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Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis.
High-grade serous ovarian cancer (or HGSOC for short) is the fifth leading cause of cancer-related deaths in women. It is generally diagnosed at an advanced stage of disease when the cancer has already spread to other parts of the body. Surgical removal of tumors and subsequent treatment with chemotherapy often reduces the signs and symptoms of the disease for a time but some cancer cells tend to survive so that patients eventually relapse. The HGSOC cells typically spread from the ovaries by moving through the liquid surrounding organs in the abdomen. The cells clump together and enter an inactive state known as dormancy that allows them to survive chemotherapy and low-nutrient conditions. Understanding how to develop new drug therapies that target dormant cancer cells is thought to be an important step in prolonging the life of HGSOC patients. Cancer cells are hardwired to multiply and grow, so Perampalam et al. reasoned that becoming dormant poses challenges for HGSOC cells, which may create unique vulnerabilities not shared by proliferating cancer cells. To find out more, the researchers used HGSOC cells that had been isolated from patients and grown in the laboratory. The team used a gene editing technique to screen HGSOC cells for genes required by the cells to survive when they are dormant. The experiments found that genes involved in a cell signaling pathway, known as Netrin signaling, were critical for the cells to survive. Previous studies have shown that Netrin signaling helps the nervous system form in embryos and inhibits a program of controlled cell death in some cancers. Perampalam et al. discovered that Netrins were present in the environment immediately surrounding dormant HGSOC cells. Human HGSOC patients with higher levels of Netrin gene expression had poorer prognoses than patients with lower levels of Netrin gene expression. Further experiments demonstrated that Netrins help dormant HGSOC cells to spread around the body. These findings suggest that Netrin signalling may provide useful targets for future drug therapies against dormant cells in some ovarian cancers. This could include repurposing drugs already in development or creating new inhibitors of this pathway.
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Carcinoma Epitelial de Ovario , Supervivencia Celular , Netrinas , Neoplasias Ováricas , Transducción de Señal , Humanos , Femenino , Animales , Línea Celular Tumoral , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Netrinas/metabolismo , Netrinas/genética , Ratones , Netrina-1/metabolismo , Netrina-1/genética , Proliferación Celular , Receptores de Netrina/metabolismo , Receptores de Netrina/genéticaRESUMEN
Liver Kinase B1 (LKB1), encoded by Serine-Threonine Kinase 11 (STK11), is a master kinase that regulates cell migration, polarity, proliferation, and metabolism through downstream adenosine monophosphate-activated protein kinase (AMPK) and AMPK-related kinase signalling. Since genetic screens identified STK11 mutations in Peutz-Jeghers Syndrome, STK11 mutants have been implicated in tumourigenesis labelling it as a tumour suppressor. In support of this, several compounds reduce tumour burden through upregulating LKB1 signalling, and LKB1-AMPK agonists are cytotoxic to tumour cells. However, in certain contexts, its role in cancer is paradoxical as LKB1 promotes tumour cell survival by mediating resistance against metabolic and oxidative stressors. LKB1 deficiency has also enhanced the selectivity and cytotoxicity of several cancer therapies. Taken together, there is a need to develop LKB1-specific pharmacological compounds, but prior to developing LKB1 inhibitors, further work is needed to understand LKB1 activity and regulation. However, investigating LKB1 activity is strenuous as cell/tissue type, mutations to the LKB1 signalling pathway, STE-20-related kinase adaptor protein (STRAD) binding, Mouse protein 25-STRAD binding, splicing variants, nucleocytoplasmic shuttling, post-translational modifications, and kinase conformation impact the functional status of LKB1. For these reasons, guidelines to standardize experimental strategies to study LKB1 activity, associate proteins, spliced isoforms, post-translational modifications, and regulation are of upmost importance to the development of LKB1-specific therapies. Therefore, to assess the therapeutic relevancy of LKB1 inhibitors, this review summarizes the importance of LKB1 in cell physiology, highlights contributors to LKB1 activation, and outlines the benefits and risks associated with targeting LKB1.
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Quinasas de la Proteína-Quinasa Activada por el AMP , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Animales , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Epithelial ovarian cancer (EOC) is highly lethal due to its unique metastatic characteristics. EOC spheroids enter a non-proliferative state, with hypoxic cores and reduced oncogenic signaling, all of which contribute to tumour dormancy during metastasis. We investigated the metabolomic states of EOC cells progressing through the three steps to metastasis. Metabolomes of adherent, spheroid, and re-adherent cells were validated by isotopic metabolic flux analysis and mitochondrial functional assays to identify metabolic pathways that were previously unknown to promote EOC metastasis. Although spheroids were thought to exist in a dormant state, metabolomic analysis revealed an unexpected upregulation of energy production pathways in spheroids, accompanied by increased abundance of tricarboxylic acid (TCA) cycle and electron transport chain proteins. Tracing of 13C-labelled glucose and glutamine showed increased pyruvate carboxylation and decreased glutamine anaplerosis in spheroids. Increased reductive carboxylation suggests spheroids adjust redox homeostasis by shuttling cytosolic NADPH into mitochondria via isocitrate dehydrogenase. Indeed, we observed spheroids have increased respiratory capacity and mitochondrial ATP production. Relative to adherent cells, spheroids reduced serine consumption and metabolism, processes which were reversed upon spheroid re-adherence. The data reveal a distinct metabolism in EOC spheroids that enhances energy production by the mitochondria while maintaining a dormant state with respect to growth and proliferation. The findings advance our understanding of EOC metastasis and identify the TCA cycle and mitochondrional activity as novel targets to disrupt EOC metastasis, providing new approaches to treat advanced disease.
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Eukaryotic elongation factor 1A1 (EEF1A1) is canonically involved in protein synthesis but also has noncanonical functions in diverse cellular processes. Previously, we identified EEF1A1 as a mediator of lipotoxicity and demonstrated that chemical inhibition of EEF1A1 activity reduced mouse liver lipid accumulation. These findings suggested a link between EEF1A1 and metabolism. Therefore, we investigated its role in regulating metabolic substrate preference. EEF1A1-deficient Chinese hamster ovary (2E2) cells displayed reduced media lactate accumulation. These effects were also observed with EEF1A1 knockdown in human hepatocyte-like HepG2 cells and in WT Chinese hamster ovary and HepG2 cells treated with selective EEF1A inhibitors, didemnin B, or plitidepsin. Extracellular flux analyses revealed decreased glycolytic ATP production and increased mitochondrial-to-glycolytic ATP production ratio in 2E2 cells, suggesting a more oxidative metabolic phenotype. Correspondingly, fatty acid oxidation was increased in 2E2 cells. Both 2E2 cells and HepG2 cells treated with didemnin B exhibited increased neutral lipid content, which may be required to support elevated oxidative metabolism. RNA-seq revealed a >90-fold downregulation of a rate-limiting glycolytic enzyme, hexokinase 2, which we confirmed through immunoblotting and enzyme activity assays. Pathway enrichment analysis identified downregulations in TNFA signaling via NFKB and MYC targets. Correspondingly, nuclear abundances of RELB and MYC were reduced in 2E2 cells. Thus, EEF1A1 deficiency may perturb glycolysis by limiting NFKB- and MYC-mediated gene expression, leading to decreased hexokinase expression and activity. This is the first evidence of a role for a translation elongation factor, EEF1A1, in regulating metabolic substrate utilization in mammalian cells.
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Hexoquinasa , Factor 1 de Elongación Peptídica , Animales , Cricetinae , Humanos , Adenosina Trifosfato , Línea Celular , Cricetulus , Hexoquinasa/genética , Hexoquinasa/metabolismo , Lípidos , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/química , Factor 1 de Elongación Peptídica/metabolismo , Glucólisis , Oxidación-Reducción , Movimiento Celular , Proliferación Celular , Metabolismo de los LípidosRESUMEN
Ovarian cancers exhibit high rates of recurrence and poor treatment response. Preclinical models that recapitulate human disease are critical to develop new therapeutic approaches. Syngeneic mouse models allow for the generation of tumours comprising the full repertoire of non-malignant cell types but have expanded in number, varying in the cell type of origin, method for transformation, and ultimately, the properties of the tumours they produce. Here we have performed a comparative analysis of high-grade serous ovarian cancer models based on transcriptomic profiling of 22 cell line models, and intrabursal and intraperitoneal tumours from 12. Among cell lines, we identify distinct signalling activity, such as elevated inflammatory signalling in STOSE and OVE16 models, and MAPK/ERK signalling in ID8 and OVE4 models; metabolic differences, such as reduced glycolysis-associated expression in several engineered ID8 subclones; and relevant functional properties, including differences in EMT activation, PD-L1 and MHC class I expression, and predicted chemosensitivity. Among tumour samples, we observe increased variability and stromal content among intrabursal tumours. Finally, we predict differences in the microenvironment of ID8 models engineered with clinically relevant mutations. We anticipate that this work will serve as a valuable resource, providing new insight to help select models for specific experimental objectives.
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Neoplasias Ováricas , Animales , Ratones , Humanos , Femenino , Neoplasias Ováricas/patología , Perfilación de la Expresión Génica , Transducción de Señal , Microambiente Tumoral/genéticaRESUMEN
High-grade serous carcinoma (HGSC) is the most common and aggressive subtype of epithelial ovarian cancer, characterized by gain-of-function TP53 mutations originating in the fallopian tube epithelium. Therapeutic intervention occurs at advanced metastatic disease, due to challenges in early-stage diagnosis, with common disease recurrence and therapy resistance despite initial therapy success. The mevalonate pathway is exploited by many cancers and is potently inhibited by statin drugs. Statins have shown anti-cancer activity in many, but not all cancers. Here, we investigated the role of p53 status in relation to mevalonate pathway signaling in murine oviductal epithelial (OVE) cells and identified OVE cell sensitivity to statin inhibition. We found that p53R175H mutant and Trp53 knockout OVE cells have increased mevalonate pathway signaling compared to p53 wild-type OVE cells. Through orthotopic implantation to replicate the fallopian tube origin of HGSC, p53R175H mutant cells upregulated the mevalonate pathway to drive progression to advanced-stage ovarian cancer, and simvastatin treatment abrogated this effect. Additionally, simvastatin was more efficacious at inhibiting cell metabolic activity in OVE cells than atorvastatin, rosuvastatin and pravastatin. In vitro, simvastatin demonstrated potent effects on cell proliferation, apoptosis, invasion and migration in OVE cells regardless of p53 status. In vivo, simvastatin induced ovarian cancer disease regression through decreased primary ovarian tumor weight and increased apoptosis. Simvastatin also significantly increased cytoplasmic localization of HMG-CoA reductase in ovarian tumors. Downstream of the mevalonate pathway, simvastatin had no effect on YAP or small GTPase activity. This study suggests that simvastatin can induce anti-tumor effects and could be an important inhibitor of ovarian cancer progression.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Neoplasias Ováricas , Femenino , Ratones , Animales , Humanos , Trompas Uterinas/metabolismo , Simvastatina/farmacología , Simvastatina/metabolismo , Simvastatina/uso terapéutico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Ácido Mevalónico/metabolismo , Ácido Mevalónico/uso terapéutico , Células Epiteliales/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Carcinoma Epitelial de Ovario/patologíaRESUMEN
Epithelial ovarian cancer (EOC) is a high-risk cancer presenting with heterogeneous tumors. The high incidence of EOC metastasis from primary tumors to nearby tissues and organs is a major driver of EOC lethality. We used cellular models of spheroid formation and readherence to investigate cellular signaling dynamics in each step toward EOC metastasis. In our system, adherent cells model primary tumors, spheroid formation represents the initiation of metastatic spread, and readherent spheroid cells represent secondary tumors. Proteomic and phosphoproteomic analyses show that spheroid cells are hypoxic and show markers for cell cycle arrest. Aurora kinase B abundance and downstream substrate phosphorylation are significantly reduced in spheroids and readherent cells, explaining their cell cycle arrest phenotype. The proteome of readherent cells is most similar to spheroids, yet greater changes in the phosphoproteome show that spheroid cells stimulate Rho-associated kinase 1 (ROCK1)-mediated signaling, which controls cytoskeletal organization. In spheroids, we found significant phosphorylation of ROCK1 substrates that were reduced in both adherent and readherent cells. Application of the ROCK1-specific inhibitor Y-27632 to spheroids increased the rate of readherence and altered spheroid density. The data suggest ROCK1 inhibition increases EOC metastatic potential. We identified novel pathways controlled by Aurora kinase B and ROCK1 as major drivers of metastatic behavior in EOC cells. Our data show that phosphoproteomic reprogramming precedes proteomic changes that characterize spheroid readherence in EOC metastasis.
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Neoplasias Ováricas , Humanos , Femenino , Carcinoma Epitelial de Ovario , Neoplasias Ováricas/metabolismo , Aurora Quinasa B , Proteómica , Esferoides Celulares/metabolismo , Línea Celular Tumoral , Metástasis de la Neoplasia , Quinasas Asociadas a rhoRESUMEN
Ovarian high-grade serous carcinoma (HGSC) is a highly lethal malignancy for which early detection is a challenge and treatment of late-stage disease is ineffective. HGSC initiation involves exfoliation of fallopian tube epithelial (FTE) cells which form multicellular clusters called spheroids that colonize and invade the ovary. HGSC contains universal mutation of the tumour suppressor gene TP53. However, not all TP53 mutations are the same, as specific p53 missense mutants contain gain-of-function (GOF) properties that drive tumour formation. Additionally, the role of GOF p53 in spheroid-mediated spread is poorly understood. In this study, we developed and characterized an in vitro model of HGSC based on mutation of TP53 in mouse oviductal epithelial cells (OVE). We discovered increased bulk spheroid survival and increased anchorage-independent growth in OVE cells expressing the missense mutant p53R175H compared to OVE parental and Trp53ko cells. Transcriptomic analysis on spheroids identified decreased apoptosis signaling due to p53R175H. Further assessment of the apoptosis pathway demonstrated decreased expression of intrinsic and extrinsic apoptosis signaling molecules due to Trp53 deletion and p53R175H, but Caspase-3 activation was only decreased in spheroids with p53R175H. These results highlight this model as a useful tool for discovering early HGSC transformation mechanisms and uncover a potential anti-apoptosis GOF mechanism of p53R175H.
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Cistadenocarcinoma Seroso , Neoplasias Ováricas , Ratones , Femenino , Humanos , Animales , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias Ováricas/patología , Mutación con Ganancia de Función , Cistadenocarcinoma Seroso/patologíaRESUMEN
Epithelial ovarian cancer (EOC) research has become more complex as researchers try to fully understand the metastatic process. Especially as we delve into the concept of tumour dormancy, where cells transition between proliferative and dormant states to survive during disease progression. Thus, the in vitro models used to conduct this research need to reflect this vast biological complexity. The innovation behind the many three-dimensional (3D) spheroid models has been refined to easily generate reproducible spheroids so that we may understand the various molecular signaling changes of cells during metastasis and determine therapeutic efficacy of treatments. This ingenuity was then used to develop the 3D ex vivo patient-derived organoid model, as well as multiple co-culture model systems for EOC research. Although, researchers need to continue to push the boundaries of these current models for in vitro and even in vivo work in the future. In this review, we describe the 3D models already in use, where these models can be developed further and how we can use these models to gain the most knowledge on EOC pathogenesis and discover new targeted therapies.
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Neoplasias Glandulares y Epiteliales , Neoplasias Ováricas , Femenino , Humanos , Neoplasias Ováricas/patología , Esferoides Celulares , Línea Celular Tumoral , Carcinoma Epitelial de OvarioRESUMEN
In cancer, dormancy refers to a clinical state in which microscopic residual disease becomes non-proliferative and is largely refractory to chemotherapy. Dormancy was first described in breast cancer where disease can remain undetected for decades, ultimately leading to relapse and clinical presentation of the original malignancy. A long latency period can be explained by withdrawal from cell proliferation (cellular dormancy), or a balance between proliferation and cell death that retains low levels of residual disease (tumor mass dormancy). Research into cellular dormancy has revealed features that define this state. They include arrest of cell proliferation, altered cellular metabolism, and unique cell dependencies and interactions with the microenvironment. These characteristics can be shared by dormant cells derived from disparate primary disease sites, suggesting common features exist between them.High-grade serous ovarian cancer (HGSOC) disseminates to locations throughout the abdominal cavity by means of cellular aggregates called spheroids. These growth-arrested and therapy-resistant cells are a strong contributor to disease relapse. In this review, we discuss the similarities and differences between ovarian cancer cells in spheroids and dormant properties reported for other cancer disease sites. This reveals that elements of dormancy, such as cell cycle control mechanisms and changes to metabolism, may be similar across most forms of cellular dormancy. However, HGSOC-specific aspects of spheroid biology, including the extracellular matrix organization and microenvironment, are obligatorily disease site specific. Collectively, our critical review of current literature highlights places where HGSOC cell dormancy may offer a more tractable experimental approach to understand broad principles of cellular dormancy in cancer.
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High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy often diagnosed at an advanced stage. Although most HGSOC patients respond initially to debulking surgery combined with cytotoxic chemotherapy, many ultimately relapse with platinum-resistant disease. Thus, improving outcomes requires new ways of limiting metastasis and eradicating residual disease. We identified previously that Liver kinase B1 (LKB1) and its substrate NUAK1 are implicated in EOC spheroid cell viability and are required for efficient metastasis in orthotopic mouse models. Here, we sought to identify additional signalling pathways altered in EOC cells due to LKB1 or NUAK1 loss-of-function. Transcriptome analysis revealed that inflammatory signalling mediated by NF-κB transcription factors is hyperactive due to LKB1-NUAK1 loss in HGSOC cells and spheroids. Upregulated NF-κB signalling due to NUAK1 loss suppresses reactive oxygen species (ROS) production and sustains cell survival in spheroids. NF-κB signalling is also activated in HGSOC precursor fallopian tube secretory epithelial cell spheroids, and is further enhanced by NUAK1 loss. Finally, immunohistochemical analysis of OVCAR8 xenograft tumors lacking NUAK1 displayed increased RelB expression and nuclear staining. Our results support the idea that NUAK1 and NF-κB signalling pathways together regulate ROS and inflammatory signalling, supporting cell survival during each step of HGSOC pathogenesis. We propose that their combined inhibition may be efficacious as a novel therapeutic strategy for advanced HGSOC.
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Quinasas de la Proteína-Quinasa Activada por el AMP/genética , Quinasas de la Proteína-Quinasa Activada por el AMP/fisiología , Mutación con Pérdida de Función , FN-kappa B/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Esferoides Celulares , Animales , Supervivencia Celular , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Terapia Molecular Dirigida , Trasplante de Neoplasias , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia , Especies Reactivas de Oxígeno/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/fisiología , Transcriptoma/genética , Células Tumorales CultivadasRESUMEN
The low mutational burden of epithelial ovarian cancer (EOC) is an impediment to immunotherapies that rely on conventional MHC-restricted, neoantigen-reactive T lymphocytes. Mucosa-associated invariant T (MAIT) cells are MR1-restricted T cells with remarkable immunomodulatory properties. We sought to characterize intratumoral and ascitic MAIT cells in EOC. Single-cell RNA sequencing of six primary human tumor specimens demonstrated that MAIT cells were present at low frequencies within several tumors. When detectable, these cells highly expressed CD69 and VSIR, but otherwise exhibited a transcriptomic signature inconsistent with overt cellular activation and/or exhaustion. Unlike mainstream CD8+ T cells, CD8+ MAIT cells harbored high transcript levels of TNF, PRF1, GZMM and GNLY, suggesting their arming and cytotoxic potentials. In a congenic, MAIT cell-sufficient mouse model of EOC, MAIT and invariant natural killer T cells amassed in the peritoneal cavity where they showed robust IL-17A and IFN-γ production capacities, respectively. However, they gradually lost these functions with tumor progression. In a cohort of 23 EOC patients, MAIT cells were readily detectable in all ascitic fluids examined. In a sub-cohort in which we interrogated ascitic MAIT cells for functional impairments, several exhaustion markers, most notably VISTA, were present on the surface. However, ascitic MAIT cells were capable of producing IFN-γ, TNF-α and granzyme B, but neither IL-17A nor IL-10, in response to an MR1 ligand, bacterial lysates containing MR1 ligands, or a combination of IL-12 and IL-18. In conclusion, ascitic MAIT cells in EOC possess inducible effector functions that may be modified in future immunotherapeutic strategies.
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Células T Invariantes Asociadas a Mucosa , Neoplasias Ováricas , Animales , Ascitis , Linfocitos T CD8-positivos , Carcinoma Epitelial de Ovario , Señales (Psicología) , Citocinas , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Interleucina-17 , Ligandos , Ratones , Antígenos de Histocompatibilidad MenorRESUMEN
Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in the developed world. EOC metastasis is unique since malignant cells detach directly from the primary tumor site into the abdominal fluid and form multicellular aggregates, called spheroids, that possess enhanced survival mechanisms while in suspension. As such, altered cell adhesion properties are paramount to EOC metastasis with cell detachment from the primary tumor, dissemination as spheroids, and reattachment to peritoneal surfaces for secondary tumor formation. The ability for EOC cells to establish and maintain cell-cell contacts in spheroids is critical for cell survival in suspension. Integrins are a family of cell adhesion receptors that play a crucial role in cell-cell and cell-extracellular matrix interactions. These glycoprotein receptors regulate diverse functions in tumor cells and are implicated in multiple steps of cancer progression. Altered integrin expression is detected in numerous carcinomas, where they play a role in cell migration, invasion, and anchorage-independent survival. Like that observed for other carcinomas, epithelial-mesenchymal transition (EMT) occurs during metastasis and integrins can function in this process as well. Herein, we provide a review of the evidence for integrin-mediated cell adhesion mechanisms impacting steps of EOC metastasis. Taken together, targeting integrin function may represent a potential therapeutic strategy to inhibit progression of advanced EOC.
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Neoplasias Glandulares y Epiteliales , Neoplasias Ováricas , Carcinoma Epitelial de Ovario , Adhesión Celular , Línea Celular Tumoral , Humanos , Integrinas/uso terapéutico , Metástasis de la Neoplasia , Neoplasias Ováricas/patología , Esferoides Celulares/metabolismoRESUMEN
Ovarian clear cell carcinoma (OCCC) is a rare subtype of gynecological cancer for which well-characterized and authenticated model systems are scarce. We provide an extensive characterization of '105C', a cell line generated from an adenocarcinoma of the clear cell histotype using targeted next-generation sequencing, cytogenetic microarrays, along with analyses of AKT/mTOR signaling. We report that that the 105C cell line is a bona fide OCCC cell line, carrying PIK3CA, PTEN, and ARID1A gene mutations, consistent with OCCC, yet maintain a stable genome as reflected by low copy number variation. Unlike KOC-7c, TOV-21G, and RMG-V OCCC lines also mutated for the above genes, the 105C cells do not carry mutations in mismatch repair genes. Importantly, we show that 105C cells exhibit greater resistance to mTOR inhibition and carboplatin treatment compared to 9 other OCCC cell lines in 3D spheroid cultures. This resistance may be attributed to 105C cells remaining dormant in suspension culture which surprisingly, contrasts with several other OCCC lines which continue to proliferate in long-term suspension culture. 105C cells survive xenotransplantation but do not proliferate and metastasize. Collectively, we show that the 105C OCCC cell line exhibits unique properties useful for the pre-clinical investigation of OCCC pathobiology.
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Antineoplásicos/uso terapéutico , Inestabilidad Genómica , Mutación/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Esferoides Celulares/patología , Animales , Antineoplásicos/farmacología , Adhesión Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromosomas Humanos/genética , Variaciones en el Número de Copia de ADN/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano , Inestabilidad Genómica/efectos de los fármacos , Humanos , Ratones Endogámicos NOD , Ratones SCID , Morfolinas/farmacología , Morfolinas/uso terapéutico , Tasa de Mutación , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metastasis in high-grade serous ovarian cancer (HGSOC) occurs through an unconventional route that involves exfoliation of cancer cells from primary tumors and peritoneal dissemination via multicellular clusters or spheroids. Previously, we demonstrated autophagy induction in HGSOC spheroids grown in vitro and in spheroids collected from ovarian cancer patient ascites; thus, we speculate that autophagy may contribute to spheroid cell survival and overall disease progression. Hence, in this study we sought to evaluate whether ULK1 (unc-51-like kinase-1), a serine-threonine kinase critical for stress-induced autophagy, is important for autophagy regulation in HGSOC spheroids. We demonstrate that HGSOC spheroids have increased ULK1 protein expression that parallels autophagy activation. ULK1 knockdown increased p62 accumulation and decreased LC3-II/I ratio in HGSOC spheroids. In addition, knocking down ATG13, a protein that regulates ULK1 activity via complex formation, phenocopied our ULK1 knockdown results. HGSOC spheroids were blocked in autophagic flux due to ULK1 and ATG13 knockdown as determined by an mCherry-eGFP-LC3B fluorescence reporter. These observations were recapitulated when HGSOC spheroids were treated with an ULK1 kinase inhibitor, MRT68921. Autophagy regulation in normal human fallopian tube epithelial FT190 cells, however, may bypass ULK1, since MRT68921 reduced viability in HGSOC spheroids but not in FT190 cells. Interestingly, ULK1 mRNA expression is negatively correlated with patient survival among stage III and stage IV serous ovarian cancer patients. As we observed using established HGSOC cell lines, cultured spheroids using our new, patient-derived HGSOC cells were also sensitive to ULK1 inhibition and demonstrated reduced cell viability to MRT68921 treatment. These results demonstrate the importance of ULK1 for autophagy induction in HGSOC spheroids and therefore justifies further evaluation of MRT68921, and other novel ULK1 inhibitors, as potential therapeutics against metastatic HGSOC.
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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.
RESUMEN
BACKGROUND: A hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids. RESULTS: We observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence microscopy using the mCherry-eGFP-LC3B reporter. A complementary approach using pharmacologic agents Compound C and CAMKKß inhibitor STO-609 to inhibit AMPK activity both yielded a potent blockade of autophagic flux as well. However, direct activation of AMPK in EOC cells using oligomycin and metformin was insufficient to induce autophagy. STO-609 treatment of EOC spheroids resulted in reduced viability in 7 out of 9 cell lines, but with no observed effect in non-malignant FT190 cell spheroids. CONCLUSIONS: Our results support the premise that CAMKKß-mediated AMPK activity is required, at least in part, to regulate autophagy induction in EOC spheroids and support cell viability in this in vitro model of EOC metastasis.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia/genética , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Neoplasias Ováricas/genética , Línea Celular Tumoral , Femenino , Humanos , Metástasis de la Neoplasia , Transducción de Señal , Esferoides CelularesRESUMEN
AIM: Obesity has been associated with changes in autophagy and its increasing prevalence among pregnant women is implicated in higher rates of placental-mediated complications of pregnancy such as pre-eclampsia and intrauterine growth restriction. Autophagy is involved in normal placentation, thus changes in autophagy may lead to impaired placental function and development. The aim of this study was to investigate the connection between obesity and autophagy in the placenta in otherwise uncomplicated pregnancies. METHODS: Immunohistochemistry and western blot analysis were done on placental and omental samples from obese (body mass index [BMI] ≥30 kg/m2 ) and normal weight (BMI <25 kg/m2 ) pregnant women with singleton pregnancies undergoing planned Caesarean delivery without labor at term. Samples were analyzed for autophagic markers LC3B and p62 in the peripheral, middle and central regions of the placenta and in omental adipocytes, milky spots and vasculature. RESULTS: As pre-pregnancy BMI increased, there was an increase in both placental and fetal weight as well as decreased levels of LC3B in the central region of the placenta (P = 0.0046). Within the obese patient group, LC3B levels were significantly decreased in the placentas of male fetuses compared to females (P < 0.0001). Adipocytes, compared to milky spots and vasculature, had lower levels of p62 (P = 0.0127) and LC3B (P = 0.003) in obese omenta and lower levels of LC3B in control omenta (P = 0.0071). CONCLUSION: Obesity leads to reduced placental autophagy in uncomplicated pregnancies; thus, changes in autophagy may be involved in the underlying mechanisms of obesity-related placental diseases of pregnancy.
Asunto(s)
Obesidad Materna , Autofagia , Índice de Masa Corporal , Femenino , Humanos , Masculino , Placenta , Placentación , EmbarazoRESUMEN
Tumor cells must alter their antioxidant capacity for maximal metastatic potential. Yet the antioxidant adaptations required for ovarian cancer transcoelomic metastasis, which is the passive dissemination of cells in the peritoneal cavity, remain largely unexplored. Somewhat contradicting the need for oxidant scavenging are previous observations that expression of SIRT3, a nutrient stress sensor and regulator of mitochondrial antioxidant defenses, is often suppressed in many primary tumors. We have discovered that this mitochondrial deacetylase is specifically upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase (SOD2). This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent increases in SOD2 mRNA during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 upregulation and SIRT3-mediated oxidant scavenging are required for anoikis resistance in vitro following matrix detachment, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.
Asunto(s)
Neoplasias Ováricas/patología , Sirtuina 3/metabolismo , Supervivencia Celular , Activación Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucólisis , Humanos , Mitocondrias/metabolismo , Metástasis de la Neoplasia , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 3/deficiencia , Sirtuina 3/genética , Superóxido Dismutasa/metabolismoRESUMEN
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 STK11 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 STK11-knockout cell lines using normal human FT190 cells and three EOC cell lines, OVCAR8, HeyA8, and iOvCa147. STK11KO 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-STK11KO cells, and respective controls. LKB1 loss exhibited a dramatic reduction on tumor burden and metastatic potential; in particular, OVCAR8-STK11KO 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, DUSP4 knockdown in HeyA8-STK11KO cells partially restored spheroid formation and viability. IMPLICATIONS: LKB1 possesses key tumor-promoting activity independent of downstream AMPK signaling during EOC metastasis.