Inactivation of GPR30 reduces growth of triple-negative breast cancer cells: possible application in targeted therapy

Abstract

Triple-negative breast cancers lack estrogen receptor α (ERα), progesterone receptor, and do not overexpress human epidermal growth factor receptor 2 (Her-2). They are neither susceptible to endocrine therapy nor to a therapy using the anti-Her-2 antibody, trastuzumab. Therefore, an efficient targeted therapy is warranted. Triple-negative breast tumors frequently express membrane bound estrogen receptor G-protein coupled receptor (GPR30). As proof of principle, we analyzed the consequences of a knock-down of GPR30 expression on the growth regulation of triple-negative breast cancer cell lines. Cells of triple-negative breast cancer cell lines were transfected with siRNA against GPR30 or control siRNA, and cell growth was stimulated either with 10−9 M 17β-estradiol or 10−6 M 4-hydroxytamoxifen. Cell proliferation was measured using Alamar blue staining. Activation of c-Src and epidermal growth factor (EGF)-receptor was assessed using western blot. Expression of c-fos was quantified by reverse transcription polymerase chain reaction. Seven days after transfection with siRNA, GPR30 mRNA in triple-negative breast cancer cell lines MDA-MB-435 and HCC1806 was reduced by 74 and 90%, respectively. 10−8 M 17β-estradiol enhanced proliferation of MDA-MB-435 to 129.6 ± 5.4% of control (p < 0.05) and HCC1806 to 156.9 ± 15.4% of control (p < 0.05), respectively. 10−6 M 4-hydroxytamoxifen increased cell number of MDA-MB-435 to 121.0 ± 6.9% of control (p < 0.05) and HCC1806 to 124.5 ± 12.1% of control (n.s.), respectively. This increased proliferation by the two estrogenic compounds was completely prevented by knock-down of GPR30 expression in both cell lines. In control cells, activity of Src kinase was increased 3-fold by estradiol and 3.8-fold using 4-hydroxytamoxifen. Transactivation of the EGF-receptor was similarly increased in both cell lines by 17β-estradiol and 4-hydroxytamoxifen. Both compounds increased c-fos expression 1.5- and 3.1-fold, respectively. Knock-down of GPR30 expression completely abolished activation of all these signaling pathways responsible for enhanced proliferation. A pharmacological inhibition of GPR30 by specific small molecular inhibitors might prove to be an appropriate targeted therapy of triple-negative breast cancer in the future.

Inhibition of growth hormone receptor by Somavert reduces expression of GPER and prevents growth stimulation of triple‑negative breast cancer by 17β‑estradiol

“…Following stimulation of GPER with 17β-estradiol c-src and EGFR are activated by phosphorylation (8,19). As reported earlier (8) this growth stimulation is dependent on the presence of GPER in TNBC.…”

“…Cyclin D1 regulates of the transition from G1-phase to S-phase of the cell cycle. As we reported earlier expression of cyclin D1 and aromatase is induced by 17β-estradiol in a GPER dependent manner (8). Expression of cyclin D1 was analyzed in Somavert treated TNBC cells by RT-PCR after stimulation with 10 -8 M 17β-estradiol (Fig.…”

“…Our recent observation, that 17β-estradiol stimulates growth of TNBC cell lines, despite the lack of ERα expression, points to an involvement of GPER in malignant transformation of TNBC. A knock-down of GPER using specific siRNA completely prevented this growth stimulation of TNBC by 17β-estradiol (8). A pharmacological inhibition of GPER by the specifically developed inhibitor (G15) or with estriol was also successful in TNBC cell lines but super-physiologically high concentrations of these compounds were needed to achieve a sufficient inhibition of growth (9).…”

The Effect of Activating Fibroblast Growth Factor Receptor 3 Mutations on Osteogenic Differentiation and Ectopic Bone Formation by Human Periosteal Derived Cells

“…Upon investigation of metabolic activity, previously used as a measure of cell proliferation [20], cells carrying the N540K mutation displayed a high profile within the cultures until day 11. The increased metabolic activity in the N540K mutant, when compared to all other cell populations, appears to be the opposite of what has previously been shown in activating FGFR3 mutants in the growth plate in vivo [6].…”

Adeno-associated virus type 2 infection of nude mouse human breast cancer xenograft induces necrotic death and inhibits tumor growth

“…22 This cell line is categorized as being representative of the triple-negative (ER -/PR -/HER2 -) breast disease type. 23 Additionally, the MDA-MB-435 cell line has also been shown to form tumors when injected into the mammary fat pads of immuno-compromised mice. 22 In the current study, AAV2 infection induced 100% cell death of the MDA-MB-435 cell line and was achieved via activation of caspases which regulate both the intrinsic as well as the extrinsic pathways of apoptosis.…”