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. 2018 Oct 9;9(1):4180.
doi: 10.1038/s41467-018-06309-8.

LEM4 Confers Tamoxifen Resistance to Breast Cancer Cells by Activating Cyclin D-CDK4/6-Rb and ERα Pathway

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Free PMC article

LEM4 Confers Tamoxifen Resistance to Breast Cancer Cells by Activating Cyclin D-CDK4/6-Rb and ERα Pathway

Ang Gao et al. Nat Commun. .
Free PMC article

Abstract

The elucidation of molecular events that confer tamoxifen resistance to estrogen receptor α (ER) positive breast cancer is of major scientific and therapeutic importance. Here, we report that LEM4 overexpression renders ER+ breast cancer cells resistant to tamoxifen by activating the cyclin D-CDK4/6 axis and the ERα signaling. We show that LEM4 overexpression accelerates tumor growth. Interaction with LEM4 stabilizes CDK4 and Rb, promotes Rb phosphorylation and the G1/S phase transition. LEM4 depletion or combined tamoxifen and PD0332991 treatment significantly reverses tamoxifen resistance. Furthermore, LEM4 interacts with and stabilizes both Aurora-A and ERα, promotes Aurora-A mediated phosphorylation of ERα-Ser167, leading to increase in ERα DNA-binding and transactivation activity. Elevated levels of LEM4 correlates with poorer relapse-free survival in patients with ER+ breast cancer undergoing endocrine therapy. Thus, LEM4 represents a prognostic marker and an attractive target for breast cancer therapeutics. Functional antagonism of LEM4 could overcome tamoxifen resistance.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
High LEM4 expression correlates with poor survival of patients with breast tumors. a The Cancer Genome Atlas analysis showed the expression levels of LEM4 across different subtypes of breast cancer and normal tissues. Normal, n = 106, luminal A, n = 372, luminal B, n = 174, HER2, n = 49, basal-like, n = 127. b TMA analysis of LEM4 protein expression in breast cancers. Representative IHC images of LEM4 expression in matched normal and primary tumors from two patients are shown. Scale bars, 100 μm. c Scatter plot showing LEM4 protein expression in 16 paired normal and matched primary tumors, indicated by the blue arrows. d Scatter plots showing the LEM4 immunohistochemical staining results for 259 breast tumors in relation to cancer progression. eh Kaplan–Meier analysis with median cutoff values of LEM4 expression and overall survival (e) in all 284 patients and patients who had luminal B or HER2 positive subtypes of breast cancer (f, g) or stratified according to ERα status (h). P-values were calculated by the log–rank test. i Kaplan–Meier analysis with median cutoff values of LEM4 expression for breast cancer from GEO datasets. P-values were calculated by the log–rank test. **P < 0.01, ***P < 0.001, n.s., not significant. Tukey’s multiple comparisons test for a, d. Paired t-tests for c
Fig. 2
Fig. 2
LEM4 promotes cell proliferation and tumorigenesis. a Growth curve of MCF7-vec and MCF7-LEM4 cells were measured by SRB assay in monolayer culture. Immunoblot was performed with anti-LEM4 antibody. b Growth curves of shControl-T47D, shLEM4#1-T47D, and shLEM4#2-T47D cells were measured by SRB assay in monolayer culture. Immunoblot was performed with anti-LEM4 antibody. c MCF7-LEM4 cells were transfected with LEM4 siRNA and control siRNA. Western blot was performed with anti-LEM4 antibody. Growth curves were measured by SRB assay in monolayer culture. d, e Soft agar colony formation by MCF7-vec and MCF7-LEM4 cells (d) or by T47D-shcontrol and T47D-shLEM4 cells (e). f, g Tumor growth of MCF7 and T47D cells implanted subcutaneously in athymic mice in the presence of an exogenous slow release estrogen implant. Mean ± s.e.m, n = 4 or 6 for MCF7 cells (f), n = 6 for T47D cells (g). h, i IHC for ki-67 in subcutaneous xenograft tumors from Figs. 2f, g. Mean ± s.d. for three independent replicates. Scale bars, 50 μm. *P < 0.05, **P < 0.01, and ***P < 0.001. Repeated measures ANOVA for f (volume), g (volume). Student’s t-test for f (weight), g (weight), h, i
Fig. 3
Fig. 3
LEM4 overexpression promotes the G1 to S phase transition. ac Depletion of LEM4 in T47D (a) and BT474 (b) cells and overexpression of LEM4 in MCF7 cells (c) altered the proportion of cells in G1, S, and G2/M phase by FACS analysis. d, e Real-time RT-PCR analysis of the cell cycle-related gene expression in T47D cells with LEM4-depleted (d) and LEM4-overexpressing MCF7 cells (e). f, g Immunoblot of cell cycle-related gene expression using the indicated antibodies in LEM4-depleted T47D cells (f) and MCF7-LEM4 cells (g). h, i Immunohistochemical analysis of the expression of cyclin D1, p-CDK4 (T172), p-Rb (S780), and CDK1 in tumors (Fig. 2f, g). Sizes of cell populations averaged from three independent experiments with standard deviations. Scale bars, 50 μm. *P < 0.05, **P < 0.01, *** P < 0.001. Tukey’s multiple comparisons test for a, b, d, e. Student’s t-test for c
Fig. 4
Fig. 4
LEM4 promotes tamoxifen resistance in ER positive breast cancers. a Western blot was performed with LEM4 antibody in MCF7 and MCF7-TAMR cells. b The GEO GSE100075 was downloaded from Gene Expression Omnibus (GEO).The expression level of LEM4 in MCF7, MCF7-LTED-ESR1(WT), and MCF7-LTED-ESR1(Y537C) cells was measured by transcription per million (TPM). c LEM4 siRNA and control siRNA treated MCF7-TAMR cells were treated with or without tamoxifen (1.0 μΜ) for 5 days. Total cell viability were assessed by SRB assays. Experiments were repeated three times, each experiment was triplicates. d A cellular viability assay was performed in MCF7-LEM4 and MCF7-vec cells treated with various concentrations of 4-OHT for 7 days. e BT474-shcontrol and BT474-shLEM4 cells were treated with various concentrations of 4-OHT for 7 days. Cellular viability was assessed by SRB assays. f Tumor growth and tumor weight of MCF7-vec and MCF7-LEM4 cells as subcutaneous xenografts in athymic mice with E2 supplementation until tumors reached 100 mm3, then treated with or without tamoxifen (TAM) pellets implanted subcutaneously for 30 days. Mean ± s.e.m., n = 8. g Tumor growth of BT474-shControl and BT474-shLEM4 cells as subcutaneous xenografts in athymic mice with E2 supplementation until tumors reached ~200 mm3, then treated with tamoxifen pellets implanted subcutaneously. Mean ± s.e.m., n = 7. h Three breast cancer datasets (GSE2990, GSE3494 and GSE9195) were from the KM Plotter database (www.kmplot.com). Kaplan–Meier analysis of recurrence-free survival in the cohorts of patients treated with adjuvant tamoxifen monotherapy (exclude all chemotherapy). Samples were stratified into “high” and “low” LEM4 expression based on median cutoff value in each dataset. P-values were calculated by the log–rank (Mantel–Cox) test. n.s., not significant, *P < 0.05, **P < 0.01 Tukey’s multiple comparisons test for b, f (weight). Repeated measures ANOVA for g (volume), Student’s t-test for g (weight)
Fig. 5
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)
Fig. 6
Fig. 6
LEM4 induces ERα transactivation activity. a Immunoblot analysis of the phosphorylation of ER, Cyclin D1, and c-Myc in MCF-LEM4 and MCF7-TAMR cells. b Immunoblot analysis of the phosphorylation of ER, Cyclin D1, and c-Myc in LEM4-depleted MCF7-LEM4 and LEM4 knocked-down MCF7-TAMR cells. c Luciferase assay. ER+ MCF7 and ERα-negative MDA-MB-231 cells were transfected with ERE-Luc and other indicated plasmids. Following incubation for 48 h, luciferase activity was measured and normalized to Renilla. Results are the mean ± s.e.m. of three independent experiments performed in triplicate. d ERα ChIP assay of known ER-binding sites in ERα target genes was performed in MCF7-LEM4 cells incubated in estrogen-depleted medium (5% charcoal-stripped serum in phenol red-free DMEM) for 72 h before treatment with vehicle, 10 nmol L−1 E2 for 45 min. e Time course ChIP study of the endogenous ERα with the estrogen response elements in the promoter region of the ERα target genes. MCF7, MCF7-LEM4, and LEM4-depleted MCF7-LEM4 cells incubated in estrogen-depleted medium (5% charcoal-stripped serum in phenol red-free DMEM) for 72 h before treatment with vehicle, 10 nmol L−1 E2. ChIP analysis was conducted by using anti-ERα antibody. *P < 0.05, **P < 0.01, ***P < 0.001. Tukey’s multiple comparisons test for c. Student’s t-test for d
Fig. 7
Fig. 7
LEM4 interacts with and stabilizes ERα. a Immunoblot analysis of ERα in MCF7 and MCF7-LEM4 cells grown under estrogen-deprived conditions in DMEM phenol-free medium containing 5% dextran charcoal-stripped serum and treated with 10 nmol L−1 E2 for 30 min. b MCF7-shControl and MCF7-shLEM4 cells were treated with 50 μg mL−1 CHX for 0, 1, 2, and 4 h and ERα analyzed by immunoblot. c For endogenous LEM4 and ERα interaction, BT474 cells were immunoprecipitated with anti-ERα antibody and detected with anti-LEM4 antibody. d HEK293T cells were transfected with FLAG-LEM4 and GFP-ERα or the empty vector pCMV6. After incubation for 48 h, cell lysates were precipitated with anti-FLAG affinity gel and immunoblotted with anti-GFP and anti-FLAG antibody. e GST alone or GST-LEM4 immobilized on glutathione-agarose beads was incubated with the cell extract of HEK293T cells transfected with GFP-ERα or various mutants of ERα tagged with GFP. Bound proteins were separated by SDS-PAGE and immunoblotted with anti-ERα antibody. f MCF7-LEM4 cells were immunostained with anti-FLAG (indicated LEM4, red) and anti-ERα (green) antibody, and counterstained with DAPI (blue). Scale bars, 7.5 μm. g MCF7-LEM4 cells were transfected with siRNA of ESR1 or treated with fulvestrant for 6 days. Total cell viability were assessed by SRB assays. Results are the mean ± s. d. of three independent experiments performed in triplicate. Western blot was performed with anti-ERα antibody. h Compared the relative mRNA LEM4 level between pre-treatment and post-treatment in samples from GEO GSE33658. ***P < 0.001. Tukey’s multiple comparisons test for g
Fig. 8
Fig. 8
LEM4 mediated the phosphorylation of ERα-Ser167 by Aurora-A. a Immunoblot analysis of the phosphorylation of Aurora-A, Aurora-A, AKT, and S6K1 in MCF7-LEM4 and MCF7-TAMR cells. b LEM4 siRNA-treated and control siRNA-treated MCF7-LEM4 and MCF7-TAMR cells were treated for 48 h. Western blot was performed with indicated antibodies. c BT474 cells were transfected with LEM4 siRNA or control siRNA for 48 h. Western blot was performed with indicated antibodies. d MCF7-LEM4 cells were transfected with LEM4 siRNA or AKT siRNA or Aurora-A siRNA for 48 h. Western blot was performed with indicated antibodies. e HEK293T cells were transfected with FLAG-LEM4 and GFP-Aurora-A. After incubating for 48 h, cell lysates were precipitated with anti-FLAG affinity gel and immunoblotted with anti-GFP antibody. f HEK293T-shLEM4 cells were transfected with FLAG-ERα and GFP-Aurora-A. After incubating for 48 h, cell lysates were precipitated with anti-FLAG affinity gel and immunoblotted with anti-GFP antibody. g MCF7-shControl and MCF7-shLEM4 cells, or BT474-shControl and BT474-shLEM4 cells were treated with 50 μg mL−1 CHX for 0, 1, 2, and 4 h and analyzed for Aurora-A by immunoblot. h Model of LEM4 regulation of the ERα signaling leading to tamoxifen resistance in ER+ breast cancer

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