Fig. 5.

LEM4 confers tamoxifen resistance by activating the cyclin D-CDK4/6-Rb axis. a MCF7, MCF7-LEM4, and MCF7-TAMR cells were treated with 5% DCC-FBS (vehicle), 4-OHT (1 μM), PD0332991 (PD) (0.2 μM), or a combination of 4-OHT and PD0332991. Adherent cells were tested by SRB after 9 days. Data are presented as % parental control. Mean ± s.d. for three independent replicates. b Immunoblots of lysates from cells treated as in a with indicated antibodies. c Tumor growth of MCF7-LEM4 cells as subcutaneous xenografts in athymic mice with E2 pellets when tumors reached an approximate volume of 100 mm³, then treated with tamoxifen pellet implanted subcutaneously, 100 mg kg⁻¹ PD0332991 (tricubic weekly), or a combination of tamoxifen pellet and PD0332991. Mean ± s.e.m., n = 8. d H&E staining and IHC for Ki-67, p-CDK4, p-Rb, and cleaved caspase 3/7 from c. Scale bars for H&E, 150 μm. Scale bars for IHC, 50 μm. e GST alone or recombinant GST-LEM4 immobilized on glutathione-agarose beads was incubated with the MCF7 cell extract. The pulled-down proteins were analyzed by immunoblotting with CDK4 and Rb antibodies. f HEK293T cells were transfected with GFP-LEM4 and pCMV6-FLAG-CDK4 or the empty vector pCMV6. The interaction of FLAG-CDK4 with GFP-LEM4 was analyzed by immunoprecipitation of the cell lysate with anti-FLAG affinity gel and immunoblotted with anti-GFP antibody. g HEK293T cells were transfected with pCMV6-FLAG-LEM4 or the empty vector pCMV6. The interaction of FLAG-LEM4 with Rb was analyzed by immunoprecipitation of the cell lysate with anti-FLAG affinity gel and immunoblotted with Rb antibody. h MCF7-shControl and MCF7-shLEM4 cells were treated with 50 μg mL⁻¹ CHX for 0, 1, 2, and 4 h and Western blotting was performed. i Model of LEM4 regulation of the cyclin D-CDK4/6-Rb axis leading to tamoxifen resistance in ER+ breast cancer. n.s., not significant. **P < 0.01, ***P < 0.001. Tukey’s multiple comparisons test for a, c (weight). Repeated measures ANOVA for c (volume)