Publications by authors named "Jean-Michel Bidart"

48 Publications

DNA FISH Diagnostic Assay on Cytological Samples of Thyroid Follicular Neoplasms.

Cancers (Basel) 2020 Sep 6;12(9). Epub 2020 Sep 6.

Department of Endocrinology, Gustave Roussy, Université Paris-Saclay, 94805 Villejuif, France.

Although fine-needle aspiration cytology (FNAC) is helpful in determining whether thyroid nodules are benign or malignant, this distinction remains a cytological challenge in follicular neoplasms. Identification of genomic alterations in cytological specimens with direct and routine techniques would therefore have great clinical value. A series of 153 cases consisting of 72 and 81 histopathologically confirmed classic follicular adenomas (cFAs) and classic follicular thyroid carcinomas (cFTCs), respectively, was studied by means of different molecular techniques in three different cohorts of patients (pts). In the first cohort (training set) of 66 pts, three specific alterations characterized by array comparative genomic hybridization (aCGH) were exclusively found in half of cFTCs. These structural abnormalities corresponded to losses of 1p36.33-35.1 and 22q13.2-13.31, and gain of whole chromosome X. The second independent cohort (validation set) of 60 pts confirmed these data on touch preparations of frozen follicular neoplasms by triple DNA fluorescent in situ hybridization using selected commercially available probes. The third cohort, consisting of 27 archived cytological samples from an equal number of pts that had been obtained for preoperative FNAC and morphologically classified as and histologically verified to be follicular neoplasms, confirmed our previous findings and showed the feasibility of the DNA FISH (DNA fluorescent in situ hybridization) assay. All together, these data suggest that our triple DNA FISH diagnostic assay may detect 50% of cFTCs with a specificity higher than 98% and be useful as a low-cost adjunct to cytomorphology to help further classify follicular neoplasms on already routinely stained cytological specimens.
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http://dx.doi.org/10.3390/cancers12092529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564487PMC
September 2020

Insights into significance of combined inhibition of MEK and m-TOR signalling output in KRAS mutant non-small-cell lung cancer.

Br J Cancer 2016 08 21;115(5):549-52. Epub 2016 Jul 21.

The Institute of Cancer Research, Clinical Pharmacology and Trials Team, London, SM2 5PT, UK.

Background: We aimed to understand the dependence of MEK and m-TOR inhibition in EGFR(WT)/ALK(non-rearranged) NSCLC cell lines.

Methods: In a panel of KRAS(M) and KRAS(WT) NSCLC cell lines, we determined growth inhibition (GI) following maximal reduction in p-ERK and p-S6RP caused by trametinib (MEK inhibitor) and AZD2014 (m-TOR inhibitor), respectively.

Results: GI caused by maximal m-TOR inhibition was significantly greater than GI caused by maximal MEK inhibition in the cell line panel (52% vs 18%, P<10(-4)). There was no significant difference in GI caused by maximal m-TOR compared with maximal m-TOR+MEK inhibition. However, GI caused by the combination was significantly greater in the KRAS(M) cell lines (79% vs 61%, P=0.017).

Conclusions: m-TOR inhibition was more critical to GI than MEK inhibition in EGFR(WT)/ALK(non-rearranged) NSCLC cells. The combination of MEK and m-TOR inhibition was most effective in KRAS(M) cells.
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http://dx.doi.org/10.1038/bjc.2016.220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997545PMC
August 2016

NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation.

Proc Natl Acad Sci U S A 2015 Apr 6;112(16):5051-6. Epub 2015 Apr 6.

UMR 8200, CNRS, Villejuif F-94805, France; Institut Gustave Roussy, Villejuif F-94805, France; University Paris-Sud, Orsay F-91400, France;

Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H2O2, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H2O2 production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H2O2 production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect.
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http://dx.doi.org/10.1073/pnas.1420707112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413347PMC
April 2015

Epigenetic-related gene expression profile in medullary thyroid cancer revealed the overexpression of the histone methyltransferases EZH2 and SMYD3 in aggressive tumours.

Mol Cell Endocrinol 2014 Jul 9;392(1-2):8-13. Epub 2014 May 9.

Dipartimento di Medicina Interna e Specialità Mediche, Università di Roma "Sapienza", 00161 Roma, Italy. Electronic address:

Epigenetic control of gene expression plays a major influence in the development and progression of many cancer types. Aim of the present study was to investigate the expression of epigenetic regulators in a large cohort of medullary thyroid carcinomas (MTC), correlating the data with the clinical outcome and mutational status of the patients. Taqman Low Density Arrays (TLDAs) were used to analyze expression levels of several genes involved in the epigenetic control of transcription in a series of 54 MTCs. The patients cohort included 13 familial MTCs and 41 sporadic forms; 33 hosted a RET mutation and 13 a RAS somatic mutation. The expression profiling revealed in the more aggressive diseases (i.e. occurrence of metastases; persistent disease; disease-related death) a significant increase of EZH2 and SMYD3 gene expression. The increased levels of EZH2 and SMYD3 did not correlate significantly with mutational status of RET or RAS genes. Thus, the histone methyltransferases EZH2 and SMYD3 mRNA expression may represent useful prognostic biomarkers tailoring the most appropriate follow-up and timing of therapeutic approaches.
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http://dx.doi.org/10.1016/j.mce.2014.04.016DOI Listing
July 2014

Changes in signaling pathways induced by vandetanib in a human medullary thyroid carcinoma model, as analyzed by reverse phase protein array.

Thyroid 2014 Jan 24;24(1):43-51. Epub 2013 Dec 24.

1 Genetic Stability and Oncogenesis Research Unit, National Center for Scientific Research (CNRS UMR 8200), Gustave Roussy and University of Paris-Sud , Villejuif, France .

Background: Medullary thyroid carcinoma (MTC) is a rare tumor that is caused by activating mutations in the proto-oncogene RET. Vandetanib, a tyrosine-kinase inhibitor, has been recently approved to treat adult patients with metastatic MTC. The aim of this study was to investigate changes in signaling pathways induced by vandetanib treatment in preclinical MTC models, using the reverse-phase protein array method (RPPA).

Methods: The human TT cell line was used to assess in vitro and in vivo activity of vandetanib. Protein extracts from TT cells or TT xenografted mice, treated by increasing concentrations of vandetanib for different periods of time, were probed with a set of 12 antibodies representing major signaling pathways, using RPPA. Results were validated using two distinct protein detection methods: Western immunoblotting and immunohistochemistry.

Results: Vandetanib displays antiproliferative and antiangiogenic activities and inhibits RET autophosphorylation. The MAPK and AKT pathways were the two major signaling pathways inhibited by vandetanib. Interestingly, phosphorylated levels of NFκB-p65 were significantly increased by vandetanib. Comparable results were obtained in both the in vitro and in vivo approaches, as well as for the protein detection methods. However, some discrepancies were observed between RPPA and Western immunoblotting, possibly due to lack of specificity of the primary antibodies used.

Conclusions: Overall, our results confirmed the interest of RPPA for screening global changes induced in signaling pathways by kinase inhibitors. MAPK and AKT were identified as the main pathways involved in vandetanib response in MTC models. Our results also suggest alternative routes for controlling the disease, and provide a rationale for the development of therapeutic combinations based on the comprehensive identification of molecular events induced by inhibitors.
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http://dx.doi.org/10.1089/thy.2013.0514DOI Listing
January 2014

Changes in signaling pathways induced by vandetanib in a human medullary thyroid carcinoma model, as analyzed by reverse phase protein array.

Thyroid 2013 Jul 3. Epub 2013 Jul 3.

Institut Gustave-Roussy, CNRS UMR8200, Villejuif, France ;

Background: Medullary thyroid carcinoma (MTC) is a rare tumor that is due to activating mutations in the proto-oncogene RET. Vandetanib, a tyrosine-kinase inhibitor, has been recently approved to treat adult patients with metastatic MTC. The aim of this study was to investigate changes in signaling pathways induced by vandetanib treatment in preclinical MTC models, using the reverse-phase protein array method (RPPA).

Methods: The human TT cell line was used to assess in vitro and in vivo activity of vandetanib. Protein extracts from TT cells or TT xenografted mice, treated by increasing concentrations of vandetanib for different periods of time, were probed with a set of 12 antibodies representing major signaling pathways, using RPPA. Results were validated using two distinct protein detection methods, western-immunoblotting and immunohistochemistry.

Results: Vandetanib displays antiproliferative and antiangiogenic activities and inhibits RET auto-phosphorylation. MAPK and AKT pathways were the two major signaling pathways inhibited by vandetanib. Interestingly, phosphorylated levels of NFκB-p65 were significantly increased by vandetanib. Comparable results were obtained in both the in vitro and in vivo approaches as well as for the protein detection methods, although some discrepancies were observed between RPPA and western-immunoblotting.

Conclusions: Results confirmed the reliability and the utility of RPPA for screening global changes induced in signaling pathways by kinase inhibitors. MAPK and AKT were identified as the main pathways involved in vandetanib response in MTC models. Our results also suggest alternative routes for controlling the disease and provide a rationale for the development of therapeutic combinations based on the comprehensive identification of molecular events induced by inhibitors.
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http://dx.doi.org/10.1089/thy.2012.0224DOI Listing
July 2013

Tumor-associated macrophages (TAMs) form an interconnected cellular supportive network in anaplastic thyroid carcinoma.

PLoS One 2011 21;6(7):e22567. Epub 2011 Jul 21.

Department of Biopathology, Institut Gustave Roussy, Villejuif, France.

Background: A relationship between the increased density of tumor-associated macrophages (TAMs) and decreased survival was recently reported in thyroid cancer patients. Among these tumors, anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumors in humans. TAMs (type M2) have been recognized as promoting tumor growth. The purpose of our study was to analyze with immunohistochemistry the presence of TAMs in a series of 27 ATC.

Methodology/principal Findings: Several macrophages markers such as NADPH oxidase complex NOX2-p22phox, CD163 and CD 68 were used. Immunostainings showed that TAMs represent more than 50% of nucleated cells in all ATCs. Moreover, these markers allowed the identification of elongated thin ramified cytoplasmic extensions, bestowing a "microglia-like" appearance on these cells which we termed "Ramified TAMs" (RTAMs). In contrast, cancer cells were totally negative. Cellular stroma was highly simplified since apart from cancer cells and blood vessels, RTAMs were the only other cellular component. RTAMs were evenly distributed and intermingled with cancer cells, and were in direct contact with other RTAMs via their ramifications. Moreover, RTAMs displayed strong immunostaining for connexin Cx43. Long chains of interconnected RTAMs arose from perivascular clusters and were dispersed within the tumor parenchyma. When expressed, the glucose transporter Glut1 was found in RTAMs and blood vessels, but rarely in cancer cells.

Conclusion: ATCs display a very dense network of interconnected RTAMs in direct contact with intermingled cancer cells. To our knowledge this is the first time that such a network is described in a malignant tumor. This network was found in all our studied cases and appeared specific to ATC, since it was not found in differentiated thyroid cancers specimens. Taken together, these results suggest that RTAMs network is directly related to the aggressiveness of the disease via metabolic and trophic functions which remain to be determined.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022567PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3141071PMC
December 2011

Hes1 is required for appropriate morphogenesis and differentiation during mouse thyroid gland development.

PLoS One 2011 Feb 25;6(2):e16752. Epub 2011 Feb 25.

INSERM U845, Université Paris-Descartes, Paris, France.

Notch signalling plays an important role in endocrine development, through its target gene Hes1. Hes1, a bHLH transcriptional repressor, influences progenitor cell proliferation and differentiation. Recently, Hes1 was shown to be expressed in the thyroid and regulate expression of the sodium iodide symporter (Nis). To investigate the role of Hes1 for thyroid development, we studied thyroid morphology and function in mice lacking Hes1. During normal mouse thyroid development, Hes1 was detected from E9.5 onwards in the median anlage, and at E11.5 in the ultimobranchial bodies. Hes1(-/-) mouse embryos had a significantly lower number of Nkx2-1-positive progenitor cells (p<0.05) at E9.5 and at E11.5. Moreover, Hes1(-/-) mouse embryos showed a significantly smaller total thyroid surface area (-40 to -60%) compared to wild type mice at all study time points (E9.5-E16.5). In both Hes1(-/-) and wild type mouse embryos, most Nkx2-1-positive thyroid cells expressed the cell cycle inhibitor p57 at E9.5 in correlation with low proliferation index. In Hes1(-/-) mouse embryos, fusion of the median anlage with the ultimobranchial bodies was delayed by 3 days (E16.5 vs. E13.5 in wild type mice). After fusion of thyroid anlages, hypoplastic Hes1(-/-) thyroids revealed a significantly decreased labelling area for T4 (-78%) and calcitonin (-65%) normalized to Nkx2-1 positive cells. Decreased T4-synthesis might be due to reduced Nis labelling area (-69%). These findings suggest a dual role of Hes1 during thyroid development: first, control of the number of both thyrocyte and C-cell progenitors, via a p57-independent mechanism; second, adequate differentiation and endocrine function of thyrocytes and C-cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016752PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045378PMC
February 2011

Identification of soluble candidate biomarkers of therapeutic response to sunitinib in medullary thyroid carcinoma in preclinical models.

Clin Cancer Res 2011 Apr 15;17(7):2044-54. Epub 2011 Feb 15.

Unité CNRS UMR8200, Institut de Cancérologie Gustave Roussy and Univ Paris-Sud, Villejuif, France.

Purpose: Medullary thyroid carcinoma (MTC), an aggressive rare tumor due to activating mutations in the proto-oncogene RET, requires new therapeutic strategies. Sunitinib, a potent inhibitor of RET, VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor (PDGFR)α/β, has been reported as clinically effective in some patients with advanced MTC. In this study, we examine molecular mechanisms of action of sunitinib and identify candidate soluble biomarkers of response.

Experimental Design: Both in vitro and in vivo assays, using the human TT RET(C634W) MTC cell line, were done to assess the activity of sunitinib. Kinetic microarray studies were used to analyze molecular pathways modified by sunitinib and to identify candidate biomarkers that were subsequently investigated in the serum of patients.

Results: Sunitinib displayed antiproliferative and antiangiogenic activities and inhibited RET autophosphorylation and activation of downstream signaling pathways. We showed that sunitinib treatment induced major changes in the expression of genes involved in tissue invasion and metastasis including vimentin (VIM), urokinase plasminogen (PLAU), tenascin-C (TN-C), SPARC, and CD44. Analyzing downregulated genes, we identified those encoding secreted proteins and, among them, interleukin (IL)-8 was found to be modulated in the serum of xenografted mice under sunitinib treatment. Furthermore, we demonstrated that metastatic MTC patients presented increased serum levels of IL-8 and TGF-β2.

Conclusions: Experimental models confirm the clinical efficacy of sunitinib observed in a few studies. Molecular pathways revealed by genomic signatures underline the impact of sunitinib on tissue invasion. Selected soluble candidate biomarkers could be of value for monitoring sunitinib response in metastatic MTC patients.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-2041DOI Listing
April 2011

Gene expression signature discriminates sporadic from post-radiotherapy-induced thyroid tumors.

Endocr Relat Cancer 2011 Feb 19;18(1):193-206. Epub 2011 Jan 19.

CEA, DSV, IRCM, LCE, BP6, Fontenay-aux-Roses F-92265, France.

Both external and internal exposure to ionizing radiation are strong risk factors for the development of thyroid tumors. Until now, the diagnosis of radiation-induced thyroid tumors has been deduced from a network of arguments taken together with the individual history of radiation exposure. Neither the histological features nor the genetic alterations observed in these tumors have been shown to be specific fingerprints of an exposure to radiation. The aim of our work is to define ionizing radiation-related molecular specificities in a series of secondary thyroid tumors developed in the radiation field of patients treated by radiotherapy. To identify molecular markers that could represent a radiation-induction signature, we compared 25K microarray transcriptome profiles of a learning set of 28 thyroid tumors, which comprised 14 follicular thyroid adenomas (FTA) and 14 papillary thyroid carcinomas (PTC), either sporadic or consecutive to external radiotherapy in childhood. We identified a signature composed of 322 genes which discriminates radiation-induced tumors (FTA and PTC) from their sporadic counterparts. The robustness of this signature was further confirmed by blind case-by-case classification of an independent set of 29 tumors (16 FTA and 13 PTC). After the histology code break by the clinicians, 26/29 tumors were well classified regarding tumor etiology, 1 was undetermined, and 2 were misclassified. Our results help shed light on radiation-induced thyroid carcinogenesis, since specific molecular pathways are deregulated in radiation-induced tumors.
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http://dx.doi.org/10.1677/ERC-10-0205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023880PMC
February 2011

Functional consequences of dual oxidase-thyroperoxidase interaction at the plasma membrane.

J Clin Endocrinol Metab 2010 Dec 8;95(12):5403-11. Epub 2010 Sep 8.

Instituto de Biofisica Carlos Chagas Filho, CCS-Bloc G-Cidade Universitaria, Rio de Janeiro, Brazil.

Context: Thyroperoxidase (TPO) and dual oxidase (DUOX) are present at the apical membrane of thyrocytes, where TPO catalyzes thyroid hormone biosynthesis in the presence of H2O2 produced by DUOX. Both enzymes are colocalized and associated, but the consequences of this interaction remain obscure.

Objective: The objective of this study was to evaluate the functional consequences of TPO-DUOX interaction at the plasma membrane.

Design: The functional consequences of DUOX-TPO interaction were studied by measuring extracellular H2O2 concentration and TPO activity in a heterologous system. For this purpose, HEK293 cells were transiently transfected with a combination of human TPO with human DUOX1 or DUOX2 in the presence of their respective maturation factors, DUOXA1 or DUOXA2. The effect of human DUOX2 mutants in which cysteine residues in the N-terminal domain were replaced by glycines was also analyzed.

Results: We observed that production of H2O2 decreases both TPO and DUOX activities. We show that TPO presents a catalase-like effect that protects DUOX from inhibition by H2O2. This catalase-like effect depends on the association between both enzymes, which probably occurs through the DUOX peroxidase-like domain because this effect was not observed with human DUOX2 mutants.

Conclusion: The DUOX-TPO association at the plasma membrane is relevant for normal enzyme properties. Normally, TPO consumes H2O2 produced by DUOX, decreasing the availability of this substance at the apical membrane of thyrocytes and, in turn, probably decreasing the oxidative damage of macromolecules.
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http://dx.doi.org/10.1210/jc.2010-1085DOI Listing
December 2010

Role of H2O2 in RET/PTC1 chromosomal rearrangement produced by ionizing radiation in human thyroid cells.

Cancer Res 2010 May 27;70(10):4123-32. Epub 2010 Apr 27.

UBRM, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, Rabat, Morocco.

During childhood, the thyroid gland is one of the most sensitive organs to the carcinogenetic effects of ionizing radiation that may lead to papillary thyroid carcinoma (PTC) associated with RET/PTC oncogene rearrangement. Exposure to ionizing radiation induces a transient "oxidative burst" through radiolysis of water, which can cause DNA damage and mediates part of the radiation effects. H(2)O(2) is a potent DNA-damaging agent that induces DNA double-strand breaks, and consequently, chromosomal aberrations. Irradiation by 5 Gy X-ray increased extracellular H(2)O(2). Therefore, we investigated the implication of H(2)O(2) in the generation of RET/PTC1 rearrangement after X-ray exposure. We developed a highly specific and sensitive nested reverse transcription-PCR method. By using the human thyroid cell line HTori-3, previously found to produce RET/PTC1 after gamma-irradiation, we showed that H(2)O(2), generated during a 5 Gy X-ray irradiation, causes DNA double-strand breaks and contributes to RET/PTC1 formation. Pretreatment of cells with catalase, a scavenger of H(2)O(2), significantly decreased RET/PTC1 rearrangement formation. Finally, RET/PTC chromosomal rearrangement was detected in HTori-3.1 cells after exposure of cells to H(2)O(2) (25 micromol/L), at a dose that did not affect the cell viability. This study shows for the first time that H(2)O(2) is able to cause RET/PTC1 rearrangement in thyroid cells and consequently highlights that oxidative stress could be responsible for the occurrence of RET/PTC1 rearrangement found in thyroid lesions even in the absence of radiation exposure.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-4336DOI Listing
May 2010

Differential gene expression profiles of invasive and non-invasive non-functioning pituitary adenomas based on microarray analysis.

Endocr Relat Cancer 2010 Jun 21;17(2):361-71. Epub 2010 Apr 21.

Hôpital de Bicêtre, France.

Non-functioning pituitary adenomas (NFPAs) may be locally invasive. Markers of invasiveness are needed to guide patient management and particularly the use of adjuvant radiotherapy. To examine whether invasive NFPAs display a specific gene expression profile relative to non-invasive tumors, we selected 40 NFPAs (38 of the gonadotroph type) and classified them as invasive (n=22) or non-invasive (n=18) on the basis of magnetic resonance imaging and surgical findings. We then performed pangenomic analysis with the 44k Agilent human whole genome expression oligonucleotide microarray in order to identify genes with differential expression between invasive and non-invasive NFPAs. Candidate genes were then tested in qRT-PCR. Prediction class analysis showed that the expression of 346 genes differed between invasive and non-invasive NFPAs (P<0.001), of which 233 genes were up-regulated and 113 genes were down-regulated in invasive tumors. On the basis of Ingenuity networks and the degree of up- or down-regulation in invasive versus non-invasive tumors, 35 genes were selected for expression quantification by qRT-PCR. Overexpression of only four genes was confirmed, namely IGFBP5 (P=0.02), MYO5A (P=0.04), FLT3 (P=0.01), and NFE2L1 (P=0.02). At the protein level, only myosin 5A (MYO5A) immunostaining was stronger in invasive than in non-invasive NFPAs. Molecular signature allows to differentiate 'grossly' invasive from non-invasive NFPAs. The product of one of these genes, MYO5A, may be a useful marker of tumor invasiveness.
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http://dx.doi.org/10.1677/ERC-10-0018DOI Listing
June 2010

Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues.

Endocr Relat Cancer 2010 Mar 29;17(1):27-37. Epub 2010 Jan 29.

CNRS, FRE2939, Villejuif F-94805, France.

NADPH oxidase 4 (NOX4) belongs to the NOX family that generates reactive oxygen species (ROS). Function and tissue distribution of NOX4 have not yet been entirely clarified. To date, in the thyroid gland, only DUOX1/2 NOX systems have been described. NOX4 mRNA expression, as shown by real-time PCR, was present in normal thyroid tissue, regulated by TSH and significantly increased in differentiated cancer tissues. TSH increased the protein level of NOX4 in human thyroid primary culture and NOX4-dependent ROS generation. NOX4 immunostaining was detected in normal and pathologic thyroid tissues. In normal thyroid tissue, staining was heterogeneous and mostly found in activated columnar thyrocytes but absent in quiescent flat cells. Papillary and follicular thyroid carcinomas displayed more homogeneous staining. The p22(phox) protein that forms a heterodimeric enzyme complex with NOX4 displayed an identical cellular expression pattern and was also positively regulated by TSH. ROS may have various biological effects, depending on the site of production. Intracellular NOX4-p22(phox) localization suggests a role in cytoplasmic redox signaling, in contrast to the DUOX localization at the apical membrane that corresponds to an extracellular H(2)O(2) production. Increased NOX4-p22(phox) in cancer might be related to a higher proliferation rate and tumor progression but a role in the development of tumors has to be further studied and established in the future.
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http://dx.doi.org/10.1677/ERC-09-0175DOI Listing
March 2010

Aggressive inherited and sporadic medullary thyroid carcinomas display similar oncogenic pathways.

Endocr Relat Cancer 2009 Dec 12;16(4):1261-72. Epub 2009 Aug 12.

CNRS FRE 2939, Institut de Cancérologie Gustave-Roussy, Villejuif, France.

RET oncogene mutations are found in familial medullary thyroid carcinomas (MTC) and in one-third of sporadic cases. Oncogenic mechanisms involved in non-RET mutated sporadic MTC remain unclear. To study alterations associated with the development of both inherited and sporadic MTC, pangenomic DNA microarrays were used to analyze the transcriptome of 13 MTCs (four familial and nine sporadic). By using an ANOVA test, a list of 173 gene sequences with at least a twofold change expression was obtained. A subset of differentially expressed genes was controlled by real-time quantitative PCR and immunohistochemistry on a larger collection of MTCs. The expression pattern of those genes allowed us to distinguish two groups of sporadic tumors. The first group displays an expression profile similar to that expressed by inherited RET634 tumors. The second presents an expression profile close to that displayed by inherited RET918 tumors and includes tumors from patients with distant metastases. It is characterized by the overexpression of genes involved in proliferation and invasion (PTN, ESM1, and CEACAM6) or matrix remodeling (COL1A1, COL1A2, and FAP). Interestingly, RET918 tumors showed overexpression of the PTN gene, encoding pleiotrophin, a protein associated with metastasis. Using a MTC cell line, silencing of RET induced the inhibition of PTN gene expression. Overall, our results suggest that familial MTC and sporadic MTC could activate similar oncogenic pathways.
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http://dx.doi.org/10.1677/ERC-08-0289DOI Listing
December 2009

Differences in recognition of the 1st WHO international reference reagents for hCG-related isoforms by diagnostic immunoassays for human chorionic gonadotropin.

Clin Chem 2009 Aug 18;55(8):1484-91. Epub 2009 Jun 18.

Department of Clinical Biochemistry, Royal Infirmary, Edinburgh, UK.

Background: The 1st WHO International Reference Reagents (IRRs) for 6 human chorionic gonadotropin (hCG)-related molecular variants, highly purified and calibrated in substance concentrations by the IFCC Working Group for hCG, permit experimental elucidation of what commercially available hCG methods measure in molar terms and enable assessment of their fitness for clinical purposes.

Methods: Pools containing known amounts of the IRRs spiked into normal human serum were issued to participants through the UK National External Quality Assessment Service for hCG for a period of 7 years. Among 16 assays used, 4 recognized only hCG, whereas 6 recognized hCG and its free beta-subunit (hCGbeta), and 6 recognized hCG, hCGbeta, and the beta core fragment.

Results: Differences in calibration of current hCG assays are moderate. Mean recovery of the current International Standard (IS), hCG IS 75/589, was 107% (range 93% to 126%), whereas that of the IRR 99/688 for hCG was 139% (range 109%-164%). Between-method variation for the latter (CV 12.3%) was also greater than for IS 75/589 (CV 8.8%). Recognition of hCGbeta varied markedly (CV 37%). Most assays overestimated it, but 2 RIAs produced results that were slight underestimations. Recognition of the beta core fragment was even more variable (CV 57%) and was closest to equimolarity for the RIAs.

Conclusions: Assays for hCG show considerable variation in their recognition of various forms of hCG, and this variability is the most important cause of method-related differences in hCG results in serum and an even more important cause of method-related differences in urine measurements. Equimolar recognition of the major hCG isoforms is essential if between-method comparability for hCG is to be improved.
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http://dx.doi.org/10.1373/clinchem.2009.124578DOI Listing
August 2009

Chromatofocusing fails to separate hFSH isoforms on the basis of glycan structure.

Biochemistry 2008 Feb 16;47(6):1708-20. Epub 2008 Jan 16.

Department of Biological Sciences, Wichita State University, Wichita, Kansas 67260, USA.

Follicle-stimulating hormone (FSH) glycosylation is regulated by feedback from the gonads, resulting in an array of glycans associated with FSH preparations derived from pools of pituitary or urine extracts. FSH glycosylation varies due to inhibition of FSHbeta N-glycosylation, elaboration of 1-4 branches possessed by mature N-glycans, and the number and linkage of terminal sialic acid residues. To characterize FSH glycosylation, FSH isoforms in pituitary gland extracts and a variety of physiological fluids are commonly separated by chromatofocusing. Variations in the ratios of immunological and biological activities in the resulting FSH isoform preparations are generally attributed to changes in glycosylation, which are most often defined in terms of sialic acid content. Using Western blotting to assess human FSHbeta glycosylation inhibition revealed 30-47% nonglycosylated hFSHbeta associated with four of six hFSH isoform preparations derived by chromatofocusing. Glycopeptide mass spectrometry assessment of glycan branching in these isoforms extensively characterized two N-glycosylation sites, one at alphaAsn52, the critical glycan for FSH function, and the other at betaAsn24. With two to four N-glycans per FSH molecule, many combinations of charges distributed over these sites can provide the same isoelectric point. Indeed, several glycans were common to all isoform fractions that were analyzed. There was no trend showing predominantly monoantennary glycans associated with the high-pI fractions, nor were predominantly tri- and tetra-antennary glycans associated with low-pI fractions. Thus, differences in receptor binding activity could not be associated with any specific glycan type or location in the hormone. FSH aggregation was associated with reduced receptor binding activity but did not affect immunological activity. However, as gel filtration indicated sufficient heterodimer was present in each isoform preparation to generate complete inhibition curves, the near total loss of receptor binding activity in several preparations could not be explained by aggregation alone, and the mechanism remains unknown.
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http://dx.doi.org/10.1021/bi701764wDOI Listing
February 2008

New therapeutic approaches to treat medullary thyroid carcinoma.

Nat Clin Pract Endocrinol Metab 2008 Jan;4(1):22-32

University Paris-Sud 11, Paris, France.

Medullary thyroid carcinoma (MTC) accounts for up to 8% of all thyroid cancers. Although primary surgery is curative in the vast majority of patients treated at an early stage, disease can persist or recur with deleterious effects on quality of life. Local and distant metastases can occur and are the major causes of mortality. Reoperation, embolization, and perhaps radiotherapy can improve the outcome for some patients who are not cured by primary surgery, but there is a need for novel treatments. No comprehensive clinical trial data are available on conventional cytotoxic agents for the treatment of MTC. Patients with distant metastases, in particular, might benefit from several novel compounds directed against angiogenesis and molecular targets in tumor cells, such as products of the proto-oncogene RET and mutants of it, and other signaling components. Well-conducted clinical trials are needed to assess and optimize these treatment strategies, and this article outlines how such trials should be conducted. Although RET mutations are common in hereditary MTC and can occur in some cases of sporadic MTC, knowledge of other molecular defects associated with the development of MTC should reveal new targets for therapy.
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http://dx.doi.org/10.1038/ncpendmet0717DOI Listing
January 2008

[Circulating endothelial cells: biomarkers for monitoring activity of antiangiogenic therapy].

Bull Cancer 2007 Jul;94 Spec No:S254-9

Laboratoire de recherche translationnelle, Institut de cancérologie Gustave-Roussy, 94805 Villejuif.

Tumor vessel formation is largely dependent on the recruitment of endothelial cells. Rare in healthy individuals, circulating endothelial cells (CEC) are shed from vessel walls and enter the circulation reflecting endothelial damage or dysfunction. Increased numbers of CEC have been documented in different types of cancer. Recent studies have suggested the role for CEC in tumor angiogenesis, but whose presence could also reflect normal endothelium perturbation in cancer. Originating from the bone marrow rather than from vessel walls, endothelial progenitor cells (EPC) are mobilized following tissue ischemia and may be recruited to complement local angiogenesis supplied by existing endothelium. Recently, studies in mouse models suggest that the circulating fraction of endothelial progenitors (CEP) is involved in tumor angiogenesis but their contribution is less clear in humans. The detection of CEC and CEP is difficult and impeded by the rarity of these cells. They may have important clinical implication as novel biomarkers susceptible to predict more efficiently and rapidly the therapeutic response to anti-angiogenic treatments. However, a methodological consensus would be necessary in order to correctly evaluate the clinical interest of CEC and CEP in patients.
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July 2007

Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1alpha-mediated tumor apoptotic switch.

J Clin Invest 2007 Jul;117(7):1844-55

CNRS-UMR 8121, Laboratoire de vectorologie et transfert de gènes, Département de Médecine Nucléaire, Villejuif, France.

Tumor radioresponsiveness depends on endothelial cell death, which leads in turn to tumor hypoxia. Radiation-induced hypoxia was recently shown to trigger tumor radioresistance by activating angiogenesis through hypoxia-inducible factor 1-regulated (HIF-1-regulated) cytokines. We show here that combining targeted radioiodide therapy with angiogenic inhibitors, such as canstatin, enhances direct tumor cell apoptosis, thereby overcoming radio-induced HIF-1-dependent tumor survival pathways in vitro and in vivo. We found that following dual therapy, HIF-1alpha increases the activity of the canstatin-induced alpha(v)beta(5) signaling tumor apoptotic pathway and concomitantly abrogates mitotic checkpoint and tetraploidy triggered by radiation. Apoptosis in conjunction with mitotic catastrophe leads to lethal tumor damage. We discovered that HIF-1 displays a radiosensitizing activity that is highly dependent on treatment modalities by regulating key apoptotic molecular pathways. Our findings therefore support a crucial role for angiogenesis inhibitors in shifting the fate of radiation-induced HIF-1alpha activity from hypoxia-induced tumor radioresistance to hypoxia-induced tumor apoptosis. This study provides a basis for developing new biology-based clinically relevant strategies to improve the efficacy of radiation oncology, using HIF-1 as an ally for cancer therapy.
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http://dx.doi.org/10.1172/JCI30269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1884687PMC
July 2007

Functional characterization of human thyroid tissue with immunohistochemistry.

Thyroid 2007 Mar;17(3):203-11

Department of Pathology, Commissariat à l'Energie Atomique LRC29V, Institut Gustave Roussy, University Paris Sud, Villejuif Cedex, France.

Immunohistochemistry provides insights in the expression of functional proteins and of their localization in normal thyroid tissue and in thyroid diseases. In hyperfunctional thyroid tissues, staining for sodium/iodide symporter (NIS), pendrin, thyroid peroxidase (TPO), and thyroglobulin (Tg) is increased. In hypofunctioning thyroid tissues, NIS staining is markedly decreased; in benign hypofunctioning adenomas, the expression of the other functional proteins is unmodified or slightly decreased, whereas their expression is profoundly decreased or absent in differentiated thyroid carcinoma.
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http://dx.doi.org/10.1089/thy.2006.0174DOI Listing
March 2007

Defects in iodide metabolism in thyroid cancer and implications for the follow-up and treatment of patients.

Nat Clin Pract Endocrinol Metab 2007 Mar;3(3):260-9

Oncology at University Paris Sud, Paris, France.

The two major steps of iodine metabolism--uptake and organification--are altered in thyroid cancer tissues. Organification defects result in a rapid discharge of radioiodine from thyroid cells, a short effective half-life of iodine, and a low rate of thyroid hormone synthesis. These defects are mainly due to decreased expression of functional genes encoding the sodium-iodide symporter and thyroid peroxidase and could result in a low radiation dose to thyroid cancer cells. TSH stimulation that is achieved with injections of recombinant human TSH, or long-term withdrawal of thyroid hormone treatment increases iodine-131 uptake in two-thirds of patients with metastatic disease and increases thyroglobulin production in all patients with metastases, even in the absence of detectable uptake. Serum thyroglobulin determination obtained following TSH stimulation and neck ultrasonography is the most sensitive combination for the detection of small tumor foci. Radioiodine treatment is effective when a high radiation dose can be delivered (in patients with high uptake and retention of radioiodine) and when tumor foci are sensitive to the effects of radiation therapy (younger patients, with a well-differentiated tumor and/or with small metastases). The other patients rarely respond to radioiodine treatment, and when progression occurs, other treatment modalities should be considered. Novel strategies are currently being explored to restore iodine uptake in cancer cells that are unable to concentrate radioiodine.
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http://dx.doi.org/10.1038/ncpendmet0449DOI Listing
March 2007

Sodium/iodide symporter (NIS) gene expression is the limiting step for the onset of thyroid function in the human fetus.

J Clin Endocrinol Metab 2007 Jan 31;92(1):70-6. Epub 2006 Oct 31.

Faculty of Medicine René Descartes, Paris V, Site Necker, Institut National de la Santé et de la Recherche Médicale Equipe Mixte 0363, Pediatric Endocrine Unit, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Necker Enfants-Malades, Paris, France.

Context: Terminal differentiation of the human thyroid is characterized by the onset of follicle formation and thyroid hormone synthesis at 11 gestational weeks (GW).

Objective: This study aimed to investigate the ontogeny of thyroglobulin (Tg), thyroid peroxidase (TPO), sodium/iodide symporter (NIS), pendrin (PDS), dual oxidase 2 (DUOX2), thyroid-stimulating hormone receptor (TSHR), and thyroid transcription factor 1 (TITF1), forkhead box E1 (FOXE1), and paired box gene 8 (PAX8) in the developing human thyroid.

Design: Thyroid tissues from human embryos and fetuses (7-33 GW; n = 45) were analyzed by quantitative PCR to monitor mRNA expression for each gene and by immunohistochemistry to determine the cellular distribution of TITF1, TSHR, Tg, TPO, NIS, and the onset of T4 production. A broken line regression model was fitted for each gene to compare the loglinear increase in expression before and after the onset of T4 synthesis.

Results: TITF1, FOXE1, PAX8, TSHR, and DUOX2 were stably expressed from 7 to 33 GW. Tg, TPO, and PDS expression was detectable as early as 7 GW and was correlated with gestational age (all, P < 0.01), and the slope of the regression line was significantly different before and after the onset of T4 synthesis at 11 GW (all, P < 0.01). NIS expression appeared last and showed the highest fit by the broken line regression model of all genes (correlation age P < 0.0001, broken line regression P < 0.0001). Immunohistochemical studies detected TITF1, TSHR, and Tg in unpolarized thyrocytes before follicle formation. T(4) and NIS labeling were only found in developing follicles from 11 GW on.

Conclusion: These results imply a key role of NIS for the onset of human thyroid function.
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http://dx.doi.org/10.1210/jc.2006-1450DOI Listing
January 2007

HEX, PAX-8 and TTF-1 gene expression in human thyroid tissues: a comparative analysis with other genes involved in iodide metabolism.

Clin Endocrinol (Oxf) 2006 Apr;64(4):398-404

Department of Clinical Biology, Institut Gustave-Roussy and Commissariat à l'Energie Atomique LRC 29V, Villejuif, France.

Objective: Benign and malignant thyroid tumours are characterized by alterations of the expression level of thyroid-specific genes involved in the iodide metabolism. Imbalance in the levels of transcription factors has been recognized as a critical molecular event in the development of neoplasm. The delineation of eventual correlations existing between the expression of transcription factors and of putative target genes in physiological and pathological conditions could be relevant to better understand tumorigenesis.

Patients And Methods: We examined the expression levels of transcription factors involved in thyroid development [thyroid transcription factor 1 (TTF-1), paired box gene 8 (PAX-8) and haematopoietically expressed homeobox (HEX)] in 101 thyroid tissues, including 14 normal thyroid tissues, 13 hyperfunctioning tissues, 27 benign adenomas and 47 follicular or papillary carcinomas. Then, we compared their expression levels with those of thyroid-specific genes involved in iodide metabolism.

Results: In benign tumours, PAX-8 and TTF-1 gene expression levels were not significantly different from the expression levels in normal tissues. However, a significant decrease was found in carcinomas. Interestingly, HEX gene expression was significantly decreased in both hyper- and hypofunctioning benign tissues and also in carcinomas. Expression levels of Pendred syndrome (PDS), natrium iodine symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO) and dual oxidase 1 or 2 (DUOX2) genes were significantly correlated with the expression of PAX-8 and with that of HEX. Expression level of TTF-1 was weakly correlated only with the expression levels of PDS and DUOX2.

Conclusion: Our findings suggest that alterations in the transcription factors PAX-8, TTF-1 and HEX gene expression, by acting individually or together, have a role in both thyroidal tumorigenesis and in the dedifferentiation process.
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http://dx.doi.org/10.1111/j.1365-2265.2006.02477.xDOI Listing
April 2006

Prognostic factors for persistent or recurrent disease of papillary thyroid carcinoma with neck lymph node metastases and/or tumor extension beyond the thyroid capsule at initial diagnosis.

J Clin Endocrinol Metab 2005 Oct 19;90(10):5723-9. Epub 2005 Jul 19.

Department of Nuclear Medicine and Endocrine Tumors, Institut National de la Santé et de la Recherche Médicale U605, Institut Gustave Roussy, Rue Camille Desmoulins, 94805 Villejuif Cédex, France.

Context: Reliable prognostic factors are needed in papillary thyroid cancer patients to adapt initial therapy and follow-up schemes to the risks of persistent and recurrent disease. OBJECTIVE AND SETTINGS: To evaluate the respective prognostic impact of the extent of lymph node (LN) involvement and tumor extension beyond the thyroid capsule, we studied a group of 148 consecutive papillary thyroid cancer patients with LN metastases and/or extrathyroidal tumor extension. Initial treatment, performed at the Institut Gustave Roussy between 1987 and 1997, included in all patients a total thyroidectomy with central and ipsilateral en bloc neck dissection followed by radioactive iodine ablation.

Results: Uptake outside the thyroid bed, demonstrating persistent disease, was found on the postablation total body scan (TBS) in 22% of the patients. With a mean follow-up of 8 yr, eight patients (7%) with a normal postablation TBS experienced a recurrence. Ten-year disease-specific survival rate was 99% (confidence interval, 97-100%). Significant risk factors for persistent disease included the numbers of LN metastases (>10) and LN metastases with extracapsular extension (ECE-LN >3), tumor size (>4 cm), and LN metastases location (central). Significant risk factors for recurrent disease included the numbers of LN metastases (>10), ECE-LN (>3), and thyroglobulin level measured 6-12 months after initial treatment after T4 withdrawal.

Conclusion: We highlight an excellent survival rate and suggest risk classifications of persistent and recurrent disease based on the numbers of LN metastases and ECE-LN, LN metastases location, tumor size, and thyroglobulin level.
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http://dx.doi.org/10.1210/jc.2005-0285DOI Listing
October 2005

Follicular thyroid tumors with the PAX8-PPARgamma1 rearrangement display characteristic genetic alterations.

Am J Pathol 2005 Jul;167(1):223-31

Functional Genomic Unit, Institut Gustave-Roussy, Villejuif Cedex, France.

Follicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) gamma1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARgamma1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARgamma target genes were up-regulated in PAX8-PPARgamma-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARgamma expression profile presented a putative PPARgamma-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARgamma and as members of novel molecular pathways involved in the development of thyroid carcinomas.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1603430PMC
http://dx.doi.org/10.1016/s0002-9440(10)62967-7DOI Listing
July 2005

Canstatin acts on endothelial and tumor cells via mitochondrial damage initiated through interaction with alphavbeta3 and alphavbeta5 integrins.

Cancer Res 2005 May;65(10):4353-61

UMR 8121 Laboratoire de vectorologie et transfert de gènes, Institut Gustave Roussy, Villejuif cedex, France.

Canstatin, the noncollagenous domain of collagen type IV alpha-chains, belongs to a series of collagen-derived angiogenic inhibitors. We have elucidated the functional receptors and intracellular signaling induced by canstatin that explain its strong antitumor efficacy in vivo. For this purpose, we generated a canstatin-human serum albumin (CanHSA) fusion protein, employing the HSA moiety as an expression tag. We show that CanHSA triggers a crucial mitochondrial apoptotic mechanism through procaspase-9 cleavage in both endothelial and tumor cells, which is mediated through cross-talk between alphavbeta3- and alphavbeta5-integrin receptors. As a point of reference, we employed the first three kringle domains of angiostatin (K1-3), fused with HSA, which, in contrast to CanHSA, act only on endothelial cells through alphavbeta3-integrin receptor-mediated activation of caspase-8 alone, without ensuing mitochondrial damage. Taken together, these results provide insights into how canstatin might exert its strong anticancer effect.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-3536DOI Listing
May 2005