VCA nanobodies target N-WASp to reduce invadopodium formation and functioning

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.

Fig 1. VCA nanobody—N-WASp binding.

Pull down assay of endogenous N-WASp from MDA-MB-231 breast cancer cell lysate with HA-tagged VCA Nb (1, 2, 5, 6, 7 and 12, 13, 14, 15, 16) by means of anti-HA agarose beads. Two negative controls were included, anti-HA agarose beads were incubated with no nanobody (C1) or with a cortactin NTA nanobody against the N-terminal acidic (NTA) region of cortactin (C2). N-WASp was blotted and detected by anti-N-WASp antibody. To see the pull down of the 4 selected VCA Nbs using HNSCC61 cancer cells, which express EGFP-tagged VCA Nbs, see S1 Fig.

Fig 2. VCA Nb2, 7, 13 and 14 capture endogenous N-WASp at mitochondria.

Representative epifluorescence images of MDA-MB-231 breast cancer cells transiently expressing VCA Nbs equipped with a MOM-tag. MOM-tagged EGFP nanobody was used as a negative control (upper panel). Nuclei were visualized with DAPI (blue), nanobodies with anti-V5 antibody (green) and N-WASp with anti-N-WASp antibody (red). Right: the intensity profiles for N-WASp (red) and MOM-tagged nanobodies (green) are very similar, indicative of co-localisation, except for the EGFP nanobody (negative control). (Scale bar = 10 μm).

Fig 3. VCA Nb effect on actin or Arp2/3 binding to N-WASp using a nanobody concentration range.

Pull down was performed by using biotin-tagged VCA peptide, MDA-MB-231 breast cancer cell lysate, recombinant VCA Nbs and STREPTactin beads. The two controls (no nanobody (No Nb) or EGFP Nb) show maximal Arp2/3 binding. A concentration range was used (VCA Nb: VCA domain stoichiometry of 0.5x, 1x, 2x, 4x). For each repeat, actin as well as Arp2/3 were analysed on western blot and quantification was done using ImageJ (bars represent mean and SEM, n = 3). Kruskal-Wallis and Duns post test was performed. On top the influence of each VCA Nb on actin—N-WASp binding (A-D) is shown and at the bottom the effect of each VCA Nb on Arp2/3 –N-WASp binding (E-H) is shown.

Fig 4. N-WASp colocalizes with invadopodia markers.

Representative epifluorescence images of HNSCC61 head and neck squamous cancer cells. Nuclei were visualized with DAPI (blue) and N-WASp (green) with anti-N-WASp antibody. Invadopodia were visualized by using an invadopodium marker; (A) actin (red) with Alexa Fluor labelled phalloidin and (B) cortactin (red) with anti-cortactin antibody. (Scale bar = 10 μm).

Fig 5. Expression of VCA Nbs in cancer cells reduces invadopodia number.

Representative epifluorescence images of HNSCC61 head and neck squamous cancer cells which express EGFP-tagged VCA Nbs (green) (A). Nuclei were visualized with DAPI (blue) and actin (red) with phalloidin. Bar plots of MDA-MB-231 breast (B) and HNSCC61 head and neck squamous (C) cancer cells, which inducible express EGFP- tagged VCA Nbs, were used to count invadopodia per cell. Quantification was done using ImageJ and Kruskal-Wallis and Dunns post tests were performed. As negative control EGFP-only expressing stable cells were used. (* p < 5%, ** p < 1%, *** p < 0.01%).

Fig 6. Effects of VCA intrabodies on matrix degradation.

(A) Representative epifluorescence images of PC-3 prostate cancer cells, which inducibly express EGFP-tagged VCA Nbs, were seeded on Cy3-gelatin matrix (scale bar = 10 μm). As negative control EGFP-only expressing stable cells were used. Quantification was done using ImageJ and Kruskal-Wallis test and Dunns post tests were performed. Degrading capacity of PC-3 cells was analyzed by 3 parameters. For the first parameter ‘degradation index’ (B) and the second parameter ‘degradation area per cell’ (C), a comparison between ‘not induced’ and ‘doxycycline induced’ was made for each stable cell line. The third parameter ‘degradation area per total cell area’ (D) was compared to the EGFP-only cell line. (ns = not significant, * p < 5%, ** p < 1%, *** p < 0.01%).