RESUMO
Olfactory sensory neurons (OSNs) convert the stochastic choice of one of >1,000 olfactory receptor (OR) genes into precise and stereotyped axon targeting of OR-specific glomeruli in the olfactory bulb. Here, we show that the PERK arm of the unfolded protein response (UPR) regulates both the glomerular coalescence of like axons and the specificity of their projections. Subtle differences in OR protein sequences lead to distinct patterns of endoplasmic reticulum (ER) stress during OSN development, converting OR identity into distinct gene expression signatures. We identify the transcription factor Ddit3 as a key effector of PERK signaling that maps OR-dependent ER stress patterns to the transcriptional regulation of axon guidance and cell-adhesion genes, instructing targeting precision. Our results extend the known functions of the UPR from a quality-control pathway that protects cells from misfolded proteins to a sensor of cellular identity that interprets physiological states to direct axon wiring.
Assuntos
Axônios/metabolismo , Estresse do Retículo Endoplasmático , Receptores Odorantes , Animais , Camundongos , Bulbo Olfatório , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes play critical roles in governing genomic architecture and gene expression and are frequently perturbed in human cancers. Transcription factors (TFs), including fusion oncoproteins, can bind to BAF complex surfaces to direct chromatin targeting and accessibility, often activating oncogenic gene loci. Here, we demonstrate that the FUS::DDIT3 fusion oncoprotein hallmark to myxoid liposarcoma (MLPS) inhibits BAF complex-mediated remodeling of adipogenic enhancer sites via sequestration of the adipogenic TF, CEBPB, from the genome. In mesenchymal stem cells, small-molecule inhibition of BAF complex ATPase activity attenuates adipogenesis via failure of BAF-mediated DNA accessibility and gene activation at CEBPB target sites. BAF chromatin occupancy and gene expression profiles of FUS::DDIT3-expressing cell lines and primary tumors exhibit similarity to SMARCB1-deficient tumor types. These data present a mechanism by which a fusion oncoprotein generates a BAF complex loss-of-function phenotype, independent of deleterious subunit mutations.
Assuntos
Lipossarcoma Mixoide , Animais , Linhagem Celular Tumoral , Cromatina/genética , Lipossarcoma Mixoide/genética , Lipossarcoma Mixoide/metabolismo , Lipossarcoma Mixoide/patologia , Mamíferos/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Myxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. METHODS: To study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. RESULTS: Characterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. CONCLUSIONS: Our experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.
Assuntos
Lipossarcoma Mixoide , Proteínas de Fusão Oncogênica , Proteína FUS de Ligação a RNA , Microambiente Tumoral , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Proliferação de Células , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Lipossarcoma Mixoide/patologia , Lipossarcoma Mixoide/metabolismo , Lipossarcoma Mixoide/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Alicerces Teciduais/química , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
OBJECTIVE: DNA damage-inducible transcript 3 (DDIT3), as a downstream transcription factor of endoplasmic reticulum stress, is reported to regulate chondrogenic differentiation under physiological and pathological state. However, the specific involvement of DDIT3 in the degradation of condylar cartilage of temporomandibular joint osteoarthritis (TMJOA) is unclarified. DESIGN: The expression patterns of DDIT3 in condylar cartilage from monosodium iodoacetate-induced TMJOA mice were examined to uncover the potential role of DDIT3 in TMJOA. The Ddit3 knockout (Ddit3-/-) mice and their wildtype littermates (Ddit3+/+) were used to clarify the effect of DDIT3 on cartilage degradation. Primary condylar chondrocytes and ATDC5 cells were applied to explore the mechanisms of DDIT3 on autophagy and extracellular matrix (ECM) degradation in chondrocytes. The autophagy inhibitor chloroquine (CQ) was used to determine the effect of DDIT3-inhibited autophagy in vivo. RESULTS: DDIT3 were highly expressed in condylar cartilage from TMJOA mice. Ddit3 knockout alleviated condylar cartilage degradation and subchondral bone loss, compared with their wildtype littermates. In vitro study demonstrated that DDIT3 exacerbated ECM degradation in chondrocytes induced by TNF-α through inhibiting autophagy. The intraperitoneal injection of CQ further confirmed that Ddit3 knockout alleviated cartilage degradation in TMJOA through activating autophagy in vivo. CONCLUSIONS: Our findings identified the crucial role of DDIT3-inhibited autophagy in condylar cartilage degradation during the development of TMJOA.
Assuntos
Autofagia , Cartilagem Articular , Condrócitos , Camundongos Knockout , Osteoartrite , Fator de Transcrição CHOP , Animais , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genética , Autofagia/fisiologia , Cartilagem Articular/metabolismo , Camundongos , Osteoartrite/metabolismo , Osteoartrite/genética , Condrócitos/metabolismo , Transtornos da Articulação Temporomandibular/metabolismo , Transtornos da Articulação Temporomandibular/genética , Côndilo Mandibular/metabolismo , Côndilo Mandibular/patologia , Proteínas de Membrana , Fator 2 Relacionado a NF-E2 , Heme Oxigenase-1RESUMO
DDIT3 is a tightly regulated basic leucine zipper (bZIP) transcription factor and key regulator in cellular stress responses. It is involved in a variety of pathological conditions and may cause cell cycle block and apoptosis. It is also implicated in differentiation of some specialized cell types and as an oncogene in several types of cancer. DDIT3 was originally believed to act as a dominant-negative inhibitor by forming heterodimers with other bZIP transcription factors, preventing their DNA binding and transactivating functions. DDIT3 has, however, been reported to bind DNA and regulate target genes. Here, we employed ChIP sequencing combined with microarray-based expression analysis to identify direct binding motifs and target genes of DDIT3. The results reveal DDIT3 binding to motifs similar to other bZIP transcription factors, known to form heterodimers with DDIT3. Binding to a class III satellite DNA repeat sequence was also detected. DDIT3 acted as a DNA-binding transcription factor and bound mainly to the promotor region of regulated genes. ChIP sequencing analysis of histone H3K27 methylation and acetylation showed a strong overlap between H3K27-acetylated marks and DDIT3 binding. These results support a role for DDIT3 as a transcriptional regulator of H3K27ac-marked genes in transcriptionally active chromatin.
Assuntos
Genômica , Fatores de Transcrição , Sítios de Ligação , Fatores de Transcrição/genética , Fatores de Transcrição de Zíper de Leucina Básica , DNARESUMO
BACKGROUND: Liposarcomas are among the most common mesenchymal malignancies. However, the therapeutic options are still very limited and so far, targeted therapies had not yet been established. Immunotherapy, which has been a breakthrough in other oncological entities, seems to have no efficacy in liposarcoma. Complicating matters further, classification remains difficult due to the diversity of morphologies and nonspecific or absent markers in immunohistochemistry, leaving molecular pathology using FISH or sequencing as best options. Many liposarcomas harbor MDM2 gene amplifications. In close relation to the gene locus of MDM2, HER3 (ERBB3) gene is present and co-amplification could occur. Since the group of HER/EGFR receptor tyrosine kinases and its inhibitors/antibodies play a role in a broad spectrum of oncological diseases and treatments, and some HER3 inhibitors/antibodies are already under clinical investigation, we hypothesized that in case of HER3 co-amplifications a tumor might bear a further potential therapeutic target. METHODS: We performed FISH analysis (MDM2, DDIT3, HER3) in 56 archived cases and subsequently performed reclassification to confirm the diagnosis of liposarcoma. RESULTS: Next to 16 out of 56 cases needed to be re-classified, in 20 out of 54 cases, a cluster-amplification of HER3 could be detected, significantly correlating with MDM2 amplification. Our study shows that the entity of liposarcomas show specific molecular characteristics leading to reclassify archived cases by modern, established methodologies. Additionally, in 57.1% of these cases, HER3 was cluster-amplified profusely, presenting a putative therapeutic target for targeted therapy. CONCLUSION: Our study serves as the initial basis for further investigation of the HER3 gene as a putative therapeutic target in liposarcoma.
Assuntos
Amplificação de Genes , Lipossarcoma , Proteínas Proto-Oncogênicas c-mdm2 , Receptor ErbB-3 , Humanos , Lipossarcoma/genética , Lipossarcoma/patologia , Lipossarcoma/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Hibridização in Situ Fluorescente , Feminino , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Masculino , Prognóstico , Pessoa de Meia-Idade , Idoso , Terapia de Alvo Molecular/métodos , AdultoRESUMO
Mesenchymal neoplasms with GLI1 alterations (rearrangements and/or amplification) have been reported recently in several anatomic locations, which include head and neck, soft tissue, and gastrointestinal tract. Herein, to the best of our knowledge, we describe the first three cases of superficial/subcutaneous mesenchymal neoplasm with GLI1 amplification. The neoplasms exhibited low-grade cytologic features with predominant round cell morphology, glomangioma-like areas and a rich background capillary network. There were two to three mitotic figures per 10 HPF and focal necrosis in one case. The tumors exhibited variable expression of CDK4, MDM2, STAT6, D2-40, CD56 and cyclin D1. p16 had strong and diffuse nuclear and cytoplasmic expression in two cases. Numerous other stains were negative. Fluorescence in situ hybridization detected GLI1, DDIT3, and CDK4 coamplification in all cases, while next generation sequencing did not detect a GLI1 gene fusion. The overall features were compatible with a GLI1-amplified mesenchymal neoplasm. In Case 1 a new distant skin lesion appeared 1 month after the surgery exhibiting similar morphology albeit with a higher mitotic index. In Cases 2 and 3, there is no evidence of local recurrence or systemic disease after 8 years and 1 month of follow-up, respectively. These new cases of superficial GLI1-amplified neoplasm expand its clinical spectrum and enter the realm of dermatopathology. The combination of CDK4, cyclin D1, D2-40, and p16 expression with variable MDM2, STAT6, CD56, and S100 immunoreactivity in a low-grade neoplasm with round/ovoid cytomorphology resembling a vascular or adnexal neoplasm may suggest the possibility of GLI1-amplified neoplasm.
Assuntos
Amplificação de Genes , Tumor Glômico , Mesenquimoma , Neoplasias Cutâneas , Proteína GLI1 em Dedos de Zinco , Humanos , Masculino , Feminino , Adulto , Idoso , Proteína GLI1 em Dedos de Zinco/genética , Mesenquimoma/genética , Mesenquimoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Tumor Glômico/genética , Tumor Glômico/patologia , Mitose , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologiaRESUMO
Endoplasmic reticulum stress (ERS) plays an important role in regulating the reproductive process of female mammals, mainly involved in follicular atresia and corpus luteum regression. DNA damage induced transcript 3 (DDIT3) is a marker gene of ERS. The objectives of the present study were to clone and analyze the sequence and tissue expression characteristics of DDIT3 gene in female yaks. By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length 507-bp DDIT3-cDNA, encoding for 168-aa protein. Yak DDIT3 exhibited highest and least identity with that of bison and horse, respectively. Real-time PCR analyses revealed that the expression level of DDIT3 gene in ovary was higher than that in heart, liver, kidney, spleen, lung, uterus and oviduct (p < 0.05). DDIT3 expression level in ovary and uterus during pregnancy was higher than that in follicular phase, luteal phase and fetus stage. DDIT3 was highly expressed in metaphase II oocytes and granulosa cells than that in germinal vesicle and metaphase I oocytes (p < 0.05), respectively. This is the first molecular characterization and expression patterns of DDIT3 gene in female yaks. These results indicated that the DDIT3 gene possibly plays an important role in regulating ovary function and pregnancy maintenance in yaks.
Assuntos
Atresia Folicular , Ovário , Gravidez , Bovinos , Feminino , Animais , Cavalos , Clonagem Molecular , Ovário/metabolismo , Oócitos , Reação em Cadeia da Polimerase em Tempo Real , MamíferosRESUMO
OBJECTIVE: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma. METHODS: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features. RESULTS: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis. CONCLUSION: Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.
Assuntos
Lipoma , Lipossarcoma , Masculino , Feminino , Humanos , Hibridização in Situ Fluorescente/métodos , Quinase 4 Dependente de Ciclina/análise , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Lipossarcoma/diagnóstico , Lipossarcoma/genética , Lipossarcoma/patologia , Lipoma/diagnóstico , Lipoma/genética , Lipoma/patologia , Amplificação de Genes , Fator de Transcrição CHOP/genética , Proteínas Proto-Oncogênicas c-mdm2/análise , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismoRESUMO
DNA damage-inducible transcript 3 (DDIT3) plays important roles in endoplasmic reticulum (ER) stress-induced apoptosis and autophagy, but its role in innate immunity is not clear. Here, we report that DDIT3 inhibits the antiviral immune response during bovine viral diarrhea virus (BVDV) infection by targeting mitochondrial antiviral signaling (MAVS) in Madin-Darby bovine kidney (MDBK) cells and in mice. BVDV infection induced high DDIT3 mRNA and protein expression. DDIT3 overexpression inhibited type I interferon (IFN-I) and IFN-stimulated gene production, thereby promoting BVDV replication, while DDIT3 knockdown promoted the antiviral innate immune response to suppress viral replication. DDIT3 promoted NF-κB-dependent ovarian tumor (OTU) deubiquitinase 1 (OTUD1) expression. Furthermore, OTUD1 induced upregulation of the E3 ubiquitin ligase Smurf1 by deubiquitinating Smurf1, and Smurf1 degraded MAVS in MDBK cells in a ubiquitination-dependent manner, ultimately inhibiting IFN-I production. Moreover, knocking out DDIT3 promoted the antiviral innate immune response to reduce BVDV replication and pathological changes in mice. These findings provide direct insights into the molecular mechanisms by which DDIT3 inhibits IFN-I production by regulating MAVS degradation.IMPORTANCE Extensive studies have demonstrated roles of DDIT3 in apoptosis and autophagy during viral infection. However, the role of DDIT3 in innate immunity remains largely unknown. Here, we show that DDIT3 is positively regulated in bovine viral diarrhea virus (BVDV)-infected Madin-Darby bovine kidney (MDBK) cells and could significantly enhance BVDV replication. Importantly, DDIT3 induced OTU deubiquitinase 1 (OTUD1) expression by activating the NF-κB signaling pathway, thus increasing intracellular Smurf1 protein levels to degrade MAVS and inhibit IFN-I production during BVDV infection. Together, these results indicate that DDIT3 plays critical roles in host innate immunity repression and viral infection facilitation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Vírus da Diarreia Viral Bovina Tipo 1/fisiologia , Imunidade Inata , Fator de Transcrição CHOP/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Replicação Viral , Animais , Antivirais/antagonistas & inibidores , Antivirais/imunologia , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/patogenicidade , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais , Fator de Transcrição CHOP/deficiência , Fator de Transcrição CHOP/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/genética , UbiquitinaçãoRESUMO
Vinigrol is a natural diterpenoid with unprecedented chemical structure, driving great efforts into its total synthesis in the past decades. Despite anti-hypertension and anti-clot ever reported, comprehensive investigations on bioactions and molecular mechanisms of Vinigrol are entirely missing. Here we firstly carried out a complete functional prediction of Vinigrol using a transcriptome-based strategy coupled with multiple bioinformatic analyses and identified "anti-cancer" as the most prominent biofunction ahead of anti-hypertension and anti-depression/psychosis. Broad cytotoxicity was subsequently confirmed on multiple cancer types. Further mechanistic investigation on several breast cancer cells revealed that its anti-cancer effect was mainly through activating PERK/eIF2α arm of unfolded protein response (UPR) and subsequent non-apoptotic cell death independent of caspase activities. The other two branches of UPR, IRE1α and ATF6, were functionally irrelevant to Vinigrol-induced cell death. Using CRISPR/Cas9-based gene activation, repression, and knockout systems, we identified the essential contribution of ATF4 and DDIT3, not ATF6, to the death process. This study unraveled a broad anti-cancer function of Vinigrol and its underlying targets and regulatory mechanisms. It paved the way for further inspection on the structure-efficacy relationship of the whole compound family, making them a novel cluster of PERK-specific stress activators for experimental and clinical uses.
Assuntos
Fator 4 Ativador da Transcrição , Neoplasias da Mama , Diterpenos , Fator de Transcrição CHOP , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Neoplasias da Mama/tratamento farmacológico , Diterpenos/farmacologia , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Feminino , Humanos , Proteínas Serina-Treonina Quinases , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/metabolismoRESUMO
AIMS: Myxoid liposarcoma (MLPS) is characterised by DNA damage-inducible transcript 3 (DDIT3) gene rearrangements, confirmation of which is commonly used diagnostically. Recently, DDIT3 immunohistochemistry (IHC) has been reported to be highly sensitive and, when strict criteria are employed, specific for the diagnosis of MLPS. The aim of this study was to independently investigate DDIT3 IHC as a diagnostic marker for MLPS. METHODS AND RESULTS: DDIT3 IHC was performed on 52 MLPS and on 152 mimics on whole sections, and on 515 non-MLPS sarcomas in tissue microarray format. Only one MLPS (which had undergone acid-based decalcification) was completely negative. With inclusion of this case if any nuclear expression is considered to indicate positivity, the overall sensitivity of DDIT3 is 98% (51 of 52 cases) and the specificity is 94% (633 of 667 non-MLPS cases are negative). If a cut-off of >10% of neoplastic cells is required for positivity, then the sensitivity remains 98% (51/52) and the specificity is 98.5% (657 of 667 non-MLPS cases are negative). If a cut-off of >50% of cells is required for positivity, then the sensitivity is 96% (50 of 52 cases) but the specificity improves to 100%. CONCLUSIONS: Diffuse nuclear DDIT3 expression occurs in the overwhelming majority of MLPSs, and can be used to confirm the diagnosis in most cases without the need for molecular testing. A complete absence of expression argues strongly against MLPS, and almost completely excludes this diagnosis, particularly if there is consideration of technical factors such as decalcification. The significance of focal DDIT3 expression should be interpreted in the morphological and clinical context, although most tumours showing only focal expression are not MLPS.
Assuntos
Biomarcadores Tumorais/metabolismo , Lipossarcoma Mixoide/diagnóstico , Neoplasias de Tecidos Moles/diagnóstico , Fator de Transcrição CHOP/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/análise , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Lipossarcoma Mixoide/patologia , Masculino , Pessoa de Meia-Idade , Sarcoma/diagnóstico , Sensibilidade e Especificidade , Neoplasias de Tecidos Moles/patologia , Fator de Transcrição CHOP/análiseRESUMO
BACKGROUND: Colorectal cancers develop through several pathways, including the adenoma-carcinoma sequence and colitis-associated carcinogenesis. An altered intestinal microflora has been reported to be associated with the development and progression of colorectal cancer via these pathways. We identified Lactobacillus casei-derived ferrichrome as a mediator of the bacterial anti-tumor effect of colorectal cancer cells through the upregulation of DDIT3. In this study, we investigated the anti-tumor effects of ferrichrome on precancerous conditions and cancer cells associated with sporadic as well as colitis-associated colorectal cancer. METHODS: SRB and MTT assays were performed to assess growth inhibition in vitro. Eighteen organoids were prepared from biopsy specimens obtained by colonoscopy. An AOM-DSS carcinogenesis model and xenograft model of colorectal cancer cells were generated for the assessment of the tumor suppressive effect of ferrichrome in vivo. RESULTS: Ferrichrome inhibited the cell growth of colorectal cancer cells in vitro and in in vivo xenograft models. Ferrichrome exerted a strong tumor-suppressive effect that was superior to that of currently available anti-tumor agents, including 5-FU and cisplatin, both in vitro and in vivo. The tumor-suppressive effect of the combination of ferrichrome and 5-FU was superior to that of single treatment with either drug. The tumor suppressive effects of ferrichrome were confirmed through the upregulation of DDIT3 in patient-derived organoids of adenoma and carcinoma. Ferrichrome inhibited the tumor progression in the AOM-DSS model while exhibiting no anti-inflammatory effect in the DSS-colitis model, suggesting that ferrichrome inhibited cancer cells, but not a precancerous condition, via the colitis-associated pathway. CONCLUSIONS: Ferrichrome exerts a tumor suppressive effect on precancerous conditions and cancer cells associated with sporadic as well as colitis-associated colorectal cancer. The anti-tumor effect of ferrichrome was mediated by the upregulation of DDIT3, and was superior to that of 5-FU or cisplatin. These results suggest that Lactobacillus brevis-derived ferrichrome may be a candidate anti-tumor drug for the treatment of colorectal neoplasms.
RESUMO
Pulmonary hypertension (PH) is a life-threatening disease characterized by vascular remodeling. Exploring new therapy target is urgent. The purpose of the present study is to investigate whether and how spliced x-box binding protein 1 (xbp1s), a key component of endoplasmic reticulum stress (ERS), contributes to the pathogenesis of PH. Forty male SD rats were randomly assigned to four groups: Control, Monocrotaline (MCT), MCT+AAV-CTL (control), and MCT+AAV-xbp1s. The xbp1s protein levels were found to be elevated in lung tissues of the MCT group. Intratracheal injection of adeno-associated virus serotype 1 carrying xbp1s shRNA (AAV-xbp1s) to knock down the expression of xbp1s effectively ameliorated the MCT-induced elevation of right ventricular systolic pressure (RVSP), total pulmonary resistance (TPR), right ventricular hypertrophy and medial wall thickness of muscularized distal pulmonary arterioles. The abnormally increased positive staining rates of proliferating cell nuclear antigen (PCNA) and Ki67 and decreased positive staining rates of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) in pulmonary arterioles were also reversed in the MCT+AAV-xbp1s group. For mechanistic exploration, bioinformatics prediction of the protein network was performed on the STRING database, and further verification was performed by qRT-PCR, Western blots and co-immunoprecipitation (Co-IP). DNA damage-inducible transcript 3 (Ddit3) was identified as a downstream protein that interacted with xbp1s. Overexpression of Ddit3 restored the decreased proliferation, migration and cell viability caused by silencing of xbp1s. The protein level of Ddit3 was also highly consistent with xbp1s in the animal model. Taken together, our study demonstrated that xbp1s-Ddit3 may be a potential target to interfere with vascular remodeling in PH.
Assuntos
Pressão Arterial , Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator de Transcrição CHOP/metabolismo , Remodelação Vascular , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Apoptose , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/induzido quimicamente , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Masculino , Monocrotalina , Músculo Liso Vascular/fisiopatologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Transcrição CHOP/genética , Disfunção Ventricular Direita/induzido quimicamente , Disfunção Ventricular Direita/metabolismo , Disfunção Ventricular Direita/fisiopatologia , Função Ventricular Direita , Proteína 1 de Ligação a X-Box/genéticaRESUMO
Members of the human FET family of RNA-binding proteins, comprising FUS, EWSR1, and TAF15, are ubiquitously expressed and engage at several levels of gene regulation. Many sarcomas and leukemias are characterized by the expression of fusion oncogenes with FET genes as 5' partners and alternative transcription factor-coding genes as 3' partners. Here, we report that the N terminus of normal FET proteins and their oncogenic fusion counterparts interact with the SWI/SNF chromatin remodeling complex. In contrast to normal FET proteins, increased fractions of FET oncoproteins bind SWI/SNF, indicating a deregulated and enhanced interaction in cancer. Forced expression of FET oncogenes caused changes of global H3K27 trimethylation levels, accompanied by altered gene expression patterns suggesting a shift in the antagonistic balance between SWI/SNF and repressive polycomb group complexes. Thus, deregulation of SWI/SNF activity could provide a unifying pathogenic mechanism for the large group of tumors caused by FET fusion oncoproteins. These results may help to develop common strategies for therapy.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Metilação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/genética , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Choriocarcinoma cells BeWo b30 are used to model human placental trophoblast hypoxia using cobalt (II) chloride and hydroxyquinoline derivative (HD) as chemical inducers of hypoxia-inducible factor (HIF). In this study, it was shown that both substances activate the hypoxic pathway and the epithelial-mesenchymal transition and inhibit the pathways of cell proliferation. However, CoCl2 caused activation of the apoptosis pathway, increased the activity of effector caspases 3 and 7, and increased the expression of the unfolded protein response target DDIT3. The mTORC1 pathway was activated upon exposition to CoCl2, while HD suppressed this pathway, as it happens during real trophoblast hypoxia. Thus, effect of CoCl2 on BeWo cells can be a model of severe hypoxia with activation of apoptosis, while HD mimics moderate hypoxia.
Assuntos
Hipóxia Celular/efeitos dos fármacos , Cobalto/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Gravidez , Fator de Transcrição CHOP/metabolismoRESUMO
A 20-year-old female patient presented with the complaint of pelvic pain. Radiological studies showed a cystic mass in the left ovary. Histological examination revealed a myxoid tumor with a chicken wire-like, thin vascularization. The diagnosis of the lesion, morphologically resembling myxoid liposarcoma was supported by demonstration of rearrangement of the DDIT3 gene by fluorescence in situ hybridization.
Assuntos
Lipossarcoma Mixoide , Feminino , Humanos , Hibridização in Situ Fluorescente , Lipossarcoma Mixoide/diagnóstico por imagem , Ovário/diagnóstico por imagem , Fator de Transcrição CHOP , Adulto JovemRESUMO
Micro RNAs (miRNAs) are major players in cellular responses to xenobiotic compounds and toxins. However, their functions in organophosphate-induced cytotoxicity remain unclear. This study investigated the involvement of miR-96-5p in the non-cholinergic toxicity of malathion in normal human kidney cells (HK-2 cells). Malathion decreased HK-2 cell viability and the expression of miR-96-5p in a dose- and time-dependent manner. In addition, transfection with miR-96-5p mimics attenuated malathion-induced HK-2 cell apoptosis, whereas transfection with a miR-96-5p inhibitor increased HK-2 cell apoptosis. Luciferase assays indicated that miR-96-5p could bind directly to the 3'-untranslated region of DDIT3, a well-known marker of endoplasmic reticulum stress. Further analyses of the expression of apoptosis-related genes and proteins indicated that miR-96-5p may function to reduce malathion-induced HK-2 cell apoptosis via regulation of the DDIT3/B-cell lymphoma (BCL)-2/caspase-3 signaling pathway. In summary, the results of the present study indicate that miR-96-5p protects HK-2 cells from malathion-induced ER stress-dependent apoptosis by targeting DDIT3.
Assuntos
Apoptose/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Malation/toxicidade , MicroRNAs/genética , Fator de Transcrição CHOP/genética , Apoptose/genética , Biomarcadores/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inseticidas/toxicidade , Túbulos Renais Proximais/citologia , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo , Xenobióticos/toxicidadeRESUMO
Endochondral ossification is the process by which long bones are formed; the process of long bone formation is regulated by numerous factors such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone Nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. Whole genome microarray data from our previous study revealed that most hHNR's are up-regulated during osteoblast differentiation in hMSCS. NR2F1 was among the highest expressed hHNR during osteogenesis, NR2F1 belongs to the steroid/thyroid hormone nuclear receptor superfamily. NR2F1 is designated as an orphan nuclear receptor because its ligands are unknown. NR2F1 plays a wide range of roles, including cell differentiation, cancer progression, and central and peripheral neurogenesis. Identifying signaling networks involved in osteoblast differentiation is important in orchestrating new therapeutic and clinical applications in bone biology. This study aimed to identify alterations in signaling networks mediated by NR2F1 in osteoblast differentiation. siRNA-mediated down-regulation of NR2F1 leads to impairment in the differentiation of hBMSC-TERT to osteoblast; gene-expression results confirmed the down-regulation of osteoblast markers such as RUNX2, ALPL, OSC, and BSP. Global whole gene expression analysis revealed that most down-regulated genes were associated with osteoblast differentiation (DDIT3, BMP2). Pathway analysis revealed prominent signaling pathways that were down-regulated, including the TGFß pathway and MAPK pathway. Functional studies on NR2F1 transfected cells, during osteoblast differentiation in combination with TGFß1 and BMP-2, showed that TGFß1 does not recover osteoblast differentiation, whereas BMP-2 rescues osteoblast differentiation in NR2F1 siRNA transfected cells. Thus, our results showed that BMP-2 could intervene in NR2F1 down-regulated signaling pathways to recover osteoblast differentiation.
Assuntos
Proteína Morfogenética Óssea 2/genética , Fator I de Transcrição COUP/genética , Diferenciação Celular/genética , Fator de Crescimento Transformador beta1/genética , Desenvolvimento Ósseo/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , TransfecçãoRESUMO
UNLABELLED: The PKR-like endoplasmic reticulum kinase (PERK) pathway of the unfolded protein response (UPR) is protective against toxic accumulations of misfolded proteins in the endoplasmic reticulum, but is thought to drive cell death via the transcription factor, CHOP. However, in many cell types, CHOP is an obligate step in the PERK pathway, which frames the conundrum of a prosurvival pathway that kills cells. Our laboratory and others have previously demonstrated the prosurvival activity of the PERK pathway in oligodendrocytes. In the current study, we constitutively overexpress CHOP in myelinating cells during development and into adulthood under normal or UPR conditions. We show that this transcription factor does not drive apoptosis. Indeed, we observe no detriment in mice at multiple levels from single cells to mouse behavior and life span. In light of these data and other studies, we reinterpret PERK pathway function in the context of a stochastic vulnerability model, which governs the likelihood that cells undergo cell death upon cessation of UPR protection and while attempting to restore homeostasis. SIGNIFICANCE STATEMENT: Herein, we tackle the biggest controversy in the UPR literature: the function of the transcription factor CHOP as a protective or a prodeath factor. This manuscript is timely in light of the 2014 Lasker award for the UPR. Our in vivo data show that CHOP is not a prodeath protein, and we demonstrate that myelinating glial cells function normally in the presence of high CHOP expression from development to adulthood. Further, we propose a simplified view of UPR-mediated cell death after CHOP induction. We anticipate our work may turn the tide of the dogmatic view of CHOP and cause a reinvestigation of its function in different cell types. Accordingly, we believe our work will be a watershed for the UPR field.