eULK1\u003c/em\u003eKO cells were grown in suspension culture to assay for differences in spheroid morphology, density, integrity, and viable cell number. We found that HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids showed obvious differences in morphology with decreased density and cell number, and impaired integrity compared to parental spheroids, whereas OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids retained overall morphology but displayed reduced viable cell number also (Supplementary Fig.\u0026nbsp;3A). A significant reduction in viable cells was observed by Trypan Blue exclusion cell counting in both OVCAR8 and HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids on days 7 and 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Although all EOC spheroids grew in cell number up to day 3, growth plateaued in OVCAR8 spheroids after this time; HEYA8 parental spheroids continued to expand in cell number, yet \u003cem\u003eULK1\u003c/em\u003eKO spheroids failed to do so (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Interestingly, loss of \u003cem\u003eULK1\u003c/em\u003e in FT190 spheroids resulted in a further reduction in viable cells as compared with parental spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). EOC parental and \u003cem\u003eULK1\u003c/em\u003eKO cells expressing nuclear-localized GFP were generated to facilitate fluorescence imaging as an indirect indicator of spheroid growth. However, no differences in growth rate were observed by fluorescence imaging between EOC parental and \u003cem\u003eULK1\u003c/em\u003eKO spheroids (Supplementary Fig.\u0026nbsp;3B). To determine whether reduced viable cell number in \u003cem\u003eULK1\u003c/em\u003eKO spheroids occurs via cytostasis or cell death, we assessed markers for proliferation and apoptosis. Since EOC spheroids display features of quiescent cells compared to adherent cells (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), we evaluated the expression of tumor suppressor proteins p21 and p27, which are established markers of tumor cell dormancy (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). In line with this, we have shown previously that cellular quiescence in EOC spheroids is associated with increased p27 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Herein, we observed significant reductions in p21 and p27 expression in HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids, yet in contrast these markers exhibited significant increases in OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We measured apoptosis activity in spheroids at multiple time points using the Caspase 3/7 Glo assay. We observed a significant surge in apoptosis within 24\u0026ndash;48 h of spheroid formation, with elevated activity persisting for up to 72 h in HEYA8, OVCAR8 and FT190 spheroids due to \u003cem\u003eULK1\u003c/em\u003e ablation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings demonstrate that the primary mechanism whereby ULK1 loss impairs spheroid cell viability is through apoptosis induction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eULK1 is required for EOC tumor growth and spread in xenograft models\u003c/h2\u003e \u003cp\u003eGiven our findings that ULK1 is critical for autophagy activation and spheroid viability, we sought to investigate its role in EOC tumor formation and metastasis directly, areas that have not been explored previously. To initiate these studies in cell culture, we grew parental and \u003cem\u003eULK1\u003c/em\u003eKO cells as matrix-embedded organoids for up to 18 days and assessed expansion over time. Although total organoid number was similar between parental and \u003cem\u003eULK1\u003c/em\u003eKO lines, there was a significant reduction organoid size over time due to \u003cem\u003eULK1\u003c/em\u003e loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B; Supplementary Fig.\u0026nbsp;4A). To investigate ULK1 function in spheroid attachment, invasion and migration, we used the mesothelial clearance assay, an experimental model that mimics the early steps of EOC metastasis (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). We transferred pre-formed EOC spheroids expressing tdTomato onto either standard tissue culture plastic or GFP-expressing ZT human mesothelial cells. This allowed us to separately quantify spheroid attachment and dispersion from mesothelial cell displacement properties. OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO cells displayed significantly reduced ability to displace mesothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and disperse on tissue culture plastic (Supplementary Fig.\u0026nbsp;5B), whereas the displacement capacity of HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO cells was impaired in the presence of mesothelial cells only (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; Supplementary Fig.\u0026nbsp;4B). These findings of altered cell motility properties were recapitulated using a scratch wound closure assay, where \u003cem\u003eULK1\u003c/em\u003e loss significantly decreased wound closure rate in OVCAR8 cells but not in HEYA8 cells (Supplementary Fig.\u0026nbsp;4C). These cell culture-based results suggest ULK1 may have additional functions to promote EOC spheroid cell invasiveness and metastatic capacity \u003cem\u003ein vivo.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo model EOC metastasis, whereby malignant cells from the primary tumor are shed directly into the peritoneal cavity (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), we injected luciferase/tdTomato-expressing cells intraperitoneally into female NOD/SCID mice and monitored tumor progression over time via BLI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and D). Mice injected with OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO cells showed reduced tumor burden at all time points, with significant decreases observed during the mid-to-late stage of disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In contrast, HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO cells had a significant decrease only at very early stages of disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), which was lost at later time points. At experimental endpoint, EOC cells with ULK1 loss resulted in fewer tumor lesions observed at several metastatic sites, with notable decreases in ascites formation and omental metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and F), two canonical features of metastatic EOC. Despite reduced tumor growth and metastatic spread, no significant differences were observed in survival rates (Supplementary Fig.\u0026nbsp;5A). In addition, no differences in either Ki67- or Caspase-3-positive IHC staining on tumor samples were seen (Supplementary Fig.\u0026nbsp;5C and D). Taken together, our findings suggest that ULK1 impacts EOC progression by affecting intrinsic tumor cell growth, and spheroid adhesion and invasion at later steps of metastasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eULK1\u003c/b\u003e \u003cb\u003eknockout does not synergize with standard-of-care treatment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eElevated autophagy levels have been linked to poor prognostic outcomes in cancer patients due to cytotoxic drug resistance (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Since we observed reduced tumor burden and metastasis due to \u003cem\u003eULK1\u003c/em\u003e ablation in injected EOC cells, yet this did not alter overall survival, we tested whether \u003cem\u003eULK1\u003c/em\u003e loss and autophagy disruption would sensitize EOC spheroids to chemotherapy. Using our \u003cem\u003ein vitro\u003c/em\u003e spheroid model system, we assessed cell viability by treating spheroids with either carboplatin or paclitaxel as single agents. Carboplatin treatment resulted in a significant increase in viable cells in OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids and no difference in HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids. No significant differences in viability were observed between parental and \u003cem\u003eULK1\u003c/em\u003eKO spheroids under paclitaxel treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). We also treated spheroids with Olaparib, a poly ADP ribose polymerase (PARP) inhibitor used as maintenance therapy in select HGSOC patients (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Both parental and \u003cem\u003eULK1\u003c/em\u003eKO spheroids showed significant sensitivity to Olaparib, yet ULK1 loss did not alter this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results suggest that while \u003cem\u003eULK1\u003c/em\u003e knockout reduces spheroid cell viability, its combination with standard-of-care therapies elicited no further improvement, underscoring our subsequent studies to identify alternative strategies to improve efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eULK1\u003c/b\u003e \u003cb\u003eloss disrupts key cell survival pathways in EOC spheroids\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn addition to its well-established role as a primary regulator of autophagy, ULK1 plays critical roles in energy metabolism, mitochondrial homeostasis, and vesicular trafficking (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). However, its non-canonical functions in EOC remain largely unexplored yet may be implicated in driving EOC progression and thus serve as new therapeutic targets to improve the limited efficacy of standard chemotherapies as seen in our findings. To this end, we performed proteomic mass spectrometry and bioinformatic analyses on OVCAR8 and OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids to identify potential ULK1-regulated pathways in our experimental system. Through KEGG and Reactome pathway analyses of our resultant dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), we found significant changes biological pathways related to cell survival, including apoptosis, and PI3K-AKT-mTOR and MAPK signaling that were shared between both analyses. Since we had already observed enhanced apoptosis in ULK1KO spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), we sought to validate members of PI3K-AKT-mTOR and MAPK signaling pathways, given their critical roles in regulating tumor progression (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). We observed significant reductions in p-MEK (S217/221) and p-p38 (Thr180/Tyr182) levels, while no changes in downstream p-ERK (Thr202/Tyr204) were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C; Supplemental Fig.\u0026nbsp;6A). This trend extended to the PI3K-AKT-mTOR pathway, with a universal decrease in p-AKT (S473) in \u003cem\u003eULK1\u003c/em\u003eKO spheroids. Further downstream analysis of AKT revealed contrasting effects, with increased P70S6K phosphorylation (T389) yet decreased 4EBP1 phosphorylation (Thr37/46) in \u003cem\u003eULK1\u003c/em\u003eKO spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C; Supplementary Fig.\u0026nbsp;6B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFemale mice xenografted with OVCAR8 and OVCAR-\u003cem\u003eULK1\u003c/em\u003eKO cells developed malignant ascites with reduced prevalence due to \u003cem\u003eULK1\u003c/em\u003e loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These ascites samples were returned to cell culture to study whether their inherent pathobiology had changed during disease progression in mice. OVCAR8 \u003cem\u003eULK1KO\u003c/em\u003e spheroids from ascites-derived lines remained autophagy deficient, as evidenced by a decreased LC3II:I ratio, increased p62, suppressed ULK1 activity with reduced p-Beclin-1 S30, and lower ULK2 expression. Phosphorylated-MEK1/2 was reduced, which was consistent with original OVCAR8-\u003cem\u003eULK1\u003c/em\u003eKO spheroid cells. However, the changes in p-AKT (S473), p-P70S6K (T389), and p-p38 MAPK seen in pre-injection OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids were not observed in ascites-derived lines, although total AKT protein decreased and total P70S6K increased in ascites-derived lines (Supplementary Fig.\u0026nbsp;7A and B). Despite the observed changes in MEK-MAPK and PI3K-AKT-mTOR signaling proteins, ascites-derived OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids retained impaired metastatic potential in mesothelial clearance and reattachment assays, indicating persistent functional defects post-xenografting (Supplementary Fig.\u0026nbsp;7C and D). Collectively, these observations suggest that ULK1 disruption leads to reprogramming of key signaling pathways known to impact tumor progression and cancer cell survival.\u003c/p\u003e \u003cp\u003e \u003cb\u003eULK1\u003c/b\u003e \u003cb\u003eablation enhances efficacy of MEK and mTOR inhibition\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore novel synergistic treatment strategies in the context of \u003cem\u003eULK1\u003c/em\u003e loss, we targeted the dysregulated PI3K-AKT-mTOR and MEK-MAPK pathways in \u003cem\u003eULK1\u003c/em\u003eKO spheroids using specific inhibitors. Treatment with AKTi-1/2 (AKT inhibitor) and ralimetinib (p38 inhibitor) resulted in significantly increased cell viability in OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids, while no differences were observed in HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). However, treatment with either trametinib (MEK inhibitor) or AZD-8055 (mTORC1/2 inhibitor) resulted in significantly reduced cell viability in \u003cem\u003eULK1\u003c/em\u003eKO spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), indicating that \u003cem\u003eULK1\u003c/em\u003e loss may sensitize EOC spheroid cells to MEK and mTOR inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe conducted spheroid reattachment assays to evaluate the effect of these inhibitors on this key step in the metastatic process (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Untreated OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids had significantly reduced reattachment, which was further decreased due to trametinib and AZD-8055 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). So too did treatment with trametinib and AZD-8055 significantly reduce HEYA8 \u003cem\u003eULK1\u003c/em\u003eKO spheroid reattachment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Our findings demonstrate PI3K-AKT-mTOR and MEK-MAPK signaling pathways contribute to EOC spheroid viability and metastatic properties and may represent important therapeutic targets particularly when combined with \u003cem\u003eULK1\u003c/em\u003e ablation and autophagy blockade.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eEpithelial ovarian cancer is a highly lethal gynecologic cancer characterized by late-stage diagnosis, high relapse rates, and the formation of chemo-resistant spheroids that contribute to peritoneal metastasis. To the best of our knowledge, this is the first study to elucidate the role of ULK1 function using the combination of \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003eex vivo\u003c/em\u003e, and \u003cem\u003ein vivo\u003c/em\u003e models of EOC metastasis. Our findings revealed that beyond its role in regulating autophagy, ULK1 deficiency significantly impacted tumor progression, leading to reduced spheroid viability, diminished invasive capacity, and impaired organoid growth. Additionally, our tumor xenograft models demonstrated that \u003cem\u003eULK1\u003c/em\u003e loss significantly decreases tumor growth and spread, highlighting its critical role in supporting key processes in EOC metastasis and tumor development. Our study is underscored by our proteomic mass spectrometry analysis, which revealed dysregulated mTOR-PI3K-AKT and MAPK signaling and increased sensitivity to MEK and mTOR inhibition in \u003cem\u003eULK1\u003c/em\u003eKO spheroids. The findings provide new insights into potential autophagy-independent functions of ULK1 and highlight novel therapeutic strategies in EOC.\u003c/p\u003e \u003cp\u003eAs we expected, \u003cem\u003eULK1\u003c/em\u003e loss ablated LC3II:I processing and elevated p62 levels in EOC spheroids. Although our previous research suggests that Beclin-1 is not required for autophagy activation in EOC spheroids (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), the absence of p-Beclin-1 (S30) in \u003cem\u003eULK1\u003c/em\u003eKO cells highlights its utility as a specific biomarker for ULK1 activity in EOC, particularly if assessing on-target activity of ULK1 inhibitors. Interestingly, ULK1 loss did not impair autophagy activation in the FT190 HGSOC precursor cell line. Instead, there was a substantial increase in ULK2 protein expression in FT190 \u003cem\u003eULK1\u003c/em\u003eKO cells suggesting a compensatory mechanism for autophagy activation. Consistent with our findings, a previous study reported that ULK1 inhibition did not alter ULK2 expression in HGSOC cells (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Additionally, ULK2 has been shown to compensate for \u003cem\u003eULK1\u003c/em\u003e loss in mouse embryonic fibroblasts, but not in cerebellar granule neurons (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Our observations suggest a unique requirement for ULK1 to activate autophagy in malignant EOC cells.\u003c/p\u003e \u003cp\u003eEOC metastasis occurs by direct dissemination of tumor cells into the peritoneal cavity, where spheroid clusters suspended in ascites promote secondary tumor formation through enhanced survival, adhesion, and invasiveness. Using spheroid models that mimic these unique metastasis mechanisms, our results highlight ULK1's pivotal role in EOC progression. \u003cem\u003eULK1\u003c/em\u003e loss significantly impaired viability, increased apoptosis, and reduced invasive capacity of EOC spheroids. These reductions in spheroid cell viability appear to be driven by increased apoptosis rather than altered cell growth. Anoikis, a programmed cell death triggered by the loss of cell attachment to the extracellular matrix, serves as a critical barrier to metastasis (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Spheroids, however, resist anoikis by activating autophagy (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) and PI3K-AKT-mTOR and MAPK signaling to promote apoptosis resistance (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Given our findings of decreased PI3K-AKT-mTOR and MAPK signaling in \u003cem\u003eULK1\u003c/em\u003eKO spheroids and the role of autophagy as a defense mechanism preceding apoptosis (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), the disruption of these survival pathways likely compromises the ability of spheroids to resist anoikis, leading to increased apoptosis and reduced viability.\u003c/p\u003e \u003cp\u003eIn addition to reduced tumor cell dissemination, we observed fewer metastases and reduced ascites formation. We initially speculated that this reduction in secondary tumors was primarily due to ULK1\u0026rsquo;s role in regulating cell viability in suspension rather than directly on secondary growth or altered invasive capacities. However, we observed significant reductions in mesothelium invasion of spheroids lacking ULK1. Our findings corroborate a previous study demonstrating that inhibiting autophagy restricts the invasiveness of ovarian cancer cells via the negative regulation of p62 on ERK1/2 activity for invadopodium formation (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). In addition, ULK1 loss significantly impaired the growth of EOC cells in organoid culture, highlighting potential ULK1 function in tumor development. Patient-derived organoids (PDOs) represent another three-dimensional culture system similar to spheroids where PDOs serve to replicate the structural and functional characteristics of the original tissue, providing an accurate \u003cem\u003eex vivo\u003c/em\u003e model for studying tumor growth (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Taken together, these data suggest that metastatic cells reaching secondary sites may exhibit compromised invasiveness and impaired re-initiation of tumor growth.\u003c/p\u003e \u003cp\u003eIn xenograft models, \u003cem\u003eULK1\u003c/em\u003e knockout reduced tumor burden and limited metastatic spread, demonstrating its importance in systemic disease progression. Similarly, evidence from both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e investigations indicate that ULK1 depletion significantly reduces pancreatic and hepatocellular carcinoma growth (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). However, ULK1\u0026rsquo;s role in cancer development might be context-specific among different malignancies. For example, in breast cancer models, \u003cem\u003eULK1\u003c/em\u003e loss has been associated with an increased likelihood of osseous metastasis (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). In contrast, EOC rarely metastasizes to distant sites such as the lungs, skin, bones, or brain via hematogenous dissemination highlighting the underlying mechanisms driving EOC metastasis are biologically different from many other carcinomas (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Despite significant reductions in tumor burden, mice injected with \u003cem\u003eULK1\u003c/em\u003e knockout cells did not exhibit improved survival. These mice developed distended abdomens, jaundice, and significant weight loss, ultimately requiring sacrifice in accordance with approved guidelines. This underscores the lethality of EOC, as tumor cells, even with \u003cem\u003eULK1\u003c/em\u003e loss, can still disseminate to vital abdominal organs, driving disease progression and mortality.\u003c/p\u003e \u003cp\u003eWe reported that autophagy levels and \u003cem\u003eULK1\u003c/em\u003e mRNA overexpression are correlated with poor survival outcomes in advanced-stage ovarian cancer (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), making it a promising therapeutic target. While combining ULK1 inhibition with standard-of-care chemotherapeutics might seem promising to enhance anti-tumor effects, the results are not uniformly positive. Previous studies have suggested that \u003cem\u003eULK1\u003c/em\u003e loss can enhance chemotherapy sensitivity in OVCAR8 cells (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e); however, our findings, along with unpublished \u003cem\u003ein vitro\u003c/em\u003e data (Johnston \u0026amp; Shepherd, in preparation), indicate that ULK1 inhibition may instead reduce the efficacy of standard first-line chemotherapeutics used in EOC. Previous studies in gastric cancer have shown that elevated p62 expression activates the transcription of Chemokine C-C motif ligand 2 (CCL2), a cytokine associated with drug resistance and contributes to cisplatin resistance (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Perhaps the significant increase in p62 expression resulting from \u003cem\u003eULK1\u003c/em\u003e loss decreased chemotherapeutic efficacy in our system as well.\u003c/p\u003e \u003cp\u003eWhile ULK1 is widely recognized as a critical regulator of autophagy, its autophagy-independent functions, especially in the context of EOC, have been less studied. As such, we performed protein mass spectrometry on OVCAR8 and OVCAR8 \u003cem\u003eULK1\u003c/em\u003eKO spheroids, which verified our findings of increased apoptosis and revealed significant alterations in critical signaling pathways, including PI3K- AKT- mTOR and MAPK signaling. The PI3K-AKT-mTOR pathway is a hallmark cancer promoter that governs essential processes such as cell growth, motility, survival, and metabolism (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Similarly, MAPK pathways play a central role in regulating fundamental processes such as cell proliferation, differentiation, and stress responses (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). We demonstrated a universal decrease in p-MEK (S217/22), p-p38 (T180/Thr182), and p-AKT (S473), and an increase in p-p70S6K (T389) in EOC spheroids lacking ULK1. This aligns with previous studies implicating MAPK signaling in ovarian cancer, where it has been shown to regulate autophagy and inhibit apoptosis (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e), and promote invasion and proliferation through combined AKT and MAPK signaling activities (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Therefore, we were intrigued to assess the therapeutic potential of targeting these pathways in our \u003cem\u003eULK1\u003c/em\u003e deficient system. While AKT and p38 inhibition significantly decreased EOC spheroid viability, \u003cem\u003eULK1\u003c/em\u003e loss did not increase drug sensitivity and, in some cases, exhibited potential antagonistic effects. However, \u003cem\u003eULK1\u003c/em\u003e loss significantly enhanced the sensitivity of EOC spheroids to MEK inhibition via trametinib and mTORC1/2 inhibition via AZD-8055. Many preclinical and clinical studies have explored targeting the PI3K-AKT-mTOR pathway, including the use of mTOR inhibitors, in EOC (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). However, the clinical application of AZD-8055 is limited by its pharmacokinetics, inadequate intratumoral concentrations, and dose-limiting toxicities (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). Its successor, AZD-2014 (vistusertib), initially showed reduced liver toxicity (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e), but recent studies, such as those in meningiomas, reported poor tolerance, with most participants discontinuing the trial (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). Interestingly, combination therapy with vistusertib and anastrozole in advanced hormone receptor-positive endometrial cancer demonstrated manageable adverse events and improved overall response rates and progression-free survival (NCT02730923) (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). Trametinib, represents a new standard-of-care option for relapsed or persistent low-grade serous ovarian cancer (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e), and has shown success in a patient with recurrent HGSOC who had several lines of prior therapy (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). These findings suggest that blocking autophagy via ULK1 inhibition combined with MEK inhibition via trametinib or mTORC1/2 inhibition could be effective in EOC. It would be worthwhile to investigate the efficacy of ULK1 inhibitors combined with these targeted agents in PDOs to reveal therapeutic potential (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, our findings suggest that by targeting ULK1 and its autophagy-dependent and -independent pathways would offer a promising new therapeutic strategy to disrupt EOC metastatic progression.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOur comprehensive analysis underscores ULK1's multifaceted role in EOC, where its influence extends beyond autophagy regulation to impact key cell survival pathways, particularly apoptosis and MAPK and PI3K-AKT-mTOR signaling networks. Although \u003cem\u003eULK1\u003c/em\u003e loss did not enhance the efficacy of standard-of-care chemotherapeutics, it significantly sensitized EOC spheroids to MEK and mTORC1/2 inhibition. Ultimately, our research establishes that targeting ULK1 could offer a promising strategy for controlling tumor growth and reducing metastasis in EOC, providing a new avenue for therapeutic intervention aimed at improving patient outcomes in ovarian cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEOC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial ovarian cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eULK1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUnc51-like kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eATG13\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAutophagy-related gene 13\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFIP200\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFocal adhesion kinase interacting protein 200\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003emTOR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMammalian target of rapamycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAMPK\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAMP-activated protein kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePARP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePoly (ADP-ribose) polymerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMAPK1/2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitogen-activated protein kinase 1/2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMEK1/2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitogen-activated protein kinase kinase 1/2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eERK1/2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular signal-regulated kinase 1/2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHGSOC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-grade serous ovarian carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePDO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePatient-derived organoid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFACS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluorescence-activated cell sorting\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGFP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGreen fluorescent protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eULA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUltra-low attachment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIHC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Western Ontario (London, Ontario, Canada) and were performed in accordance with approved guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the funding support from the Cancer Research Society to TGS and the London Health Sciences Foundation through donations to the Mary and John Knight Translational Ovarian Cancer Research Unit. JDW was supported by an Obstetrics \u0026amp; Gynecology Graduate Scholarship from the Department of Obstetrics and Gynecology at the Western University and a\u0026nbsp;Queen Elizabeth II Graduate Scholarship in Science and Technology (Ontario Government).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJDW and TGS conceptualized and designed the study. JDW, ET, LV, YRV, and AB acquired data. TGS supervised and obtained funding for this study. BS, MB, and YRV provided additional resources. JDW wrote the original draft of the manuscript and TGS edited the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Ronny Drapkin and Marcin Iwanicki for providing us with the FT190 and ZT-GFP cell lines, respectively. We are also grateful to the many donors of the Mary and John Knight Translational Ovarian Cancer Research Unit through the London Health Sciences Foundation for additional infrastructure funding, including the Leica DMI 400 B inverted microscope, Bio-Rad Chemidoc, IncuCyte S3, and IVIS Lumina imaging system used in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrenner DR, Weir HK, Demers AA, Ellison LF, Louzado. Cheryl, Shaw A, et al. Projected estimates of cancer in Canada in 2020. CMAJ [Internet]. 2020;192:199\u0026ndash;205. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003c/span\u003e\u003cspan address=\"http://www.cmaj.ca/lookup/suppl/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLheureux S, Gourley C, Vergote I, Oza AM. Epithelial ovarian cancer. 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John Wiley and Sons Inc; 2023. p. 19714\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Autophagy, ovarian cancer, metastasis, CRISPR/Cas9, spheroids, organoids, xenografts, mass spectrometry, proteome","lastPublishedDoi":"10.21203/rs.3.rs-6148090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6148090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEpithelial ovarian cancer (EOC) is a leading cause of gynecological cancer mortality, driven largely by late diagnosis and chemo-resistant disease. While autophagy plays a critical role in the survival of EOC spheroids during metastasis, the role of ULK1, a key regulator of autophagy, in EOC progression remains unclear. To investigate this, we utilized CRISPR/Cas9 technology to delete \u003cem\u003eULK1\u003c/em\u003e in EOC cell lines OVCAR8 and HEYA8, and the immortalized fallopian tube epithelial cell line FT190. Immunoblotting confirmed \u003cem\u003eULK1\u003c/em\u003e loss and its associated autophagy disruption in EOC spheroids, evidenced by reduced Beclin-1 phosphorylation, impaired LC3 processing, and p62 accumulation. Culture-based assays revealed that \u003cem\u003eULK1\u003c/em\u003e knockout decreased EOC spheroid cell viability due to increased apoptosis and, notably, impaired matrix-bound organoid growth, offering new insights into the potential role of ULK1 in affecting EOC tumor growth and spread. These findings were further demonstrated by \u003cem\u003ein vivo\u003c/em\u003e xenograft models, in which \u003cem\u003eULK1\u003c/em\u003e loss significantly reduced tumor burden and metastatic potential. The potential for ULK1 requirement in metastatic properties was supported by diminished invasive capacity of \u003cem\u003eULK1\u003c/em\u003e knockout spheroid cells in mesothelial clearance assays. To investigate the mechanisms by which ULK1 contributes EOC tumor progression and metastasis, we conducted proteomic analyses of OVCAR8 spheroids, which revealed that \u003cem\u003eULK1\u003c/em\u003e loss disrupted critical signaling pathways, including MEK-MAPK, PI3K-AKT-mTOR, and apoptosis regulation. Although \u003cem\u003eULK1\u003c/em\u003e knockout failed to synergize with standard-of-care chemotherapeutics, it significantly enhanced sensitivity to MEK and mTOR inhibition, revealing potential therapeutic combinations to target autophagy via ULK1 and MAPK and PI3K-AKT-mTOR pathway vulnerabilities in EOC. Overall, this study highlights ULK1 as a critical regulator of multiple steps of EOC growth and metastasis, underscoring its potential as a novel therapeutic target in advanced ovarian cancer.\u003c/p\u003e","manuscriptTitle":"ULK1 promotes metastatic progression in epithelial ovarian cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 11:37:47","doi":"10.21203/rs.3.rs-6148090/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-07-31T15:34:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-27T06:41:57+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-14T17:50:06+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-04-19T02:47:17+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-04-10T04:00:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-03-30T17:43:32+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-03-28T09:15:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-04T13:26:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-03T16:49:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2025-03-03T16:49:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"20040695-b85f-4899-8cbd-21c86752af78","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46345988,"name":"Biological sciences/Cancer/Gynaecological cancer/Ovarian cancer"},{"id":46345989,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2026-03-11T12:30:53+00:00","versionOfRecord":{"articleIdentity":"rs-6148090","link":"https://doi.org/10.1038/s41388-026-03702-2","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2026-03-05 05:00:00","publishedOnDateReadable":"March 5th, 2026"},"versionCreatedAt":"2025-04-16 11:37:47","video":"","vorDoi":"10.1038/s41388-026-03702-2","vorDoiUrl":"https://doi.org/10.1038/s41388-026-03702-2","workflowStages":[]},"version":"v1","identity":"rs-6148090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6148090","identity":"rs-6148090","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}