Extracellular trafficking of Wnt signals in gastric cancer

Wnt proteins are secreted glycoproteins which signal in a tissue to regulate multiple cellular processes, such as cell differentiation, migration, and proliferation. However, post-translational modifications result in Wnt ligands being hydrophobic in nature. Thus, their ability to freely diffuse in the aqueous extracellular environment is restricted, and alternative mechanisms of transport have been proposed. In this thesis, I investigate and characterise the use of signalling filopodia – termed cytonemes – in the intercellular transport of Wnt3 ligands by gastric cancer cells, which display overactivated Wnt/β-catenin signalling. Additionally, I identify the membrane scaffolding protein Flotillin-2 (Flot2), which is overexpressed in gastric cancers, as a novel positive regulator of Wnt cytoneme formation and consequently proliferation. Mechanistically, I show that Flot2 is required for the intracellular transport, membrane localisation and thus signalling of the Wnt co-receptor Ror2; a known regulator of Wnt cytonemes. In parallel, I show that Flot2 also has a function in transducing signals in the Wnt- receiving cell. Here, Flot2 co-localises with the Wnt co-receptor Lrp6 and is involved in its endocytic uptake. Additionally, Flot2 knockdown results in the perinuclear accumulation of Lrp6 and its absence from recycling endosomes. Therefore, I suggest Flot2 may also be involved in the endosomal transport of Lrp6 following internalisation. Finally, following my observed co-localisations of both Ror2 and Lrp6 with Flot2, I found that these Wnt co-receptors co-localise with one another, as well as the cognate Wnt receptor Frizzled 7, in Flot2 microdomains. Expression of a mutant Ror2 missing its cysteine-rich domain, however, causes loss of co-localisation with Lrp6 and perturbed Wnt/β-catenin signalling. Together, these findings led me to propose a model whereby Frizzled 7, Ror2 and Lrp6 all interact and form one large complex, which I have termed the Wnt Receptor Supercomplex (WRS). I hypothesise that these receptors may interact, even in the absence of Wnt ligands, to regulate one another’s binding affinities for either Wnt/β-catenin or Wnt/PCP ligands. Here, I propose that flotillin microdomains provide the scaffold necessary for these interactions