MASTER EN ONCOLOGÍA MOLECULAR 2011 TGF-BETA

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1 MASTER EN ONCOLOGÍA MOLECULAR 2011 TGF-BETA Joan Seoane ICREA Research Professor Vall d Hebron Institute of Oncology Vall d Hebron University Hospital Barcelona jseoane@vhio.net

2 Glioma Stupp et al. New Engl. J. Med. 2005

3 Sathornsumetee et al. Cancer 2007

4 Inter-tumor heterogeneity Personalized medicine GBM GBM Verhaak et al. Cancer Cell 2010

5 Intra-tumor heterogeneity Cell type specific treatment

6 Detailed knowledge of molecular oncology is IMPERATIVE: Biomarkers to stratify patients Assess differential sensitivity among tumor cells Biomarkers of response Determine drug dose Determine schedules Combination with other compounds New and more specific therapeutic targets

7 Immune Cells Inhibits T-cell proliferation Impairs NK cell function Impairs antigen presentation TGFβ controls development and homeostasis Epithelium Cell cycle arrest Apoptosis ECM production Adhesion TGFβ Endothelium Migration Morphogenesis Proliferation Fibroblasts ECM production Proliferation Cytokine secretion

8 The basis of the pleiotropic response TGFβ Type II receptor Type I receptor Smad4 Smad2/3 Activated Smad Complex Cell-specific DNA-binding cofactors Co-activator or Co-repressor

9 TGFβ gene responses Cytostatic Program p15ink4b, p21cip1 c-myc Extracellular Matrix PAI-1, upa, Col VI-A1, ADAM19 Integrin α5, integrin β6 Paracrine Network IL11, VEGF,CTGF, Jagged1, Follistatin3 Angiopoietin4, IL1β, BMP4 Signaling Network BMPR-II, VDR, EphB2, RhoGEF114, Mek4, LDLR, PGE-R4, βar-2 Cell-specific DNA-binding cofactors Co-activator or Co-repressor >500 cell-specific target genes Transcription Network Ets2, c-jun, JunB, ATF3, Gadd45β, Pim1 Mad2, Mad4, C/EBPδ, MRG1, TRIP-Br2 Other Responses T-box3, MN1, Igλ, Syalyl tranf.4a Sprouty 2, IAP3, UDPG-ceramide GT Negative Feedback Smuf1, Smurf2, Smad7, SnoN

10 TGFβ duality in cancer TGF-β TGF-β Normal Epithelium Adenoma Invasive Carcinoma TGF-β TGF-β Metastasis adapted from Siegel et al. Nature Rev. Cancer, 2003

11 Normal epithelial cells Cancer cells lacking TGFβ receptor or Smad Cancer cells lacking cell cycle arrest response TGFβ TGFβ TGFβ cell cycle arrest genes other target genes cell cycle arrest genes other target genes cell cycle arrest genes other target genes Homeostasis Uncontrolled proliferation Uncontrolled proliferation Invasion Metastasis

12 Escaping from the TGFβ anti-proliferative response TGFβ mutated in colon cancer hypermethylated in NSCLC mutated in ovarian, breast, pancreatic cancer, HNSCC hypermethylated in gastric cancer mutated in ~50% pancreatic cancer ~10% colon cancer Smad4 TβRII P P P TβRI R-Smad mutated in colorectal, lung cancer (Smad2) low expression in gastric cancer (Smad3) P Cell cycle arrest Other responses

13 Loss of the TGFβ anti-proliferative response TGFβ TβRII TβRI P P TGFβ Stat NFκB Smad7 R-Smad Smad4 P Tert Ras/MAPK Ras Myc AKT TGIF FoxG1 P Cdk4/2 Ski/SnoN Evi-1 p21cip1 p15ink4b Other responses Cell cycle arrest Seoane, Carcinogenesis 2006

14 Cancer cells PTEN FoxG1 Myc AKT cell cycle arrest genes TGFβ? other target genes Uncontrolled proliferation Invasion Metastasis

15 inmunosuppression T cell NK cell TGFβ PDGF VEGF MMPs proliferation angiogenesis invasion EMT metastasis

16 TGFβ as a therapeutic target AP AP TGFβ mrna TGFβ soluble TβRII-Fc Lederlimumab Metelimumab GC-1008 Type II receptor Type I receptor LY SB SD-208 Smad4 Smad2/3

17 Seoane. Clin Transl Oncol, 2008

18 Patient No Age Diagnosis Grade Surgery Treatment Time to progression Overall survival p-smad 2 Ki67 PDGF-B years days days HScore % HScore 1 31 FA II CR S FA II CR S GBM IV CR S+Ct+Rt AA III CR S+Ct+Rt AA III PR S+Ct+Rt+T GBM IV CR S AA III PR S+Ct+Rt AA III CR S+Ct+Rt GBM IV S+Ct+Rt+T GBM IV CR S+Rt GBM IV CR S+Ct+Rt AA III CR S+Rt FA II CR S+Rt GBM IV PR S+Ct+Rt PA I CR S AA III PR S GBM IV S PA I CR S FA II CR S AA III PR S+Ct+Rt FA II CR S+Ct+Rt GBM IV PR S+Rt GBM IV CR S+Ct+Rt PA I PR S GBM IV PR S+Ct+Rt PA I CR S GBM IV PR S PA I CR S FA II PR S+Ct+Rt GBM IV CR S+Rt GBM IV CR S GBM IV PR S+Rt GBM IV CR S+Ct+Rt PA I CR S PA I PR S AA III PR S+Ct+Rt+T PA I S FA II S PA I CR S GBM IV CR S+Ct+Rt GBM IV CR S GBM IV CR S+Ct+Rt PA I CR S GBM IV CR S+Ct+Rt GBM IV CR S+Ct+Rt GBM IV CR S FA II CR S+Rt FA II PR S+Rt PA I CR S PA I CR S AA III S+Ct+Rt GBM IV CR S+Ct+Rt

19 p-smad SMAD TGFβ p-smad2 Smad2 Smad2

20 High p-smad2 level is a poor prognostic factor Progression-free Survival Low p-smad2 p= High p-smad Time (days) 2000 Overall Survival Low p-smad2 p=0.012 High p-smad Time (days) 2000

21 Primary culture of tumor cells

22 % changein BrdU incorpotation PCTC TGFβ TGFβ+ TβRI INH

23 PDGF-B correlates with TGFβ-induced proliferation Change in BrdU incorporation PCTC * * TGFβ TGFβ + TβRI Inh ** ** * * * * PDGF-B PDGFRα PDGFRβ PAI-1 18S TGFβ PCTC

24 CpG islands in the PDGF-B gene PDGF-B gene CpG island CpG island (-400) (+1000)

25 The methylation status of the PDGF-B gene dictates the proliferative response to TGFβ Change in BrdU incorporation PCTC * * TGFβ TGFβ + TβRI Inh ** ** * * * * PDGF-B PDGFRα PDGFRβ PAI-1 18S TGFβ PCTC UNMETHYLATED CpG METHYLATED CpG Average of aprox. 10 clons per cell type PCTC2 PCTC3 PCTC4 PCTC5 PCTC6 PCTC7 PCTC8 PCTC9 PCTC10

26 Role of TGFβ in Glioma Not aggressive Low proliferation Low TGFβ activity TGFβ P P TGFβ P P Highly aggressive High proliferation High TGFβ activity Smad P Smad P Smad P M M M M PDGF-B Smad P PDGF-B Proliferation Tumor progression Bruna et al. Cancer Cell, 11, , 2007

27 Animal model

28 Mixed tumors Oligoastrocytoma GFAP staining

29 Lobo et al Anny Rev. Cell Dev. Biol. 2007

30 NORMAL BRAIN CANCER Clarke, Nature 2004

31 Therapeutic implications of cancer-initiating cells

32 Patient-derived glioma cells GBM1 GBM2 GBM3 neurospheres PCTC 200 µm Peñuelas et al. Cancer Cell 2009

33 Neurospheres from patient-derived glioma samples. neuroprogenitor markers GBM1 PCTC Nsph GBM2 PCTC Nsph GBM3 PCTC Nsph nestin sox2 msi-1 actin

34 Neurospheres from patient-derived glioma samples. multi-lineage differentiation

35 Animal model

36 Patient GBM Mouse GBM Patient GBM Mouse GBM patient mouse patient mouse H&E Msi-1 Sox2 Nestin

37 Effect of TGFβ on glioma initiating cell (GIC) self-renewal 120 GBM1 treatment Number of neurospheres TGFß TβRI inh control TGFß TßRI inh TGFß + TßRI inh

38 The LIF-JAK-STAT pathway TGFβ TGFβ + TβR Inh. TβR Inh. LIF Heinrich et al., Biochem J. 2003

39 LIF is a mediator of TGFβ-induced GIC self-renewal neurospheres GBM1 * ** - IgG TGFβ IgG αlif TGFβ αlif control TGFβ LIF α LIF TGFβ + α LIF % of nsph. >75µm β LIF

infection 1500 Tumor area (arbitrary units) 1200 900

40 Expression of TGFβ, TβRI-CA and LIF promote the oncogenic capacity of GBM neurospheres Lentiviral infection 1500 Tumor area (arbitrary units) Lenticontrol Lenti- TGFβ Lenti- TβRI-CA Lenti- LIF

41 LIF LIF TGFβ LIF Peñuelas et al. Cancer Cell 2009

42 Animal model

Tumor area (a.u.) treated untreated 1200

43 Tumor area (a.u.) treated untreated untreated treated

44 Neurospheres PCTC Patient Analysis and Screening of compounds Biomarkers of response Selection clinical trial

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