Characterisation of the direct antiproliferative effects of a gonadotrophin-releasing hormone analogue

Abstract

Gonadotrophin-releasing hormone (GnRH) can inhibit proliferation of multiple reproductive tissue cancer cell lines through direct interaction with GnRH receptors (GnRHR) on tumour cells. GnRH analogues may therefore have a role in treating some cancers. The signalling pathways associated with these inhibitory effects are poorly defined, and characterising them may help to understand therapeutic sensitivity. To elucidate these pathways, transcriptomic and proteomic approaches were used to compare the effects of the GnRH agonist Triptorelin in responsive GnRHR-transfected HEK293 cells (SCL60) and unresponsive (HEK293) cells both in vitro for up to 24h and in vivo for up to 7 days. Gene expression profiling demonstrated that SCL60 gene expression was temporally regulated with Triptorelin treatment, with expression of some genes increased at one time point but decreased at another. Early and mid-phase gene expression changes comprised mainly transcription factors and late changes included the hormonal signalling component CGA. Pathway analysis implicated mitogen-activated protein kinase and cell cycle pathways, supporting the detection of G2/M arrest. Signalling effects within SCL60 xenografts, 4 and 7 days following Triptorelin treatment, were investigated using a phosphoproteomic antibody array. Changes included cell cycle and apoptosis regulators, as well as cell surface receptors and NFκB signalling pathway members. Reverse-phase protein arrays and western blotting also showed that pAkt was decreased and pNFκB-p65 was increased after Triptorelin treatment in vitro. An NFκB inhibitor enhanced the anti-proliferative effect of Triptorelin in SCL60 cells in vitro, suggesting that NFκB acts as a survival factor in the response to GnRHR stimulation. A range of GnRHR expression was observed in breast cancer tumours by immunohistochemistry, and on average GnRHR expression was significantly higher in the Triple Negative Phenotype (TNP) subgroup and in grade 3 tumours. A GnRHR-transfected breast cancer cell line, MCF7-h14, was developed. Despite this expressing a similar level of GnRHR to responsive SCL60 cells, MCF7-h14 cells were not inhibited by GnRHR activation, indicating that a high level of GnRHR is insufficient for the antiproliferative effects of Triptorelin.