RESUMEN
Following a period of slow progress, the completion of genome sequencing and the paradigm shift relative to the cell of origin for high grade serous ovarian cancer (HGSOC) led to a new perspective on the biology and therapeutic solutions for this deadly cancer. Experimental models were revisited to address old questions, and improved tools were generated. Additional pathways emerging as drivers of ovarian tumorigenesis and key dependencies for therapeutic targeting, in particular, VEGF-driven angiogenesis and homologous recombination deficiency, were discovered. Molecular profiling of histological subtypes of ovarian cancer defined distinct genetic events for each entity, enabling the first attempts toward personalized treatment. Armed with this knowledge, HGSOC treatment was revised to include new agents. Among them, PARP inhibitors (PARPis) were shown to induce unprecedented improvement in clinical benefit for selected subsets of patients. Research on mechanisms of resistance to PARPis is beginning to discover vulnerabilities and point to new treatment possibilities. This Review highlights these advances, the remaining challenges, and unsolved problems in the field.
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Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , BiologíaRESUMEN
Platinum (Pt)-based chemotherapy is the main treatment for ovarian cancer (OC); however, most patients develop Pt resistance (Pt-R). This work shows that Pt-R OC cells increase intracellular cholesterol through uptake via the HDL receptor, scavenger receptor type B-1 (SR-B1). SR-B1 blockade using synthetic cholesterol-poor HDL-like nanoparticles (HDL NPs) diminished cholesterol uptake leading to cell death and inhibition of tumor growth. Reduced cholesterol accumulation in cancer cells induces lipid oxidative stress through the reduction of glutathione peroxidase 4 (GPx4) leading to ferroptosis. In turn, GPx4 depletion induces decreased cholesterol uptake through SR-B1 and re-sensitizes OC cells to Pt. Mechanistically, GPx4 knockdown causes lower expression of the histone acetyltransferase EP300, leading to reduced deposition of histone H3 lysine 27 acetylation (H3K27Ac) on the sterol regulatory element binding transcription factor 2 (SREBF2) promoter and suppressing expression of this key transcription factor involved in the regulation of cholesterol metabolism. SREBF2 downregulation leads to decreased SR-B1 expression and diminished cholesterol uptake. Thus, chemoresistance and cancer cell survival under high ROS burden obligates high GPx4 and SR-B1 expression through SREBF2. Targeting SR-B1 to modulate cholesterol uptake inhibits this axis and causes ferroptosis in vitro and in vivo in Pt-R OC.
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Nanopartículas , Neoplasias Ováricas , Humanos , Femenino , Receptores Depuradores de Clase B/metabolismo , Colesterol/metabolismo , Factores de Transcripción/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Oxidación-ReducciónRESUMEN
PURPOSE: DNA methylation causes silencing of tumor-suppressor and differentiation-associated genes, being linked to chemoresistance. Previous studies demonstrated that hypomethylating agents (HMA) resensitize ovarian cancer to chemotherapy. NTX-301 is a highly potent and orally bioavailable HMA, in early clinical development. EXPERIMENTAL DESIGN: The antitumor effects of NTX-301 were studied in ovarian cancer models by using cell viability, stemness and ferroptosis assays, RNA sequencing, lipidomic analyses, and stimulated Raman spectroscopy. RESULTS: Ovarian cancer cells (SKOV3, IC50 = 5.08 nmol/L; OVCAR5 IC50 = 3.66 nmol/L) were highly sensitive to NTX-301 compared with fallopian tube epithelial cells. NTX-301 downregulated expression of DNA methyltransferases 1-3 and induced transcriptomic reprogramming with 15,000 differentially expressed genes (DEG, P < 0.05). Among them, Gene Ontology enrichment analysis identified regulation of fatty acid biosynthesis and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase, known features of cancer stem cells. Low-dose NTX-301 reduced the ALDH(+) cell population and expression of stemness-associated transcription factors. Stearoyl-coenzyme A desaturase 1 (SCD), which regulates production of unsaturated fatty acids (UFA), was among the top DEG downregulated by NTX-301. NTX-301 treatment decreased levels of UFA and increased oxidized lipids, and this was blunted by deferoxamine, indicating cell death via ferroptosis. NTX-301-induced ferroptosis was rescued by oleic acid. In vivo, monotherapy with NTX-301 significantly inhibited ovarian cancer and patient-derived xenograft growth (P < 0.05). Decreased SCD levels and increased oxidized lipids were detected in NTX-301-treated xenografts. CONCLUSIONS: NTX-301 is active in ovarian cancer models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.
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Neoplasias Ováricas , Humanos , Femenino , Línea Celular Tumoral , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inhibidores Enzimáticos/uso terapéutico , Aldehído Deshidrogenasa/genética , ADN , Lípidos/uso terapéuticoRESUMEN
Reprogramming of cellular metabolism is a hallmark of cancer. Cancer cells undergo metabolic adaptations to maintain tumorigenicity and survive under the attack of immune cells and chemotherapy in the tumor microenvironment. Metabolic alterations in ovarian cancer in part overlap with findings from other solid tumors and in part reflect unique traits. Altered metabolic pathways not only facilitate ovarian cancer cells' survival and proliferation but also endow them to metastasize, acquire resistance to chemotherapy, maintain cancer stem cell phenotype and escape the effects of anti-tumor immune defense. In this review, we comprehensively review the metabolic signatures of ovarian cancer and their impact on cancer initiation, progression, and resistance to treatment. We highlight novel therapeutic strategies targeting metabolic pathways under development.
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Glucólisis , Neoplasias Ováricas , Humanos , Femenino , Glucólisis/genética , Neoplasias Ováricas/genética , Metabolismo Energético , Redes y Vías Metabólicas , Microambiente TumoralRESUMEN
Development of resistance to platinum (Pt) in ovarian cancer remains a major clinical challenge. Here we focused on identifying epitranscriptomic modifications linked to Pt resistance. Fat mass and obesity-associated protein (FTO) is a N6-methyladenosine (m6A) RNA demethylase that we recently described as a tumor suppressor in ovarian cancer. We hypothesized that FTO-induced removal of m6A marks regulates the cellular response of ovarian cancer cells to Pt and is linked to the development of resistance. To study the involvement of FTO in the cellular response to Pt, we used ovarian cancer cells in which FTO was knocked down via short hairpin RNA or overexpressed and Pt-resistant (Pt-R) models derived through repeated cycles of exposure to Pt. We found that FTO was significantly downregulated in Pt-R versus sensitive ovarian cancer cells. Forced expression of FTO, but not of mutant FTO, increased sensitivity to Pt in vitro and in vivo (P < 0.05). Increased numbers of γ-H2AX foci, measuring DNA double-strand breaks, and increased apoptosis were observed after exposure to Pt in FTO-overexpressing versus control cells. Through integrated RNA sequencing and MeRIP sequencing, we identified and validated the enzyme nicotinamide N-methyltransferase (NNMT), as a new FTO target linked to Pt response. NNMT was upregulated and demethylated in FTO-overexpressing cells. Treatment with an NNMT inhibitor or NNMT knockdown restored sensitivity to Pt in FTO-overexpressing cells. Our results support a new function for FTO-dependent m6A RNA modifications in regulating the response to Pt through NNMT, a newly identified RNA methylated gene target.
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Neoplasias Ováricas , Platino (Metal) , ARN , Femenino , Humanos , Adenosina/química , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Nicotinamida N-Metiltransferasa , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Platino (Metal)/farmacología , Platino (Metal)/uso terapéutico , ARN/química , ARN/metabolismoRESUMEN
Cancer stem cells (CSC) represent a population of cancer cells responsible for tumor initiation, chemoresistance, and metastasis. Here, we identified the H3K79 methyltransferase disruptor of telomeric silencing-1-like (DOT1L) as a critical regulator of self-renewal and tumor initiation in ovarian CSCs. DOT1 L was upregulated in ovarian CSCs versus non-CSCs. shRNA-mediated DOT1 L knockdown decreased the aldehyde dehydrogenase (ALDH)+ cell population, impaired the tumor initiation capacity (TIC) of ovarian CSCs, and blocked the expression of stemness-associated genes. Inhibition of DOT1L's methyltransferase activity by the small-molecule inhibitor (DOT1Li) EPZ-5676 also effectively targeted ovarian CSCs. Integrated RNA-sequencing analyses of ovarian cancer cells in which DOT1 L was knocked down versus control cells and of ovarian CSCs versus non-CSCs, identified Wnt signaling as a shared pathway deregulated in both CSCs and in DOT1L-deficient ovarian cancer cells. ß-catenin, a key transcription factor regulated by Wnt, was downregulated in ovarian cancer cells in which DOT1 L was knocked down and upregulated in DOT1 L overexpressing ovarian cancer cells. Chromatin immunoprecipitation (ChIP) revealed enrichment of the H3K79Me3 mark at the ß-catenin promoter, suggesting that its transcription is regulated by DOT1L. Our results suggest that DOT1 L is critical for the self-renewal and TIC of ovarian CSCs by regulating ß-catenin signaling. Targeting DOT1 L in ovarian cancer could be a new strategy to eliminate CSCs. IMPLICATIONS: This study found that the histone methyltransferase DOT1 L regulates the self-renewal and tumor initiation capacity of ovarian CSCs and suggests DOT1 L as a new cancer target.
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Neoplasias Ováricas , beta Catenina , Humanos , Femenino , beta Catenina/genética , beta Catenina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Células Madre Neoplásicas/metabolismo , Transformación Celular Neoplásica/metabolismo , Vía de Señalización Wnt , Neoplasias Ováricas/patologíaRESUMEN
Fatty acids are an important source of energy and a key component of phospholipids in membranes and organelles. Saturated fatty acids (SFAs) are converted into unsaturated fatty acids (UFAs) by stearoyl Co-A desaturase (SCD), an enzyme active in cancer. Here, we studied how the dynamics between SFAs and UFAs regulated by SCD impacts ovarian cancer cell survival and tumor progression. SCD depletion or inhibition caused lower levels of UFAs vs. SFAs and altered fatty acyl chain plasticity, as demonstrated by lipidomics and stimulated Raman scattering (SRS) microscopy. Further, increased levels of SFAs resulting from SCD knockdown triggered endoplasmic reticulum (ER) stress response with brisk activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes. Disorganized ER membrane was visualized by electron microscopy and SRS imaging in ovarian cancer cells in which SCD was knocked down. The induction of long-term mild ER stress or short-time severe ER stress by the increased levels of SFAs and loss of UFAs led to cell death. However, ER stress and apoptosis could be readily rescued by supplementation with UFAs and reequilibration of SFA/UFA levels. The effects of SCD knockdown or inhibition observed in vitro translated into suppression of intraperitoneal tumor growth in ovarian cancer xenograft models. Furthermore, a combined intervention using an SCD inhibitor and an SFA-enriched diet initiated ER stress in tumors growing in vivo and potently blocked their dissemination. In all, our data support SCD as a key regulator of the cancer cell fate under metabolic stress and point to treatment strategies targeting the lipid balance.
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Supervivencia Celular , Endorribonucleasas , Ácidos Grasos Insaturados , Neoplasias Ováricas , Progresión de la Enfermedad , Ácido Graso Desaturasas , Ácidos Grasos/farmacología , Ácidos Grasos Insaturados/farmacología , Femenino , Humanos , Fosfolípidos , Proteínas Serina-Treonina Quinasas , Estearoil-CoA Desaturasa/metabolismoRESUMEN
Increased glycolysis is considered as a hallmark of cancer. Yet, cancer cell metabolic reprograming during therapeutic resistance development is under-studied. Here, through high-throughput stimulated Raman scattering imaging and single cell analysis, we find that cisplatin-resistant cells exhibit increased fatty acids (FA) uptake, accompanied by decreased glucose uptake and lipogenesis, indicating reprogramming from glucose to FA dependent anabolic and energy metabolism. A metabolic index incorporating glucose derived anabolism and FA uptake correlates linearly to the level of cisplatin resistance in ovarian cancer (OC) cell lines and primary cells. The increased FA uptake facilitates cancer cell survival under cisplatin-induced oxidative stress by enhancing beta-oxidation. Consequently, blocking beta-oxidation by a small molecule inhibitor combined with cisplatin or carboplatin synergistically suppresses OC proliferation in vitro and growth of patient-derived xenografts in vivo. Collectively, these findings support a rapid detection method of cisplatin-resistance at single cell level and a strategy for treating cisplatin-resistant tumors.
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Antineoplásicos , Neoplasias Ováricas , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Cisplatino/farmacología , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos , Ácidos Grasos/farmacología , Femenino , Glucosa/metabolismo , Glucólisis , Humanos , Neoplasias Ováricas/patología , Platino (Metal)/farmacologíaRESUMEN
Understanding the regulatory programs enabling cancer stem cells (CSCs) to self-renew and drive tumorigenicity could identify new treatments. Through comparative chromatin-state and gene expression analyses in ovarian CSCs versus non-CSCs, we identified FOXK2 as a highly expressed stemness-specific transcription factor in ovarian cancer. Its genetic depletion diminished stemness features and reduced tumor initiation capacity. Our mechanistic studies highlight that FOXK2 directly regulated IRE1α (encoded by ERN1) expression, a key sensor for the unfolded protein response (UPR). Chromatin immunoprecipitation and sequencing revealed that FOXK2 bound to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a catalytically inactive CRISPR/Cas9 (dCas9) diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 splicing and activation of stemness pathways, while genetic or pharmacological blockade of this sensor of the UPR inhibited ovarian CSCs. Collectively, these data establish what we believe is a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in CSCs.
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Factores de Transcripción Forkhead , Neoplasias Ováricas , Respuesta de Proteína Desplegada , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Femenino , Factores de Transcripción Forkhead/genética , Humanos , Neoplasias Ováricas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
BACKGROUND: Tissue transglutaminase (TG2), an enzyme overexpressed in cancer cells, promotes metastasis and resistance to chemotherapy. Its distinct effects in cancer versus the host compartments have not been elucidated. METHODS: Here, by using a TG2-/- syngeneic ovarian cancer mouse model, we assessed the effects of TG2 deficiency in the host tissues on antitumor immunity and tumor progression. Multicolor flow cytometry was used to phenotype immune cell populations in the peritoneal environment. Cancer cells recovered from malignant ascites were characterized by RNA sequencing, proliferation, and apoptosis assays. RESULTS: We observed that host TG2 loss delayed tumor growth and ascites accumulation and caused increased infiltration of CD8+ T cells and decreased numbers of myeloid cells in the peritoneal fluid. Tumor antigen-specific CD8+ T cell cytotoxic responses were enhanced in ascites from TG2-/- versus TG2+/+ mice and CD8+ T cell depletion caused accelerated ascites accumulation in TG2-/- mice. CD8+ T cells from tumor-bearing TG2-/- mice displayed an effector T cell phenotype, differentiated toward effector memory (Tem). Mechanistically, absence of TG2 augmented signals promoting T cell activation, such as increased cytokine-induced STAT1 and attenuated STAT3 phosphorylation in T cells. Additionally, immune-suppressive myeloid cell populations were reduced in the peritoneal milieu of TG2-/- tumor-bearing mice. In response to the more robust immune response caused by loss of TG2, cancer cells growing intraperitoneally exhibited an interferon-γ(IFN-γ) responsive gene signature and underwent apoptosis. In human specimens, stromal, not tumor, TG2 expression correlated indirectly with numbers of tumor-infiltrating lymphocytes. CONCLUSIONS: Collectively, our data demonstrate decreased tumor burden, increased activation and effector function of T cells, and loss of immunosuppressive signals in the tumor microenvironment of TG2-/- mice. We propose that TG2 acts as an attenuator of antitumor T cell immunity and is a new immunomodulatory target.
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Neoplasias Ováricas/genética , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Femenino , Humanos , Ratones , Neoplasias Ováricas/patología , FosforilaciónRESUMEN
Ovarian cancer remains one of the deadliest gynecologic malignancies affecting women, and development of resistance to platinum remains a major barrier to achieving a cure. Multiple mechanisms have been identified to confer platinum resistance. Numerous miRNAs have been linked to platinum sensitivity and resistance in ovarian cancer. miRNA activity occurs mainly when the guide strand of the miRNA, with its seed sequence at position 2-7/8, is loaded into the RNA-induced silencing complex (RISC) and targets complementary short seed matches in the 3' untranslated region of mRNAs. Toxic 6mer seeds, which target genes critical for cancer cell survival, have been found in tumor-suppressive miRNAs. Many siRNAs and short hairpin RNAs (shRNA) can also kill cancer cells via toxic seeds, the most toxic of which carry G-rich 6mer seed sequences. We showed here that treatment of ovarian cancer cells with platinum led to increased RISC-bound miRNAs carrying toxic 6mer seeds and decreased miRNAs with nontoxic seeds. Platinum-tolerant cells did not exhibit this toxicity shift but retained sensitivity to cell death mediated by siRNAs carrying toxic 6mer seeds. Analysis of RISC-bound miRNAs in tumors from patients with ovarian cancer revealed that the ratio between miRNAs with toxic versus nontoxic seeds was predictive of treatment outcome. Application of the 6mer seed toxicity concept to cancer relevant miRNAs provides a new framework for understanding and predicting cancer therapy responses. SIGNIFICANCE: These findings demonstrate that the balance of miRNAs that carry toxic and nontoxic 6mer seeds contributes to platinum resistance in ovarian cancer.
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MicroARNs/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Platino (Metal)/uso terapéutico , Femenino , Humanos , Platino (Metal)/farmacologíaRESUMEN
Defining traits of platinum-tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum-tolerant cells and tumors were identified using in vitro and in vivo ovarian cancer models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were enriched in ALDH+ cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared with parental cells. Additionally, platinum-tolerant cells and tumors exhibited expression of the Wnt receptor Frizzled-7 (FZD7). Knockdown of FZD7 improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7+ from FZD7- cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation-enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protected cells from chemotherapy-induced oxidative stress. FZD7+ platinum-tolerant ovarian cancer cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63, and glutathione metabolism gene sets were strongly correlated in the ovarian cancer Tumor Cancer Genome Atlas (TCGA) database and in residual human ovarian cancer specimens after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial cancer stem cell features, characterized by FZD7 expression and dependent on the FZD7-ß-catenin-Tp63-GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum-tolerant cancer cells and provide new insight into a potential "persister cancer cell" phenotype. SIGNIFICANCE: Frizzled-7 marks platinum-tolerant cancer cells harboring stemness features and altered glutathione metabolism that depend on GPX4 for survival and are highly susceptible to ferroptosis.
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Biomarcadores de Tumor/metabolismo , Cisplatino/farmacología , Resistencia a Antineoplásicos , Ferroptosis , Receptores Frizzled/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Femenino , Receptores Frizzled/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
N 6-Methyladenosine (m6A) is the most abundant modification of mammalian mRNAs. RNA methylation fine tunes RNA stability and translation, altering cell fate. The fat mass- and obesity-associated protein (FTO) is an m6A demethylase with oncogenic properties in leukemia. Here, we show that FTO expression is suppressed in ovarian tumors and cancer stem cells (CSC). FTO inhibited the self-renewal of ovarian CSC and suppressed tumorigenesis in vivo, both of which required FTO demethylase activity. Integrative RNA sequencing and m6A mapping analysis revealed significant transcriptomic changes associated with FTO overexpression and m6A loss involving stem cell signaling, RNA transcription, and mRNA splicing pathways. By reducing m6A levels at the 3'UTR and the mRNA stability of two phosphodiesterase genes (PDE1C and PDE4B), FTO augmented second messenger 3', 5'-cyclic adenosine monophosphate (cAMP) signaling and suppressed stemness features of ovarian cancer cells. Our results reveal a previously unappreciated tumor suppressor function of FTO in ovarian CSC mediated through inhibition of cAMP signaling. SIGNIFICANCE: A new tumor suppressor function of the RNA demethylase FTO implicates m6A RNA modifications in the regulation of cyclic AMP signaling involved in stemness and tumor initiation.
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Adenosina/análogos & derivados , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Ováricas/metabolismo , Sistemas de Mensajero Secundario , Proteínas Supresoras de Tumor/metabolismo , Regiones no Traducidas 3'/genética , Adenosina/genética , Adenosina/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Empalme Alternativo , Animales , Ascitis/metabolismo , Carcinogénesis/metabolismo , Línea Celular Tumoral , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Regulación hacia Abajo , Trompas Uterinas/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Ováricas/patología , Ovario/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Análisis de Secuencia de ARN , Esferoides Celulares , Análisis de Matrices Tisulares , Transcriptoma , Proteínas Supresoras de Tumor/genéticaRESUMEN
A small of population of slow cycling and chemo-resistant cells referred to as cancer stem cells (CSC) have been implicated in cancer recurrence. There is emerging interest in developing targeted therapeutics to eradicate CSCs. Aldehyde-dehydrogenase (ALDH) activity was shown to be a functional marker of CSCs in ovarian cancer (OC). ALDH activity is increased in cells grown as spheres versus monolayer cultures under differentiating conditions and in OC cells after treatment with platinum. Here, we describe the activity of CM37, a newly identified small molecule with inhibitory activity against ALDH1A1, in OC models enriched in CSCs. Treatment with CM37 reduced OC cells' proliferation as spheroids under low attachment growth conditions and the expression of stemness-associated markers (OCT4 and SOX2) in ALDH+ cells fluorescence-activated cell sorting (FACS)-sorted from cell lines and malignant OC ascites. Likewise, siRNA-mediated ALDH1A1 knockdown reduced OC cells' proliferation as spheres, expression of stemness markers, and delayed tumor initiation capacity in vivo. Treatment with CM37 promoted DNA damage in OC cells, as evidenced by induction of γH2AX. This corresponded to increased expression of genes involved in DNA damage response, such as NEIL3, as measured in ALDH+ cells treated with CM37 or in cells where ALDH1A1 was knocked down. By inhibiting ALDH1A1, CM37 augmented intracellular ROS accumulation, which in turn led to increased DNA damage and reduced OC cell viability. Cumulatively, our findings demonstrate that a novel ALDH1A1 small molecule inhibitor is active in OC models enriched in CSCs. Further optimization of this new class of small molecules could provide a novel strategy for targeting treatment-resistant OC.
RESUMEN
In high-grade serous ovarian cancer (OC), chemotherapy eliminates the majority of tumor cells, leaving behind residual tumors enriched in OC stem cells (OCSC). OCSC, defined as aldehyde dehydrogenase-positive (ALDH+), persist and contribute to tumor relapse. Inflammatory cytokine IL-6 is elevated in residual tumors after platinum treatment, and we hypothesized that IL-6 plays a critical role in platinum-induced OCSC enrichment. We demonstrate that IL-6 regulates stemness features of OCSC driven by ALDH1A1 expression and activity. We show that platinum induces IL-6 secretion by cancer-associated fibroblasts in the tumor microenvironment, promoting OCSC enrichment in residual tumors after chemotherapy. By activating STAT3 and upregulating ALDH1A1 expression, IL-6 treatment converted non-OCSC to OCSC. Having previously shown altered DNA methylation in OCSC, we show here that IL-6 induces DNA methyltransferase 1 (DNMT1) expression and the hypomethylating agent (HMA) guadecitabine induced differentiation of OCSC and reduced - but did not completely eradicate - OCSC. IL-6 neutralizing antibody (IL-6-Nab) combined with HMA fully eradicated OCSC, and the combination blocked IL-6/IL6-R/pSTAT3-mediated ALDH1A1 expression and eliminated OCSC in residual tumors that persisted in vivo after chemotherapy. We conclude that IL-6 signaling blockade combined with an HMA can eliminate OCSC after platinum treatment, supporting this strategy to prevent tumor recurrence after standard chemotherapy.
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Interleucina-6/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Ováricas/metabolismo , Platino (Metal)/farmacología , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Azacitidina/análogos & derivados , Azacitidina/metabolismo , Línea Celular Tumoral , Citocinas/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Progresión de la Enfermedad , Quimioterapia , Epigenómica , Femenino , Humanos , Inmunoterapia , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales , Neoplasias Ováricas/tratamiento farmacológico , Retinal-Deshidrogenasa , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral , Regulación hacia ArribaRESUMEN
Long non-coding RNAs (lncRNAs) play key roles in human diseases, including cancer. Functional studies of the lncRNA HOTAIR (HOX transcript antisense RNA) provide compelling evidence for therapeutic targeting of HOTAIR in cancer, but targeting lncRNAs in vivo has proven to be difficult. In the current study, we describe a peptide nucleic acids (PNA)-based approach to block the ability of HOTAIR to interact with EZH2 and subsequently inhibit HOTAIR-EZH2 activity and resensitize resistant ovarian tumors to platinum. Treatment of HOTAIR-overexpressing ovarian and breast cancer cell lines with PNAs decreased invasion and increased chemotherapy sensitivity. Furthermore, the mechanism of action correlated with reduced nuclear factor-kappaB (NF-κB) activation and decreased expression of NF-κB target genes matrix metalloprotease 9 and interleukin 6. To deliver the anti-lncRNA to the acidic (pH approximately 6) tumor microenvironment, PNAs were conjugated to pH-low insertion peptide (pHLIP). Treatment of mice harboring platinum-resistant ovarian tumor xenografts with pHLIP-PNA constructs suppressed HOTAIR activity, reduced tumor formation and improved survival. This first report on pHLIP-PNA lncRNA targeting solid tumors in vivo suggests a novel cancer therapeutic approach.
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Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Ácidos Nucleicos de Péptidos/uso terapéutico , ARN Largo no Codificante/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Cisplatino/administración & dosificación , Cisplatino/farmacología , Cisplatino/uso terapéutico , Sinergismo Farmacológico , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , FN-kappa B/metabolismo , Ácidos Nucleicos de Péptidos/administración & dosificación , Ácidos Nucleicos de Péptidos/farmacologíaRESUMEN
PURPOSE: To investigate SGI-110 as a "chemosensitizer" in ovarian cancer and to assess its effects on tumor suppressor genes (TSG) and chemoresponsiveness-associated genes silenced by DNA methylation in ovarian cancer. EXPERIMENTAL DESIGN: Several ovarian cancer cell lines were used for in vitro and in vivo platinum resensitization studies. Changes in DNA methylation and expression levels of TSG and other cancer-related genes in response to SGI-110 were measured by pyrosequencing and RT-PCR. RESULTS: We demonstrate in vitro that SGI-110 resensitized a range of platinum-resistant ovarian cancer cells to cisplatin (CDDP) and induced significant demethylation and reexpression of TSG, differentiation-associated genes, and putative drivers of ovarian cancer cisplatin resistance. In vivo, SGI-110 alone or in combination with CDDP was well tolerated and induced antitumor effects in ovarian cancer xenografts. Pyrosequencing analyses confirmed that SGI-110 caused both global (LINE1) and gene-specific hypomethylation in vivo, including TSGs (RASSF1A), proposed drivers of ovarian cancer cisplatin resistance (MLH1 and ZIC1), differentiation-associated genes (HOXA10 and HOXA11), and transcription factors (STAT5B). Furthermore, DNA damage induced by CDDP in ovarian cancer cells was increased by SGI-110, as measured by inductively coupled plasma-mass spectrometry analysis of DNA adduct formation and repair of cisplatin-induced DNA damage. CONCLUSIONS: These results strongly support further investigation of hypomethylating strategies in platinum-resistant ovarian cancer. Specifically, SGI-110 in combination with conventional and/or targeted therapeutics warrants further development in this setting.
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Azacitidina/análogos & derivados , Metilación de ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Azacitidina/administración & dosificación , Azacitidina/farmacología , Línea Celular Tumoral , Cisplatino/administración & dosificación , Cisplatino/farmacología , Aductos de ADN , Modelos Animales de Enfermedad , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Histonas/metabolismo , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Combination therapy with decitabine, a DNMTi and carboplatin resensitized chemoresistant ovarian cancer (OC) to platinum inducing promising clinical activity. We investigated gene-expression profiles in tumor biopsies to identify decitabine-reactivated pathways associated with clinical response. Gene-expression profiling was performed using RNA from paired tumor biopsies before and 8 days after decitabine from 17 patients with platinum resistant OC. Bioinformatic analysis included unsupervised hierarchical-clustering, pathway and GSEA distinguishing profiles of "responders" (progression-free survival, PFS>6 months) and "non-responders" (PFS< 6 months). Functional validation of selected results was performed in OC cells/tumors. Pre-treatment tumors from responders expressed genes associated with enhanced glycosphingolipid biosynthesis, translational misregulation, decreased ABC transporter expression, TGF-ß signaling, and numerous metabolic pathways. Analysis of post-treatment biopsies from responders revealed overexpression of genes associated with reduced Hedgehog pathway signaling, reduced DNA repair/replication, and cancer-associated metabolism. GO and GSEA analyses revealed upregulation of genes associated with glycosaminoglycan binding, cell-matrix adhesion, and cell-substrate adhesion. Computational findings were substantiated by experimental validation of expression of key genes involved in two critical pathways affected by decitabine (TGF-ß and Hh). Gene-expression profiling identified specific pathways altered by decitabine and associated with platinum-resensitization and clinical benefit in OC. Our data could influence patient stratification for future studies using epigenetic therapies.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Azacitidina/análogos & derivados , Carboplatino/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/administración & dosificación , Azacitidina/farmacología , Carboplatino/administración & dosificación , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Decitabina , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Neoplasias Ováricas/genéticaRESUMEN
Emerging results indicate that cancer stem-like cells contribute to chemoresistance and poor clinical outcomes in many cancers, including ovarian cancer. As epigenetic regulators play a major role in the control of normal stem cell differentiation, epigenetics may offer a useful arena to develop strategies to target cancer stem-like cells. Epigenetic aberrations, especially DNA methylation, silence tumor-suppressor and differentiation-associated genes that regulate the survival of ovarian cancer stem-like cells (OCSC). In this study, we tested the hypothesis that DNA-hypomethylating agents may be able to reset OCSC toward a differentiated phenotype by evaluating the effects of the new DNA methytransferase inhibitor SGI-110 on OCSC phenotype, as defined by expression of the cancer stem-like marker aldehyde dehydrogenase (ALDH). We demonstrated that ALDH(+) ovarian cancer cells possess multiple stem cell characteristics, were highly chemoresistant, and were enriched in xenografts residual after platinum therapy. Low-dose SGI-110 reduced the stem-like properties of ALDH(+) cells, including their tumor-initiating capacity, resensitized these OCSCs to platinum, and induced reexpression of differentiation-associated genes. Maintenance treatment with SGI-110 after carboplatin inhibited OCSC growth, causing global tumor hypomethylation and decreased tumor progression. Our work offers preclinical evidence that epigenome-targeting strategies have the potential to delay tumor progression by reprogramming residual cancer stem-like cells. Furthermore, the results suggest that SGI-110 might be administered in combination with platinum to prevent the development of recurrent and chemoresistant ovarian cancer.