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

Tim Hebbrecht (UGent) , Isabel Van Audenhove (UGent) , Olivier Zwaenepoel (UGent) , Adriaan Verhelle (UGent) and Jan Gettemans (UGent)
(2017) PLOS ONE. 12(9).
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Abstract
Invasive cancer cells develop small actin-based protrusions called invadopodia, which perform a primordial role in metastasis and extracellular matrix remodelling. Neural WiskottAldrich 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 D Arp2/3 complex interaction, indicating that this region of NWASp plays an important role in these processes.
Keywords
ALDRICH-SYNDROME PROTEIN, CANCER-CELL INVASION, ARP2/3 COMPLEX, ACTIN POLYMERIZATION, FAMILY PROTEINS, WASP/WAVE PROTEINS, TUMOR CELLS, CORTACTIN, DOMAIN, ACTIVATION

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Citation

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Chicago
Hebbrecht, Tim, Isabel Van Audenhove, Olivier Zwaenepoel, Adriaan Verhelle, and Jan Gettemans. 2017. “VCA Nanobodies Target N-WASp to Reduce Invadopodium Formation and Functioning.” Plos One 12 (9).
APA
Hebbrecht, T., Van Audenhove, I., Zwaenepoel, O., Verhelle, A., & Gettemans, J. (2017). VCA nanobodies target N-WASp to reduce invadopodium formation and functioning. PLOS ONE, 12(9).
Vancouver
1.
Hebbrecht T, Van Audenhove I, Zwaenepoel O, Verhelle A, Gettemans J. VCA nanobodies target N-WASp to reduce invadopodium formation and functioning. PLOS ONE. 2017;12(9).
MLA
Hebbrecht, Tim, Isabel Van Audenhove, Olivier Zwaenepoel, et al. “VCA Nanobodies Target N-WASp to Reduce Invadopodium Formation and Functioning.” PLOS ONE 12.9 (2017): n. pag. Print.
@article{8547145,
  abstract     = {Invasive cancer cells develop small actin-based protrusions called invadopodia, which perform a primordial role in metastasis and extracellular matrix remodelling. Neural WiskottAldrich 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 D Arp2/3 complex interaction, indicating that this region of NWASp plays an important role in these processes.},
  articleno    = {0185076},
  author       = {Hebbrecht, Tim and Van Audenhove, Isabel and Zwaenepoel, Olivier and Verhelle, Adriaan and Gettemans, Jan},
  issn         = {1932-6203},
  journal      = {PLOS ONE},
  language     = {eng},
  number       = {9},
  pages        = {19},
  title        = {VCA nanobodies target N-WASp to reduce invadopodium formation and functioning},
  url          = {http://dx.doi.org/10.1371/journal.pone.0185076},
  volume       = {12},
  year         = {2017},
}

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