ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis
Thomas Worzfeld, Jakub M. Swiercz, Mario Looso, Beate K. Straub, Kishor K. Sivaraj, Stefan Offermanns
Thomas Worzfeld, Jakub M. Swiercz, Mario Looso, Beate K. Straub, Kishor K. Sivaraj, Stefan Offermanns
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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.