RESUMO
BACKGROUND: Neuroendocrine prostate cancer (NEPC) is an aggressive form of prostate cancer, arising from resistance to androgen-deprivation therapies. However, the molecular mechanisms associated with NEPC development and invasiveness are still poorly understood. Here we investigated the expression and functional significance of Fascin-1 (FSCN1), a pro-metastasis actin-bundling protein associated with poor prognosis of several cancers, in neuroendocrine differentiation of prostate cancer. METHODS: Differential expression analyses using Genome Expression Omnibus (GEO) database, clinical samples and cell lines were performed. Androgen or antagonist's cellular treatments and knockdown experiments were used to detect changes in cell morphology, molecular markers, migration properties and in vivo tumour growth. Chromatin immunoprecipitation-sequencing (ChIP-Seq) data and ChIP assays were analysed to decipher androgen receptor (AR) binding. RESULTS: We demonstrated that FSCN1 is upregulated during neuroendocrine differentiation of prostate cancer in vitro, leading to phenotypic changes and NEPC marker expression. In human prostate cancer samples, FSCN1 expression is restricted to NEPC tumours. We showed that the androgen-activated AR downregulates FSCN1 expression and works as a transcriptional repressor to directly suppress FSCN1 expression. AR antagonists alleviate this repression. In addition, FSCN1 silencing further impairs in vivo tumour growth. CONCLUSION: Collectively, our findings identify FSCN1 as an AR-repressed gene. Particularly, it is involved in NEPC aggressiveness. Our results provide the rationale for the future clinical development of FSCN1 inhibitors in NEPC patients.
Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Humanos , Masculino , Antagonistas de Androgênios/uso terapêutico , Androgênios , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologiaRESUMO
Older age is one of the strongest risk factors for severe COVID-19. In this study, we determined whether age-associated cellular senescence contributes to the severity of experimental COVID-19. Aged golden hamsters accumulate senescent cells in the lungs, and the senolytic drug ABT-263, a BCL-2 inhibitor, depletes these cells at baseline and during SARS-CoV-2 infection. Relative to young hamsters, aged hamsters had a greater viral load during the acute phase of infection and displayed higher levels of sequelae during the post-acute phase. Early treatment with ABT-263 lowered pulmonary viral load in aged (but not young) animals, an effect associated with lower expression of ACE2, the receptor for SARS-CoV-2. ABT-263 treatment also led to lower pulmonary and systemic levels of senescence-associated secretory phenotype factors and to amelioration of early and late lung disease. These data demonstrate the causative role of age-associated pre-existing senescent cells on COVID-19 severity and have clear clinical relevance.
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COVID-19 , SARS-CoV-2 , Cricetinae , Animais , Carga Viral , Pulmão , Mesocricetus , Inflamação , Senescência CelularRESUMO
Coronavirus disease 2019 (COVID-19, caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)) is primarily a respiratory illness. However, various extrapulmonary manifestations have been reported in patients with severe forms of COVID-19. Notably, SARS-CoV-2 was shown to directly trigger white adipose tissue (WAT) dysfunction, which in turn drives insulin resistance, dyslipidemia, and other adverse outcomes in patients with COVID-19. Although advanced age is the greatest risk factor for COVID-19 severity, published data on the impact of SARS-CoV-2 infection on WAT in aged individuals are scarce. Here, we characterized the response of subcutaneous and visceral WAT depots to SARS-CoV-2 infection in young adult and aged golden hamsters. In both age groups, infection was associated with a decrease in adipocyte size in the two WAT depots; this effect was partly due to changes in tissue's lipid metabolism and persisted for longer in aged hamsters than in young-adult hamsters. In contrast, only the subcutaneous WAT depot contained crown-like structures (CLSs) in which dead adipocytes were surrounded by SARS-CoV-2-infected macrophages, some of them forming syncytial multinucleated cells. Importantly, older age predisposed to a unique manifestation of viral disease in the subcutaneous WAT depot during SARS-CoV-2 infection; the persistence of very large CLSs was indicative of an age-associated defect in the clearance of dead adipocytes by macrophages. Moreover, we uncovered age-related differences in plasma lipid profiles during SARS-CoV-2 infection. These data suggest that the WAT's abnormal response to SARS-CoV-2 infection may contribute to the greater severity of COVID-19 observed in elderly patients.
Assuntos
Tecido Adiposo Branco , COVID-19 , Animais , Cricetinae , Tecido Adiposo Branco/patologia , COVID-19/patologia , Modelos Animais de Doenças , Mesocricetus , SARS-CoV-2RESUMO
Mounting evidence suggests that the gut-to-lung axis is critical during respiratory viral infections. We herein hypothesized that disruption of gut homeostasis during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may associate with early disease outcomes. To address this question, we took advantage of the Syrian hamster model. Our data confirmed that this model recapitulates some hallmark features of the human disease in the lungs. We further showed that SARS-CoV-2 infection associated with mild intestinal inflammation, relative alteration in intestinal barrier property and liver inflammation and altered lipid metabolism. These changes occurred concomitantly with an alteration of the gut microbiota composition over the course of infection, notably characterized by a higher relative abundance of deleterious bacterial taxa such as Enterobacteriaceae and Desulfovibrionaceae. Conversely, several members of the Ruminococcaceae and Lachnospiraceae families, including bacteria known to produce the fermentative products short-chain fatty acids (SCFAs), had a reduced relative proportion compared to non-infected controls. Accordingly, infection led to a transient decrease in systemic SCFA amounts. SCFA supplementation during infection had no effect on clinical and inflammatory parameters. Lastly, a strong correlation between some gut microbiota taxa and clinical and inflammation indices of SARS-CoV-2 infection severity was evidenced. Collectively, alteration of the gut microbiota correlates with disease severity in hamsters making this experimental model valuable for the design of interventional, gut microbiota-targeted, approaches for the control of COVID-19.Abbreviations: SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; COVID-19, coronavirus disease 2019; SCFAs, short-chain fatty acids; dpi, day post-infection; RT-PCR, reverse transcription polymerase chain reaction; IL, interleukin. ACE2, angiotensin converting enzyme 2; TMPRSS2, transmembrane serine protease 2.
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COVID-19/microbiologia , COVID-19/fisiopatologia , Modelos Animais de Doenças , Microbioma Gastrointestinal , Mesocricetus , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/metabolismo , COVID-19/patologia , Cricetinae , Ácidos Graxos Voláteis/administração & dosagem , Ácidos Graxos Voláteis/metabolismo , Humanos , Masculino , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Tratamento Farmacológico da COVID-19RESUMO
Post-transcriptional modifications of RNA constitute an emerging regulatory layer of gene expression. The demethylase fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), has been shown to play a role in cancer, but its contribution to tumor progression and the underlying mechanisms remain unclear. Here, we report widespread FTO downregulation in epithelial cancers associated with increased invasion, metastasis and worse clinical outcome. Both in vitro and in vivo, FTO silencing promotes cancer growth, cell motility and invasion. In human-derived tumor xenografts (PDXs), FTO pharmacological inhibition favors tumorigenesis. Mechanistically, we demonstrate that FTO depletion elicits an epithelial-to-mesenchymal transition (EMT) program through increased m6A and altered 3'-end processing of key mRNAs along the Wnt signaling cascade. Accordingly, FTO knockdown acts via EMT to sensitize mouse xenografts to Wnt inhibition. We thus identify FTO as a key regulator, across epithelial cancers, of Wnt-triggered EMT and tumor progression and reveal a therapeutically exploitable vulnerability of FTO-low tumors.
Assuntos
Neoplasias Epiteliais e Glandulares , RNA , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Humanos , CamundongosRESUMO
HIC1 (Hypermethylated In Cancer 1) a tumor suppressor gene located at 17p13.3, is frequently deleted or epigenetically silenced in many human tumors. HIC1 encodes a transcriptional repressor involved in various aspects of the DNA damage response and in complex regulatory loops with P53 and SIRT1. HIC1 expression in normal prostate tissues has not yet been investigated in detail. Here, we demonstrated by immunohistochemistry that detectable HIC1 expression is restricted to the stroma of both normal and tumor prostate tissues. By RT-qPCR, we showed that HIC1 is poorly expressed in all tested prostate epithelial lineage cell types: primary (PrEC), immortalized (RWPE1) or transformed androgen-dependent (LnCAP) or androgen-independent (PC3 and DU145) prostate epithelial cells. By contrast, HIC1 is strongly expressed in primary PrSMC and immortalized (WMPY-1) prostate myofibroblastic cells. HIC1 depletion in WPMY-1 cells induced decreases in α-SMA expression and contractile capability. In addition to SLUG, we identified stromal cell-derived factor 1/C-X-C motif chemokine 12 (SDF1/CXCL12) as a new HIC1 direct target-gene. Thus, our results identify HIC1 as a tumor suppressor gene which is poorly expressed in the epithelial cells targeted by the tumorigenic process. HIC1 is expressed in stromal myofibroblasts and regulates CXCL12/SDF1 expression, thereby highlighting a complex interplay mediating the tumor promoting activity of the tumor microenvironment. Our studies provide new insights into the role of HIC1 in normal prostatic epithelial-stromal interactions through direct repression of CXCL12 and new mechanistic clues on how its loss of function through promoter hypermethylation during aging could contribute to prostatic tumors.
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Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFß3. Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGFß signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. SIGNIFICANCE: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/prevenção & controle , Neoplasias da Mama/prevenção & controle , Receptores de Estrogênio/metabolismo , Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Quimiocina CCL17/genética , Quimiocina CCL17/metabolismo , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Receptores de Estrogênio/genética , Transdução de Sinais , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
The current health crisis caused by COVID-19 is a challenge for oncology treatment, especially when it comes to radiotherapy. Cancer patients are already known to be very fragile and COVID-19 brings about the risk of severe respiratory complications. In order to treat patients safely while protecting medical teams, the entire health care system must optimize the way it approaches prevention and treatment at a time when social distancing is key to stemming this pandemic. All indications and treatment modalities must be re-discussed. This is particularly the case for radiotherapy of bone metastases for which it is possible to reduce the number of sessions, the frequency of transport and the complexity of treatments. These changes will have to be discussed according to the organization of each radiotherapy department and the health situation, while medical teams must remain vigilant about the risks of complications of bone metastases, particularly spinal metastases. In this short piece, the members of the GEMO (the European Study Group of Bone Metastases) offer a number of recommendations to achieve the above objectives, both in general and in relation to five of the most common situations on radiation therapy for bone metastases.
RESUMO
Bone metastasis (BM) in cancer remains a critical issue because of its associated clinical and biological complications. Moreover, BM can alter the quality of life and survival rate of cancer patients. Growing evidence suggests that bones are a fertile ground for the development of metastasis through a "vicious circle" of bone resorption/formation and tumor growth. This review aims to outline the current major issues in the diagnosis and management of BM in the most common types of osteotropic cancers and describe the mechanisms and effects of BM. First, we discuss the incidence of BM through the following questions: Are we witnessing an increase in incidence, and are we now better equipped with modern imaging techniques? Is the advent of efficient bone resorption inhibitors affecting the bigger picture of BM management? Second, we discuss the potential effects of cancer progression and well-prescribed drugs, such as multitarget tyrosine kinase inhibitors, inhibitors of the mammalian target of rapamycin, and immune checkpoint inhibitors, on BM. Finally, we examine the duality of the effects of some therapies that may help in cancer treatment but may also contribute to further BM.
RESUMO
Prostate cancers have a strong propensity to metastasize to bone and promote osteoblastic lesions. TMPRSS2:ERG is the most frequent gene rearrangement identified in prostate cancer, but whether it is involved in prostate cancer bone metastases is largely unknown. We exploited an intratibial metastasis model to address this issue and we found that ectopic expression of the TMPRSS2:ERG fusion enhances the ability of prostate cancer cell lines to induce osteoblastic lesions by stimulating bone formation and inhibiting the osteolytic response. In line with these in vivo results, we demonstrate that the TMPRSS2:ERG fusion protein increases the expression of osteoblastic markers, including Collagen Type I Alpha 1 Chain and Alkaline Phosphatase, as well as Endothelin-1, a protein with a documented role in osteoblastic bone lesion formation. Moreover, we determined that the TMPRSS2:ERG fusion protein is bound to the regulatory regions of these genes in prostate cancer cell lines, and we report that the expression levels of these osteoblastic markers are correlated with the expression of the TMPRSS2:ERG fusion in patient metastasis samples. Taken together, our results reveal that the TMPRSS2:ERG gene fusion is involved in osteoblastic lesion formation induced by prostate cancer cells.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Osteoblastos/metabolismo , Neoplasias da Próstata/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Cadeia alfa 1 do Colágeno Tipo I , Endotelina-1/genética , Endotelina-1/metabolismo , Humanos , Masculino , Camundongos SCID , Proteínas de Fusão Oncogênica/metabolismo , Osteoblastos/patologia , Células PC-3 , Fenótipo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transplante Heterólogo , Carga Tumoral/genéticaRESUMO
Bone metastasis is the major deleterious event in prostate cancer (PCa). TMPRSS2-ERG fusion is one of the most common chromosomic rearrangements in PCa. However, its implication in bone metastasis development is still unclear. Since bone metastasis starts with the tropism of cancer cells to bone through specific migratory and invasive processes involving osteomimetic capabilities, it is crucial to better our understanding of the influence of TMPRSS2-ERG expression in the mechanisms underlying the bone tropism properties of PCa cells. We developed bioluminescent cell lines expressing the TMPRSS2-ERG fusion in order to assess its role in tumor growth and bone metastasis appearance in a mouse model. First, we showed that the TMPRSS2-ERG fusion increases cell migration and subcutaneous tumor size. Second, using intracardiac injection experiments in mice, we showed that the expression of TMPRSS2-ERG fusion increases the number of metastases in bone. Moreover, TMPRSS2-ERG affects the pattern of metastatic spread by increasing the incidence of tumors in hind limbs and spine, which are two of the most frequent sites of human PCa metastases. Finally, transcriptome analysis highlighted a series of genes regulated by the fusion and involved in the metastatic process. Altogether, our work indicates that TMPRSS2-ERG increases bone tropism of PCa cells and metastasis development.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Proteínas de Fusão Oncogênica/biossíntese , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Animais , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , TransfecçãoRESUMO
Bone metastases are one of the main complications of prostate cancer and they are incurable. We investigated whether and how estrogen receptor-related receptor alpha (ERRα) is involved in bone tumor progression associated with advanced prostate cancer. By meta-analysis, we first found that ERRα expression is correlated with castration-resistant prostate cancer (CRPC), the hallmark of progressive disease. We then analyzed tumor cell progression and the associated signaling pathways in gain-of-function/loss-of-function CRPC models in vivo and in vitro. Increased levels of ERRα in tumor cells led to rapid tumor progression, with both bone destruction and formation, and direct impacts on osteoclasts and osteoblasts. VEGF-A, WNT5A and TGFß1 were upregulated by ERRα in tumor cells and all of these factors also significantly and positively correlated withERRα expression in CRPC patient specimens. Finally, high levels of ERRα in tumor cells stimulated the pro-metastatic factor periostin expression in the stroma, suggesting that ERRα regulates the tumor stromal cell microenvironment to enhance tumor progression. Taken together, our data demonstrate that ERRα is a regulator of CRPC cell progression in bone. Therefore, inhibiting ERRα may constitute a new therapeutic strategy for prostate cancer skeletal-related events.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Neoplasias Ósseas/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias de Próstata Resistentes à Castração/genética , Receptores de Estrogênio/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Wnt-5a/metabolismo , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
The expression gradient of the morphogen Sonic Hedgehog (SHH) is crucial in establishing the number and the identity of the digits during anteroposterior patterning of the limb. Its anterior ectopic expression is responsible for preaxial polydactyly (PPD). Most of these malformations are due to the gain-of-function of the Zone of Polarizing Activity Regulatory Sequence, the only limb-specific enhancer of SHH known to date. We report a family affected with a novel condition associating PPD and hypertrichosis of the upper back, following an autosomal dominant mode of inheritance. This phenotype is consistent with deregulation of SHH expression during limb and follicle development. In affected members, we identified a 2 kb deletion located ~240 kb upstream from the SHH promoter. The deleted sequence is capable of repressing the transcriptional activity of the SHH promoter in vitro, consistent with a silencer activity. We hypothesize that the deletion of this silencer could be responsible for SHH deregulation during development, leading to a PPD-hypertrichosis phenotype.
Assuntos
Sequência de Bases , Proteínas Hedgehog/genética , Hipertricose/genética , Polidactilia/genética , Deleção de Sequência , Elementos Silenciadores Transcricionais , Regiões 5' não Traduzidas , Adolescente , Adulto , Idoso , Padronização Corporal/genética , Criança , Feminino , Dedos/anormalidades , Regulação da Expressão Gênica no Desenvolvimento , Genes Dominantes , Haplótipos , Humanos , Hipertricose/etnologia , Hipertricose/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Fenótipo , Polidactilia/etnologia , Polidactilia/patologia , Análise de Sequência de DNA , População BrancaRESUMO
Nine new 17-(piperazin-1-yl)pyridin-5-yl)steroids as abiraterone analogues were synthesized. Compounds 5d and 5g showed selective activities against 17α-hydroxylase/C17,20-lyase (CYP17A1) and aromatase (CYP19), respectively. IC50 values of 5d were 5.09 and >50ï µm, whereas these values for 5g were >50 ï µm and 7.40 µm, respectively, for CYP17A1 and CYP19. Molecular modelling highlighted that the inhibitor designed to bind cytochrome P450 haem iron is a necessary condition but not the only rationale to explain inhibitory activity. These abiraterone analogues were then evaluated on hormone-independent prostate cancer cell lines DU-145 and PC-3 and on hormone-dependent breast and prostate cancer cell lines MCF-7 and LNCaP, respectively. Compounds 5e, 5g and 5i have showed potent activities only on hormone-independent prostate cancer cell lines DU-145 and PC-3 with 60-85% inhibition of both cell viability and growth at 10 nm with pro-apoptotic mechanism as illustrated in PC-3 cells by DNA ladder assay and Western blotting of Bax, Casp-3 and its substrate, the poly (ADP-ribose) polymerase. We conclude that hybrid heterocycle steroids could be good lead compounds in the drug design especially against hormone-independent prostate cancer.
Assuntos
Androstenóis/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Esteroides/química , Esteroides/farmacologia , Androstenos , Androstenóis/síntese química , Androstenóis/farmacologia , Antineoplásicos/síntese química , Aromatase/química , Aromatase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Clivagem do DNA/efeitos dos fármacos , Humanos , Células MCF-7 , Masculino , Piperazinas/química , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Piridinas/química , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroides/síntese químicaRESUMO
Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site.
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Amidinas/farmacologia , Antineoplásicos/farmacologia , Tiofenos/farmacologia , Transativadores/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Amidinas/química , Amidinas/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Tiofenos/química , Tiofenos/metabolismo , Transativadores/metabolismo , Regulador Transcricional ERGRESUMO
In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the Erg gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg) during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation. Along with the in vitro studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.
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Adipogenia/genética , Células da Medula Óssea/metabolismo , Condrócitos/metabolismo , Condrogênese/genética , Proteínas Proto-Oncogênicas c-ets/genética , Animais , Células da Medula Óssea/citologia , Cartilagem/citologia , Cartilagem/metabolismo , Condrócitos/citologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-ets/metabolismoRESUMO
Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-ß pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-ß pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond to mechanical forces.
Assuntos
Cartilagem Articular/fisiologia , Condrócitos/fisiologia , Sefarose/metabolismo , Animais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Regulação para Baixo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Mecanotransdução Celular/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Estresse Mecânico , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Osteopontin (OPN) is an extracellular matrix glycophosphoprotein that plays a key role in the metastasis of a wide variety of cancers. The high level of OPN expression in prostate cells is associated with malignancy and reduced survival of the patient. Recent studies on prostate cancer (PCa) tissue have revealed recurrent genomic rearrangements involving the fusion of the 5' untranslated region of a prostate-specific androgen-responsive gene with a gene coding for transcription factors from the ETS family. The most frequently identified fusion gene is TMPRSS2:ERG, which causes ERG protein overexpression in PCa cells. ERG is a transcription factor linked to skeletogenesis. This study was designed to test whether ERG and the product of the TMPRSS2:ERG fusion gene modulate OPN gene expression in PCa cells. To characterize ERG and TMPRSS2:ERG transcriptional activity of OPN, we focused on ETS binding sites (EBS) localized in conserved regions of the promoter. Using in vitro and in vivo molecular assays, we showed that ERG increases OPN expression and binds to an EBS (nt -115 to -118) in the OPN promoter. Moreover, stable transfection of prostate tumor cell lines by TMPRSS2:ERG upregulates endogenous OPN expression. Finally, in human prostate tumor samples, detection of the TMPRSS2:ERG fusion gene was significantly associated with OPN overexpression. Taken together, these data suggest that OPN is an ERG-target gene in PCa where the abnormal expression of the transcription factor ERG, due to the TMPRSS2:ERG fusion, disturbs the expression of genes that play an important role in PCa cells and associated metastases.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/metabolismo , Osteopontina/genética , Neoplasias da Próstata/patologia , Transativadores/metabolismo , Ativação Transcricional , Idoso , Sítios de Ligação , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Regulador Transcricional ERGRESUMO
The Ucma protein (Upper zone of growth plate and cartilage matrix associated protein) has recently been described as a novel secretory protein mainly expressed in cartilage and also as a novel vitamin-K-dependent protein named GRP (Gla-rich protein). This protein has the highest Gla content of any protein known to date. In this article, we identify four alternatively spliced variants of Ucma/GRP gene transcripts in mouse chondrocytes. We show that the expression of all four isoforms is associated with the early stages of chondrogenesis. The Ucma/GRP gene encodes four proteins named Ucma/GRP-F1, -F2, -F3, and -F4, which differ by exon 2, exon 4, or both. Among them, only Ucma/GRP-F1 and -F3 were secreted into the culture medium of transfected chondrocytes, while Ucma/GRP-F2 and -F4 accumulated in the cells. Using HeLa cells or freshly isolated embryonic mouse chondrocytes transfected with enhanced green fluorescent protein fusions, microscopy analysis revealed that Ucma/GRP-F1 and -F3 were localized in the Golgi complex, whereas Ucma/GRP-F2 and -F4 formed aggregates. This aggregation was microtubule-dependent since disruption of microtubules with nocodazole reduced Ucma/GRP-F2 and -F4 aggregation in a reversible manner. Using biochemical fractionation and Western blot analysis, Ucma/GRP-F1 and -F3 isoforms were detected in the soluble fraction while Ucma/GRP-F2 and -F4 were found in an insoluble-enriched fraction. We conclude that the co-expression of soluble and insoluble isoforms also Gla-rich and Gla-deleted isoforms may be finely tuned. Imbalance in isoform expression may therefore be involved in skeletal pathology.
Assuntos
Ácido 1-Carboxiglutâmico/análise , Processamento Alternativo/genética , Proteínas/genética , Animais , Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/fisiologia , Condrócitos/metabolismo , Condrogênese/fisiologia , Citoplasma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Éxons/genética , Proteínas da Matriz Extracelular , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Nocodazol/farmacologia , Organelas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/química , Proteína Proto-Oncogênica c-fli-1/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND: Radical resection of primary tracheal tumors may be challenging when more than one-half of the tracheal length is concerned. The present study evaluated the use of cryopreserved aortic allografts (CAAs) to replace long tracheal segments. METHODS: Sixteen adult minipigs underwent tracheal replacement with a CAA. A silicone stent was used to splint the CAA for the first 12 months. Animals were followed-up using bronchoscopic evaluation and killed at predetermined times, for a period up to 18 months long. RESULTS: Intense inflammation and progressive disappearance of typical histologic structures of the aorta were seen within the first 3 months. All animals studied for more than 3 months showed progressive transformation of the graft into a chimerical conduit sharing aortic and tracheal histologic patterns (eg, islands of disorganized elastic fibers/mature respiratory ciliated epithelium, respiratory glands, islets of cartilage). Stent removal was attempted after 12 months in 10 animals, and critical tracheal stenosis was found in six animals and moderate asymptomatic stenosis in four. Clinical course in these latter animals was uneventful until they were killed at 15 to 18 months. In situ hybridization showed that collagen2a1 mRNA was expressed in the cartilage islets at 1 year. Polymerase chain reaction analysis of the SRY gene demonstrated that the newly formed cartilage cells derived from the host. CONCLUSIONS: CAA may be considered as a valuable tracheal substitute for patients with extensive tracheal tumors. Prolonged stenting will be probably mandatory for the clinical application of the procedure in humans.