ABSTRACT
Post-transcriptional regulation plays a central role in cell differentiation and proliferation. Among the regulatory factors involved in this mechanism, Tristetraprolin (ZFP36 or TTP) is the prototype of a family of RNA-binding proteins that bind to adenylate and uridylate (AU)-rich sequences in the 3'UTR of mRNAs, which promotes their physiological decay. Here, we investigated whether TTP correlates with tumor aggressiveness in breast cancer and is a novel prognostic factor for this neoplasia. By immunoblot analysis, we determined the amount of TTP protein in different breast cancer cell lines and found an inverse correlation between aggressiveness and metastatic potential. TTP mRNA levels were very variable among cells lines and did not correlate with protein levels. Interestingly, by sequencing the entire TTP coding region in Hs578T cells that do not express the TTP protein, we identified a synonymous polymorphism (rs3746083) that showed a statistically significant association with a lack of response to Herceptin/Trastuzumab in HER2-positive-breast cancer patients. Even though this genetic change did not modify the corresponding amino acid, we performed functional studies and showed an effect on protein translation associated with the variant allele with respect to the wild-type. These data underline the importance of synonymous variants on gene expression and the potential role of TTP genetic polymorphisms as a prognostic marker for breast cancer.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Breast Neoplasms/drug therapy , Polymorphism, Single Nucleotide/genetics , Protein Biosynthesis/genetics , RNA-Binding Proteins/genetics , Tristetraprolin/genetics , Angiogenesis Inducing Agents/metabolism , Antibodies, Monoclonal, Humanized/pharmacology , Base Sequence , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Case-Control Studies , Cell Line, Tumor , Cell Proliferation/drug effects , Clone Cells , Female , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , Humans , Interleukin-8/genetics , Interleukin-8/metabolism , Mutant Proteins/metabolism , Neoplasm Invasiveness , Protein Biosynthesis/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Transfection , Trastuzumab , Tristetraprolin/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Most melanoma cells are characterized by the V600E mutation in B-Raf kinase. This mutation leads to increased expression of interleukin (CXCL8), which plays a key role in cell growth and angiogenesis. Thus CXCL8 appears to be an interesting therapeutic target. Hence, we performed vaccination of mice with GST-CXCL8, which results in a reduced incidence of syngenic B16 melanoma cell xenograft tumors. We next addressed the molecular mechanisms responsible for aberrant CXCL8 expression in melanoma. The CXCL8 mRNA contains multiples AU-rich sequences (AREs) that modulate mRNA stability through the binding of tristetraprolin (TTP). Melanoma cell lines express very low TTP levels. We therefore hypothesized that the very low endogenous levels of TTP present in different melanoma cell lines might be responsible for the relative stability of CXCL8 mRNAs. We show that TTP is actively degraded by the proteasome and that extracellular-regulated kinase inhibition results in TTP accumulation. Conditional expression of TTP in A375 melanoma cells leads to CXCL8 mRNA destabilization via its 3' untranslated regions (3'-UTR), and TTP overexpression reduces its production. In contrast, downregulation of TTP by short hairpin RNA results in upregulation of CXCL8 mRNA. Maintaining high TTP levels in melanoma cells decreases cell proliferation and autophagy and induces apoptosis. Sorafenib, a therapeutic agent targeting Raf kinases, decreases CXCL8 expression in melanoma cells through reexpression of TTP. We conclude that loss of TTP represents a key event in the establishment of melanomas through constitutive expression of CXCL8, which constitutes a potent therapeutic target.
Subject(s)
Down-Regulation/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Interleukin-8/metabolism , Melanoma/metabolism , RNA Stability/physiology , Tristetraprolin/metabolism , Animals , Antibodies/immunology , Antibodies/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/physiology , Autophagy/physiology , Benzamides/pharmacology , Benzenesulfonates/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Chemokine CXCL1/immunology , Chemokine CXCL1/pharmacology , Chemokine CXCL5/immunology , Chemokine CXCL5/pharmacology , Dichlororibofuranosylbenzimidazole/pharmacology , Female , Gene Expression/drug effects , Gene Expression/genetics , Genes, Reporter/genetics , Half-Life , Humans , Immunotherapy, Active/methods , Interleukin-8/genetics , Interleukin-8/immunology , Interleukin-8/pharmacology , Leupeptins/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/metabolism , Melanoma/prevention & control , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Microtubule-Associated Proteins/metabolism , Niacinamide/analogs & derivatives , Phenylurea Compounds , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Pyridines/pharmacology , RNA Stability/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Receptors, Interleukin-8B/metabolism , Sorafenib , Transfection , Tristetraprolin/geneticsABSTRACT
DUSP6/MKP-3 is a cytoplasmic dual-specificity phosphatase specific for the MAP kinases ERK1/2. Previous data have shown that the MEK/ERK axis exerts a retro-control on its own signaling through transcriptional and post-translational regulation of DUSP6. We first confirm the key role of MEK/ERK in maintaining the levels of dusp6 mRNA, while PI3K/mTOR, p38 MAPK, and JNK signaling pathways had no significant effects. We further show that regulation of dusp6 mRNA stability plays a critical role in ERK-dependent regulation of dusp6 expression. Luciferase reporter constructs indicated that MEK/ERK signaling increased the half-life of dusp6 mRNA in a 3'untranslated region (3'UTR)-dependent manner. In addition, hypoxia, a hallmark of tumor growth, was found to increase both endogenous levels of dusp6 mRNA and the stability of the luciferase reporter constructs containing its 3'UTR, in a HIF-1-dependent manner. Nevertheless, a basal ERK activity was required for the response to hypoxia. Finally, Tristetraprolin (TTP), a member of the TIS11 CCCH zinc finger protein family, and PUM2, an homolog of drosophila pumilio, two proteins regulating mRNA stability reduced the levels of endogenous dusp6 mRNA and the activity of the dusp6/3'UTR luciferase reporter constructs. This study shows that post-transcriptional regulation is a key process in the control of DUSP6 expression.
Subject(s)
Dual Specificity Phosphatase 6/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/physiology , MAP Kinase Kinase Kinases/metabolism , RNA Processing, Post-Transcriptional/physiology , Cell Line , Dual Specificity Phosphatase 6/genetics , Humans , Hypoxia-Inducible Factor 1/genetics , Hypoxia-Inducible Factor 1/metabolism , MAP Kinase Kinase Kinases/genetics , Protein Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Signal Transduction/physiology , Tristetraprolin/genetics , Tristetraprolin/metabolismABSTRACT
Bevacizumab (Bvz), a Vascular Endothelial Growth Factor (VEGF)-targeted humanised monoclonal antibody, provides clinical benefit in combination with docetaxel (DXL), a microtubule-stabilising agent, in the treatment of metastatic breast and prostate cancers. Since VEGF and their receptors are expressed by tumour cells, we hypothesised that Bvz, in addition to its impact on neo-vascularisation, could have an impact on tumour cells and enhance the DXL activity. Hence, we studied the effect of DXL and Bvz on metastatic breast (MDA MB-231) and prostate (PC3) cancer cells lines. Bvz alone did not decrease cell proliferation but in combination with DXL, Bvz enhanced the anti-proliferative activity of DXL. Other anti-angiogenic factors Sunitinib, Sorafenib and Gefitinib enhanced the anti-proliferative effect of DXL. qPCR experiments showed that DXL significantly increased the VEGF and VEGF receptor 2 (VEGF-R2) mRNA levels. Activation of VEGF and VEGF-R2 promoters demonstrated that enhanced mRNA levels are partly due to transcriptional activation. ELISA assays showed that DXL induced accumulation of cytoplasmic VEGF but decreased extracellular levels by 39% (MDA) and 48% (PC3). Cell surface localisation of VEGF-R2 was increased by DXL alone, but decreased after combined treatment of DXL plus Bvz. Abnormal expression of VEGF-R2 was also shown on breast and prostate tumour samples reinforcing the results obtained on cellular models. In conclusion, DXL and Bvz in combination decreased extracellular VEGF and VEGF-R2 levels at the plasma membrane thereby blocking an important growth/survival loop. Thus, the combined therapeutic impact of Bvz and DXL observed in clinical trials is associated with enhanced anti-proliferative activity and inhibition of the vascular network.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Prostatic Neoplasms/drug therapy , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized , Bevacizumab , Breast Neoplasms/pathology , Cell Division , Cell Survival/drug effects , Cytoplasm/chemistry , Docetaxel , Female , Humans , Male , Phosphorylation , Prostatic Neoplasms/pathology , Sp1 Transcription Factor/physiology , Taxoids/administration & dosage , Transcription, Genetic , Treatment Outcome , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Extracellular signal-regulated kinases (ERK) regulate cellular functions in response to a variety of external signals. However, the specific functions of individual ERK isoforms are largely unknown. Hence, we have investigated the specific function of ERK1 in skin homeostasis and tumorigenesis in ERK1 knockout mice. They spontaneously develop cutaneous lesions and hyperkeratosis with epidermis thickness. Skin hyperproliferation and inflammation induced by application of 12-O-tetradecanoylphorbol-13-acetate (TPA) is strongly reduced in mutant mice. ERK1(-/-) mice are resistant to development of skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by TPA. Tumor appearance was delayed, their formation was less frequent, and their number and size were reduced. Keratinocytes obtained from knockout mice showed reduced growth and resistance to apoptotic signals, accompanied by an impaired expression of genes implicated in growth control and invasiveness. These results highlight the importance of ERK1 in skin homeostasis and in the process of skin tumor development.
Subject(s)
MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 3/deficiency , Skin Neoplasms/enzymology , 9,10-Dimethyl-1,2-benzanthracene , Animals , Apoptosis/physiology , Carcinogens , Cell Growth Processes/physiology , Cocarcinogenesis , Keratinocytes/cytology , Keratinocytes/enzymology , Male , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Skin/cytology , Skin/enzymology , Skin Neoplasms/chemically induced , Skin Neoplasms/pathology , Tetradecanoylphorbol AcetateABSTRACT
Increased angiogenesis in bone marrow (BM) is one of the characteristics of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder that expresses a chimeric Bcr/Abl protein. Recently, the therapeutic strategy in CML has been totally modified with the development of a new drug: imatinib mesylate (STI571), a specific inhibitor of Bcr/Abl tyrosine kinase activity. The aim of our study was to determine, in patients with CML, the capacity of imatinib mesylate to modulate one of the most potent regulators of angiogenesis, the vascular endothelial growth factor (VEGF). In newly diagnosed CML, we observed significantly increased VEGF secretion by CML BM cells and significantly increased VEGF plasma concentrations. We showed that low plasma VEGF concentrations could be one of the characteristics of complete cytogenetic remission. To understand the molecular mechanisms leading to the inhibition of VEGF production by imatinib, we focused our experiments on the human cell line K562, which is Bcr/Abl positive. We demonstrated that imatinib inhibits VEGF gene transcription by targeting the Sp1 and Sp3 transcription factors. Taken together, our results highlight the potential prognostic value of VEGF concentrations in evaluating the evolution of CML patients treated with imatinib.
Subject(s)
Antineoplastic Agents/administration & dosage , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/blood , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/administration & dosage , Pyrimidines/administration & dosage , Vascular Endothelial Growth Factor A/blood , Antineoplastic Agents/pharmacology , Benzamides , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , DNA-Binding Proteins/metabolism , Gene Expression/drug effects , Humans , Imatinib Mesylate , K562 Cells , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Piperazines/pharmacology , Promoter Regions, Genetic , Pyrimidines/pharmacology , RNA, Messenger/analysis , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor , Transcription Factors/metabolism , Transcriptional Activation/drug effects , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.
Subject(s)
Cell Differentiation/physiology , Endothelium, Vascular/growth & development , Lysophospholipids , Neovascularization, Physiologic/physiology , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Thrombin/metabolism , rho GTP-Binding Proteins/metabolism , src-Family Kinases/metabolism , Actins/metabolism , Cell Compartmentation/drug effects , Cell Compartmentation/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cell Size/drug effects , Cell Size/physiology , Cells, Cultured , Cortactin , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Enzyme Inhibitors/pharmacology , Humans , Infant, Newborn , Microfilament Proteins/metabolism , Protein Transport/drug effects , Protein Transport/physiology , Pseudopodia/drug effects , Pseudopodia/metabolism , Pseudopodia/ultrastructure , Pyrimidines/pharmacology , Recombinant Fusion Proteins/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Sphingosine/pharmacology , Thrombin/pharmacology , Umbilical Veins , rac1 GTP-Binding Protein/metabolismABSTRACT
The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).
Subject(s)
Capillary Permeability/drug effects , Neovascularization, Physiologic/drug effects , Proteins/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Signal Transduction , Viper Venoms/chemistry , Amino Acid Sequence , Animals , Cell Line , Enzyme Inhibitors/pharmacology , Humans , Mice , Molecular Sequence Data , Molecular Weight , Proteins/antagonists & inhibitors , Proteins/pharmacology , Receptors, Vascular Endothelial Growth Factor , Sequence Homology, Amino Acid , ViperidaeABSTRACT
Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation, reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study, we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter, tie-1. Using EGFP as a reporter gene, we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently, tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected, puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers, including CD31, CD34, VEGFR-1, VEGFR-2, Tie-1, VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of alpha-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-beta1, two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 and alpha-smooth muscle actin markers. Finally, we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together, these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies.