Growth Factor Signalling

TGF-β is a pleiotropic cytokine that can act as both a tumour suppressor and a tumour promoter in a context-dependent fashion. TGF-β can function as a potent promoter of invasion and metastasis by acting on the tumour cells themselves, the surrounding stroma, and by functioning as an immunosuppressor. We are studying the spatial, temporal and tissue specific regulation of TGF-β signal transduction pathways in the normal and diseased state with a view to understanding how TGF-β acts as an immunosuppressor and a tumour promoter. Our ultimate goal is to identify and develop specific molecular targets for pharmaceutical intervention in cancer therapy.

TGF-β tumour suppressor/tumour promoter
TGF-β is a peptide growth factor that regulates many fundamental biological processes both in the developing embryo and the adult. These processes include specification of cell fate, movement, proliferation and death. Under normal conditions the tumour suppressor activities of TGF-β predominate by restricting the proliferative capacity of the epithelial cell compartment through induction of growth arrest and apoptosis. As tumours develop to invasive metastatic cancers many epigenetic and genetic changes take place. During this process the tumour cells lose their anti-proliferative response to TGF-β and produce active TGF-β (Figure 1). This autocrine production of TGF-β potently promotes cell motility, metastasis and cell survival. The acquisition of the mesenchymal phenotype may form the basis for progression of cancers towards a more invasive and malignant state and TGF-β has been shown to be a potent inducer of the epithelial to mesenchymal transition (EMT). Tumour produced TGF-β can also act in a paracrine pro-oncogenic fashion by switching off the host immune response through inducing growth arrest and apoptosis in B and T lymphocytes and by promoting angiogenesis.

To simply block all TGF-β signalling would also block the tumour suppressive and other normal roles of TGF-β. Ultimately we aim to design targeted therapeutics that block the tumour promoting aspects of TGF-β signalling selectively during pathogenesis whilst maintaining normal functions. This goal requires a detailed understanding of TGF-β signalling and biological responses in normal and tumour tissues.

TGF-β signalling
TGF-β signalling, transduced from the plasma membrane to the nucleus, ultimately results in an alteration of the gene expression programme with each step in this process acting as a point of control and a potential target for therapeutic intervention. TGF-β signals via transmembrane serine/threonine kinase receptors. Several pathways are known to operate downstream of TGF-β receptors, but by far the best characterised is the Smad pathway (Figure 2). These proteins are direct substrates of the TGF-β receptors and act as intracellular transducers and sensors of the TGF-β signal by shuttling between the cytoplasm and the nucleus (Inman et al., Mol Cell 2002; 10:283). Smad proteins, once in the nucleus, regulate transcription of target genes in concert with other sequence specific transcription factors (Randall et al., EMBO J 2002, 21 (1-2): 145; Howell et al., Development 2002, 129:2823; Inman and Hill, J Biol Chem 2002, 277: 51008.). The switch of tumour cells' biological response to TGF-β is likely to involve modulation of the Smad pathway and so we are employing protein purification techniques to study the regulation of Smad transcription factor complexes.

Fig. 2: Schematic representation of TGF-β signalling. Following activation TGF-β induces the formation of a heteromeric complex of TGF-β receptors. The type II receptor activates the type I receptor which in turn phosphorylates Smads 2 and 3. These form heteromeric complexes with Smad 4 and translocate to the nucleus where in conjunction with transcription factors (TFs) and co-activators or co-repressors they modulate target gene expression. The molecular mechanisms of how TGF-β regulates other signalling pathways remain to be determined.

TGF-β also activates the ERK, JNK, p38MAPK, RhoA and PI3K signalling pathways in a tissue specific fashion and these may be important in TGF-β mediated tumour promotion. The precise mechanism of activation of these pathways remains unclear. Signal propagation from the TGF-β receptors must involve physical interaction with target molecules and we are identifying TGF-β receptor substrates and regulators utilising modified yeast two-hybrid and proteomic approaches.

TGF-β mediated immunoregulation
Knockout studies demonstrate that TGF-β signalling plays a critical role in the stringent control of both B and T cell activation and development. Little however is known about how TGF-β regulates the growth of lymphocytes. Normal B cell development and activation takes place in a highly temporally and spatially regulated fashion and TGF-β may play a crucial role in these processes. In collaboration with Dr Louise Clark (Southern General Hospital) we are determining the biological response of B cell subsets isolated from human tonsils to exogenous and endogenous TGF-β . Using the recently characterised TGF-β receptor inhibitor (Inman et al., Mol Pharmacol 2002; 62(1):65) we are determining the duration of TGF-β signalling required to induce and maintain cytostasis in B cells. We are examining the effects of TGF-β treatment on the activity of key cell cycle regulatory molecules and we will seek new genes involved in this process using microarray analysis.

We have previously shown that many Burkitt's lymphoma (BL) cell lines are exquisitely sensitive to TGF-β induced apoptosis (Inman and Allday, J Immunol 2000; 165(5):2500). In parallel with the experiments described above, we will determine if different B cell subsets apoptose in response to endogenous and exogenous TGF-β. We will perform kinetic analysis of the apoptosis cascade in BL cells following TGF-β addition. Having established the duration of TGF-β signal required to induce apoptosis novel target genes involved in this process will be identified using microarray analysis. Future work will then focus on the regulation, roles and mechanisms of action of these genes and their products.

Fig. 1: Diagram illustrating the tumour suppressor and tumour promoter activities of TGF-β.

Towards targeted therapy: Switching the TGF-β response
The response of tumour cells to TGF-β is modulated relative to the response of the normal cellular counterpart. These changes can take place at the epigenetic and genetic level (Figure 1). In collaboration with Dr Tim Crook (Ludwig Institute, London) we are screening for changes in genes that modulate TGF-β signalling during tumourigenesis and assessing their effects on TGF-β-mediated biological responses.

TGF-β has been shown to be a potent inducer of EMT but it is unclear how TGF-β signalling contributes to the mesenchymal state. We are developing a system designed to characterise which molecules and signalling pathways are required to maintain the mesenchymal phenotype. It is hoped that these studies will lead to the identification of target molecules for pharmaceutical intervention with the aim of reverting the invasive and metatstatic phenotype of some tumours.