ErbB-2 overexpression leads Plexin-B1 to act downstream and activate prometastatic small GTPases.
Scientists have identified the semaphorin receptor Plexin-B1 as a potential therapeutic target in ErB-2-overexpressing breast cancers. A research team at the Max Planck Institute for Heart and Lung Research, and the Universities of Frankfurt, and Heidelberg in Germany, have found that overexpression of ErbB-2 in human breast cancer cells results in phosphorylation and activation of Plexin-B1, which is needed for ErbB-2-dependent activation of prometastatic small GTPases.
Their studies in a mouse model of ErbB-2-overexpressing breast cancer showed that knocking out the Plexin-B1 gene reduced the occurrence of lung metastases. In human patients with ErbB2-overexpressing breast cancer, low levels of Plexin-1 expression were associated with good prognosis. Lead authors Stefan Offermans, Ph.D., and Thomas Worzfeld, Ph.D., and colleagues report their findings in the Journal of Clinical Investigation, in a paper titled “ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis”.
The receptor tyrosine kinase ErbB-2 is overexpressed in about 30% of all breast cancers, and these are characterized by high metastatic potential and poor prognosis. However, the researchers point out, signaling pathways that mediate cancer cell invasion and metastasis in ErbB-2–overexpressing tumors aren’t yet well understood.
Meanwhile, recent studies have implicated the transmembrane receptor Plexin-B1 in cancer, and demonstrated its interaction with ErbB-2. Plexin-B1 binding to its ligand semaphorin 4D (Sema4D) stimulates ErbB-2 kinase activity, resulting in Plexin-B1 phosphorylation at two tyrosine residues. This phosphorylation allows Plexin-B1 to interact with proteins that trigger activation of GTPases that are already the subject of study in terms of invasion and metastasis of cancer cells, including breast cancer.
So, given that Plexin-B1 interacts with both ErbB-2 and the Rho-activating proteins, it may represent the link between ErbB-2 overexpression, the activation of Rho GTPases, and cancer cell invasiveness. To confirm whether Plexin-B1 acts downstream of ErbB-2 and mediates its oncogenic potential, the team carried out a series of studies to see whether ErbB-2 overexpression directly leads to activation of Plexin-B1, and whether the latter plays a role in the progression or even initiation of ErbB-2-positive breast cancer.
Initial in vitro studies confirmed that overexpression of constitutively active ErbB-2 in HEK293 cells was enough to lead to phosphorylation of Plexin-B1 and activation of RhoA and RhoC. Conversely, expression of a Plexin-B1 mutant lacking its intracellular domain blocked RhoA and RhoC activation, “indicating that Plexin-B1 signaling is indeed required for RhoA and RhoC activation downstream of ErbB-2,” the researchers note.
Closer analysis of a number of human breast cancer cell lines showed that basal Plexin-B1 phosphorylation and RhoA activity were only evident in cell lines overexpressing ErbB-2, supporting the notion that the pathway is active in breast cancer cells with high but not low levels of ErbB-2 expression. Indeed, knockdown of ErbB-2 in cells that would naturally express high levels of ErbB-2 led to a marked reduction of Plexin-B1 phosphorylation and RhoA/RhoC activity. “Thus, Plexin-B1 links ErbB-2 overexpression to the activation of RhoA and RhoC,” the investigators write. In fact, knockdown of Plexin-B1 didn’t impact on the proliferation of ErbB-2 overexpressing cancer cells, but it did reduce their migratory and invasive capacity, a finding which ties in with previous research associating RhoA and RhoC with increased cancer cell invasiveness, they add.
The crucial role of ErbB-2-mediated phosphorylation of Plexin-B1 for cancer cell invasiveness was demonstrated in cells that expressed a phosphorylation site-deficient Plexin-B1 mutant instead of endogenous Plexin-B1. These cells also had strongly reduced levels of active RhoA/RhoC and were markedly less capable of invasion than cells expressing wild-type Plexin-B1. Similarly, using Plexin-B1-targeting monoclonal antibodies or the purified Plexin-B1 extracellular domain (PlxB1ext) to block interaction with ErbB-2 in breast cancer cells strongly reduced phosphorylation of Plexin-B1, inhibited RhoA and RhoC activity, and decreased tumor cell invasiveness. This confirmed requirement for the ErbB-2/Plexin-B1 receptor complex in breast cancer cells, the authors state.
Interestingly, treating tumor cells with the anti-ErbB-2 antibody trastuzumab—which doesn’t impact on ErbB-2-Plexin-B1 interaction—inhibited tumor cell invasion to a similar degree as treating the cells with PlxB1ext. The inhibitory effects of PlxB1ext and trastuzumab on tumor cell invasion were additive. “These data indicate that the additional inhibition of Plexin-B1 signaling downstream of ErbB-2 is likely to increase the efficacy of conventional anti–ErbB-2 therapy,” the authors suggest.
To test the relevance of their findings in vivo, the investigators used a mouse model of metastatic breast cancer that overexpresses wild-type ErbB-2 in mammary glands, and expresses Plexin-B1 in primary and lung metastases. These mice were crossed with a viable, fertile Plexin-B1-deficient mouse model. The resulting Plexin-B1 deficient, ErbB-2-overexpressing animals demonstrated the same primary tumor size, vascularization, and tumor-free survival as their wild-type Plexin-B1-expressing parents, and there was no effect of Plexin-B1 loss on ErbB-2 tyrosine phosphorylation of cancer cells. This finding again supported the notion that Pelxin-B1 signals downstream of ErbB-2 phosphorylation.
However, the Pelxin-B1-deficient tumors did demonstrate reduced local invasiveness, and a striking reduction in macroscopically visible lung metastases. “These results indicate that Plexin-B1 has no effect on tumorigenesis or tumor growth but is required for metastasis of ErbB-2–dependent breast cancers in vivo,” the authors state.
Importantly, evaluation of human breast cancer tissue samples confirmed that those overexpressing ErbB-2 exhibited tyrosine phosphorylated Plexin-B1, whereas ErbB-2-negative cancers showed no evidence of Plexin-B1 phosphorylation. Further analysis of breast cancer microarray datasets found no direct correlation between Plexin-B1 and ErbB-2 expression levels. However, among patients with ErbB-2–negative breast cancer, low levels of Plexin-B1 expression was associated with a trend towards shorter disease-free survival than high Plexin-B1 expression levels. Even more strikingly, among patients with ErbB-2–overexpressing breast cancer, low expression levels of Plexin-B1 correlated strongly with longer disease-free survival than high Plexin-B1 expression levels. And for the small number of patients for whom overall survival data were available, there was a trend towards low Plexin-B1 expression correlating with increased survival.
“Our data show that Plexin-B1 couples ErbB-2 overexpression to Rho signaling and tumor cell invasiveness and that Plexin-B1 is centrally involved in the metastasis of ErbB-2–overexpressing breast cancer,” the authors conclude. “Therefore, inhibition of the ErbB-2/Plexin-B1 interaction or of Plexin-B1–mediated signaling may reduce the risk of metastasis in patients with ErbB-2–overexpressing breast cancer and therefore represent a promising new therapeutic principle.”