Abstract B51: Stress management: LKB1 controls growth and survival of dormant epithelial ovarian cancer spheroid cells.

Most epithelial ovarian cancer (EOC) patients with metastatic disease initially respond to cytotoxic chemotherapy, yet almost all will relapse with resistant disease. Thus, improving patient outcomes will require novel approaches to limit metastasis and overcome chemo-resistance. Liver Kinase B1 (LKB1), encoded by the STK11 gene, is a key intracellular regulator of metabolic stress and is considered a putative tumor suppressor in some cancers. However, we have demonstrated that LKB1 is intact and required for EOC cell viability and growth in an in vitro spheroid model of ovarian cancer metastasis. We propose that LKB1 signaling enables malignant EOC cells to maintain viability and survive in metabolically challenging environments like that encountered during intraperitoneal metastasis. To further investigate the therapeutic potential of targeting LKB1 activity in metastatic EOC, we generated STK11-knockout cell lines—normal FT190 cells, and EOC cell lines OVCAR8, HeyA8, and iOvCa147—using CRISPR technology. STK11KO resulted in decreased malignant properties of EOC cells in vitro, including clonogenicity and anchorage-independent growth; however, loss of LKB1 in FT190 cells had no effect on cell proliferation, clonogenicity, or anchorage-independent growth, indicating LKB1 does not likely act as a tumor suppressor in EOC. Loss of LKB1 sensitized EOC cells to the growth-inhibiting effects of specific metabolic stresses. OVCAR8-STK11KO cells were more sensitive to nutrient deprivation in adherent culture, and to carboplatin and paclitaxel treatment in spheroid culture, as compared with OVCAR8 cells. Among the three EOC cell lines, STK11KO yielded variable sensitivity to inhibition of mitochondrial ATP production via oligomycin treatment. Interestingly, STK11KO did not affect induction of AMP-activated protein kinase (AMPK) phosphorylation in EOC spheroids, indicating that metabolic stress signaling to support EOC cell survival in spheroids during metastasis may occur via alternative pathways. In support of our previous knockdown results, EOC spheroids completely lacking LKB1 had markedly impaired growth in suspension culture compared to parental cell controls. In contrast, FT190 spheroids exhibited rapid cell attrition in spheroid culture regardless of LKB1 status. These results indicate that LKB1 may be specifically required in EOC cells to evade anoikis during metastatic spread. Finally, to test directly whether loss of LKB1 activity affects the metastatic potential of EOC cells, we performed intraperitoneal injections of OVCAR8-STK11KO and HeyA8-STK11KO cells with their respective parental cell controls. Loss of LKB1 in both aggressive EOC cell lines exhibited a dramatic reduction on tumor burden. STK11KO significantly decreased the establishment of large, solid tumor masses, reduced adhesion of OVCAR8 tumor nodules, and even changed the metastatic trajectory of HeyA8 cells with evidence of tumor cell growth only as a thin layer on peritoneal walls. Histologic analysis revealed evidence of extensive necrosis in STK11KO tumors, which was likely the major contributor to reduced tumor burden. These results strongly indicate that loss of LKB1 activity abrogates the metabolic stress response necessary during EOC metastasis both in spheroids and establishment of intraperitoneal tumors. Overall, LKB1 or its AMPK-independent signaling mediators represent unique yet very potent therapeutic vulnerabilities in metastatic EOC. Citation Format: Adrian Buensuceso, Yudith R. Valdes, Rene Figueredo, Gabriel E. DiMattia, Trevor G. Shepherd. The metabolic stress mediator LKB1 is required for ovarian cancer metastasis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A12.

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse throughout the peritoneum via ascites to seed secondary lesions. We hypothesize that EOC spheroids are key mediators of metastasis, which use specific signaling pathways to alter cell metabolism for increased survival. Our lab discovered that AKT signaling is reduced during spheroid formation leading to cellular quiescence and autophagy. Given the induction of quiescence and autophagy in EOC spheroids, we are studying the 5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response and dormant phenotype of EOC spheroids. We demonstrate AMPK activity and its upstream kinase LKB1 are increased in quiescent EOC spheroids compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Targeted knockdown of STK11, encoding LKB1, reduces cell viability in ovarian cancer cell line spheroids; PRKAA1 (AMPKα1) knockdown has little to no effect on EOC cell or spheroid viability. Combination of STK11 knockdown with carboplatin treatment leads to a synergistic enhancement in EOC spheroid cell death. In contrast, AICAR treatment of proliferating adherent ovarian cancer cell lines and primary EOC cells induces AMPK activity and causes either cytostasis or cell death. In addition, AICAR treatment of spheroids during reattachment decreases the dispersion capacity of migrating EOC cells. These results offer a glimpse of the potential important contributions of LKB1-AMPK pathway in stress signaling related to EOC cell survival during metastasis. In addition, downstream effectors of upregulated LKB1-AMPK signalling may provide additional mechanisms by which EOC evades chemotherapy. It is foreseeable that these findings will allow us to identify new therapeutic targets within this pathway critical to EOC progression for ultimate translation to the clinic. Citation Format: Teresa Peart, Elena Fazio, Yudith Ramos-Valdes, Monique Bertrand, Jacob McGee, Michel Prefontaine, Akira Sugimoto, Gabriel E. DiMattia, Trevor G. Shepherd. The LKB1-AMPK pathway mediates the metabolic stress response of dormant ovarian cancer spheroids. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A47.

<div>Abstract<p>Epithelial ovarian cancer (EOC) spreads by direct dissemination of malignant cells and multicellular clusters, known as spheroids, into the peritoneum followed by implantation and growth on abdominal surfaces. Using a spheroid model system of EOC metastasis, we discovered that Liver kinase B1 (LKB1), encoded by the <i>STK11</i> gene, and its canonical substrate AMP-activated protein kinase (AMPK) are activated in EOC spheroids, yet only LKB1 is required for cell survival. We have now generated <i>STK11</i>-knockout cell lines using normal human FT190 cells and three EOC cell lines, OVCAR8, HeyA8, and iOvCa147. <i>STK11</i>KO did not affect growth and viability in adherent culture, but it decreased anchorage-independent growth of EOC cells. EOC spheroids lacking LKB1 had markedly impaired growth and viability, whereas there was no difference in normal FT190 spheroids. To test whether LKB1 loss affects EOC metastasis, we performed intraperitoneal injections of OVCAR8-, HeyA8-, and iOvCa147-<i>STK11</i>KO cells, and respective controls. LKB1 loss exhibited a dramatic reduction on tumor burden and metastatic potential; in particular, OVCAR8-<i>STK11</i>KO tumors had evidence of extensive necrosis, apoptosis, and hypoxia. Interestingly, LKB1 loss did not affect AMPKα phosphorylation in EOC spheroids and tumor xenografts, indicating that LKB1 signaling to support EOC cell survival in spheroids and metastatic tumor growth occurs via other downstream mediators. We identified the dual-specificity phosphatase DUSP4 as a commonly upregulated protein due to LKB1 loss; indeed, <i>DUSP4</i> knockdown in HeyA8-<i>STK11</i>KO cells partially restored spheroid formation and viability.</p>Implications:<p>LKB1 possesses key tumor-promoting activity independent of downstream AMPK signaling during EOC metastasis.</p></div>

<div>Abstract<p>Epithelial ovarian cancer (EOC) spreads by direct dissemination of malignant cells and multicellular clusters, known as spheroids, into the peritoneum followed by implantation and growth on abdominal surfaces. Using a spheroid model system of EOC metastasis, we discovered that Liver kinase B1 (LKB1), encoded by the <i>STK11</i> gene, and its canonical substrate AMP-activated protein kinase (AMPK) are activated in EOC spheroids, yet only LKB1 is required for cell survival. We have now generated <i>STK11</i>-knockout cell lines using normal human FT190 cells and three EOC cell lines, OVCAR8, HeyA8, and iOvCa147. <i>STK11</i>KO did not affect growth and viability in adherent culture, but it decreased anchorage-independent growth of EOC cells. EOC spheroids lacking LKB1 had markedly impaired growth and viability, whereas there was no difference in normal FT190 spheroids. To test whether LKB1 loss affects EOC metastasis, we performed intraperitoneal injections of OVCAR8-, HeyA8-, and iOvCa147-<i>STK11</i>KO cells, and respective controls. LKB1 loss exhibited a dramatic reduction on tumor burden and metastatic potential; in particular, OVCAR8-<i>STK11</i>KO tumors had evidence of extensive necrosis, apoptosis, and hypoxia. Interestingly, LKB1 loss did not affect AMPKα phosphorylation in EOC spheroids and tumor xenografts, indicating that LKB1 signaling to support EOC cell survival in spheroids and metastatic tumor growth occurs via other downstream mediators. We identified the dual-specificity phosphatase DUSP4 as a commonly upregulated protein due to LKB1 loss; indeed, <i>DUSP4</i> knockdown in HeyA8-<i>STK11</i>KO cells partially restored spheroid formation and viability.</p>Implications:<p>LKB1 possesses key tumor-promoting activity independent of downstream AMPK signaling during EOC metastasis.</p></div>

AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine kinase involved in metabolic regulation. It is phosphorylated by the upstream liver kinase B1 (LKB1) or calcium/calmodulin-dependent kinase kinase 2 (CaMKKβ). In cultured cells, AMPK activation correlates with LKB1 activity. The phosphorylation activates AMPK, shifting metabolism toward catabolism and promoting mitogenesis. In muscles, inactivity reduces AMPK activation, shifting the phenotype of oxidative muscles toward a more glycolytic profile. Here, we compared the basal level of AMPK activation in glycolytic and oxidative muscles and analyzed whether this relates to LKB1 or CaMKKβ. Using Western blotting, we assessed AMPK expression and phosphorylation in soleus, gastrocnemius (GAST), extensor digitorum longus (EDL), and heart from C57BL6J mice. We also assessed LKB1 and CaMKKβ expression, and CaMKKβ activity in tissue homogenates. AMPK activation was higher in oxidative (soleus and heart) than in glycolytic muscles (gastrocnemius and EDL). This correlated with AMPK α1-isoform expression, but not LKB1 and CaMKKβ. LKB1 expression was sex dependent and lower in male than female muscles. CaMKKβ expression was very low in skeletal muscles and did not phosphorylate AMPK in muscle lysates. The higher AMPK activation in oxidative muscles is in line with the fact that activated AMPK maintains an oxidative phenotype. However, this could not be explained by LKB1 and CaMKKβ. These results suggest that the regulation of AMPK activation is more complex in muscle than in cultured cells. As AMPK has been proposed as a therapeutic target for several diseases, future research should consider AMPK isoform expression and localization, and energetic compartmentalization.NEW & NOTEWORTHY It is important to understand how AMP-activated kinase, AMPK, is regulated, as it is a potential therapeutic target for several diseases. AMPK is activated by liver kinase B1, LKB1, and calcium/calmodulin-dependent kinase kinase 2, CaMKKβ. In cultured cells, AMPK activation correlates with LKB1 expression. In contrast, we show that AMPK-activation was higher in oxidative than glycolytic muscle, without correlating with LKB1 or CaMKKβ expression. Thus, AMPK regulation is more complex in highly compartmentalized muscle cells.

MiR-206 inhibits epithelial ovarian cancer cells growth and invasion via blocking c-Met/AKT/mTOR signaling pathway

Glycosylated antitumor ether lipids (GAELs) are a promising class of investigational anticancer agents with potent antitumor activity against a range of cancers, including platinum-resistant high-grade serous ovarian cancer (HGSOC). We previously demonstrated that epithelial ovarian cancer (EOC) cell lines (HGSOC and endometrioid) and primary cells derived from patient ascites (HGSOC and clear cell) were sensitive to the cell-killing effects of D-glucosamine-derived GAELs via an apoptosis-independent mechanism. However, D-linked carbohydrates are metabolized by endogenous glucosidases in vivo, rendering the GAEL inactive. Thus, the aim of the current study was to synthesize a novel class of GAELs with L-linked carbohydrates and to evaluate their efficacy in EOC cell lines and patient samples. Out of seven novel compounds tested, L-rhamnose-GAEL was identified as the compound with the greatest efficacy for killing EOC cell lines and patient cells that were grown under adherent or nonadherent (3D) conditions. The drug-sensitive and drug-resistant syngeneic endometrioid EOC cell lines, A2780s and A2780cp, respectively, as well as the NIH:OVCAR-3 and COV362 HGSOC lines, were incubated with L-rhamnose-GAEL for 48 hours and the CC50 determined as 15 μM for A2780s, 22.5 μM for A2780cp, and 5 μM for NIH:OVCAR-3 and COV362 cells. Similar experiments were conducted with primary EOC cells, chemo-naïve EOC126 (clear-cell adenocarcinoma), and platinum-resistant EOC 183I (HGSOC). Cell viability decreased in a dose-dependent manner with CC50 of 12 μM for EOC126 and 22.5 μM for EOC183l. For comparison, when EOC126 and EOC183I were grown as 3D cultures the CC50for cisplatin was 20 μM and 40 μM, respectively. To evaluate the effect of using low-dose treatment of L-rhamnose-GAEL on primary EOC cells (chemo-naive and chemoresistant), cells were exposed to L-rhamnose-GAEL for 96 hours. These studies revealed that prolonged incubation led to low CC50 of 1-5 μM. These results showed that primary EOC cells derived from chemo-naïve and platinum-resistant patients were sensitive to the L-rhamnose-GAEL and offer a novel drug class capable of killing chemotherapy-resistant EOC cells. Citation Format: Mark W. Nachtigal, Frank Schweizer, Gilbert Arthur. Novel L-sugar linked glycosylated antitumor ether lipids for killing platinum-resistant human epithelial ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A56.

Myxoma virus-mediated oncolysis of ascites-derived human ovarian cancer cells and spheroids is impacted by differential AKT activity

Late-stage epithelial ovarian cancer (EOC) involves the widespread dissemination of malignant disease throughout the peritoneal cavity, often accompanied by ascites. EOC metastasis relies on the formation of multicellular aggregates, called spheroids. Given that Liver Kinase B1 (LKB1) is required for EOC spheroid viability and LKB1 loss in EOC cells decreases tumor burden in mice, we investigated whether the LKB1 complex controls the invasive properties of human EOC spheroids. LKB1 signalling was antagonized through the CRISPR/Cas9 genetic knockout of LKB1 and/or the RNAi-dependent targeting of STE20-related kinase adaptor protein (STRAD, an LKB1 activator). EOC spheroids expressing nuclear GFP (green) or mKate2 (red) constructs were embedded in Matrigel for real-time live-cell invasion monitoring. Migration and invasion were also assessed in spheroid culture using Transwell chambers, spheroid reattachment, and mesothelial clearance assays. The loss of LKB1 and STRAD signalling decreased cell invasion through Matrigel and Transwell membranes, as well as mesothelial cell clearance. In the absence of LKB1, zymographic assays identified a loss of matrix metalloproteinase (MMP) activity, whereas spheroid reattachment assays found that coating plates with fibronectin restored their invasive potential. A three-dimensional EOC organoid model demonstrated that organoid area was greatly reduced by LKB1 loss. Overall, our data indicated that LKB1 and STRAD facilitated EOC metastasis by promoting MMP activity and fibronectin expression. Given that LKB1 and STRAD are crucial for EOC metastasis, targeting LKB1 and/or STRAD could disrupt the dissemination of EOC, making inhibitors of the LKB1 pathway an alternative therapeutic strategy for EOC patients.

Approximately 90% of ovarian cancer (OC) is Epithelial ovarian Cancer (EOC) subtype and claims ~15,000 lives in the United States annually, making it the deadliest reproductive cancer in women. There are few treatment options for EOC, amongst them include surgical resection of tumors or debulking, and chemotherapy alone or combination. In general, the survival rate of patients with EOC is about 48% and this has not changed in last few decades. According to recent reports from American Cancer Society and SEER, racial disparity in ovarian cancer not only exists for African American (AA) patients, but is worsening over the past few years. African American (AA) patients presents with more advanced disease and develop chemoresistance frequently, and as such, they experience worse survival. Thus identifying the cause of this discrepancy, or more importantly, describing which AA patients are at the highest risk of therapeutic relapse would alter our current treatment strategies and improve overall disease free survival rate. Therefore, the central focus of this proposal is to delineate the molecular and genetic mechanisms contributing to racial disparity of AA patients. Therefore, the overall objective of our studies are to identify the etiology of racial disparity in ovarian cancer and define the molecular networks that contribute this discrepancy in outcomes. Lymphoblastic Leukemia-Derived Sequence 1 (LYL1) is a polypeptide that harbors basic helix-loop-helix transcription factor, a DNA binding motif and dysregulated in many cancers including EOC. Analysis of TCGA data for EOC revealed that LYL1 gene amplification in about 12% patients and associated with poor prognosis. Interestingly, further analysis of LYL1 copy number alteration in different ethnicities disclosed LYL1 amplification in about 36% of the African American (AA) EOC patients. Importantly, EOC patients with low LYL1 (n=1640) expression has better survival probability compared to patients with overexpressed LYL1 (n=202). This discrepancy in survival probability is much more prominent in AA EOC patients. As this is an intriguing observation, we evaluated the levels of LYL1 expression in different EOC cell lines in comparison with fallopian tube epithelial cells. Our data shows, upregulation of LYL1 in most of the EOC cell lines compared to normal fallopian tube epithelial cells. To examine the upregulated LYL1 in EOC cell lines, we performed siRNA mediated downregulation, and evaluated their clonogenic, migration invasion potential. Consistent with the TCGA data, knocking down LYL1 in EOC cells significantly attenuated their clongenic, migration and invasion potential. Furthermore, ectopic overexpression of LYL1 in EOC cells that shows deep deletion of the gene, exhibited increased clonogenicity, invasion and migration. Collectively, our studies indicate an important role for LYL1 in EOC tumor progression and metastatic phenotypes, and could be a biomarker for disparities in EOC outcomes. Citation Format: Damieanus Ochola, Shirisha Jonnalagadda, Swetha Peddibhotla, Tasmin Omy, Mark Reedy, Palle Komaraiah. Upregulated LYL1 promotes epithelial ovarian cancer (EOC) cell growth and metastasis [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-132.

Epithelial ovarian cancer (EOC) cells form multicellular aggregates, or spheroids, and enter a dormant state during intraperitoneal metastasis. Dormant spheroids reduce anabolic metabolism and cell proliferation, which are linked to chemo-resistance. Liver kinase B1 (LKB1), encoded by the STK11 gene, is a critical regulator of stress metabolic signaling; LKB1 phosphorylates the downstream substrates AMP-activated protein kinase (AMPK) and AMPK-related kinases (ARKs) to mediate stress signaling. We have demonstrated that LKB1 expression and activity is required for EOC spheroid cell survival, yet knockdown of AMPKα1/α2 has no effect on viability. In addition, we have preliminary data demonstrating that phosphorylated AMPK is still maintained in EOC spheroids generated from CRISPR-mediated STK11-deleted cells, which completely lack LKB1 expression. Taken together, these results implicate the importance of other AMPK-independent effectors of LKB1 signaling in spheroid cell viability. Therefore, to identify critical downstream targets of LKB1-mediated signaling in EOC spheroids, we employed multiplex inhibitor beads/mass spectrometry (MIB/MS) using OVCAR8 STK11-knockout cells and OVCAR8 control cells grown in adherent and spheroid culture conditions. Using this proteomic approach, we identified NUAK1 as the sole ARK out of 12 different family members that is negatively affected by LKB1 loss. In fact, both phosphorylated and total NUAK1 protein expression are decreased in STK11-knockout cells and spheroids. NUAK1 can negatively control cell growth and proliferation by direct regulation of cell cycle checkpoint proteins in several cell systems; however, it has been relatively understudied in EOC. In analysis of the serous ovarian carcinoma data from TCGA, NUAK1 is infrequently altered (<2% of tumors) and it has the highest mean mRNA expression level as compared with the other 11 ARK genes. However, using immunoblot analysis, we show that NUAK1 protein is largely underexpressed in many established (n=15) and new ascites-derived EOC cell lines (n=22). In fact, further NUAK1 knockdown increased spheroid cell viability, size, and reattachment capability, whereas knockdown of its closely-related family member NUAK2 had no effect compared with the non-targeting siRNA control. Likewise, treatment with the pharmacologic NUAK1/2 inhibitor WZ4003 increased EOC cell growth and clonogenicity. Conversely, ectopic overexpression of NUAK1 reduced EOC cell growth and clonogenicity. Collectively, our data indicate that NUAK1 acts as a newly identified growth suppressor downstream of LKB1 metabolic stress signaling in EOC. We are currently investigating mechanisms regulating NUAK1 protein stability and activity in EOC cells, and whether activated LKB1-NUAK1 signaling promotes stress metabolic signaling during tumor dormancy in the context of EOC metastasis. Citation Format: Parima Saxena, Olga Collins, Yudith Ramos Valdes, Adrian Buensuceso, Kyle Francis, Kevin Brown, Karen Colwill, Anne-Claude Gingras, Robert Rottapel, Gabriel E. DiMattia, Trevor G. Shepherd. NUAK1 acts as a growth suppressor in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A17.

IntroductionLong non-coding RNAs (lncRNAs) have been shown to have great importance in cancer development and progression. However, the mechanism of lncRNAs in epithelial ovarian cancer remains unclear. In the present study, we aimed to explore the role of the lncRNA myocardial infarction-associated transcript (MIAT) in epithelial ovarian cancer tumorigenesis.Material and methodsQuantitative real-time PCR (qRT-PCR) was used to determine MIAT expression in human epithelial ovarian cancer tissues and cell lines, and the effects of MIAT on cell proliferation and cell apoptosis were determined by CCK-8 assay or flow cytometry analysis. Dual-Luciferase Reporter assay and Western blot assay were used to explore the molecular mechanisms of MIAT in epithelial ovarian cancer cells progression.ResultsOur data showed that the expression of lncRNA MIAT was remarkably increased in human epithelial ovarian cancer tissues and cell lines (p < 0.05). High MIAT expression was associated with poor overall survival of epithelial ovarian cancer patients (p < 0.05). Function assays showed that knockdown of MIAT expression significantly inhibited epithelial ovarian cancer cell proliferation and promoted cell apoptosis in vitro (p < 0.05). Moreover, we revealed that MIAT might function as an endogenous miR-330-5p sponge to regulate the target gene of miR-330-5p in epithelial ovarian cancer progression.ConclusionsLncRNA MIAT was found to be a tumor oncogenic lncRNA in epithelial ovarian cancer tumorigenesis. LncRNA MIAT promoted cell proliferation and inhibited cell apoptosis by negative regulation of miR-330-5p in epithelial ovarian cancer cells. Our findings suggested that MIAT might act as a candidate prognostic biomarker and new therapeutic target for treating epithelial ovarian cancer patients.

Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-NUAK1KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-NUAK1KO spheroids. In fact, the FN1 gene, encoding fibronectin, exhibited a 745-fold decreased expression in NUAK1KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in NUAK1KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-NUAK1KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production.

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

Aromatase expression in ovarian epithelial cancers