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1.
Life Sci Alliance ; 5(2)2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34819356

RESUMO

In melanoma, a switch from a proliferative melanocytic to an invasive mesenchymal phenotype is based on dramatic transcriptional reprogramming which involves complex interactions between a variety of signaling pathways and their downstream transcriptional regulators. TGFß/SMAD, Hippo/YAP/TAZ, and Wnt/ß-catenin signaling pathways are major inducers of transcriptional reprogramming and converge at several levels. Here, we report that TGFß/SMAD, YAP/TAZ, and ß-catenin are all required for a proliferative-to-invasive phenotype switch. Loss and gain of function experimentation, global gene expression analysis, and computational nested effects models revealed the hierarchy between these signaling pathways and identified shared target genes. SMAD-mediated transcription at the top of the hierarchy leads to the activation of YAP/TAZ and of ß-catenin, with YAP/TAZ governing an essential subprogram of TGFß-induced phenotype switching. Wnt/ß-catenin signaling is situated further downstream and exerts a dual role: it promotes the proliferative, differentiated melanoma cell phenotype and it is essential but not sufficient for SMAD or YAP/TAZ-induced phenotype switching. The results identify epistatic interactions among the signaling pathways underlying melanoma phenotype switching and highlight the priorities in targets for melanoma therapy.

2.
Nat Commun ; 12(1): 6468, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34753908

RESUMO

Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors in improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta (TOP2B), not RNA-POL I. This is important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity-often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.

3.
EMBO Mol Med ; : e14351, 2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34664408

RESUMO

Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In this study, a combination of shRNA-mediated synthetic lethality screening and transcriptomic analysis revealed the transcription factors YAP/TAZ as key drivers of Sorafenib resistance in hepatocellular carcinoma (HCC) by repressing Sorafenib-induced ferroptosis. Mechanistically, in a TEAD-dependent manner, YAP/TAZ induce the expression of SLC7A11, a key transporter maintaining intracellular glutathione homeostasis, thus enabling HCC cells to overcome Sorafenib-induced ferroptosis. At the same time, YAP/TAZ sustain the protein stability, nuclear localization, and transcriptional activity of ATF4 which in turn cooperates to induce SLC7A11 expression. Our study uncovers a critical role of YAP/TAZ in the repression of ferroptosis and thus in the establishment of Sorafenib resistance in HCC, highlighting YAP/TAZ-based rewiring strategies as potential approaches to overcome HCC therapy resistance.

4.
J Cell Sci ; 134(21)2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34633031

RESUMO

The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFß-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.

5.
Oncogene ; 40(43): 6195-6209, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34545187

RESUMO

Canonical Wnt/ß-catenin signaling is an established regulator of cellular state and its critical contributions to tumor initiation, malignant tumor progression and metastasis formation have been demonstrated in various cancer types. Here, we investigated how the binding of ß-catenin to the transcriptional coactivators B-cell CLL/lymphoma 9 (Bcl9) and Bcl9-Like (Bcl9L) affected mammary gland carcinogenesis in the MMTV-PyMT transgenic mouse model of metastatic breast cancer. Conditional knockout of both Bcl9 and Bcl9L resulted into tumor cell death. In contrast, disrupting the interaction of Bcl9/Bcl9L with ß-catenin, either by deletion of their HD2 domains or by a point mutation in the N-terminal domain of ß-catenin (D164A), diminished primary tumor growth and tumor cell proliferation and reduced tumor cell invasion and lung metastasis. In comparison, the disruption of HD1 domain-mediated binding of Bcl9/Bcl9L to Pygopus had only moderate effects. Interestingly, interfering with the ß-catenin-Bcl9/Bcl9L-Pygo chain of adapters only partially impaired the transcriptional response of mammary tumor cells to Wnt3a and TGFß treatments. Together, the results indicate that Bcl9/Bcl9L modulate but are not critically required for canonical Wnt signaling in its contribution to breast cancer growth and malignant progression, a notion consistent with the "just-right" hypothesis of Wnt-driven tumor progression.

6.
Nature ; 597(7876): 415-419, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34471287

RESUMO

Inflammasomes are important sentinels of innate immune defence, sensing pathogens and inducing cell death in infected cells1. There are several inflammasome sensors that each detect and respond to a specific pathogen- or damage-associated molecular pattern (PAMP or DAMP, respectively)1. During infection, live pathogens can induce the release of multiple PAMPs and DAMPs, which can simultaneously engage multiple inflammasome sensors2-5. Here we found that AIM2 regulates the innate immune sensors pyrin and ZBP1 to drive inflammatory signalling and a form of inflammatory cell death known as PANoptosis, and provide host protection during infections with herpes simplex virus 1 and Francisella novicida. We also observed that AIM2, pyrin and ZBP1 were members of a large multi-protein complex along with ASC, caspase-1, caspase-8, RIPK3, RIPK1 and FADD, that drove inflammatory cell death (PANoptosis). Collectively, our findings define a previously unknown regulatory and molecular interaction between AIM2, pyrin and ZBP1 that drives assembly of an AIM2-mediated multi-protein complex that we term the AIM2 PANoptosome and comprising multiple inflammasome sensors and cell death regulators. These results advance the understanding of the functions of these molecules in innate immunity and inflammatory cell death, suggesting new therapeutic targets for AIM2-, ZBP1- and pyrin-mediated diseases.


Assuntos
Apoptose/imunologia , Proteínas de Ligação a DNA/metabolismo , Necroptose/imunologia , Pirina/metabolismo , Piroptose/imunologia , Proteínas de Ligação a RNA/metabolismo , Animais , Caspase 1/metabolismo , Células Cultivadas , Citocinas/metabolismo , Feminino , Francisella , Herpesvirus Humano 1 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células THP-1
7.
Proc Natl Acad Sci U S A ; 118(34)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34408016

RESUMO

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, ß-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of ß-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of ß-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of ß-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of ß-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by ß-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-ß-induced EMT. Our results uncouple the signaling from the adhesion function of ß-catenin and underline the importance of Wnt/ß-catenin-dependent transcription in malignant tumor progression of breast cancer.

8.
Oncogenesis ; 10(7): 52, 2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272356

RESUMO

Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

9.
Circulation ; 143(16): 1614-1628, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33682422

RESUMO

BACKGROUND: Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart. METHODS: Single-nucleus RNA sequencing of 54 140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA sequencing and the Assay for Transposase-Accessible Chromatin using sequencing were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice. RESULTS: Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate that the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties. CONCLUSIONS: These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.

10.
Nat Struct Mol Biol ; 28(3): 240-248, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33558761

RESUMO

Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase holocomplex, which consists of catalytic components (SPTLC1 and SPTLC2) and regulatory components (ssSPTa and ORMDL3). However, the assembly, substrate processing and regulation of the complex are unclear. Here, we present 8 cryo-electron microscopy structures of the human serine palmitoyltransferase holocomplex in various functional states at resolutions of 2.6-3.4 Å. The structures reveal not only how catalytic components recognize the substrate, but also how regulatory components modulate the substrate-binding tunnel to control enzyme activity: ssSPTa engages SPTLC2 and shapes the tunnel to determine substrate specificity. ORMDL3 blocks the tunnel and competes with substrate binding through its amino terminus. These findings provide mechanistic insights into sphingolipid biogenesis governed by the serine palmitoyltransferase complex.


Assuntos
Microscopia Crioeletrônica , Serina C-Palmitoiltransferase/metabolismo , Serina C-Palmitoiltransferase/ultraestrutura , Sítios de Ligação , Biocatálise , Domínio Catalítico , Humanos , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Mutação , Reprodutibilidade dos Testes , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/genética , Especificidade por Substrato
11.
Oncogene ; 40(1): 12-27, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33046799

RESUMO

Despite major progress in breast cancer research, the functional contribution of distinct cancer cell clones to malignant tumor progression and metastasis remains largely elusive. We have assessed clonal heterogeneity within individual primary tumors and metastases and also during the distinct stages of malignant tumor progression using clonal tracking of cancer cells in the MMTV-PyMT mouse model of metastatic breast cancer. Comparative gene expression analysis of clonal subpopulations reveals a substantial level of heterogeneity across and also within the various stages of breast carcinogenesis. The intra-stage heterogeneity is primarily manifested by differences in cell proliferation, also found within invasive carcinomas of luminal A-, luminal B-, and HER2-enriched human breast cancer. Surprisingly, in the mouse model of clonal tracing of cancer cells, chemotherapy mainly targets the slow-proliferative clonal populations and fails to efficiently repress the fast-proliferative populations. These insights may have considerable impact on therapy selection and response in breast cancer patients.


Assuntos
Neoplasias da Mama/patologia , Rastreamento de Células/métodos , Perfilação da Expressão Gênica/métodos , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/patogenicidade , Receptor ErbB-2/genética , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Evolução Clonal , Progressão da Doença , Feminino , Redes Reguladoras de Genes , Humanos , Microdissecção e Captura a Laser , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/virologia , Camundongos , Metástase Neoplásica , Estadiamento de Neoplasias , Análise de Sequência de RNA
13.
Nature ; 588(7839): 688-692, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33268895

RESUMO

Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)1. Activation of the inflammasome provides host defence against aspergillosis2,3, which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatus PAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatus is a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillus mutant (Δgt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response.


Assuntos
Aspergilose/prevenção & controle , Aspergillus fumigatus/metabolismo , Inflamassomos/metabolismo , Padrões Moleculares Associados a Patógenos/metabolismo , Polissacarídeos/metabolismo , Animais , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergillus fumigatus/imunologia , Biofilmes , Colite/induzido quimicamente , Colite/prevenção & controle , Sulfato de Dextrana , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Imunidade Inata , Inflamassomos/imunologia , Masculino , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Polissacarídeos/imunologia , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo
14.
Sci Rep ; 10(1): 11444, 2020 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-32632219

RESUMO

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

15.
Cancer Res ; 80(17): 3631-3648, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32586983

RESUMO

Pygopus 2 (Pygo2) is a coactivator of Wnt/ß-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer, we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller, differentiated, and less metastatic tumors, due, in part, to decreased canonical Wnt/ß-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGFß signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically, the Pygo2-histone interaction potentiated Wnt/ß-catenin signaling, in part, by repressing the expression of Wnt signaling antagonists. Furthermore, Pygo2 and ß-catenin regulated the expression of miR-29 family members, which, in turn, repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively, these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer, as its histone-binding capability promotes ß-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation, EMT, and metastasis.


Assuntos
Desdiferenciação Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Mamárias Experimentais/patologia , Animais , Progressão da Doença , Feminino , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL
16.
Oncogene ; 39(7): 1498-1513, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31666683

RESUMO

An epithelial-mesenchymal transition (EMT) represents a basic morphogenetic process of high cell plasticity underlying embryogenesis, wound healing, cancer metastasis and drug resistance. It involves a profound transcriptional and epigenetic reprogramming of cells. A critical role of epigenetic modifiers and their specific chromatin modifications has been demonstrated during EMT. However, it has remained elusive whether epigenetic control differs between the dynamic cell state transitions of reversible EMT and the fixed differentiation status of irreversible EMT. We have employed varying EMT models of murine breast cancer cells to identify the key players establishing epithelial-mesenchymal cell plasticity during reversible and irreversible EMT. We demonstrate that the Mbd3/NuRD complex and the activities of histone deacetylases (HDACs), and Tet2 hydroxylase play a critical role in keeping cancer cells in a highly metastatic mesenchymal state. Combinatorial interference with their functions leads to mesenchymal-epithelial transition (MET) and efficiently represses metastasis formation by invasive murine and human breast cancer cells in vivo.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Histona Desacetilases/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Metástase Neoplásica
17.
Nat Commun ; 10(1): 5755, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848340

RESUMO

Autophagy perturbation represents an emerging therapeutic strategy in cancer. Although LATS1 and LATS2 kinases, core components of the mammalian Hippo pathway, have been shown to exert tumor suppressive activities, here we report a pro-survival role of LATS1 but not LATS2 in hepatocellular carcinoma (HCC) cells. Specifically, LATS1 restricts lethal autophagy in HCC cells induced by sorafenib, the standard of care for advanced HCC patients. Notably, autophagy regulation by LATS1 is independent of its kinase activity. Instead, LATS1 stabilizes the autophagy core-machinery component Beclin-1 by promoting K27-linked ubiquitination at lysine residues K32 and K263 on Beclin-1. Consequently, ubiquitination of Beclin-1 negatively regulates autophagy by promoting inactive dimer formation of Beclin-1. Our study highlights a functional diversity between LATS1 and LATS2, and uncovers a scaffolding role of LATS1 in mediating a cross-talk between the Hippo signaling pathway and autophagy.


Assuntos
Autofagia/imunologia , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/imunologia , Neoplasias Hepáticas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/imunologia , Humanos , Estimativa de Kaplan-Meier , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/mortalidade , Lisina/metabolismo , Camundongos , Camundongos Knockout , Organoides , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Proteínas Supressoras de Tumor/imunologia , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Sci Rep ; 9(1): 7258, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31076619

RESUMO

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.


Assuntos
Expressão Gênica/fisiologia , Heparina/metabolismo , Células Estromais/metabolismo , Plaquetas/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Soro/metabolismo , Transdução de Sinais/fisiologia , Cordão Umbilical/metabolismo
19.
Cell Chem Biol ; 26(6): 863-877.e7, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31031142

RESUMO

Necroptosis is an inflammatory form of programmed cell death executed through plasma membrane rupture by the pseudokinase mixed lineage kinase domain-like (MLKL). We previously showed that MLKL activation requires metabolites of the inositol phosphate (IP) pathway. Here we reveal that I(1,3,4,6)P4, I(1,3,4,5,6)P5, and IP6 promote membrane permeabilization by MLKL through directly binding the N-terminal executioner domain (NED) and dissociating its auto-inhibitory region. We show that IP6 and inositol pentakisphosphate 2-kinase (IPPK) are required for necroptosis as IPPK deletion ablated IP6 production and inhibited necroptosis. The NED auto-inhibitory region is more extensive than originally described and single amino acid substitutions along this region induce spontaneous necroptosis by MLKL. Activating IPs bind three sites with affinity of 100-600 µM to destabilize contacts between the auto-inhibitory region and NED, thereby promoting MLKL activation. We therefore uncover MLKL's activating switch in NED triggered by a select repertoire of IP metabolites.


Assuntos
Fosfatos de Inositol/metabolismo , Proteínas Quinases/metabolismo , Animais , Sobrevivência Celular , Células HT29 , Humanos , Proteínas Quinases/isolamento & purificação , Células Sf9 , Spodoptera
20.
Cancer Cell ; 35(1): 17-32.e6, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30645973

RESUMO

Cancer cell plasticity facilitates the development of therapy resistance and malignant progression. De-differentiation processes, such as an epithelial-mesenchymal transition (EMT), are known to enhance cellular plasticity. Here, we demonstrate that cancer cell plasticity can be exploited therapeutically by forcing the trans-differentiation of EMT-derived breast cancer cells into post-mitotic and functional adipocytes. Delineation of the molecular pathways underlying such trans-differentiation has motivated a combination therapy with MEK inhibitors and the anti-diabetic drug Rosiglitazone in various mouse models of murine and human breast cancer in vivo. This combination therapy provokes the conversion of invasive and disseminating cancer cells into post-mitotic adipocytes leading to the repression of primary tumor invasion and metastasis formation.


Assuntos
Adipócitos/citologia , Neoplasias da Mama/tratamento farmacológico , Transdiferenciação Celular/efeitos dos fármacos , Flavonoides/administração & dosagem , Metástase Neoplásica/tratamento farmacológico , Rosiglitazona/administração & dosagem , Células 3T3-L1 , Adipogenia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Flavonoides/farmacologia , Humanos , Camundongos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-met/metabolismo , Rosiglitazona/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
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