Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 194
Filtrar
1.
Cancer Cell ; 39(11): 1439-1441, 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34752751

RESUMO

Gut colonization by colibactin-producing bacteria is associated with colorectal cancer. A mutational signature of this genotoxin in human cancer indicates causality but only partially accounts for cell transformation. Instead, the failure of adequately resolving DNA damage causes genomic aberrations and chromosomal instability, constituting the main starting point for colibactin-driven cancer.

2.
EMBO Rep ; 22(9): e52878, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34328245

RESUMO

Helicobacter pylori infection constitutes one of the major risk factors for the development of gastric diseases including gastric cancer. The activation of nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) via classical and alternative pathways is a hallmark of H. pylori infection leading to inflammation in gastric epithelial cells. Tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA) was previously suggested to trigger classical NF-κB activation, but its role in alternative NF-κB activation remains unexplored. Here, we identify TRAF6 and TRAF2 as binding partners of TIFA, contributing to the formation of TIFAsomes upon H. pylori infection. Importantly, the TIFA/TRAF6 interaction enables binding of TGFß-activated kinase 1 (TAK1), leading to the activation of classical NF-κB signaling, while the TIFA/TRAF2 interaction causes the transient displacement of cellular inhibitor of apoptosis 1 (cIAP1) from TRAF2, and proteasomal degradation of cIAP1, to facilitate the activation of the alternative NF-κB pathway. Our findings therefore establish a dual function of TIFA in the activation of classical and alternative NF-κB signaling in H. pylori-infected gastric epithelial cells.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Helicobacter pylori/metabolismo , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo
3.
Nat Commun ; 12(1): 3818, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155207

RESUMO

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 µM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , SARS-CoV-2/metabolismo , Animais , Antinematódeos/farmacologia , Autofagossomos/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , COVID-19/tratamento farmacológico , COVID-19/patologia , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Modelos Animais de Doenças , Humanos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Metaboloma , Niclosamida/farmacologia , Organoides , SARS-CoV-2/isolamento & purificação , Espermidina/farmacologia , Espermina/farmacologia
4.
Gastroenterology ; 161(2): 623-636.e16, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33957136

RESUMO

BACKGROUND & AIMS: The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organized inside glands and crypts. Regeneration depends on Wnt/ß-catenin pathway activation, but to understand homeostasis and its dysregulation in disease, we need to identify the signaling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen-, and gastric acid-producing cells. METHODS: We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief, and parietal cells. We localized the source of the growth factors in the tissue of origin. RESULTS: We show that epidermal growth factor is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with bone morphogenetic factor/Noggin signals, epidermal growth factor controls the differentiation of foveolar cells vs parietal or chief cells. We also show that epidermal growth factor is likely to underlie alteration of the gastric mucosa in the precancerous condition atrophic gastritis. CONCLUSIONS: Use of our recently established mucosoid cultures in combination with analysis of the tissue of origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signaling microenvironment in the human gastric glands.

5.
Nat Commun ; 12(1): 1003, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579932

RESUMO

Genotoxic colibactin-producing pks+ Escherichia coli induce DNA double-strand breaks, mutations, and promote tumor development in mouse models of colorectal cancer (CRC). Colibactin's distinct mutational signature is reflected in human CRC, suggesting a causal link. Here, we investigate its transformation potential using organoids from primary murine colon epithelial cells. Organoids recovered from short-term infection with pks+ E. coli show characteristics of CRC cells, e.g., enhanced proliferation, Wnt-independence, and impaired differentiation. Sequence analysis of Wnt-independent organoids reveals an enhanced mutational burden, including chromosomal aberrations typical of genomic instability. Although we do not find classic Wnt-signaling mutations, we identify several mutations in genes related to p53-signaling, including miR-34a. Knockout of Trp53 or miR-34 in organoids results in Wnt-independence, corroborating a functional interplay between the p53 and Wnt pathways. We propose larger chromosomal alterations and aneuploidy as the basis of transformation in these organoids, consistent with the early appearance of chromosomal instability in CRC.


Assuntos
Células Epiteliais/metabolismo , Escherichia coli/metabolismo , Genômica , Peptídeos/metabolismo , Policetídeos/metabolismo , Animais , Aberrações Cromossômicas , Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/psicologia , Dano ao DNA , Células Epiteliais/patologia , Escherichia coli/genética , Masculino , Camundongos , Camundongos Knockout , Mutação , Organoides , Peptídeos/genética
6.
iScience ; 24(3): 102151, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33585804

RESUMO

Detailed knowledge of the molecular biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is crucial for understanding of viral replication, host responses, and disease progression. Here, we report gene expression profiles of three SARS-CoV- and SARS-CoV-2-infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes, a pro-inflammatory cytokines such as IL-6, were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells.

7.
J Virol Methods ; 290: 114085, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33545196

RESUMO

Emerging mosquito-borne RNA viruses cause massive health complications worldwide. The Zika virus (ZIKV), in particular, has spread dramatically since 2007 and has provoked epidemics in the Americas and the South Pacific. The lack of antiviral therapy and vaccination has focused research on the investigation of ZIKV-host interactions, in order to understand underlying molecular infection mechanisms. We have established an approach for the analysis of ZIKV host dependency factors in a human trophoblast cell line and applied genome-wide CRISPR/Cas9 knockout mutagenesis. The presented method is especially of value for the identification of factors that are essential for placental infection with the potential to serve as targets for antiviral treatment.


Assuntos
Sistemas CRISPR-Cas , Infecção por Zika virus , Zika virus , Animais , Feminino , Humanos , Placenta/virologia , Gravidez , Trofoblastos , Replicação Viral , Zika virus/genética , Infecção por Zika virus/diagnóstico
8.
Nat Cell Biol ; 23(2): 184-197, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462395

RESUMO

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.


Assuntos
Colo do Útero/patologia , Epitélio/patologia , Homeostase , Via de Sinalização Wnt , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Linhagem da Célula , Microambiente Celular , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinas/metabolismo , Metaplasia , Camundongos Endogâmicos C57BL , Organoides/patologia , Receptores Notch/metabolismo , Células-Tronco/patologia , Células Estromais/patologia , Transcrição Genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
9.
Nat Commun ; 11(1): 5117, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037203

RESUMO

Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate ß-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of ß-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Helicobacter pylori/patogenicidade , NF-kappa B/metabolismo , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , DNA/química , DNA/genética , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Floxuridina , Glicosiltransferases/metabolismo , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Lipopolissacarídeos/metabolismo , Mutação , NF-kappa B/genética , Proteínas Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia
10.
mBio ; 11(5)2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963006

RESUMO

Carcinoma of the gallbladder (GBC) is the most frequent tumor of the biliary tract. Despite epidemiological studies showing a correlation between chronic infection with Salmonella enterica Typhi/Paratyphi A and GBC, the underlying molecular mechanisms of this fatal connection are still uncertain. The murine serovar Salmonella Typhimurium has been shown to promote transformation of genetically predisposed cells by driving mitogenic signaling. However, insights from this strain remain limited as it lacks the typhoid toxin produced by the human serovars Typhi and Paratyphi A. In particular, the CdtB subunit of the typhoid toxin directly induces DNA breaks in host cells, likely promoting transformation. To assess the underlying principles of transformation, we used gallbladder organoids as an infection model for Salmonella Paratyphi A. In this model, bacteria can invade epithelial cells, and we observed host cell DNA damage. The induction of DNA double-strand breaks after infection depended on the typhoid toxin CdtB subunit and extended to neighboring, non-infected cells. By cultivating the organoid derived cells into polarized monolayers in air-liquid interphase, we could extend the duration of the infection, and we observed an initial arrest of the cell cycle that does not depend on the typhoid toxin. Non-infected intoxicated cells instead continued to proliferate despite the DNA damage. Our study highlights the importance of the typhoid toxin in causing genomic instability and corroborates the epidemiological link between Salmonella infection and GBC.IMPORTANCE Bacterial infections are increasingly being recognized as risk factors for the development of adenocarcinomas. The strong epidemiological evidence linking Helicobacter pylori infection to stomach cancer has paved the way to the demonstration that bacterial infections cause DNA damage in the host cells, initiating transformation. In this regard, the role of bacterial genotoxins has become more relevant. Salmonella enterica serovars Typhi and Paratyphi A have been clinically associated with gallbladder cancer. By harnessing the stem cell potential of cells from healthy human gallbladder explant, we regenerated and propagated the epithelium of this organ in vitro and used these cultures to model S. Paratyphi A infection. This study demonstrates the importance of the typhoid toxin, encoded only by these specific serovars, in causing genomic instability in healthy gallbladder cells, posing intoxicated cells at risk of malignant transformation.


Assuntos
Dano ao DNA , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Vesícula Biliar/citologia , Salmonella paratyphi A/patogenicidade , Adulto , Idoso , Animais , Células Cultivadas , Feminino , Vesícula Biliar/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Sorogrupo , Virulência/genética
11.
Protein Expr Purif ; 176: 105742, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32866611

RESUMO

Cdc-like kinase 1 (CLK1) is a dual-specificity kinase capable of autophosphorylation on tyrosine residues and Ser/Thr phosphorylation of its substrates. CLK1 belongs to the CLK kinase family that regulates alternative splicing through phosphorylation of serine-arginine rich (SR) proteins. Recent studies have demonstrated that CLK1 has an important role in the replication of influenza A and chikungunya viruses. Furthermore, CLK1 was found to be relevant for the replication of HIV-1 and the West Nile virus, making CLK1 an interesting cellular candidate for the development of a host-directed antiviral therapy that might be efficient for treatment of newly emerging viruses. We describe here our attempts and detailed procedures to obtain the recombinant kinase domain of CLK1 in suitable amounts for crystallization in complex with specific inhibitors. The key solution for the reproducibility of crystals resides in devising and refining expression and purification protocols leading to homogeneous protein. Co-expression of CLK1 with λ-phosphatase and careful purification has yielded crystals of CLK1 complexed with the KH-CB19 inhibitor that diffracted to 1.65 Å. These results paved the path to the screening of more structures of CLK1 complexed compounds, leading to further optimization of their inhibitory activity. Moreover, since kinases are desired targets in numerous pathologies, the approach we report here, the co-expression of kinases with λ-phosphatase, previously used in other kinases, can be adopted as a general protocol in numerous kinase targets for obtaining reproducible and homogenic non-phosphorylated (inactive) forms suitable for biochemical and structural studies thus facilitating the development of novel inhibitors.


Assuntos
Expressão Gênica , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Antivirais/uso terapêutico , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos , Humanos , Domínios Proteicos , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Viroses/tratamento farmacológico , Viroses/enzimologia , Fenômenos Fisiológicos Virais , Replicação Viral , Vírus/metabolismo
12.
mBio ; 11(4)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843556

RESUMO

Multiple studies have demonstrated rapid bacterial genome evolution during chronic infection with Helicobacter pylori In contrast, little was known about genetic changes during the first stages of infection, when selective pressure is likely to be highest. Using single-molecule, real-time (SMRT) and Illumina sequencing technologies, we analyzed genome and methylome evolution during the first 10 weeks of infection by comparing the cag pathogenicity island (cagPAI)-negative H. pylori challenge strain BCS 100 with pairs of H. pylori reisolates from gastric antrum and corpus biopsy specimens of 10 human volunteers who had been infected with this strain as part of a vaccine trial. Most genetic changes detected in the reisolates affected genes with a surface-related role or a predicted function in peptide uptake. Apart from phenotypic changes of the bacterial envelope, a duplication of the catalase gene was observed in one reisolate, which resulted in higher catalase activity and improved survival under oxidative stress conditions. The methylomes also varied in some of the reisolates, mostly by activity switching of phase-variable methyltransferase (MTase) genes. The observed in vivo mutation spectrum was remarkable for a very high proportion of nonsynonymous mutations. Although the data showed substantial within-strain genome diversity in the challenge strain, most antrum and corpus reisolates from the same volunteers were highly similar to each other, indicating that the challenge infection represents a major selective bottleneck shaping the transmitted population. Our findings suggest rapid in vivo selection of H. pylori during early-phase infection providing adaptation to different individuals by common mechanisms of genetic and epigenetic alterations.IMPORTANCE Exceptional genetic diversity and variability are hallmarks of Helicobacter pylori, but the biological role of this plasticity remains incompletely understood. Here, we had the rare opportunity to investigate the molecular evolution during the first weeks of H. pylori infection by comparing the genomes and epigenomes of H. pylori strain BCS 100 used to challenge human volunteers in a vaccine trial with those of bacteria reisolated from the volunteers 10 weeks after the challenge. The data provide molecular insights into the process of establishment of this highly versatile pathogen in 10 different human individual hosts, showing, for example, selection for changes in host-interaction molecules as well as changes in epigenetic methylation patterns. The data provide important clues to the early adaptation of H. pylori to new host niches after transmission, which we believe is vital to understand its success as a chronic pathogen and develop more efficient treatments and vaccines.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Epigenoma , Evolução Molecular , Genoma Bacteriano , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Adaptação Fisiológica , Ilhas Genômicas , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Virulência
13.
Nat Med ; 26(7): 1063-1069, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483361

RESUMO

The mucosal epithelium is a common target of damage by chronic bacterial infections and the accompanying toxins, and most cancers originate from this tissue. We investigated whether colibactin, a potent genotoxin1 associated with certain strains of Escherichia coli2, creates a specific DNA-damage signature in infected human colorectal cells. Notably, the genomic contexts of colibactin-induced DNA double-strand breaks were enriched for an AT-rich hexameric sequence motif, associated with distinct DNA-shape characteristics. A survey of somatic mutations at colibactin target sites of several thousand cancer genomes revealed notable enrichment of this motif in colorectal cancers. Moreover, the exact double-strand-break loci corresponded with mutational hot spots in cancer genomes, reminiscent of a trinucleotide signature previously identified in healthy colorectal epithelial cells3. The present study provides evidence for the etiological role of colibactin in human cancer.


Assuntos
Neoplasias Colorretais/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Peptídeos/farmacologia , Policetídeos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Células Epiteliais/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Mutação/efeitos dos fármacos , Motivos de Nucleotídeos/efeitos dos fármacos
14.
Cell Rep ; 31(7): 107667, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32433976

RESUMO

Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of GTPases and conveys host defense against intracellular bacteria and parasites. During infection, hGBP1 is recruited to pathogen-containing vacuoles, such as Chlamydia trachomatis inclusions, restricts pathogenic growth, and induces the activation of the inflammasome pathway. hGBP1 has a unique catalytic activity to hydrolyze guanosine triphosphate (GTP) to guanosine monophosphate (GMP) in two consecutive cleavage steps. However, the functional significance of this activity in host defense remains elusive. Here, we generate a structure-guided mutant that specifically abrogates GMP production, while maintaining fast cooperative GTP hydrolysis. Complementation experiments in human monocytes/macrophages show that hGBP1-mediated GMP production is dispensable for restricting Chlamydia trachomatis growth but is necessary for inflammasome activation. Mechanistically, GMP is catabolized to uric acid, which in turn activates the NLRP3 inflammasome. Our study demonstrates that the unique enzymology of hGBP1 coordinates bacterial growth restriction and inflammasome signaling.


Assuntos
Infecções por Chlamydia/imunologia , Chlamydia trachomatis/crescimento & desenvolvimento , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Inflamassomos/metabolismo , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/microbiologia , GMP Cíclico , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Nucleotídeos de Guanina/metabolismo , Humanos , Hidrólise , Inflamassomos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais , Células THP-1 , Ácido Úrico/metabolismo
15.
Am J Cancer Res ; 10(2): 610-629, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32195031

RESUMO

The Hippo pathway has been initially discovered by screening genes that regulate organ size in Drosophila. Recent studies have highlighted the role of the Hippo pathway in controlling organ size, tissue homeostasis and regeneration, and signaling dysregulation, especially the overactivation of the transcriptional coactivator YAP/TAZ, which leads to uncontrolled cell growth and malignant transformation. The core components of the Hippo pathway may initiate tumorigenesis by inducing tumor stem cells and proliferation, ultimately leading to metastasis and drug resistance, which occurs extensively in gynecological malignancies, including cervical cancer, ovarian cancer, and endometrial cancer. In this review, we attempt to systematically summarize recent progress in our understanding of the mechanism of Hippo pathway regulation in tumorigenesis and the mechanisms that underlie alterations during gynecological malignancies, as well as new therapeutic strategies.

16.
EMBO J ; 39(6): e104013, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32009247

RESUMO

High-grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the mutational profile and phenotype of the parental tumor. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for the generation of HGSOC organoids, while healthy fallopian tube organoids depend on BMP suppression by Noggin. Fallopian tube organoids modified by stable shRNA knockdown of p53, PTEN, and retinoblastoma protein (RB) also require a low-Wnt environment for long-term growth, while fallopian tube organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotypes of normal vs. loss-of-function organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation.


Assuntos
Neoplasias Ovarianas/genética , Proteínas Supressoras de Tumor/genética , Via de Sinalização Wnt/genética , Carcinogênese/genética , Diferenciação Celular , Progressão da Doença , Epitélio/patologia , Tubas Uterinas/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Organoides/patologia , Neoplasias Ovarianas/patologia , Fenótipo , Nicho de Células-Tronco
17.
Nat Commun ; 10(1): 4368, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554819

RESUMO

The colonic epithelial turnover is driven by crypt-base stem cells that express the R-spondin receptor Lgr5. Signals that regulate epithelial regeneration upon stem cell injury are largely unknown. Here, we explore the dynamics of Wnt signaling in the colon. We identify two populations of cells with active Wnt signaling: highly proliferative Lgr5+/Axin2+ cells, as well as secretory Lgr5-/Axin2+ cells. Upon Lgr5+ cell depletion, these cells are recruited to contribute to crypt regeneration. Chemical injury induced by DSS leads to a loss of both Lgr5+ cells and Axin2+ cells and epithelial regeneration is driven by Axin2- cells, including differentiated Krt20+ surface enterocytes. Regeneration requires stromal Rspo3, which is present at increased levels upon injury and reprograms Lgr5- but Lgr4+ differentiated cells. In contrast, depletion of stromal Rspo3 impairs crypt regeneration, even upon mild injury. We demonstrate that Rspo3 is essential for epithelial repair via induction of Wnt signaling in differentiated cells.


Assuntos
Colo/fisiologia , Mucosa Intestinal/fisiologia , Regeneração/fisiologia , Células-Tronco/metabolismo , Trombospondinas/metabolismo , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Diferenciação Celular/genética , Colite/genética , Colite/metabolismo , Colo/metabolismo , Enterócitos/metabolismo , Perfilação da Expressão Gênica/métodos , Mucosa Intestinal/metabolismo , Queratina-20/genética , Queratina-20/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regeneração/genética , Células-Tronco/citologia , Trombospondinas/genética , Via de Sinalização Wnt/genética
18.
Exp Ther Med ; 18(4): 2401-2412, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31555351

RESUMO

Breast cancer is the most common cancer in women worldwide. Despite recent developments in breast cancer detection and treatment, 1.38 million women each year are still affected. Breast cancer heterogeneity at the population and single-cell level, complexity and developing different metastases are setting several challenges to develop efficient breast cancer therapies. RNA interference (RNAi) represents an opportunity to silence gene expression and inhibit specific pathways in cancer cells. In order to reap the full advantages of RNAi-based therapy, different pathways that sustain cancer cells growth have been targeted using specific siRNAs. The present study investigated the ability of a set of cytotoxic siRNAs to inhibit growth of breast cancer cells. These siRNAs are targeting eukaryotic elongation factor 2 (EEF2), polo-like kinase 1 (PLK1), G protein-coupled receptor kinase 4 (GRK4) and sphingosine kinase interacting protein (SKIP5). To facilitate their targeted delivery, the human epidermal growth factor receptor-3 (HER3)-specific aptamer A30 was used. The in vitro results described in this work indicate that combining the highly specific HER3 aptamer with cytotoxic siRNAs targeting (EEF2, PLK1, GRK4 and SKIP5) can inhibit its activity and ultimately suppress proliferation of HER3 positive breast cancer cells.

19.
Antiviral Res ; 168: 187-196, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176694

RESUMO

Influenza A virus carries eight negative single-stranded RNAs and uses spliced mRNAs to increase the number of proteins produced from them. Several genome-wide screens for essential host factors for influenza A virus replication revealed a necessity for splicing and splicing-related factors, including Cdc-like kinase 1 (CLK1). This CLK family kinase plays a role in alternative splicing regulation through phosphorylation of serine-arginine rich (SR) proteins. To examine the influence that modulation of splicing regulation has on influenza infection, we analyzed the effect of CLK1 knockdown and inhibition. CLK1 knockdown in A549 cells reduced influenza A/WSN/33 virus replication and increased the level of splicing of segment 7, which encodes the viral M1 and M2 proteins. CLK1-/- mice infected with influenza A/England/195/2009 (H1N1pdm09) virus supported lower levels of virus replication than wild-type mice. Screening of newly developed CLK inhibitors revealed several compounds that have an effect on the level of splicing of influenza A gene segment M in different models and decrease influenza A/WSN/33 virus replication in A549 cells. The promising inhibitor KH-CB19, an indole-based enaminonitrile with unique binding mode for CLK1, and its even more selective analogue NIH39 showed high specificity towards CLK1 and had a similar effect on influenza mRNA splicing regulation. Taken together, our findings indicate that targeting host factors that regulate splicing of influenza mRNAs may represent a novel therapeutic approach.


Assuntos
Processamento Alternativo , Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/virologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Processamento Alternativo/efeitos dos fármacos , Animais , Antivirais/farmacologia , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos
20.
Nat Cell Biol ; 21(7): 812-823, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235935

RESUMO

Wnt signalling stimulated by binding of R-spondin (Rspo) to Lgr-family members is crucial for gastrointestinal stem cell renewal. Infection of the stomach with Helicobacter pylori stimulates increased secretion of Rspo by myofibroblasts, leading to an increase in proliferation of Wnt-responsive Axin2+Lgr5- stem cells in the isthmus of the gastric gland and finally gastric gland hyperplasia. Basal Lgr5+ cells are also exposed to Rspo3, but their response remains unclear. Here, we demonstrate that-in contrast to its known mitogenic activity-Rspo3 induces differentiation of basal Lgr5+ cells into secretory cells that express and secrete antimicrobial factors, such as intelectin-1, into the lumen. The depletion of Lgr5+ cells or the knockout of Rspo3 in myofibroblasts leads to hypercolonization of the gastric glands with H. pylori, including the stem cell compartment. By contrast, systemic administration or overexpression of Rspo3 in the stroma clears H. pylori from the gastric glands. Thus, the Rspo3-Lgr5 axis simultaneously regulates both antimicrobial defence and mucosal regeneration.


Assuntos
Mucosa Gástrica/metabolismo , Trombospondinas/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Autorrenovação Celular/fisiologia , Camundongos Transgênicos , Miofibroblastos/metabolismo , Organoides/citologia , Receptores Acoplados a Proteínas G/genética , Células-Tronco/metabolismo , Estômago/efeitos dos fármacos , Trombospondinas/genética , Trombospondinas/farmacologia , Via de Sinalização Wnt/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...