Hormones and Cancer Research Unit Department of Medicine, McGill University Dr. Jean-Jacques Lebrun

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1 Hormones and Cancer Research Unit Department of Medicine, McGill University Dr. Jean-Jacques Lebrun TGF /Smad signaling pathways CELLULAR AND MOLECULAR BIOLOGY Institut de recherches cliniques de Montréal TGF Superfamily Members and Structure Monomer Dimer 1

2 Schematic representation of inhibins and activins The subunit peptides are synthesized initially as precursors, which are then processed to release the mature C-terminal portions. An a-subunit can associate with either b-subunits to form inhibin A or inhibin B. The b-subunits can also dimerize to form the activins. The dimerization of the bc, bd and be-subunits have not yet been demonstrated. Reproduction(2001) 121, TGFβ targets and its actions in mature tissues -Cell Growth -Apoptosisp pt -Immortalization Tumor Suppression -Immunosuppression -Angiogenesis -Cell Adhesion -Cell migration -ECM/metalloproteinases -Invasion/metastasisI i / i Tumor rogression Nature Reviews Cancer 3, (2003) 2

3 Mammary Gland lacenta Cell growth Milk proteins hcg rogesterone GRF Gonads Estrogens Androgens Bone marrow Erythropoiesis Cell growth Activins ituitary FSH, LH, GnRHR ACTH, GH, RL Cell growth Embryos Dorsal mesoderm Blood Cell growth Apoptosis Liver Cell growth Apoptosis Tumor Cells Cell growth Apoptosis Myostatin null mice Sections of distal hindlimb stained with haematoxylin and eosin (Nature , 1997) 3

4 S Structure -Function Relationships of Receptors for Activin and the TGF Superfamily Ligand TM point mutants: GS T to D S: Signal eptide TM: transmembrane domain GS: glycine/serine rich Ser/Thr kinase I II III IV V VI A VIB VII VIII IX X XI K to R 1. Loss-of-Function mutants Ser/Thr kinase domain deletion mutant Ser/Thr kinase domain point mutant Lys/Arg (K to R) 2. Gain-of-Function mutants GS domain point mutant Thr/Asp (T to D) Serine Kinase Receptors Mechanism of Action Activin S-S Type II Type I S-S S-S S-S Inhibin Ligand Type II Type I I: ALK4 Wb:ActRII Smad2/3 Wb: ALK4 eptide: Human leukemia cells Gene Transcription Cell Differentiation Cell Growth Inhibition Apoptosis Smad4 4

5 The Smad family hosphosmad2 Tumor Suppressors Eur J Biochem. 2000, 267: The Smad Domains GRK2 phosphorylation Tumor Suppressors Adapted from Derynck & Zhang, Nature :577 5

6 Signal Transduction by TGF family members Tissue and cell specificity Cell specific target genes Growth Inhibition Apoptosis (Annu Rev Cell Dev Biol. 2005;21:659-93) Activin & TGF in Tumor Suppression 1- Cell cycle arrest 6

7 Cell Cycle Regulation Mitogenic Signals Cdk4/6 Cyclin D Cyclin E Cdk2 CoR CoR E2F inactive Rb E2F D E2F Rb D E2F active DNA pol C-myc G1 arrest S phase Cell Cycle Arrest Anti-proliferative Signals INK4 Family (Inhibitor of CDK4) -p15 INK4b -p16 INK4a -p18 INK4c -p19 INK4d CI/KI Family -p21 CI1/WAF1 -p27 KI1 -p57 KI2 Cdk4/6 Cyclin D Cyclin E Cdk2 CoR E2F inactive CoR Rb E2F D E2F active Rb E2F D G1 arrest S phase 7

8 TGF tumor suppressive effects Ligand Cdc25A ATF3 II Mad2/4 I Id1 Id2 G1 Id3 Smad S2/3 E2F4/5 Sp1 p S4 Sp1 myc Cell Cycle S M G2 p15 15 p21 27, p57 others Bone marrow Mammary Gland Cell growth proteins Milk p lacenta hcg rogesterone GRF Embryos Erythropoiesis Cell growth Dorsal mesoderm Blood Cell growth Apoptosis Activins A, C Liver Cell growth Apoptosis Gonads Estrogens Androgens ituitary FSH GH, RL ACTH Hepatocarcinomas Smad mutation (10-20%) Transplantation Hepatectomy/Liver regeneration 8

9 Activin/TGF -induced cell growth arrest and apoptosis in human Hepatocarcinoma cells Activin 0 8 hrs 24 hrs HepG 2 HepG 2 HuH 7 Annexin V staining HuH 7 Flow Cytometry Cell cycle arrest Apoptosis EMBO J (18): Activin/TGF induce cell growth arrest through up-regulation of the CDKI p15 INK4B rotein RNA (Cell Signaling, 16, ) 9

10 Activin induces p15 INK4B gene promoter Role of the Smad pathway Luciferase Activity Fold Induction Activin: CTL Smad2 Smad3 DN Smad2 DN Smad3 Regulation of p15 INK4B gene promoter by activin and TGF (J Biol Chem, 270: , 1995) (Cell Signaling, 16, ) 10

11 Role of the transcription factor Sp1 in p15 gene activation (J Biol Chem, 270: , 1995) (Cell Signaling, 16, ) Ligand Regulation of p15 by Activin/TGF Type II Type I S S S S Smad2/3 S S Smad4 Leukemia 2003 (9): Sp1 S S S S S S S S4 S2/3 Sp1 p15 gene expression p21 gene expression CYT. NUC. 11

12 Activin & TGF in Tumor Suppression 2- Apoptosis Cell growth arrest by activin/tgf HuH7 cells + Activin TGF Affymetrix microarrays - Tumor Suppressor Anti-apoptotic TGF : 0 16 h 24 h p15 Bax ro-apoptotic roto-oncogenes BCL-2 c-myc Tubulin G1 arrest - Apoptosis Western Others (Ids, Apaf-1, Caspases, ) 12

13 TGF can trigger programmed cell death SHI Several mechanisms for TGF -induced apoptosis have been proposed, including the JNK/p38 cascade and/or the modulation of the balance between anti- and proapoptotic proteins of the Bcl family as well as the regulation of phospholipid metabolism. Adapted from J Leukoc Biol : and Nature Cell Biology, 4: , 2002 Activin & TGF in Tumor Suppression 3- Implications in human cancer 13

14 Loss or mutation of TGF signaling in human cancers The New England Journal of Medicine 342, , 2000 Dual role of TGF as a tumor suppressor and tumor promoter NEJM 342, , 2000 Cell growth arrest Inhibition proliferation Limited replication Failure to arrest growth Uncontrolled proliferation Limitless replication Immunosuppression Angiogenesis Invasion/Metastasis (cell adhesion, migration, matrix) EMT (Zeb2, Twist) Tumor Metastasis TGF antagonists/signaling pathways 14

15 Activin & TGF in Tumor Suppression 4- Shutting off Signaling Shutting off signaling kinases 15

16 The Smad Domains MH1 domain -DNA binding -Interaction with transcription factors Linker -Negative regulation MH2 domain -Receptor interaction -Smad oligomerization -Interaction with transcription factors R-Smad -SSXS Tumor Suppressors Receptor phosphorylation MAK phosphorylation CamKII phosphorylation CDK4 phosphorylation GRK2 phosphorylation GRK2 kinase domain is required to antagonize activin/tgf induced G1 arrest and apoptosis Activin: 0 8 hrs WT GRK2 K220M EMBO J 24(18): ,

17 GRK2 blocks activin-induced Smad phosphorylation and nuclear translocation Mock Activin: i p-smad2 Flag-GRK GF-GRK2 Smad3 Overlay CTL Smad2/3 Smad4 TGF GRK2 Nuclear extracts EMBO J 24(18): , 2005 GRK2 phosphorylates Smad linker domain in vitro GST- MH1 Linker MH2 GST- GST- GST- urified: urified GRK2: wt K/M wt K/M wt K/M wt K/M GST-Smad2 Autoradiography GST-Smad3 EMBO J 24(18): ,

18 GRK2 phosphorylates a single S/T residue in the linker domain of Smad2/3 GST MH1 MH T T S T S S T S T T T GRK2: EMBO J 24(18): , 2005 TGF /Activin antagonists hsmad2 hsmad3 hsmad1 hsmad5 hsmad8 hsmad4 hsmad6 hsmad7 VLVRHT---E ILTE-L LDDYTHSI VLVRHT---E IAE-F LDDYSHSI VLVRHS---EYN-----QHS-LL---AQFRNLGQNEHM----LNATF VLVRHN---EFN-----QHS-LL---VQFRNLSHNEHM----QNATF VLVRHS---EYN-----QLS-LL---AKFRSASLHSELM SSMMVKDEYVHDFEGQSLSTEG-HSIQTIQHSNRASTETY----STAL DTLLEAVES---RGG-----VGGCVL---VRADLRLGGQAQLLLGRLF GRLDCRLG---GA-----AGA-Q---AQ--SSYSLLL----LCKVF Smad2 C elegans Drosophila Mouse Rat Human VLVRYSEKQEVTLAKFQLMEMSGSRMQNV VKSLTDSIV-DY--LDNHTHQINNTDYNAAI VLVRHTEILT-EL-LDDYTHSIENTNFAGI VLVRHTEILT-EL-LDDYTHSIENTNFAGI VLVRHTEILT-EL-LDDYTHSIENTNFAGI 18

19 GRK2 inhibits activin/tgf -regulated target gene expression ex vivo Ad-LacZ Ad-GRK2 Activin: 0 16 h 0 16 h Bax p15 GRK2 tubulin Activin: 0 4 h 8 h otent antagonist Negative feed-back loop Ad-LacZ EMBO J 24(18): , 2005 Ad-GRK2 Activin and TGF specifically induce GRK2 expression levels Negative feedback loop Activin: 0 1 h 2 h 4 h 8 h 16 h 24 h GRK2 HuH7 cells Activin: i 0 4 h 8 h 16 h 24 h GRK2 28s 18s Northern tubulin HepG2 cells Activin: 0 4 h 8 h 16 h 24 h VSMC MCF-7 Activin: GRK2 tubulin GRK2 tubulin Western Western EMBO J 24(18): ,

20 ligand GCR Type II Type I SMAD2 SARA cml GRK2 SMAD3 SMAD4 GRK2 SMAD2 SMAD3 SMAD2 SMAD3 G1 ARREST AOTOSIS CO-FACTOR Antagonist pathway p15, myc Bax, Bcl2 SMAD2 SMAD4 SMAD3 CO-FACTOR Activin & TGF in Tumor Suppression 5- Immortality 20

21 Acquired abilities of cancer cells Activin/TGF Cell Cycle - + Apoptosis - Immortalization Immunosuppression Tumor Suppression Cell, Vol. 100, 57 70, 2000 Fluorescence in situ hybridization (FISH) Human Somatic Cells Limited proliferative potential (Hayflick limit) Telomeres shortening Loss of structural integrity Cellular senescence and crisis Human Cancer Cells Infinite Replication and Immortalization Reactivation Telomerase activity Addition telomeric repeat Cancer Diagnosis marker Reverse Transcriptase RNA template rotein htert Add telomeric repeat (TTAGGG) 21

22 TGF Represses htert Gene Expression RNA rotein htert Gene promoter Cell Signal, 20(1): Smad3 but not Smad2 is required for TGF to inhibit htert expression in human keratinocytes TGF : 0 1/2 h 1 h 2 h 4 h 0 1/2 h 1 h 2 h 4 h hospho-smad2 hospho-smad3 h Smad2/3 Smad2/3 Cell Signal, 20(1):

23 The 252 bp proximal region of the htert gene promoter is critical for the TGF response The 252bp proximal htert romoter E-boxes: DNA binding elements for Myc GC-boxes: DNA binding elements for Sp1 4 E2F1 DNA binding elements No Smad DNA binding elements 23

24 Fold Induction * The transcription factor E2F1 is required for TGF -mediated inhibition of htert Fold Induction * Fold Induction * TGFβ - E2F-1 (1-374) TGFβ - E2F-1 Y411C DBD B TA DBD B TA 24

25 E2F1 is required for TGF -mediated inhibition of mtert in normal fibroblasts TGFβ: psmad3 wt E2F1 -/- 0 h 1/2 h 1 h 0 h 1/2 h 1 h Ligand Type II Type I tubulin wt E2F1 -/- TGFβ: 0 h 2 h 4 h 8 h 16 h 24 h 0 h 2 h 4 h 8 h 16 h 24 h Smad3 Smad4 mtert GADH RNA wt E2F1 -/- E2F1 HDAC? TGFβ: 0 h 8 h 16 h 24 h 0 h 8 h 16 h 24 h htert mtert tubulin rotein Immortalization TGF tumor suppressive effects Ligand G1 Cell Cycle M S II I G2 Smad 2/3 Apoptosis 4 Immortality 25