Abstract:
Kisspeptin receptor, also known as KISS1R, is the endogenous receptor for the ligand peptide, kisspeptin. It is a G-protein coupled receptor that couples through the Gαq/11 pathway. It has been shown that KISS1R activation by kisspeptin results in the activation of phospholipase C, calcium mobilization and ERK1/2 phosphorylation. Moreover, kisspeptin production has been linked to inhibition of metastasis in many cancers. However, in breast cancer, conflicting studies have shown that kisspeptin and KISS1R may play pro-metastatic or anti-metastatic roles. The aim of this study was to decipher the role of endogenous KISS1R in breast cancer cell proliferation, invasion and migration. Firstly, endogenous protein expression of the receptor was determined in 3 breast cancer cell lines by western blotting. Secondly, the signalling potential of the receptor was determined by assessing ERK1/2 phosphorylation after stimulation of cells with the ligand, KP-10. The effects of KP-10 stimulation on the growth rate and migration speed of the cell lines were assessed using crystal violet staining and scratch assay, respectively. Lastly, the last exon, exon 5 of the KISS1R gene in the BT-20 and MDA-MB-231 cell lines was assessed by Sanger sequencing. The results show that KISS1R protein is expressed in all the breast cancer cell lines tested albeit at significantly different levels. The non-metastatic BT-20 cell line express a much higher level of KISS1R compared to the metastatic MDA-MB-231 cell line or the migratory, MCF-7 cell line. Furthermore, ERK1/2 phosphorylation analysis after ligand stimulation suggests that only BT-20 cells muster a KISS1R response while no ERK1/2 phosphorylation is seen in MDA-MB-231 cells. The temporal nature of the ERK1/2 response suggests that it is a β-arrestin related activation of ERK1/2. Interestingly, β-arrestin1/2 expression analysis shows high levels of β-arrestin1/2 expression in BT-20 cells but very little in MDA-MB-231 cells. Physiologically, treatment with the KISS1R ligand, KP-10, had no significant impact on cell growth and migration. However, under serum free culture conditions, there was an increase in the migration of MDA-MB-231 cell line treated with KP-10, compared to the untreated control. In the BT-20 and MDA-MB-231 cell lines, a KISS1R variant was identified which was characterized by a c.1091T>A in exon 5, resulting in the substitution of leucine to histidine (p.L364H) in the C-terminus or cytoplasmic tail of the receptor. Overall, our data suggest that KISS1R expression correlates with the migratory potential of the cells and the KISS1R in the BT-20 cells activates ERK1/2 through a G-protein independent mechanism.
