PMC

Expression of HRGβ1, erbB3 and erbB4 in tamoxifen-resistant MCF-7 cells. (a) RT-PCR analysis of basal HRGβ1 mRNA in DU145 prostate and tamoxifen-resistant MCF-7 (Tam-R) breast cancer cells. (b) Total erbB3 and erbB4 protein expression in Tam-R cells by western blotting and immunocytochemistry (original magnification, ×20). β-Actin protein expression was also assessed to demonstrate equivalent sample loading. Tamoxifen was present in Tam-R media. Data are representative of at least three separate experiments.

Effects of HRGβ1 and gefitinib on tamoxifen-resistant MCF-7 cell growth and invasion. (a) Growth and (b) invasive capacity of tamoxifen-resistant MCF-7 (Tam-R) cells, on day 7 for growth and on day 3 for invasion, following treatment with either HRGβ1 (10 ng/ml) or gefitinib (1 mM) or a combination of the two agents. Results expressed as the mean ± standard error of the mean of triplicate wells and are representative of at least three separate experiments. **P < 0.01 versus control, ***P < 0.001 versus control, ^†P < 0.05 versus gefitinib, ^†††P < 0.001 versus gefitinib.

Effects of drug combinations on HRGβ1-driven tamoxifen-resistant MCF-7 cell growth and invasion. (a) Growth and (b) invasive capacity of tamoxifen-resistant MCF-7 (Tam-R) cells, on day 7 for growth and on day 3 for invasion, following treatment with either HRGβ1 (10 ng/ml), gefitinib (1 μM), trastuzumab (100 nM), LY294002 (10 μM) or a combination of these agents. Results expressed as the mean ± standard error of the mean of triplicate wells and are representative of three separate experiments. Tamoxifen was present in all studies. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control; ^††P < 0.01 and ^†††P < 0.001 versus HRGβ1; ^§P < 0.05 and ^§§§P < 0.001 versus gefitinib + HRGβ1.

Effects of combining gefitinib with trastuzumab or LY294002 on HRGβ1-driven signalling in tamoxifen-resistant MCF-7 cells. Western analysis of total and phosphorylated epidermal growth factor receptor (EGFR), erbB2, AKT and ERK1/2 protein expression in tamoxifen-resistant MCF-7 (Tam-R) cells prior to and following incubation with either (a) trastuzumab (100 nM) or vehicle control for 7 days, (b) LY294002 (10 μM) or vehicle control for 1 hour, (c) gefitinib (1 μM), gefitinib in combination with trastuzumab or vehicle control for 7 days, and (d) gefitinib, gefitinib in combination with LY294002 or vehicle control for 1 hour, all followed by either HRGβ1 (10 ng/ml) or vehicle control for 5 minutes. Tamoxifen was also present in all studies. Data are representative of at least three separate experiments.

Immunohistochemical staining for HRGβ1 in primary breast cancer specimens. (a) Examples of high and low HRGβ1 expression. Scale bars = 20 μm. (b) Box-plots illustrating cytoplasmic HRGβ1 immunohistochemistry assessed by H-score analysis in membrane (mem) epidermal growth factor receptor (EGFR)-negative, erbB2-negative and erbB3-negative primary breast cancer versus membrane EGFR-positive, erbB2-positive and erbB3-positive primary breast cancer. A significant positive correlation between cytoplasmic HRGβ1 expression and membrane erbB receptor positivity was only seen with EGFR (Mann–Whitney U test, P = 0.04). (c) Box-plots illustrating cytoplasmic HRGβ1 immunohistochemistry assessed by H-score analysis in membrane (mem) phosphorylated erbB2-negative versus membrane phosphorylated erbB2-positive primary breast cancer and in nuclear (nuc) phosphorylated ERK1/2-negative versus nuclear phosphorylated ERK1/2-positive primary breast cancer (Mann–Whitney U test, P = 0.006 and P = 0.017, respectively), and scatter plot illustrating the significant positive correlation between expression levels of nuclear phosphorylated AKT and cytoplasmic HRGβ1 in the same samples (Spearman rank test, P = 0.044).

Effects of gefitinib on erbB receptor dimerization patterns and activity of associated downstream signalling elements. (a) Western blot (WB) analysis of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated erbB2, phosphorylated tyrosine (Tyr) and total erbB3 protein expression following immunoprecipitation with total erbB3 antibody in tamoxifen-resistant MCF-7 (Tam-R) cells prior to and following incubation of Tam-R cells with gefitinib (1 μM) for 1 hour. (b) WB analysis of total and phosphorylated EGFR, erbB2, AKT and ERK1/2 protein expression in Tam-R cells prior to and following incubation with gefitinib (1 μM) for 1 hour. (c) WB analysis of total and phosphorylated EGFR, erbB2 and erbB3 protein expression following immunoprecipitation with total EGFR antibody in Tam-R cells prior to and following incubation with gefitinib (1 μM) for 1 hour. Tamoxifen (100 nM) was present in all studies. Data are representative of at least three separate experiments.

Effects of HRGβ1 and gefitinib on erbB receptor dimerization patterns and associated downstream signalling activity. (a) Western blot (WB) analysis of phosphorylated epidermal growth factor receptor (EGFR), phosphorylated erbB2, phosphorylated tyrosine (Tyr) and total erbB3 protein expression following immunoprecipitation with total erbB3 antibody in tamoxifen-resistant MCF-7 (Tam-R) cells prior to and following treatment with either gefitinib (1 μM) or vehicle control for 1 hour followed by HRGβ1 (10 ng/ml) for 5 minutes. (b) WB analysis of total and phosphorylated EGFR, erbB2, AKT and ERK1/2 protein expression in Tam-R cells prior to and following incubation with either gefitinib (1 μM) or vehicle control for 1 hour followed by either HRGβ1 (10 ng/ml) or vehicle control for 5 minutes. Tamoxifen was also present in all studies. Data are representative of at least three separate experiments.