RESUMEN
Tight coordination of growth regulatory signaling is required for intestinal epithelial homeostasis. Protein kinase C α (PKCα) and transforming growth factor ß (TGFß) are negative regulators of proliferation with tumor suppressor properties in the intestine. Here, we identify novel crosstalk between PKCα and TGFß signaling. RNA-Seq analysis of nontransformed intestinal crypt-like cells and colorectal cancer cells identified TGFß receptor 1 (TGFßR1) as a target of PKCα signaling. RT-PCR and immunoblot analysis confirmed that PKCα positively regulates TGFßR1 mRNA and protein expression in these cells. Effects on TGFßR1 were dependent on Ras-extracellular signal-regulated kinase 1/2 (ERK) signaling. Nascent RNA and promoter-reporter analysis indicated that PKCα induces TGFßR1 transcription, and Runx2 was identified as an essential mediator of the effect. PKCα promoted ERK-mediated activating phosphorylation of Runx2, which preceded transcriptional activation of the TGFßR1 gene and induction of Runx2 expression. Thus, we have identified a novel PKCαâERKâRunx2âTGFßR1 signaling axis. In further support of a link between PKCα and TGFß signaling, PKCα knockdown reduced the ability of TGFß to induce SMAD2 phosphorylation and cell cycle arrest, and inhibition of TGFßR1 decreased PKCα-induced upregulation of p21Cip1 and p27Kip1 in intestinal cells. The physiological relevance of these findings is also supported by The Cancer Genome Atlas data showing correlation between PKCα, Runx2, and TGFßR1 mRNA expression in human colorectal cancer. PKCα also regulated TGFßR1 in endometrial cancer cells, and PKCα, Runx2, and TGFßR1 expression correlates in uterine tumors, indicating that crosstalk between PKCα and TGFß signaling may be a common mechanism in diverse epithelial tissues.
Asunto(s)
Neoplasias Colorrectales , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Proteína Quinasa C-alfa , Receptor Tipo I de Factor de Crecimiento Transformador beta , Humanos , Neoplasias Colorrectales/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Células Epiteliales/metabolismo , Intestinos , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , ARN Mensajero/genética , Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Ovarian cancer is the deadliest gynecologic malignancy. The majority of patients diagnosed with advanced ovarian cancer will relapse, at which point additional therapies can be administered but, for the most part, these are not curative. As such, a need exists for the development of novel therapeutic options for ovarian cancer patients. Research in the field of targeted protein degradation (TPD) through the use of proteolysis-targeting chimeras (PROTACs) has significantly increased in recent years. The ability of PROTACs to target proteins of interest (POI) for degradation, overcoming limitations such as the incomplete inhibition of POI function and the development of resistance seen with other inhibitors, is of particular interest in cancer research, including ovarian cancer research. This review provides a synopsis of PROTACs tested in ovarian cancer models and highlights PROTACs characterized in other types of cancers with potential high utility in ovarian cancer. Finally, we discuss methods that will help to enable the selective delivery of PROTACs to ovarian cancer and improve the pharmacodynamic properties of these agents.
Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Proteolisis , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Femenino , Proteolisis/efectos de los fármacos , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Animales , Terapia Molecular Dirigida/métodos , Quimera Dirigida a la ProteólisisRESUMEN
The unfolded protein response (UPR) is an adaptation mechanism activated to resolve transient accumulation of unfolded/misfolded proteins in the endoplasmic reticulum. Failure to resolve the transient accumulation of such proteins results in UPR-mediated programmed cell death. Loss of tumor suppressor gene or oncogene addiction in cancer cells can result in sustained higher basal UPR levels; however, it is not clear if these higher basal UPR levels in cancer cells can be exploited as a therapeutic strategy. We hypothesized that covalent modification of surface-exposed cysteine (SEC) residues could simulate unfolded/misfolded proteins to activate the UPR, and that higher basal UPR levels in cancer cells would provide the necessary therapeutic window. To test this hypothesis, here we synthesized analogs that can covalently modify multiple SEC residues and evaluated them as UPR activators. We identified a spirocyclic dimer, SpiD7, and evaluated its effects on UPR activation signals, that is, XBP1 splicing, phosphorylation of eIF2α, and a decrease in ATF 6 levels, in normal and cancer cells, which were further confirmed by RNA-Seq analyses. We found that SpiD7 selectively induced caspase-mediated apoptosis in cancer cells, whereas normal cells exhibited robust XBP1 splicing, indicating adaptation to stress. Furthermore, SpiD7 inhibited the growth of high-grade serous carcinoma cell lines ~3-15-fold more potently than immortalized fallopian tube epithelial (paired normal control) cells and reduced clonogenic growth of high-grade serous carcinoma cell lines. Our results suggest that induction of the UPR by covalent modification of SEC residues represents a cancer cell vulnerability and can be exploited to discover novel therapeutics.
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Apoptosis , Carcinoma , Respuesta de Proteína Desplegada , Carcinoma/tratamiento farmacológico , Carcinoma/metabolismo , Línea Celular Tumoral , Descubrimiento de Drogas , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/metabolismo , HumanosRESUMEN
Evidence for active DNA demethylation in vertebrates is accumulating, but the mechanisms and enzymes remain unclear. Using zebrafish embryos we provide evidence for 5-methylcytosine (5-meC) removal in vivo via the coupling of a 5-meC deaminase (AID, which converts 5-meC to thymine) and a G:T mismatch-specific thymine glycosylase (Mbd4). The injection of methylated DNA into embryos induced a potent DNA demethylation activity, which was attenuated by depletion of AID or the non enzymatic factor Gadd45. Remarkably, overexpression of the deaminase/glycosylase pair AID/Mbd4 in vivo caused demethylation of the bulk genome and injected methylated DNA fragments, likely involving a G:T intermediate. Furthermore, AID or Mbd4 knockdown caused the remethylation of a set of common genes. Finally, Gadd45 promoted demethylation and enhanced functional interactions between deaminase/glycosylase pairs. Our results provide evidence for a coupled mechanism of 5-meC demethylation, whereby AID deaminates 5-meC, followed by thymine base excision by Mbd4, promoted by Gadd45.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Glicosilasas/metabolismo , Metilación de ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Timina ADN Glicosilasa/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Línea Celular , Citidina Desaminasa/metabolismo , Embrión no Mamífero/metabolismo , Humanos , Neuropéptidos/metabolismo , Regulación hacia Arriba , Proteinas GADD45RESUMEN
Effective cancer prevention requires the discovery and intervention of a factor critical to cancer development. Here we show that ovarian progesterone is a crucial endogenous factor inducing the development of primary tumors progressing to metastatic ovarian cancer in a mouse model of high-grade serous carcinoma (HGSC), the most common and deadliest ovarian cancer type. Blocking progesterone signaling by the pharmacologic inhibitor mifepristone or by genetic deletion of the progesterone receptor (PR) effectively suppressed HGSC development and its peritoneal metastases. Strikingly, mifepristone treatment profoundly improved mouse survival (â¼18 human years). Hence, targeting progesterone/PR signaling could offer an effective chemopreventive strategy, particularly in high-risk populations of women carrying a deleterious mutation in the BRCA gene.
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Proteína BRCA1/genética , Cistadenocarcinoma Seroso/prevención & control , Mifepristona/farmacología , Neoplasias Ováricas/prevención & control , Progesterona/antagonistas & inhibidores , Adulto , Animales , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Cistadenocarcinoma Seroso/química , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Modelos Animales de Enfermedad , Estradiol/administración & dosificación , Femenino , Humanos , Ratones , Persona de Mediana Edad , Mifepristona/uso terapéutico , Mutación , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Neoplasias Experimentales/prevención & control , Neoplasias Ováricas/inducido químicamente , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ovario/patología , Ovario/cirugía , Progesterona/administración & dosificación , Progesterona/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Salpingooforectomía , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.
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Antineoplásicos/farmacología , Cistadenocarcinoma Seroso/genética , Neoplasias Ováricas/patología , Neoplasias Peritoneales/genética , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Inestabilidad Cromosómica , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/secundario , ARN Helicasas DEAD-box/genética , Reparación del ADN , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales/métodos , Estudios de Factibilidad , Femenino , Humanos , Ratones , Ratones Noqueados , Mutación , Clasificación del Tumor , Metástasis de la Neoplasia/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Fosfohidrolasa PTEN/genética , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/secundario , Cultivo Primario de Células , Ribonucleasa III/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Several studies have suggested that global DNA methylation in circulating white blood cells (WBC) is associated with breast cancer risk. METHODS: To address conflicting results and concerns that the findings for WBC DNA methylation in some prior studies may reflect disease effects, we evaluated the relationship between global levels of WBC DNA methylation in white blood cells and breast cancer risk in a case-control study nested within the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO) cohort. A total of 428 invasive breast cancer cases and 419 controls, frequency matched on age at entry (55-59, 60-64, 65-69, ≥70 years), year of entry (on/before September 30, 1997, on/after October 1, 1997) and period of DNA extraction (previously extracted, newly extracted) were included. The ratio of 5-methyl-2' deoxycytidine [5-mdC] to 2'-deoxyguanine [dG], assuming [dG] = [5-mdC] + [2'-deoxycytidine [dC]] (%5-mdC), was determined by liquid chromatography-electrospray ionization-tandem mass spectrometry, an especially accurate method for assessing total genomic DNA methylation. RESULTS: Odds ratio (OR) estimates and 95% confidence intervals (CI) for breast cancer risk adjusted for age at entry, year of entry, and period of DNA extraction, were 1.0 (referent), 0.89 (95% CI, 0.6-1.3), 0.88 (95% CI, 0.6-1.3), and 0.84 (95% CI, 0.6-1.2) for women in the highest compared to lowest quartile levels of %5md-C (p for trend = .39). Effects did not meaningfully vary by time elapsed from WBC collection to diagnosis. DISCUSSION: These results do not support the hypothesis that global DNA hypomethylation in WBC DNA is associated with increased breast cancer risk prior to the appearance of clinical disease.
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Neoplasias de la Mama Masculina/epidemiología , Neoplasias de la Mama/epidemiología , Metilación de ADN/genética , Leucocitos , Células Neoplásicas Circulantes , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/etiología , Neoplasias de la Mama/patología , Neoplasias de la Mama Masculina/etiología , Neoplasias de la Mama Masculina/patología , Ensayos Clínicos como Asunto , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/complicaciones , Neoplasias Colorrectales/epidemiología , Neoplasias Colorrectales/genética , Femenino , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/genética , Masculino , Neoplasias Ováricas/sangre , Neoplasias Ováricas/complicaciones , Neoplasias Ováricas/epidemiología , Neoplasias Ováricas/genética , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/complicaciones , Neoplasias de la Próstata/epidemiología , Neoplasias de la Próstata/genética , Factores de RiesgoRESUMEN
The drivers of abnormal DNA methylation in human cancers include widespread aberrant splicing of the DNMT3B gene, producing abnormal transcripts that encode truncated proteins that may act as dominant negative isoforms. To test whether reduced Dnmt3b dosage can alter tumorigenesis, we bred Dnmt3b(+/-) mice to Eµ-Myc mice, a mouse model susceptible to B-cell lymphomas. Eµ-Myc/Dnmt3b(+/-) mice showed a dramatic acceleration of lymphomagenesis, greater even than that observed in Eµ-Myc mice that express a truncated DNMT3B isoform found in human tumors, DNMT3B7. This finding indicates that Dnmt3b can act as a haploinsufficient tumor suppressor gene. Although reduction in both Dnmt3b dosage and expression of DNMT3B7 within the Eµ-Myc system had similar effects on tumorigenesis and DNA hypermethylation, different molecular mechanisms appear to underlie these changes. This study offers insight into how de novo DNA methyltransferases function as tumor suppressors and the sensitivity of Myc-induced lymphomas to DNA methylation.
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Transformación Celular Neoplásica/genética , ADN (Citosina-5-)-Metiltransferasas/fisiología , Genes Supresores de Tumor/fisiología , Haploinsuficiencia/fisiología , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , Animales , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Haploinsuficiencia/genética , Linfoma de Células B/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Regiones Promotoras Genéticas/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ADN Metiltransferasa 3BRESUMEN
OBJECTIVE: We determined whether DNA methylation of repetitive elements (RE) is altered in epithelial ovarian cancer (EOC) patient tumors and white blood cells (WBC), compared to normal tissue controls. METHODS: Two different quantitative measures of RE methylation (LINE1 and Alu bisulfite pyrosequencing) were used in normal and tumor tissues from EOC cases and controls. Tissues analyzed included: i) EOC, ii) normal ovarian surface epithelia (OSE), iii) normal fallopian tube surface epithelia (FTE), iv) WBC from EOC patients, obtained before and after treatment, and v) WBC from demographically-matched controls. RESULTS: REs were significantly hypomethylated in EOC compared to OSE and FTE, and LINE1 and Alu methylation showed a significant direct association in these tissues. In contrast, WBC RE methylation was significantly higher in EOC cases compared to controls. RE methylation in patient-matched EOC tumors and pre-treatment WBC did not correlate. CONCLUSIONS: EOC shows robust RE hypomethylation compared to normal tissues from which the disease arises. In contrast, RE are generally hypermethylated in EOC patient WBC compared to controls. EOC tumor and WBC methylation did not correlate in matched patients, suggesting that RE methylation is independently controlled in tumor and normal tissues. Despite the significant differences observed over the population, the range of RE methylation in patient and control WBC overlapped, limiting their specific utility as an EOC biomarker. However, our data demonstrate that DNA methylation is deranged in normal tissues from EOC patients, supporting further investigation of WBC DNA methylation biomarkers suitable for EOC risk assessment.
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Elementos Alu , Metilación de ADN , Leucocitos/metabolismo , Elementos de Nucleótido Esparcido Largo , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/sangre , Neoplasias Ováricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Epitelial de Ovario , Femenino , Humanos , Leucocitos/citología , Persona de Mediana Edad , Medición de RiesgoRESUMEN
The DNA replication stress (DRS) response is a crucial homeostatic mechanism for maintaining genome integrity in the face of intrinsic and extrinsic barriers to DNA replication. Importantly, DRS is often significantly increased in tumor cells, making tumors dependent on the cellular DRS response for growth and survival. Rad9-Hus1-Rad1 Interacting Nuclear Orphan 1 (RHNO1), a protein involved in the DRS response, has recently emerged as a potential therapeutic target in cancer. RHNO1 interacts with the 9-1-1 checkpoint clamp and TopBP1 to activate the ATR/Chk1 signaling pathway, the crucial mediator of the DRS response. Moreover, RHNO1 was also recently identified as a key facilitator of theta-mediated end joining (TMEJ), a DNA repair mechanism implicated in cancer progression and chemoresistance. In this literature review, we provide an overview of our current understanding of RHNO1, including its structure, function in the DRS response, and role in DNA repair, and discuss its potential as a cancer therapeutic target. Therapeutic targeting of RHNO1 holds promise for tumors with elevated DRS as well as tumors with DNA repair deficiencies, including homologous recombination DNA repair deficient (HRD) tumors. Further investigation into RHNO1 function in cancer, and development of approaches to target RHNO1, are expected to yield novel strategies for cancer treatment.
RESUMEN
BACKGROUND: Genetic studies implicate the oncogenic transcription factor Forkhead Box M1 (FOXM1) as a potential therapeutic target in high-grade serous ovarian cancer (HGSOC). We evaluated the activity of different FOXM1 inhibitors in HGSOC cell models. RESULTS: We treated HGSOC and fallopian tube epithelial (FTE) cells with a panel of previously reported FOXM1 inhibitors. Based on drug potency, efficacy, and selectivity, determined through cell viability assays, we focused on two compounds, NB-73 and NB-115 (NB compounds), for further investigation. NB compounds potently and selectively inhibited FOXM1 with lesser effects on other FOX family members. NB compounds decreased FOXM1 expression via targeting the FOXM1 protein by promoting its proteasome-mediated degradation, and effectively suppressed FOXM1 gene targets at both the protein and mRNA level. At the cellular level, NB compounds promoted apoptotic cell death. Importantly, while inhibition of apoptosis using a pan-caspase inhibitor rescued HGSOC cells from NB compound-induced cell death, it did not rescue FOXM1 protein degradation, supporting that FOXM1 protein loss from NB compound treatment is specific and not a general consequence of cytotoxicity. Drug washout studies indicated that FOXM1 reduction was retained for at least 72 h post-treatment, suggesting that NB compounds exhibit long-lasting effects in HGSOC cells. NB compounds effectively suppressed both two-dimensional and three-dimensional HGSOC cell colony formation at sub-micromolar concentrations. Finally, NB compounds exhibited synergistic activity with carboplatin in HGSOC cells. CONCLUSIONS: NB compounds are potent, selective, and efficacious inhibitors of FOXM1 in HGSOC cells and are worthy of further investigation as HGSOC therapeutics.
Asunto(s)
Antineoplásicos , Apoptosis , Proteína Forkhead Box M1 , Neoplasias Ováricas , Proteína Forkhead Box M1/metabolismo , Proteína Forkhead Box M1/antagonistas & inhibidores , Humanos , Femenino , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Línea Celular Tumoral , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/patología , Cistadenocarcinoma Seroso/metabolismo , Supervivencia Celular/efectos de los fármacos , Clasificación del TumorRESUMEN
Cancer germline (CG) genes are normally expressed in germ cells and aberrantly expressed in a variety of cancers; their immunogenicity has led to the widespread development of cancer vaccines targeting these antigens. BORIS/CTCFL is an autosomal CG antigen and promising cancer vaccine target. BORIS is the only known paralog of CTCF, a gene intimately involved in genomic imprinting, chromatin insulation, and nuclear regulation. We have previously shown that BORIS is expressed in epithelial ovarian cancer (EOC) and that its expression coincides with promoter and global DNA hypomethylation. Recently, 23 different BORIS mRNA variants have been described, and have been functionally grouped into six BORIS isoform families (sf1-sf6). In the present study, we have characterized the expression of BORIS isoform families in normal ovary (NO) and EOC, the latter of which were selected to include two groups with widely varying global DNA methylation status. We find selective expression of BORIS isoform families in NO, which becomes altered in EOC, primarily by the activation of BORIS sf1 in EOC. When comparing EOC samples based on methylation status, we find that BORIS sf1 and sf2 isoform families are selectively activated in globally hypomethylated tumors. In contrast, CTCF is downregulated in EOC, and the ratio of BORIS sf1, sf2, and sf6 isoform families as a function of CTCF is elevated in hypomethylated tumors. Finally, the expression of all BORIS isoform families was induced to varying extents by epigenetic modulatory drugs in EOC cell lines, particularly when DNMT and HDAC inhibitors were used in combination.
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Proteínas de Unión al ADN/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Carcinoma Epitelial de Ovario , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Masculino , Persona de Mediana Edad , Ovario/metabolismo , Ovario/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Testículo/metabolismoRESUMEN
Combination chemotherapeutic drugs administered via a single nanocarrier for cancer treatment provides benefits in reducing dose-limiting toxicities, improving the pharmacokinetic properties of the cargo and achieving spatial-temporal synchronization of drug exposure for maximized synergistic therapeutic effects. In an attempt to develop such a multi-drug carrier, our work focuses on functional multimodal polypeptide-based polymeric nanogels (NGs). Diblock copolymers poly (ethylene glycol)-b-poly (glutamic acid) (PEG-b-PGlu) modified with phenylalanine (Phe) were successfully synthesized and characterized. Self-assembly behavior of the resulting polymers was utilized for the synthesis of NGs with hydrophobic domains in cross-linked polyion cores coated with inert PEG chains. The resulting NGs were small (ca. 70 nm in diameter) and were able to encapsulate the combination of drugs with different physicochemical properties such as cisplatin and neratinib. Drug combination-loaded NGs exerted a selective synergistic cytotoxicity towards EGFR overexpressing ovarian cancer cells. Moreover, we developed ligand-installed EGFR-targeted NGs and tested them as an EGFR-overexpressing tumor-specific delivery system. Both in vitro and in vivo, ligand-installed NGs displayed preferential associations with EGFR (+) tumor cells. Ligand-installed NGs carrying cisplatin and neratinib significantly improved the treatment response of ovarian cancer xenografts. We also confirmed the importance of simultaneous administration of the dual drug combination via a single NG system which provides more therapeutic benefit than individual drug-loaded NGs administered at equivalent doses. This work illustrates the potential of our carrier system to mediate efficient delivery of a drug combination to treat EGFR overexpressing cancers.
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Antineoplásicos , Nanopartículas , Neoplasias Ováricas , Femenino , Humanos , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Cisplatino , Portadores de Fármacos/química , Quimioterapia Combinada , Receptores ErbB , Ligandos , Nanogeles , Nanopartículas/química , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Polietilenglicoles/química , Polímeros/química , AnimalesRESUMEN
Although DNA methylation is critical for proper embryonic and tissue-specific development, how different DNA methyltransferases affect tissue-specific development and their targets remains unknown. We address this issue in zebrafish through antisense-based morpholino knockdown of Dnmt3 and Dnmt1. Our data reveal that Dnmt3 is required for proper neurogenesis, and its absence results in profound defects in brain and retina. Interestingly, other organs such as intestine remain unaffected suggesting tissue-specific requirements of Dnmt3. Further, comparison of Dnmt1 knockdown phenotypes with those of Dnmt3 suggested that these two families have distinct functions. Consistent with this idea, Dnmt1 failed to complement Dnmt3 deficiency, and Dnmt3 failed to complement Dnmt1 deficiency. Downstream of Dnmt3 we identify a neurogenesis regulator, lef1, as a Dnmt3-specific target gene that is demethylated and up-regulated in dnmt3 morphants. Knockdown of lef1 rescued neurogenesis defects resulting from Dnmt3 absence. Mechanistically, we show cooperation between Dnmt3 and an H3K9 methyltransferase G9a in regulating lef1. Further, like Dnmt1-Suv39h1 cooperativity, Dnmt3 and G9a seemed to function together for tissue-specific development. G9a knockdown, but not Suv39h1 loss, phenocopied dnmt3 morphants and G9a overexpression provided a striking rescue of dnmt3 morphant phenotypes, whereas Suv39h1 overexpression failed, supporting the notion of specific DNMT-histone methyltransferase networks. Consistent with this model, H3K9me3 levels on the lef1 promoter were reduced in both dnmt3 and g9a morphants, and its knockdown rescued neurogenesis defects in g9a morphants. We propose a model wherein specific DNMT-histone methyltransferase networks are utilized to silence critical regulators of cell fate in a tissue-specific manner.
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ADN (Citosina-5-)-Metiltransferasas/genética , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Inmunoprecipitación de Cromatina , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , N-Metiltransferasa de Histona-Lisina/metabolismo , Hibridación in Situ , Modelos Biológicos , Neurogénesis/genética , Retina/citología , Retina/embriología , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Forkhead box M1 (FOXM1) is a member of the conserved forkhead box (FOX) transcription factor family. Over the last two decades, FOXM1 has emerged as a multifunctional oncoprotein and a robust biomarker of poor prognosis in many human malignancies. In this review article, we address the current knowledge regarding the mechanisms of regulation and oncogenic functions of FOXM1, particularly in the context of ovarian cancer. FOXM1 and its associated oncogenic transcriptional signature are enriched in >85% of ovarian cancer cases and FOXM1 expression and activity can be enhanced by a plethora of genomic, transcriptional, post-transcriptional, and post-translational mechanisms. As a master transcriptional regulator, FOXM1 promotes critical oncogenic phenotypes in ovarian cancer, including: (1) cell proliferation, (2) invasion and metastasis, (3) chemotherapy resistance, (4) cancer stem cell (CSC) properties, (5) genomic instability, and (6) altered cellular metabolism. We additionally discuss the evidence for FOXM1 as a cancer biomarker, describe the rationale for FOXM1 as a cancer therapeutic target, and provide an overview of therapeutic strategies used to target FOXM1 for cancer treatment.
RESUMEN
The FOXM1 transcription factor is an oncoprotein and a top biomarker of poor prognosis in human cancer. Overexpression and activation of FOXM1 is frequent in high-grade serous carcinoma (HGSC), the most common and lethal form of human ovarian cancer, and is linked to copy number gains at chromosome 12p13.33. We show that FOXM1 is co-amplified and co-expressed with RHNO1, a gene involved in the ATR-Chk1 signaling pathway that functions in the DNA replication stress response. We demonstrate that FOXM1 and RHNO1 are head-to-head (i.e., bidirectional) genes (BDG) regulated by a bidirectional promoter (BDP) (named F/R-BDP). FOXM1 and RHNO1 each promote oncogenic phenotypes in HGSC cells, including clonogenic growth, DNA homologous recombination repair, and poly-ADP ribosylase inhibitor resistance. FOXM1 and RHNO1 are one of the first examples of oncogenic BDG, and therapeutic targeting of FOXM1/RHNO1 BDG is a potential therapeutic approach for ovarian and other cancers.
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Proteínas Portadoras/genética , Proteína Forkhead Box M1/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Quísticas, Mucinosas y Serosas/genética , Neoplasias Ováricas/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Carboplatino/farmacología , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proliferación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Bases de Datos Genéticas , Resistencia a Antineoplásicos , Femenino , Proteína Forkhead Box M1/metabolismo , Humanos , Neoplasias Quísticas, Mucinosas y Serosas/tratamiento farmacológico , Neoplasias Quísticas, Mucinosas y Serosas/metabolismo , Neoplasias Quísticas, Mucinosas y Serosas/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Regiones Promotoras Genéticas , Reparación del ADN por Recombinación , Transducción de SeñalRESUMEN
Anti-tubulin agents, such as paclitaxel, have been used extensively for treatment of several types of cancer, including ovarian, lung, breast, and pancreatic cancers. Despite their wide use in cancer treatment, however, patient response is highly variable and drug resistance remains a major clinical issue. Protein kinase RNA-activated (PKR) plays a critical role in immune response to viral infection. We identified PKR as a phospho-protein in response to anti-tubulin agents and this phosphorylation occurs independent of its own kinase activity. PKR is phosphorylated by cyclin-dependent kinase 1 (CDK1) during anti-tubulin treatment and unperturbed mitosis and that PKR regulates mitotic progression in a phosphorylation-dependent manner. Furthermore, inactivation of PKR confers resistance to paclitaxel in ovarian and breast cancer cells in vitro and in vivo. PKR expression levels and activity are decreased in chemotherapeutic recurrent ovarian cancer patients. Mechanistically, our findings suggest that PKR controls paclitaxel chemosensitivity through repressing Bcl2 expression. Pharmacological inhibition of Bcl2 with FDA-approved agent venetoclax overcomes paclitaxel resistance in preclinical animal models of ovarian cancer. Our results suggest that PKR is a critical determinant of paclitaxel cytotoxicity and that PKR-Bcl2 axis as a potential therapeutic target for the treatment of recurrent drug-resistant ovarian tumors.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mitosis , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , eIF-2 Quinasa/genéticaRESUMEN
CCNE1-amplified ovarian cancers (OVCAs) and endometrial cancers (EMCAs) are associated with platinum resistance and poor survival, representing a clinically unmet need. We hypothesized that dysregulated cell-cycle progression promoted by CCNE1 overexpression would lead to increased sensitivity to low-dose WEE1 inhibition and ataxia telangiectasia and Rad3-related (ATR) inhibition (WEE1i-ATRi), thereby optimizing efficacy and tolerability. The addition of ATRi to WEE1i is required to block feedback activation of ATR signaling mediated by WEE1i. Low-dose WEE1i-ATRi synergistically decreases viability and colony formation and increases replication fork collapse and double-strand breaks (DSBs) in a CCNE1 copy number (CN)-dependent manner. Only upon CCNE1 induction does WEE1i perturb DNA synthesis at S-phase entry, and addition of ATRi increases DSBs during DNA synthesis. Inherent resistance to WEE1i is overcome with WEE1i-ATRi, with notable durable tumor regressions and improved survival in patient-derived xenograft (PDX) models in a CCNE1-level-dependent manner. These studies demonstrate that CCNE1 CN is a clinically tractable biomarker predicting responsiveness to low-dose WEE1i-ATRi for aggressive subsets of OVCAs/EMCAs.
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Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Ciclina E/genética , Neoplasias Endometriales/genética , Dosificación de Gen , Modelos Biológicos , Proteínas Oncogénicas/genética , Neoplasias Ováricas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , Replicación del ADN , Neoplasias Endometriales/patología , Femenino , Humanos , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Ováricas/patología , Proteínas Tirosina Quinasas/metabolismo , Fase S , Transducción de Señal , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Breast cancer, the most common malignancy in women, emerges through a multistep process, encompassing the progressive sequential evolution of morphologically distinct stages from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma and metastasis. The success of treatment of breast cancer could be greatly improved by the detection at early stages of cancer. In the present study, we investigated the underlying molecular mechanisms involved in breast carcinogenesis in Augustus and Copenhagen-Irish female rats, a cross between the ACI strains, induced by continuous exposure to 17beta-estradiol. The results of our study demonstrate that early stages of estrogen-induced breast carcinogenesis are characterized by altered global DNA methylation, aberrant expression of proteins responsible for the proper maintenance of DNA methylation pattern and epigenetic silencing of the critical Rassf1a (Ras-association domain family 1, isoform A) tumor suppressor gene. Interestingly, transcriptional repression of the Rassf1a gene in mammary glands during early stages of breast carcinogenesis was associated with an increase in trimethylation of histones H3 lysine 9 and H3 lysine 27 and de novo CpG island methylation and at the Rassf1a promoter and first exon. In conclusion, we demonstrate that epigenetic alterations precede formation of preneoplastic lesions indicating the significance of epigenetic events in induction of oncogenic pathways in early stages of carcinogenesis.
Asunto(s)
Transformación Celular Neoplásica/genética , Estradiol/toxicidad , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genes Supresores de Tumor , Neoplasias Mamarias Experimentales/genética , Animales , Islas de CpG , Metilación de ADN , Exones/genética , Femenino , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Hiperplasia , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/inducido químicamente , Proteínas de Neoplasias/metabolismo , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Regiones Promotoras Genéticas/genética , Distribución Aleatoria , Ratas , Ratas Endogámicas ACI , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genéticaRESUMEN
Melanoma antigen gene protein-A11 (MAGE-11) of the MAGE family of cancer germ-line antigens increases androgen receptor (AR) transcriptional activity through its interaction with the AR NH(2)-terminal FXXLF motif. The present study investigated the regulatory mechanisms that control MAGE-11 expression during androgen deprivation therapy and prostate cancer progression. Studies include the CWR22 xenograft model of human prostate cancer, clinical specimens of benign and malignant prostate, and prostate cancer cell lines. MAGE-11 mRNA levels increased 100- to 1,500-fold during androgen deprivation therapy and prostate cancer progression, with highest levels in the castration-recurrent CWR22 xenograft and clinical specimens of castration-recurrent prostate cancer. Pyrosequencing of genomic DNA from prostate cancer specimens and cell lines indicated the increase in MAGE-11 resulted from DNA hypomethylation of a CpG island in the 5' promoter of the MAGE-11 gene. Sodium bisulfite sequencing of genomic DNA from benign and malignant prostate tumors and prostate cancer cell lines revealed DNA hypomethylation at individual CpG sites at the transcription start site were most critical for MAGE-11 expression. Cyclic AMP (cAMP) also increased MAGE-11 expression and AR transcriptional activity in prostate cancer cell lines. However, cAMP did not alter DNA methylation of the promoter and its effects were inhibited by extensive DNA methylation in the MAGE-11 promoter region. Increased expression of the AR coregulator MAGE-11 through promoter DNA hypomethylation and cAMP provides a novel mechanism for increased AR signaling in castration-recurrent prostate cancer.