SMAD4 is a tumour suppressor gene that is mutated in up to 60% of pancreatic ductal adenocarcinoma (PDAC) tumours. Activins are members of the TGF-b signalling superfamily which signal through SMAD4 and are generally associated with an oncogenic process, with differing effects on tumour and stromal cells, such as cancer associated fibroblasts (CAFs). We sought to further elucidate the role of activin within PDAC and perturb activin signalling in vitro and in vivo. Analysis was performed of publicly available human datasets alongside human and murine single-cell RNA-sequencing datasets to study the effect of activin expression on survival and tumourigenesis. We developed anti-activin monoclonal antibodies to perturb activin signalling in PDAC mice, with deep analysis of tumour composition including immune cells and CAFs via a multitude of basic science techniques. We demonstrated that circulating activin levels are higher in PDAC patients than in healthy volunteers, whilst activin overexpression is associated with reduced overall survival in PDAC patients. Activin was shown to be predominantly expressed by contractile (collagen-depositing) CAFs, as opposed to inflammatory CAFs. Activin expression co-localises with fibroblasts, and anti-activin antibodies can reduce the collagen content of dense PDAC tumours. Additionally, anti-activin antibodies led to a statistically significant increase in CD4 and CD8 T-cell infiltration into murine PDAC tumours. We have shown that activin is overexpressed in PDAC, and high expression is associated with a more aggressive disease process. Anti-activin antibodies can alter the composition of desmoplastic PDAC tumours and increase the immune infiltrate of these typically “immune cold” tumours.

Deregulation of TGF‐β family signalling underlies some serious human diseases, including cancer, the Marfan syndromes, and other connective tissue disorders. Recent work in my lab has focused on working out how mutations in key components of TGF‐β family signalling pathways cause these diseases.

Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are debilitating diseases that share causal mutations in ACVR1, a TGF‐β family type I receptor. ACVR1R206H is a frequent mutation in both diseases. Pathogenic signaling via the SMAD1/5 pathway is mediated by Activin A, but how the mutation triggers aberrant signaling is not known. We show that ACVR1 is essential for Activin A‐mediated SMAD1/5 phosphorylation and is activated by two distinct mechanisms. Wild‐type ACVR1 is activated by the Activin type I receptors, ACVR1B/C. In contrast, ACVR1R206H activation does not require upstream kinases, but is predominantly activated via Activin A‐dependent receptor clustering, which induces its auto‐activation. We use optogenetics and live‐imaging approaches to demonstrate Activin A‐induced receptor clustering and show it requires the type II receptors ACVR2A/B. Our data provide molecular mechanistic insight into the pathogenesis of FOP and DIPG by linking the causal activating genetic mutation to disrupted signaling.

Introduction: Lung cancer is the result of a multistep accumulation of genetic and/or epigenetic alterations; therefore, a better understanding of the molecular mechanism by which these alterations ...

Hyaluronan is a ubiquitous glycosaminoglycan which is an important constituent of the extracellular matrix (ECM). In addition to organizing the extracellular matrix and regulating tissue homeostasi ...

Background: The hyaluronan receptor CD44 interacts with the PDGF β-receptor and the TGFβ type I receptor. Results: CD44, PDGF β-receptor and TGFβ type I receptor affect each other's signaling, stability and function. Conclusion: Cross-talk between PDGF β-receptor and TGFβ type I receptor occurs in human dermal fibroblasts. Significance: This study reveals novel modulatory mechanisms of PDGF and TGFβ signaling. Growth factors, such as platelet-derived growth factor BB (PDGF-BB) and transforming growth factor β (TGFβ), are key regulators of cellular functions, including proliferation, migration, and differentiation. Growth factor signaling is modulated by context-dependent cross-talk between different signaling pathways. We demonstrate in this study that PDGF-BB induces phosphorylation of Smad2, a downstream mediator of the canonical TGFβ pathway, in primary dermal fibroblasts. The PDGF-BB-mediated Smad2 phosphorylation was dependent on the kinase activities of both TGFβ type I receptor (TβRI) and PDGF β-receptor (PDGFRβ), and it was prevented by inhibitory antibodies against TGFβ. Inhibition of the activity of the TβRI kinase greatly reduced the PDGF-BB-dependent migration in dermal fibroblasts. Moreover, we demonstrate that the receptors for PDGF-BB and TGFβ interact physically in primary dermal fibroblasts and that stimulation with PDGF-BB induces internalization not only of PDGFRβ but also of TβRI. In addition, silencing of PDGFRβ by siRNA decreased the stability of TβRI and delayed TGFβ-induced signaling. We further show that the hyaluronan receptor CD44 interacts with both PDGFRβ and TβRI. Depletion of CD44 by siRNA increased signaling via PDGFRβ and TβRI by stabilizing the receptor proteins. Our data suggest that cross-talk between PDGFRβ and TβRI occurs in dermal fibroblasts and that CD44 negatively modulates signaling via these receptors.

In order for solid tumors to metastasize, tumor cells must acquire the ability to invade the surrounding tissue and intravasate into blood- or lymph vessels, survive in the circulation and then extravasate at a distant site to form a new tumor. Overexpression of the glycosaminoglycan hyaluronan, and its adhesion receptor CD44, correlate with breast cancer progression. This thesis focuses on the role of hyaluronan in tumor invasion and metastasis.In paper I, we demonstrated that upregulation of the hyaluronan synthesizing enzyme hyaluronan synthase 2 (HAS2) was crucial for transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells. In paper II, we further demonstrated that silencing of HAS2 decreased the invasive behavior of bone-metastasizing breast cancer cells, via upregulation of tissue inhibitor for metalloproteinase 1 (TIMP1), and dephosphorylation of focal adhesion kinase (FAK).During tumorigenesis, stromal cells, such as fibroblasts, play important roles and several growth factors are synthesized, promoting crosstalk between different cell surface receptors. In paper III, we investigated the crosstalk between the hyaluronan receptor CD44 and the receptors for TGFβ and platelet-derived growth factor BB (PDGF-BB) in dermal fibroblasts. We found that the receptors for the three molecules form a ternary complex, and that PDGF-BB can activate the Smad pathway downstream of TGFβRI. Importantly, CD44 negatively modulated the signaling of both PDGF-BB and TGFβ.In paper IV, we studied the process by which breast cancer cells invade blood-vessels and the role of hyaluronan and CD44 in angiogenesis. Importantly, CD44, or the hyaluronan degrading enzyme hyaluronidase 2 (HYAL2), decreased the capacity of endothelial cells to form tubes in a 3D in vivo-like assay. Collectively, our studies add to the understanding of the role of hyaluronan in tumor progression.