Cancer du sein et micro-environnement tumoral: rôle de la protéase ...

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INTRODUCTIONLysosomal aspartic protease cathepsin-D (cath-D) is overexpressed and highlysecreted by human epithelial breast cancer cells (1-4). Its overexpression in breastcancer is correlated with poor prognosis (5-7). Cath-D is synthesized as a 52-kDa,catalytically-inactive precursor. It is present in endosomes as an active 48-kDa,single-chain intermediate that is subsequently converted in the lysosomes into thefully active mature protease, composed of a 34-kDa heavy chain, and a 14-kDa lightchain. The 52-kDa pro-cath-D hypersecreted by breast cancer cells into theextracellular environment is endocytosed by both cancer cells and fibroblasts (1, 8,9). While cath-D endocytosis is mainly performed by the mannose-6-phosphatereceptors (M6PR), the existence of alternative cath-D receptors has been postulated(10, 11). Cath-D affects both cancer cells and stromal cells in the tumor microenvironmentby increasing cell proliferation, metastasis, angiogenesis and fibroblastoutgrowth (1, 12-16). We previously observed that a mutated catalytically-inactiveversion of cath-D (D231N) remains mitogenic for tumor cells and fibroblasts (12, 14,15), suggesting that cath-D acts as an extracellular messenger that interacts eitherdirectly or indirectly with an as-yet unidentified cell surface receptor.Here, we identify the fibroblast receptor that mediates the cath-D-inducedparacrine fibroblast outgrowth, LDL receptor-related protein-1 (LRP1). LRP1 iscomposed of a 515-kDa extracellular a chain, and an 85-kDa b chain (17, 18). Theb chain contains an extracellular domain, a trans-membrane region, and acytoplasmic tail. The extracellular a chain contains binding-sites for many structurallyunrelated ligands, including lipoprotein particles, proteases and protease-inhibitorcomplexes. At present, the ligands of the extracellular domain of the LRP1b chain are3
still unknown. In this study, we report for the first time the interaction between cath-Dand the extracellular domain of the b chain of LRP1.MATERIALS AND METHODSMaterials. Human mammary fibroblasts (HMFs), kindly provided by J. Piette (IGM,Montpellier, France), were obtained from reduction mammoplasty tissues from apatient without cancer.cath-D-/- MEF-cath-D,cath-D-/- MEF- D231N cath-D and cath-Dtransfected 3Y1-Ad12 cells were previously described (15). LRP1-/- MEF and LRP1+/-MEFs were purchased from ATCC. B-41 cells, LRP1-/- MEF cells stably transfected withthe full-length human LRP1, were kindly provided by D. Strickland (UniversityMaryland School of Medicine, Baltimore, USA). Cells were cultured in DMEMmedium with 10% fetal calf serum (FCS, GibcoBRL). The 11H4 hybridoma directedagainst the C-terminal part of LRP1b chain was purchased at ATCC. Polyclonal antihumanLRP1b-chain antiserum was previously described (19). The anti-human cath-D monoclonal antibody (BD Biosciences) used for immuno-blotting recognized 52-,48- and 34-kDa forms of cath-D. The anti-human cath-D M1G8 monoclonal antibodyused for immuno-precipitation and purification recognized 52-, 48- and 34-kDa formsof cath-D. The anti-human, cath-D monoclonal antibody M2E8, used for immunofluorescence,interacts only with 52-kDa pro-cath-D. Anti-b actin polyclonal antibodywas purchased from Sigma.Plasmids. pcDNA3.1(+)Myc-tagged LRP1b chain, pcDNA3.1(-)cath-D andpcDNA3.1(-) D231N cath-D expression plasmids have previously been described (12).The pcDNA3.1(+)LRP1b extracellular domain (1-476) expression plasmid wascreated by inserting the PCR-amplified cDNA encoding LRP1b (1-476) from4
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Université Montpellier I UFR Méd
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Un grand merci à tous mes collabor
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TitleBreast cancer and tumoral micr
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Cancer du sein et micro-environneme
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C. PRESENTATION DU TRAVAIL DE THESE
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But de la thèseLa cathepsine-D (ca
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I. Le tissu adipeux1) Généralité
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(Adenosine triphosphate). Cette pro
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Cette synthèse de novo a lieu dans
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ABFigure 6 : Schéma de la oxydati
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d. Ladipocyte : une cellule sécré
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3) Ladipogenèsea. Les différentes
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. Les facteurs de transcriptionLa d
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Lexpression de C/EBP est quant à e
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adipeux. Les analyses histologiques
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adipocyteFigure 10 : Schéma repré
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II. La cathepsine-D1) Synthèse, ma
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les cath-B et L, en une forme matur
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Il existe deux RM6P : le RM6P/IGFII
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2) Fonctions de la cath-Da. Dans la
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. Dans les pathologiesEn plus de se
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c. Rôles et mécanismes daction da
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carcinome de prostate serait respon
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La cath-D joue donc un rôle import
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Une fois le marquage des peptides e
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Afin de réaliser ce projet, nous a
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III. Le récepteur LRP11) Organisat
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2) Trafic intra-cellulaireComme le
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LRP1αLRP1Membrane associatedprotea
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Tableau 3 : Ligands connus du LRP1(
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I. Etude du rôle de la cathepsine
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Cathepsin-D, a key protease in brea
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IntroductionConsumption of meals ri
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(Fig. 1A, panel a). This differenti
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differentiated adipocyte (Fig. 4B).
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DiscussionOur results demonstrate t
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from normal and peri-al breast adip
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mouse RS9 (sens 5CGGCCCGGGAGCTGTTGA
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agreement of local ethic committee.
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Statistical analysis. Results are e
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Loncarek J, Freiss G, Vignon F and
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Aa3000 *b4000*B8000**cath-D mRNA(ar
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ABcath-D mRNA(ratio RS9)PPARg mRNA(
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A D3 D7 D14D0BaD0 D3 D7 D14D0 D3 D7
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AshLucshcath-DF442A C34 C37 A4 D10c
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Page 94 and 95: II. Etude du rôle du LRP1 dans les
Page 96 and 97: 2) Article 2:LRP1 receptor controls
Page 98 and 99: LRP1, Adipogenesis, Obesityrelative
Page 100 and 101: LRP1, Adipogenesis, ObesityFigure 3
Page 102 and 103: LRP1, Adipogenesis, ObesityFigure 5
Page 104 and 105: LRP1, Adipogenesis, ObesitySome ins
Page 106 and 107: LRP1, Adipogenesis, ObesityLipolysi
Page 108 and 109: CONCLUSIONLa cathepsine D (cath-D)
Page 110 and 111: la souris obèses. Enfin, linhibiti
Page 112 and 113: carcinogénique sont encore mal con
Page 114 and 115: Ce travail de thèse a étudié, po
Page 116 and 117: REFERENCESAhima, R. S. (2006). Adip
Page 118 and 119: implicates them as antigen presenti
Page 120 and 121: Cataldo, A. M., Barnett, J. L., Ber
Page 122 and 123: Folkman, J. (2003). Fundamental con
Page 124 and 125: Hofmann, S. M., Zhou, L., Perez-Til
Page 126 and 127: Langin, D. (2006a). Adipose tissue
Page 128 and 129: Ludwig, T., Ovitt, C. E., Bauer, U.
Page 130 and 131: Nirde, P., Derocq, D., Maynadier, M
Page 132 and 133: Rozanov, D. V., Hahn-Dantona, E., S
Page 134 and 135: Taleb, S., Cancello, R., Clement, K
Page 136 and 137: Xiao, Y., Junfeng, H., Tianhong, L.
Page 138 and 139: F. ANNEXE98
Page 140 and 141: Cathepsin D is a new ligand for ext
Page 144 and 145: pcDNA3.1(+)Myc-tagged LRP1b into pc
Page 146 and 147: (Protein refolding kit, Novagen) fo
Page 148 and 149: anti-mouse-gold (Aurion). Sections
Page 150 and 151: not secrete detectable levels of pr
Page 152 and 153: using cath-D-/- MEF transfected wit
Page 154 and 155: co-culture outgrowth assays with ca
Page 156 and 157: REFERENCES1. Vignon, F., Capony, F.
Page 158 and 159: steps in vivo: proliferation, angio
Page 160 and 161: 28. Laurent-Matha, V., Lucas, A., H
Page 162 and 163: 42. Zurhove, K., Nakajima, C., Herz
Page 164 and 165: Figure 1. Cath-D interacts with the
Page 166 and 167: Figure 2. Cath-D binds to residues
Page 168 and 169: Figure 3. Cath-D interacts with LRP
Page 170 and 171: Figure 5. Silencing LRP1 in cath-D
Page 172 and 173: Figure 1. Cath-D interacts with the
Page 174 and 175: Figure 6. LRP1 is the receptor medi
Page 176 and 177: Beaujouin, Figure Sup. 2cath-D-/-ME
Page 178: RésuméLaspartyl protéase catheps
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