Selective G-Protein Estrogen Receptor (Gper) Activation Triggers Anti-Multiple Myeloma Activity And Synergizes With Mir-29b-Inducing Drugs

Multiple myeloma (MM) is a plasma cell malignancy which remains incurable despite novel therapeutic approaches targeting both myeloma cells and their bone marrow milieu (BMM). MM cells express estrogen receptors (ER) belonging to both α and β isotypes and selective ER modulators or pure anti-estrogens have demonstrated therapeutic activity against this malignancy. GPER, formerly known as GPR30, is an orphan membrane-associated ER previously described to mediate non-genomic effects of estrogens and whose involvement in the pathophysiology of solid tumors is currently emerging. Here, we studied the expression pattern of GPER and the biological effects triggered by GPER activation using the synthetic compound G-1 ((±)-1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H cyclopenta[c]quinolin-8-yl] ethanone), a selective GPER agonist (Tocris). We detected GPER expression in 9 out of 9 MM cell lines either at mRNA and protein level, as assessed by qRT-PCR and western blotting, respectively. By analysis of our microarray dataset based on plasma cells from 4 normal donors, 11 MGUS, 133 MM and 9 plasma cell leukemias (PCLs), we observed that GPER mRNA levels progressively declined during MM progression, since lower levels were found in PCL and MM samples as compared to healthy controls or MGUS. Interestingly, adhesion of MM cells to bone marrow stromal cells (BMSCs) reduced GPER mRNA levels, supporting a potential role of the BMM in regulating GPER expression. To address the relevance of GPER in modulating MM cell proliferation and/or death mechanisms, first we tested the GPER agonist G-1 in vitro. We found that G-1 inhibited, in a dose-dependent manner, proliferation of IL-6 dependent (INA-6) and independent (MM1R, MM1S, U266, RPMI-8226, NCI-H929, OPM2) MM cell lines, with an IC50 ranging from 2 to 5 microM, while did not affect the survival of peripheral blood mononuclear cells from healthy donors. G-1 treatment caused cell cycle arrest by increasing cells in G0 phase; moreover, it induced a significant and dose-dependent apoptotic cell death in all MM cell lines tested, as assessed by Annexin V/7AAD staining and western blot analysis of active caspases 3, 7 and 9. G-1 promoted the expression of autophagic markers like Beclin-1 and LC3A/B, the cytosolic punctate pattern of LC3B and down-regulated p62/SQSTM-1 expression, indicating functional involvement of GPER in autophagy. Moreover, GPER transduced rapid non-genomic signaling through MAPKs, since G-1-mediated GPER activation triggered phosphorylation of ERK1/2 already after 15’ treatment in MM1S and U266 cells. Importantly, i.p. injection of G-1 (2mg/kg) in SCID mice significantly reduced the growth of subcutaneous MM1S xenografts, as compared to vehicle-treated animals.