MET exon 14 skipping mutation is a hepatocyte growth factor (HGF)-dependent oncogenic driver in vitro and in humanised HGF knock-in mice.

Exon skipping mutations of the MET receptor tyrosine kinase (METex14), increasingly reported in cancers, occur in 3-4% of non-small-cell lung cancer (NSCLC). Only 50% of patients have a beneficial response to treatment with MET-tyrosine kinase inhibitors (TKIs), underlying the need to understand the mechanism of METex14 oncogenicity and sensitivity to TKIs. Whether METex14 is a driver mutation and whether it requires hepatocyte growth factor (HGF) for its oncogenicity in a range of in vitro functions and in vivo has not been fully elucidated from previous preclinical models. Using CRISPR/Cas9, we developed a METex14/WT isogenic model in nontransformed human lung cells and report that the METex14 single alteration was sufficient to drive MET-dependent in vitro anchorage-independent survival and motility and in vivo tumorigenesis, sensitising tumours to MET-TKIs. However, we also show that human HGF (hHGF) is required, as demonstrated in vivo using a humanised HGF knock-in strain of mice and further detected in tumour cells of METex14 NSCLC patient samples. Our results also suggest that METex14 oncogenicity is not a consequence of an escape from degradation in our cell model. Thus, we developed a valuable model for preclinical studies and present results that have potential clinical implication.

Quantitative Analysis of Integrin Trafficking.

The last two decades of research into integrin trafficking has revealed fascinating insight into the function of integrin receptors, particularly in the context of cell invasion and migration in cancer. Deregulation in the trafficking pathways of integrin receptors contributes to a variety of pathological conditions including cancer, and in fact, altered endocytic trafficking of these receptors has been shown to drive transformation and tumor progression. Being able to experimentally measure integrin internalization, recycling and cell surface levels are vital for determining the role integrins play in health and disease. Surface-labeling based endocytic trafficking assays provide a way to experimentally measure changes in the rate of internalization of cell surface proteins, and the recycling of internalized proteins back to the cell surface, with high accuracy. This chapter will focus on quantitative approaches based on cell surface biotinylation and capture ELISA to measure endocytosis, recycling, and cell surface levels of integrin receptors.