The bioactive peptide hormone adrenomedullin (AM) is a potent vasodilator and plays a pivotal role in several physiological mechanisms such as hormone secretion, vasculogenesis, lymphangiogenesis, and angiogenesis. AM mediates its function via two receptors;​ AM1 (CLR/RAMP2) and AM2 (CLR/RAMP3) receptors. AM signalling has been shown to impart a beneficial effect on cancer cells by enhancing proliferation, evading apoptosis, potentiating EMT, inducing angiogenesis, and tumour development. Although the pro-tumourigenic effect of AM has been established in several solid tumours, the effect of RAMP3 on cell growth, survival, and metastasis of hormone dependent and hormone independent breast carcinoma are still unexplored. We hypothesize that AM signalling via CLR/RAMP3 is critical for cancer progression and tumour development of hormone-negative breast cancer. To test this hypothesis, triple negative metastatic human breast cancer cell line MDA-MB-231 was chosen. The preliminary receptor expression results displayed that AM, CLR, RAMP1, RAMP2, and RAMP3 were observed in MDA-MB-231 cells validated by end-point RT-PCR, DNA sequencing, qRT-PCR and western blotting. The cells were then transfected with RAMP3 using shRNA mediated lentiviral transfection by Lipofectamine®. Knockdown of RAMP3 was significantly reduced and the expression confirmed by western blotting. Amongst the transfected RAMP3 shRNA clones, only co-transfected RAMP3 significantly inhibited RAMP3 expression by 50% compared to control cells and was taken further for subsequent in vitro and in vivo studies. RAMP3 knockdown displayed increased AM expression and did not affect the expression of CLR, RAMP1, and RAMP2 validated by end-point RT-PCR, qRT-PCR and western blotting.

In vitro cell-based functional assays such as cell proliferation and viability assays revealed that RAMP3 knockdown did not affect cellular growth. AM agonist at 1 µM did not induce proliferation and viability of both control and knockdown cells, while AM antagonist at 1 µM significantly suppressed proliferation and viability of only control cells. Contrary to growth assay results, RAMP3 knockdown significantly decreased migration, invasion, clonogenicity and enhanced apoptosis compared to control cells. AM agonist and antagonist did not alter migration and apoptosis of control and knockdown cells. However, the cAMP assay results showed no effect on both MDA-MB-231 and RAMP3 knockdown cells stimulated with exogenous AM (1-52) on cAMP level. Interestingly, in vivo model using BALB/c nude mice revealed that RAMP3 knockdown tumours had significantly higher necrosis, and CD31 expression compared to control cells. Despite an increase in tumour volume, RAMP3 knockdown did not induce a significant change compared to control cells. Based on in vivo and in vitro results, RAMP3 knockdown may not function as a growth factor in initiating tumour proliferation.

Overall, RAMP3 has been shown to play an important role in migration, invasion, apoptosis, clonogenicity, necrosis, and angiogenesis of hormone-negative breast cancer.