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. 2020 Mar 15;146(6):1674-1685.
doi: 10.1002/ijc.32588. Epub 2019 Aug 7.

GPER-induced signaling is essential for the survival of breast cancer stem cells

Yu-Tzu Chan  1 , Alan C-Y Lai  2   3 , Ruey-Jen Lin  1 , Ya-Hui Wang  1 , Yi-Ting Wang  4 , Wen-Wei Chang  5 , Hsin-Yi Wu  6 , Yu-Ju Lin  1 , Wen-Ying Chang  1 , Jen-Chine Wu  1 , Jyh-Cherng Yu  7 , Yu-Ju Chen  4 , Alice L Yu  1   8   9
Affiliations

GPER-induced signaling is essential for the survival of breast cancer stem cells

Yu-Tzu Chan et al. Int J Cancer. .

Erratum in

  • Erratum.
    [No authors listed] [No authors listed] Int J Cancer. 2020 Oct 15;147(8):E7. doi: 10.1002/ijc.33105. Epub 2020 Jun 5. Int J Cancer. 2020. PMID: 32421855 Free PMC article. No abstract available.

Abstract

G protein-coupled estrogen receptor-1 (GPER), a member of the G protein-coupled receptor (GPCR) superfamily, mediates estrogen-induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non-BCSCs of three patient-derived xenografts of ER- /PR+ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER-mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD-Ser118 to sustain BCSC characteristics. Transfection with a dominant-negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs.

Keywords: BAD; GPER; breast cancer stem cell; phosphoproteomics; tamoxifen.

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Figures

Figure 1
Figure 1
Greater expression of GPER in BCSCs than in non‐BCSCs. (a) Clinical characteristics of the three patients with breast cancer BC0145, BC0244 and BC0634, from whom the xenografts were derived. (b) Total cell lysates from BCSCs and non‐BCSCs sorted from xenografts of BC0145, BC0244 and BC0634 were subjected to western blotting. GPERs were detected at the bands with a molecular weight in the 50–55 kDa region. GAPDH protein served as the internal control for normalization. For normalization, total GPER expressions in BCSCs of BC0145, BC0244 and BC0634 were each set as 1.0 for comparison to values in non‐BCSCs. N‐BCSC: non‐BCSC. (c) Tumor cells from BC0145 and BC0244 xenografts were analyzed for the expression of surface antigens CD24, CD44 and total GPER by flow cytometry. After cells staining for CD24/CD44, cells were fixed and permeabilized for GPER staining.
Figure 2
Figure 2
GPER regulation of the survival and stemness of BCSCs. (a) Left panel: AS‐B145 or AS‐B634 cells were infected with lentiviral shRNAs and the total proteins were harvested 6 days after infection for western blotting. GAPDH protein served as the internal control for normalization. The normalized total GPER expression of shLuc cells was set as 1.0 for comparison to values of shGPER infected cells (sh‐A, sh‐B and sh‐C). Center and Right panels: the growth curve of shRNA infected AS‐B145 (shLuc and sh‐B) and AS‐B634 (shLuc and sh‐B) cells was determined using the xCELLigence system over a period of 72 hr. (b) H2Kd−/CD24−/CD44+ BC0145 cells (left panel) and H2Kd−BC0634 cells (right panel) were infected with shLuc, sh‐B or sh‐C. GPER mRNA and protein levels were determined by real‐time qPCR and western blotting at 6 days after infection, respectively. The expression of GPER was presented as fold expression relative to shLuc. (c) The cell proliferation of shRNAs infected H2Kd−/CD24−/CD44+ BC0145 or H2Kd− BC0634 cells were monitored by xCelligence system over a period of 120 hr. (d) Mammosphere formation was assessed for freshly sorted BCSCs from the BC0145 (left) and BC0634 xenograft (right) (2,000 cells/well in a 96‐well plate format). Both cell types were infected with shLuc control or shGPER‐B. ***p < 0.001 as compared to the control group using the Student's t‐test.
Figure 3
Figure 3
Functional analysis of differentially expressed phosphoproteins in BCSCs and non‐BCSCs. (a) Workflow diagram for the phosphoproteomic study, which quantitatively compared the phosphoproteomes of the BCSCs and non‐BCSCs sorted from the BC0145 xenograft. Equal amounts of BCSC and non‐BCSC lysates, each containing the internal standard β‐casein, were enzymatically digested, enriched in phosphopeptides by IMAC and subjected to triplicate LC–MS/MS. Quantification of the phosphopeptides was performed using IDEAL‐Q software. The phosphoproteins having at least twofold greater expression in BCSCs than in non‐BCSCs from two independent experiments performed in triplicate are shown. (b) The top 10 GeneGo pathway maps (p < 0.001). (c) The top 10 GeneGo process networks (d) GoMiner analysis showing the distribution of the classified molecular functions of differentially expressed phosphoproteins. (e) Heat maps for the differentially expressed phosphoproteins identified in duplicated experiments. Quantitative comparison revealed that most of the differentially expressed phosphoproteins are involved in cell adhesion, cell migration, secretion, cell motility or the cell cycle. (f) Validation of selected phosphoproteins. Phosphorylation states of selected phosphoproteins in BCSCs and non‐BCSCs from BC0145 and BC0244 xenografts. Western blot analysis of total cell lysates from BC0145 and BC0244 BCSCs and non‐BCSCs was performed with antibodies against BAD, BAD pSer118, PRKACA and PRKACA pThr197. The ratios of expression levels were calculated for the BCSC and non‐BCSC proteins after normalization of their respective signals to the corresponding GAPDH signal. The blots of GAPDH were identical to those in Figure 1 b since the western blot were derived from the same experiment.
Figure 4
Figure 4
GPER signaling included BAD phosphorylation in BCSCs. (a) The growth curve of AS‐B145 cells expressing the dominant‐negative BAD Ser118Ala mutant. AS‐B145 cells were infected with a doxycycline‐inducible lentiviral vector that expressed the mutant BAD or WT BAD. Cell growth was measured using the xCELLigence system. Values represent mean ± SD, n = 3, ***p < 0.001 for BAD S118A compared to WT BAD, the cell index at 96 hr was analyzed by one‐way ANOVA. (b) Total cell lysates prepared from AS‐B145 cells that had been treated with 4‐OHT (2,500 nM) for 15, 30, 45 or 60 min were immunoblotted. The amount of BAD p‐Ser118, p‐Ser112 and p‐Ser136 was normalized to the corresponding amount of GAPDH, and the fold increase in phosphorylation was normalized to the amount at 0 min. (c) AS‐B145 cells were treated with 4‐OHT (2,500 nM), E2 (200 nM), E3 (100 nM) or G1 (100 nM) for 72 hr. Cell proliferation was assessed using the xCELLigence system. The cell index of treated cells at 0 hr was set as 1.0, and the cell index of treated cells at 72 hr relative to the time zero value. Value represents mean ± SD, n = 3, *p < 0.05, **p < 0.01 and ***p < 0.001. The statistic was determined by one‐way ANOVA. (d) AS‐B634 cells were treated with 4‐OHT (2000 nM), E2 (50 nM), E3 (100 nM) or G1 (100 nM) for 48 hr. Data presentation as for Figure 4 c. (e) GPER expression of permeabilized AS‐B634 cells was assessed by flow cytometry 3 days after treatment with 4‐OHT (2000 nM), E2 (50 nM), E3 (100 nM) or G1 (100nM). The GPER+ population of treated cells was presented as fold increase relative to the control group (DMSO). The statistic was determined by one‐way ANOVA, n = 3, *p < 0.05, **p < 0.01. (f) ALDH activity in AS‐B145 cells was assessed by ALDEFLUOR assay after treatment with 4‐OHT (2,500 nM), E2 (200 nM), E3 (100 nM) or G1 (100 nM) for 3 days. Cell treated with diethylaminobenzaldehyde (DEAB), which is the inhibitor of ALDH, was used to distinguish ALDH+ from ALDH− cells. The ALDH+ populations of treated cells were presented as fold increase relative to the control group. The statistic was determined by on way ANOVA, n = 3 for 4‐OHT, n = 3 for E2, n = 6 for E3 and n = 2 for G1, *p < 0.05, **p < 0.01.
Figure 5
Figure 5
The tumorigenicity of GPER in BCSCs. (a) AS‐B145 cells were infected with lentiviral shLuc or shGPER for 6 days. Cells (1 × 105) infected with shRNAs were implanted into the mammary fat pads of 6‐ to 8‐week‐old female NSG mice (n = 6). Photos show the tumors of individual mice after sacrifice. The black line represents 1 cm. (b) The tumor growth curves in individual mice of each group during the 2 months after implantation. (c) Due to the small tumor sizes, six shLuc and four shGPER tumors were digested into single cells suspension and stained with H2Kd/CD24/CD44 antibodies to determine the BCSC subpopulation. The mean percentages of H2Kd−/CD24−/CD44+ cells in the total tumor cell population in the shLuc and shGPER are shown. Values represent mean ± SD, p = 0.2, Student's t‐test. (d and e) AS‐B634 cells (5 × 104) infected with shRNAs were implanted into the mammary fat pads of 6‐ to 8‐week‐old NSG mice (n = 5). The tumor growth curves in individual mice during the 4 weeks after implantation are shown (five shLuc and five shGPER tumors). (f) Tumors (five shLuc and four shGPER tumors) were digested and stained with using the ALDEFLUOR assay. The mean percentages of ALDH+ cells in the total tumor cell population in the shLuc and shGPER groups are shown. Values represent mean ± SD, *p < 0.05, Student's t‐test. (g) Schematic illustration of GPER signal transduction pathway involved in the regulation of survival and stemness of BCSCs. Treatment of GPER+ breast cancer cells with GPER agonists (TMX, E2 and E3) triggers cAMP production, which in turn induces phosphorylation of PKA and BAD, thereby promoting cell proliferation and sustaining CSCs.
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