Distinctive functions of p160 steroid receptor coactivators in proliferation of an estrogen-independent, tamoxifen-resistant breast cancer cell line

Abstract

Elevated expression of steroid receptor coactivator-3 (SRC-3), a member of the p160 family of nuclear receptor coactivators, has been implicated in tamoxifen resistance of breast tumors while the involvement of the two other members of this family, SRC-1 and SRC-2, is less well characterized. In this study, using small interfering RNA-based silencing, the role of each SRC coactivator in the growth of the LCC2 estrogen-independent and tamoxifen-resistant breast cancer cell line was evaluated. The loss of SRC-1, SRC-2, or SRC-3 did not significantly alter LCC2 proliferation or cell cycle distribution of 4-hydroxytamoxifen- versus vehicle-treated cells. However, depletion of SRC-2 and SRC-3, but not SRC-1, decreased basal cell proliferation and increased apoptosis. Cell cycle analyses further illustrated the divergent contributions of SRC-2 and SRC-3 with depletion of the former increasing the percentage of cells in the G(0)G(1) and sub-G(0)G(1) phases of cell cycle yet maintaining sensitivity to estradiol and ICI 182 780 antiestrogen, while SRC-3 depletion increased cells in the sub-G(0)G(1) phase and ablated response to estrogen receptor α (ERα) ligands. Surprisingly, the effects of SRC coactivator depletion on ERα transcriptional activity, as measured by luciferase reporter gene, did not correspond to the observed effects on proliferation (e.g. SRC-1 knockdown increases ERα activity). Collectively, these data indicate that SRC control of basal and hormone-regulated proliferations is not solely mediated by ERα, and suggest that targeting growth inhibition by disrupting SRC-2 and SRC-3 function may be an effective approach to inhibit the growth of tamoxifen-resistant breast cancer.

Figure 1. Comparison of cell proliferation and expression of SRC coactivators and estrogen receptors in MCF-7 and LCC2 breast cancer cells

MCF-7 (A) and LCC2 (B) cells cultured in phenol red-free DMEM containing 10% sFBS and phenol red-free IMEM containing 5% sFBS, respectively, were treated with Veh (0.1% ethanol), E2 (1 nM) or 4HT (100 nM) and allowed to grow for six days. Cells were harvested from parallel sets of cultures and counted on day 0 (on the day of hormone addition), day 4 and day 6. The experiment was repeated three times in triplicate and data were plotted as relative change of cell number with time, setting the number corresponding to E2 treated cells on day 6 as 100. Data are reported as the average ± SEM. Statistical analyses by student’s t-test indicate *p<0.05, **p<0.001 and ^ap=0.052 versus each group’s vehicle control. Expression of (C) SRC coactivators and (D) ERα and ERβ were assessed for each cell type by Western blot. Actin was used as a loading control and recombinant ERβ (rERβ) was employed in panel D as a positive control. Expression analyses were conducted three times and representative blots are shown.

Figure 2. Efficient silencing of p160 family coactivators five days after siRNA transfection

LCC2 cells were transfected with 10 pmol of control siRNA or siRNA directed against SRC-1, SRC-2 or SRC-3 in each well of six well plates. Four hours after transfection, cells were treated with VEH (0.1% ethanol), E2 (1 nM), 4HT (100 nM) or ICI (100 nM) and allowed to grow for five days. Thereafter, cells were harvested, lysed and total proteins were resolved by SDS-PAGE followed by Western blotting for SRC coactivators and actin. (B – D) Blots from two independent experiments were quantitated by densitometry and normalized to actin. Bars represent the average ± the range of individual values.

Figure 3. Regulation of LCC2 cell proliferation by SRC family coactivators

(A) LCC2 cells were transfected with siRNAs as indicated and 24 h post-transfection, cells were treated with VEH (0.1% ethanol), E2 (1 nM), 4HT (100 nM) or ICI (100 nM) for five days and counted. Data represent the average ± SEM (n=3) and are expressed as relative change in cell number, setting the number corresponding to the E2-treated siControl group as 100. Statistical analyses were done by students’ t-test where ‘*’ indicates p<0.05 and ‘a’ indicates p=0.053 versus the respective treatment (VEH, E2, 4HT or ICI) group in the siControl cells; ‘#’ indicates p<0.05 and ‘b’ indicates p=0.090 versus the vehicle control within the group. (B) LCC2 cells were transfected with plasmids for GFP control (con), wild-type GFP-SRC-3 (wt) or a GFP-SRC-3 mutant (mt) in which each of the 3 receptor interacting motifs of SRC3 were mutated (LXXLL→LXXAA), allowed to grow for five days and counted. Results are plotted as relative cell number, setting the value for wild type SRC-3 transfected cells as 100 (n=4). *, p < 0.05; or **, p < 0.001 vs. vector control group. (C) Transcriptional activity of wild-type and mutated GFP-SRC-3 in comparison to control GFP plasmid was determined in a trans-activation assay in HeLa cells co-transfected with ERα and ERE-e1b-Luc. Data represent the average ± SEM of three experiments. *, P < 0.05; or **, P < 0.001 vs. vector control group. (D) LCC2 cells transfected as in panel B were assessed for expression of wt SRC-3 and GFP-SRC3 by Western blot using antibody specific for SRC-3. Actin was employed as a loading control.

Figure 4. Down-regulation of SRCs differentially affects the cell cycle distribution of LCC2 cells

LCC2 cells were subjected to siRNA treatment and 48 h after transfection were treated with VEH (0.1% ethanol), 1 nM estradiol (E2), 100 nM tamoxifen (4HT) or 100 nM ICI for an additional 24 h. Thereafter, cells were collected, fixed and stained with propidium iodide followed by flow-cytometric analysis. Data represents average of four independent experiments ± SEM where ‘#’ indicates p<0.05 versus the corresponding Veh-treated control and ‘*’ indicates significance at p<0.05 versus the siControl vehicle group. Statistical analyses were done by student’s t-test.

Figure 5. Depletion of SRC-2 and SRC-3 but not SRC-1 induces apoptosis in LCC2 cells

(A) LCC2 cells were subjected to siRNA transfection and then allowed to grow 48 h in phenol-red free IMEM containing 5% stripped serum followed by exposure to VEH (0.1% ethanol) for an additional 24 h. Thereafter cells (both adherent and non-adherent) were collected and assayed by Cell Death ELISA. The experiment was repeated six times and data represent the average ± SEM, setting the number corresponding to the control group as 100. Statistical analyses was done by t-test and indicates *p<0.05 versus siControl cells. (B) LCC2 cells were transfected with control or SRC-3 siRNA as above and 48 h thereafter were treated with VEH (0.1% ethanol), E2 (1 nM), 4HT (100 nM) or ICI (100 nM) for 24 h followed by assessment by Cell Death ELISA. Data represent average ± SEM of three experiments. ‘*’ indicates p<0.05 versus VEH treated siControl cells.

Figure 6. ERα and PR transcriptional activity and endogenous ERα target gene expression is differentially altered by down regulation of SRCs in LCC2 cells

Twenty-four hours after the indicated siRNA transfection, LCC2 cells were further transfected with ERE-e1b-Luc (A) or PR_B and PRE-e1b-Luc (C) reporter genes and 24 h thereafter were subjected to treatment with vehicle (VEH; 0.1% ethanol), 1 nM E2 or 100 nM progesterone (P4) for an additional 24 h. Cells were then harvested, lysed and measured for luciferase activity followed by normalization against total protein. The experiment was conducted three times in duplicate and data are expressed as the average ± SEM. Statistical analyses were carried out using student’s t-test, where *p<0.05 and **p<0.001 versus the siControl E2 group and ‘#’ indicates p<0.05 versus siControl vehicle groups. (B) Parallel sets of samples from experiment (A) were assessed by 765 Western blot to determine the impact of E2 treatment on ERα expression. (D) Expression of PR_A and PR_B in LCC2 and MCF-7 cells treated with VEH or 1 nM E2 for 24 h was determined by Western blot. A representative blot from three experiments is shown. The expression of the ERα target genes PR (panel E) and pS2 (panel F) was assessed by RT-qPCR for LCC2 cells transfected with the indicated siRNA and treated 48 h later with VEH or 1 nM E2 for 24 h. Data are normalized to 18S and E2-treated control siRNA samples were set to 100. Values represent the mean ± SEM of four experiments. *, p < 0.05; or **, p < 0.001 vs. E2 treated control siRNA-transfected cells.