Abstract
Nectin-4 cell adhesion molecule and ErbB2 tyrosine kinase receptor are upregulated in many cancers, including breast cancer, and promote cancer cell proliferation and metastasis. Using human breast cancer cell lines T47D and SUM190-PT, in which both nectin-4 and ErbB2 were upregulated, we showed here that nectin-4 cis-interacted with ErB2 and enhanced its dimerization and activation, followed by the activation of the phosphoinositide 3-kinase-AKT signalling pathway for DNA synthesis. The third immunoglobulin-like domain of nectin-4 cis-interacted with domain IV of ErbB2. This region differs from the trastuzumab-interacting region but is included in the trastuzumab-resistant splice variants of ErbB2, p95-ErbB2 and ErbB2ΔEx16. Nectin-4 also cis-interacted with these trastuzumab-resistant splice variants and enhanced the activation of the phosphoinositide 3-kinase-AKT signalling pathway for DNA synthesis. In addition, nectin-4 enhanced the activation of the p95-ErbB2-induced JAK-STAT3 signalling pathway, but not the ErbB2- or ErbB2ΔEx16-induced JAK-STAT3 signalling pathway. These results indicate that nectin-4 cis-interacts with ErbB2 and its trastuzumab-resistant splice variants and enhances the activation of these receptors and downstream signalling pathways in a novel mechanism.
Highlights
Nectin-4 cell adhesion molecule and ErbB2 tyrosine kinase receptor are upregulated in many cancers, including breast cancer, and promote cancer cell proliferation and metastasis
When endogenous nectin-4 was immunoprecipitated using an anti-nectin-4 polyclonal Ab in SUM190-PT breast cancer cells, endogenous ErbB2 was co-immunoprecipitated with endogenous nectin-4 (Fig. 2b)
These results indicate that nectin-4 cis-interacts with ErbB2 among the ErbB family members and that this cis-interaction occurs on the same plasma membrane
Summary
The threonine-phosphorylation of AKT was reduced in the SUM190-PT cells in which endogenous nectin-4 was knocked down compared with that in the control cells (Fig. 4c) These results indicate that nectin-4 mainly enhances the ErbB2-mediated PI3K-AKT signalling pathway, but not the Ras-Raf-MEK-ERK1/2 signalling pathway or the JAK-STAT3 signalling pathway. The incorporation of EdU into DNA in the T47D cells stably expressing FLAG-Nectin-4 and the control cells was inhibited by the PI3K inhibitors wortmannin and LY294002 and the MEK inhibitor U0126, but not by the JAK1/2 inhibitor ruxolitinib (Fig. 5i) These results indicate that nectin-4 enhances the ErbB2-mediated DNA synthesis through the activation of the PI3K-AKT signalling pathway, and that the nectin-4-independent ErbB2-mediated activation of the Ras-Raf-MEK-ERK1/2 pathway, but not the JAK-STAT3 pathway, is involved in the ErbB2-mediated DNA synthesis. When ErbB2-GFP was immunoprecipitated with the anti-GFP pAb, ErbB2-HA was co-immunoprecipitated with ErbB2-GFP and FLAG-Nectin-4-Ig3-TM, but not FLAG, and the amount of ErbB2-HA from the cells co-expressing www.nature.com/scientificreports a IB: IP: GFP Cell lysates (kDa)
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