Tumor development is characterized by uncontrolled cell growth due to genetic and environmental factors. Cancer mortality is mainly caused by the spread of cancer cells from the primary tumor to other parts of the body. Cancer cells break away from the original tumor, invade the surrounding tissue, enter blood or lymph vessels, and extravasate at distant sites to form secondary tumors, or metastases. During the different stages of metastasis, the actin cytoskeleton is hijacked by cancer cells to increase their motility. Different types of actin-mediated cellular protrusions assist in cancer cell motility including lamellipodia and filopodia at the leading edge, and proteolytic active invadopodia at the ventral side of invading cells. In physiological conditions, the actin cytoskeleton is essential for the maintenance of cell shape, response to environmental changes and protrusive force during cell movement. The actin binding, multidomain protein cortactin is one of the many regulators of the shape and dynamics of actin filaments: cortactin binds to and activates the Arp2/3 complex, leading to the formation of a branched actin network, and stabilizes actin filaments. The cortactin gene is overexpressed in different cancer types, and is associated with increased invadopodia formation, cell motility, invasion and metastasis formation.
The objective of this thesis is to delineate the role of different cortactin domains during invadopodia formation and function. Three different cortactin nanobody sets are described, each targeting a different cortactin region: two NTA nanobodies against the N-terminal acidic (NTA) domain, two FHP nanobodies targeting the central regions (the F-actin binding repeats, helical and proline rich domain), and one SH3 nanobody against the SH3 domain. The NTA and FHP nanobody sets were generated and characterized, and their effect on invadopodia and invasion was compared to the previously characterized SH3 nanobody. Additionally, the interaction of the SH3 nanobody with the SH3 domain was studied in more detail.
In the first section, two nanobodies against the NTA domain are characterized and compared with a nanobody that targets the SH3 domain. The NTA nanobodies both prevent cortactin-mediated Arp2/3 activation (Table 1). The two nanobody sets, i.e. the NTA and SH3 nanobodies, similarly decrease invadopodia formation, protease secretion, matrix degradation and protease-dependent invasion, illustrated by protease inhibitor GM6001. One NTA nanobody also inhibits the cortactin-Arp2/3 complex interaction and reduces invadopodium stability. Interestingly, inhibition of Arp2/3 complex activation, either by cortactin NTA nanobodies or by Arp2/3 inhibitor CK-666, causes a larger decrease in invadopodia formation in head and neck squamous cell carcinoma versus breast cancer cells. This suggests that invadopodia formation and composition is a cell type-specific process.
In the second section, the central regions of cortactin are targeted by two FHP nanobodies and their effects are again compared with the SH3 nanobody. Both cortactin nanobody sets equally decrease invadopodia formation, invadopodia-mediated matrix degradation and invasion (Table 1). One FHP nanobody binds to the proline rich domain and decreases cortactin tyrosine phosphorylation; the other FHP nanobody binds the actin binding repeats but has no effect on F-actin interaction. This indicates that the cortactin actin binding repeats also have a role in invadopodia formation and function that is independent from their ability to bind and stabilize actin filaments.
Finally, we present a structural analysis of the interaction between the SH3 nanobody and SH3 domain by means of X-ray crystallography. The first co-crystal structure was obtained at acidic pH, therefore a second structure was determined at physiological pH. However, the degree of acidity has no major influence on the thermodynamic parameters or structure of the cortactin-nanobody interaction. The SH3 nanobody/domain crystal structure explains two observations: first, the specificity of the SH3 nanobody for cortactin and the lack of cross reactivity with cortactin homologue HS1; and second, the inhibition of cortactin-WIP interaction by the SH3 nanobody without affecting other SH3 domain interaction partners.
Overall, nanobodies are an excellent tool to scrutinize cortactin domain specific functions in invadopodia and invasion. Targeting of distinct cortactin domains and their functions causes different effects on invadopodia number, stability or function, but ultimately results in a similar decrease of cancer cell invasion.