Selective Activation of the Non-Classical Estrogen Receptor Gper Elicits Potent Anti-Tumor Activity in Multiple Myeloma

Within the hematopoietic compartment, estrogens influence the differentiation, proliferation and survival of B cells and also increase the number of plasmacells (PCs) and their capacity to produce immunoglobulins. In addition to the classical estrogen receptors, an extensive body of literature has shed light on a 7-transmembrane spanning G-protein coupled estrogen receptor, GPER (also known as GPR30). The discovery of GPER-selective agonists and antagonists has led to the comprehension of the pathophysiological role of GPER and its involvement in cancer biology. However, the functional role of GPER in hematological malignancies has not been previously investigated.

We detected GPER expression in 12 MM cell lines, including dexamethasone, bortezomib and carfilzomib-resistant cell lines, as well as in Waldestrom Macroglobulinemia (WM) cell lines either at mRNA and protein level, as assessed by qRT-PCR and western blotting, respectively. Importantly, MM patients' expression dataset analysis revealed a progressive decline of GPER mRNA levels during MM progression. Moreover, adhesion of MM cell lines (NCI-H929, U266) to bone marrow stromal cells (BMSCs) reduced GPER mRNA and protein levels even further, supporting a potential role of the BM microenvironment in regulating GPER expression.

To address the role of GPER-mediated signaling on MM cell survival and cell death, first we tested the selective GPER agonist 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) in vitro. G-1 inhibited, in a dose-dependent manner, the survival of WM (n=2) and MM cell lines (n=9), as well as of primary MM cells (n=3), with an IC50 at 48 h ranging from 2 to 3 μM, while it did not affect the viability of healthy donors' PBMCs (n=3); moreover, G-1 synergized with bortezomib in MM1S MM cells and with ibrutinib in BCMW1 WM cells. Both in MM and WM cell lines, G-1 treatment increased cells in G2/M phase and induced a potent and dose-dependent apoptotic cell death, as assessed by Annexin V/7AAD staining and western blot analysis of active caspases 3, 7 and 9. G-1-induced apoptosis was reverted by the pan-caspase inhibitor ZVAD-FMK and was not impaired by co-colture with BMSCs or by exogenous IL-6, IGF-1 or HGF. 17β-estradiol (10nM) and the selective GPER-antagonist G-15 (0.5µM) slightly increased the survival of MM and WM cells, and this effect was overcome by G-1 treatment. Moreover, G-1 induced the occurrence of a cytoprotective autophagy, as shown by the expression of autophagic markers, such as beclin-1 and LC3A/B, the cytosolic punctate pattern of LC3B and down-regulation of p62/SQSTM1 expression, and treatment of MM and WM cell lines with the autophagy inhibitor chloroquine potentiated G-1-triggered anti-survival effects. Importantly, intraperitoneal injection of G-1 (2mg/kg) in SCID mice significantly reduced the growth of subcutaneous MM1S and bortezomib-resistant AMO-abzb xenografts, and prolonged survival of treated animals.

Since GPER-dependent molecular effects in solid tumors have been attributed to modulation of MAPK activity, by using an antibody array we analyzed the phosphorylation status of 24 protein kinases after GPER activation. Notably, treatment with G-1 (2µM) reduced the phosphorylation of ERK1/2, p38α-γ, AKT and GSK3α-β.

Finally, on the light of the emerging role of miRNAs in MM pathobiology, we investigated by TaqMan qPCR the effects of G-1 on the miRNA profile and found that G-1 induced the expression of tumor suppressor miR-29b by down-regulating Sp1, the major miR-29b-negative regulator, thus disrupting the previously reported miR-29b/Sp1 negative feedback loop. Consistently, an inverse correlation between GPER and Sp1 mRNA levels emerged in MM patient PCs. In addition, miR-29b canonical targets, such as CDK6 and MCL-1, were downregulated in G-1-treated MM cells.

Altogether, our results indicate that GPER is expressed in MM and WM and its selective activation via G-1 triggers potent anti-tumor activity through inhibition of oncogenic protein kinases and activation of miR-29b, providing the preclinical rationale for clinical investigation of GPER-agonists to treat these hematological malignancies.