GPER1 Activation Exerts Anti-Tumor Activity in Multiple Myeloma

Abstract

G protein-coupled estrogen receptor 1 (GPER1) activation is emerging as a promising therapeutic strategy against several cancer types. While GPER targeting has been widely studied in the context of solid tumors, its effect on hematological malignancies remains to be fully understood. Here, we show that GPER1 mRNA is down-regulated in plasma cells from overt multiple myeloma (MM) and plasma cell leukemia patients as compared to normal donors or pre-malignant conditions (monoclonal gammopathy of undetermined significance and smoldering MM); moreover, lower GPER1 expression associates with worse overall survival of MM patients. Using the clinically applicable GPER1-selective agonist G-1, we demonstrate that the pharmacological activation of GPER1 triggered in vitro anti-MM activity through apoptosis induction, also overcoming the protective effects exerted by bone marrow stromal cells. Noteworthy, G-1 treatment reduced in vivo MM growth in two distinct xenograft models, even bearing bortezomib-resistant MM cells. Mechanistically, G-1 upregulated the miR-29b oncosuppressive network, blunting an established miR-29b-Sp1 feedback loop operative in MM cells. Overall, this study highlights the druggability of GPER1 in MM, providing the first preclinical framework for further development of GPER1 agonists to treat this malignancy.

Keywords: G-1; GPER; multiple myeloma; plasma cell dyscrasias.

Figure 1

Low GPER1 expression is observed in MM and PCL and associates with poor overall survival. (A) Violin plot showing expression levels of GPER1 mRNA in normal samples (n = 24), MGUS (n = 34), SMM (n = 24), MM (n = 172), PCL (n = 9), and HMCLs (n = 23) from GSE6477, GSE6691, GSE13591, and GSE6205 datasets. Each dot represents a single sample. Expression levels were compared using an unpaired Student’s t-test. ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. N = normal donors; MGUS = monoclonal gammopathy of undetermined significance; MM = multiple myeloma; SMM = smoldering MM; PCL = plasma cell leukemia; HMCLs = human MM cell lines. (B) Kaplan-Meyer analysis of the overall survival of 264 MM patients in the Mulligan dataset (GSE9782) with low (blue) and high (red) GPER1 expression levels. Survival analysis was performed using the log-rank test. (C) WB analysis of GPER1 protein expression in MM cell lines. Densitometric analysis of protein expression in three different experiments is reported on the right; α-tubulin was used as a loading control.

Figure 2

G-1 triggers anti-MM activity in vitro. (A) Cell viability was assessed by CTG assay in MM cell lines as well as in three different NDMM patient-derived plasma cells (MM pt#1, MM pt#2, and MM pt#3), 48 h after G-1 treatment. (B) BrdU incorporation assay was performed in U266 and INA-6 MM cell lines after 48 h of G-1 exposure. Data represent the average of at least three independent experiments. * p < 0.05. (C) Cell viability was assessed by CTG assay on PBMCs derived from three different healthy donors (HD), 48 h after G-1 treatment.

Figure 3

G-1 triggers apoptosis of MM cells. (A) FACS analysis of Annexin V/7-AAD stained RPMI-8226, NCI-H929, MM1S, OPM2, and INA-6 MM cell lines after 48 h of G-1 treatment. Histogram bars are representative of the percentage of apoptotic cells in at least three independent experiments. * p < 0.05. (B) FACS detection of JC-1 fluorescence of NCI-H929 cells, 8 h after treatment with 2 µM G-1 or vehicle. Dot plots are representative of an independent biological replicate (n = 3). (C) Histogram bars represent the percentage of apoptotic cells assessed by FACS analysis of Annexin V/7-AAD-labeled U266 and NCI-H929 cells, 48 h after 2 µM G-1 treatment, alone or in combination with 20 µM Z-VAD. * p < 0.05. (D) FACS analysis of Annexin V-positive NCI-H929 cells, alone or co-cultured with BMSCs, 48 h after 2 µM G-1 treatment. (E) WB analysis of Caspase-3/7 and PARP in NCI-H929 cells treated with 2 µM G-1; * indicates the cleaved active form; GAPDH was used as a loading control. (F) Cell viability and Caspase 3/7 activities were assessed by CTG assay and Caspase 3/7 Glo assay, respectively, in primary PCs derived from a PCL patient ex-vivo exposed to different concentrations of G-1 for 48 h. * p < 0.05.

Figure 4

G-1 regulates the miR-29b/Sp1 transcriptional feedback loop. (A) qPCR for GPER promoter performed after ChIP with Sp1 antibody, in NCI-H929 cell line treated for 48 h with 2 µM of G-1, or vehicle as control. Data are representative of at least three independent experiments. * p < 0.05. (B) GSE17498 dataset interrogation showing an inverse correlation between mRNA levels of GPER and Sp1 in MM patients (p = 0.009967; R² = 0.0423); linear regression is shown as dotted line. (C) WB of GPER1 and SP1 protein levels in NCI-H929 cells, 24 h after G-1 treatment. GAPDH was used as a loading control. (D) q-RT-PCR of hsa-miR-29b expression in NCI-H929 cells after 2 µM G-1 exposure; results represent the average of miR-29b expression levels after normalization using RNU44 housekeeping and ΔΔCt calculations; * p < 0.05. (E) WB of MCL1 and CDK6 protein levels in NCI-H929 cells, 24 h after G-1 treatment. GAPDH was used as a loading control.

Figure 5

In vivo anti-MM activity of G-1. (A) MM1S cells (5 × 10⁶) were subcutaneously implanted in the flank of NOD.SCID mice. When tumors became palpable, mice were randomized and intraperitoneally treated with G-1 (1 mg/kg), 4 days a week for 3 consecutive weeks, or vehicle. Tumor size was measured by using an electron caliper every other day. Averaged tumor volume of each group ±SD is shown (* p < 0.05). (B) In vivo tumor growth inhibition of luciferase-engineered AMO-BZB subcutaneous xenografts, intraperitoneally treated with G-1 (1 mg/kg), bortezomib (1 mg/kg), or vehicle (NaCl 0.9%). G-1 treatments were performed daily (4 days a week, 3 consecutive weeks), while bortezomib on days 1, 4, 8, and 11. BLI measurements on days 11 and 22 are reported (* p < 0.05). (C) BLI pictures of a representative G-1-, bortezomib- or vehicle-treated mouse, at day 0 and day 7 after the start of treatments.