Selective G-Protein Estrogen Receptor (GPER) Activation Triggers Anti-Multiple Myeloma Activity and Synergizes with MiR-29b-Inducing Drugs
Authors: Amodio Nicola , Leotta Marzia , Morelli Eugenio , Calimeri Teresa , Gullà Anna Maria , Stamato Maria Angelica , Romeo Enrica , Foresta Umberto , Neri Antonino , Di Martino Maria Teresa , Tagliaferri Pierosandro , Tassone Pierfrancesco 
Published: October 2019

Abstract
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.
We next evaluated whether G-1-induced effects could be associated to modulation of miRNA levels in MM cells. Indeed, we found that G-1 up-regulated the tumor suppressor miR-29b; this effect was likely a consequence of the down-regulation of the miR-29b transcriptional inhibitor Sp1, whose mRNA and protein levels were reduced after G-1 treatment. Consistently, an inverse correlation between GPER and Sp1 mRNA levels in MM patient plasma cells could be gathered from our microarray dataset. In addition, miR-29b canonical targets, like CDK6 and MCL-1, were down-regulated at protein level in G-1-treated MM cell lines.
Finally, we demonstrated that G-1 synergizes with established miR-29b-inducing compounds, like bortezomib and vorinostat, in the inhibition of MM cell survival.
Taken together, our results indicate that the GPER agonist G-1 is a novel powerful anti-tumor compound enriching the repertoire of investigative anti-MM agents, and further strengthen the role of miR-29b as effector of anti-MM drugs.

Disclosures
No relevant conflicts of interest to declare.