Tyrosine phosphorylation of the BCR-ABL SH3 domain results in the recruitment of SH2 domain containing proteins

Bei der Chronischen Myeolischen Leukämie (CML) handelt es sich um eine umfassend charakterisierte myeloproliferative Erkrankung, die aus der anhaltenden Aktivität der Tyrosinkinase BCR-ABL resultiert. Trotz der Vielzahl an bereits gewonnen Erkenntnissen sind weiter Untersuchungen notwendig um die der Krankheit zugrunde liegenden Veränderungen in Signaltransduktion und Genexpression gänzlich zu verstehen. In der vorliegenden Studie wurden zwei Aspekte der BCR-ABL abhängigen Signaltransduktion genauer untersucht. Einen Schwerpunkt legten wir auf die Charakterisierung jener Änderungen im Interaktionsprofil von BCR-ABL, die durch die Phosphorylierung der BCR-ABL eigenen SH3 Domäne verursacht werden. Im zweiten Projekt suchten wir nach mikroRNAs, deren Expressionsprofil von der Aktivität der BCR-ABL Tyrosinkinase bestimmt wird. Bereits in einer früheren Studie wurde gezeigt, dass bei CML die SH3 Domäne von BCR-ABL am Tyr134 phosphoryliert wird. Wir führten verschiedene biochemische Analysen durch, mit dem Ziel Proteine zu identifizieren die über eine etwaige SH2 Domäne mit BCR-ABL über eben diesen phosphorylierten Tyrosinrest interagieren. Wir behalfen uns dabei eines sogenannten Peptid Pull-downs. Kurze synthetische Peptide wurde designt deren Sequenz dem Tyr134 umgebenden Bereich der SH3 Domäne entspricht. Eines der beiden Peptide wurde durch das kovalente Anhängen einer Phosphatgruppe and Tyr134 modifiziert. Im zweiten Peptid, dem Kontrollpeptid, wurde das Tyrosin durch einen Phenylalaninrest ersetzt. Die massenspektrometrische Analyse beider Pull-downs führte zur Identifizierung von PLCG1 und SHP2, zwei Proteine die spezifisch das Tyrosin-phosphorylierte Peptid binden, jedoch nicht an das Kontrollpeptid. Beiden Kanditaten wurden bereits in frühern Studien eine Rolle in der Entwicklung verschiedener Tumorarten zugeschrieben. In einem nächsten Schritt klonierten wir die SH3 Domäne BCR-ABLs als GST-Fusionsprotein in einen bakteriellen Expressionsvektor. Nach der Aufreinigung der in E. coli exprimierten SH3 Domäne mittels FPLC führten wir weiter Pull-down Experimente durch. Diesmal verglichen wir die vollständige SH3 Domäne mit einem Kontrolkonstrukt bei dem wiederum Tyr134 durch ein Phenylalanin subsituiert wurde. Die abschließende Verifizierung erfolgte über einen Co-Immunoprezipitationsansatz, bei welchem wir verschiedene BCR-ABL Konstrukte hinsichtlich einer spezifischen Interaktion zwischen PLCG1 und SHP2 und der Tyrosin phosphorylierten SH3 Domäne untersuchten. Es dürfte sich hierbei um die erste Studie halten, die eine Phosphotyrosin abhäbgige Interaktion zwischen eine SH2 Domäne und einer SH3 Domäne zeigt. In Anbetracht der wachstumsbegünstigenden Natur von sowohl PLCG1 als auch SHP2 ist es nicht auszuschließen, dass die Phosphorylierung von Tyr134 einen weiteren Mechanismus der BCR-ABL vermittelten Onkogenese darstellt. MikroRNAs (miRNAs) sind neuartige, kurze nicht kodierende, RNAs, die die Expression von Genen auf posttranskriptioneller Ebene modulieren. Die aberrante Expression von miRNAs wurde in Verbindung zu zahlreichen bösartigen zellulären Veränderungen, einschließlich CML gebracht. Im zweiten Teil unserer Studie wurden 31 miRNAs identifiziert deren Expressionprofile durch die Inkubation von K562 Zellen mit Dasatinib und Nilotinib signifikant verändert wurden. Wir konnten in Übereinstimmung mit früheren Studien zeigen, dass die Expression von miRNAs der miR-17-92 Familie durch Inhibierung der BCR-ABL Aktivität negativ reguliert wird. Ferner beobachteten wir eine Reduktion der miR-21 Expression durch sowohl Dasatinib als auch Niltoinib, und verifizierten dadurch das Ergebnis einer schon länger zurückliegenden Mikroarray basierenden Genexpressionsanalyse. Eine weitere Bestätigung der Runterregulierung von miR-21 wurde durch eine miR-21 spezifische miR-qRT-PCR Analyse erzielt. Anhand der TargetScan Plattform bestimmten wir abschließend die Zielgene der idenfizierten miRNAs und konzentrierten uns dabei auf jene Gene deren Expressionslevel umgekehrt proportional zu den Expressionsleveln der jeweiligen miRNAs verlaufen. In der vorliegenden Studie wird eine umfassende Analyse der BCR-ABL abhängigen miRNA Expression durchgeführt. Außerdem liefern wir weitere Hinweise dafür, dass die onkogene miRNAs miR-17-92 und miR-21 an der Pathophysiologie von CML beteiligt sind.Chronic myelogenous leukemia (CML) is a myeloproliferative disorder that is characterized by the presence of the constitutively active BCR-ABL fusion tyrosine kinase. In general, CML is a well-studied disease, but the underlying changes in signal transduction networks and gene expression patterns that lead to oncogenic reprogramming of haematopoietic cells by the expression of BCR-ABL are only incompletely understood. In the present study we are focusing on two different aspects of BCR-ABL signalling. One aim was to assess the consequences of BCR-ABL SH3 domain phosphorylation for the interaction pattern of the fusion protein. In a second approach, we sought to identify microRNAs whose expression profile is dictated by the tyrosine kinase activity of BCR-ABL. The SH3 domain of BCR-ABL was previously found to be phosphorylated on Tyr134 in CML. We conducted biochemical analyses to investigate if Tyr134 phosphorylation of the BCR-ABL SH3 domain causes the recruitment of phospho-tyrosyl specific, SH2 domain harboring, proteins. We started with a peptide pull-down using two synthetic peptides whose sequence corresponded to the region of interest of the BCR-ABL SH3 domain. One peptide was modified by the covalent attachment of a phosphate group to the tyrosine, while in the other peptide the tyrosine was substituted by a phenylalanine. LS-ESI-MS/MS analysis of the pull-downs yielded two proteins, PLCG1 and SHP2, which both specifically bind to the tyrosine-phosphorylated peptide but not to the control peptide. Both candidates have proto-oncogenic attributes and have already been described to be involved in the manifestation of various malignancies. We subsequently coned the BCR-ABL SH3 domain as a GST fusion protein into a bacterial expression vector, and after expression in E. coli and FPLC-purification we performed additional pull-downs, this time comparing the fully folded SH3 domain with a control construct where the tyrosine residue of interest was mutated to phenylalanine. Finally, overexpression of various full-length BCR-ABL constructs in HEK-null cells was performed to confirm the phosphotyrosine dependent interaction between the BCR-ABL SH3 domain and the potential interactors in co-immunoprecipitation experiments. To our knowledge this is the first study showing a phosphotyrosine dependent interaction between an SH2 domain and an SH3 domain. Considering the pro-growth nature of PLCG1 and SHP2 it is entirely thinkable that Tyr134 phosphorylation represents a further mechanism by which BCR-ABL exerts its oncogenic function. MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that modulate the expression of genes at the posttranscriptional level. Aberrant miRNA expression has been described in several human malignancies, including CML. In the second part of this study we provide an informative profile of the expression of miRNAs that are deregulated upon Dasatinib or Nilotinib treatment of K562 cells. We identified 31 miRNAs that were differentially expressed in the presence of tyrosine kinase activity suppressed BCR-ABL. In line with previous findings, expression of miRNAs belonging to the miR-17-92 family was specifically downregulated by both Dasatinib and Nilotinib treatment. In addition we detected a marked decrease of miR-21 in the drug treated samples, confirming earlier microarray gene expression data. Furthermore, we observed miR-21 downregulation also by a miR-21 specific miR-qRT-PCR assay. Finally, we used the TargetScan platform for the identification of predicted miRNA target genes whose expression levels inversely correlate with any of the 31 miRNA candidates following BCR-ABL inhibition in K562 cells. In summary, the results of this study offer a comprehensive and quantitative profile of BCR-ABL dependent miRNA expression in CML and further implicate the oncogenic miRNAs miR-17-92 and miR-21 in the pathophysiology of the disease

Loss of microRNA-7a2 induces hypogonadotropic hypogonadism and infertility

MicroRNAs (miRNAs) are negative modulators of gene expression that fine-tune numerous biological processes. miRNA loss-of-function rarely results in highly penetrant phenotypes, but rather, influences cellular responses to physiologic and pathophysiologic stresses. Here, we have reported that a single member of the evolutionarily conserved miR-7 family, miR7a2, is essential for normal pituitary development and hypothalamic-pituitary-gonadal (HPG) function in adulthood. Genetic deletion of mir-7a2 causes infertility, with low levels of gonadotropic and sex steroid hormones, small testes or ovaries, impaired spermatogenesis, and lack of ovulation in male and female mice, respectively. We found that miR-7a2 is highly expressed in the pituitary, where it suppresses golgi glycoprotein 1 (GLG1) expression and downstream bone morphogenetic protein 4 (BMP4) signaling and also reduces expression of the prostaglandin F2a receptor negative regulator (PTGFRN), an inhibitor of prostaglandin signaling and follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion. Our results reveal that miR-7a2 critically regulates sexual maturation and reproductive function by interconnecting miR-7 genomic circuits that regulate FSH and LH synthesis and secretion through their effects on pituitary prostaglandin and BMP4 signaling

Foxa1 is essential for development and functional integrity of the subthalamic nucleus

Inactivation of transcription factor Foxa1 in mice results in neonatal mortality of unknown cause. Here, we report that ablation of Foxa1 causes impaired development and loss of the subthalamic nucleus (STN). Functional deficits in the STN have been implicated in the etiology of Huntington's and Parkinson's disease. We show that neuronal ablation by Synapsin1-Cre-mediated Foxa1 deletion is sufficient to induce hyperlocomotion in mice. Transcriptome profiling of STN neurons in conditional Foxa1 knockout mice revealed changes in gene expression reminiscent of those in neurodegenerative diseases. We identified Ppargc1a, a transcriptional co-activator that is implicated in neurodegeneration, as a Foxa1 target. These findings were substantiated by the observation of Foxa1-dependent demise of STN neurons in conditional models of Foxa1 mutant mice. Finally, we show that the spontaneous firing activity of Foxa1-deficient STN neurons is profoundly impaired. Our data reveal so far elusive roles of Foxa1 in the development and maintenance of STN function

Foxa1 is essential for development and functional integrity of the subthalamic nucleus

Inactivation of transcription factor Foxa1 in mice results in neonatal mortality of unknown cause. Here, we report that ablation of Foxa1 causes impaired development and loss of the subthalamic nucleus (STN). Functional deficits in the STN have been implicated in the etiology of Huntington’s and Parkinson’s disease. We show that neuronal ablation by Synapsin1-Cre-mediated Foxa1 deletion is sufficient to induce hyperlocomotion in mice. Transcriptome profiling of STN neurons in conditional Foxa1 knockout mice revealed changes in gene expression reminiscent of those in neurodegenerative diseases. We identified Ppargc1a, a transcriptional co-activator that is implicated in neurodegeneration, as a Foxa1 target. These findings were substantiated by the observation of Foxa1-dependent demise of STN neurons in conditional models of Foxa1 mutant mice. Finally, we show that the spontaneous firing activity of Foxa1-deficient STN neurons is profoundly impaired. Our data reveal so far elusive roles of Foxa1 in the development and maintenance of STN function.ISSN:2045-232

Cytotoxic, antioxidant, and cytoprotective properties of polyphenol‐enriched extracts from pecan nutshells in MDA‐MB‐231 breast cancer cells

Phenolic compounds present in plants have demonstrated several biological properties such as antioxidant, antitumor, cardioprotective, and antiproliferative. On the other hand, doxorubicin, a chemotherapeutic widely used to treat breast cancer, usually exhibits chronic cardiotoxicity associated with oxidative stress. Therefore, we aimed to study the effects of phenolic compound-enriched extract (PCEE) with doxorubicin in breast cancer. To achieve this, after an SPE-C18-column purification process of crude extracts obtained from pecan nutshells (Carya illinoinensis), the resulting PCEE was used to evaluate the cytotoxicity and antioxidant properties against the human breast cancer cell line MDA-MB-231 and the normal-hamster ovary cell line CHO-K1. PCEE was selectively cytotoxic against both cell lines, with an IC50 value (≈26.34 mg/L) for MDA-MB-231 lower than that obtained for CHO-K1 (≈55.63 mg/L). As a cytotoxic mechanism, PCEE inhibited cell growth by G2/M cell cycle arrest in MDA-MB-231 cells. Simultaneously, the study of the antioxidant activity showed that PCEE had a cytoprotective effect, evidenced by reduced ROS production in cells with oxidative stress caused by doxorubicin. The results highlight PCEE as a potential antitumor agent, thus revaluing it as an agro-industrial residue.Fil: Ribas, Lucas Emanuel. Universidad Nacional del Litoral. Escuela Universitaria de Analisis de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Gasser, Fátima Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Baravalle, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Renna, Maria Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Perello, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Escuela Universitaria de Analisis de Alimentos; ArgentinaFil: Savino, Graciela. Universidad Nacional del Litoral. Escuela Universitaria de Analisis de Alimentos; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Van de Velde, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología de los Alimentos; ArgentinaFil: Hein, Gustavo Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentin

Extraction of phenolic compounds from the shells of pecan nuts with cytotoxic activity through apoptosis against the colon cancer cell line HT-29

The water extraction of phenolic compounds from two varieties (“Mahan” and “Marameck”) of pecan nutshells (Carya illinoinensis) without and with sonication, varying the solvent/solid ratio (S), the pH, and the refluxing time (t), was studied. Additionally, the in vitro cytotoxicity and the determination of the cell death mechanism of the extracts against the colon cancer cell line HT-29 were investigated. The content of total phenolic compounds (TPC) of “Marameck” nutshells resulted higher than for the “Mahan” variety, and the pH increase resulted in higher TPC contents for both cultivars. The optimized conditions for TPC extraction without and with sonication resulted: S = 33 ml/g, pH = 12, and t = 9.6 min, and yielded ≈ 70 and 90 mg/g of TPC for “Mahan” and “Marameck” nutshells, respectively. The optimized extracts of pecan nutshells without sonication from both cultivars presented similar cytotoxicity against HT-29 colon cancer cells (IC50≈ 50 µg/ml), higher than for sonicated extracts (IC50 ≈ 88 and 138 µg/ml for “Mahan” and “Marameck,” respectively). Cell death through apoptosis was the main mechanism of cell death induced by the nutshell extracts. Practical Application: The extraction of phenolic compounds (TPC) from the residues of two varieties of pecan nutshells (“Mahan” and “Marameck”) was studied. An optimal combination of variables within the pH range that minimizes the solvent-to-solid ratio (S) and the time of refluxing (t), saving at the same time, water and energy, was set up. The phenolic compound extracts obtained from the residues of the pecan nuts exhibit cytotoxic effects against colon cancer cells and could be of interest as an alternative treatment of different types of cancer. Additionally, these extracts may be of importance to the food industry as they can be used as antioxidant agents in food formulation. Also, the high levels of anthocyanidins obtained from the pecan nut extracts after proanthocyanidins’ strong acid hydrolysis can be purified and employed as natural red dyes.Fil: Ribas, Lucas Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Baravalle, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Gasser, Fátima Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Renna, Maria Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Addona, Silvina Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Savino, Graciela Hilda. Universidad Nacional del Litoral. Escuela Universitaria de Analisis de Alimentos; ArgentinaFil: Van de Velde, Franco. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología de los Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hein, Gustavo Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentin

FXR Regulates Intestinal Cancer Stem Cell Proliferation

Increased levels of intestinal bile acids (BAs) are a risk factor for colorectal cancer (CRC). Here, we show that the convergence of dietary factors (high-fat diet) and dysregulated WNT signaling (APC mutation) alters BA profiles to drive malignant transformations in Lgr5-expressing (Lgr5+) cancer stem cells and promote an adenoma-to-adenocarcinoma progression. Mechanistically, we show that BAs that antagonize intestinal farnesoid X receptor (FXR) function, including tauro-β-muricholic acid (T-βMCA) and deoxycholic acid (DCA), induce proliferation and DNA damage in Lgr5+ cells. Conversely, selective activation of intestinal FXR can restrict abnormal Lgr5+ cell growth and curtail CRC progression. This unexpected role for FXR in coordinating intestinal self-renewal with BA levels implicates FXR as a potential therapeutic target for CRC