Detailed Study Notes on TGF-β Signalling

TGF-β Signalling

Lecture Overview

  • This lecture focuses on TGF-β (Transforming Growth Factor beta) signalling, covering its canonical pathway, roles in tissue development and homeostasis, dysregulation in diseases (Chagas disease and carcinogenesis), cooperation with Hippo signalling in the liver, and potential treatment strategies.

Learning Outcomes

  • Outline the canonical TGF-β signal pathway, including key receptors and effector molecules.
  • Describe TGF-β signalling's contribution to normal tissue development and homeostasis.
  • Explain TGF-β signalling dysregulation in pathological conditions, focusing on Chagas disease and carcinogenesis, and potential treatments.
  • Explain the cooperation between TGF-β and Hippo signalling in liver development, regeneration, and disease.

The TGF-β Superfamily

  • Comprises approximately 30 factors and 12 receptors that form heteromeric complexes.
  • Regulates cell fate during development and maintains tissue homeostasis in adults.
  • Signals canonically through SMAD proteins (8 types) to drive transcription of various genes.
  • Also signals through non-canonical routes (via DAXX proteins), often interacting with growth factor targets.
  • TGF-β is the most relevant member of the superfamily in the context of cancer.
  • There are three TGF-β isoforms: TGF-β1, TGF-β2, and TGF-β3.

TGF-β Signalling Pathway

  • TGF-β dimer binding promotes the assembly of a tetrameric receptor complex, including two copies each of type-I and type-II receptors which are serine/threonine kinases.
  • Type-II receptors phosphorylate specific sites on type-I receptors, activating their kinase domains, which leads to phosphorylation of R-SMADs (SMAD2 and SMAD3).
  • Phosphorylated R-SMADs form a trimeric SMAD complex with the co-SMAD, SMAD4.
  • This complex translocates to the nucleus to control the transcription of specific target genes with other cofactors.

Non-Canonical Signalling

  • TGF-β can signal through non-canonical pathways to activate various pathways, often overlapping with growth factor signalling.
  • Includes MAPK (ERK) signalling via Ras, p38 MAPK, c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB), PI3K/AKT, and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling.
  • These non-canonical pathways participate actively in a broad range of cellular events.

TGF-β Signalling in Health and Disease

  • TGF-β signalling plays crucial roles in development and tissue homeostasis.
  • When dysregulated, it can lead to many diseases.

Epithelial-Mesenchymal Transition (EMT)

  • During embryonic development, TGF-β regulates cell differentiation, epithelial/endothelial-mesenchymal transition, and apoptosis for proper histogenesis and organogenesis.
  • In developmental contexts like gastrulation, EMT driven by Nodal TGF-β signalling occurs alongside mesendodermal cell differentiation.
  • In regenerative contexts (wound healing, fibrosis, and cancer), TGF-β driven EMT occurs alongside fibrogenic effects that remodel the ECM.

TGF-β in Epithelial Homeostasis and Repair

  • In response to injury, epithelial progenitors undergo EMT to migrate to niches with appropriate ECM support and signals to orchestrate injury repair.
  • TGF-β is a major inducer of EMTs, often requiring cooperation with RAS-activated MAPK signals.
  • RREB1 (RAS-responsive element binding protein 1) links the TGF-β-SMAD and RAS-MAPK pathways, coordinating developmental and fibrogenic EMT programmes.
  • TGF-β modulates the proliferation of epithelial progenitors and regulates their differentiation, often with countervailing WNT, BMP, and other signals, contributing to tissue homeostasis.

EMT in Wound Healing and Cancer

  • TGFβ promotes EMT in epithelial cells, showing decreased E-cadherin, and accumulated striated fibres upon TGFβ stimulation, which is necessary for the response to injury.
  • TGFβ also promotes the migration of human breast cancer cells.

TGF-β Regulation of Fibroblasts

  • TGF-β induces the recruitment, proliferation, and activation of fibroblasts that produce collagens, fibronectin, integrins, and other components required for ECM assembly, which is essential to reconstitute the tissue barrier after injury.
  • Tissue fibrosis is characterised by chronic inflammation and accumulation of fibrillar collagens and other ECM components, resulting from imbalanced ECM production by tissue-resident fibroblasts, contributed to by feed-forward loops involving TGF-β.
  • TGF-β regulates fibroblast activity throughout the tissue response to injury and during chronic fibrosis; collagen is key.

TGF-β in Chagas Disease

  • Chagas disease (CD) is a chronic systemic parasitosis caused by Trypanosoma cruzi, affecting 6-7 million people worldwide, with approximately 30% developing cardiac alterations.
  • It is the main cause of cardiomyopathy in endemic areas of Latin America.
  • TGF-β influences the development of myocardiopathy in Chagas disease by inducing inflammation, heart fibrosis and remodelling, and affecting gap junction modulation.

Therapeutic Target in Chagas Disease

  • Current drugs against CD have serious side effects and do not reverse fibrosis.
  • Pirfenidone, which inhibits TGF-β, has shown effectiveness in preventing the increase in collagen type I triggered by T. cruzi infection, both at the gene and protein expression levels.

TGF-β in Tissue & Immune Homeostasis

  • TGF-β is indispensable for tissue homeostasis, suppressing cell proliferation and inducing cell apoptosis to preserve tissue integrity.
  • TGF-β is a critical modulator of both adaptive and innate immunity arms, acting as an enforcer of immune tolerance and a suppressor of inflammation to maintain immune homeostasis.

TGF-β Signalling in Disease

  • Dysfunctional TGF-β signalling exacerbates tissue injuries in inflammatory and infectious diseases by promoting inflammation, pathogen infection, and tissue remodelling.
  • Aberrant TGF-β signalling is implicated in all aspects of tumour development, including tumorigenesis, tumour growth, tumour invasion, tumour metastasis, and tumour microenvironment (TME) remodelling.

TGF-β Mutations and Cancer Signalling

  • Aberrations in signalling pathways that control development are associated with many cancers.
  • TGF-β normally inhibits proliferation of many cell types to control growth.
  • Activated Smads translocate to the nucleus and promote the expression of:
    • p15 inhibitor of cyclin-dependent kinase 4 (CDK4), which causes cells to arrest in G1.
    • Plasminogen activator inhibitor 1 (PAI-1), which promotes the expression of genes encoding extracellular matrix proteins and reduces plasmin-catalysed ECM degradation.
  • TGF-β receptor/Smad loss-of-function mutations:
    • Promote cell proliferation and contribute to invasiveness and metastasis of tumor cells.
    • Found in a variety of human cancers (retinoblastoma, pancreatic, colon).

TGF-β Signalling During Carcinogenesis

  • To escape TGF-β dependent apoptosis, RAS-mutant cells must acquire TGF-β pathway inactivating mutations or alterations that decouple TGF-β signalling from apoptosis, enabling carcinoma progression.
  • Tumour-promoting effects include generation of an immune evasive TME, induction of cancer cell EMTs, and promotion of cancer cell growth, invasion, angiogenesis, and stromal cooperation for metastasis.
  • During early stages of carcinogenesis, TGF-β exerts tumour suppressive effects by inhibiting tumorigenic inflammation or triggering EMT-coupled apoptosis in pre-malignant progenitors harbouring RAS mutations.

Poll Answer

  • Which mutations might enable TGF-β signalling to de-couple from apoptosis?
    • Bcl2 hyperactivation will inhibit apoptosis.
    • Lowered Fas death receptor expression will inhibit apoptosis.
    • p53 loss will inhibit apoptosis.
    • Hyperactivation of PKB will inhibit apoptosis via Bad protein phosphorylation.
    • Mst1 (Hippo) hyperactivation will stimulate apoptosis.

Functions of TGF-β in the Tumour Microenvironment

  • TGF-β-rich TME promotes survival mechanisms, including angiogenesis, immune suppression, fibrosis, and tumour cell plasticity, which promotes invasion and metastasis.
  • This prevents antitumour immune responses, limits drug and immune cell access to the tumour, and promotes resistance to therapy.
  • Increasing concentrations of TGF-β in the TME will modify the EMT status of carcinoma cells and induce a reorganisation of the TME.
  • TGF-β will also impact metabolism in all carcinoma cells and create a hypoxic environment.

Liver Development, Regeneration & Carcinogenesis

  • The liver plays a crucial role in maintaining whole-body homeostasis in health and disease, requiring the coordination of multiple signalling pathways.
  • TGF-β and Hippo signalling pathways cooperate in liver development, size, and regeneration by regulating proliferation, differentiation, and apoptosis.
  • De-regulation of the HIPPO pathway is associated with metabolic diseases such as T2D and NAFLD, and overexpression of YAP/TAZ in the liver promotes cell proliferation that may lead to hepatomegaly and hepatocarcinogenesis.
  • In liver cancer, the TME dictates that TGF-β functions as a tumour suppressor in early stages but becomes a tumour promoter in advanced stages.

Hippo and TGFβ Signalling in Liver

  • Transient TGF-β signalling promotes differentiation of hepatoblasts to biliary epithelial cells and EMT of hepatocytes during liver development, in cooperation with Hippo signalling, and operates similarly during regeneration.
  • Sustained activation of these pathways during disease promotes fibrosis, inhibits the immune system, and promotes EMT and invasion of cancerous cells.

HIPPO/YAP/TAZ and TGF-β/SMAD Crosstalk

  • A major crosstalk between the HIPPO and TGF-β pathways controls liver size and regeneration; both pathways inhibit cell proliferation and maintain hepatocyte homeostasis, acting as tumour suppressors.
  • HIPPO downstream effectors, such as TEAD and TAZ/YAP, can function as cofactors of the TGF-β/SMAD canonical pathway, since some gene promoters contain both SBE and TBE.
  • SMAD/YAP/TAZ complexes can synergise transcriptionally.
  • In contexts such as cancer cells, where TGF-β/SMAD signalling is active, this pathway may cooperate with YAP/TAZ to induce fibrogenic factors.

Strategies Targeting TGFβ Signalling

  • TGF-β inhibitory agents include antisense oligonucleotides targeting TGF-β expression, antibodies targeting multiple points of the pathway, and small-molecule compounds targeting TGF-β-activating integrins and TGF-β receptors.
  • TGF-β receptor ectodomains fused to immune checkpoint antibodies are engineered to increase the efficacy of immunotherapeutic agents by trapping TGF-β near target cells.
  • Dominant-negative TGF-β receptor constructs are overexpressed in engineered various types of anti-cancer T cells (CAR T cells, autologous CTLs) to protect them from inhibition by TGF-β.

Synergistic Combination Therapies

  • (a) Chemo/radio/targeted therapy alone inhibits the growth of epithelial-like tumour cells, and in combination with anti-TGFβ therapy, invasive escape and resistance to these therapies are attenuated, and metastasis of mesenchymal tumour cells is restrained.
  • (b) Activated CAFs mediated by high TGFβ activity suppresses immunotherapy efficacy by blocking T cell infiltration into tumours and inducing T cell dysfunction.
  • In combination with anti-TGFβ therapy however, T cell exclusion is inhibited, and the antitumour efficacy of the immunotherapy is improved.

Key Points

  • The TGF-β family regulates cell fate during development and the maintenance of tissue homeostasis in adults, EMT during embryogenesis, wound healing and immune regulation, and various neurogenic processes.
  • TGF-β receptor subunits include a constitutively active S/Tkinase that, when bound by TGF-β, phosphorylates and activates a serine domain that phosphorylates an R-smad (2 or 3) that then bind a co-Smad (4) and translocate to the nucleus to act as transcription factors.
  • TGF-β promotes wound healing by participating in inflammation, re-epithelialisation, angiogenesis, and fibroblast activation, but may lead to chronic inflammation and fibrosis, characterised by excess TGF-β and collagen deposition.
  • TGF-β influences the development of myocardiopathy in Chagas disease through inducing inflammation, heart fibrosis and remodelling, and affecting gap junction modulation: TGF-β inhibitors represent a potential therapeutic target.
  • TGF-β is a critical modulator of both adaptive and innate immunity arms, acting as an enforcer of immune tolerance and a suppressor of inflammation to maintain immune homeostasis.
  • Aberrant TGF-β signalling is implicated in all aspects of tumour development including tumorigenesis, tumour growth, tumour invasion, tumour metastasis, as well as tumour microenvironment (TME) remodelling.
  • TGF-β receptor/Smad loss-of-function mutations promote cell proliferation and contribute to tumour invasiveness: TGF-β inhibitors include agents targeting the receptor and TGF-β expression, including via linked integrins.
  • TGF-β and Hippo signalling pathways cooperate in the process of liver development, size and regeneration: both pathways inhibit cell proliferation and maintain hepatocyte homeostasis, by promoting differentiation of hepatoblasts to biliary epithelial cells and hepatocyte EMT.
  • HIPPO downstream effectors, such as TEAD and TAZ/YAP, can function as cofactors of the TGF-β/SMAD canonical pathway and SMAD/YAP/TAZ complexes can synergise transcriptionally.
  • Overexpression of YAP/TAZ in the liver promotes cell proliferation that may lead to hepatomegaly and eventually hepatocarcinogenesis, and in liver cancer, the TME dictates that TGF-β functions as a tumour suppressor in early stages but a tumour promoter in advanced stages: sustained activation of these pathways promotes fibrosis, inhibits the immune system, and promotes EMT and invasion of cancerous cells.