RESUMEN
In response to energy and nutrient shortage, the liver triggers several catabolic processes to promote survival. Despite recent progress, the precise molecular mechanisms regulating the hepatic adaptation to fasting remain incompletely characterized. Here, we report the identification of hydroxysteroid dehydrogenase-like 2 (HSDL2) as a mitochondrial protein highly induced by fasting. We show that the activation of PGC1α-PPARα and the inhibition of the PI3K-mTORC1 axis stimulate HSDL2 expression in hepatocytes. We found that HSDL2 depletion decreases cholesterol conversion to bile acids (BAs) and impairs FXR activity. HSDL2 knockdown also reduces mitochondrial respiration, fatty acid oxidation, and TCA cycle activity. Bioinformatics analyses revealed that hepatic Hsdl2 expression positively associates with the postprandial excursion of various BA species in mice. We show that liver-specific HSDL2 depletion affects BA metabolism and decreases circulating cholesterol levels upon refeeding. Overall, our report identifies HSDL2 as a fasting-induced mitochondrial protein that links nutritional signals to BAs and cholesterol homeostasis.
Asunto(s)
Ácidos y Sales Biliares , Colesterol , Homeostasis , Animales , Colesterol/metabolismo , Ácidos y Sales Biliares/metabolismo , Ratones , Ayuno/metabolismo , Hígado/metabolismo , Humanos , Mitocondrias/metabolismo , Transducción de Señal , Hepatocitos/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismoRESUMEN
ETV6 transcriptional activity is critical for proper blood cell development in the bone marrow. Despite the accumulating body of evidence linking ETV6 malfunction to hematological malignancies, its regulatory network remains unclear. To uncover genes that modulate ETV6 repressive transcriptional activity, we performed a specifically designed, unbiased genome-wide shRNA screen in pre-B acute lymphoblastic leukemia cells. Following an extensive validation process, we identified 13 shRNAs inducing overexpression of ETV6 transcriptional target genes. We showed that the silencing of AKIRIN1, COMMD9, DYRK4, JUNB, and SRP72 led to an abrogation of ETV6 repressive activity. We identified critical modulators of the ETV6 function which could participate in cellular transformation through the ETV6 transcriptional network.
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Ursodeoxycholic acid (UDCA) is the first line therapy for the treatment of cholestatic and autoimmune liver diseases. Its clinical use is currently limited by a significant proportion of non-responder patients. Polyunsaturated fatty acids (n-3 PUFAs) possess important anti-inflammatory properties and protect liver cells against bile acid (BA)-induced toxicity. The present study was designed to rapidly evaluate whether combining n-3 PUFAs (i.e., eicosapentaenoic [EPA] and docosahexaenoic [DHA] acids) to UDCA would provide additional benefits when compared to the drug alone. The parameters evaluated were (i) the expression of genes governing BA synthesis, transport, and metabolism; (ii) the prevention of BA-induced apoptosis and endoplasmic reticulum (ER)-stress; and (iii) the control of BA- and LPS-dependent inflammation. In the absence of n-3 PUFAs, most of the parameters investigated were unaffected by UDCA or were only altered by the higher dose (500 µM) of the drug. By contrast, in the presence of EPA/DHA (50/50 µM), all parameters showed a strongly improved response and the lowest UDCA dosage (50 µM) provided equal or better benefits than the highest dose used alone. For example, the combination EPA/DHA + UDCA 50 µM caused comparable down-regulation of the CYP7A1 gene expression and of the BA-induced caspase 3 activity as observed with UDCA 500 µM. In conclusion, these results suggest that the addition of n-3 PUFAs to UDCA may improve the response to the drug, and that such a pharmaco-nutraceutical approach could be used in clinic to open the narrow therapeutic dose of UDCA in cholestatic liver diseases.
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Suplementos Dietéticos , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-3/farmacología , Ácido Ursodesoxicólico/farmacología , Apoptosis/efectos de los fármacos , Enfermedades Autoinmunes , Ácidos y Sales Biliares/metabolismo , Ácidos y Sales Biliares/toxicidad , Carcinoma Hepatocelular , Caspasa 3 , Colangitis Esclerosante , Colestanotriol 26-Monooxigenasa/genética , Colestasis , Colesterol 7-alfa-Hidroxilasa/genética , Regulación hacia Abajo/efectos de los fármacos , Quimioterapia Combinada , Estrés del Retículo Endoplásmico/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Inflamación , Hígado/metabolismo , Cirrosis Hepática Biliar , Hepatopatías , Células THP-1RESUMEN
A novel tumor suppressing agent was discovered against PC-3 prostate cancer cells from the screening of a 1,4-benzodiazepin-3-one library. In this study, 96 highly diversified 2,4,5-trisubstituted 1,4-benzodiazepin-3-one derivatives were prepared by a two-step approach using sequential Ugi multicomponent reaction and simultaneous deprotection and cyclization to afford pure compounds bearing a wide variety of substituents. The most promising compound showed a potent and selective antiproliferative activity against prostate cancer cell line PC-3 (GI50 = 10.2 µM), but had no effect on LNCAP, LAPC4 and DU145 cell lines. The compound was initially prepared as a mixture of two diastereomers and after their separation by HPLC, similar antiproliferative activities against PC-3 cells were observed for both diastereomers (2S,5S: GI50 = 10.8 µM and 2S,5R: GI50 = 7.0 µM). Additionally, both diastereomers showed comparable stability profiles after incubation with human liver microsomes. Finally, in vivo evaluation of the hit compound with the chick chorioallantoic membrane xenograft assay revealed a good toxicity profile and significant antitumor activity after intravenous injection.
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Antineoplásicos/farmacología , Benzodiazepinas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Benzodiazepinas/síntesis química , Benzodiazepinas/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Hígado/química , Hígado/metabolismo , Masculino , Estructura Molecular , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
RAS proteins are GTPases that lie upstream of a signaling network impacting cell fate determination. How cells integrate RAS activity to balance proliferation and cellular senescence is still incompletely characterized. Here, we identify ZNF768 as a phosphoprotein destabilized upon RAS activation. We report that ZNF768 depletion impairs proliferation and induces senescence by modulating the expression of key cell cycle effectors and established p53 targets. ZNF768 levels decrease in response to replicative-, stress- and oncogene-induced senescence. Interestingly, ZNF768 overexpression contributes to bypass RAS-induced senescence by repressing the p53 pathway. Furthermore, we show that ZNF768 interacts with and represses p53 phosphorylation and activity. Cancer genomics and immunohistochemical analyses reveal that ZNF768 is often amplified and/or overexpressed in tumors, suggesting that cells could use ZNF768 to bypass senescence, sustain proliferation and promote malignant transformation. Thus, we identify ZNF768 as a protein linking oncogenic signaling to the control of cell fate decision and proliferation.
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Senescencia Celular/genética , Genes ras/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Carcinogénesis , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Transformación Celular Neoplásica , Replicación del ADN , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genómica , Células HeLa , Humanos , Oncogenes , Fenotipo , Fosfoproteínas , Fosforilación , Represión Psicológica , Transducción de Señal , Proteínas ras/genéticaRESUMEN
High concentrations of the damage-associated molecular patterns S100A8 and S100A9 are found in skin and serum from patients suffering from psoriasis, an IL-17-related disease. Notably, although the expression of these proteins correlates with psoriatic disease severity, the exact function of S100A8 and S100A9 in psoriasis pathogenesis remains unclear. In this study, we investigated the role of S100A8 and S100A9 in psoriasis-associated skin hyperplasia and immune responses using S100a8-/- and S100a9-/- mice in an imiquimod-induced model of psoriasis. We found that S100a8-/- and S100a9-/- psoriatic mice exhibit worsened clinical symptoms relative to wild-type mice and increased expression of S100A9 and S100A8 proteins in keratinocytes, respectively. In addition, the loss of S100A8 enhances proliferation of keratinocytes and disrupts keratinocyte differentiation. We further detected elevated production of IL-17A and -F from CD4+ T cells in the absence of S100A8 and S100A9, as well as increased infiltration of neutrophils in the skin. In addition, treatment with anti-IL-17A and -F was found to reduce psoriasis symptoms and skin hyperplasia in S100a8-/- and S100a9-/- mice. These data suggest that S100A8 and S100A9 regulate psoriasis by inhibiting production of IL-17A and -F, thereby, to our knowledge, providing new insights into their biological functions.
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Calgranulina A/metabolismo , Calgranulina B/metabolismo , Interleucina-17/metabolismo , Psoriasis/metabolismo , Psoriasis/patología , Piel/patología , Células Th17/inmunología , Animales , Anticuerpos Bloqueadores/metabolismo , Calgranulina A/genética , Calgranulina B/genética , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Hiperplasia , Imiquimod , Interleucina-17/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Psoriasis/inducido químicamenteRESUMEN
OBJECTIVES: In this study, the antiproliferative activity of 3 phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) was assessed in a time-dependent manner together with their hepatic stability and metabolism using human, mouse and rat liver microsomes. METHODS: CEU-818, -820 and -913 were selected as promising hit compounds. Their antiproliferative activity on human breast carcinoma MCF-7 cells was evaluated using escalating concentrations of drugs at 24, 36 and 48 h and the sulforhodamine B assay. Their hepatic stability was evaluated by HPLC-UV of extracts obtained from human, mouse and rat liver microsomes. KEY FINDINGS: The antiproliferative activity of PAIB-SOs is concentration and time-dependent and requires between 24 and 36 h of contact with MCF-7 cells to detect a significant antiproliferative activity. PAIB-SOs stability in microsomes usually decreases following this order: human ≈ (rat > mouse). The CEU-913 exhibits the longest half-life in rat and human liver microsomes while the CEU-820 exhibits the longest half-life in mouse liver microsomes. CONCLUSIONS: Our in vitro results suggest that PAIB-SOs should have a minimum contact time of 24 h with the tumour to trigger significant antitumoural activity. The activity of mouse liver microsomes towards PAIB-SOs is higher than rat microsomes and tends to be higher than human liver microsomes.
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Antineoplásicos/farmacología , Bencenosulfonatos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Microsomas Hepáticos/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Bencenosulfonatos/administración & dosificación , Bencenosulfonatos/química , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Femenino , Semivida , Humanos , Células MCF-7 , Ratones , Profármacos , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Factores de TiempoRESUMEN
Epithelial ovarian cancer (EOC) represents the most lethal gynecologic malignancy; a better understanding of the molecular mechanisms associated with EOC etiology could substantially improve EOC management. Aberrant O-glycosylation in cancer is attributed to alteration of N-acetylgalactosaminyltransferases (GalNAc-Ts). Reports suggest a genetic and functional redundancy between GalNAc-Ts, and our previous data are indicative of an induction of GALNT6 expression upon GALNT3 suppression in EOC cells. We performed single GALNT3 and double GALNT3/T6 suppression in EOC cells, using a combination of the CRISPR-Cas9 system and shRNA-mediated gene silencing. The effect of single GALNT3 and double GALNT3/T6 inhibition was monitored both in vitro (on EOC cells roliferation, migration, and invasion) and in vivo (on tumor formation and survival of experimental animals). We confirmed that GALNT3 gene ablation leads to strong and rather compensatory GALNT6 upregulation in EOC cells. Moreover, double GALNT3/T6 suppression was significantly associated with stronger inhibitory effects on EOC cell proliferation, migration, and invasion, and accordingly displayed a significant increase in animal survival rates compared with GALNT3-ablated and control (Ctrl) EOC cells. Our data suggest a possible functional redundancy of GalNAc-Ts (GALNT3 and T6) in EOC, with the perspective of using both these enzymes as novel EOC biomarkers and/or therapeutic targets.
Asunto(s)
Carcinoma Epitelial de Ovario/genética , Proliferación Celular/genética , N-Acetilgalactosaminiltransferasas/genética , Animales , Sistemas CRISPR-Cas/genética , Carcinoma Epitelial de Ovario/patología , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Glicosilación , Humanos , Ratones , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Ovario/patología , ARN Interferente Pequeño/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
The HOX genes are transcription factors that are expressed in coordinated spatiotemporal patterns to ensure normal development. Ectopic expression may instead lead to the development and progression of tumors. Genetic polymorphisms in the regions of four HOX gene clusters were tested for association with lung cancer in 420 cases and 3,151 controls. The effect of these variants on lung gene expression (expression quantitative trait loci, eQTL) was tested in a discovery set of 409 non-tumor lung samples and validated in two lung eQTL replication sets (n = 287 and 342). The expression levels of HOXB2 were evaluated at the mRNA and protein levels by quantitative real-time PCR and immunohistochemistry in paired tumor and non-tumor lung tissue samples. The most significant SNP associated with lung cancer in the HOXB cluster was rs10853100 located upstream of the HOXB cluster. HOXB2 was the top eQTL-regulated gene with several polymorphisms associated with its mRNA expression levels in lung tissue. This includes the lung cancer SNP rs10853100 that was significantly associated with HOXB2 expression (P=3.39E-7). In the lung eQTL discovery and replication sets, the lung cancer risk allele (T) for rs10853100 was associated with lower HOXB2 expression levels. In paired normal-tumor samples, HOXB2 mRNA and protein levels were significantly reduced in tumors when compared to non-tumor lung tissues. Genetic variants in the HOXB cluster may confer susceptibility to lung cancer by modulating the expression of HOXB2 in the lung.
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Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/genética , Neoplasias Pulmonares/genética , Pulmón/metabolismo , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Anciano , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Sitios de Carácter Cuantitativo , Factores de Transcripción/metabolismoRESUMEN
The aminosteroid derivative RM-133 is an effective anticancer molecule for which proof of concept has been achieved in several mouse xenograph models (HL-60, MCF-7, PANC-1 and OVCAR-3). To promote this new family of molecules toward a clinical phase 1 trial, the mechanism of action governing the anticancer properties of the representative candidate RM-133 needs to be characterized. In vitro experiments were first used to determine that RM-133 causes apoptosis in cancer cells. Then, using proteomic and transcriptomic experiments, RM-133 cytotoxicity was proven to be achieved via the endoplasmic reticulum (ER)-related apoptosis, which characterizes RM-133 as an endoplasmic reticulum stress aggravator (ERSA) anticancer drug. Furthermore, an shRNA-genome-wide screening has permitted to identify the steroidogenic acute regulator-related lipid transfer protein 5 (STARD5) as a major player in the RM-133 ER-related apoptosis mechanism, which was validated by an in vitro binding experiment. Altogether, the results presented herein suggest that RM-133 provokes a disturbance of cholesterol homeostasis via the implication of STARD5, which delivers an ERSA molecule to the ER. These results will be a springboard for RM-133 in its path toward clinical use.
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Androstenos/farmacología , Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Estrés del Retículo Endoplásmico , Proteínas Adaptadoras del Transporte Vesicular , Apoptosis/efectos de los fármacos , Proteínas Portadoras/genética , Línea Celular Tumoral , Homeostasis/efectos de los fármacos , HumanosRESUMEN
The molecular basis of epithelial ovarian cancer (EOC) dissemination is still poorly understood. We have previously identified the hydrogen peroxide-inducible clone-5 (Hic-5) gene as hypomethylated in high-grade (HG) serous EOC tumors, compared to normal ovarian tissues. Hic-5 is a focal adhesion scaffold protein and has been primarily studied for its role as a key mediator of TGF-ß-induced epithelial-to-mesenchymal transition (EMT) in epithelial cells of both normal and malignant origin; however, its role in EOC has been never investigated. Here we demonstrate that Hic-5 is overexpressed in advanced EOC, and that Hic-5 is upregulated upon TGFß1 treatment in the EOC cell line with epithelial morphology (A2780s), associated with EMT induction. However, ectopic expression of Hic-5 in A2780s cells induces EMT independently of TGFß1, accompanied with enhancement of cellular proliferation rate and migratory/invasive capacity and increased resistance to chemotherapeutic drugs. Moreover, Hic-5 knockdown in the EOC cells with mesenchymal morphology (SKOV3) was accompanied by induction of mesenchymal-to-epithelial transition (MET), followed by a reduction of their proliferative, migratory/invasive capacity, and increased drugs sensitivity in vitro, as well as enhanced tumor cell colonization and metastatic growth in vivo. The modulation of Hic-5 expression in EOC cells resulted in altered regulation of numerous EMT-related canonical pathways and was indicative for a possible role of Hic-5 in controlling EMT through a RhoA/ROCK mediated mechanism. To our knowledge, this is the first report examining the role of Hic-5 in EOC, and its role in maintaining the mesenchymal phenotype of EOC cells independently of exogenous TGFß1 treatment.
RESUMEN
Prodrug-mediated utilization of the cytochrome P450 (CYP) 1A1 to obtain the selective release of potent anticancer products within cancer tissues is a promising approach in chemotherapy. We herein report the rationale, preparation, biological evaluation, and mechanism of action of phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) that are antimicrotubule prodrugs activated by CYP1A1. Although PAIB-SOs are inert in most cells tested, they are highly cytocidal toward several human breast cancer cells, including hormone-independent and chemoresistant types. PAIB-SOs are N-dealkylated into cytotoxic phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) in CYP1A1-positive cancer cells, both in vitro and in vivo. In conclusion, PAIB-SOs are novel chemotherapeutic prodrugs with no equivalent among current antineoplastics and whose selective action toward breast cancer is tailored to the characteristic pattern of CYP1A1 expression observed in a large percentage of human breast tumors.
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Antimitóticos/farmacología , Bencenosulfonatos/química , Neoplasias de la Mama/tratamiento farmacológico , Citocromo P-450 CYP1A1/metabolismo , Profármacos/farmacología , Animales , Antimitóticos/farmacocinética , Bencenosulfonatos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Embrión de Pollo , Citocromo P-450 CYP1A1/genética , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Humanos , Profármacos/farmacocinéticaRESUMEN
Previously, we have identified the Grainyhead transcription factor 2 gene (GRHL2) as notably hypomethylated in high-grade (HG) serous epithelial ovarian tumors, compared with normal ovarian tissues. GRHL2 is known for its functions in normal tissue development and wound healing. In the context of cancer, the role of GRHL2 is still ambiguous as both tumorigenic and tumor suppressive functions have been reported for this gene, although a role of GRHL2 in maintaining the epithelial status of cancer cells has been suggested. In this study, we report that GRHL2 is strongly overexpressed in both low malignant potential (LMP) and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Suppression of the GRHL2 expression led to a sharp decrease in cell proliferation, migration and invasion and induced G1 cell cycle arrest in epithelial ovarian cancer (EOC) cells displaying either epithelial (A2780s) or mesenchymal (SKOV3) phenotypes. However, no phenotypic alterations were observed in these EOC cell lines following GRHL2 silencing. Gene expression profiling and consecutive canonical pathway and network analyses confirmed these data, as in both these EOC cell lines, GRHL2 ablation was associated with the downregulation of various genes and pathways implicated in cell growth and proliferation, cell cycle control and cellular metabolism. Taken together, our data are indicative for a strong oncogenic potential of the GRHL2 gene in EOC progression and support recent findings on the role of GRHL2 as one of the major phenotypic stability factors (PSFs) that stabilize the highly aggressive/metastatic hybrid epithelial/mesenchymal (E/M) phenotype of cancer cells.
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Puntos de Control del Ciclo Celular/genética , Movimiento Celular/genética , Proteínas de Unión al ADN/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Factores de Transcripción/genética , Sistemas CRISPR-Cas/genética , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Proliferación Celular/genética , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Inmunohistoquímica , Invasividad Neoplásica , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
Despite progress in our comprehension of the mechanisms regulating adipose tissue development, the nature of the factors that functionally characterize adipose precursors is still elusive. Defining the early steps regulating adipocyte development is needed for the generation of tools to control adipose tissue size and function. Here, we report the discovery of V-set and transmembrane domain containing 2A (VSTM2A) as a protein expressed and secreted by committed preadipocytes. VSTM2A expression is elevated in the early phases of adipogenesis in vitro and adipose tissue development in vivo. We show that VSTM2A-producing cells associate with the vasculature and express the common surface markers of adipocyte progenitors. Overexpression of VSTM2A induces adipogenesis, whereas its depletion impairs this process. VSTM2A controls preadipocyte determination at least in part by modulating BMP signaling and PPARγ2 activation. We propose a model in which VSTM2A is produced to preserve and amplify the adipogenic capability of adipose precursors.
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Adipogénesis , Linaje de la Célula , Proteínas de la Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Tejido Adiposo Blanco/irrigación sanguínea , Tejido Adiposo Blanco/citología , Animales , Biomarcadores/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Células 3T3 NIH , Neovascularización Fisiológica , PPAR gamma/metabolismo , Transducción de SeñalRESUMEN
Controlling the transcriptional program is essential to maintain the identity and the biological functions of a cell. The Mediator and Cohesin complexes have been established as central cofactors controlling the transcriptional program in normal cells. However, the distribution, recruitment and importance of these complexes in cancer cells have not been fully investigated. Here we show that FOXA and master transcription factors are part of the core transcriptional regulatory circuitry of cancer cells and are essential to recruit M ediator and Cohesin. Indeed, Mediator and Cohesin occupied the enhancer and promoter regions of actively transcribed genes and maintained the proliferation and colony forming potential. Through integration of publically available ChIP-Seq datasets, we predicted the core transcriptional regulatory circuitry of each cancer cell. Unexpectedly, for all cells investigated, the pioneer transcription factors FOXA1 and/or FOXA2 were identified in addition to cell-specific master transcription factors. Loss of both types of transcription factors phenocopied the loss of Mediator and Cohesin. Lastly, the master and pioneer transcription factors were essential to recruit Mediator and Cohesin to regulatory regions of actively transcribed genes. Our study proposes that maintenance of the cancer cell state is dependent on recruitment of Mediator and Cohesin through FOXA and master transcription factors.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Complejo Mediador/metabolismo , Neoplasias/metabolismo , Células A549 , Proliferación Celular , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Células Hep G2 , Humanos , Células MCF-7 , Análisis de Componente Principal , Regiones Promotoras Genéticas , Transcripción Genética , CohesinasRESUMEN
Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.
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Técnicas de Cultivo de Célula/métodos , Movimiento Celular/fisiología , Matriz Extracelular , Invasividad Neoplásica , Animales , Colágeno , Fibrina , Humanos , Imagenología Tridimensional , NeoplasiasRESUMEN
Epithelial cells that lose attachment to the extracellular matrix undergo a specialized form of apoptosis called anoikis. Here, using large-scale RNA interference (RNAi) screening, we find that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase, plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. We find that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis we show that knockdown of Kdm3a enhances metastatic potential. Finally, we find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, our results reveal a novel transcriptional regulatory program that mediates anoikis.
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Anoicis , Células Epiteliales/fisiología , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Neoplasias de la Mama/patología , Neoplasias de la Mama/secundario , Adhesión Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Pruebas Genéticas , Humanos , Ratones , Interferencia de ARNRESUMEN
Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used in vivo and in vitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), the key hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression in murine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions.
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Transdiferenciación Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Músculo Liso Vascular/patología , Fosfatos/metabolismo , Insuficiencia Renal Crónica/complicaciones , Calcificación Vascular/metabolismo , Animales , Biomarcadores/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Hipoxia/metabolismo , Inmunohistoquímica , Isoquinolinas/farmacología , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Ratas , Ratas Wistar , Insuficiencia Renal Crónica/metabolismo , Transducción de Señal , Calcificación Vascular/etiología , Rigidez VascularRESUMEN
Approximately half of the familial aggregation of breast cancer remains unexplained. This proportion is less for early-onset disease where familial aggregation is greater, suggesting that other susceptibility genes remain to be discovered. The majority of known breast cancer susceptibility genes are involved in the DNA double-strand break repair pathway. ABRAXAS is involved in this pathway and mutations in this gene impair BRCA1 recruitment to DNA damage foci and increase cell sensitivity to ionizing radiation. Moreover, a recurrent germline mutation was reported in Finnish high-risk breast cancer families. To determine if ABRAXAS could be a breast cancer susceptibility gene in other populations, we conducted a population-based case-control mutation screening study of the coding exons and exon/intron boundaries of ABRAXAS in the Breast Cancer Family Registry. In addition to the common variant p.Asp373Asn, sixteen distinct rare variants were identified. Although no significant difference in allele frequencies between cases and controls was observed for the identified variants, two variants, p.Gly39Val and p.Thr141Ile, were shown to diminish phosphorylation of gamma-H2AX in MCF7 human breast adenocarcinoma cells, an important biomarker of DNA double-strand breaks. Overall, likely damaging or neutral variants were evenly represented among cases and controls suggesting that rare variants in ABRAXAS may explain only a small proportion of hereditary breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Estudios de Asociación Genética/métodos , Polimorfismo de Nucleótido Simple , Edad de Inicio , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Humanos , Células MCF-7 , Linaje , QuebecRESUMEN
The molecular basis of epithelial ovarian cancer (EOC) dissemination is still poorly understood. Previously, we identified the mannose receptor LY75 gene as hypomethylated in high-grade (HG) serous EOC tumors, compared to normal ovarian tissues. LY75 represents endocytic receptor expressed on dendritic cells and so far, has been primarily studied for its role in antigen processing and presentation. Here we demonstrate that LY75 is overexpressed in advanced EOC and that LY75 suppression induces mesenchymal-to-epithelial transition (MET) in EOC cell lines with mesenchymal morphology (SKOV3 and TOV112), accompanied by reduction of their migratory and invasive capacity in vitro and enhanced tumor cell colonization and metastatic growth in vivo. LY75 knockdown in SKOV3 cells also resulted in predominant upregulation of functional pathways implicated in cell proliferation and metabolism, while pathways associated with cell signaling and adhesion, complement activation and immune response were mostly suppressed. Moreover, LY75 suppression had an opposite effect on EOC cell lines with epithelial phenotype (A2780s and OV2008), by directing epithelial-to-mesenchymal transition (EMT) associated with reduced capacity for in vivo EOC cell colonization, as similar/identical signaling pathways were reversely regulated, when compared to mesenchymal LY75 knockdown EOC cells.To our knowledge, this is the first report of a gene displaying such pleiotropic effects in sustaining the cellular phenotype of EOC cells and points to novel functions of this receptor in modulating EOC dissemination. Our data also support previous findings regarding the superior capacity of epithelial cancer cells in metastatic colonization of distant sites, compared to cancer cells with mesenchymal-like morphology.