GPR30 as an initiator of tamoxifen resistance in hormone-dependent breast cancer

Abstract

Introduction: Tamoxifen is widely used to treat hormone-dependent breast cancer, but its therapeutic benefit is limited by the development of drug resistance. Here, we investigated the role of estrogen G-protein coupled receptor 30 (GPR30) on Tamoxifen resistance in breast cancer.

Methods: Primary tumors (PTs) of breast cancer and corresponding metastases (MTs) were used to evaluate the expression of GPR30 and epidermal growth factor receptor (EGFR) immunohistochemically. Tamoxifen-resistant (TAM-R) subclones derived from parent MCF-7 cells were used to investigate the role of GPR30 in the development of tamoxifen resistance, using MTT assay, western blot, RT-PCR, immunofluorescence, ELISA and flow cytometry. TAM-R xenografts were established to assess anti-tumor effects of combination therapy with GPR30 antagonist G15 plus 4-hydroxytamoxifen (Tam), using tumor volume measurement and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).

Results: In 53 human breast cancer specimens, GPR30 expression in MTs increased compared to matched PTs; in MTs, the expression patterns of GPR30 and EGFR were closely related. Compared to parent MCF-7 cells, TAM-R cells had greater growth responses to 17β-estradiol (E2), GPR30 agonist G1 and Tam, and significantly higher activation of Mitogen-activated protein (MAP) kinases; but this increased activity was abolished by G15 or AG1478. In TAM-R cells, GPR30 cell-surface translocation facilitated crosstalk with EGFR, and reduced cAMP generation, attenuating inhibition of EGFR signaling. Combination therapy both promoted apoptosis in TAM-R cells and decreased drug-resistant tumor progression.

Conclusions: Long-term endocrine treatment facilitates the translocation of GPR30 to cell surfaces, which interferes with the EGFR signaling pathway; GPR30 also attenuates the inhibition of MAP kinases. These factors contribute to tamoxifen resistance development in breast cancer. Combination therapy with GPR30 inhibitors and tamoxifen may provide a new therapeutic option for drug-resistant breast cancer.

Figure 1

Paraffin-embedded breast cancer tissue immunostained with GPR30 or EGFR antibodies. The predominant staining pattern of GPR30 was cytoplasmic in carcinoma tissue, whereas EGFR was mainly expressed on plasma membrane (A). Cytoplasmic GPR30 immunostaining of breast tumors was (a) negative, (b) weak, (c) moderate or (d) strong. EGFR staining of breast tumors was (e) no staining, (f) moderate and complete staining, or (g) strong and complete staining. GPR30 moderately stained primary tumors (h), but strongly stained corresponding tissue during tamoxifen treatment (i). Quantitative (B) and paired (C) expression of GPR30 was compared in 53 matched tissues from primary tumors (PTs) and their corresponding metastases (MTs). Pair-wise scatter plots showed the correlation of GPR30 and EGFR expression in MTs (D). EGFR, epidermal growth factor receptor; GPR30, G-protein coupled receptor 30.

Figure 2

Effects of 17ß-estradiol (E2), GPR30 agonist G1, and Tam on proliferation of parental MCF-7 cells and tamoxifen-resistant (TAM-R) cells. (A) Cells were counted five days after treatment with the indicated concentrations of E2 and G1. (B) Cells were counted five days after treatment with different concentrations of Tam. (C) Cells were treated with 1 μM Tam in the presence of 10 nM E2 and counted after five days. Each experiment was repeated at least three times; results are expressed as means ± SD from three independent experiments. GPR30, G-protein coupled receptor; Tam, 4-hydroxytamoxifen.

Figure 3

Activation of Erk1/2 responses to E2, G1 or Tam in MCF-7 and TAM-R cells. Erk1/2 expression was investigated by western blot using specific antibodies against phosphorylated (p) and total (t) Erk1/2 protein. Cells were cultured for the indicated times with 1 nM E2 (A), 10 nM G1 (B) or 1 μM Tam (C) before preparation of cell lysates and western blot analysis. t-Erk1/2 expression was used as loading control. E2, 17β-estradiol; Tam, 4-hydroxytamoxifen; TAM-R, tamoxifen resistant.

Figure 4

Role of GPR30/EGFR signaling pathway in phosphorylation of Erk1/2 and EGFR in TAM-R cells. Cells were treated with ethanol (control), 1 μM Tam or 10 ng/ml EGF alone or in combination with the GPR30 antagonist G15 or the EGFR inhibitor AG1478 for 10 minutes. Levels of p-Erk1/2 and p-EGFR were detected by western blot using specific antibodies (A). Fold changes of expression were quantified, normalized to β-actin (B). EGFR, epidermal growth factor receptor; GPR30, G-protein coupled receptor 30; Tam, 4-hydroxytamoxifen; TAM-R, tamoxifen resistant.

Figure 5

Distribution and expression of GPR30 in MCF-7 and TAM-R cells. The GPR30 subcellular location was detected using immunofluorescent staining. (A) Cells were stained with DAPI, GPR30-GFP alone or a combination (Merge). GPR30 mRNA and protein expressions in MCF-7 and TAM-R cells were quantified by qPCR (B) and western blot (C). Fold changes of GPR30 in total protein and membrane-enriched protein fractions of MCF-7 and TAM-R cells were normalized to β-actin (C). Each experiment was repeated at least three times. Results are expressed as means ± SD. *P <0.05 versus MCF-7 cells. DAPI, 4′, 6-diamidino-2-phenylindole; GPR30, G-protein coupled receptor 30; TAM-R, tamoxifen resistant.

Figure 6

Generation of cAMP mediated by GPR30 in MCF-7 and TAM-R cells. Cells were incubated for five minutes with 1 nM E2, 10 nM G1 or 1 μM Tam; ELISA was then performed after preparation of cell lysates. Each experiment was repeated at least three times. Data show means ± SD. *P <0.05 versus similarly treated MCF-7 cells. E2, 17β-estradiol; GPR30, G-protein coupled receptor 30; Tam, 4-hydroxytamoxifen; TAM-R, tamoxifen resistant.

Figure 7

Synergistic effects of GPR30 antagonist G15 and Tam on apoptosis. Cells were treated with ethanol (control), 1 μM Tam, 10 nM G15 alone or 10 nM G15 plus 1 μM Tam for 48 hours using Annexin V-FITC flow cytometry. Scattergrams show numbers of MCF-7 (A) and TAM-R (B) cells in early-phase apoptosis (EA) using the indicated treatments. (C) Histograms show percentages of MCF-7 (A) and TAM-R (B) cells in early-phase apoptosis 48 hours after using the indicated treatments. Data show means ± SD from three independent experiments. * P<0.05, versus control; ** P<0.05 versus Tam treatment. GPR30, G-protein coupled receptor 30; Tam, 4-hydroxytamoxifen; TAM-R, tamoxifen resistant.

Figure 8

Assessing the therapeutic effect of combination therapy with Tam plus G15 in a TAM-R xenograft. Nude mice bearing TAM-R tumors were randomized on day 0 to receive ethanol alone, Tam (50 μg) alone, G15 (4 μg) alone or G15 (4 μg) in combination with Tam (50 μg) (A). Images represent the xenograft tumors in monotherapy and combination therapy groups (B). Paraffin-embedded sections from ethanol alone (a), Tam alone (b), G15 alone (c) or G15 in combination with Tam (d) were labeled for cellular apoptosis using Tunel (C). Histogram shows percentage of apoptotic tumor cells induced by monotherapy or combination therapy for xenograft experiments (D). *P<0.05 versus ethanol alone; ** P <0.05 versus Tam alone. Results are expressed as means ± SD. Tam, 4-hydroxytamoxifen; TAM-R, tamoxifen resistant.

Figure 9

Role of GPR30 in the development of tamoxifen resistance. Long-term endocrine therapy can inhibit ERα-regulated gene transcription in hormone-dependent breast cancer; whereas tamoxifen-facilitated translocation of GPR30 to the cell membrane enhances crosstalk with EGFR signaling through the Gβγ subunit of GPR30. However, when treated with GPR30 plus tamoxifen, GPR30’s Gα subunit attenuates cAMP suppression of Erk1/2 phosphorylation of an EGF downstream factor. As tamoxifen is an agonist for GPR30, endocrine therapy can stimulate GPR30/EGFR crosstalk, leading to cell growth. When this activation effect exceeds ERα inhibition, breast cancer progresses under tamoxifen treatment. Interrupting direct crosstalk between GPR30 and EGFR by use of a GPR30-specific antagonist (G15) induces both cytocidal action in vitro and an antitumor effect in vivo. Targeted therapy with GPR30 could restore endocrine therapy response in tamoxifen-resistant breast cancer. EGFR, epidermal growth factor receptor; ER, estrogen receptor; GPR30, G-protein coupled receptor 30.