Transformující růstový faktor – b: rozmanitost přenosu signálu a funkce část 2. Karel Souček Bi6051 Molekulární fyziologie živočichů Transforming growth factor -b (TGF-b) TGF-b rodina ~ TGF-bs, activins, bone morphogenic proteins (BMP) TGF-b1 • pleiotropní cytokin • negativní regulátor Epstein, F.H., N Engl J Med 2000; 342:1350-1358, 2000 Growth factors in cancer cell signaling ncancer is not single cell disease; ntissue microenvironment plays an important role in tumor initiation and progression; ngrowth factors - cytokines - play crucial role in cancer development and some of them belong to the significant autocrine/paracrine factors produced by various cell types in tumor microenvironment; nmodulation of their signal transduction represent potential target for therapy. n Growth factors in cancer cell signaling nWhat is a role of TGF-b family cytokines in cancer progression? nHow we can effectively modify pathological plasticity of the cancer cells? n figure 14-01 Figure 14.1 The Biology of Cancer (© Garland Science 2007) figure 14-03 Figure 14.3 The Biology of Cancer (© Garland Science 2007) figure 14-04 Figure 14.4 The Biology of Cancer (© Garland Science 2007) figure 14-42 Figure 14.42 The Biology of Cancer (© Garland Science 2007) Organ-specific barriers Transforming growth factor-b nRole v rozvoji patologických stavů n Biologické funkce TGF-b nHraje klíčovou úlohu během embryogeneze; nreguluje proliferaci, diferenciaci, buněčnou smrt, motilitu, adhezi (v závislosti na buněčném typu) = ovlivňuje homeostázu; n reguluje expresi extracelulární matrix; –indukuje fibrilární kolagen a fibronectin; –inhibuje degradaci ECM (inhibicí MMPs a indukci TIMPs). Role TGF-b v rozvoji patologických stavů nFibróza –deregulace exprese ECM prostřednictvím indukce proliferace fibroblastů a jejich myofibroblastového fenotypu. nNádorová onemocnění –ztráta citlivosti epiteliálních buněk k inhibičnímu působeni TGF-b; –indukce angiogeneze. – [USEMAP] [USEMAP] Role TGF-b v carcinogenezi NEJM 342, 18 (2000) 1350-1358 Role TGF-b v carcinogenezi SMAD3 TGFR2 Prostate – normal vs. cancer normal, hyperplasia vs. cancer Role TGF-b v carcinogenezi NEJM 342, 18 (2000) 1350-1358 > Role TGF-b v carcinogenezi TRENDS in Cell Biology Vol.11 No.11 2001 Epithelial-Mesenchymal Transition (EMT) nZměna buněčného fenotypu spojená se ztrátou adheze a zvýšením motility figure 14-17b Figure 14.17b The Biology of Cancer (© Garland Science 2007) EMT & Cancer Epithelial-to-mesenchymal transition (EMT) nReversible acquisition of migratory and invasive properties by epithelial cells nRole in embryonic development, fibrosis, cancer Thiery, J.P., et al., Epithelial-mesenchymal transitions in development and disease. Cell, 2009. 139(5): p. 871-90. Kalluri, R. and R.A. Weinberg, The basics of epithelial-mesenchymal transition. J Clin Invest, 2009. 119(6): p. 1420-8. M S Simonson Kidney International 71, 846-854 (May (1) 2007) Markers and regulators of EMT nrc2620-f1 Kornelia Polyak & Robert A. Weinberg Nature Reviews Cancer 9, 265-273 (April 2009) Transforming growth factor-b (TGF-b) Massagué J, Gomis RR. FEBS Lett. 2006 May 22;580(12):2811-20. Jian Xu, Samy Lamouille, Rik Derynck Cell Research (2009) 19:156-172. Introduction EMT in benign cells EMT in tumorigenic cells Slug and Twist in EMT Summary ●●●○○○ ○○○○ ○○○ ○○ ○○ Recent discoveries in the EMT field nEMT creates cells with cancer stem cell characteristics Mani SA, et al., Cell. 2008 May 16;133(4):704-15. nCross-regulation between ZEB1/2 and miR-200 family Gregory PA, et al., Nat Cell Biol. 2008 May;10(5):593-601. nCross-regulation between Twist and Slug onc2009406f1 H Zhang, Y Li and M Lai Oncogene 29, 937-948 (18 February 2010) Esmeralda Casas, Jihoon Kim, Andrés Bendesky, et al. Cancer Res; 71(1) January 1, 2011 fig1 Prachi Jain, Suresh K. Alahari Frontiers in Bioscience 16, 1824-1832, January 1, 2011 Experimental approach BPH par ctrl Benign (BPH-1) EMT TGF-b Cancer associated fibroblast - tumor derived (CAFTD) EMT TGF-b EMT • EMT markers • EMT regulators • Cell shape and behavior xCELLigence – analýza migračního potenciálu Floating cell Protect ring Attached cell Matrix Electrodes Invading cell Dvojí úloha TGF-b v carcinogenezi nDeregulace inhibice proliferace epiteliálních buněk; nEpithelia-mesenchymal transition npodpora migrace, metastázování a angiogeneze. Role TGF-b v diagnóze, prognóze a léčbě nVysoká sérová hladina TGF-b1 je spojena s nádory tlustého střeva, prostaty a rozvojem fibrózy; npolymorfismus genu pro TGF-b1 vedoucí k jeho zvýšené produkci určuje predispozici k fibróze, hypertenzi a osteoporéze; nblokování produkce a aktivity TGF-b má velký potenciál pro léčbu fibrózy; nprotektivní účinek retinoidů a vitamínu D3 může být způsoben prostřednictvím TGF-b. GDF-15: tumor promoter or supresor? nKarel Souček, Eva Slabáková, Zuzana Pernicová, Eva Slavíčková, Radek Fedr nDepartment of Cytokinetics nInstitute of Biophysics AS CR n nCenter of Biomolecular and Cellular Engineering nInternational Clinical Research Center FNUSA-ICRC n nBrno, Czech Republic n n n IBP-logo •Membrane receptor(s) – not identified •Signal transduction – not full understood •Target genes – not identified •Function – not clear 9/10/2013 ~ 1261 records GDF15 gene Homo sapiens Mus musculus Rattus norvegicus Pan troglodytes Control of GDF15 expression GDF-15 regulation M, Mimeualt, JCP 2010 human GDF15 mRNA expression singleGene Varambally, S. et al., Cancer Cell 8 (5), 393 (2005). GDF15 mRNA normal vs. prostate adenocarcinoma Various forms of GDF-15 pro-GDF-15 monomer ~40kDa pro-GDF-15 dimer ~80kDa pro-GDF-15 hemidimer ~55kDa propeptide ~28kDa mature GDF-15 dimer ~30kDa mature GDF-15 monomer ~15kDa Bauskin AR, Zhang HP, Fairlie WD, He XY, Russell PK, Moore AG, et al. The propeptide of macrophage inhibitory cytokine (MIC-1), a TGF-beta superfamily member, acts as a quality control determinant for correctly folded MIC-1. EMBO J 2000;19:2212-20. GDF-15 Concentration Reference Normal 450 ± 50 pg/ml Tanno, T. et al., Nat Med 13 (9), 1096 (2007) beta-thalassemia syndrome 66,000 ± 9,600 pg/ml CSF - non-neoplastic 156 pg/m Sophie Shnaper et al., International Journal of Cancer in press (2009). CSF - gliosblastoma 229 pg/ml normal 495 pg/ml Brown, D. A. et al., Clin Cancer Res 9 (7), 2642 (2003). Adenomatous polyps 681 pg/ml High-grade dysplasia 1114 pg/ml colorectal carcinoma 783 pg/ml Congenital dyserythropoietic anemia 10 239 ± 3049 pg/ml Tamary, H. et al., Blood 112 (13), 5241 (2008). Normal 16.1 ± 23.4 pg/ml Baek, K. E. et al., Clinica Chimica Acta 401 (1-2), 128 (2009). Gastric cancer 164.5 ± 183.7 pg/ml Prostate cancer Grade 3 2,326.1 pg/ml Selander, K. S. et al., Cancer Epidemiology Biomarkers & Prevention 16 (3), 532 (2007). Prostate cancer Grade 2 2,054.1 pg/ml Prostate cancer Grade 1 761.5 pg/ml normal 859 ± 619 pg/ml Brown, D. A. et al., Clin Cancer Res 12 (1), 89 (2006). BPH 983 ± 850 pg/ml Prostate cancer 731 ± 500 pg/mL Women with cardiovascular events 618 pg/mL Brown, D. A. et al., The Lancet 359 (9324), 2159 (2002). Women w/o cardiovascular events 538 pg/mL Thalassemia (from θάλασσα, thalassa, sea + αἷμα, haima, blood; British spelling, "thalassaemia") is an inherited autosomal recessive blood disease. In thalassemia, the genetic defect results in reduced rate of synthesis of one of the globin chains that make up hemoglobin. Reduced synthesis of one of the globin chains can cause the formation of abnormal hemoglobin molecules, thus causing anemia, the characteristic presenting symptom of the thalassemias. Beta thalassemias are due to mutations in the HBB gene on chromosome 11,[6] also inherited in an autosomal-recessive fashion. The severity of the disease depends on the nature of the mutation. Mutations are characterized as (βo or β thalassemia major) if they prevent any formation of β chains (which is the most severe form of beta Thalassemia); they are characterized as (β+ or β thalassemia intermedia) if they allow some β chain formation to occur. In either case there is a relative excess of α chains, but these do not form tetramers: rather, they bind to the red blood cell membranes, producing membrane damage, and at high concentrations they form toxic aggregates. K. Souček et al., Human Reproduction 2010 Growth/differentiation factor-15 is an abundant cytokine in human seminal plasma normal 0.36 ± 0.04 ng/mL first trimester 6.3 ± 0.02 ng/mL second trimester 12.2 ± 0.5 ng/mL third trimester 15.3 ± 1.3 ng/mL ? x E-02 mg/ml ? GDF-15/MIC-1 in cancer GDF-15 & NSAIDs E. Lincová (Slabáková) et al., Biochem. Pharmacol. 2009 LNCaP GDF-15 & NSAIDs E. Lincová (Slabáková) Biochem. Pharmacol. 2009 P. Vaňhara & E. Lincová (Slabáková) Differentiation 2009 Growth-differentiation factor-15 inhibits differentiation into osteoclasts - A novel factor involved in control of osteoclast differentiation RAW264.7 - primary bone marrow - derived osteoclasts ……Both seminal plasma and sperm components of the seminal fluid are necessary to confer full tolerance and elicit the Treg cell response, potentially through provision of immunedeviating cytokines and antigens, respectively CD4+ T Cell Differentiation CD3-coated plates + in vitro treatment cells quantification - CyQuant Naïve T-cells activation > CD4+CD25+ in vitro treatment FACS flow cytometry FOXP3 +/- qRT-PCR FOXP3 FOXP3 – qRT-PCR CD4+CD25+ human cells treated for 24h > FACS analysis – CD4+CD25+FOXP3+ in human cells treated for 24h n Male (CBAxC57Bl/10)F1 thymus ts26 1 See full size image in vitro treatment pan-T MACS flow cytometry CD4+CD25+FOXP3 +/- FACS analysis – CD4+CD25+FOXP3+ in mouse cells treated for 48h GDF-15 & mice prostate cancer •The TRAMP-C1 (ATCC - CRL-2730) cell line was derived in 1996 by Norman Greenberg from a heterogeneous 32 week primary tumor in the prostate of a PB-Tag C57BL/6 (TRAMP) mouse. • • TRAMP is a transgenic line of C57BL/6 mice harboring a construct comprised of the minimal -426/+28 rat probasin promoter (426 base pairs of the rat probasin (PB) gene promoter and 28 base pairs of 5'-untranslated region) to target expression of the SV40 large T antigen to prostatic epithelium. • •TRAMP-C1 cells are tumorigenic when grafted into syngeneic C57BL/6 hosts. GDF-15 stimulates growth of syngeneic grafts of TRAMP-C1 cells > GDF-15 signaling GDF-15 signaling GDF-15 signaling LAPC-4 > Summary nGDF-15: ninhibits naïve T-cells activation (proliferation) and induce Treg in vitro; nits overexpression potentiate tumor graft progression in syngeneic hosts; ntumor graft progression is significantly potentiate by ectopic overexpression of mGDF-15 and inhibited in GDF-15 knockout mice; nGDF-15 binds ACVR2B receptors and activates SMAD2 in LAPC-4 cells Shrnutí přednášky nTGF-b hraje významnou roli v rozvoji karcinogeneze a dalších patologických stavů. nEMT je významný proces ovlivňující schopnost nádorových buněk diseminovat nGDF-15 hraje důležitou úlohu v nádorové progresi Na konci dnešní přednášky byste měli: 1. 1.být schopni vysvětlit úlohu TGF-b v karcinogenezi; 2.charakterizovat proces EMT včetně hlavních znaků a regulátorů; 3.popsat známé vlastnosti GDF-15.