Sema3E–Plexin D1 signaling drives human cancer cell invasiveness and metastatic spreading in mice
Andrea Casazza, Veronica Finisguerra, Lorena Capparuccia, Andrea Camperi, Jakub M. Swiercz, Sabrina Rizzolio, Charlotte Rolny, Claus Christensen, Andrea Bertotti, Ivana Sarotto, Mauro Risio, Livio Trusolino, Jurgen Weitz, Martin Schneider, Massimilano Mazzone, Paolo M. Comoglio, Luca Tamagnone
Andrea Casazza, Veronica Finisguerra, Lorena Capparuccia, Andrea Camperi, Jakub M. Swiercz, Sabrina Rizzolio, Charlotte Rolny, Claus Christensen, Andrea Bertotti, Ivana Sarotto, Mauro Risio, Livio Trusolino, Jurgen Weitz, Martin Schneider, Massimilano Mazzone, Paolo M. Comoglio, Luca Tamagnone
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Research Article

Sema3E–Plexin D1 signaling drives human cancer cell invasiveness and metastatic spreading in mice

Abstract

Semaphorin 3E (Sema3E) is a secreted molecule implicated in axonal path finding and inhibition of developmental and postischemic angiogenesis. Sema3E is also highly expressed in metastatic cancer cells, but its mechanistic role in tumor progression was not understood. Here we show that expression of Sema3E and its receptor Plexin D1 correlates with the metastatic progression of human tumors. Consistent with the clinical data, knocking down endogenous expression of either Sema3E or Plexin D1 in human metastatic carcinoma cells hampered their metastatic potential when injected into mice, while tumor growth was not markedly affected. Conversely, overexpression of exogenous Sema3E in cancer cells increased their invasiveness, transendothelial migration, and metastatic spreading, although it inhibited tumor vessel formation, resulting in reduced tumor growth in mice. The proinvasive and metastatic activity of Sema3E in tumor cells was dependent on transactivation of the Plexin D1–associated ErbB2/Neu oncogenic kinase. In sum, Sema3E–Plexin D1 signaling in cancer cells is crucially implicated in their metastatic behavior and may therefore be a promising target for strategies aimed at blocking tumor metastasis.

Authors

Andrea Casazza, Veronica Finisguerra, Lorena Capparuccia, Andrea Camperi, Jakub M. Swiercz, Sabrina Rizzolio, Charlotte Rolny, Claus Christensen, Andrea Bertotti, Ivana Sarotto, Mauro Risio, Livio Trusolino, Jurgen Weitz, Martin Schneider, Massimilano Mazzone, Paolo M. Comoglio, Luca Tamagnone

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Figure 5

Molecular mechanisms of Sema3E-dependent endothelial cell repulsion.

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Molecular mechanisms of Sema3E-dependent endothelial cell repulsion. (A)...
(A) The haptotactic migration of HUVECs was analyzed in the presence of 7 nM purified Sema3E (or equal concentration of Sema3A, as positive control) in the lower chamber. Migrated cells were stained with crystal violet and quantified by absorbance at 595 nm (see Methods for details). The graphs show the average ± SD of at least 2 independent experiments throughout the figure. **P < 0.005. See also Supplemental Figure 6, A and B. (B) Plexin D1 expression was knocked down in HUVECs (verified by Q-PCR), and the haptotactic migration of these cells was assayed in Transwell inserts. Both processable wild-type Sema3E (yielding a p87/p61 mix) and the recombinant p61-Sema3E fragment (7 nM each) inhibited the migration of controls but not of Plexin D1–deficient cells. Migrated cells were quantified by staining with crystal violet and measuring absorbance at 595 nm (see Methods). Data are given as average ± SD of 2 different experiments. **P < 0.005. (C) HUVEC migration assay as above, upon expression of dominant-activated R-Ras-L61 mutant (verified by Western blotting). *P < 0.02. (D) Rnd2 expression was knocked down in HUVECs by siRNA transfection (verified by Q-PCR), and cell migration was assayed as above. **P < 0.005. (E) Rnd2-depleted HUVECs (same as above) were incubated with 7 nM p61-Sema3E for 4 hours, then fixed and stained with FITC-Phalloidin. The percentage of collapsed cells (defined as having a diameter shorter than 30 μm) is indicated on the graph. Data are representative of at least 3 experiments, displaying consistent results. **P < 0.005.