Relaxin reduces xenograft tumour growth of human MDA-MB-231 breast cancer cells

Abstract

IntroductionRelaxin levels are increased in cases of human breast cancer and has been shown to promote cancer cell migration in carcinoma cells of the breast, prostate gland and thyroid gland. In oestrogen receptor alpha-negative MDA-MB-231 human breast cancer cells, relaxin was shown to down-regulate the metastasis-promoting protein S100A4 (metastasin), a highly significant prognostic factor for poor survival in breast cancer patients. The cellular mechanisms of relaxin exposure in breast cancer cells are not fully understood. The aim of this study was to investigate short-term and long-term effects of relaxin on cancer cell motility and S100A4 expression and to determine the long-term effects of relaxin on in vivo tumour growth in an oestrogen-independent context.MethodWe have established stable transfectants of highly invasive oestrogen-receptor alpha-negative MDA-MB-231 human breast cancer cells with constitutive expression of bioactive H2-relaxin (MDA/RLN2). RLN2 secretion was determined by ELISA. Relaxin receptor RXFP1 (Relaxin-family-peptide) was detected by reverse transcription (RT) PCR and its activation was assessed by induction of cyclic adenosine monophosphate (cAMP). Stable MDA/RLN2 clones and RLN2 treated MDA-MB-231 cells were subjected to motility and in vitro-invasion assays. Proliferation was assessed in bromodeoxyuridine (BrdU) and MTT assays. S100A4 expression was determined by RT-PCR and Western blot. Specific small interfering RNA was employed to down-regulate relaxin receptor and S100A4. MDA/EGFP vector control and two MDA/RLN2 clones were injected subcutaneously in nude mice to determine tumour growth and cancer cell invasiveness in vivo. Xenograft tumour tissues were assessed by histology and immunohistochemistry and frozen tissues were used for the detection of S100A4 and RLN2.ResultsShort-term exposure to relaxin for 24 hours increased cell motility in a relaxin receptor-dependent manner. This increase in cell motility was mediated by S100A4. Long-term exposure to relaxin secreted from stable transfectants reduced cell motility and in vitro invasiveness. Relaxin decreased cell proliferation and down-regulated cellular S100A4 levels in MDA-MB-231 and T47D breast cancer cells. Stable MDA/RLN2 transfectants produced smaller xenograft tumours containing reduced S100A4 protein levels in vivo.ConclusionOur results indicate that long-term exposure to relaxin confers growth inhibitory and anti-invasive properties in oestrogen-independent tumours in vivo, which may in part be mediated through a down-regulation of S100A4.