RESUMEN
Aberrant cell proliferation is a hallmark of cancer, including glioblastoma (GBM). Here we report that protein arginine methyltransferase (PRMT) 6 activity is required for the proliferation, stem-like properties, and tumorigenicity of glioblastoma stem cells (GSCs), a subpopulation in GBM critical for malignancy. We identified a casein kinase 2 (CK2)-PRMT6-regulator of chromatin condensation 1 (RCC1) signaling axis whose activity is an important contributor to the stem-like properties and tumor biology of GSCs. CK2 phosphorylates and stabilizes PRMT6 through deubiquitylation, which promotes PRMT6 methylation of RCC1, which in turn is required for RCC1 association with chromatin and activation of RAN. Disruption of this pathway results in defects in mitosis. EPZ020411, a specific small-molecule inhibitor for PRMT6, suppresses RCC1 arginine methylation and improves the cytotoxic activity of radiotherapy against GSC brain tumor xenografts. This study identifies a CK2α-PRMT6-RCC1 signaling axis that can be therapeutically targeted in the treatment of GBM.
Asunto(s)
Neoplasias Encefálicas , Carcinogénesis , Proteínas de Ciclo Celular , Glioblastoma , Factores de Intercambio de Guanina Nucleótido , Mitosis/efectos de la radiación , Proteínas de Neoplasias , Proteínas Nucleares , Proteína-Arginina N-Metiltransferasas , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/efectos de la radiación , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Femenino , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Mitosis/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The pyruvate kinase M2 isoform (PKM2) is preferentially expressed in cancer cells to regulate anabolic metabolism. Although PKM2 was recently reported to regulate lipid homeostasis, the molecular mechanism remains unclear. Herein, we discovered an ER transmembrane protein 33 (TMEM33) as a downstream effector of PKM2 that regulates activation of SREBPs and lipid metabolism. Loss of PKM2 leads to up-regulation of TMEM33, which recruits RNF5, an E3 ligase, to promote SREBP-cleavage activating protein (SCAP) degradation. TMEM33 is transcriptionally regulated by nuclear factor erythroid 2-like 1 (NRF1), whose cleavage and activation are controlled by PKM2 levels. Total plasma cholesterol levels are elevated by either treatment with PKM2 tetramer-promoting agent TEPP-46 or by global PKM2 knockout in mice, highlighting the essential function of PKM2 in lipid metabolism. Although depletion of PKM2 decreases cancer cell growth, global PKM2 knockout accelerates allografted tumor growth. Together, our findings reveal the cell-autonomous and systemic effects of PKM2 in lipid homeostasis and carcinogenesis, as well as TMEM33 as a bona fide regulator of lipid metabolism.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo de los Lípidos/fisiología , Proteínas de la Membrana/metabolismo , Hormonas Tiroideas/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/genética , Línea Celular Tumoral , Colesterol/sangre , Femenino , Regulación Neoplásica de la Expresión Génica , Homeostasis , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones Noqueados , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Hormonas Tiroideas/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión a Hormona TiroideRESUMEN
Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular â¼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.
Asunto(s)
Modelos Animales de Enfermedad , GTP Fosfohidrolasas/genética , Leucemia Mieloide Aguda/patología , Leucemia Mielomonocítica Crónica/patología , Proteínas de la Membrana/genética , Mutación , Proteínas Represoras/genética , Microambiente Tumoral , Animales , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/inmunología , Ratones , Proteínas de Unión al GTP Monoméricas/genética , Fenotipo , Transducción de SeñalRESUMEN
As a popular sample preparation approach, filter-aided sample preparation (FASP) has been widely used in proteomic analysis. However, several limitations have been noted, including sample loss during filtration, repetitive centrifugation steps, and the possibility of breakage of filtration membrane. Extraction bias among different sample preparation strategies presents another challenge. To overcome these limitations and address remaining challenges, we developed a novel surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion (SCAD) protocol. The new strategy resulted in higher protein yield and improved peptide recovery and protein coverage compared to two conventional sample preparation methods (FASP and urea). In combination of three strategies, more than 10,000 distinct protein groups were identified with 1% FDR from MDA-MB-231 cells without any prefractionation. This in-depth proteome analysis was accomplished by optimization of protein extraction, enzymatic digestion, LC gradient, and peptide sequencing method. Ingenuity Pathways Analysis (IPA) of proteins exclusively identified in SCAD revealed several crucial signaling pathways that regulate breast cancer progression. SCAD also enabled an unbiased extraction of different categories of proteins (membrane, intracellular, nuclear) associated with tumorigenesis, which integrates the advantages of FASP and urea extraction. This novel strategy expedites comprehensive protein identification, which is applicable for biomarker discovery in various types of cancers.
Asunto(s)
Proteínas/metabolismo , Proteómica/métodos , Manejo de Especímenes/métodos , Tensoactivos/química , Secuencia de Aminoácidos , Neoplasias de la Mama/metabolismo , Carcinogénesis , Línea Celular Tumoral , Femenino , Humanos , Proteínas/análisis , Proteínas/aislamiento & purificación , Proteolisis , Transducción de Señal , UreaRESUMEN
The biologic effects of estrogens are transduced by two estrogen receptors (ERs), ERα and ERß, which function in dimer forms. The ERα/α homodimer promotes and the ERß/ß inhibits estrogen-dependent growth of mammary epithelial cells; the functions of ERα/ß heterodimers remain elusive. Using compounds that promote ERα/ß heterodimerization, we have previously shown that ERα/ß heterodimers appeared to inhibit tumor cell growth and migration in vitro. Further dissection of ERα/ß heterodimer functions was hampered by the lack of ERα/ß heterodimer-specific ligands. Herein, we report a multistep workflow to identify the selective ERα/ß heterodimer-inducing compound. Phytoestrogenic compounds were first screened for ER transcriptional activity using reporter assays and ER dimerization preference using a bioluminescence resonance energy transfer assay. The top hits were subjected to in silico modeling to identify the pharmacophore that confers ERα/ß heterodimer specificity. The pharmacophore encompassing seven features that are potentially important for the formation of the ERα/ß heterodimer was retrieved and subsequently used for virtual screening of large chemical libraries. Four chemical compounds were identified that selectively induce ERα/ß heterodimers over their respective homodimers. Such ligands will become unique tools to reveal the functional insights of ERα/ß heterodimers.
Asunto(s)
Biología Computacional/métodos , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Glándulas Mamarias Humanas/citología , Fitoestrógenos/farmacología , Transferencia de Energía por Resonancia de Bioluminiscencia , Línea Celular , Evaluación Preclínica de Medicamentos , Receptor alfa de Estrógeno/química , Receptor beta de Estrógeno/química , Femenino , Células HEK293 , Humanos , Ligandos , Células MCF-7 , Glándulas Mamarias Humanas/metabolismo , Modelos Moleculares , Fitoestrógenos/química , Multimerización de ProteínaRESUMEN
The system-wide site-specific analysis of intact glycopeptides is crucial for understanding the exact functional relevance of protein glycosylation. A dedicated workflow with the capability to simultaneously characterize and quantify intact glycopeptides in a site-specific and high-throughput manner is essential to reveal specific glycosylation alteration patterns in complex biological systems. In this study, an enhanced, dedicated, large-scale site-specific quantitative N-glycoproteomics workflow has been established, which includes improved specific extraction of membrane-bound glycoproteins using the filter aided sample preparation (FASP) method, enhanced enrichment of N-glycopeptides using sequential hydrophilic interaction liquid chromatography (HILIC) and multi-lectin affinity (MLA) enrichment, site-specific N-glycopeptide characterization enabled by EThcD, relative quantitation utilizing isobaric N,N-dimethyl leucine (DiLeu) tags and automated FDR-based large-scale data analysis by Byonic. For the first time, our study shows that HILIC complements to a very large extent to MLA enrichment with only 20% overlapping in enriching intact N-glycopeptides. When applying the developed workflow to site-specific N-glycoproteome study in PANC1 cells, we were able to identify 1067 intact N-glycopeptides, representing 311 glycosylation sites and 88 glycan compositions from 205 glycoproteins. We further applied this approach to study the glycosylation alterations in PKM2 knockout cells vs. parental breast cancer cells and revealed altered N-glycoprotein/N-glycopeptide patterns and very different glycosylation microheterogeneity for different types of glycans. To obtain a more comprehensive map of glycoprotein alterations, N-glycopeptides after treatment with PNGase F were also analyzed. A total of 484 deglycosylated peptides were quantified, among which 81 deglycosylated peptides from 70 glycoproteins showed significant changes. KEGG pathway analysis revealed that the PI3K/Akt signaling pathway was highly enriched, which provided evidence to support the previous finding that PKM2 knockdown cancer cells rely on activation of Akt for their survival. With glycosylation being one of the most important signaling modulators, our results provide additional evidence that signaling pathways are closely regulated by metabolism.
Asunto(s)
Neoplasias de la Mama/química , Glicopéptidos/análisis , Glicoproteínas/análisis , Proteínas Portadoras/genética , Línea Celular Tumoral , Electrones , Técnicas de Inactivación de Genes , Glicosilación , Humanos , Leucina , Proteínas de la Membrana/genética , Hormonas Tiroideas/genética , Proteínas de Unión a Hormona TiroideRESUMEN
The oncoprotein hepatitis B X-interacting protein (HBXIP) results in the dysregulation of lipid metabolism to enhance the development of breast cancer. Acyl-CoA synthetase long-chain family member 1 (ACSL1) is required for thioesterification of long-chain fatty acids into their acyl-CoA derivatives. In this study, we present a hypothesis that HBXIP might be involved in the regulation of ACSL1 in breast cancer. Interestingly, we found that the overexpression of HBXIP was able to up-regulate ACSL1 at the levels of mRNA and protein in a dose-dependent manner in breast cancer cells. Conversely, silencing of HBXIP led to the opposite results. Mechanistically, HBXIP as a coactivator interacted with transcriptional factor Sp1 through binding to the promoter of ACSL1 by ChIP assays analysis, leading to the transcription of ACSL1 in breast cancer cells. Immunohistochemistry staining revealed that the positive rate of ACSL1 was 71.4% (35/49) in clinical breast cancer tissues, HBXIP 79.6% (39/49), in which the positive rate of ACSL1 was 76.9% (30/39) in the HBXIP-positive specimens. But, few positive rate of ACSL1 10% (1/10) was observed in normal breast tissues. The mRNA levels of ACSL1 were significantly higher in clinical breast cancer tissues than those in their corresponding peritumor tissues. The mRNA levels of ACSL1 were positively associated with those of HBXIP in clinical breast cancer tissues. Thus, we conclude that the oncoprotein HBXIP is able to up-regulate ACSL1 through activating the transcriptional factor Sp1 in breast cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Coenzima A Ligasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Factor de Transcripción Sp1/metabolismo , Línea Celular Tumoral , Humanos , Transducción de Señal , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Tumor angiogenesis plays an important role in the development of cancer. Previously, we reported that hepatitis B X-interacting protein (HBXIP) functioned as an oncoprotein in breast cancer. However, the role of HBXIP in angiogenesis in breast cancer remains poorly understood. In the present study, we show that the oncoprotein HBXIP plays crucial roles in the event. We observed that the expression levels of HBXIP were positively correlated with those of fibroblast growth factor 8 (FGF8) or vascular endothelial growth factor (VEGF) in clinical breast cancer tissues. Then, we demonstrated that HBXIP was able to upregulate FGF8 through activation of its promoter involving direct binding to cAMP response element-binding protein (CREB) in breast cancer cells and thereby increased its secretion. Strikingly, we identified another pathway that HBXIP upregulated FGF8 and VEGF through inhibiting miRNA-503, which directly targeted 3' untranslated region of FGF8 or VEGF mRNA in the cells. Moreover, we revealed that HBXIP-induced FGF8 could upregulate VEGF expression through activating phosphoinositide 3-kinase (PI3K)/Akt/hypoxia-inducible factor 1-alpha (HIF1α) signaling and increase its secretion. In function, matrigel angiogenesis assay and hemoglobin content analysis uncovered that HBXIP-enhanced FGF8/VEGF boosted tumor angiogenesis and growth in breast cancer in vitro and in vivo in a paracrine/autocrine manner. Thus, we conclude that HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF. Our finding provides new insights into the mechanism of tumor angiogenesis in breast cancer. Therapeutically, HBXIP may serve as a novel target of tumor angiogenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Neovascularización Patológica/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Factor 8 de Crecimiento de Fibroblastos/genética , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , MicroARNs/genética , Neovascularización Patológica/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Activación Transcripcional , Carga Tumoral/genéticaRESUMEN
BACKGROUND: Hepatitis B virus X protein (HBx) plays crucial roles in hepatocarcinogenesis. However, the underlying mechanism remains elusive. We have reported that HBx is able to up-regulate survivin in hepatocellular carcinoma tissues. The oncopreotein hepatitis B X-interacting protein (HBXIP), a target of miR-520b, is involved in the development of cancer. In this study, we focus on the investigation of hepatocarcinogenesis mediated by HBx. METHODS: The expression of HBx and survivin was examined in the liver tissues of HBx-Tg mice. The effect of HBx/survivin on the growth of LO2-X-S cells was determined by colony formation and transplantation in nude mice. The effect of HBx/survivin on promoter of miR-520b was determined by Western blot analysis, luciferase reporter gene assay, co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP), respectively. The expression of HBx, survivin and HBXIP was detected by immunohistochemistry and real-time PCR in clinical HCC tissues, respectively. The DNA demethylation of HBXIP promoter was examined. The functional influence of miR-520b and HBXIP on proliferation of hepatoma cells was analyzed by MTT, colony formation, EdU and transplantation in nude mice in vitro and in vivo. RESULTS: In this study, we provided evidence that HBx up-regulated survivin in the liver cancer tissues of HBx-Tg mice aged 18 M. The engineered LO2 cell lines with survivin and/or HBx were successfully established, termed LO2-X-S. MiR-520b was down-regulated in LO2-X-S cells and clinical HCC tissues. Our data revealed that HBx survivin-dependently down-regulated miR-520b through interacting with Sp1 in the cells. HBXIP was highly expressed in LO2-X-S cells, liver cancer tissues of HBx-Tg mice aged 18 M and clinical HCC tissues (75.17%, 112/149). The expression level of HBXIP was positively associated with those of HBx or survivin in clinical HCC tissues. In addition, we showed that HBx survivin-dependently up-regulated HBXIP through inducing demethylation of HBXIP promoter in LO2-X-S cells and clinical HCC tissues. In function, low level miR-520b and high level HBXIP mediated by HBx with partner survivin contributed to the growth of LO2-X-S cells in vitro and in vivo. CONCLUSION: HBx accelerates hepatocarcinogenesis with partner survivin through modulating tumor suppressor miR-520b and oncoprotein HBXIP.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma Hepatocelular/genética , Hepacivirus/genética , Proteínas Inhibidoras de la Apoptosis/genética , Neoplasias Hepáticas/genética , MicroARNs/genética , Transactivadores/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinogénesis/genética , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular , Metilación de ADN , Regulación de la Expresión Génica , Hepacivirus/metabolismo , Hepatitis B/complicaciones , Hepatitis B/genética , Hepatitis B/metabolismo , Hepatitis B/patología , Interacciones Huésped-Patógeno , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Ratones Transgénicos , MicroARNs/metabolismo , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Transducción de Señal , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Survivin , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3'UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.
Asunto(s)
Proliferación Celular , MicroARNs/genética , Mutación , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Transactivadores/genética , Regiones no Traducidas 5'/genética , Animales , Secuencia de Bases , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Interferencia de ARN , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias Virales , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
AIM: MiR-506 is a miRNA involved in carcinogenesis of several kinds of cancer. In this study, we explored whether miR-506 played a critical role in hepatocellular carcinoma (HCC). METHODS: Twenty HCC and adjacent normal liver tissue samples were collected. Human hepatoma cell lines HepG2 and H7402 were used for in vitro studies. The expression of miR-506 and transcriptional co-activator YAP was examined using qRT-PCR. Western blot analysis was used to measure the expression of YAP and its target genes. Luciferase reporter gene assay was used to identify YAP as a target gene of miR-506. MTT and EdU assays were carried out for functional analysis. RESULTS: The expression of miR-506 was significantly lower in HCC than in adjacent normal liver tissues. Bioinformatics analysis revealed that YAP mRNA might be one of the targets of miR-506, and miR-506 in HCC tissues was found to be negatively correlated with YAP (r=-0.605). In both HepG2 and H7402 cells, miR-506 dose-dependently down-regulated YAP and its target genes c-Myc and the connective tissue growth factor (CTGF). Luciferase reporter gene assays demonstrated that miR-506 targeted the wild type 3'UTR of YAP mRNA, but not a 3'UTR with a mutant seed site. Furthermore, miR-506 significantly inhibited the proliferation of HepG2 and H7402 cells, while anti-miR-506 enhanced the cell proliferation, which was blocked by YAP siRNA. CONCLUSION: MiR-506 suppresses the proliferation of hepatoma cells by targeting YAP mRNA.
Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma Hepatocelular/genética , Proliferación Celular/genética , Neoplasias Hepáticas/genética , MicroARNs/genética , Fosfoproteínas/genética , ARN Mensajero/genética , Línea Celular Tumoral , Células Hep G2 , Humanos , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Hepatitis B virus X protein (HBx) contributes to the development of hepatocellular carcinoma (HCC) through inducing dysregulation of lipogenesis. However, the mechanism by which HBx induces the abnormal lipogenesis is not well known. In this study, we report that the oncogene Rab18, a member of Ras family, enhances the HBx-induced hepatocarcinogenesis through inducing dysregulation of lipogenesis and proliferation. Our data showed that the expression levels of Rab18 were positively associated with those of HBx in clinical HCC tissues. HBx was able to upregulate the expression of Rab18 in p21-HBx transgenic mice and hepatoma cell lines. Next, we identified the mechanism by which HBx upregulated Rab18. The results demonstrated that cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) were able to stimulate Rab18 promoter through activating transcription factor activator protein 1 (AP-1) and cyclic adenosine 3',5'-monophosphate response element-binding (CREB). In addition, we identified another pathway that HBx activated Rab18. We found that miR-429 was able to directly target the 3' untranslated region of Rab18, suggesting that Rab18 is one of the target genes of miR-429. Then, we found that HBx was able to downregulate miR-429 in hepatoma cells. The oil red O staining showed that HBx resulted in the dysregulation of lipogenesis through Rab18. Moreover, Rab18 contributed to the HBx-enhanced proliferation of hepatoma cells in vitro and in vivo. HBx enhances hepatocarcinogenesis through leading to the dysregulation of lipogenesis and proliferation of hepatoma cells, involving two pathways such as HBx/COX-2/5-LOX/AP-1/CREB/Rab18 and HBx/miR-429/Rab18.
Asunto(s)
Carcinoma Hepatocelular/patología , Proliferación Celular , Lipogénesis , Transactivadores/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Sitios de Unión , Carcinoma Hepatocelular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Femenino , Regulación Viral de la Expresión Génica , Células HEK293 , Células Hep G2 , Virus de la Hepatitis B , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Regiones Promotoras Genéticas , Transactivadores/genética , Regulación hacia Arriba , Proteínas Reguladoras y Accesorias Virales , Proteínas de Unión al GTP rab/genéticaRESUMEN
Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) is a prevalent post-translational modification (PTM) that regulates diverse cellular processes. Aberrant expression of type I PRMTs that catalyze asymmetric arginine dimethylation (ADMA) is often found in cancer, though little is known about the ADMA status of substrate proteins in tumors. Using LC-MS/MS along with pan-specific ADMA antibodies, we performed global mapping of ADMA in five patient-derived xenograft (PDX) tumors representing different subtypes of human breast cancer. In total, 403 methylated sites from 213 proteins were identified, including 322 novel sites when compared to the PhosphositesPlus database. Moreover, using peptide arrays in vitro, approximately 70% of the putative substrates were validated to be methylated by PRMT1, PRMT4, and PRMT6. Notably, when compared with our previously identified ADMA sites from breast cancer cell lines, only 75 ADMA sites overlapped between cell lines and PDX tumors. Collectively, this study provides a useful resource for both PRMT and breast cancer communities for further exploitation of the functions of PRMT dysregulation during breast cancer progression.
Asunto(s)
Neoplasias de la Mama , Proteoma , Humanos , Femenino , Cromatografía Liquida , Arginina/metabolismo , Espectrometría de Masas en Tándem , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Proteínas NuclearesRESUMEN
PURPOSE: Bile acids are steroid synthesized in liver, which are essential for fat emulsification, cholesterol excretion and gut microbial homeostasis. However, the role of bile acids in leukemia progression remains unclear. We aim at exploring the effects and mechanisms of chenodeoxycholic acid (CDCA), a type of bile acids, on acute myeloid leukemia (AML) progression. RESULTS: Here, we found that CDCA was decreased in feces and plasma of AML patients, positively correlated with the diversity of gut microbiota, and negatively associated with AML prognosis. We further demonstrated that CDCA suppressed AML progression both in vivo and in vitro. Mechanistically, CDCA bound to mitochondria to cause mitochondrial morphology damage containing swelling and reduction of cristae, decreased mitochondrial membrane potential and elevated mitochondrial calcium level, which resulted in the production of excessive reactive oxygen species (ROS). Elevated ROS further activated p38 MAPK signaling pathway, which collaboratively promoted the accumulation of lipid droplets (LDs) through upregulating the expression of the diacylglycerol O-acyltransferase 1 (DGAT1). As the consequence of the abundance of ROS and LDs, lipid peroxidation was enhanced in AML cells. Moreover, we uncovered that CDCA inhibited M2 macrophage polarization and suppressed the proliferation-promoting effects of M2 macrophages on AML cells in co-cultured experiments. CONCLUSION: Our findings demonstrate that CDCA suppresses AML progression through synergistically promoting LDs accumulation and lipid peroxidation via ROS/p38 MAPK/DGAT1 pathway caused by mitochondrial dysfunction in leukemia cells and inhibiting M2 macrophage polarization.
Asunto(s)
Leucemia Mieloide Aguda , Proteínas Quinasas p38 Activadas por Mitógenos , Ácidos y Sales Biliares/metabolismo , Calcio/metabolismo , Ácido Quenodesoxicólico/metabolismo , Ácido Quenodesoxicólico/farmacología , Colesterol/metabolismo , Diacilglicerol O-Acetiltransferasa/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Peroxidación de Lípido , Macrófagos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Protein arginine methyltransferases (PRMTs) introduce arginine methylation, a post-translational modification with the increasingly eminent role in normal physiology and disease. PRMT4 or coactivator-associated arginine methyltransferase 1 (CARM1) is a propitious target for cancer therapy; however, few CARM1 substrates are known, and its mechanism of substrate recognition is poorly understood. Here we employed a quantitative mass spectrometry approach to globally profile CARM1 substrates in breast cancer cell lines. We identified >130 CARM1 protein substrates and validated in vitro >90% of sites they encompass. Bioinformatics analyses reveal enrichment of proline-containing motifs, in which both methylation sites and their proximal sequences are frequently targeted by somatic mutations in cancer. Finally, we demonstrate that the N-terminus of CARM1 is involved in substrate recognition and nearly indispensable for substrate methylation. We propose that development of CARM1-specific inhibitors should focus on its N-terminus and predict that other PRMTs may employ similar mechanism for substrate recognition.
Asunto(s)
Secuencias de Aminoácidos/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteína-Arginina N-Metiltransferasas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Arginina/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Cromatografía Líquida de Alta Presión/métodos , Biología Computacional , Inhibidores Enzimáticos , Femenino , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Células MCF-7 , Metilación , Terapia Molecular Dirigida/métodos , Mutación , Prolina/química , Prolina/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Especificidad por Sustrato/fisiología , Espectrometría de Masas en Tándem/métodosRESUMEN
Metabolic reprogramming is a hallmark of cancer. Herein we discover that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator-associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondrial calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol-1,4,5-trisphosphate receptors (InsP3Rs), methylated PKM2 inhibits the influx of calcium from the endoplasmic reticulum to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticles perturbs the metabolic energy balance in cancer cells, leading to a decrease in cell proliferation, migration and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to InsP3R-dependent mitochondrial functions.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Carcinogénesis/metabolismo , Carcinogénesis/patología , Proteínas Portadoras/metabolismo , Glucólisis/fisiología , Guanilato Ciclasa/metabolismo , Proteínas de la Membrana/metabolismo , Hormonas Tiroideas/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Retículo Endoplásmico/metabolismo , Femenino , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Células MCF-7 , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/metabolismo , Metástasis de la Neoplasia/patología , Fosforilación Oxidativa , Proteínas de Unión a Hormona TiroideRESUMEN
This corrects the article DOI: 10.1038/ncb3630.
RESUMEN
Abnormal lipid metabolism is a hallmark of tumorigenesis. Accumulating evidence demonstrates that fatty acid synthase (FAS, FASN) is a metabolic oncogene that supports the growth and survival of tumor cells and is highly expressed in many cancers. Here, we report that the oncoprotein, hepatitis B X-interacting protein (HBXIP, LAMTOR5) contributes to abnormal lipid metabolism. We show that high expression of HBXIP in 236 breast cancer patients was significantly associated with decreased overall survival and progression-free survival. Interestingly, the expression of HBXIP was positively related to that of FAS in clinical breast cancer tissues, and HBXIP overexpression in breast cancer cells resulted in FAS upregulation. Mechanistically, HBXIP upregulated SREBP-1c (SREBF1), which activates the transcription of FAS, by directly interacting with and coactivating nuclear receptor (NR) liver X receptors (LXR). Physiologically, LXRs are activated via a coactivator containing NR motif in a ligand-dependent manner. However, in breast cancer cells, HBXIP containing the corepressor/nuclear receptor motif with special flanking sequence could coactivate LXRs independent of ligand. Moreover, overexpressed SREBP-1c was able to activate the transcription of HBXIP, forming a positive-feedback loop. Functionally, HBXIP enhanced lipogenesis, resulting in the growth of breast cancer cells in vitro and in vivo Thus, we conclude that the oncoprotein HBXIP contributes to the abnormal lipid metabolism in breast cancer through LXRs/SREBP-1c/FAS signaling, providing new insights into the mechanisms by which cancer cells reprogram lipid metabolism in their favor. Cancer Res; 76(16); 4696-707. ©2016 AACR.