RESUMO
Post-transcriptional regulation by RNA binding proteins can determine gene expression levels and drive changes in cancer cell proteomes. Identifying mechanisms of protein-RNA binding, including preferred sequence motifs bound in vivo, provides insights into protein-RNA networks and how they impact mRNA structure, function, and stability. In this review, we will focus on proteins that bind to AU-rich elements (AREs) in nascent or mature mRNA where they play roles in response to stresses encountered by cancer cells. ARE-binding proteins (ARE-BPs) specifically impact alternative splicing, stability, decay and translation, and formation of RNA-rich biomolecular condensates like cytoplasmic stress granules (SGs). For example, recent findings highlight the role of ARE-BPs - like TIAR and HUR - in chemotherapy resistance and in translational regulation of mRNAs encoding pro-inflammatory cytokines. We will discuss emerging evidence that different modes of ARE-BP activity impact leukaemia and lymphoma development, progression, adaptation to microenvironment and chemotherapy resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Hematológicas , Proteínas de Ligação a RNA , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/genética , Elementos Ricos em Adenilato e Uridilato , Regulação Neoplásica da Expressão Gênica , Animais , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Estabilidade de RNA , Ligação ProteicaRESUMO
Excision of introns during splicing regulates gene expression. In this issue, work by Sung et al. (https://doi.org/10.1083/jcb.202111151) demonstrates that the timing of intron removal in response to stress is coordinated in nuclear speckles, adding a component of spatial regulation to co-/post-transcriptional splicing.
Assuntos
Processamento Alternativo , Splicing de RNA , Íntrons/genética , Splicing de RNA/genética , Salpicos NuclearesRESUMO
Chronic myeloid leukemia (CML) cells circulate between blood and bone marrow niche, representing different microenvironments. We studied the role of the two RNA-binding proteins, T-cell-restricted intracellular antigen (TIAR), and the fragile X mental retardation protein (FMRP) in the regulation of protein translation in CML cells residing in settings mimicking peripheral blood microenvironment (PBM) and bone marrow microenvironment (BMM). The outcomes showed how conditions shaped the translation process through TIAR and FMRP activity, considering its relevance in therapy resistance. The QuaNCAT mass-spectrometric approach revealed that TIAR and FMRP have a discrete modulatory effect on protein synthesis and thus affect distinct aspects of leukemic cells functioning in the hypoxic niche. In the BMM setup, FMRP impacted metabolic adaptation of cells and TIAR substantially supported the resistance of CML cells to translation inhibition by homoharringtonine. Overall, our results demonstrated that targeting post-transcriptional control should be considered when designing anti-leukemia therapeutic solutions.
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Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect.
Assuntos
Proteína BRCA1 , Dano ao DNA , Leucemia , Animais , Camundongos , Proteína BRCA2 , DNA/metabolismo , Leucemia/enzimologia , Leucemia/genética , DNA Polimerase tetaRESUMO
The advanced development of synthetic lethality has opened the doors for specific anti-cancer medications of personalized medicine and efficient therapies against cancers. One of the most popular approaches being investigated is targeting DNA repair pathways as the implementation of the PARP inhibitor (PARPi) into individual or combinational therapeutic schemes. Such treatment has been effectively employed against homologous recombination-defective solid tumors as well as hematopoietic malignancies. However, the resistance to PARPi has been observed in both preclinical research and clinical treatment. Therefore, elucidating the mechanisms responsible for the resistance to PARPi is pivotal for the further success of this intervention. Apart from mechanisms of acquired resistance, the bone marrow microenvironment provides a pre-existing mechanism to induce the inefficiency of PARPi in leukemic cells. Here, we describe the pre-existing and acquired mechanisms of the resistance to PARPi-induced synthetic lethality. We also discuss the potential rationales for developing effective therapies to prevent/repress the PARPi resistance in cancer cells.
RESUMO
Atherosclerosis, a common age-related disease, is characterized by intense immunological activity. Atherosclerotic plaque is composed of endothelial cells, vascular smooth muscle cells (VSMCs), lipids and immune cells infiltrating from the blood. During progression of the disease, VSMCs undergo senescence within the plaque and secrete SASP (senescence-associated secretory phenotype) factors that can actively modulate plaque microenvironment. We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs). Based on unbiased proteomic analysis of VMSC-derived EVs and of the soluble fraction of SASP (sSASP), more than 900 proteins were identified in each of SASP compartments. Comparison of the composition of VMSC-derived EVs with the SASP atlas revealed several proteins, including Serpin Family F Member 1 (SERPINF1) and Thrombospondin 1 (THBS1), as commonly upregulated components of EVs secreted by senescent VSMCs and fibroblasts. Among soluble SASP factors, only Growth Differentiation Factor 15 (GDF15) was universally increased in the secretome of senescent VSMCs, fibroblasts, and epithelial cells. Bioinformatics analysis of EV proteins distinguished functionally organized protein networks involved in immune cell function regulation. Accordingly, EVs released by senescent VSMCs induced secretion of IL-17, INFγ, and IL-10 by T cells and of TNFα produced by monocytes. Moreover senEVs influenced differentiation of monocytes favoring mix M1/M2 polarization with proinflammatory characteristics. Altogether, our studies provide a complex, unbiased analysis of VSMC SASP and prove that EVs derived from senescent VSMCs influence the cytokine milieu by modulating immune cell activity. Our results strengthen the role of senescent cells as an important inducer of inflammation in atherosclerosis.
Assuntos
Aterosclerose , Vesículas Extracelulares , Humanos , Músculo Liso Vascular , Senescência Celular/fisiologia , Proteômica , Células Endoteliais , Vesículas Extracelulares/metabolismo , Aterosclerose/metabolismo , Miócitos de Músculo LisoRESUMO
Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFßR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-ß1). Genetic and/or pharmacological targeting of the TGF-ß1-TGFßR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFßR inhibitor in patients receiving PARPis.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad3/metabolismo , Animais , Humanos , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Microambiente TumoralAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Células-Tronco Neoplásicas/patologia , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The unique bone marrow microenvironment is created by stromal cells and such physical conditions as hypoxia. Both hypoxia and interactions with stromal cells have a significant impact on the biology of leukemia cells, changing their sensitivity to antileukemic therapies. Thus, it is crucial to introduce biological systems, which enable the investigation of leukemia-stroma cross-talk and verification of novel therapies effectiveness under such bone marrow niche-mimicking conditions. Here, we have established an experimental setup based on the hypoxic co-culture of stromal cells with different cell lines derived from various leukemia patients. Flow cytometry enables simultaneous fluorescent tracking of viable cells and analysis of fundamental cellular processes, also to monitor the basal vital state of cells in the hypoxic co-culture. This is critically important, as the stromal cells deliver a big variability of signals to protect leukemia cells and provide drug resistance. Therefore, keeping stromal cells at the healthy state is crucial during experimental procedures. In the proposed studies, viability, apoptosis, proliferation, ROS production, and mitochondrial membrane potential were monitored in both cell types, which were separated on the basis of the fluorescence of a cell tracker. We have shown that the proposed hypoxic co-culture conditions do not affect basal live parameters of stromal cells, indicating the relevance of proposed model. Finally, we utilized this experimental setup to monitor the stroma-mediated protection of leukemia cells from the imatinib-induced cell death, which contributes to the leukemia progression and development of therapy resistance. Altogether, we recommend such flow cytometric strategy as an elementary screen of the vital state of stromal cells, which should be performed when using the co-culture hypoxic models. The proposed approach can also be broadly used for other studies of the leukemia-stroma cross-talk and of the part played by the leukemic microenvironment in drug screening studies.
Assuntos
Células da Medula Óssea/patologia , Medula Óssea/patologia , Leucemia/patologia , Células Estromais/patologia , Apoptose/fisiologia , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Citometria de Fluxo/métodos , Células HL-60 , Humanos , Hipóxia/patologia , Células K562 , Microambiente Tumoral/fisiologiaRESUMO
Mutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Reparo do DNA/efeitos dos fármacos , Leucemia Mieloide Aguda , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Mutação , Compostos de Fenilureia/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
BRCA1 (breast cancer 1 susceptibility protein) is one of main regulators of cellular genomic stability. It is responsible for proper segregation of chromatides to daughter cells during mitosis as well as DNA double strand breaks repair by homologous recombination (HR). Genetic alterations of BRCA1 gene are cancer predisposition markers. Mutations or epigenetic alterations have been noticed in breast, ovarian and prostate cancers, significantly increasing risk of cancer development. Such gene alterations are not connected with leukemias. Importantly, BRCA1 deficiency is a factor which makes patients susceptible for personalized therapy with PARP1 inhibitors, which is based on the phenomenon called synthetic lethality. In this review we present our discoveries of novel mechanism leading to BRCA1 deficiency in leukemia, which is not connected with BRCA1 gene mutations or epigenetic alterations, but with attenuated translation of BRCA1 protein linked to the cellular stress response and controlled by RNA binding proteins. Moreover, we found that some treatments or genetic alterations in leukemias might also result in BRCA1 deficits. Our studies provide evidence that PARP1 inhibitors should be considered as efficient treatment in BRCA1-deficient leukemias, leading to elimination of cancer cells, including stem and progenitor cells. Finally we propose a strategy to select leukemia patients which might be sensitive to therapy with PARP1 inhibitors.
Assuntos
Proteína BRCA1/biossíntese , Proteína BRCA1/deficiência , Leucemia/tratamento farmacológico , Leucemia/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Mutações Sintéticas Letais , Proteína BRCA1/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Leucemia/patologiaRESUMO
Burkitt lymphoma/leukemia cells carry t(8;14)(q24;q32) chromosomal translocation encoding IGH/MYC, which results in the constitutive expression of the MYC oncogene. Here, it is demonstrated that untreated and cytarabine (AraC)-treated IGH/MYC-positive Burkitt lymphoma cells accumulate a high number of potentially lethal DNA double-strand breaks (DSB) and display low levels of the BRCA2 tumor suppressor protein, which is a key element of homologous recombination (HR)-mediated DSB repair. BRCA2 deficiency in IGH/MYC-positive cells was associated with diminished HR activity and hypersensitivity to PARP1 inhibitors (olaparib, talazoparib) used alone or in combination with cytarabine in vitro Moreover, talazoparib exerted a therapeutic effect in NGS mice bearing primary Burkitt lymphoma xenografts. In conclusion, IGH/MYC-positive Burkitt lymphoma/leukemia cells have decreased BRCA2 and are sensitive to PARP1 inhibition alone or in combination with other chemotherapies.Implications: This study postulates that IGH/MYC-induced BRCA2 deficiency may predispose Burkitt lymphoma cells to synthetic lethality triggered by PARP1 inhibitors.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/15/8/967/F1.large.jpgMol Cancer Res; 15(8); 967-72. ©2017 AACR.
Assuntos
Proteína BRCA2/genética , Linfoma de Burkitt/tratamento farmacológico , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Animais , Proteína BRCA2/deficiência , Linfoma de Burkitt/genética , Citarabina/administração & dosagem , Reparo do DNA/efeitos dos fármacos , Genes myc/genética , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Poli(ADP-Ribose) Polimerase-1/genética , Mutações Sintéticas Letais/genética , Translocação Genética/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células , Leucemia/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Cricetinae , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Genes BRCA1 , Genes BRCA2 , Genes Letais , Genes abl , Humanos , Leucemia/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Embrionárias Murinas/fisiologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The recently announced new methodologies to detect mRNA molecules in single cells offer opportunities for research, medicine and molecular diagnostics. The NanoFlare RNA Detection Probes are tools for characterizing RNA content (not localization) using fluorescence-based approaches in living cells. Combined with flow cytometry, NanoFlares have expanded the available possibilities of quantitative analysis of mRNA level in a single cell. Herein we present that in some cases, the specific NanoFlare probes (SmartFlares) detect different amounts of mRNA compared to qPCR. Using the previously published model, in which we studied influence of BCR-ABL oncogene on BRCA1 mRNA translation, we found that the NanoFlare-mediated measurement of mRNA was affected by the assembly of stress granules, structures which store mRNA in complexes with RNA binding proteins. With the usage of chemical compounds we confirmed that under conditions supporting assembly of stress granules, the detection of mRNAs by these probes was decreased, whereas disassembly resulted in the increased mRNAs detection. Altogether, we showed that assembly of stress granules could interfere with mRNA accessibility to the NanoFlare RNA Detection Probes, indicating that the SmartFlares could recognize only the translationally active pool of mRNA, contrary to qPCR. This can significantly influence the quality of obtained data and should be taken into consideration while planning the analysis of mRNA markers using NanoFlares.
Assuntos
Grânulos Citoplasmáticos/fisiologia , RNA Mensageiro/metabolismo , Animais , Proteína BRCA1/metabolismo , Linhagem Celular , Fluorescência , Genes abl/genética , Camundongos , Biossíntese de Proteínas/fisiologia , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Recent studies underscore the role of the microenvironment in therapy resistance of chronic myeloid leukemia (CML) cells and leukemia progression. We previously showed that sustained mild activation of endoplasmic reticulum (ER) stress in CML cells supports their survival and resistance to chemotherapy. We now demonstrate, using dominant negative non-phosphorylable mutant of eukaryotic initiation factor 2 α subunit (eIF2α), that phosphorylation of eIF2α (eIF2α-P), which is a hallmark of ER stress in CML cells, substantially enhances their invasive potential and modifies their ability to secrete extracellular components, including the matrix-modifying enzymes cathepsins and matrix metalloproteinases. These changes are dependent on the induction of activating transcription factor-4 (ATF4) and facilitate extracellular matrix degradation by CML cells. Conditioned media from CML cells with constitutive activation of the eIF2α-P/ATF4 pathway induces invasiveness of bone marrow stromal fibroblasts, suggesting that eIF2α-P may be important for extracellular matrix remodeling and thus leukemia cells-stroma interactions. Our data show that activation of stress response in CML cells may contribute to the disruption of bone marrow niche components by cancer cells and in this way support CML progression.
Assuntos
Catepsinas/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Matriz Extracelular/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Metaloproteinases da Matriz Secretadas/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Células da Medula Óssea/enzimologia , Células da Medula Óssea/patologia , Movimento Celular , Fator de Iniciação 2 em Eucariotos/genética , Matriz Extracelular/patologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Invasividade Neoplásica , Comunicação Parácrina , Fosforilação , Proteólise , Transdução de Sinais , Células Estromais/enzimologia , Células Estromais/patologia , Transfecção , Microambiente TumoralRESUMO
BRCA1 tumor suppressor regulates crucial cellular processes involved in DNA damage repair and cell cycle control. We showed that expression of BCR-ABL1 correlates with decreased level of BRCA1 protein, which promoted aberrant mitoses and aneuploidy as well as altered DNA damage response. Using polysome profiling and luciferase-BRCA1 3'UTR reporter system here we demonstrate that downregulation of BRCA1 protein in CML is caused by inhibition of BRCA1 mRNA translation, but not by increased protein degradation or reduction of mRNA level and half-life. We investigated 2 mRNA-binding proteins - HuR and TIAR showing specificity to AU-Rich Element (ARE) sites in 3'UTR of mRNA. BCR-ABL1 promoted cytosolic localization of TIAR and HuR, their binding to BRCA1 mRNA and formation of the TIAR-HuR complex. HuR protein positively regulated BRCA1 mRNA stability and translation, conversely TIAR negatively regulated BRCA1 translation and was found localized predominantly in the cytosolic stress granules in CML cells. TIAR-dependent downregulation of BRCA1 protein level was a result of ER stress, which is activated in BCR-ABL1 expressing cells, as we previously shown. Silencing of TIAR in CML cells strongly elevated BRCA1 level. Altogether, we determined that TIAR-mediated repression of BRCA1 mRNA translation is responsible for downregulation of BRCA1 protein level in BCR-ABL1 -positive leukemia cells. This mechanism may contribute to genomic instability and provide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in "BRCAness" CML and BCR-ABL1 -positive ALL cells.
Assuntos
Proteína BRCA1/metabolismo , Regulação para Baixo/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteína BRCA1/genética , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Neoplasias/imunologia , Inibidores de Proteínas Quinases/imunologia , Quinases da Família src/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD20/genética , Antígenos CD20/metabolismo , Western Blotting , Linhagem Celular Tumoral , Dasatinibe , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Células HEK293 , Humanos , Células K562 , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/imunologia , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rituximab , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Tiazóis/imunologia , Tiazóis/farmacologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Osteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis). RESULTS: In this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization. CONCLUSIONS: Our study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma.
Assuntos
Ácido Ascórbico/farmacologia , Neoplasias Ósseas/metabolismo , Extensões da Superfície Celular/metabolismo , Glicerofosfatos/farmacologia , Osteossarcoma/metabolismo , Neoplasias Ósseas/patologia , Calcificação Fisiológica/efeitos dos fármacos , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Colágeno Tipo I/metabolismo , Humanos , Invasividade Neoplásica , Osteossarcoma/patologia , Fenótipo , ProteóliseRESUMO
BACKGROUND: The bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants. RESULTS: In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation. CONCLUSIONS: We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.
Assuntos
Processamento Alternativo/fisiologia , Histona Desacetilases/metabolismo , Fatores de Transcrição NFATC/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/biossíntese , Animais , Histona Desacetilases/genética , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Fatores de Transcrição NFATC/genética , Células PC12 , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , RatosRESUMO
There is significant evidence for an involvement of reactive oxygen species (ROS) in the pathogenesis of diabetic vascular complications through many metabolic and structural derangements. However, despite the advanced knowledge on the crucial role of ROS in cardiovascular damage, their intracellular source in endothelial cells exposed to high concentrations of glucose has not been precisely defined. Moreover, the molecular mechanism of action of elevated glucose on mitochondria has not been fully elucidated. The main aim of this study was to describe changes in the mitochondrial metabolism of human umbilical vein endothelial cells (HUVECs) treated with high glucose concentrations and to indicate the actual source of ROS in these cells. HUVECs exposed to 30 mM glucose exhibited an increased content of vascular adhesive molecule-1 (VCAM-1) and an excessive ROS production. Faster oxygen consumption and increased abundance of selected respiratory complexes coexist with slightly declined mitochondrial membrane potential and substantially elevated amount of uncoupling protein-2 (UCP2). Inhibition of NADPH oxidase (NOX) and modification of mitochondrial ROS generation with a mitochondrial uncoupler or respiratory chain inhibitors allowed concluding that the major source of ROS in HUVECs exposed to hyperglycaemic conditions is NOX. The mitochondrial respiratory chain seems not to participate in this phenomenon.