The Role of Adrenomedullin signalling through RAMP3 in Cancer

Adrenomedullin is a pluripotent peptide hormone (AM) involved in many physiological actions. Also, AM is considered to be an important signal transduction hormone in 80% of malignant cells regardless of cell type. AM plays an important role in many processes such as metastasis, proliferation, invasion, angiogenesis and acts as an apoptosis survival factor in cancer cells. AM functions are mediated by two different receptors which are complexes of receptor activity-modifying proteins (RAMPs) and the calcitonin like receptor (CLR), where AM1 (RAMP2+CLR) and AM2 (RAMP3+CLR). The majority of the current studies suggest AM1 is responsible for the physiological actions of AM, while AM2 is suggested to be involved in pathophysiological conditions. Since AM plays an important role and is involved in tumour progression, it was hypothesised that a component of the AM2 receptor which is RAMP3 is involved in signalling between the tumour and stromal tissues, promoting tumour growth and development. The BMA-178-2 cell line expressed AM1 (RAMP2 and CLR) and AM2 receptor components (RAMP3 and CLR). Possibly the expression of the AM2 receptor has contributed to the proliferation, migration and invasion of BMA178-2 cells. The expression of RAMP3 was inhibited using lentiviral particles containing shRNA of 70% at the gene and protein level. This result was confirmed using RT-PCR, Q-PCR and western blotting. In vitro, the proliferation, migration, invasion and cAMP production of the RAMP3 knockdown cells were decreased significantly compared to the control cells. Also, the clonogenic assay revealed that the RAMP3 knockdown cells formed smaller and fewer colonies than the control cells. Interestingly, the chemosensitivity of the RAMP3 knockdown cells to an anti-cancer drug called 5-Fluorouracil was increased compared to the control cells. The effects of knocking down RAMP3 on the proliferation, metastasis and invasion of the cells in vivo was determined by injecting the RAMP3 stable knockdown cells and the control cells into the prostate of wild type mice 129S6/SvEv as well as RAMP3 knockout mice. The histology sections of the lung, bladder and bone shows that the RAMP3 knockout mice injected either with control cells or RAMP3 stable knockdown cells have smaller and fewer metastasised tumours compared to the wild type mice injected either with control cells or RAMP3 stable knockdown cells. Collectively, RAMP3 plays an important role in cancer development as a result of its role in the communication between cells and tumours and communication between tumours and the host cells via the AM2 receptor.