RESUMO
Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in many physiological functions including embryonic development and immune responses and is often activated under pathological conditions such as cancer. Strategies to inactivate STAT3 are being pursued as potential anticancer therapies and have led to the identification of Stattic (6-nitrobenzo[b]thiophene-1,1-dioxide) as a "specific" STAT3 inhibitor that is often used to interrogate STAT3-mediated gene expression in vitro and in vivo. Here, we show that Stattic exerts many STAT3-independent effects on cancer cells, calling for reassessment of results previously ascribed to STAT3 functions. Studies of the STAT3-deficient prostate cancer cell line PC-3 (PC3) along with STAT3-proficient breast cancer cell lines (MDA-MB-231, SUM149) revealed that Stattic attenuated histone acetylation and neutralized effects of the histone deacetylase (HDAC) inhibitor romidepsin. In PC3 cells, Stattic alone inhibited gene expression of CCL20 and CCL2, but activated expression of TNFA, CEBPD, SOX2, and MYC. In addition, we found that Stattic promoted autophagy and caused cell death. These data point to profound epigenetic effects of Stattic that are independent of its function as a STAT3 inhibitor. Our results demonstrate that Stattic directly or indirectly reduces histone acetylation and suggest reevaluation of Stattic and related compounds as polypharmacological agents through multipronged cytotoxic effects on cancer cells.
Assuntos
Antineoplásicos/farmacologia , Óxidos S-Cíclicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT3/genética , Acetilação/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína delta de Ligação ao Facilitador CCAAT/agonistas , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL20/antagonistas & inibidores , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Células PC-3 , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/agonistas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/agonistas , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/agonistas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína Vermelha FluorescenteRESUMO
The complex cross-talk of intricate intercellular signaling networks between the tumor and stromal cells promotes cancer progression. Hypoxia is one of the most common conditions encountered within the tumor microenvironment that drives tumorigenesis. Most responses to hypoxia are elicited by a family of transcription factors called hypoxia-inducible factors (HIFs), which induce expression of a diverse set of genes that assist cells to adapt to hypoxic environments. Among the three HIF protein family members, the role of HIF-1 is well established in cancer progression. HIF-1 functions as a signaling hub to coordinate the activities of many transcription factors and signaling molecules that impact tumorigenesis. This mini review discusses the complex role of HIF-1 and its context-dependent partners under various cancer-promoting events including inflammation and generation of cancer stem cells, which are implicated in tumor metastasis and relapse. In addition, the review highlights the importance of therapeutic targeting of HIF-1 for cancer prevention.
Assuntos
Hipóxia Celular , Fator 1 Induzível por Hipóxia/fisiologia , Inflamação/etiologia , Neoplasias/etiologia , Animais , Transição Epitelial-Mesenquimal , Humanos , Fator 1 Induzível por Hipóxia/análise , Células-Tronco Neoplásicas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Microambiente TumoralRESUMO
Inflammation and hypoxia are known to promote the metastatic progression of tumours. The CCAAT/enhancer-binding protein-δ (C/EBPδ, CEBPD) is an inflammatory response gene and candidate tumour suppressor, but its physiological role in tumourigenesis in vivo is unknown. Here, we demonstrate a tumour suppressor function of C/EBPδ using transgenic mice overexpressing the Neu/Her2/ERBB2 proto-oncogene in the mammary gland. Unexpectedly, this study also revealed that C/EBPδ is necessary for efficient tumour metastasis. We show that C/EBPδ is induced by hypoxia in tumours in vivo and in breast tumour cells in vitro, and that C/EBPδ-deficient cells exhibit reduced glycolytic metabolism and cell viability under hypoxia. C/EBPδ supports CXCR4 expression. On the other hand, C/EBPδ directly inhibits expression of the tumour suppressor F-box and WD repeat-domain containing 7 gene (FBXW7, FBW7, AGO, Cdc4), encoding an F-box protein that promotes degradation of the mammalian target of rapamycin (mTOR). Consequently, C/EBPδ enhances mTOR/AKT/S6K1 signalling and augments translation and activity of hypoxia-inducible factor-1α (HIF-1α), which is necessary for hypoxia adaptation. This work provides new insight into the mechanisms by which metastasis-promoting signals are induced specifically under hypoxia.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas F-Box/biossíntese , Regulação da Expressão Gênica , Hipóxia , Neoplasias Mamárias Animais/secundário , Metástase Neoplásica/patologia , Ubiquitina-Proteína Ligases/biossíntese , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Proteína 7 com Repetições F-Box-WD , Glicólise , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/fisiopatologia , Camundongos , Camundongos Transgênicos , Metástase Neoplásica/fisiopatologiaRESUMO
Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor of BRCA2 whose overexpression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described estrogen as inducing MLH1 expression through estrogen receptor α (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER-positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and show how it may contribute to the establishment of BRCA2-deficient breast tumors.
Assuntos
Proteína BRCA2 , Neoplasias Mamárias Animais , Animais , Camundongos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Reparo de Erro de Pareamento de DNA , Replicação do DNARESUMO
The transcription factor CCAAT/enhancer binding protein delta (C/EBPdelta, CEBPD, NFIL-6beta) has tumor suppressor function; however, the molecular mechanism(s) by which C/EBPdelta exerts its effect are largely unknown. Here, we report that C/EBPdelta induces expression of the Cdc27 (APC3) subunit of the anaphase promoting complex/cyclosome (APC/C), which results in the polyubiquitination and degradation of the prooncogenic cell cycle regulator cyclin D1, and also down-regulates cyclin B1, Skp2, and Plk-1. In C/EBPdelta knockout mouse embryo fibroblasts (MEF) Cdc27 levels were reduced, whereas cyclin D1 levels were increased even in the presence of activated GSK-3beta. Silencing of C/EBPdelta, Cdc27, or the APC/C coactivator Cdh1 (FZR1) in MCF-10A breast epithelial cells increased cyclin D1 protein expression. Like C/EBPdelta, and in contrast to cyclin D1, Cdc27 was down-regulated in several breast cancer cell lines, suggesting that Cdc27 itself may be a tumor suppressor. Cyclin D1 is a known substrate of polyubiquitination complex SKP1/CUL1/F-box (SCF), and our studies show that Cdc27 directs cyclin D1 to alternative degradation by APC/C. These findings shed light on the role and regulation of APC/C, which is critical for most cellular processes.
Assuntos
Neoplasias da Mama/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase , Western Blotting , Proteína delta de Ligação ao Facilitador CCAAT/genética , Linhagem Celular Tumoral , Ciclina B1/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Imunoprecipitação , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases Associadas a Fase S/metabolismo , Quinase 1 Polo-LikeRESUMO
Metastatic progression of epithelial cancers can be associated with epithelial-mesenchymal transition (EMT) including transcriptional inhibition of E-cadherin (CDH1) expression. Recently, EM plasticity (EMP) and E-cadherin-mediated, cluster-based metastasis and treatment resistance have become more appreciated. However, the mechanisms that maintain E-cadherin expression in this context are less understood. Through studies of inflammatory breast cancer (IBC) and a 3D tumor cell "emboli" culture paradigm, we discovered that cyclooxygenase 2 (COX-2; PTGS2), a target gene of C/EBPδ (CEBPD), or its metabolite prostaglandin E2 (PGE2) promotes protein stability of E-cadherin, ß-catenin, and p120 catenin through inhibition of GSK3ß. The COX-2 inhibitor celecoxib downregulated E-cadherin complex proteins and caused cell death. Coexpression of E-cadherin and COX-2 was seen in breast cancer tissues from patients with poor outcome and, along with inhibitory GSK3ß phosphorylation, in patient-derived xenografts (PDX) including triple negative breast cancer (TNBC).Celecoxib alone decreased E-cadherin protein expression within xenograft tumors, though CDH1 mRNA levels increased, and reduced circulating tumor cell (CTC) clusters. In combination with paclitaxel, celecoxib attenuated or regressed lung metastases. This study has uncovered a mechanism by which metastatic breast cancer cells can maintain E-cadherin-mediated cell-to-cell adhesions and cell survival, suggesting that some patients with COX-2+/E-cadherin+ breast cancer may benefit from targeting of the PGE2 signaling pathway.
Assuntos
Dinoprostona , Neoplasias de Mama Triplo Negativas , Humanos , Caderinas/genética , Caderinas/metabolismo , Celecoxib/farmacologia , Celecoxib/uso terapêutico , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Transdução de Sinais/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , FemininoRESUMO
Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Quimiocina CCL20/metabolismo , Estresse do Retículo Endoplasmático , Imunomodulação , Interleucina-8/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Linhagem Celular Tumoral , Quimiocina CCL20/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Interleucina-8/genética , Janus Quinases/metabolismo , Modelos Biológicos , Comunicação Parácrina/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genéticaRESUMO
Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence (547)CNCTNCKCDQTKSCHGGDC(565) are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1Delta strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu(4)-S(6)] cage structure, characterized by a core of trigonally S(3) coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu(4)-L(6) (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I).
Assuntos
Cobre/toxicidade , Cisteína/química , Proteínas de Ligação a DNA/química , Drosophila melanogaster/metabolismo , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Cobre/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/efeitos dos fármacos , Análise Espectral , Enxofre/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios X , Fator MTF-1 de TranscriçãoRESUMO
During physiological epithelial-mesenchymal transition (EMT), which is important for embryogenesis and wound healing, epithelial cells activate a program to remodel their structure and achieve a mesenchymal fate. In cancer cells, EMT confers increased invasiveness and tumor-initiating capacity, which contribute to metastasis and resistance to therapeutics. However, cellular plasticity that navigates between epithelial and mesenchymal states and maintenance of a hybrid or partial E/M phenotype appears to be even more important for cancer progression. Besides other core EMT transcription factors, the well-characterized Snail-family proteins Snail (SNAI1) and Slug (SNAI2) play important roles in both physiological and pathological EMT. Often mentioned in unison, they do, however, differ in their functions in many scenarios. Indeed, Slug expression does not always correlate with complete EMT or loss of E-cadherin (CDH1). For example, Slug plays important roles in mammary epithelial cell progenitor cell lineage commitment and differentiation, DNA damage responses, hematopoietic stem cell self-renewal, and in pathologies such as pulmonary fibrosis and atherosclerosis. In this Perspective, we highlight Slug functions in mammary epithelial cells and breast cancer as a "non-EMT factor" in basal epithelial cells and stem cells with focus reports that demonstrate co-expression of Slug and E-cadherin. We speculate that Slug and E-cadherin may cooperate in normal mammary gland and breast cancer/stem cells and advocate for functional assessment of such Slug+/E-cadherinlow/+ (SNAI2+/CDH1low/+) "basal-like epithelial" cells. Thus, Slug may be regarded as less of an EMT factor than driver of the basal epithelial cell phenotype.
RESUMO
Epithelial-mesenchymal transition (EMT) is a cellular biological process involved in migration of primary cancer cells to secondary sites facilitating metastasis. Besides, EMT also confers properties such as stemness, drug resistance and immune evasion which can aid a successful colonization at the distant site. EMT is not a binary process; recent evidence suggests that cells in partial EMT or hybrid E/M phenotype(s) can have enhanced stemness and drug resistance as compared to those undergoing a complete EMT. Moreover, partial EMT enables collective migration of cells as clusters of circulating tumor cells or emboli, further endorsing that cells in hybrid E/M phenotypes may be the 'fittest' for metastasis. Here, we review mechanisms and implications of hybrid E/M phenotypes, including their reported association with hypoxia. Hypoxia-driven activation of HIF-1α can drive EMT. In addition, cyclic hypoxia, as compared to acute or chronic hypoxia, shows the highest levels of active HIF-1α and can augment cancer aggressiveness to a greater extent, including enriching for a partial EMT phenotype. We also discuss how metastasis is influenced by hypoxia, partial EMT and collective cell migration, and call for a better understanding of interconnections among these mechanisms. We discuss the known regulators of hypoxia, hybrid EMT and collective cell migration and highlight the gaps which needs to be filled for connecting these three axes which will increase our understanding of dynamics of metastasis and help control it more effectively.
RESUMO
Metallothioneins are ubiquitous, small, cysteine-rich proteins with the ability to bind heavy metals. In spite of their biochemical characterization, their in vivo function remains elusive. Here, we report the generation of a metallothionein gene family knockout in Drosophila melanogaster by targeted disruption of all four genes (MtnA to -D). These flies are viable if raised in standard laboratory food. During development, however, they are highly sensitive to copper, cadmium, and (to a lesser extent) zinc load. Metallothionein expression is particularly important for male viability; while copper load during development affects males and females equally, adult males lacking metallothioneins display a severely reduced life span, possibly due to copper-mediated oxidative stress. Using various reporter gene constructs, we find that different metallothioneins are expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midgut's "copper cells." The same expression pattern is observed with a synthetic minipromoter consisting only of four tandem metal response elements. From these and other experiments, we conclude that tissue specificity of metallothionein expression is a consequence, rather than a cause, of metal distribution in the organism. The bright orange luminescence of copper accumulated in copper cells of the midgut is severely reduced in the metallothionein gene family knockout, as well as in mutants of metal-responsive transcription factor 1 (MTF-1), the main regulator of metallothionein expression. This indicates that an in vivo metallothionein-copper complex forms the basis of this luminescence. Strikingly, metallothionein mutants show an increased, MTF-1-dependent induction of metallothionein promoters in response to copper, cadmium, silver, zinc, and mercury. We conclude that free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and that metallothioneins regulate their own expression by a negative feedback loop.
Assuntos
Cobre/metabolismo , Homeostase/genética , Inativação Metabólica/genética , Metalotioneína/genética , Metalotioneína/metabolismo , Envelhecimento/genética , Animais , Animais Geneticamente Modificados , Cádmio/metabolismo , Cobre/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica , Luminescência , Masculino , Mercúrio/metabolismo , Mercúrio/farmacologia , Família Multigênica , Recombinação Genética , Elementos de Resposta , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator MTF-1 de TranscriçãoRESUMO
To improve cancer patient outcome significantly, we must understand the mechanisms regulating stem-like cancer cells, which have been implicated as a cause of metastasis and treatment resistance. The transcription factor C/EBPδ can exhibit pro- and anti-tumorigenic activities, but the mechanisms underlying the complexity of its functions are poorly understood. Here we identify a role for breast cancer cell intrinsic C/EBPδ in promoting phenotypes that have been associated with cancer stem cells (CSCs). While C/EBPδ expression is not abundant in most metastatic breast cancers, our data support a pro-tumorigenic role of C/EBPδ when expressed in subsets of tumor cells and/or through transient activation by the tumor microenvironment or loss of substrate adhesion. Using genetic mouse models and human breast cancer cell lines, we show that deletion or depletion of C/EBPδ reduced expression of stem cell factors and stemnness markers, sphere formation and self-renewal, along with growth of tumors and established experimental metastases in vivo. C/EBPδ is also known as a mediator of the innate immune response, which is enhanced by hypoxia and interleukin-6 (IL-6) signaling, two conditions that also play important roles in cancer progression. Our mechanistic data reveal C/EBPδ as a link that engages two positive feedback loops, in part by directly targeting the IL-6 receptor (IL6RA) gene, and, thus, amplifying IL-6 and HIF-1 signaling. This study provides a molecular mechanism for the synergism of tumor microenvironmental conditions in cancer progression with potential implications for the targeting of CSCs.
Assuntos
Neoplasias da Mama/patologia , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-6/metabolismo , Células-Tronco Neoplásicas/patologia , Animais , Neoplasias da Mama/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Linhagem Celular Tumoral , Proteína 7 com Repetições F-Box-WD/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , Receptores de Interleucina-6/genética , Transdução de Sinais/genética , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A mismatch between metabolic demand and oxygen delivery leads to microenvironmental changes in solid tumors. The resulting tumor hypoxia is associated with malignant progression, therapy resistance and poor prognosis. However, the molecular mechanisms underlying therapy resistance in hypoxic tumors are not fully understood. The hypoxia-inducible factor (HIF) is a master transcriptional activator of oxygen-regulated gene expression. Transformed mouse embryonic fibroblasts (MEFs) derived from HIF-1alpha-deficient mice are a popular model to study HIF function in tumor progression. We previously found increased chemotherapy and irradiation susceptibility in the absence of HIF-1alpha. Here, we show by single-cell electrophoresis, histone 2AX phosphorylation and nuclear foci formation of gammaH2AX and 53BP1, that the number of DNA double-strand breaks (DSB) is increased in untreated and etoposide-treated HIF-deficient MEFs. In etoposide-treated cells, cell cycle control and p53-dependent gene expression were not affected by the absence of HIF-1alpha. Using a candidate gene approach to screen 17 genes involved in DNA repair, messenger RNA (mRNA) and protein of three members of the DNA-dependent protein kinase complex were found to be decreased in HIF-deficient MEFs. Of note, residual HIF-1alpha protein in cancer cells with a partial HIF-1alpha mRNA knockdown was sufficient to confer chemoresistance. In summary, these data establish a novel molecular link between HIF and DNA DSB repair. We suggest that selection of early, non-hypoxic tumor cells expressing low levels of HIF-1alpha might contribute to HIF-dependent tumor therapy resistance.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Animais , Antineoplásicos/farmacologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Ensaio Cometa , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Embrião de Mamíferos , Etoposídeo/farmacologia , Fibroblastos/efeitos dos fármacos , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Camundongos , Interferência de RNA , RNA Mensageiro/análise , TransfecçãoRESUMO
The transition metal copper is an essential trace element for both prokaryotes and eukaryotes. However, intracellular free copper has to be strictly limited due to its toxic side effects, not least the generation of reactive oxygen species (ROS) via redox cycling. Thus, all organisms have sophisticated copper homeostasis mechanisms that regulate uptake, distribution, sequestration and export of copper. From insects to mammals, metal-responsive transcription factor (MTF-1), a zinc finger transcription factor, controls expression of metallothioneins and other components involved in heavy metal homeostasis. In the fruit fly Drosophila, MTF-1 paradoxically acts as an activator under both high and low copper concentrations. Namely, under high copper conditions, MTF-1 activates metallothioneins in order to protect the cell, while under low copper conditions MTF-1 activates the copper importer Ctr1B in order to acquire scarce copper from the surroundings. This review highlights the current knowledge of copper homeostasis in eukaryotes with a focus on Drosophila and the role of MTF-1.
Assuntos
Cobre/metabolismo , Células Eucarióticas/metabolismo , Homeostase , AnimaisRESUMO
The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.
Assuntos
Pró-Colágeno-Prolina Dioxigenase/biossíntese , Pró-Colágeno-Prolina Dioxigenase/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Cromatografia em Camada Fina , Descarboxilação , Glutaratos/química , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Peptídeos/química , Peptídeos/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/isolamento & purificação , Extratos de Tecidos/químicaRESUMO
Apoptosis is an active process induced by a variety of physiological and external stimuli, in which elimination of damaged cells are effected through a genetically controlled process. In this study, we have examined the mechanism of chromium(III) [Cr(III)]-induced cytotoxicity with respect to its relationship to oxidative stress. Morphology, flow cytometry, and DNA fragmentation studies show that tris-(1,10-phenanthroline)chromium(III) [Cr(III)-phen], tris-(2,2'-bipyridyl)chromium(III) [Cr(III)-bpy], trans-diaqua[1,2-bis(salicylideneamino)ethanechromium(III)] [Cr(III)-salen], and trans-diaqua[1,3-bis(salicylideneamino)propanechromium(III)] [Cr(III)-salprn] induced apoptosis of lymphocytes. Pentaammineaquachromium(III) [Cr(III)-hpa] does not induce apoptosis. Apoptosis induced by these complexes involves the generation of reactive oxygen species (ROS) as seen by increased fluorescence of dichloroflourescein (DCF) observed through flow cytometry. Pretreatment of lymphocytes with antioxidants completely abrogate apoptosis. Cr(III) treatment also increased the expression and activation of Src-family tyrosine kinases viz. p56lck, p59fyn, and p53/56lyn, as seen by immunoblotting and immune complex kinase assay. PP2, a selective Src-family tyrosine kinase inhibitor, abolishes apoptosis, indicating that Src-family tyrosine kinases are directly involved in eliciting apoptosis. Interestingly, a one-to-one correlation between the expression of Src-family tyrosine kinases and ROS is observed, since antioxidants pretreatment inhibits the expression and the activation of these kinases. These results further indicate that Cr(III)-induced apoptosis is mediated through production of ROS, which in turn activates the Src-family tyrosine kinases. The increased activation of Src-family tyrosine kinases may be a mechanism involved in apoptosis of lymphocytes elicited by various other physiological stimuli that exploit ROS as a second messenger.
Assuntos
Apoptose/efeitos dos fármacos , Cromo/farmacologia , Linfócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Quinases da Família src/metabolismo , Trifosfato de Adenosina/metabolismo , Antioxidantes/farmacologia , Transporte Biológico , Western Blotting , Tamanho Celular , Cromo/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
The CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ) is a transcription factor that modulates many biological processes including cell differentiation, motility, growth arrest, proliferation, and cell death. The diversity of C/EBPδ's functions depends in part on the cell type and cellular context and can have opposing outcomes. For example, C/EBPδ promotes inflammatory signaling, but it can also inhibit pro-inflammatory pathways, and in a mouse model of mammary tumorigenesis, C/EBPδ reduces tumor incidence but promotes tumor metastasis. This review highlights the multifaceted nature of C/EBPδ's functions, with an emphasis on pathways that are relevant for cancer and inflammation, and illustrates how C/EBPδ emerged from the shadow of its family members as a fascinating "jack of all trades." Our current knowledge on C/EBPδ indicates that, rather than being essential for a specific cellular process, C/EBPδ helps to interpret a variety of cues in a cell-type and context-dependent manner, to adjust cellular functions to specific situations. Therefore, insights into the roles and mechanisms of C/EBPδ signaling can lead to a better understanding of how the integration of different signaling pathways dictates normal and pathological cell functions and physiology.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/fisiologia , Inflamação/genética , Neoplasias/genética , Transdução de Sinais , Animais , Proteína delta de Ligação ao Facilitador CCAAT/genética , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular/genética , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , RatosRESUMO
Toll-like receptor 4 (Tlr4) has a pivotal role in innate immune responses, and the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ, Cebpd) is a Tlr4-induced gene. Here we identify a positive feedback loop in which C/EBPδ activates Tlr4 gene expression in macrophages and tumour cells. In addition, we discovered a negative feedback loop whereby the tumour suppressor FBXW7α (FBW7, Cdc4), whose gene expression is inhibited by C/EBPδ, targets C/EBPδ for degradation when C/EBPδ is phosphorylated by GSK-3ß. Consequently, FBXW7α suppresses Tlr4 expression and responses to the ligand lipopolysaccharide. FBXW7α depletion alone is sufficient to augment pro-inflammatory signalling in vivo. Moreover, as inflammatory pathways are known to modulate tumour biology, Cebpd null mammary tumours, which have reduced metastatic potential, show altered expression of inflammation-associated genes. Together, these findings reveal a role for C/EBPδ upstream of Tlr4 signalling and uncover a function for FBXW7α as an attenuator of inflammatory signalling.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/fisiologia , Regulação para Baixo , Proteínas F-Box/fisiologia , Inflamação/fisiopatologia , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Linhagem Celular Tumoral , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamação/genética , Camundongos , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Organisms from insects to mammals respond to heavy metal load (copper, zinc, cadmium, and mercury) by activating the metal-responsive transcription factor 1 (MTF-1). MTF-1 binds to short DNA sequence motifs, termed metal response elements, and boosts transcription of a number of genes, notably those for metallothioneins. In Drosophila, MTF-1 somewhat counter-intuitively also activates transcription of a copper importer gene (Ctr1B) in response to copper starvation. Here, we report that mutant flies lacking Ctr1B are extremely sensitive to cadmium and mercury treatment, but can be rescued by excess copper in the food. We thus propose that copper, by competing for binding sites on cellular proteins, alleviates the toxic effects of mercury and cadmium. Such a scenario also explains a seemingly fortuitous metal response, namely, that cadmium and mercury strongly induce the expression of a Ctr1B reporter gene. Thus, the transcription enhancer/promoter region of the Ctr1B copper importer gene is subject to three modes of regulation. All of them depend on MTF-1 and all make biological sense, namely, (i) induction by copper starvation, (ii) repression by copper abundance, and (iii), as shown here, induction by cadmium or mercury at normal copper supply.