Stimulation of proliferation of MCF-7 breast cancer cells by a transfected splice variant of growth hormone-releasing hormone receptor

Abstract

Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as an autocrine/paracrine growth factor for various human cancers. A splice variant (SV) of the full-length receptor for GHRH (GHRHR) is widely expressed in various primary human cancers and established cancer cell lines and appears to mediate the proliferative effects of GHRH. To investigate in greater detail the role of SV1 in tumorigenesis, we have expressed the full-length GHRHR and its SV1 in MCF-7 human breast cancer cells that do not possess either GHRHR or SV1. In accordance with previous findings, the expression of both GHRHR and SV1 restored the sensitivity to GHRH-induced stimulation of cell proliferation, with SV1 being more potent than the GHRHR. Furthermore, MCF-7 cells transfected with SV1 proliferated more quickly than the controls, even in the absence of exogenously added GHRH, suggesting the existence of intrinsic, ligand-independent activity of SV1 after its transfection. In agreement with the stimulation of cell proliferation, the levels of proliferation markers cyclin D1, cyclin E, and proliferating cell nuclear antigen were elevated in MCF-7 cells treated with GHRH, cultured in both serum-free and serum-containing media. In addition, SV1 caused a considerable stimulation of the ability of MCF-7 cells to grow in semisolid medium, an assay considered diagnostic for cell transformation. Collectively, our findings show that the expression of SV1 confers oncogenic activity and provide further evidence that GHRH operates as a growth factor in breast cancer and probably other cancers as well.

Fig. 1.

Expression of GHRHR(s) in MCF-7 cells. (a) Immunocytochemical detection of SV1 receptor expression in MCF-7 pcDNA3-SV1 transfected cells. (b) Proliferation rate of MCF-7 cells exposed to different concentrations of GHRH following transfection with pcDNA3 (controls), pcDNA3-SV1, and pcDNA3-GHRHR. Ectopic expression of both SV1 and GHRHR restored sensitivity to GHRH with a peak at 0.1 μM GHRH. In the absence of GHRH, a significant stimulation of cell proliferation was noted in the SV1-transfected cells that is consistent with the ligand-independent activity of this alternatively spliced form of GHRHR. ∗, P < 0.01; ∗∗, P < 0.001.

Fig. 2.

Representative results of the immunocytochemical analysis of MCF-7 cells transfected with pcDNA3, pcDNA3-SV1, and pcDNA3-GHRHR for cyclin D1 (Left), cyclin E (Center), and PCNA (Right), in the presence (+GHRH) or the absence (−GHRH) of exogenously added GHRH. Cells were cultured in media containing 10% FBS. The arrows indicate positive cells.

Fig. 3.

Representative results of the immunocytochemical analysis of MCF-7 cells transfected with pcDNA3, pcDNA3-SV1, and pcDNA3-GHRHR for cyclin D1 (Left), cyclin E (Center), and PCNA (Right) in the presence (+GHRH) or the absence (−GHRH) of exogenously added GHRH. Cells were cultured in serum-free media. The arrows indicate positive cells.

Fig. 4.

Graphic presentation of the fraction of MCF-7 cells transfected with pcDNA3, pcDNA3-SV1, and pcDNA3-GHRHR that was positive for cyclin D1 expression. The presence or absence of exogenously added GHRH in the culture media is indicated. (a) Cyclin D1 (+FBS). Shows the experiment performed with media-containing serum (FBS). (b) Cyclin D1 (−FBS). Shows the experiment performed with serum-free media. ∗, P < 0.05; ∗∗, P < 0.01.

Fig. 5.

Graphic presentation of the fraction of MCF-7 cells transfected with pcDNA3, pcDNA3-SV1, and pcDNA3-GHRHR that was positive for cyclin E expression. The presence or absence of exogenously added GHRH in the culture media is indicated. (a) Cyclin E (+FBS). Shows the experiment performed with media-containing serum (FBS). (b) Cyclin E (−FBS). Shows the experiment performed with serum-free media. ∗, P < 0.05; ∗∗, P < 0.01.

Fig. 6.

Graphic presentation of the fraction of MCF-7 cells transfected with pcDNA3, pcDNA3-SV1, and pcDNA3-GHRHR that was positive for PCNA expression. The presence or absence of exogenously added GHRH in the culture media is indicated. (a) PNCA (+FBS). Shows the experiment performed with media-containing serum (FBS). (b) PCNA (−FBS). Shows the experiment performed with serum-free media. ∗, P < 0.05; ∗∗, P < 0.01.

Fig. 7.

Growth in soft agar of MCF-7 cells transfected with pcDNA3 or pcDNA3-SV1. (a) Graphic presentation of the results. (b and c) Representative microphotographs. The expression of SV1 stimulated the ability of MCF-7 cells to grow in soft agar. (b) Colonies formed by SV1 expressing cells are larger and noncanonical in shape. ∗, P < 0.05.