ULK1 is required for autophagy and promotes metastatic progression in epithelial ovarian cancer

doi:10.21203/rs.3.rs-5153449/v1

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ULK1 is required for autophagy and promotes metastatic progression in epithelial ovarian cancer

https://doi.org/10.21203/rs.3.rs-5153449/v1

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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 deletion, which disrupted autophagy by blocking LC3 processing, causing p62 accumulation, and decreasing Beclin-1 phosphorylation. Culture-based assays revealed that ULK1 knockout decreased EOC spheroid cell viability due to increased apoptosis, and its loss impaired organoid growth. In vivo xenograft models demonstrated that ULK1 loss significantly reduced tumor burden and metastatic potential. These in vivo findings were supported by results from mesothelial clearance assays, which showed reduced spheroid invasion by ULK1 knockout cells. Proteomic analyses of OVCAR8 spheroids revealed dysregulation due to ULK1 loss in key signaling pathways, including MAPK, mTOR-PI3K-AKT, and apoptosis regulation. 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/Autophagy

Biological sciences/Molecular biology/Proteomics

Biological sciences/Cell biology/Cell signalling

Autophagy

ovarian cancer

metastasis

CRISPR/Cas9

spheroids

organoids

xenografts

mass spectrometry

proteome

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 the survival 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 the ultimate emergence of chemoresistance.

Autophagy is a well-preserved and tightly controlled metabolic degradation process in which proteins and organelles are broken down in the lysosomes (13). This process releases metabolic by-products, including amino acids and other molecules, from lysosomes, providing vital nutrients and energy for essential cellular functions during nutrient scarcity or metabolic stress (14). Autophagy typically operates at low baseline 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); the ULK1 complex initiates the formation of phagophores (16). ULK1 is a serine-threonine kinase that responds to upstream signals of nutrient and energy availability to trigger autophagy. Under nutrient-abundant conditions, mTORC1 phosphorylates ULK1, thereby inhibiting its activity and initiating autophagy. Conversely, the absence of nutrients leads to mTORC1 deactivation and ULK1 dephosphorylation, while AMPK phosphorylates and activates ULK1 (17). Consequently, ULK1 and autophagy are essential for maintaining cellular equilibrium by eliminating impaired proteins and organelles and preserving intracellular energy supplies to meet cellular demands.

Autophagy plays a complex role in cancer by balancing tumor suppression and promoting resistance to therapy. It helps maintain cellular function and genome integrity, potentially inhibiting early tumor development (18–20). Conversely, in advanced tumors, cancer cells often exploit autophagy for growth (18) and acquire nutrients necessary for survival, particularly under metabolic stress induced by chemotherapy and radiation (15,21). Preclinical studies have indicated that chemotherapy and targeted treatments can induce autophagy, which may improve treatment efficacy via cell death (22,23). However, autophagy also contributes to therapy resistance by preventing cell death pathways, supporting cancer cell metabolism, and limiting cell death (24,25). The dual role of autophagy in cancer highlights its potential as a therapeutic target.

Given the persistent challenges of late-stage diagnosis and chemoresistance in patients with EOC, we investigated autophagy and the ULK1 complex as therapeutic vulnerabilities. We have previously demonstrated a coordinated response of AMPK activation (10) and AKT-mTORC1 downregulation (26) in 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 (27). Based on our prior work on autophagy in EOC spheroids and utilizing CRISPR/CAS9 technology for ULK1 ablation here, we aimed to elucidate ULK1's role as a potential therapeutic target in EOC metastasis.

To the best of our knowledge, this is the first study to elucidate the essential role of ULK1 in autophagy activation and metastatic progression in in vitro, ex vivo, and in vivo models of EOC metastasis. Our study is underscored by proteomic mass spectrometry analysis, which provides new insights into potential autophagy-independent functions of ULK1 in EOC. Our findings revealed that ULK1 is crucial for initiating autophagy in EOC spheroids, and its deficiency leads to impaired autophagic flux, reduced spheroid viability and invasion, organoid growth and decreased MAPK and AKT signaling. Additionally, our tumor xenograft models demonstrated that ULK1 significantly influences tumor spread at both early and advanced stages, highlighting its pivotal role in various stages of tumor progression.

Cultured cell lines

The cell lines OVCAR8, OVCAR8-ULK1KO, HeyA8, and HeyA8-ULK1KO were grown in RPMI-1640 medium (Wisent), whereas FT190 and FT190-ULK1KO 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 (28) was generously provided by R. Drapkin from the University of Pennsylvania, Philadelphia, PA, USA. ZT cells were generously provided by Marcin Iwanicki from the Stevens Institute of Technology (6). 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 ULK1KO 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 1-1.5x10⁵ 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 fluorescence-activated cell sorting (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 ULK1KO cells were transfected with mCherry-eGFP-LC3B autophagy reporter plasmid (Addgene, #22418). 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 fluorescence-activated cell sorting (FACS) to identify double-positive cells (GFP+, mCherry+).

Generation of luc2tdTomato cell lines

Cells were transduced with pCDH-EF1-Luc2-P2A-tdTomato (plasmid #72486, Addgene), according to the manufacturer’s instructions. 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 select cells with varying (i.e., medium and high) tdTomato expression intensities.

Antibodies and reagents

Antibodies against ULK1 (#8054S), p62 (#5114S), LC3B (#2775S), Beclin1 S30 (#5410S), Beclin1 (#3738S), PTEN (#5384S), MTOR S2448 (#2971S), MTOR (#2983S), AKT S473 (#9271), AKT (#9272S), MEK1/2 S217/221 (#9154S), MEK (#8727), ERK1/2 (#9101S), ERK1/2 (#9102), cleaved-PARP (#9541S), P70S6K Thr389 (#9234S), P70S6K (#2708S), P38 MAPK Thr180/Y182 (#4511S), 4EBP1 T37/46 (#2855S), 4EBP1 (#9452S), PI3K110\(\:\alpha\:\) (#4255S) were purchased from Cell Signaling Technology. Anti-ULK2 antibody (AB97695), ATG16L1 (AB187671), p-ATG16L1 S30 (AB19016), and mCherry (AB167453; 1:500) was 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. 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).

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× 10⁶ cells in 10 mL medium (10 cm dish), or spheroid cells cultured at a density of 1–3× 10⁶ 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 RIPA buffer. Spheroids (minimum of 1.5 × 10⁶ 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).

Microscopy

Fluorescent images of tdTomato spheroids, along with mesothelial clearance and standard reattachment assays, were obtained using a Leica DMI4000B inverted microscope. Fluorescent images of mCherry-eGFP-LC3 and Nuclight GFP spheroids were captured using the Incucyte® S3 System.

Spheroid viability assays

For bulk spheroids. Cells were placed in 24-well ultra-low attachment (ULA) cluster plates at a density of 0.5-1 × 10⁵ 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 with 500 µL of PBS (to avoid cell uptake into the pipette tip), 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 (in a volume equal to that of the combined trypsin/EDTA and FBS) and gentle mixing via pipetting. Cell counting was performed using the TC20 Automated Cell Counter (Bio-Rad Laboratories).

For single 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 for 4, 24, or 48-hours and fluorescence was measured using a Biotek plate reader. For the CellTiter-Glo (Promega, G7572) and Caspase-Glo 3/7 (Promega, G8092) assays, 100 µL of reagent was added, and the plate was frozen at -80°C. After 24-hours, the plates were incubated in the dark for 60 min on a plate rocker. The wells were transferred to 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-hour 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 the green and green mean intensity features, respectively.

Proteomic Mass Spectrometry

Protein extraction and mass spectrometer analysis were performed on OVCAR8 wild-type and OVCAR8 ULK1KO spheroids that were cultured for 24 hours post-seeding. Spheroids were lysed using three 5-second pulse rounds of sonication at 35% amplitude lysed OVCAR8 wild-type and OVCAR8 ULK1KO spheroids at 24 hours 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 Na₂MoO₄, 1 mM Na₄P₂O₇, 4 mM Na₂C₄H₄O₆, 2 mM Na₃VO₄, 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.

Mesothelial Clearance Assay

ZT-GFP mesothelial cells + tdTomato spheroids. Mesothelial cells (1-1.5 × 10⁵ 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. Spheroids (2000 cells per well) were seeded into 96-well ULA plates and incubated for 24 h. Ascites-derived 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).

Patient-derived mesothelial cells + 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 cells. To generate green-expressing patient-derived mesothelial cells, 3.5µL of Ad-GFP per 100,000 cells was added to the cell suspension, which was then seeded on top of the collagen layer at a density of 1-1.5 × 10⁵ cells and incubated for 24 h. Ascites-derived spheroids were seeded 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, and 1mL of fresh media was added to each well, including control wells without mesothelial cells. Using a P200, ascites-derived spheroids were transferred to a 24-well plate at 5 per well. Green and red fluorescent images were captured 24 h later using a Leica microscope and spheroid displacement was quantified using Fiji.

Organoids

Organoid Size and Number Analysis. Cells were seeded at a density of 5000 cells/well as droplets in 50µL of Cultrex BME 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 hours with the Incucyte® S3 System. The total organoid area (µm²) and number of organoids per well were quantified using the Organoid Analysis Software.

Xenotransplantation assays

NOD/SCID female mice (8–10 weeks old; Charles River Laboratories) were intraperitoneally injected with 150 µL PBS containing the following cells: OVCAR8/OVCAR8-ULK1KO, 2 × 10⁶; HeyA8/HeyA8-ULK1KO, 1 × 10⁶). For survival analyses, mice were monitored daily after intraperitoneal injection and euthanized using standard criteria for humane endpoints (lethargy, hunched posture, impaired breathing, extreme weight loss, and excessive ascites). Mice received weekly injections of D-luciferin (Perkin Elmer, #122799) at 75 mg/kg in 100µL PBS to monitor tumor progression via bioluminescent imaging using the IVIS Lumina S5 system (PerkinElmer). 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.

IHC quantification and scoring

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 reflects “high-positive” and “positive” as defined by the IHC profiler.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software). Specific analysis details are described in the figure legends.

ULK1 is required for autophagy activation in EOC spheroids, but not in HGSOC precursor spheroids

Autophagy ensures homeostasis under challenging conditions by facilitating the degradation of intracellular components and the subsequent replenishment of vital biomolecules. Autophagy induction is controlled by the ULK complex, notably by ULK1 kinase activity (16). Our previous studies revealed elevated ULK1 expression in EOC spheroids, which corresponded to increased autophagy activation (32). To further elucidate the role of ULK1 in autophagy activation, here in we ablated ULK1 in EOC cells using CRISPR/Cas9 and pooled multiple independent clones to generate a population of ULK1 knockout (ULK1KO) cells. Upon examining parental OVCAR8 and HEYA8 spheroids, we observed increased ULK1 expression, whereas ULK1KO cells exhibited a complete absence of ULK1 (Fig. 1A). To evaluate the effect of ULK1 loss on autophagy, we examined proteins involved in the autophagic pathway. In both OVCAR8 and HEYA8 ULK1KO 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. 1B). This is important, as p62 accumulation serves as an indicator of autophagy inhibition, whereas its decrease suggests autophagy induction (33). Additionally, the LC3II:I ratio, derived from the processing of LC3I to LC3II, a marker of autophagosome membranes, reflects the activation of autophagy (34). These findings were recapitulated under serum starvation conditions in HEYA8 adherent cells (Supplementary Fig. 1A), but not in OVCAR8 adherent cells (Supplementary Fig. 1B). We observed a significant decrease in the LC3II:I ratio observed as early as 4-hours and 24-hours within OVCAR8 and HEYA8 ULK1KO spheroids, respectively (Supplementary Fig. 2A/B). Interestingly, ULK1 knockout resulted in an elevation of LC3I under adherent conditions (Fig. 1B) and a significant reduction in p-ATG16L1 (S278) (Supplementary Fig. 3A), supporting the established role of ULK1 in the processing of LC3 (35). Additionally, we verified the abrogation of ULK1 activity through its direct downstream substrate p-Beclin1 (S30). As anticipated, ULK1 loss eliminated its activity in EOC spheroids, highlighted by reductions in p-Beclin1 (S30) (Fig. 1B). We also sought to evaluate the autophagic flux, defined as the efficiency and rate at which cellular components are degraded and recycled through the autophagy pathway (36). We observed a significant reduction in mCherry:GFP and monomeric mCherry levels, indicative of impaired lysosomal degradation as early as 48-hours in OVCAR8 ULK1KO spheroids, starting at 120 h in HEYA8 ULK1KO spheroids (Supplementary Fig. 3B).

Historically, it was believed that HGSOC was derived from the ovarian surface epithelium; however, emerging evidence has shifted this paradigm, as the majority of HGSOC cases arise from the fallopian tube epithelium (37). To investigate the role of ULK1 in autophagy, specifically in fallopian tube-derived cells, we examined the expression of key proteins that constitute the canonical autophagy pathway in FT190 cells, which are immortalized human fallopian tube secretory epithelial cells (28). Significant reductions in ULK1 activity, as indicated by phosphorylated Beclin1 (S30), were observed under both ULK1KO conditions (Fig. 1B). Significant elevations in p62 expression were noted when comparing FT190 parental spheroids with ULK1KO spheroids (Fig. 1B). Additionally, significant increases in the LC3II:I ratio were observed in both parental and ULK1KO spheroid conditions. However, no notable differences in the LC3II:I ratio were detected between parental and ULK1KO spheroids (Fig. 1B; Supplementary Fig. 3C), suggesting that ULK1 activity may not be essential for autophagy activation in these 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). We observed a significant increase in ULK2 expression in FT190 ULK1KO adherent cells (Fig. 1B), whereas no significant differences were observed in the ULK1KO EOC lines. Collectively, our results suggest that ULK1 is vital for the activation of both autophagy and autophagic flux in EOC spheroids, whereas its role appears to be less critical or possibly redundant in HGSOC precursor cells.

Cell viability is significantly impaired in EOC ULK1KO spheroids

Assessment of ULK1 and autophagy during spheroid formation is crucial because spheroids enhance viability and protect EOC cells from anoikis and chemotherapeutic damage (39). To further understand how ULK1 and autophagy contribute to spheroid formation and viability, ULK1KO cells were grown in suspension culture, and viability was assessed over time. We assayed for differences in spheroid morphology, density, integrity, and cell number in OVCAR8 and HEYA8 cell lines due to ULK1 loss. We found that HEYA8 ULK1KO bulk spheroids showed obvious differences in morphology with decreased density and cell number, and impaired integrity compared to parental spheroids, whereas OVCAR8 ULK1KO spheroids retained their morphology but displayed reduced cell numbers (Supplementary Fig. 4a). To evaluate the number of viable cells over time, we used Trypan Blue exclusion on spheroids over a 10-day period and found a decrease in viable cells across all time points in both cell lines. A significant reduction in viable cells was observed in both OVCAR8 and HEYA8 ULK1KO spheroids on days 7 and 10 (Fig. 2A). Both parental and ULK1KO EOC spheroids showed significant growth from days 0 to 3. However, this growth did not continue beyond day 3 in OVCAR8 parental and ULK1KO spheroids (Fig. 2A). Interestingly, HEYA8 parental spheroids continued to grow after day 3 (Fig. 2A), unlike ULK1KO spheroids, suggesting ULK1's absence limits growth and induces dormancy, or induces cell death. Unsurprisingly, there was a significant reduction in the viable cell count in FT190 spheroids, as we have observed this previously (11). However, the absence of ULK1 further contributed to this reduction in viable cell number (Fig. 2A). To determine the cause of reduced cell viability of ULK1KO spheroids, we investigated whether this stemmed from decreased proliferation or increased apoptosis. To evaluate cell proliferation, we generated EOC parental and ULK1KO cells that expressed nuclear-localized GFP, enabling fluorescence imaging via the Incucyte Live-cell analysis system. No growth rate disparities were observed between EOC parental and ULK1KO spheroids (Supplementary Fig. 4B). Analysis of cleaved-PARP, a definitive marker of apoptotic cell death, revealed that spheroid formation triggered apoptosis, as indicated by increased cleaved PARP levels under spheroid conditions. Notably, comparing parental and ULK1KO spheroids yielded varied outcomes: a decrease in cleaved PARP levels in OVCAR8 ULK1KO spheroids, an increase in HEYA8 ULK1KO spheroids, and no change in FT190 ULK1KO spheroids (Fig. 2B). Apoptotic activity was assessed 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 ULK1KO spheroids (Fig. 2C). These findings demonstrate that ULK1 is essential for maintaining viability and regulating apoptosis in EOC cells and HGSOC precursor spheroids, indicating its pivotal role in spheroid integrity and stress response.

ULK1 loss disrupts key cell survival pathways in epithelial ovarian cancer spheroids

In addition to its role as a primary regulator of autophagy, ULK1 plays pivotal roles beyond autophagy regulation (40). To further investigate ULK1's impact on the progression of epithelial ovarian cancer, we performed proteomic mass spectrometry and bioinformatic analyses on OVCAR8 and OVCAR8 ULK1KO spheroids collected after 24 hours of culture. Through KEGG and Reactome pathway analyses (Fig. 3A), we found significant changes in crucial cell survival pathways, including apoptosis, and mTOR-PI3K-AKT and MAPK signaling pathways. We sought to validate these pathways via immunoblotting, as they are crucial for cell survival, growth, and dormancy (41,42). We observed significant alterations in protein expression and signaling due to ULK1 loss. Specifically, OVCAR8 parental spheroids exhibited a notable increase in MEK activity compared to adherent cells, as demonstrated by the enhanced phosphorylation of MEK1/2 (S217/221), an effect that was not observed in HEYA8 parental spheroids (Fig. 3C). MEK activity was significantly diminished in both EOC ULK1KO spheroid conditions and reductions were observed in HEYA8 ULK1KO adherent conditions (Fig. 3C). Further analysis of the downstream MEK pathway revealed a consistent decrease in ERK activity across parental spheroids, with a pronounced reduction in OVCAR8 ULK1KO spheroids, as indicated by the decreased phosphorylation of ERK1/2 (Thr 202/Tyr204) (Supplementary Fig. 5A). Under spheroid conditions, there was a significant increase in MAPK activity, as evidenced by elevated phosphorylation of P38 MAPK (Thr180/Tyr182). However, this activity was markedly decreased in both EOC ULK1KO spheroid conditions (Fig. 3C). These results suggest that both MEK and MAPK activities were compromised in the absence of ULK1. This trend extended to the mTOR-PI3K-AKT pathway, which was highlighted by a universal decrease in p-AKT (S473) in ULK1KO adherent and spheroid conditions (Fig. 3C). Furthermore, our analysis of the downstream targets within the mTOR-AKT pathway revealed divergent outcomes: phosphorylation of P70S6K (T389) showed a universal increase (Fig. 3C), whereas phosphorylation of 4EBP1 (Thr37/46) was consistently reduced across all ULK1KO spheroid conditions (Supplementary Fig. 5B). These observations collectively suggest that disruption of ULK1 initiates complex reprogramming of signaling pathways, impairing cellular survival mechanisms.

ULK1 affects both initial and advanced phases of tumor development in xenograft models

Based on our finding that ULK1 is important for autophagy activation, maintenance of spheroid viability, and regulation of apoptosis, we aimed to evaluate the role of ULK1 in EOC tumor formation and metastasis in ex vivo and xenograft models. To investigate this, we cultured both parental and ULK1KO cells as matrix-embedded organoids for up to 21 days. Although the number of organoids formed was similar, we observed a notable reduction in the size of organoids derived from ULK1KO cells (Fig. 4A). Additionally, we observed a significant increase in the growth of parental EOC organoids, while the growth of EOC ULK1KO organoids was attenuated (Fig. 4B), suggesting that although ULK1 loss impacts organoid growth, it does not affect initiation, as the number of organoids remained similar (Supplementary Fig. 6A). To investigate the metastatic capacity of ULK1 using an in vitro model, we replated EOC spheroids expressing tdTomato on either standard tissue culture plastic or GFP-expressing ZT human mesothelial cells and quantified the area of dispersion and mesothelial cell displacement. OVCAR8 ULK1KO cells displayed significantly reduced ability to displace mesothelial cells (Fig. 4C) and disperse on tissue culture plastic (Supplementary Fig. 6B), whereas the displacement capacity of HEYA8 ULK1KO cells was impaired in the presence of mesothelial cells only (Fig. 4D; Supplementary Fig. 6B), suggesting that ULK1 may promote invasiveness in vivo. To model the unique pattern of EOC metastasis, in which cells from primary tumor site at the ovary or fallopian tube are shed directly into the peritoneal cavity(2,3) we injected EOC cells expressing luciferase and tdTomato intraperitoneally into female NOD/SCID mice and monitored tumor progression over time (Fig. 5A/D). OVCAR8 ULK1KO cells showed a reduced tumor burden at all time points, with significant decreases observed during the mid-to-late stage of disease progression as identified by bioluminescent imaging (BLI) (Fig. 5B). In contrast, HEYA8 ULK1KO cells exhibited a significant decrease in tumor burden during the early stages of disease progression (Fig. 5E), but this difference was lost at later stages. ULK1 loss in EOC cells resulted in fewer tumor lesions across several metastatic sites, with a notable decrease in ascites formation and abdominal and omental metastasis (Fig. 5C/F). However, minimal differences were observed in both overall and average survival times (Supplementary Fig. 7A). with no significant variations in Ki67 and Caspase-3 IHC staining on endpoint tumor samples (Supplementary Fig. 7B). Our findings indicate that ULK1 directly influences the tumor burden by regulating cell viability and growth and by facilitating invasiveness.

Ascites-derived cells exhibit potential reprogramming compared to their in vitro counterparts

To investigate the impact of the tumor microenvironment and xenografting process on cellular signaling, we conducted molecular analyses on OVCAR8 and OVCAR8 ULK1KO ascites-derived cells (Fig. 6A), which showed a significant decrease in the LC3II:I ratio and suppression of ULK1 activity, as evidenced by reductions in p-Beclin1 (S30) levels (Fig. 6B). Surprisingly, we observed decreased ULK2 expression ULK1KO spheroids, which was not observed in pre-injected ULK1KO cells (Fig. 6B). Furthermore, ULK1KO spheroids showed a significant downregulation of the pro-survival marker p-MEK1/2 (S217/221) alongside an upregulation of the tumor suppressor PTEN (Fig. 6A; Supplemental Fig. 7C), reflecting the characteristics seen in pre-injected ULK1KO cells. Interestingly, the notable differences in cleaved PARP levels between parental and ULK1KO spheroids appeared to diminish in the ascites-derived cells (Fig. 6B; Supplemental Fig. 5A). Similarly, while pre-injected ULK1KO cells consistently displayed a decrease in p-AKT (S473) and an increase in p-70S6K (T389) within ULK1KO spheroids (Fig. 3B), no significant differences in p-AKT (S473) were observed in ascites-derived cells under any culture conditions (Fig. 6B). Additionally, p-P70S6K (T389) levels remained unchanged between parental and ULK1KO ascites-derived spheroids (Fig. 6B). We did, however, detect a significant reduction in overall AKT levels and an increase in total P70S6K levels in both ULK1KO adherent and spheroid cells, a pattern not seen in pre-injected cells (Fig. 6B). Moreover, no significant differences in p-P38 MAPK (Thr180/Y182) were observed in ascites-derived ULK1KO spheroids (Fig. 6B), whereas a universal decrease in p-P38 MAPK (Thr180/Y182) had previously been observed in pre-injected ULK1KO cells (Fig. 3B). To assess whether ascites-derived spheroids maintain their impaired metastatic potential in vitro, we conducted the mesothelial clearance assay using human mesothelial cells derived from patient-ascites. Similarly, ascites-derived ULK1KO cells showed impaired dispersion and reduced displacement ability, even in the presence of collagen (Fig. 6C; Supplementary Fig. 7D). These findings suggest that although the altered signaling pathways observed in pre-injected ULK1KO cells are not fully preserved in ascites-derived spheroids, their impaired metastatic capacity persists despite the injection process, indicating a possible adaptation or selection during metastasis.

To the best of our knowledge, this study is the first to elucidate the essential role of ULK1 in autophagy activation within both in vitro and in vivo models of EOC metastasis, underscored by our proteomic mass spectrometry analysis which also provides novel insights into autophagy-independent functions of ULK1 in EOC. Our findings reveal that ULK1 is crucial for initiating autophagy in EOC spheroids, with its deficiency leading to impaired autophagic flux, reduced spheroid viability and invasion, and organoid growth. Additionally, our tumor xenograft models demonstrate that ULK1 significantly influences tumor spread at both early and advanced stages, highlighting its pivotal role across various stages of tumor progression.

ULK1’s role as a central regulator of autophagy initiation is well established (43). In our study, the observed increase in ULK1 protein expression in parental spheroids are consistent with autophagy activation, as indicated by the significant rise in the LC3II:I ratio. In contrast, the unaltered LC3II:I ratio and elevated p62 levels in EOC ULK1KO spheroids indicate compromised autophagy activation. Intriguingly, the loss of ULK1 did not hinder autophagy activation in FT190 precursor cells with substantial increases in ULK2 protein expression in FT190 ULK1KO cells suggesting compensatory autophagy activation. Like our findings, one study reported that ULK1 inhibition did not alter ULK2 expression in HGSOC cells (44); however, that study did not assess whether this occurs in HGSOC precursor fallopian tube cells. This observation emphasizes that the specific requirement for ULK1 on autophagy activation could be specific to malignant cells.

To verify the loss of ULK1 activity, we interrogated the phosphorylation state of Beclin-1 and ATG16L1, both direct downstream targets of ULK1 essential for autophagosome formation (45). A dramatic reduction in p-Beclin-1 (S30) levels in ULK1KO cells compared with parental cell lines also confirmed the absence of ULK1 activity. Since our previous research indicates that Beclin-1 is not essential for autophagy in EOC spheroids (46), thus, in our present study, phosphorylated Beclin-1 (S30) served primarily to verify ULK1 activity. We observed decreased phosphorylation of ATG16L1 (S278) in OVCAR8 ULK1KO cells and a notable increase in the unlipidated LC3I in EOC ULK1KO cells. We previously showed a reduction in p-ATG4B (S316) following ULK1 knockdown and pharmacological inhibition (32), thus explaining how ULK1 loss leads to accumulation of LC3I as observed here.

Using our collective in vitro and in vivo spheroid models of EOC growth and metastasis, these results highlight ULK1's pivotal role in EOC progression. We observed a significant decrease in tumor burden at both early and late time points upon ULK1 ablation, which mirrored our in vitro experiments displaying significant reductions in spheroid cell viability over extended time in culture. In addition to reduced tumor cell dissemination, we observed fewer tumors at metastatic sites 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 with autophagy rather than directly on secondary growth or altered invasive capacities. However, we observed significant reductions in the mesothelium invasion of both pre-xenograft spheroids as compared with mouse ascites-derived spheroids. In addition, EOC organoid growth was significantly impaired due to ULK1 loss. Taken together, these data suggest that metastatic cells reaching secondary sites may exhibit compromised invasiveness and impaired growth re-initiation. Our findings corroborate a previous study demonstrating that inhibiting autophagy restricts the invasiveness of ovarian cancer cells (47). Similarly, evidence from both in vitro and in vivo investigations indicate that ULK1 depletion significantly reduces pancreatic and hepatocellular carcinoma growth (48,49). However, ULK1 function in cancer development might be context-specific among different malignancies. For example, the absence of ULK1 in breast cancer models has been linked to an increased likelihood of osseous metastasis (50). This observation differs from our previous study where we reported that autophagy levels and ULK1 mRNA overexpression are correlated with poor survival outcomes in advanced-stage ovarian cancer (27). Most evidence, particularly our own in EOC, highlights the potential of therapeutically targeting ULK1 and autophagy to slow disease progression and improve treatment responses. 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 indicated that ULK1 loss can enhance chemotherapy sensitivity in OVCAR8 cells (51), yet we have unpublished in vitro findings suggesting that ULK1 inhibition might in fact diminish the effectiveness of standard first-line chemotherapeutics used in EOC (Johnston & Shepherd, in preparation). Future work aims to uncover novel synergistic therapeutic treatments in our ULK1-ablated and autophagy-deficient EOC spheroids to further improve advanced treatment therapy.

While ULK1 is widely recognized as a critical regulator of autophagy, its autophagy-independent functions, especially in the context of cancer, have been less studied and warrant further exploration. Consequently, we conducted label-free mass spectrometry analysis on OVCAR8 and OVCAR8 ULK1KO spheroids, which uncovered significant changes in several key cell signaling pathways, including MAPK, and PI3K-mTOR-AKT signaling, and verified our results of increased apoptosis. Given the significant decrease in viability and increase in apoptosis observed in spheroids lacking ULK1, we investigated the status of these specific that are linked to cancer cell growth and survival. We observed significant alterations in both MAPK and PI3K-mTOR-AKT pathways among EOC ULK1KO lines, highlighted by a universal decrease in p-MEK (S217/22), p-P38 (T180/Thr182), and p-AKT (S473). This aligns with previous studies implicating MAPK signaling in ovarian cancer, where it has been shown to regulate autophagy and inhibit apoptosis (52), and promote invasion and proliferation through combined AKT-MAPK signaling (53). Intriguingly, when we re-assessed the status of these same proteins in ascites-derived cells from mouse xenografts, their regulation was further altered. These ascites-derived cells remained autophagy-deficient, highlighting that ULK1’s potential effects on MAPK and PI3K-AKT-mTOR pathway regulation are distinct from those regulating autophagy. These results underscore ULK1's essential role in sustaining the integrity of signaling networks and bioenergetic processes impacting survival during EOC metastasis.

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 the MAPK and PI3K-mTOR-AKT signaling networks. The significant dysregulation observed in these pathways, along with the pronounced reduction in spheroid viability, organoid growth, and tumor cell dissemination following ULK1 ablation, underscores the vital role in the progression and metastasis of EOC. 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.

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

FACS

Fluorescence-activated cell sorting

GFP

Green fluorescent protein

ULA

Ultra-low attachment

IHC

Immunohistochemistry

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.

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. JDW wrote the original draft of the manuscript and TGS edited the manuscript. All authors have read and approved the final manuscript.

Acknowledgements

We 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.

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There is NO conflict of interest to disclose.

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ULK1 is required for autophagy and promotes metastatic progression in epithelial ovarian cancer

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Abstract

Figures

BACKGROUND

METHODS

Cultured cell lines

Generation of Nuclight GFP cell lines

Generation of mCherry-eGFP-LC3 cell lines

Generation of luc2tdTomato cell lines

Antibodies and reagents

Immunoblot analysis

Preparation of wholecell lysates

Microscopy

Spheroid viability assays

Cell Proliferation

Proteomic Mass Spectrometry

Mesothelial Clearance Assay

Organoids

Xenotransplantation assays

IHC quantification and scoring

Statistical analysis

RESULTS

ULK1 is required for autophagy activation in EOC spheroids, but not in HGSOC precursor spheroids

ULK1 loss disrupts key cell survival pathways in epithelial ovarian cancer spheroids

ULK1 affects both initial and advanced phases of tumor development in xenograft models

DISCUSSION

CONCLUSION

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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