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1.
Nature ; 580(7805): E20, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32350466

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

2.
J Pathol ; 259(4): 455-467, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36695554

RESUMO

The aggressive basal/squamous (Ba/Sq) bladder cancer (BLCA) subtype is often diagnosed at the muscle-invasive stage and can progress to the sarcomatoid variant. Identification of molecular changes occurring during progression from non-muscle-invasive BLCA (NMIBC) to Ba/Sq muscle-invasive BLCA (MIBC) is thus challenging in human disease. We used the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model of Ba/Sq MIBC to study longitudinally the molecular changes leading to the Ba/Sq phenotype and to the sarcomatoid variant using IHC and microdissection followed by RNA-seq at all stages of progression. A shift to the Ba/Sq phenotype started in early progression stages. Pathway analysis of gene clusters with coordinated expression changes revealed Shh signaling loss and a shift from fatty acid metabolism to glycolysis. An upregulated cluster, appearing early in carcinogenesis, showed relevance to human disease, identifying NMIBC patients at risk of progression. Similar to the human counterpart, sarcomatoid BBN tumors displayed a Ba/Sq phenotype and epithelial-mesenchymal transition (EMT) features. An EGFR/FGFR1 signaling switch occurred with sarcomatoid dedifferentiation and correlated with EMT. BLCA cell lines with high EMT were the most sensitive to FGFR1 knockout and resistant to EGFR knockout. Taken together, these findings provide insights into the underlying biology of Ba/Sq BLCA progression and sarcomatoid dedifferentiation with potential clinical implications. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Carcinoma de Células Escamosas , Sarcoma , Neoplasias de Tecidos Moles , Neoplasias da Bexiga Urinária , Animais , Camundongos , Humanos , Bexiga Urinária , Neoplasias da Bexiga Urinária/genética , Carcinogênese/genética , Receptores ErbB
3.
Nucleic Acids Res ; 49(19): 11005-11021, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34648034

RESUMO

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium.


Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/química , Mutação com Perda de Função , Proteínas Nucleares/genética , Transcrição Gênica , Neoplasias da Bexiga Urinária/genética , Sequência de Bases , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Anotação de Sequência Molecular , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
4.
Br J Cancer ; 120(5): 555-564, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30765874

RESUMO

BACKGROUND: Muscle-invasive bladder cancer (MIBC) is an aggressive neoplasm with poor prognosis, lacking effective therapeutic targets. Oncogenic dependency on members of the TAM tyrosine kinase receptor family (TYRO3, AXL, MERTK) has been reported in several cancer types, but their role in bladder cancer has never been explored. METHODS: TAM receptor expression was evaluated in two series of human bladder tumours by gene expression (TCGA and CIT series), immunohistochemistry and western blotting analyses (CIT series). The role of the different TAM receptors was assessed by loss-of-function experiments and pharmaceutical inhibition in vitro and in vivo. RESULTS: We reported a significantly higher expression of TYRO3, but not AXL or MERTK, in both non-MIBCs and MIBCs, compared to normal urothelium. Loss-of-function experiments identified a TYRO3-dependency of bladder carcinoma-derived cells both in vitro and in a mouse xenograft model, whereas AXL and MERTK depletion had only a minor impact on cell viability. Accordingly, TYRO3-dependent bladder tumour cells were sensitive to pharmacological treatment with two pan-TAM inhibitors. Finally, growth inhibition upon TYRO3 depletion relies on cell cycle inhibition and apoptosis associated with induction of tumour-suppressive signals. CONCLUSIONS: Our results provide a preclinical proof of concept for TYRO3 as a potential therapeutic target in bladder cancer.


Assuntos
Carcinoma de Células de Transição/genética , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Bexiga Urinária/genética , Animais , Apoptose/genética , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Expressão Gênica , Humanos , Hylobatidae , Imunoquímica , Técnicas In Vitro , Camundongos , Terapia de Alvo Molecular , Músculo Liso/patologia , Invasividade Neoplásica , Transplante de Neoplasias , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Receptor Tirosina Quinase Axl
5.
Genome Res ; 23(10): 1563-79, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23893515

RESUMO

Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localization of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show an increase in both cell size and global protein levels. Strikingly, we found that in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.


Assuntos
Proliferação de Células , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Genes Essenciais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Ciclo Celular , Linhagem Celular , Senescência Celular , Perfilação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA de Transferência/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Transcrição Gênica , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
6.
Nature ; 464(7292): 1192-5, 2010 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-20414307

RESUMO

During infection, pathogenic bacteria manipulate the host cell in various ways to allow their own replication, propagation and escape from host immune responses. Post-translational modifications are unique mechanisms that allow cells to rapidly, locally and specifically modify activity or interactions of key proteins. Some of these modifications, including phosphorylation and ubiquitylation, can be induced by pathogens. However, the effects of pathogenic bacteria on SUMOylation, an essential post-translational modification in eukaryotic cells, remain largely unknown. Here we show that infection with Listeria monocytogenes leads to a decrease in the levels of cellular SUMO-conjugated proteins. This event is triggered by the bacterial virulence factor listeriolysin O (LLO), which induces a proteasome-independent degradation of Ubc9, an essential enzyme of the SUMOylation machinery, and a proteasome-dependent degradation of some SUMOylated proteins. The effect of LLO on Ubc9 is dependent on the pore-forming capacity of the toxin and is shared by other bacterial pore-forming toxins like perfringolysin O (PFO) and pneumolysin (PLY). Ubc9 degradation was also observed in vivo in infected mice. Furthermore, we show that SUMO overexpression impairs bacterial infection. Together, our results reveal that Listeria, and probably other pathogens, dampen the host response by decreasing the SUMOylation level of proteins critical for infection.


Assuntos
Listeria monocytogenes/patogenicidade , Listeriose/metabolismo , Listeriose/microbiologia , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Linhagem Celular , Células HeLa , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Camundongos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Fatores de Virulência/metabolismo
7.
Int J Biol Sci ; 19(1): 1-12, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36594099

RESUMO

Bladder cancer (BlCa) is the ninth most common cancer worldwide, associated with significant morbidity and mortality. Thus, understand the biological mechanisms underlying tumour progression is of great clinical significance. Vimentin (VIM) is (over)expressed in several carcinomas, putatively in association with EMT. We have previously found that VIM promoter methylation accurately identified BlCa and VIM expression associated with unfavourable prognosis. Herein, we sought to investigate VIM expression regulation and its role in malignant transformation of BlCa. Analysis of tissue samples disclosed higher VIM transcript, protein, and methylation levels in BlCa compared with normal urothelium. VIM protein and transcript levels significantly increased from non-muscle invasive (NMIBC) to muscle-invasive (MIBC) cases and to BlCa metastases. Inverse correlation between epithelial CDH1 and VIM, and a positive correlation between mesenchymal CDH2 and VIM were also observed. In BlCa cell lines, exposure to demethylating agent increased VIM protein, with concomitant decrease in VIM methylation. Moreover, exposure to histone deacetylases pan-inhibitor increased the deposit of active post-translational marks (PTMs) across VIM promoter. In primary normal urothelium cells, lower levels of active PTMs with concomitant higher levels of repressive marks deposit were observed. Finally, VIM knockdown in UMUC3 cell line increased epithelial-like features and decreased migration and invasion in vitro, decreasing tumour size and angiogenesis in vivo. We demonstrated that VIM promoter is epigenetically regulated in normal and neoplastic urothelium, which determine a VIM switch associated with EMT and acquisition of invasive and metastatic properties. These findings might allow for development of new, epigenetic-based, therapeutic strategies for BlCa.


Assuntos
Neoplasias da Bexiga Urinária , Humanos , Vimentina/genética , Vimentina/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Epigênese Genética/genética , Fenótipo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/genética
8.
Oncogene ; 42(19): 1524-1542, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36944729

RESUMO

Muscle-invasive bladder cancer (BLCA) is an aggressive disease. Consensus BLCA transcriptomic subtypes have been proposed, with two major Luminal and Basal subgroups, presenting distinct molecular and clinical characteristics. However, how these distinct subtypes are regulated remains unclear. We hypothesized that epigenetic activation of distinct super-enhancers could drive the transcriptional programs of BLCA subtypes. Through integrated RNA-sequencing and epigenomic profiling of histone marks in primary tumours, cancer cell lines, and normal human urothelia, we established the first integrated epigenetic map of BLCA and demonstrated the link between subtype and epigenetic control. We identified the repertoire of activated super-enhancers and highlighted Basal, Luminal and Normal-associated SEs. We revealed super-enhancer-regulated networks of candidate master transcription factors for Luminal and Basal subgroups including FOXA1 and ZBED2, respectively. FOXA1 CRISPR-Cas9 mutation triggered a shift from Luminal to Basal phenotype, confirming its role in Luminal identity regulation and induced ZBED2 overexpression. In parallel, we showed that both FOXA1 and ZBED2 play concordant roles in preventing inflammatory response in cancer cells through STAT2 inhibition. Our study furthers the understanding of epigenetic regulation of muscle-invasive BLCA and identifies a co-regulated network of super-enhancers and associated transcription factors providing potential targets for the treatment of this aggressive disease.


Assuntos
Fatores de Transcrição , Neoplasias da Bexiga Urinária , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Epigenômica , Epigênese Genética , Regulação da Expressão Gênica , Neoplasias da Bexiga Urinária/patologia , Elementos Facilitadores Genéticos/genética
9.
Gastroenterology ; 140(1): 286-96, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20951138

RESUMO

BACKGROUND & AIMS: Small ubiquitin-like modifiers (SUMOs) are attached to other proteins to regulate their function (sumoylation). We investigated the role of Ubc9, which covalently attaches SUMOs to proteins, in the gastrointestinal tract of adult mice. METHODS: We investigated the effects of decreased sumoylation in adult mammals by generating mice with an inducible knockout (by injection of 4-hydroxytamoxifen) of the E2 enzyme Ubc9 (Ubc9fl/-/ROSA26-CreERT2 mice). We analyzed the phenotypes using a range of histologic techniques. RESULTS: Loss of Ubc9 from adult mice primarily affected the small intestine. Ubc9fl/-/ROSA26-CreERT2 mice died within 6 days of 4-hydroxytamoxifen injection, losing 20% or less of their body weight and developing severe diarrhea on the second day after injection. Surprisingly, other epithelial tissues appeared to be unaffected at that stage. Decreased sumoylation led to the depletion of the intestinal proliferative compartment and to the rapid disappearance of stem cells. Sumoylation was required to separate the proliferative and differentiated compartments from the crypt and control differentiation and function of the secretory lineage. Sumoylation was required for nucleus positioning and polarized organization of actin in the enterocytes. Loss of sumoylation caused detachment of the enterocytes from the basal lamina, as observed in tissue fragility diseases. We identified the intermediate filament keratin 8 as a SUMO substrate in epithelial cells. CONCLUSIONS: Sumoylation maintains intestinal stem cells and the architecture, mechanical stability, and function of the intestinal epithelium of mice.


Assuntos
Mucosa Intestinal/metabolismo , Células-Tronco/metabolismo , Sumoilação , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Fenótipo , Células-Tronco/efeitos dos fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Enzimas de Conjugação de Ubiquitina/genética
10.
EMBO Mol Med ; 10(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29463565

RESUMO

FGFR3 alterations (mutations or translocation) are among the most frequent genetic events in bladder carcinoma. They lead to an aberrant activation of FGFR3 signaling, conferring an oncogenic dependence, which we studied here. We discovered a positive feedback loop, in which the activation of p38 and AKT downstream from the altered FGFR3 upregulates MYC mRNA levels and stabilizes MYC protein, respectively, leading to the accumulation of MYC, which directly upregulates FGFR3 expression by binding to active enhancers upstream from FGFR3 Disruption of this FGFR3/MYC loop in bladder cancer cell lines by treatment with FGFR3, p38, AKT, or BET bromodomain inhibitors (JQ1) preventing MYC transcription decreased cell viability in vitro and tumor growth in vivo A relevance of this loop to human bladder tumors was supported by the positive correlation between FGFR3 and MYC levels in tumors bearing FGFR3 mutations, and the decrease in FGFR3 and MYC levels following anti-FGFR treatment in a PDX model bearing an FGFR3 mutation. These findings open up new possibilities for the treatment of bladder tumors displaying aberrant FGFR3 activation.


Assuntos
Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Azepinas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Triazóis/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Mol Cell Biol ; 33(11): 2163-77, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23530056

RESUMO

Arkadia is a RING domain E3 ubiquitin ligase that activates the transforming growth factor ß (TGF-ß) pathway by inducing degradation of the inhibitor SnoN/Ski. Here we show that Arkadia contains three successive SUMO-interacting motifs (SIMs) that mediate noncovalent interaction with poly-SUMO2. We identify the third SIM (VVDL) of Arkadia to be the most relevant one in this interaction. Furthermore, we provide evidence that Arkadia can function as a SUMO-targeted ubiquitin ligase (STUBL) by ubiquitinating SUMO chains. While the SIMs of Arkadia are not essential for SnoN/Ski degradation in response to TGF-ß, we show that they are necessary for the interaction of Arkadia with polysumoylated PML in response to arsenic and its concomitant accumulation into PML nuclear bodies. Moreover, Arkadia depletion leads to accumulation of polysumoylated PML in response to arsenic, highlighting a requirement of Arkadia for arsenic-induced degradation of polysumoylated PML. Interestingly, Arkadia homodimerizes but does not heterodimerize with RNF4, the other STUBL involved in PML degradation, suggesting that these two E3 ligases do not act synergistically but most probably act independently during this process. Altogether, these results identify Arkadia to be a novel STUBL that can trigger degradation of signal-induced polysumoylated proteins.


Assuntos
Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arsênio/farmacologia , Linhagem Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética
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