RESUMEN
Iron is essential for normal brain development and function. Hence, understanding the mechanisms of iron efflux at the blood-brain barrier and their regulation are critical for the establishment of brain iron homeostasis. Here, we have investigated the role of exosomes in mediating the transfer of H-ferritin (FTH1)- or transferrin (Tf)-bound iron across the blood-brain barrier endothelial cells (BBBECs). Our study used ECs derived from human-induced pluripotent stem cells that are grown in bicameral chambers. When cells were exposed to 55Fe-Tf or 55Fe-FTH1, the 55Fe activity in the exosome fraction in the basal chamber was significantly higher compared to the supernatant fraction. Furthermore, we determined that the release of endogenous Tf, FTH1, and exosome number is regulated by the iron concentration of the endothelial cells. Moreover, the release of exogenously added Tf or FTH1 to the basal side via exosomes was significantly higher when ECs were iron loaded compared to when they were iron deficient. The release of exosomes containing iron bound to Tf or FTH1 was independent of hepcidin regulation, indicating this mechanism by-passes a major iron regulatory pathway. A potent inhibitor of exosome formation, GW4869, reduced exosomes released from the ECs and also decreased the Tf- and FTH1-bound iron within the exosomes. Collectively, these results indicate that iron transport across the blood-brain barrier is mediated via the exosome pathway and is modified by the iron status of the ECs, providing evidence for a novel alternate mechanism of iron transport into the brain.
Asunto(s)
Barrera Hematoencefálica , Exosomas , Hierro , Humanos , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Exosomas/metabolismo , Hierro/metabolismo , Transferrina/metabolismo , Transporte BiológicoRESUMEN
RNA-binding proteins (RBPs) regulate the expression of large cohorts of RNA species to produce programmatic changes in cellular phenotypes. To describe the function of RBPs within a cell, it is key to identify their mRNA-binding partners. This is often done by crosslinking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, which involves complex processing with attendant sample losses, thus large amounts of starting materials and prone to artifacts. To evaluate potential alternative technologies, we tested "exclusion-based" purification of immunoprecipitates (IFAST or SLIDE) and report here that these methods can efficiently, rapidly, and specifically isolate RBP-RNA complexes. The analysis requires less than 1% of the starting material required for techniques that include crosslinking. Depending on the antibody used, 50% to 100% starting protein can be retrieved, facilitating the assay of endogenous levels of RBPs; the isolated ribonucleoproteins are subsequently analyzed using standard techniques, to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA-binding partners for RBP IGF2BP1 in cultured mammary epithelial cells are enriched in mRNAs important for detoxifying superoxides (specifically glutathione peroxidase [GPX]-1 and GPX-2) and mRNAs encoding mitochondrial proteins. We show that these interactions are functionally significant, as loss of function of IGF2BP1 leads to destabilization of GPX mRNAs and reduces mitochondrial membrane potential and oxygen consumption. We speculate that this underlies a consistent requirement for IGF2BP1 for the expression of clonogenic activity in vitro.
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Glándulas Mamarias Animales , Glándulas Mamarias Humanas , Proteínas de Unión al ARN , Animales , Células Epiteliales/metabolismo , Femenino , Humanos , Inmunoprecipitación , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/metabolismo , ARN/metabolismo , ARN Mensajero , Proteínas de Unión al ARN/metabolismoRESUMEN
Excessive brain iron accumulation is observed early in the onset of Alzheimer's disease, notably prior to widespread proteinopathy. These findings suggest that increases in brain iron levels are due to a dysregulation of the iron transport mechanism at the blood-brain barrier. Astrocytes release signals (apo- and holo-transferrin) that communicate brain iron needs to endothelial cells in order to modulate iron transport. Here we use iPSC-derived astrocytes and endothelial cells to investigate how early-disease levels of amyloid-ß disrupt iron transport signals secreted by astrocytes to stimulate iron transport from endothelial cells. We demonstrate that conditioned media from astrocytes treated with amyloid-ß stimulates iron transport from endothelial cells and induces changes in iron transport pathway proteins. The mechanism underlying this response begins with increased iron uptake and mitochondrial activity by the astrocytes, which in turn increases levels of apo-transferrin in the amyloid-ß conditioned astrocyte media leading to increased iron transport from endothelial cells. These novel findings offer a potential explanation for the initiation of excessive iron accumulation in early stages of Alzheimer's disease. What's more, these data provide the first example of how the mechanism of iron transport regulation by apo- and holo-transferrin becomes misappropriated in disease that can lead to iron accumulation. The clinical benefit from understanding early dysregulation in brain iron transport in AD cannot be understated. If therapeutics can target this early process, they could possibly prevent the detrimental cascade that occurs with excessive iron accumulation.
RESUMEN
BACKGROUND: Apo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood-brain barrier. Apo-Tf indicates an iron-deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanisms of apo- and holo-Tf influence on iron release was largely unknown. METHODS: Here we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism by which apo- and holo-Tf influence cellular iron release. Given the established role of hepcidin in regulating cellular iron release, we further explored the relationship of hepcidin to transferrin in this model. RESULTS: We demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly interacts with ferroportin, whereas apo-Tf directly interacts with hephaestin. Only pathophysiological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, but similar hepcidin levels are unable to interfere with the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin's ability to more rapidly internalize ferroportin compared to holo-Tf. CONCLUSIONS: These novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein-protein interactions, and offer a model for how holo-Tf and hepcidin cooperate to suppress iron release. These results expand on our previous reports on mechanisms mediating regulation of brain iron uptake to provide a more thorough understanding of the regulatory mechanisms mediating cellular iron release in general.
Asunto(s)
Hepcidinas , Transferrina , Humanos , Transferrina/metabolismo , Hepcidinas/metabolismo , Células Endoteliales/metabolismo , Células HEK293RESUMEN
High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS' function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.
Asunto(s)
Quinasa de la Caseína II/genética , Resistencia a Antineoplásicos , Regulación Leucémica de la Expresión Génica , Factor de Transcripción Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína bcl-X/genética , Animales , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológicoRESUMEN
Igf2bp1 is an oncofetal RNA binding protein whose expression in numerous types of cancers is associated with upregulation of key pro-oncogenic RNAs, poor prognosis, and reduced survival. Importantly, Igf2bp1 synergizes with mutations in Kras to enhance signalling and oncogenic activity, suggesting that molecules inhibiting Igf2bp1 could have therapeutic potential. Here, we isolate a small molecule that interacts with a hydrophobic surface at the boundary of Igf2bp1 KH3 and KH4 domains, and inhibits binding to Kras RNA. In cells, the compound reduces the level of Kras and other Igf2bp1 mRNA targets, lowers Kras protein, and inhibits downstream signalling, wound healing, and growth in soft agar, all in the absence of any toxicity. This work presents an avenue for improving the prognosis of Igf2bp1-expressing tumours in lung, and potentially other, cancer(s).
Asunto(s)
Antineoplásicos/farmacología , Carcinogénesis/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Unión Proteica/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an mRNA-binding protein that has an oncofetal pattern of expression. It is also expressed in intestinal tissue, suggesting that it has a possible role in intestinal homeostasis. To investigate this possibility, here we generated Villin CreERT2:Igf2bp1flox/flox mice, which enabled induction of an IGF2BP1 knockout specifically in intestinal epithelial cells (IECs) of adult mice. Using gut barrier and epithelial permeability assays and several biochemical approaches, we found that IGF2BP1 ablation in the adult intestinal epithelium causes mild active colitis and mild-to-moderate active enteritis. Moreover, the IGF2BP1 deletion aggravated dextran sodium sulfate-induced colitis. We also found that IGF2BP1 removal compromises barrier function of the intestinal epithelium, resulting from altered protein expression at tight junctions. Mechanistically, IGF2BP1 interacted with the mRNA of the tight-junction protein occludin (Ocln), stabilizing Ocln mRNA and inducing expression of occludin in IECs. Furthermore, ectopic occludin expression in IGF2BP1-knockdown cells restored barrier function. We conclude that IGF2BP1-dependent regulation of occludin expression is an important mechanism in intestinal barrier function maintenance and in the prevention of colitis.
Asunto(s)
Ocludina/metabolismo , Permeabilidad , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular , Colitis/inducido químicamente , Colitis/mortalidad , Colitis/patología , Colon/patología , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ocludina/genética , Unión Proteica , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Índice de Severidad de la Enfermedad , Tasa de Supervivencia , Proteínas de Uniones Estrechas/metabolismo , Regulación hacia ArribaRESUMEN
Due to hurdles, including resistance, adverse effects, and poor bioavailability, among others linked with existing therapies, there is an urgent unmet need to devise new, safe, and more effective treatment modalities for skin cancers. Herein, a series of flavonol-based derivatives of fisetin, a plant-based flavonoid identified as an anti-tumorigenic agent targeting the mammalian targets of rapamycin (mTOR)-regulated pathways, were synthesized and fully characterized. New potential inhibitors of receptor tyrosine kinases (c-KITs), cyclin-dependent kinase-2 (CDK2), and mTOR, representing attractive therapeutic targets for melanoma and non-melanoma skin cancers (NMSCs) treatment, were identified using inverse-docking, in vitro kinase activity and various cell-based anticancer screening assays. Eleven compounds exhibited significant inhibitory activities greater than the parent molecule against four human skin cancer cell lines, including melanoma (A375 and SK-Mel-28) and NMSCs (A431 and UWBCC1), with IC50 values ranging from 0.12 to < 15 µM. Seven compounds were identified as potentially potent single, dual or multi-kinase c-KITs, CDK2, and mTOR kinase inhibitors after inverse-docking and screening against twelve known cancer targets, followed by kinase activity profiling. Moreover, the potent compound F20, and the multi-kinase F9 and F17 targeted compounds, markedly decreased scratch wound closure, colony formation, and heightened expression levels of key cancer-promoting pathway molecular targets c-Kit, CDK2, and mTOR. In addition, these compounds downregulated Bcl-2 levels and upregulated Bax and cleaved caspase-3/7/8 and PARP levels, thus inducing apoptosis of A375 and A431 cells in a dose-dependent manner. Overall, compounds F20, F9 and F17, were identified as promising c-Kit, CDK2 and mTOR inhibitors, worthy of further investigation as therapeutics, or as adjuvants to standard therapies for the control of melanoma and NMSCs.
Asunto(s)
Antineoplásicos/farmacología , Flavonoles/farmacología , Melanoma/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Neoplasias Cutáneas/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Flavonoles/síntesis química , Flavonoles/química , Humanos , Melanoma/metabolismo , Melanoma/patología , Estructura Molecular , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales CultivadasRESUMEN
Melanoma is one of the deadliest forms of skin cancer. Although BRAF inhibitors significantly enhance survival of metastatic melanoma patients, most patients relapse after less than a year of treatment. We previously reported that mRNA binding protein Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is overexpressed in metastatic melanoma and that expression of IGF2BP1 confers resistance to chemotherapeutic agents. Here we demonstrate that IGF2BP1 plays an important role in the sensitivity of melanoma to targeted therapy. Inhibition of IGF2BP1 enhances the effects of BRAF-inhibitor and BRAF-MEK inhibitors in BRAFV600E melanoma. Also, knockdown of IGF2BP1 alone is sufficient to reduce tumorigenic characteristics in vemurafenib-resistant melanoma. These findings suggest that IGF2BP1 can be a novel therapeutic target for melanoma.
Asunto(s)
Resistencia a Antineoplásicos , Melanoma/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas de Unión al ARN/genética , Vemurafenib/farmacología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Melanoma/tratamiento farmacológico , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
MicroRNAs (miRNAs), small 22-25 nucleotide non-coding RNAs, play important roles in cellular and tumor biology. However, characterizing miRNA function remains challenging due to an abundance of predicted targets and an experimental bottleneck in identifying biologically relevant direct targets. Here, we developed a novel technique (miFAST) to identify direct miRNA target genes. Using miFAST, we confirmed several previously reported miR-340 target genes and identified five additional novel direct miR-340 targets in melanoma cells. This methodology can also be efficiently applied for the global characterization of miRNA targets. Utilizing miFAST to characterize direct miRNA targetomes will further our understanding of miRNA biology and function.
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Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Biología Molecular/métodos , Línea Celular Tumoral , Perfilación de la Expresión Génica/instrumentación , Humanos , Biología Molecular/instrumentación , Reproducibilidad de los ResultadosRESUMEN
Non-melanoma skin cancers (NMSCs) are the leading cause of skin cancer-related morbidity and mortality. Effective strategies are needed to control NMSC occurrence and progression. Non-toxic, plant-derived extracts have been shown to exert multiple anti-cancer effects. Graviola (Annona muricata), a tropical fruit-bearing plant, has been used in traditional medicine against multiple human diseases including cancer. The current study investigated the effects of graviola leaf and stem extract (GLSE) and its solvent-extracted fractions on two human NMSC cell lines, UW-BCC1 and A431. GLSE was found to: (i) dose-dependently suppress UW-BCC1 and A431 cell growth, motility, wound closure, and clonogenicity; (ii) induce G0/G1 cell cycle arrest by downregulating cyclin/cdk factors while upregulating cdk inhibitors, and (iii) induce apoptosis as evidenced by cleavage of caspases-3, -8 and PARP. Further, GLSE suppressed levels of activated hedgehog (Hh) pathway components Smo, Gli 1/2, and Shh while inducing SuFu. GLSE also decreased the expression of pro-apoptotic protein Bax while decreasing the expression of the anti-apoptotic protein Bcl-2. We determined that these activities were concentrated in an acetogenin/alkaloid-rich dichloromethane subfraction of GLSE. Our data identify graviola extracts and their constituents as promising sources for new chemopreventive and therapeutic agent(s) to be further developed for the control of NMSCs.
Asunto(s)
Annona/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Extractos Vegetales/farmacología , Neoplasias Cutáneas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Transducción de Señal , Ensayo de Tumor de Célula MadreRESUMEN
miRNAs are largely known to base pair with the 3'UTR of target mRNAs, downregulating their stability and translation. mRNA of betaTrCP1 ubiquitin ligase is very unstable, but unlike the majority of mRNAs where 3'UTR determines the rate of mRNA turnover, betaTrCP1 mRNA contains cis-acting destabilizing elements within its coding region. Here we show that degradation of mRNA of betaTrCP1 is miRNA dependent and identify miR-183 as a microRNA that interacts with the coding region of betaTrCP1 mRNA. Argonaute2 interacts with the same region of betaTrCP1 mRNA in an miR-183-dependent manner. Inhibition of miR-183 function or disruption of the miR-183-binding site stabilizes betaTrCP1 mRNA and elevates betaTrCP1 levels, resulting in activation of the SCF(betaTrCP) E3 ubiquitin ligase. We previously showed that the RNA-binding protein CRD-BP binds to the coding region of betaTrCP1 mRNA and stabilizes it. Here we demonstrate that CRD-BP prevents degradation of betaTrCP1 mRNA by attenuating its miR-183-dependent interaction with Ago2.
Asunto(s)
MicroARNs/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/fisiología , Proteínas con Repetición de beta-Transducina/metabolismo , Proteínas Argonautas , Sitios de Unión , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/fisiología , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , MicroARNs/metabolismo , Sistemas de Lectura Abierta , ARN Mensajero/química , Complejo Silenciador Inducido por ARN/metabolismo , Proteínas Recombinantes de Fusión , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Ribonucleasa III/fisiología , Proteínas con Repetición de beta-Transducina/genéticaRESUMEN
Alternative cleavage and polyadenylation generates multiple transcript variants producing mRNA isoforms with different length 3'-UTRs. Alternative cleavage and polyadenylation enables differential post-transcriptional regulation via the availability of different cis-acting elements in 3'-UTRs. Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and melanogenesis. This central transcription factor is also implicated in melanoma development. Here, we show that melanoma cells favor the expression of MITF mRNA with a shorter 3'-UTR. We also establish that this isoform is regulated by a micro RNA (miRNA/miR), miR-340. miR-340 interacts with two of its target sites on the MITF 3'-UTR, causing mRNA degradation as well as decreased expression and activity of MITF. Conversely, the RNA-binding protein, coding region determinant-binding protein, was shown to be highly expressed in melanoma, directly binds to the 3'-UTR of MITF mRNA, and prevents the binding of miR-340 to its target sites, resulting in the stabilization of MITF transcripts, elevated expression, and transcriptional activity of MITF. This regulatory interplay between RNA-binding protein and miRNA highlights an important mechanism for the regulation of MITF in melanocytes and malignant melanomas.
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Regiones no Traducidas 3' , Regulación Neoplásica de la Expresión Génica , Melanocitos/metabolismo , MicroARNs/genética , Factor de Transcripción Asociado a Microftalmía/genética , Proteínas de Unión al ARN/genética , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Células HEK293 , Humanos , Melanocitos/patología , MicroARNs/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Transducción de SeñalRESUMEN
CRD-BP/IGF2BP1 has been characterized as an "oncofetal" RNA binding protein typically highly expressed in embryonic tissues, suppressed in normal adult tissues, but induced in many tumor types. In this study, we show that adult breast tissues express ubiquitous but low levels of CRD-BP protein and mRNA. Although CRD-BP mRNA expression is induced in breast tumor cells, levels remain â¼1000-fold lower than in embryonic tissues. Despite low expression levels, CRD-BP is required for clonogenic growth of breast cancer cells. We reveal that because the most common protein isoform in normal adult breast and breast tumors has an N-terminal deletion (lacking two RNA recognition motif (RRM) domains) and is therefore missing antibody epitopes, CRD-BP expression has been under-reported by previous studies. We show that a CRD-BP mutant mouse strain retains expression of the shorter transcript (ΔN-CRD-BP), which originates in intron 2, suggesting that the impact of complete ablation of this gene in mice is not yet known. Either the full-length CRD-BP or the N-terminally truncated version can rescue the clonogenicity of CRD-BP knockdown breast cancer cells, suggesting that clonogenic function is served by either CRD-BP isoform. In summary, although CRD-BP expression levels are low in breast cancer cells, this protein is necessary for clonogenic activity.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Mama/metabolismo , Proliferación Celular , Proteínas de Unión al ARN/metabolismo , Adulto , Animales , Apoptosis , Western Blotting , Mama/citología , Neoplasias de la Mama/genética , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Ratones , Persona de Mediana Edad , Isoformas de Proteínas , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Matrices TisularesRESUMEN
We have previously shown that the mRNA binding protein CRD-BP is overexpressed in human melanomas, where it promotes cell survival and resistance to chemotherapy. The present study investigates the role of hypoxia, a common characteristic of the tumor microenvironment, in the regulation of CRD-BP expression and melanoma cell responses. We found that hypoxia increases CRD-BP levels in metastatic melanoma cell lines but not in melanocytes or primary melanoma cells. Hypoxic stimulation transcriptionally regulates CRD-BP by facilitating the acetylation of histones within the CRD-BP gene and by modulating the extent of HIF1α binding to the CRD-BP promoter. Hypoxia significantly enhances the proliferative and invasive potential of metastatic melanoma cells but not that of normal or primary melanoma cells. Furthermore, inhibition of CRD-BP impairs the ability of metastatic cells to proliferate and invade in response to hypoxia. These findings identify CRD-BP as a novel effector of hypoxic responses that is relevant for the selection of metastatic cells. This work also describes a previously unknown role for CRD-BP in the regulation of melanoma cell invasion and highlights the importance of the hypoxic microenvironment in determining cell fate.
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Melanoma/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Proliferación Celular , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Melanoma/genética , Melanoma/patología , Ratones , Invasividad Neoplásica , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Células Tumorales CultivadasRESUMEN
Epigenetic regulation of gene expression by DNA methylation is a central mechanism governing the silencing of tumor suppressor genes in many forms of cancer. Current methods have not proven optimal for the quantitative analysis of DNA methylation and corresponding in situ protein expression within cells in small specimens like skin biopsies. We have overcome this limitation by combining and modifying several techniques: target cell enrichment, DNA micro-isolation, one-step denaturation/bisulphite conversion/in-column desulphonation, specially designed PCR amplification, pyrosequencing and multispectral image analysis. Using this approach optimized for small samples, we can quantify minor alterations in gene methylation and protein expression using minimal amounts of tissue. Comparative studies of fresh and processed cells showed that our method is valid for DNA in both fresh and formalin-fixed, paraffin-embedded specimens. We can measure the effects of DNA methylation inhibitors, administered in vitro or in vivo, on the promoter methylation and protein expression of selected genes in specific cells. This novel approach should prove useful for a wide variety of investigative and clinical applications in dermatology and other specialties where the collection of small, routinely processed biopsy specimens is common. We refer to this method as Q-GAME (quantitative gene analysis of methylation and expression).
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Metilación de ADN , Epigénesis Genética , Linfoma Cutáneo de Células T/genética , Línea Celular Tumoral , Islas de CpG , Citometría de Flujo , Formaldehído/química , Silenciador del Gen , Humanos , Células Jurkat , Linfoma Cutáneo de Células T/diagnóstico , Adhesión en Parafina , Reacción en Cadena de la Polimerasa , Procesamiento de Señales Asistido por Computador , Fijación del TejidoRESUMEN
microRNA (miRNA)-dependent regulation of gene expression is increasingly linked to development and progression of melanoma. In this study we evaluated the functions of miR-340 in human melanoma cells. Here, we show that miR-340 inhibits the tumorigenic phenotype of melanoma cells. We also found that miR-340 regulates RAS-RAF-Mitogen Activated Protein Kinase (MAPK) signaling by modulating the expression of multiple components of this pathway. Given the importance of MAPK signaling in melanoma, these results provide further insight into the pathogenesis of melanoma.
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Sistema de Señalización de MAP Quinasas/genética , Melanoma/genética , Melanoma/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Quinasas raf/metabolismo , Proteínas ras/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Melanoma/etiología , Mutación , Fenotipo , Transducción de Señal/genéticaRESUMEN
Neuroblastoma (NB) is the most common extracranial solid pediatric cancer, and is one of the leading causes of cancer-related deaths in children. Despite the current multi-modal treatment regimens, majority of patients with advanced-stage NBs develop therapeutic resistance and relapse, leading to poor disease outcomes. There is a large body of knowledge on pathophysiological role of small extracellular vesicles (EVs) in progression and metastasis of multiple cancer types, however, the importance of EVs in NB was until recently not well understood. Studies emerging in the last few years have demonstrated the involvement of EVs in various aspects of NB pathogenesis. In this review we summarize these recent findings and advances on the role EVs play in NB progression, such as tumor growth, metastasis and therapeutic resistance, that could be helpful for future investigations in NB EV research. We also discuss different strategies for therapeutic targeting of NB-EVs as well as utilization of NB-EVs as potential biomarkers.
Asunto(s)
Biomarcadores de Tumor , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Vesículas Extracelulares , Neuroblastoma , Humanos , Neuroblastoma/terapia , Neuroblastoma/metabolismo , Neuroblastoma/patología , Vesículas Extracelulares/metabolismo , Biomarcadores de Tumor/metabolismo , AnimalesRESUMEN
IGF2BP1 is a protein that controls the stability, localization, and translation of various mRNA targets. Poor clinical outcomes in numerous cancer types have been associated with its overexpression. As it has been demonstrated to impede tumor growth and metastasis in animal models, inhibiting IGF2BP1 function is a promising strategy for combating cancer. A lead chemical, 7773, which specifically decreased IGF2BP1 RNA binding and cellular activities, was previously identified in a high-throughput screen for effective IGF2BP1 inhibitors. Additional optimization of 7773 described in this manuscript led to the discovery of six compounds that performed equally well or better than 7773. In cell lines with high levels of endogenous IGF2BP1, one of 7773 derivatives, AVJ16, was found to be most efficient at preventing cell migration. Further, AVJ16 was found to be IGF2BP1-specific because it had no effect on cell lines that expressed little or no IGF2BP1 protein. The direct binding of AVJ16 to IGF2BP1 was validated by binding tests, with a 12-fold increase in binding efficiency over the lead compound. AVJ16 was shown to bind to a hydrophobic region at the protein's KH34 di-domain interface between the KH3 and KH4 domains. Overall, the findings imply that AVJ16 is a potent and specific inhibitor of IGF2BP1 activity.
Asunto(s)
Neoplasias , Animales , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Movimiento Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
Tumor-infiltrating regulatory T cells (TI-Tregs) elicit immunosuppressive effects in the tumor microenvironment (TME) leading to accelerated tumor growth and resistance to immunotherapies against solid tumors. Here, we demonstrate that poly-(ADP-ribose)-polymerase-11 (PARP11) is an essential regulator of immunosuppressive activities of TI-Tregs. Expression of PARP11 correlates with TI-Treg cell numbers and poor responses to immune checkpoint blockade (ICB) in human patients with cancer. Tumor-derived factors including adenosine and prostaglandin E2 induce PARP11 in TI-Tregs. Knockout of PARP11 in the cells of the TME or treatment of tumor-bearing mice with selective PARP11 inhibitor ITK7 inactivates TI-Tregs and reinvigorates anti-tumor immune responses. Accordingly, ITK7 decelerates tumor growth and significantly increases the efficacy of anti-tumor immunotherapies including ICB and adoptive transfer of chimeric antigen receptor (CAR) T cells. These results characterize PARP11 as a key driver of TI-Treg activities and a major regulator of immunosuppressive TME and argue for targeting PARP11 to augment anti-cancer immunotherapies.