TNF-TNFR interactions. Tumor necrosis factors (TNFs) Transforming growth factor- s (TGF- s) & Tumor necrosis factors (TNFs)

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1 Transforming growth factor- s (TGF- s) & Tumor necrosis factors (TNFs) Marja Mikkola Developmental Biology Program Institute of Biotechnology Growth factors and their receptors Tumor necrosis factors (TNFs) 19 TNF family members are known in humans/mice Low homology (20-30% identity) which is limited to the 150 aa long C-terminal receptor binding domain About 30 members in TNF receptor family TNF (formerly TNF cachetin) is the prototypic member of the TNF superfamily TNF was first identified in 1984 as a cytokine with the ability to induce tumor cell death (apoptosis) as well as fever and wasting type II membrane proteins (N-terminus inside the cell); however many TNFs are shed from cell surface function as trimers (usually homotrimers) Tumor necrosis factors (TNFs) Tumor necrosis factor receptors (TNFRs) TNF LT LT OX40 ligand CD40 ligand Fas ligand CD27 ligand CD30 ligand 4-1-BB ligand TRAIL RANK ligand TWEAK APRIL BAFF=BlyS LIGHT TL1A GITR ligand Eda-A1 Eda-A2 About 30 TNFRs are currently known 20 signalling receptors Some receptors are non-signalling, so called decoy receptors Typically higher homology in the extracellular ligand binding domain which consists of one to four repeats of 40 amino acids (rich in cysteines intrachain S-S bridges) some homology also in the intracellular domain, some receptors have so called death domains (DD) one ligand one receptor? often true but not always e.g. TRAIL has two signalling receptors and two decoy receptors TNF-TNFR interactions Functions of TNFs Most TNFs/TNFRs are expressed in the immune system TNFs are important (i.e. have beneficial effects) in numerous processes such as host defense inflammation Protective immune responses development and dintegrity i of lymphoid organs such as spleen, lymph nodes bone formation and remodeling development of ectodermal organs such as hairs, teeth, mammary glands, sweat glands etc TNFs also exert host-damaging effects in several diseases such as septic shock, fever syndromes, cachexia Inflammatory autoimmune diseases such as rheumatoid arthritis, proriasis, Crohn s disease TNF inhibitor (TNF antibodies) in clinical use!! 1

2 Anti-TNF drugs Three drugs approved by the US authorities all are very expensive Infliximab (Remicade) humanized (human-mouse chimeric) monoclonal Ab to TNF. i.v. infusion every 6-8 weeks. Can cost even USD a year. Etanercept (Enbrel) Fc-TNFRI ectodomain. Subcutaneous injection by patient at home (once or twice a weak). Adalimumab (brand name HUMIRA) a human monoclonal. Subcutaneous injection by patient at home (twice a week). Typically used together with other immunosuppressive drugs Signalling by TNFRs At cellular level TNFs have pleiotropic effects and may cause cell proliferation cell survival differentiation apoptosis Ligand induced trimerization/multimerization of the receptors initiates iti t the signalling cascade TNFRs have no intrinsic enzymatic activity and their intracellular domains are modest in length and function as docking sites for signalling molecules Two pivotal modes of signalling (usage of two types of adapter molecules): TRAFs 1-6 (TNF receptor-associated factors) death domain (DD) molecules ( = protein-protein interaction domain) Signalling by TNFRs Signalling by TNFRs Activated TNFR binds adapter molecules: TRAF1-6 and/or DD molecules caspase activation (apoptosis) NF- B (cell survival) JNK/p38 Often both survival and apoptotic pathways are activated Outcome depends on the cellular context Many receptors recruit only TRAFs and do not (directly) induce cell death Some receptors recruit only specific (apoptosis promoting) DD molecules activation usually leads to cell death Gaur & Aggarwal, 2003 Functions of TNFs in host defense TNF/TNFRs are fundamental in the vertebrate adaptive (antigen directed) immune system Regulation of T and B cell survival/proliferation Coordinate the social context of cells enabling lymphocytes to maximally respond to pathogens Deficiency in some TNF/TNFRs results in defective lymphoid organs (e.g. LT / LT R signalling) Role in antibody class (IgM to IgG) switching (CD40L CD40) TNF/TNFRs as mediators of cell death Cell-mediated toxicity in response to infectious agents by DD containing TNFRs Immune homeostasis to balance lymphocyte expansion in response to antigen within the limited space of lymphoid organs If disturbed (as in Fas -/- mice) autoimmunity Functions of TNFs in host defense TNF/TNFRs in innate immunity Mice deficient for TNF or TNFR1 are susceptible to infections by intracellular pathogens such as Salmonella, Listeria, Mycobacteria TNF/TNFRs in acute inflammation Host defence relies on the rapid recruitment on many inflammatory cell types to the site of an infection To prevent damage to host it is essential to have Rapid response to microbial replication AND Rapid subsidence of inflammatory response TNF has a prominent role in inflammation TNF secretion is induced by conserved structural elements common to microbial pathogens such as LPS The inflammatory cascade induced by infection can be recapitulated by injection of TNF If deregulated, TNF can cause chronic inflammation 2

3 Functions of TNFs in bone homeostasis Mice lacking RANK ligand have severe osteopetrosis (increased bone density, stome bone ) RANK-L / RANK was the first receptor-ligand pair to function outside immune system It takes three to tango: RANK ligand RANK OPG (a decoy receptor of RANK-L, osteoprotegerin, protector of bone a physiological inhibitor of RANK signalling The formation and homeostasis of bone is a result of the action of two cell types: Osteoblasts = bone forming cells (mesenchymal cells) Osteoclasts = bone resorbing cells (of haematopoietic origin) Functions of TNFs in bone homeostasis RANK ligand, RANK, and OPG are key molecules regulating bone homeostasis both during embryonic development and adulthood osteoclast: NF- B activation RANK RANK-L OPG osteoblast Binding of RANK ligand to RANK leads to activation of NF- B resulting in the proliferation, differentiation, and activation of pre-osteoclasts RANK pathway in osteopetrosis Despite increased bone density, bone may be more brittle Serious forms of osteopetrosis: Skull deformation (macrocephaly) Severe anemia (and recurrent infections) already staring during infancy due to encroachment of the bone marrow by excess bone blindness, deafness, facial paralysis due to the increased pressure put on the nerves by the extra bone Autosomal recessive osteopetrosis may be caused by mutations in either RANK or Rank ligand severe forms of osteopetrosis Treatment: bone marrow transplant Functions of TNFs in bone homeostasis Studies with transgenic mouse models confirm the proposed model for bone homeostasis: RANK ligand -/-, and RANK -/- mice have the same phenotype i.e. osteopetrosis resulting in short stature, increased bone mass, failed eruption of teeth, and total lack of osteoclasts OPG -/- mice have severe osteoporosis OPG overexpressing mice are osteopetrotic Many other factors regulate bone homeostasis (CSF-1, vitamin D3, various cytokines, several hormones) but it appears that they (all) regulate the expression/function of either RANK-L, OPG, RANK, or other molecules in the RANK signalling pathway e.g. estrogen deficiency caused increased expression of RANK ligand expression by stromal cells postmenopausal osteoporosis estrogen stimulates OPG production by osteoblasts Usage of OPG or antibodies to RANKL to treat various diseases characterized by increased bone resorption (osteoporosis) TNFs in ectodermal organ development ectodermal organs are derivatives of the embryonic ectoderm = skin appendages hair, whiskers, feathers, teeth, scales, horns, beaks, nails, sweat gland, mammary gland, etc share common developmental mechanisms regulated by interactions between two types of tissues, the epithelium (ectoderm) and the underlying mesenchyme epithelium mesenchyme TNFs in ectodermal organ development: Eda-Edar signalling Eda (ectodysplasin) and Edar: novel TNF and TNFR mutations in Eda or Edar cause defective development of a number of ectodermal organs both in mice and humans Wild type ectodysplasin Adapted from Chuong (1998) Eda -/- 3

4 Eda-Edar in ectodermal organ development: Mice overexpressing Eda have supernumerary ectodermal organs: extra teeth: extra mammary glands: embryonic wild type Eda transgenic 1 st molar 2 nd 3 rd 1 st molar 2 nd 3 rd Eda/Edar pathway is conserved in all vertebrates Edar, Xedar, Troy sequences found in all vertebrates Xedar may have been lost in some fish; lampreys??? Only one Edar -like gene in simple chordates (Ciona, sea squirts; amphioxus) role in epithelial morphogenesis?? Eda/Edar is an innovation of early vertebrates? Oi Origin i of ffirst mineralized skin appendages Eda/Edar is essential for feather formation, fish scale and tooth development, also for fin development in zebrafish wild type Edar mutant adult Harris et al PLoS Genetics 4:e Biomedical applications of TNFs: Effective cancer drugs? Current cancer treatment: surgery radiotherapy chemotherapy Limitations of conventional cancer drugs: Toxic side effects Appearance of treatment-resistant tumor cells What about activation of death receptors such as TNFR1, Fas, or TRAIL receptors in cancer cells? Many conventional cancer drugs are dependent on functional p53, a tumor suppression protein However, p53 is inactivated in more than half of human cancers resistance to many cancer drugs TNF receptor activation can trigger cell death independent of p53! TNFs as cancer drugs? TNF (little attention recently): Side effects at doses needed from treatment i.e. hypotension, septic shock Has been successfully used in locally advanced soft tissue sarcoma and melanomas Isolated limb perfusions Combined treatment with TNF & more conventional drugs may give good results Fas activation (no clinical trials done): Activation of the receptor by Fas agonists Induces apoptosis in hepatocytes Lethal liver failure in treated mice TRAIL/Apo2L may hold better promise The biological role of TRAIL is not well understood but mice deficient in Trail signaling are more susceptible to tumorigenesis (at least some tumor types) and pathogen infection A role in host defence against tumor initiation & metastasis A role in interferon-dependent host defence against viral infections TNFs as cancer drugs: TRAIL story TRAIL signalling pathways: Two signalling receptors Two decoy receptors TRAIL and its receptors are fairly widely expressed in normal tissues Benefits of TRAIL: Recombinant soluble TRAIL induces apoptosis in a variety of cancer cells in vitro whereas it seldom effects normal cells Reason not completely understood (Kelley & Ashkenazi, 2004) In vivo mouse models reveal that TRAIL has prominent anti-tumor activity in various tumor xenograft models Safety studies indicate that Trail it is well tolerated TRAIL (phase I) and anti-trailr1 agonistic antibodies (phase I and II) trials initiated for solid tumors In patients with certain advanced malignancies, only modest effect has been seen so far key question: how do those cells become resistant to Trail induced apoptosis TRAIL-receptor agonists and their clinical status. Treatment Clinical development stages Company HGS-ETR1 (anti-trail-r1 mab) Phase II completed: NHL, colorectal cancer, NSCLC Human Genome Science HGS-ETR1 _ paclitaxel _ carboplatin Phase Ib: advanced solid tumors HGS-ETR1 _ gemcitabine _ cisplatin Phase Ib: advanced solid tumors HGS-ETR1 _ bortezomib Phase II: advanced multiple myeloma HGS-ETR2 (anti-trail-r2 mab) Phase I: advanced solid tumors HGS-ETR2 _ chemotherapy Phase Ib: advanced solid tumors HGS-TR2J (anti-trail-r2 mab) Phase I: advanced solid tumors LBY135 (anti-trail-r2) Phase I/II: advanced solid tumors Novartis LBY135 _ capecitabine Phase I/II: advanced solid tumors (recruiting since 2006) Apomab (anti-trail-r2) Phase I/II: advanced solid tumors Genentech Apomab _ avastin Phase II: advanced solid tumors (initiated in 2007) TRA-8 (anti-trail-r2) Phase I: advanced solid tumors and Daiichi Sankyo Inc. lymphomas AMG655 (anti-trail-r2) Phase I: NSCLC, colorectal cancer Amgen (initiated in 2005) Apo2L/TRAIL _ rituximab Phase Ib/II: NHL (recruiting since Genentech/Amgen 2006) Ad5-TRAIL Phase Ia: organ-confined prostate cancer Mahmood & Shukla. Exp Cell Res 2010 NHL indicates non-hodgkin's lymphoma; NSCLC, non-small cell lung cancer. 4

5 TNFs as cancer drugs: TRAIL story Death receptor signaling Programmed cell death: There are two ways to induce apoptosis Effector caspases (-3, 6, -7) are key molecules in apoptosis and their activation results in the cleavage of more than 100 proteins eventually leading to cell death Diverse stress pathways cause release of mitochondrial proteins to activate the cell-intrinsic pathway TNFRs induce cell-extrinsic pathway, and may also activate cell-intrinsic pathway thus amplifying the apoptotic signal Kelley & Ashkenazi, 2004 Mahmood & Shukla. Exp Cell Res 2010 TNF pathway and inherited diseases Germline mutations in the following genes have been associated with congenital diseases (the list is not complete): Gene type of mutation phenotype/notes Eda null/x-linked hypohidrotic ectodermal dysplasia; defective development teeth, hairs, sweat & several other glands Edar recessive or same as in Eda dominant Fas heterozygous autoimmune lymhoproliferative lif i syndrome (ALPS); (defective lymphocyte apoptosis, autoimmune hemolytic anemia, lymphadenopathy, splenomegaly; high lymphoma risk) Fas ligand dominant ALPS RANK dominant, activating familial expansile osteolysis (FEO); overactivated osteoclasts, severe bone pain & fractures leading to amputations, early onset of deafness (<10years), early loss of dentition TNFR1 dominant TNFR associated syndrome (TRAPS); unexplained periods of fever & severe localized inflammation; defect in downregulation of TNFR1 (by shedding) after stimulation? CD40 recessive, null immunodeficiency with hyper-igm CD40 ligand null/x-linked same as CD40 Transforming growth factor- (TGF- ) superfamily TGF- 1 was the first member discovered 25 years ago To date over 35 structurally related members in vertebrates Divided into several subfamilies: TGF- family: TGF- in mammals Bone morphogenetic protein (BMP) family: over 20 BMPs are known BMPs (10 in mammals) GDFs (growth and differentiation factors) (11 in mammals) Activin family: activin A and B, and others such as Nodal, GDNF TGF- s are produced as precursor molecules; the C-terminal regions contains the mature peptide Are active as dimers, usually homodimers Have crucial roles during embryonic development and in tissue homeostasis; perturbations lead to developmental disorders, cancer, fibrosis, auto-immune diseases etc. Transforming growth factor- (TGF- ) superfamily Human proteins in black, Drosophila in grey Schmierer & Hill, 2007 Ducy & Karsenty,

6 TGF- receptors There are much less receptors than there are ligands Two types of receptors: Type I receptors (7 in vertebrates) Type II receptors (5 in vertebrates) Receptors have conserved serine- threonine kinase domains Signalling requires both types of receptors which act as dimers heterotetramer Receptor activation activates SMADs SMAD family of proteins: R-SMADs (SMAD1, 2, 3, 5, 8) Common SMAD = SMAD4 Inhibitory (I-) SMADs = SMAD6, 7 AMHR2 TGF- signalling TGF- signalling pathway appears simple at first glance: Ligand binding induces association of type I and type II receptors This leads to the phosphorylation of type I receptor by type II receptor thereby activating its kinase domain Activated type I receptor activates R- SMADs by phosphorylation (P) P R-SMADs associate with SMAD-4 and translocate into the nucleus where they regulate the expression of target genes The output is determined by the type I receptor, not type II However, there are several modulators of the pathway TGF- signalling Alk4,5, 7 Smad2/3 Alk123 Alk1,2,3, 6 Smad1/5/8 /8 TGF- signalling Only two modes of signal transduction: Either SMAD1/5/8 is phosphorylated (three out of 7 type I receptors) = BMP type of signalling or SMAD2/3 is phosphorylated (four out of 7 type I receptors) = TGF- like signalling (and activin & nodal) TGF- receptors can also signal independent of SMADs! Schmierer & Hill, 2007 TGF- signalling Each R-SMAD can interact with a wide array of proteins to regulate transcription; also additional complexity is achieved through cross talk with other signalling cascades As with all growth factor signalling the final outcome depends on the cellular context and often depends on the concentration of the growth factor Extracellular modulation of TGF- signalling Extracellular inhibitors play a prominent role in controlling and fine-tuning TGF- signalling A transmembrane decoy receptor called BAMBI blocks both activin, BMP & TGF- signalling by stably associating with functional receptors There are numerous extracellular inhibitors (most of which inhibit ligandreceptor interactions) which influence the local concentration of TGF- s: Inhibitor interacting ligand note! Decorin TGF- Biglycan TGF- -/- : decreased bone mass Follistatin activins, BMPs -/-: reduced size, skeletal & tooth & hair defects Noggin BMPs -/-: open neural tube, defects in body axis, cartil&bone Chordin BMPs -/-: defects in ear & cardiovascular organization Cerberus BMPs, activin, Nodal head formation (Xenopus); -/-: no obvious phenotype Dan BMPs -/-: no obvious phenotype Gremlin BMPs role in limb outgrowth Sclerostin BMPs -/- humans: excessive bone formation Ectodin BMPs modulates also WNT pathway; -/- mice: bone & tooth defects 6

7 Functions of Bone morphogenetic proteins (BMPs) BMPs were first identified due to their ability to promote ectopic bone formation, now 21 BMPs (or GFDs) known in ammals Later they have been shown to be important in numerous other developmental processes such as Body plan, left-right asymmetry Neurogenesis Mesoderm patterning Organogenesis: kidney, gut, lung, tooth, hair, limb, testis development etc. as well as in adult functions in cardiovascular, pulmonary, reproductive, urogenital organs & in the nervous system At cellular level they may promote cell Differentiation Proliferation Migration Apoptosis GDF = growth and differentiation factor Functions of BMPs: lessons from knockout mice Many of the BMP family members have been knocked out in mice Analysis hampered by Redundancy Early lethality BMP BMP-2 BMP-3 BMP-4 BMP-5 BMP-6 BMP-7 BMP-8A BMP-8B BMP-15 GDF-1 defect early lethal: heart, neural tube, amnion/chorion defects etc. skeletal phenotype: increased bone mass early lethal: no mesodermal differentiation defects in skull & skeleton & in a number of organs, yet viable and fertile no overt phenotype defective kidney, eye, skeletal development, polydactyly defects in spermatogenesis defects in spermatogenesis, male sterility defects in oogenesis, subfertile females defects in left-right asymmetry Biomedical applications of BMPs BMPs have pivotal roles in bone induction bone maintenance bone repair Therefore they have been of interest as therapeutic agents for healing fractures of bones preventing osteoporosis treating periodontal defects healing bony and cartilaginous defects followed by orthopedic surgery Bmp2 and-7 are approved for clinical use for e.g. open fractures of long bones and nonunion A limited number of clinical trials using BMPs in humans have been reported no major breakthroughs Delivery of BMPs to the target site is a problem Gene therapy? Short-term protein production is enough for many bone repair problems Functions of TGF- 1, -2, -3 TGF- 1, TGF-, and TGF- are highly similar in many of their biological activities in vitro Potent growth inhibitors of many cell lines, especially epithelial cells But usually mitogenic for cells of mesenchymal origin Stimulate the production of ECM proteins ( fibrosis in vivo) However, they differ in their in vivo expression patterns which is reflected in the phenotypes of knockout mice: TGF- TGF- 1 TGF- 2 TGF- 3 defect 50% embryonic lethal: defects in vasculogenesis & angiogenesis 50% die in 3 weeks due to excessive infiltration of inflammatory cells into multiple organs die before or at birth: cardiac, lung, craniofacial, eye, limb, urogenital, spinal column defects die shortly after birth: defective lung development, cleft palate TGF- 1 and cancer A good guy or a bad guy? A double agent? The name comes from its ability to promote malignant behaviour of normal fibroblasts Growth suppressive effects on epithelial and lymphoid cells (which form the basis of the majority of human cancers) Data from both experimental model systems and human cancers show that it is important in suppressing primary tumorigenesis However, in advanced disease it has proto-oncogenic effects Indeed, overexpression of TGF- 1 is associated in poor patient prognosis in some cancers Metastasis of many different tumors requires TGF- 1 activity E.g. in some breast cancer models required for bone metastases to form Tumor suppressor in primary tumors, proto-oncogene in metastases TGF- switches from tumor suppressor in the premalignant stages of tumorigenesis to prooncogene at later stages of disease leading to metastasis Upregulates expression of cyclindependent kinase inhibitors represses c-myc oncogene TGF- 1 and cancer Roberts, Anita B. and Wakefield, Lalage M. (2003) Proc. Natl. Acad. Sci. USA 100, Any potential for therapeutic targeting? 7

8 TGF- 1 as a tumor suppressor TGF- 1 as a tumor promoter Meulmeester & ten Dijke, 2010 With increased knowledge of TGF- signaling, it may be possible to therapeutically target specific sub-arms of the TGF- pathway (see next slide) Clinical trials with systemic TGF- signaling inhibitors in cancer patients have been initiated Meulmeester & ten Dijke, 2010 Functions of other TGF- s Activins are required for proper embryonic development Two gene products: activin A and activin B which dimerize to form Activin A Activin B Activin AB Dimerize with inhibin to form ihibi inhibin A inhibin B Essential role in mesoderm and neural induction in Xenopus but not in mammals Activin A knockout mice: Defective hair and tooth development Cleft palate Activin B knockout mice: Defects in female reproduction Functions of other TGF- s Nodal Is a distinct relative of BMPs Nodal -/- reveal that it is essential for primitive streak and mesoderm formation (no gastrulation) Required for normal placenta development Important in left-right asymmetry Lefty1, Lefty2 Leftys are actually not signalling molecules but Nodal antagonists Loss of Lefty activity results in an expansion of Nodal signalling in vertebrates Lefty1 -/-: abnormal left-right axis Lefty2 -/-: excessive mesoderm, early embryonic lethal Conditional Lefty 2 -/-: abnormal left-right axis 8