TRPV2 mediates adrenomedullin stimulation of prostate and urothelial cancer cell adhesion, migration and invasion

Abstract

Adrenomedullin (AM) is a 52-amino acid peptide initially isolated from human pheochromocytoma. AM is expressed in a variety of malignant tissues and cancer cell lines and was shown to be a mitogenic factor capable of stimulating growth of several cancer cell types. In addition, AM is a survival factor for certain cancer cells. Some data suggest that AM might be involved in the progression cancer metastasis via angiogenesis and cell migration and invasion control. The Transient Receptor Potential channel TRPV2 is known to promote in prostate cancer cell migration and invasive phenotype and is correlated with the stage and grade of bladder cancer. In this work we show that AM induces prostate and urothelial cancer cell migration and invasion through TRPV2 translocation to plasma membrane and the subsequent increase in resting calcium level.

Figure 1. Adrenomedullin increases PC-3 and T24/83 cell adhesion, migration and invasion.

(A) RT-PCR experiment showing RAMP2, RAMP3 and CLR expression in PC-3 and T24/83 cells. (B) PC-3 (left panel) and T24/83 (right panel) cell adhesion was examined by seeding 3*10⁴ and 1.5*10⁴ cells respectively per well in 96-well plates pre-coated with fibronectin, and incubated for 45 min with or without AM (200 nM) (N = 3, *P<0.05 compared with control cells). β1 integrin phosphorylation was studied by western-blotting on total proteins extracted from PC-3 and T24/83 cells seeded on fibronectin coated plates and treated with or without AM. (C) PC-3 and T24/83 cell migration was studied by Transwell assay after 8 h of treatment (N = 3, *P<0.05 compared with control cells). FAK phosphorylation was studied by western-blotting on total proteins extracted from PC-3 and T24/83 cells treated with or without AM. (D) For invasion assay, transwell membrane was pre-coated with 50 µg Matrigel, and PC-3 and T24/83 cells were let to invade for 24 h (N = 3, *P<0.05 compared with control cells).

Figure 2. Adrenomedullin effect is mediated by TRPV2.

(A) Western-blotting analysis of TRPV2 protein level in PC-3 and T24/83 cells treated with either siCTL or siTRPV2 (50 nM, 48 h). Effect of TRPV2 silencing (siTRPV2, 50 nM, 48 h) (B) on PC-3 and T24/83 cell adhesion to fibroncectin incubated or not with AM (200 nM, 45 min) (N = 3, *P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM); (C) on PC-3 and T24/83 cell migration examined by transwell assay after 8 h incubation with or without AM (N = 3 *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM); (D) on PC-3 and T24/83 cell invasion through matrigel (AM 200 nM, 24 h) (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM).

Figure 3. Adrenomedullin induces TRPV2 translocation to plasma membrane.

(A) The effect of AM (200 nM, 45 min) and TRPV2 silencing (siTRPV2, 50 nM, 48 h) on basal cytosolic calcium of PC-3 and T24-83 cells was studied by calcium imaging. (n = 120 cells, N = 4, *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM). (B) TRPV2 presence at the plasma membrane was examined by biotinylation on T24/83 cells control or either treated with AM (200 nM, 45 min) or AM and PI3K inhibitor LY294.002 (10 µM, added 5 min before AM). (C) Effect of LY294.002 on PC-3 and T24/83 cell migration examined by transwell assay after 8 h incubation with or without AM (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM). (D) Effect of LY294.002 on AM-induced migration of PC-3 and T24/83 TRPV2-silenced cells examined by transwell assay after 8 h incubation with or without AM (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM).