Figure 7

Cis-interaction of nectin-4 with trastuzumab-resistant variants of ErbB2 and enhancement of their signalling pathways for DNA synthesis. (a) Schematic diagram and amino acid sequences of ErbB2 splice variants. (b) Cis-interaction of nectin-4 with ErbB2 splice variants. HEK293E cells were co-transfected with various combinations of the indicated plasmids and cultured in suspension. FLAG-tagged nectin-4 (FLAG-Nectin-4) was immunoprecipitated using the anti-FLAG mAb. The samples were subjected to Western blotting using the indicated Abs. (c) Enhancement of the tyrosine-phosphorylation of ErbB2 splice variants and the PI3K-AKT signalling pathway by nectin-4. T47D cells stably expressing ErbB2 splice variants or T47D cells stably expressing GFP-tagged ErbB2 splice variants with FLAG-Nectin-4 were serum-starved for 24 h, and the samples were subjected to Western blotting using the indicated Abs. Ratio represents the band intensities of each phosphorylated protein on the indicated tyrosine and threonine residues that were normalized to those of each total protein, and the normalized value of each control cell (GFP-tagged ErbB2 and its splice variants, but not FLAG-Nectin-4, expressing cells) was set as 1.00. Arrowheads and square brackets indicate each of the proteins. The displayed blots were cropped, and the full-length blots are shown in Supplementary Fig. 9. Representative results from three independent experiments are shown. (d) Enhancement of DNA synthesis by nectin-4 in ErbB2 splice variant-expressing cells. T47D cells stably expressing ErbB2 splice variants or T47D cells stably expressing GFP-tagged ErbB2 splice variants with FLAG-Nectin-4 were serum-starved and treated with medium containing 10 μM EdU for 12 h. After washout of the medium, the cells were cultured in the absence of serum and stained with Hoechst33342, and EdU staining reagents. The number of EdU-incorporated cells was counted by microscopy and Hybrid Cell Counter software. (e) Reduction of DNA synthesis by the ErbB2, PI3K, and MEK inhibitors, but not the JAK inhibitor in ErbB2 splice variant-expressing cells. T47D cells stably expressing ErbB2 splice variants or T47D cells stably expressing GFP-tagged ErbB2 splice variants with FLAG-Nectin-4 were serum-starved and treated with the ErbB2 inhibitor irbinitinib at 1 μM, the PI3K inhibitor wortmannin at 1 μM or the PI3K inhibitor LY294002 at 50 μM, the MEK inhibitor U0126 at 10 μM, or the JAK1 and JAK2 inhibitor ruxolitinib at 1 μM. The assay was carried out as in (d). Bars indicate the means ± S.E. of three independent experiments. The actual P values for each test are shown in each figure. pAKT, phospho-AKT; pErbB2, phospho-ErbB2; pERK1/2, phospho-ERK1/2; pSTAT3, phospho-STAT3; IB, immunoblotting; IP, immunoprecipitation.