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Andreas Plückthun - One of the best experts on this subject based on the ideXlab platform.
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Cryo-EM structure of an activated GPCR–G protein complex in lipid nanodiscs
Nature Structural & Molecular Biology, 2021Co-Authors: Meng Zhang, Christoph Klenk, Franz Hagn, Lisa Merklinger, Lena Morstein, Miao Gui, Zi-fu Wang, Christoph Gorgulla, Zhen-yu J. Sun, Andreas PlückthunAbstract:G-protein-coupled Receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR–G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of Neurotensin, Neurotensin Receptor 1 and Gα_i1β_1γ_1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein–protein interactions at the GPCR–G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling. Structures of GPCR Neurotensin Receptor 1 (NTSR1) in complex with Neurotensin and Gα_i1β_1γ_1 in a lipid bilayer environment and without stabilizing antibodies reveal extensive interactions at the GPCR–G protein interface.
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probing the conformation states of Neurotensin Receptor 1 variants by nmr site directed methyl labeling
ChemBioChem, 2021Co-Authors: Inguna Goba, Andreas Plückthun, Matthias Hillenbrand, David Goricanec, Dominik Schum, Franz HagnAbstract:G protein-coupled Receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein-stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope-labeled methyl probe, we show that the activity and the conformation state of stabilized Neurotensin Receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist-induced activity of the Receptor. Furthermore, this assay can be used as a readout when re-introducing agonist-dependent signaling into a highly stabilized, and thus rigidified, Receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively 19 F-labeled Receptors are not sufficient to resolve signal overlap for a more detailed analysis.
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spr based fragment screening with Neurotensin Receptor 1 generates novel small molecule ligands
PLOS ONE, 2017Co-Authors: Sylwia Huber, Philipp Heine, Andreas Plückthun, Fabio Casagrande, Melanie N Hug, Lisha Wang, Lutz Kummer, Michael HennigAbstract:The Neurotensin Receptor 1 represents an important drug target involved in various diseases of the central nervous system. So far, the full exploitation of potential therapeutic activities has been compromised by the lack of compounds with favorable physicochemical and pharmacokinetic properties which efficiently penetrate the blood-brain barrier. Recent progress in the generation of stabilized variants of solubilized Neurotensin Receptor 1 and its subsequent purification and successful structure determination presents a solid starting point to apply the approach of fragment-based screening to extend the chemical space of known Neurotensin Receptor 1 ligands. In this report, surface plasmon resonance was used as primary method to screen 6369 compounds. Thereby 44 hits were identified and confirmed in competition as well as dose-response experiments. Furthermore, 4 out of 8 selected hits were validated using nuclear magnetic resonance spectroscopy as orthogonal biophysical method. Computational analysis of the compound structures, taking the known crystal structure of the endogenous peptide agonist into consideration, gave insight into the potential fragment-binding location and interactions and inspires chemistry efforts for further exploration of the fragments.
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A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional Neurotensin Receptor 1 variants from E. coli
Protein Expression and Purification, 2015Co-Authors: Pascal Egloff, Mattia Deluigi, Philipp Heine, Stefanie Balada, Andreas PlückthunAbstract:G protein-coupled Receptors (GPCRs) are key players of cell signaling, thus representing important drug targets for the treatment of human diseases. Since inherent difficulties in Receptor production and handling have precluded the application of many in vitro experiments, major questions about GPCR mechanisms and dynamics remain elusive to date. We recently used directed evolution in Escherichia coli on Neurotensin Receptor 1 (NTR1) for the generation of GPCR variants with greatly elevated functional expression levels and with excellent stability in detergent micelles. In this work we outline a highly efficient purification method for our evolved Receptor variants, which is based on the application of an inexpensive, disposable high-affinity ligand column as the initial purification step. The ligand resin allows isolation of correctly folded GPCR variants directly from whole E. coli cell lysates at the scale of 10 mg and it permits preparations of agonist- and antagonist-bound Receptor samples. The purification principle presented here was key to the first structures of signaling-active NTR1 variants (Egloff et al., 2014). Since E. coli is uniquely suitable for the production of fully deuterated proteins, our method provides the basis for an array of NMR experiments that were not feasible for GPCRs to date, but which will shed light on novel aspects of Receptor function and dynamics.
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structure of signaling competent Neurotensin Receptor 1 obtained by directed evolution in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Pascal Egloff, Philipp Heine, Stefanie Balada, Matthias Hillenbrand, Christoph Klenk, A Batyuk, Karola M Schlinkmann, Daniel Scott, Marco Schutz, Andreas PlückthunAbstract:Crystallography has advanced our understanding of G protein–coupled Receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using Neurotensin Receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three Neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 A resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled Receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many Receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled Receptor signaling, Receptor maturation, and desensitization.
Daniel Scott - One of the best experts on this subject based on the ideXlab platform.
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Expression and Purification of a Functional E. coli 13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Fabian Bumbak, Daniel Scott, Ross A D Bathgate, Paul R GooleyAbstract:Escherichia coli (E. coli) is the most widely used expression host for recombinant proteins due to high expression yields and straightforward molecular cloning. Directed evolution of G protein-coupled Receptors (GPCRs) has made several of these difficult to express membrane proteins amenable to prokaryotic expression. Here, we describe a protocol for near complete 13CH3-methionine labeling of a thermostable Neurotensin Receptor 1 (enNTS1) variant in E. coli for solution NMR-based dynamics studies. Our expression strategy utilizes methionine biosynthesis pathway inhibition forcing E. coli to incorporate exogenous methionine with 96% efficiency at expression levels of 2.6 mg enNTS1 per liter of expression culture containing 50 mg of 13CH3-methionine. We also provide a 3-step purification protocol that produces final yields of 0.6 mg of functional Apo-state enNTS1.
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optimization and 13ch3 methionine labeling of a signaling competent Neurotensin Receptor 1 variant for nmr studies
Biochimica et Biophysica Acta, 2018Co-Authors: Daniel Scott, Fabian Bumbak, Alastair C Keen, Natalie Gunn, Paul R Gooley, Ross A D BathgateAbstract:Abstract Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled Receptor (GPCR), Neurotensin Receptor 1 (NTS1). Agonist binding to GPCRs shifts the conformational equilibrium of the transmembrane helices towards distinct, thermodynamically favorable conformations that favor effector protein interactions and promotes cell signaling. The introduction of site specific labels for NMR spectroscopy has proven useful for investigating this dynamic process, but the low expression levels and poor stability of GPCRs is a hindrance to solution NMR experiments. Several thermostabilized mutants of NTS1 have been engineered to circumvent this, with the crystal structures of four of these published. The conformational dynamics of NTS1 however, has not been thoroughly investigated with NMR. It is generally accepted that stabilized GPCRs exhibit attenuated signaling, thus we thoroughly characterized the signaling characteristics of several thermostabilized NTS1 variants to identify an optimal variant for protein NMR studies. A variant termed enNTS1 exhibited the best combination of signaling capability and stability upon solubilization with detergents. enNTS1 was subsequently labeled with 13CH3-methionine in E. coli and purified to homogeneity in the absence of bound ligands. Using solution NMR spectroscopy we observed several well dispersed 13CH3-methionine resonances, many of which exhibited chemical shift changes upon the addition of the high affinity agonist peptide, NT8-13. Thus, enNTS1 represents a novel tool for investigating ligand induced conformational changes in NTS1 to gain insights into the molecular mechanisms underlying Neurotensin signaling.
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structure of signaling competent Neurotensin Receptor 1 obtained by directed evolution in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Pascal Egloff, Philipp Heine, Stefanie Balada, Matthias Hillenbrand, Christoph Klenk, A Batyuk, Karola M Schlinkmann, Daniel Scott, Marco Schutz, Andreas PlückthunAbstract:Crystallography has advanced our understanding of G protein–coupled Receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using Neurotensin Receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three Neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 A resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled Receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many Receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled Receptor signaling, Receptor maturation, and desensitization.
Maurizio Boaron - One of the best experts on this subject based on the ideXlab platform.
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Neurotensin Receptor 1 determines the outcome of non small cell lung cancer
Clinical Cancer Research, 2010Co-Authors: Marco Alifano, Frédérique Souazé, Sandra Dupouy, Mohamad Younes, Takashi Takahashi, Alessandra Cancellieri, Stefania Damiani, Sophie Camilleribroet, Sadimenad Ahmedzaid, Maurizio BoaronAbstract:Purpose: This study aimed to investigate the role of the Neurotensin/Neurotensin Receptor I (NTSR1) complex in non–small cell lung cancer (NSCLC) progression. Experimental Design: The expression of Neurotensin and NTSR1 was studied by transcriptome analysis and immunohistochemistry in two series of 74 and 139 consecutive patients with pathologic stage I NSCLC adenocarcinoma. The findings were correlated with clinic-pathologic features. Experimental tumors were generated from the malignant human lung carcinoma cell line A459, and a subclone of LNM35, LNM-R. The role of the Neurotensin signaling system on tumor growth and metastasis was investigated by small hairpin RNA–mediated silencing of NTSR1 and Neurotensin. Results: Transcriptome analysis carried out in a series of 74 patients showed that the positive regulation of NTSR1 put it within the top 50 genes related with relapse-free survival. Immunohistochemistry revealed Neurotensin- and NTSR1-positive staining in 60.4% and 59.7% of lung adenocarcinomas, respectively. At univariate analysis, NTSR1 expression was strongly associated with worse 5-year overall survival rate (P = 0.0081) and relapse-free survival (P = 0.0024). Multivariate analysis showed that patients over 65 years of age (P = 0.0018) and NTSR1 expression (P = 0.0034) were independent negative prognostic factors. Experimental tumor xenografts generated by Neurotensin- and NTSR1-silenced human lung cancer cells revealed that Neurotensin enhanced primary tumor growth and production of massive nodal metastasis via autocrine and paracrine regulation loops. Conclusion: NTSR1 expression was identified as a potential new prognostic biomarker for surgically resected stage I lung adenocarcinomas, as NTSR1 activation was shown to participate in lung cancer progression. Clin Cancer Res; 16(17); 4401–10. ©2010 AACR.
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Neurotensin Receptor 1 determines the outcome of non-small cell lung cancer.
Clinical cancer research : an official journal of the American Association for Cancer Research, 2010Co-Authors: Marco Alifano, Frédérique Souazé, Sandra Dupouy, Sophie Camilleri-broët, Mohamad Younes, Sadi-menad Ahmed-zaïd, Takashi Takahashi, Alessandra Cancellieri, Stefania Damiani, Maurizio BoaronAbstract:This study aimed to investigate the role of the Neurotensin/Neurotensin Receptor I (NTSR1) complex in non-small cell lung cancer (NSCLC) progression. The expression of Neurotensin and NTSR1 was studied by transcriptome analysis and immunohistochemistry in two series of 74 and 139 consecutive patients with pathologic stage I NSCLC adenocarcinoma. The findings were correlated with clinic-pathologic features. Experimental tumors were generated from the malignant human lung carcinoma cell line A459, and a subclone of LNM35, LNM-R. The role of the Neurotensin signaling system on tumor growth and metastasis was investigated by small hairpin RNA-mediated silencing of NTSR1 and Neurotensin. Transcriptome analysis carried out in a series of 74 patients showed that the positive regulation of NTSR1 put it within the top 50 genes related with relapse-free survival. Immunohistochemistry revealed Neurotensin- and NTSR1-positive staining in 60.4% and 59.7% of lung adenocarcinomas, respectively. At univariate analysis, NTSR1 expression was strongly associated with worse 5-year overall survival rate (P = 0.0081) and relapse-free survival (P = 0.0024). Multivariate analysis showed that patients over 65 years of age (P = 0.0018) and NTSR1 expression (P = 0.0034) were independent negative prognostic factors. Experimental tumor xenografts generated by Neurotensin- and NTSR1-silenced human lung cancer cells revealed that Neurotensin enhanced primary tumor growth and production of massive nodal metastasis via autocrine and paracrine regulation loops. NTSR1 expression was identified as a potential new prognostic biomarker for surgically resected stage I lung adenocarcinomas, as NTSR1 activation was shown to participate in lung cancer progression.
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Neurotensin Receptor 1 Determines the Outcome of Non-Small Cell Lung Cancer
Clinical Cancer Research, 2010Co-Authors: Marco Alifano, Frédérique Souazé, Sandra Dupouy, Sophie Camilleri-broët, Mohamad Younes, Sadi-menad Ahmed-zaïd, Takashi Takahashi, Alessandra Cancellieri, Stefania Damiani, Maurizio BoaronAbstract:Purpose: This study aimed to investigate the role of the Neurotensin/Neurotensin Receptor I (NTSR1) complex in non-small cell lung cancer (NSCLC) progression. Experimental Design: The expression of Neurotensin and NTSR1 was studied by transcriptome analysis and immunohistochemistry in two series of 74 and 139 consecutive patients with pathologic stage I NSCLC adenocarcinoma. The findings were correlated with clinic-pathologic features. Experimental tumors were generated from the malignant human lung carcinoma cell line A459, and a subclone of LNM35, LNM-R. The role of the Neurotensin signaling system on tumor growth and metastasis was investigated by small hairpin RNA-mediated silencing of NTSR1 and Neurotensin. Results: Transcriptome analysis carried out in a series of 74 patients showed that the positive regulation of NTSR1 put it within the top 50 genes related with relapse-free survival. Immunohistochemistry revealed Neurotensin-and NTSR1-positive staining in 60.4% and 59.7% of lung adenocarcinomas, respectively. At univariate analysis, NTSR1 expression was strongly associated with worse 5-year overall survival rate (P = 0.0081) and relapse-free survival (P = 0.0024). Multivariate analysis showed that patients over 65 years of age (P = 0.0018) and NTSR1 expression (P = 0.0034) were independent negative prognostic factors. Experimental tumor xenografts generated by Neurotensin-and NTSR1-silenced human lung cancer cells revealed that Neurotensin enhanced primary tumor growth and production of massive nodal metastasis via autocrine and paracrine regulation loops. Conclusion: NTSR1 expression was identified as a potential new prognostic biomarker for surgically resected stage I lung adenocarcinomas, as NTSR1 activation was shown to participate in lung cancer progression.
Saburo Sone - One of the best experts on this subject based on the ideXlab platform.
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the neuromedin u growth hormone secretagogue Receptor 1b Neurotensin Receptor 1 oncogenic signaling pathway as a therapeutic target for lung cancer
Cancer Research, 2006Co-Authors: Koji Takahashi, Chiyuki Furukawa, Atsushi Takano, Nobuhisa Ishikawa, Tatsuya Kato, Satoshi Hayama, Chie Suzuki, Wataru Yasui, Kouki Inai, Saburo SoneAbstract:Using a genome-wide cDNA microarray to search for genes that were specifically up-regulated in non–small cell lung cancers (NSCLC), we identified an abundant expression of neuromedin U (NMU) in the great majority of lung cancers. Immunohistochemical analysis showed a significant association of NMU expression with poorer prognosis of patients with NSCLC. Treatment of NSCLC cells with short interfering RNA against NMU suppressed its expression and inhibited the growth of the cells; on the other hand, the induction of exogenous expression of NMU conferred growth-promoting activity and enhanced cell mobility in vitro . We found that two G protein–coupled Receptors, growth hormone secretagogue Receptor 1b and Neurotensin Receptor 1, were also overexpressed in NSCLC cells, and that a heterodimer complex of these Receptors functioned as an NMU Receptor. The NMU-Receptor interaction subsequently induced the generation of a second messenger, cyclic AMP, to activate its downstream genes including transcription factors and cell cycle regulators. Treatment of NSCLC cells with short interfering RNAs for growth hormone secretagogue Receptor or Neurotensin Receptor 1 suppressed the expression of those genes and the growth of NSCLC cells. These data strongly implied that targeting the NMU signaling pathway would be a promising therapeutic strategy for the treatment of lung cancers. (Cancer Res 2006; 66(19): 9408-19)
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The neuromedin U-growth hormone secretagogue Receptor 1b/Neurotensin Receptor 1 oncogenic signaling pathway as a therapeutic target for lung cancer.
Cancer research, 2006Co-Authors: Koji Takahashi, Chiyuki Furukawa, Atsushi Takano, Nobuhisa Ishikawa, Tatsuya Kato, Satoshi Hayama, Chie Suzuki, Wataru Yasui, Kouki Inai, Saburo SoneAbstract:Using a genome-wide cDNA microarray to search for genes that were specifically up-regulated in non-small cell lung cancers (NSCLC), we identified an abundant expression of neuromedin U (NMU) in the great majority of lung cancers. Immunohistochemical analysis showed a significant association of NMU expression with poorer prognosis of patients with NSCLC. Treatment of NSCLC cells with short interfering RNA against NMU suppressed its expression and inhibited the growth of the cells; on the other hand, the induction of exogenous expression of NMU conferred growth-promoting activity and enhanced cell mobility in vitro. We found that two G protein-coupled Receptors, growth hormone secretagogue Receptor 1b and Neurotensin Receptor 1, were also overexpressed in NSCLC cells, and that a heterodimer complex of these Receptors functioned as an NMU Receptor. The NMU-Receptor interaction subsequently induced the generation of a second messenger, cyclic AMP, to activate its downstream genes including transcription factors and cell cycle regulators. Treatment of NSCLC cells with short interfering RNAs for growth hormone secretagogue Receptor or Neurotensin Receptor 1 suppressed the expression of those genes and the growth of NSCLC cells. These data strongly implied that targeting the NMU signaling pathway would be a promising therapeutic strategy for the treatment of lung cancers.
Marco Alifano - One of the best experts on this subject based on the ideXlab platform.
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Neurotensin Receptor 1 determines the outcome of non small cell lung cancer
Clinical Cancer Research, 2010Co-Authors: Marco Alifano, Frédérique Souazé, Sandra Dupouy, Mohamad Younes, Takashi Takahashi, Alessandra Cancellieri, Stefania Damiani, Sophie Camilleribroet, Sadimenad Ahmedzaid, Maurizio BoaronAbstract:Purpose: This study aimed to investigate the role of the Neurotensin/Neurotensin Receptor I (NTSR1) complex in non–small cell lung cancer (NSCLC) progression. Experimental Design: The expression of Neurotensin and NTSR1 was studied by transcriptome analysis and immunohistochemistry in two series of 74 and 139 consecutive patients with pathologic stage I NSCLC adenocarcinoma. The findings were correlated with clinic-pathologic features. Experimental tumors were generated from the malignant human lung carcinoma cell line A459, and a subclone of LNM35, LNM-R. The role of the Neurotensin signaling system on tumor growth and metastasis was investigated by small hairpin RNA–mediated silencing of NTSR1 and Neurotensin. Results: Transcriptome analysis carried out in a series of 74 patients showed that the positive regulation of NTSR1 put it within the top 50 genes related with relapse-free survival. Immunohistochemistry revealed Neurotensin- and NTSR1-positive staining in 60.4% and 59.7% of lung adenocarcinomas, respectively. At univariate analysis, NTSR1 expression was strongly associated with worse 5-year overall survival rate (P = 0.0081) and relapse-free survival (P = 0.0024). Multivariate analysis showed that patients over 65 years of age (P = 0.0018) and NTSR1 expression (P = 0.0034) were independent negative prognostic factors. Experimental tumor xenografts generated by Neurotensin- and NTSR1-silenced human lung cancer cells revealed that Neurotensin enhanced primary tumor growth and production of massive nodal metastasis via autocrine and paracrine regulation loops. Conclusion: NTSR1 expression was identified as a potential new prognostic biomarker for surgically resected stage I lung adenocarcinomas, as NTSR1 activation was shown to participate in lung cancer progression. Clin Cancer Res; 16(17); 4401–10. ©2010 AACR.
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Neurotensin Receptor 1 determines the outcome of non-small cell lung cancer.
Clinical cancer research : an official journal of the American Association for Cancer Research, 2010Co-Authors: Marco Alifano, Frédérique Souazé, Sandra Dupouy, Sophie Camilleri-broët, Mohamad Younes, Sadi-menad Ahmed-zaïd, Takashi Takahashi, Alessandra Cancellieri, Stefania Damiani, Maurizio BoaronAbstract:This study aimed to investigate the role of the Neurotensin/Neurotensin Receptor I (NTSR1) complex in non-small cell lung cancer (NSCLC) progression. The expression of Neurotensin and NTSR1 was studied by transcriptome analysis and immunohistochemistry in two series of 74 and 139 consecutive patients with pathologic stage I NSCLC adenocarcinoma. The findings were correlated with clinic-pathologic features. Experimental tumors were generated from the malignant human lung carcinoma cell line A459, and a subclone of LNM35, LNM-R. The role of the Neurotensin signaling system on tumor growth and metastasis was investigated by small hairpin RNA-mediated silencing of NTSR1 and Neurotensin. Transcriptome analysis carried out in a series of 74 patients showed that the positive regulation of NTSR1 put it within the top 50 genes related with relapse-free survival. Immunohistochemistry revealed Neurotensin- and NTSR1-positive staining in 60.4% and 59.7% of lung adenocarcinomas, respectively. At univariate analysis, NTSR1 expression was strongly associated with worse 5-year overall survival rate (P = 0.0081) and relapse-free survival (P = 0.0024). Multivariate analysis showed that patients over 65 years of age (P = 0.0018) and NTSR1 expression (P = 0.0034) were independent negative prognostic factors. Experimental tumor xenografts generated by Neurotensin- and NTSR1-silenced human lung cancer cells revealed that Neurotensin enhanced primary tumor growth and production of massive nodal metastasis via autocrine and paracrine regulation loops. NTSR1 expression was identified as a potential new prognostic biomarker for surgically resected stage I lung adenocarcinomas, as NTSR1 activation was shown to participate in lung cancer progression.
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Neurotensin Receptor 1 Determines the Outcome of Non-Small Cell Lung Cancer
Clinical Cancer Research, 2010Co-Authors: Marco Alifano, Frédérique Souazé, Sandra Dupouy, Sophie Camilleri-broët, Mohamad Younes, Sadi-menad Ahmed-zaïd, Takashi Takahashi, Alessandra Cancellieri, Stefania Damiani, Maurizio BoaronAbstract:Purpose: This study aimed to investigate the role of the Neurotensin/Neurotensin Receptor I (NTSR1) complex in non-small cell lung cancer (NSCLC) progression. Experimental Design: The expression of Neurotensin and NTSR1 was studied by transcriptome analysis and immunohistochemistry in two series of 74 and 139 consecutive patients with pathologic stage I NSCLC adenocarcinoma. The findings were correlated with clinic-pathologic features. Experimental tumors were generated from the malignant human lung carcinoma cell line A459, and a subclone of LNM35, LNM-R. The role of the Neurotensin signaling system on tumor growth and metastasis was investigated by small hairpin RNA-mediated silencing of NTSR1 and Neurotensin. Results: Transcriptome analysis carried out in a series of 74 patients showed that the positive regulation of NTSR1 put it within the top 50 genes related with relapse-free survival. Immunohistochemistry revealed Neurotensin-and NTSR1-positive staining in 60.4% and 59.7% of lung adenocarcinomas, respectively. At univariate analysis, NTSR1 expression was strongly associated with worse 5-year overall survival rate (P = 0.0081) and relapse-free survival (P = 0.0024). Multivariate analysis showed that patients over 65 years of age (P = 0.0018) and NTSR1 expression (P = 0.0034) were independent negative prognostic factors. Experimental tumor xenografts generated by Neurotensin-and NTSR1-silenced human lung cancer cells revealed that Neurotensin enhanced primary tumor growth and production of massive nodal metastasis via autocrine and paracrine regulation loops. Conclusion: NTSR1 expression was identified as a potential new prognostic biomarker for surgically resected stage I lung adenocarcinomas, as NTSR1 activation was shown to participate in lung cancer progression.
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The Neurotensin Receptor-1 pathway contributes to human ductal breast cancer progression.
PloS one, 2009Co-Authors: Sandra Dupouy, Frédérique Souazé, Marco Alifano, Véronique Viardot-foucault, Geneviève Plu-bureau, Marc Chaouat, Anne Lavaur, Danielle Hugol, Christian Gespach, Anne GompelAbstract:Background The Neurotensin (NTS) and its specific high affinity G protein coupled Receptor, the NT1 Receptor (NTSR1), are considered to be a good candidate for one of the factors implicated in neoplastic progression. In breast cancer cells, functionally expressed NT1 Receptor coordinates a series of transforming functions including cellular migration and invasion.