Abstract
Invasive cancer cells develop small actin-based protrusions called invadopodia, which perform a primordial role in metastasis and extracellular matrix remodelling. Neural Wiskott-Aldrich syndrome protein (N-WASp) is a scaffold protein which can directly bind to actin monomers and Arp2/3 and is a crucial player in the formation of an invadopodium precursor. Expression modulation has pointed to an important role for N-WASp in invadopodium formation but the role of its C-terminal VCA domain in this process remains unknown. In this study, we generated alpaca nanobodies against the N-WASp VCA domain and investigated if these nanobodies affect invadopodium formation. By using this approach, we were able to study functions of a selected functional/structural N-WASp protein domain in living cells, without requiring overexpression, dominant negative mutants or siRNAs which target the gene, and hence the entire protein. When expressed as intrabodies, the VCA nanobodies significantly reduced invadopodium formation in both MDA-MB-231 breast cancer and HNSCC61 head and neck squamous cancer cells. Furthermore, expression of distinct VCA Nbs (VCA Nb7 and VCA Nb14) in PC-3 prostate cancer cells resulted in reduced overall matrix degradation without affecting MMP9 secretion/activation or MT1-MMP localisation at invadopodial membranes. From these results, we conclude that we have generated nanobodies targeting N-WASp which reduce invadopodium formation and functioning, most likely via regulation of N-WASp—Arp2/3 complex interaction, indicating that this region of N-WASp plays an important role in these processes.
Highlights
Metastasis is the primary cause of cancer associated deaths
Prostate and head and neck cancer cell lines that inducibly express these VCA nanobodies, we show that this Neural WiskottAldrich syndrome protein (N-WASp) region contributes to invadopodium formation and extracellular matrix remodelling, suggesting that it is involved in invadopodium based spreading of cancer cells
MDA-MB-231 breast cancer cell lysate was used as a source of endogenous N-WASp
Summary
Metastasis is the primary cause of cancer associated deaths. In this process cancer cells leave a primary tumour to disseminate through the entire body [1]. In doing so, these cancer cells are able to create secondary tumours throughout the body, a lethal process for the cancer patient in almost all cases [1]. In order to leave the primary tumour, cancer cells create small actinbased protrusions to facilitate their spreading [2].
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