TGF-β Signaling Lecture Notes

TGF-β Signaling

Learning Outcomes

• Outline the canonical TGF-β signal pathway, with the key receptors and effector molecules.

• Describe in broad terms how TGF-β signaling contributes to normal tissue development and homeostasis.

• Explain how TGF-β signaling is dysregulated in pathological conditions, including Chagas disease and carcinogenesis, with potential treatment strategies.

• Explain how TGF-β and Hippo signaling cooperate 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 phosphorylating SMAD proteins (8 types) to drive transcription.

• Also signals through non-canonical routes (via DAXX proteins), often cross-talking with growth factor targets.

• TGF-β is the most relevant of the superfamily for cancer. There are three TGF-β isoforms: TGF-β1, TGF-β2, TGF-β3.

TGF-β Signaling Pathway

• Binding of a TGFβ dimer promotes the assembly of a tetrameric receptor complex containing two copies each of type-I and type-II receptors, which include a serine/threonine kinase.

• Type-II receptors phosphorylate specific sites on type-I receptors, activating their kinase domains and leading to phosphorylation of R-Smads (Smad2 and Smad3).

• Phosphorylation opens up R-Smads, exposing a binding complex, leading to the formation of a trimeric Smad complex containing two R-Smads and the co-Smad, Smad4.

• The phosphorylated Smad complex enters the nucleus to control the transcription of specific target genes with other cofactors.

Non-Canonical Signaling

• TGF-β can signal through non-canonical pathways to activate a number of different pathways, many overlapping with growth factor signalling.

• This includes MAPK (ERK) signalling via Ras, p38 MAPK, c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB), $(PI3K)/AKT$, as well as Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling.

• These non-canonical TGF-β signaling pathways are actively involved in an extensive range of cellular events.

TGF-β Signaling in Health and Disease

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

Epithelial-Mesenchymal Transition (EMT)

• During embryonic development, TGF-β regulates cell differentiation, epithelial/endothelial-mesenchymal transition and apoptosis to ensure proper histogenesis and organogenesis.

• In developmental contexts such as gastrulation, EMT driven by Nodal TGF-β signaling occurs together with mesendodermal cell differentiation.

• In regenerative contexts such as wound healing and their derivative pathologies - fibrosis and cancer - TGF-β driven EMT occurs alongside fibrogenic effects that remodels the ECM.

TGF-β in Epithelial Homeostasis and Repair

• In response to injury, epithelial progenitors undergo EMT for migration to niches that provide appropriate basal lamina ECM support and signals to orchestrate injury repair.

• TGF-β is a major inducer of EMTs, which often requires the cooperation of RAS-activated MAPK signals.

• RREB1 (RAS-responsive element binding protein 1) links the TGF-β- SMAD and RAS-MAPK pathways and coordinates the expression of developmental and fibrogenic EMT programmes.

• TGF-β modulates the proliferation of epithelial progenitors and regulates their differentiation, frequently 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, as determined via real-time imaging of a wound-healing scratch assay.

TGF-β Regulation of Fibroblasts in Health and Disease

• TGF-β potently 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 production of ECM by tissue resident fibroblasts contributed to by feed- forward loops involving TGF-β.

• TGF-β regulates fibroblast activity throughout the tissue response to injury as well as during chronic fibrosis.

TGF-β is linked to cardiac fibrosis in Chagas disease

• Chagas disease (CD), is a chronic systemic parasitosis caused by the protozoan Trypanosoma cruzi, afflicting 6-7 million people worldwide, approximately 30% of whom develop 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 through inducing inflammation, heart fibrosis and remodelling, and affecting gap junction modulation.

Inhibition of TGF-β is a therapeutic target in Chagas disease

• Currently, only two drugs are used against CD, but the long treatment course triggers serious side effects, often resulting in treatment suspension: additionally, whilst they kill the parasites, they do not reverse the fibrosis.

• A recent paper showed that pirfenidone, which inhibits TGF-β, was effective in preventing the increase in collagen type I triggered by T. cruzi infection, both at the gene and protein expression levels (several drugs were trialled).

TGF-β signalling in health: tissue & immune homeostasis

• TGF-β is indispensable for tissue homeostasis as it generally suppresses cell proliferation and induces cell apoptosis through various mechanisms: the whole tissue, more than any of the constituent cell types, is the target of TGF-β, with the ultimate goal of preserving 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: inflammation and cancer

• Dysfunctional TGF-β signalling exacerbates tissue injuries in inflammatory diseases 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, as well as tumour microenvironment (TME) remodelling.

TGF-β mutations and cancer signalling

• Aberrations in signaling 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: this enables carcinoma progression and turns TGF-β into a tumour-promoting agonist as the disease progresses.

• 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 the 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 is anti-apoptotic: hyperactivation will inhibit apoptosis

• Bax is pro-apoptotic: upregulation will stimulate apoptosis

• Lowered Fas death receptor expression will inhibit apoptosis

• p53 triggers apoptosis in response to DNA damage: loss will inhibit apoptosis

• Hyperactivation of PKB will lead to phosphorylation and inhibition of the pro- apoptotic Bad protein: this will inhibit apoptosis

• Mst1 (Hippo) is pro-apoptotic: 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 in turn 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 not only modify the EMT status of carcinoma cells but also 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 exemplifies the roles of regulated/dysfunctional cell signalling

• The liver plays a crucial role in maintaining whole-body homeostasis in both health and disease, engaging in important communication with other organs, which requires the coordination of multiple signalling pathways.

• TGF-β and Hippo signalling pathways cooperate in the process of liver development, siz e and regeneration (the only organ that can substantially regenerate in adult humans) by regulating essential processes such as proliferation, differentiation and apoptosis.

• De-regulation of the HIPPO pathway, has been 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 eventually hepatocarcinogenesis.

• In liver cancer, the tumour microenvironment 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 development & disease

• 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.

• However, sustained activation of these pathways during disease promotes fibrosis, inhibits the immune system, and promotes EMT and invasion of cancerous cells.

Gene expression is regulated by the HIPPO/YAP/TAZ and TGF- β/SMAD crosstalk in the liver

• A major crosstalk between the HIPPO and TGF-β pathways occurs to control liver size and regeneration: both pathways inhibit cell proliferation and maintain hepatocyte homeostasis, acting primarily 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 under development 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.

• For the same purpose, 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.