ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis
Thomas Worzfeld, … , Kishor K. Sivaraj, Stefan Offermanns
Thomas Worzfeld, … , Kishor K. Sivaraj, Stefan Offermanns
Published March 1, 2012
Citation Information: J Clin Invest. 2012;122(4):1296-1305. https://doi.org/10.1172/JCI60568.
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Research Article Oncology

ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis

Abstract

Diagnosis of metastatic breast cancer is associated with a very poor prognosis. New therapeutic targets are urgently needed, but their development is hampered by a lack of understanding of the mechanisms leading to tumor metastasis. Exemplifying this is the fact that the approximately 30% of all breast cancers overexpressing the receptor tyrosine kinase ErbB-2 are characterized by high metastatic potential and poor prognosis, but the signaling events downstream of ErbB-2 that drive cancer cell invasion and metastasis remain incompletely understood. Here we show that overexpression of ErbB-2 in human breast cancer cell lines leads to phosphorylation and activation of the semaphorin receptor Plexin-B1. This was required for ErbB-2–dependent activation of the pro-metastatic small GTPases RhoA and RhoC and promoted invasive behavior of human breast cancer cells. In a mouse model of ErbB-2–overexpressing breast cancer, ablation of the gene encoding Plexin-B1 strongly reduced the occurrence of metastases. Moreover, in human patients with ErbB-2–overexpressing breast cancer, low levels of Plexin-B1 expression correlated with good prognosis. Our data suggest that Plexin-B1 represents a new candidate therapeutic target for treating patients with ErbB-2–positive breast cancer.

Authors

Thomas Worzfeld, Jakub M. Swiercz, Mario Looso, Beate K. Straub, Kishor K. Sivaraj, Stefan Offermanns

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Figure 2

Plexin-B1 promotes invasion of ErbB-2–overexpressing human breast cancer cells.

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Plexin-B1 promotes invasion of ErbB-2–overexpressing human breast cancer...
(A) Human breast cancer cell lines MCF-7, T-47D, SK-BR-3, or BT-474 or (B) BT-474 cells transfected with control siRNA or siRNA against ErbB-2 were lysed; Plexin-B1 was immunoprecipitated; and precipitates were immunoblotted using anti-phosphotyrosine or anti–Plexin-B1 antibodies. In a parallel experiment, levels of active RhoA/RhoC were determined. (C–F) BT-474 cells were transfected with control or Plexin-B1 siRNA. (C) The amount of Plexin-B1 and active RhoA/RhoC was determined. (D) Cell lysates were probed with an anti–phospho–ErbB-2(Y1248) antibody. (E) BT-474 cells were counted on 5 consecutive days. (F) Cells were seeded onto Matrigel-coated filters, and invading cells were counted as described in Methods. (G and H) BT-474 cells stably expressing siRNA-insensitive wild-type Plexin-B1 or siRNA-insensitive mutant Plexin-B1(Y1708F/Y1732F) were transfected with Plexin-B1 siRNA to knock down endogenous Plexin-B1. (G) Plexin-B1 was immunoprecipitated, and precipitates were immunoblotted using anti–Plexin-B1 and anti–phosphotyrosine antibodies. In addition, levels of active RhoA/RhoC were determined. (H) In parallel, cells were seeded onto Matrigel-coated filters, and invading cells were counted. (I and J) BT-474 cells were incubated (I) without or with a mouse monoclonal anti–Plexin-B1 antibody (anti-PlxB1; clone #93, 1.8 ng/μl) or (J) without or with 150 nM PlxB1ext, and the amounts of active RhoA/RhoC were determined. (K and L) BT-474 cells were seeded onto Matrigel-coated filters in (K) the absence or presence of a mouse monoclonal anti–Plexin-B1 antibody (anti-PlxB1; clone #93, 1.8 ng/μl) or (L) the presence of 150 nM PlxB1ext, 2 μg/ml trastuzumab, or both, and invading cells were counted. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.