RESUMO
Fibroblasts have a considerable functional and molecular heterogeneity and can play various roles in the tumor microenvironment. Here we identify a pro-tumorigenic IL1R1+, IL-1-high-signaling subtype of fibroblasts, using multiple colorectal cancer (CRC) patient single cell sequencing datasets. This subtype of fibroblasts is linked to T cell and macrophage suppression and leads to increased cancer cell growth in 3D co-culture assays. Furthermore, both a fibroblast-specific IL1R1 knockout and IL-1 receptor antagonist Anakinra administration reduce tumor growth in vivo. This is accompanied by reduced intratumoral Th17 cell infiltration. Accordingly, CRC patients who present with IL1R1-expressing cancer-associated-fibroblasts (CAFs), also display elevated levels of immune exhaustion markers, as well as an increased Th17 score and an overall worse survival. Altogether, this study underlines the therapeutic value of targeting IL1R1-expressing CAFs in the context of CRC.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Colorretais , Humanos , Fibroblastos Associados a Câncer/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Fibroblastos/patologia , Tolerância Imunológica , Terapia de Imunossupressão , Microambiente Tumoral , Proliferação de Células , Receptores Tipo I de Interleucina-1/genéticaRESUMO
The canonical WNT signaling pathway is crucial for intestinal stem cell renewal and aberrant WNT signaling is an early event in colorectal cancer (CRC) development. Here, we show for the first time that WNT2 is one of the most significantly induced genes in CRC stroma as compared to normal stroma. The impact of stromal WNT2 on carcinoma formation or progression was not addressed so far. Canonical WNT/ß-catenin signaling was assessed using a 7TGP-reporter construct. Furthermore, effects of WNT2 on fibroblast migration and invasion were determined using siRNA-mediated gene silencing. Tumor cell invasion was studied using organotypic raft cultures and in vivo significance was assessed via a xenograft mouse model. We identified cancer-associated fibroblasts (CAFs) as the main source of WNT2. CAF-derived WNT2 activated canonical signaling in adenomatous polyposis coli/ß-catenin wild-type colon cancer cells in a paracrine fashion, whereas no hyperactivation was detectable in cell lines harboring mutations in the adenomatous polyposis coli/ß-catenin pathway. Furthermore, WNT2 activated autocrine canonical WNT signaling in primary fibroblasts, which was associated with a pro-migratory and pro-invasive phenotype. We identified FZD8 as the putative WNT2 receptor in CAFs. Three-dimensional organotypic co-culture assays revealed that WNT2-mediated fibroblast motility and extracellular matrix remodeling enhanced cancer cell invasion of cell lines even harboring mutations in the adenomatous polyposis coli/ß-catenin pathway. Thus, suggesting a tumor-promoting influence on a broad range of CRC. In line, WNT2 also promotes tumor growth, invasion and metastasis in vivo. Moreover, high WNT2 expression is associated with poor prognosis in human CRC. The identification of the pro-malignant function of stromal derived WNT2 in CRC classifies WNT2 and its receptor as promising stromal targets to confine cancer progression in combination with conventional or targeted therapies.
Assuntos
Comunicação Autócrina/fisiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Via de Sinalização Wnt/fisiologia , Proteína Wnt2/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Progressão da Doença , Células HCT116 , Células HT29 , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteína Wnt2/genéticaRESUMO
The insulin-like growth factor (IGF)2/IGF1 receptor (IGF1R) signaling axis has an important role in intestinal carcinogenesis and overexpression of IGF2 is an accepted risk factor for colorectal cancer (CRC) development. Genetic amplifications and loss of imprinting contribute to the upregulation of IGF2, but insufficiently explain the extent of IGF2 expression in a subset of patients. Here, we show that IGF2 was specifically induced in the tumor stroma of CRC and identified cancer-associated fibroblasts (CAFs) as the major source. Further, we provide functional evidence that stromal IGF2, via the paracrine IGF1R/insulin receptor axis, activated pro-survival AKT signaling in CRC cell lines. In addition to its effects on malignant cells, autocrine IGF2/IGF1R signaling in CAFs induced myofibroblast differentiation in terms of alpha-smooth muscle actin expression and contractility in floating collagen gels. This was further augmented in concert with transforming growth factor-ß (TGFß) signaling suggesting a cooperative mechanism. However, we demonstrated that IGF2 neither induced TGFß/smooth muscle actin/mothers against decapentaplegic (SMAD) signaling nor synergized with TGFß to hyperactivate this pathway in two dimensional and three dimensional cultures. IGF2-mediated physical matrix remodeling by CAFs, but not changes in extracellular matrix-modifying proteases or other secreted factors acting in a paracrine manner on/in cancer cells, facilitated subsequent tumor cell invasion in organotypic co-cultures. Consistently, colon cancer cells co-inoculated with CAFs expressing endogenous IGF2 in mouse xenograft models exhibited elevated invasiveness and dissemination capacity, as well as increased local tumor regrowth after primary tumor resection compared with conditions with IGF2-deficient CAFs. In line, expression of IGF2 correlated with elevated relapse rates and poor survival in CRC patients. In agreement with our results, high-level coexpression of IGF2 and TGFß was predicting adverse outcome with higher accuracy than increased expression of the individual genes alone. Taken together, we demonstrate that stroma-induced IGF2 promotes colon cancer progression in a paracrine and autocrine manner and propose IGF2 as potential target for tumor stroma cotargeting strategies.
Assuntos
Neoplasias Colorretais/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Animais , Comunicação Autócrina , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HCT116 , Xenoenxertos , Humanos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Camundongos Endogâmicos NOD , Comunicação Parácrina , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologia , TransfecçãoRESUMO
The activated tumor stroma participates in many processes that control tumorigenesis, including tumor cell growth, invasion and metastasis. Cancer-associated fibroblasts (CAFs) represent the major cellular component of the stroma and are the main source for connective tissue components of the extracellular matrix and various classes of proteolytic enzymes. The signaling pathways involved in the interactions between tumor and stromal cells and the molecular characteristics that distinguish normal 'resting' fibroblasts from cancer-associated or '-activated' fibroblasts remain poorly defined. Recent studies emphasized the prognostic and therapeutic significance of CAF-related molecular signatures and a number of those genes have been shown to serve as putative therapeutic targets. We have used immuno-laser capture microdissection and whole-genome Affymetrix GeneChip analysis to obtain transcriptional signatures from the activated tumor stroma of colon carcinomas that were compared with normal resting colonic fibroblasts. Several members of the Wnt-signaling pathway and gene sets related to hypoxia, epithelial-to-mesenchymal transition (EMT) and transforming growth factor-ß (TGFß) pathway activation were induced in CAFs. The putative TGFß-target IGFBP7 was identified as a tumor stroma marker of epithelial cancers and as a tumor antigen in mesenchyme-derived sarcomas. We show here that in contrast to its tumor-suppressor function in epithelial cells, IGFPB7 can promote anchorage-independent growth in malignant mesenchymal cells and in epithelial cells with an EMT phenotype when IGFBP7 is expressed by the tumor cells themselves and can induce colony formation in colon cancer cells co-cultured with IGFBP7-expressing CAFs by a paracrine tumor-stroma interaction.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias do Colo/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/biossíntese , Proteínas de Neoplasias/biossíntese , Comunicação Parácrina , Sarcoma/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Masculino , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Sarcoma/genética , Transcrição Gênica/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
PURPOSE: Neurolaena lobata is a Caribbean medicinal plant used for the treatment of several conditions including inflammation. Recent data regarding potent anti-inflammatory activity of the plant and isolated sesquiterpene lactones raised our interest in further pharmacological studies. The present work aimed at providing a mechanistic insight into the anti-inflammatory activity of N. lobata and eight isolated sesquiterpene lactones, as well as a structure-activity relationship and in vivo anti-inflammatory data. METHODS: The effect of the extract and its compounds on the generation of pro-inflammatory proteins was assessed in vitro in endothelial and monocytic cells by enzyme-linked immunosorbent assay. Their potential to modulate the expression of inflammatory genes was further studied at the mRNA level. In vivo anti-inflammatory activity of the chemically characterized extract was evaluated using carrageenan-induced paw edema model in rats. RESULTS: The compounds and extract inhibited LPS- and TNF-α-induced upregulation of the pro-inflammatory molecules E-selectin and interleukin-8 in HUVECtert and THP-1 cells. LPS-induced elevation of mRNA encoding for E-selectin and interleukin-8 was also suppressed. Furthermore, the extract inhibited the development of acute inflammation in rats. CONCLUSIONS: Sesquiterpene lactones from N. lobata interfered with the induction of inflammatory cell adhesion molecules and chemokines in cells stimulated with bacterial products and cytokines. Structure-activity analysis revealed the importance of the double bond at C-4-C-5 and C-2-C-3 and the acetyl group at C-9 for the anti-inflammatory activity. The effect was confirmed in vivo, which raises further interest in the therapeutic potential of the compounds for the treatment of inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Asteraceae/química , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Animais , Selectina E/metabolismo , Edema/tratamento farmacológico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Interleucina-8/metabolismo , Masculino , Estrutura Molecular , Monócitos/efeitos dos fármacos , Componentes Aéreos da Planta/química , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
BACKGROUND: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration. METHODS: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis. RESULTS: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation. CONCLUSION: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.
Assuntos
Acetoexamida/farmacologia , Neoplasias da Mama/tratamento farmacológico , Endotélio Linfático/efeitos dos fármacos , Isoxsuprina/farmacologia , Vasos Linfáticos/efeitos dos fármacos , Nifedipino/farmacologia , Proadifeno/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Adesão Celular/efeitos dos fármacos , Movimento Celular , Quimiotaxia/efeitos dos fármacos , Técnicas de Cocultura , Sinergismo Farmacológico , Endotélio Linfático/citologia , Endotélio Linfático/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Hipoglicemiantes/farmacologia , Metástase Linfática , Vasos Linfáticos/irrigação sanguínea , Vasos Linfáticos/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas , Vasodilatadores/farmacologiaRESUMO
BACKGROUND: Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics. METHODS: Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of 'circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs. RESULTS: Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively. CONCLUSION: Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Endotélio Linfático/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/química , NF-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , Esferoides Celulares/efeitos dos fármacos , Sulfonas/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Quimiotaxia/efeitos dos fármacos , Endotélio Linfático/citologia , Endotélio Linfático/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
BACKGROUND: The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts. METHODS: To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs. RESULTS: We show that tumour spheroids generate 'circular chemorepellent-induced defects' (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with â¼50% attenuation of CCID formation by treatment of cells with 10 µM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general. INTERPRETATION: These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell-cell contacts, migration, and the generation of CCID.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Neoplasias da Mama/patologia , Carcinoma/patologia , Transdiferenciação Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , NF-kappa B/fisiologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Células Endoteliais/fisiologia , Feminino , Humanos , Mesoderma/efeitos dos fármacos , Mesoderma/fisiologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Invasividade Neoplásica , Nitrilas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Células Tumorais CultivadasRESUMO
Higher eukaryotic ribosome biogenesis takes place in the nucleolus and requires the import of ribosomal proteins from the cytoplasm. The ribosomal protein S6 is essential for the formation of ribosome subunits, and in mice S6 heterozygosity triggers embryonal lethality. Downstream of the mTOR (mammalian target of rapamycin) and MAPK (mitogen-activated protein kinase) signalling pathways S6 protein is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. Here, we show that S240/244-phosphorylated S6 is predominantly nuclear but also detectable in the cytoplasm, whereas S235/236-phosphorylated S6 is almost exclusively localized to the nucleus of primary human cells and virtually undetectable in the cytoplasm. However, in transformed cells the latter can also be detected in the cytoplasm. Experiments with the mTOR inhibitor rapamycin revealed that neither blocking the phosphorylation of S6 at S235/236 and S240/244 nor arresting the cell cycle affects the cytoplasmic/nuclear localization of S6 protein. Our findings provide new insights into the regulation of S6 phosphorylation and S6 protein localization in mammalian cells.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteína S6 Ribossômica/química , Proteína S6 Ribossômica/metabolismo , Serina/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/química , Citoplasma/química , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Transporte Proteico , Serina/química , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Human amniotic fluid stem cells (hAFSCs) harbor high proliferative capacity and high differentiation potential and do not raise the ethical concerns associated with human embryonic stem cells. The formation of three-dimensional aggregates known as embryoid bodies (EBs) is the principal step in the differentiation of pluripotent embryonic stem cells. Using c-Kit-positive hAFSC lines, we show here that these stem cells harbor the potential to form EBs. As part of the two kinase complexes, mTORC1 and mTORC2, mammalian target of rapamycin (mTOR) is the key component of an important signaling pathway, which is involved in the regulation of cell proliferation, growth, tumor development and differentiation. Blocking intracellular mTOR activity through the inhibitor rapamycin or through specific small interfering RNA approaches revealed hAFSC EB formation to depend on mTORC1 and mTORC2. These findings demonstrate hAFSCs to be a new and powerful biological system to recapitulate the three-dimensional and tissue level contexts of in vivo development and identify the mTOR pathway to be essential for this process.
Assuntos
Líquido Amniótico/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Agregação Celular , Linhagem Celular , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Proteínas , Serina-Treonina Quinases TOR , Fatores de Transcrição/metabolismoRESUMO
The tuberous sclerosis gene 2 product tuberin is an important regulator of the mammalian target of rapamycin (mTOR). In addition, tuberin is known to bind to the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) and to regulate its stability and localization via mTOR-independent mechanisms. Recently, evidence has been provided that tuberin also affects p27 localization via regulating mTOR's potential to activate the serum- and glucocorticoid-inducible kinase (SGK1) to phosphorylate p27. Taken together, these findings strengthen the argument that besides mTOR-inhibitors, such as rapamycin analogues, p27 and CDKs could also be considered targets for hamartoma therapeutics in tuberous sclerosis.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Imunossupressores/uso terapêutico , Sirolimo/uso terapêutico , Esclerose Tuberosa/tratamento farmacológico , Proteínas Supressoras de Tumor/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Quinases Ciclina-Dependentes/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Serina-Treonina Quinases TOR , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/fisiopatologia , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/fisiologiaRESUMO
The tumor-stroma crosstalk is a dynamic process fundamental in tumor development. In hepatocellular carcinoma (HCC), the progression of malignant hepatocytes frequently depends on transforming growth factor (TGF)-beta provided by stromal cells. TGF-beta induces an epithelial to mesenchymal transition (EMT) of oncogenic Ras-transformed hepatocytes and an upregulation of platelet-derived growth factor (PDGF) signaling. To analyse the influence of the hepatic tumor-stroma crosstalk onto tumor growth and progression, we co-injected malignant hepatocytes and myofibroblasts (MFBs). For this, we either used in vitro-activated p19(ARF) MFBs or in vivo-activated MFBs derived from physiologically inflamed livers of Mdr2/p19(ARF) double-null mice. We show that co-transplantation of MFBs with Ras-transformed hepatocytes strongly enhances tumor growth. Genetic interference with the PDGF signaling decreases tumor cell growth and maintains plasma membrane-located E-cadherin and beta-catenin at the tumor-host border, indicating a blockade of hepatocellular EMT. We further generated a collagen gel-based three dimensional HCC model in vitro to monitor the MFB-induced invasion of micro-organoid HCC spheroids. This invasion was diminished after inhibition of TGF-beta or PDGF signaling. These data suggest that the TGF-beta/PDGF axis is crucial during hepatic tumor-stroma crosstalk, regulating both tumor growth and cancer progression.
Assuntos
Comunicação Celular/fisiologia , Transformação Celular Neoplásica/patologia , Neoplasias Hepáticas Experimentais/patologia , Animais , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Imuno-Histoquímica , Neoplasias Hepáticas Experimentais/metabolismo , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: The association between cigarette smoke exposure and allergic airway disease is a matter for debate. We sought to investigate in an in vitro system whether active smoking reduces the integrity and barrier function of the respiratory epithelium and thus facilitates allergen penetration. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate for the intact respiratory epithelium. The cell monolayer was exposed to standardized cigarette smoke extract (CSE). The extent and effects of trans-epithelial allergen penetration were measured using 125I-labelled purified major respiratory allergens (rBet v 1, rPhl p 5 and rDer p 2) and histamine release experiments. RESULTS: Exposure of cells to concentrations of CSE similar to those found in smokers induced the development of para-cellular gaps and a decrease in trans-epithelial resistance. CSE exposure induced a more than threefold increase in allergen penetration. Increased subepithelial allergen concentrations provoked a substantial augmentation of histamine release from sensitized basophils. CONCLUSIONS: Our results indicate that cigarette smoke is a potent factor capable of reducing the barrier function of the respiratory epithelium for allergens and may contribute to increased allergic inflammation, exacerbation of allergic disease and boosting of IgE memory.
Assuntos
Alérgenos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Nicotiana/efeitos adversos , Mucosa Respiratória/metabolismo , Alérgenos/imunologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/imunologia , Citocinas , Citometria de Fluxo , Liberação de Histamina/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Mucosa Respiratória/imunologia , FumaçaRESUMO
The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.
Assuntos
Diferenciação Celular , Proliferação de Células , Células Precursoras Eritroides/metabolismo , Receptores da Eritropoetina/fisiologia , Receptores de Glucocorticoides/fisiologia , Fator de Transcrição STAT5/fisiologia , Animais , Apoptose , Western Blotting , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Eritroblastos/citologia , Eritroblastos/metabolismo , Citometria de Fluxo , Humanos , Fígado/citologia , Fígado/metabolismo , Camundongos , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes v-ErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal differentiation under appropriate conditions, were established from fetal livers of p53-/- mice. Expression and activation of the EGF-receptor (HER-1/c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal differentiation. Upon inhibition of ErbB function, differentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5- and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms.
Assuntos
Transformação Celular Neoplásica , Receptores ErbB/metabolismo , Células Precursoras Eritroides/patologia , Eritropoese , Leucemia/patologia , Proteínas Oncogênicas v-erbB/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores da Eritropoetina/metabolismo , Animais , Divisão Celular , Células Cultivadas , Ativação Enzimática , Receptores ErbB/genética , Eritroblastos/citologia , Células Precursoras Eritroides/citologia , Humanos , Camundongos , Camundongos Knockout , Proteínas Oncogênicas v-erbB/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Estresse Fisiológico , Proteína Supressora de Tumor p53/genéticaAssuntos
Células Precursoras Eritroides/citologia , Perfilação da Expressão Gênica/métodos , Animais , Diferenciação Celular , Meios de Cultura Livres de Soro , Técnicas de Cultura/métodos , DNA Complementar , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Supressora de Tumor p53/deficiênciaRESUMO
Studies using genetically modified mice and ex vivo tissue culture of erythroid progenitors converge to show that generation of mature erythroid cells depends on the interplay between specific transcriptional regulators and intracellular signals controlled by cytokines and growth factors. These studies also show that terminal differentiation in the erythroid lineage is unusual since the acquisition of the phenotypic traits of mature cells occurs while the cells are still actively dividing. Furthermore, under specific stress conditions, a massive and sustained self-renewal of committed erythroid progenitors can take place to replenish the pool of terminally differentiated cells. We review here how the erythroid genetic program and its interplay with specific cytokines, growth factors and hormones controls survival, proliferation and differentiation of erythroid progenitors both in normal and stress conditions. Special emphasis is laid on our present understanding of the differences in cell cycle control, which result either in self-renewal of erythroid progenitors or in the particular cell divisions which accompany terminal differentiation. Finally, we discuss how deregulation of the various aspects of the physiological control of erythroid progenitor survival, proliferation and differentiation can lead to erythroblast transformation and erythroleukemia.
Assuntos
Ciclo Celular/fisiologia , Transformação Celular Neoplásica , Células Precursoras Eritroides/fisiologia , Leucemia Eritroblástica Aguda , Oncogenes , Transdução de Sinais , Animais , Apoptose , Diferenciação Celular , Técnicas Genéticas , Humanos , CamundongosRESUMO
In immortalized cells of the erythroid lineage, the iron-regulatory protein (IRP) has been suggested to coregulate biosynthesis of the iron storage protein ferritin and the erythroid delta-aminolevulinate synthase (eALAS), a key enzyme in heme production. Under iron scarcity, IRP binds to an iron-responsive element (IRE) located in ferritin and eALAS mRNA leaders, causing a block of translation. In contrast, IRP-IRE interaction is reduced under high iron conditions, allowing efficient translation. We show here that primary chicken erythroblasts (ebls) proliferating or differentiating in culture use a drastically different regulation of iron metabolism. Independently of iron administration, ferritin H (ferH) chain mRNA translation was massively decreased, whereas eALAS transcripts remained constitutively associated with polyribosomes, indicating efficient translation. Variations in iron supply had minor but significant effects on eALAS mRNA polysome recruitment but failed to modulate IRP-affinity to the ferH-IRE in vitro. However, leukemic ebls transformed by the v-ErbA/v-ErbB-expressing avian erythroblastosis virus showed an iron-dependent reduction of IRP mRNA-binding activity, resulting in mobilization of ferH mRNA into polysomes. Hence, we analyzed a panel of ebls overexpressing v-ErbA and/or v-ErbB oncoproteins as well as the respective normal cellular homologues (c-ErbA/TRalpha, c-ErbB/EGFR). It turned out that v-ErbA, a mutated class II nuclear hormone receptor that arrests erythroid differentiation, caused the change in ferH mRNA translation. Accordingly, inhibition of v-ErbA function in these leukemic ebls led to a switch from iron-responsive to iron-independent ferH expression.
Assuntos
Eritroblastos/metabolismo , Ferritinas/metabolismo , Ferro/metabolismo , Proteínas Oncogênicas v-erbA/metabolismo , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Galinhas , Ferritinas/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Proteínas Oncogênicas v-erbA/genética , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
Friend virus-induced erythroleukemia involves two members of the ETS family of transcriptional regulators, both activated via proviral insertion in the corresponding loci. Spi-1/PU.1 is expressed in the disease induced by the original Friend virus SFFV(F-MuLV) complex in adult mice. In contrast, FLI-1 is overexpressed in about 75% of the erythroleukemias induced by the F-MuLV helper virus in newborn mice. To analyse the consequences of the enforced expression of FLI-1 on erythroblast differentiation and proliferation and to compare its activity to that of PU.1/Spi-1, we used a heterologous system of avian primary erythroblasts previously described to study the cooperation between Spi-1/PU.1 and the other molecular alterations observed in SFFV-induced disease. FLI-1 was found: (i) to inhibit the apoptotic cell death program normally activated in erythroblasts following Epo deprivation; (ii) to inhibit the terminal differentiation program induced in these cells in response to Epo and; (iii) to induce their proliferation. However, in contrast to Spi-1/PU.1, the effects of FLI-1 on erythroblast, differentiation and proliferation did not require its cooperation with an abnormally activated form of the EpoR. Enhanced survival of FLI-1 expressing erythroblasts correlated with the upregulation of bcl2 expression. FLI-1 also prevented the rapid downregulation of cyclin D2 and D3 expression normally observed during Epo-induced differentiation and delayed the downregulation of several other genes involved in cell cycle or cell proliferation control. Our results show that overexpression of FLI-1 profoundly deregulates the normal balance between differentiation and proliferation in primary erythroblasts. Thus, the activation of FLI-1 expression observed at the onset of F-MuLV-induced erythroleukemia may provide a proliferative advantage to virus infected cells that would otherwise undergo terminal differentiation or cell death.