Targeting Dopamine Receptor D2 by Imipridone Suppresses Uterine Serous Cancer Malignant Phenotype

Abstract

Uterine serous cancer (USC) is an aggressive subtype of endometrial cancer, with poor survival and high recurrence rates. The development of novel and effective therapies specific to USC would aid in its management. However, few studies have focused solely on this rare subtype. The current study demonstrated that the orally bioavailable, investigational new drug and novel imipridone ONC206 suppressed USC cell proliferation and induced apoptosis both in vitro and in vivo. Disruption of the DRD2-mediated p38MAPK/ERK/PGC-1α network by ONC206 led to metabolic reprogramming and suppression of both glycolysis and oxidative phosphorylation. ONC206 also synergized with paclitaxel in reducing USC cell viability. In addition, DRD2 overexpression correlated with poor overall survival in patients. This study provides the first evidence that ONC206 induced metabolic reprogramming in USC cells and is a promising therapeutic agent for USC treatment. These findings support further development of ONC206 as a promising therapeutic agent and improves survival rates in patients with USC.

Keywords: dopamine receptor D2; imipridone; metabolic reprogramming; uterine serous cancer.

Figure 1

Effect of ONC206 on uterine serous cancer cell viability and apoptosis in vitro. (A) Chemical structure of imipridones ONC201 and ONC206. (B) ONC201 and ONC206 suppress cell viability of ARK1, ARK2, and HEC50 cells after 72 h of treatment, as measured using the MTT assay. Three independent experiments were performed (mean ± standard deviation). (C) ONC201 and ONC206 (0.5 and 10 μM) suppress cell proliferation of ARK1, ARK2, and HEC50 cells, as measured using the xCELLigence real-time cell analysis assay. (D) ONC201 and ONC206 (0.5, 10 and 100 μM) induce cell apoptosis of ARK1, ARK2, and HEC50 cells after 72 h of treatment, as measured using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. Representative images are shown. Results in the bar charts were averaged from three independent experiments and are shown as mean ± standard deviation. *** p < 0.001, ** p < 0.01, * p < 0.05, two-tailed Student t test.

Figure 2

Effect of ONC206 on uterine tumor growth and apoptosis in vivo. (A) Schema shows the use of an in vivo model to evaluate the effect of ONC206 on uterine tumor growth. i.p., intraperitoneal; PBS, phosphate-buffered saline. (B) Images show lower bioluminescence signals in ARK1 cell-bearing nude mice treated with ONC201 (n = 10) or ONC206 (n = 10) than in untreated PBS controls (n = 10). (C) Box plot shows a significantly lower bioluminescence from ARK1 cell-bearing nude mice treated with ONC206 (n = 10) than those treated with ONC201 (n = 10; p < 0.05, Mann–Whitney U test) or untreated PBS controls (n = 10; p < 0.001, Mann–Whitney U test). (D,E) Representative microscopic images of paraffinized sections of tumor tissues collected from ARK1 cell-bearing nude mice 6 weeks after treatment with ONC201 or ONC206, showing significantly (D) lower Ki-67 expression and (E) higher apoptosis in the ONC206 treatment group compared with the ONC201 treatment group or untreated controls. Bar = 100 μm. Quantification of cell staining positively for Ki-67 or apoptotic cells for each group is shown in the box plot. *** p < 0.001, ** p < 0.01, * p < 0.05, n. s. = not significant (p > 0.05), Mann-Whitney U test.

Figure 3

Effect of ONC206 on mitochondrial functions and glycolysis in uterine serous cancer cells. (A) Heat maps obtained using reverse phase protein array analysis shows differentially expressed proteins related to the p38MAPK/ERK signaling network and mitochondrial functions in ARK1 cells with (n = 2) or without (n = 2) 48 h of treatment with 50 μM ONC206. (B) Western blot analyses show decreased protein levels of p-ERK1/2, p-p38MAPK, p-S6, p-CREB, and PGC-1α in ONC206-treated ARK1, ARK2 and HEC50 cells compared with control cells without treatment. β-actin served as a loading control. Ratios between phosphorylated and total protein levels are presented. Three independent experiments were performed. (C) Western blot analyses show lower protein levels of TFAM, SDHA, MT-CO1, COX-IV, SOD1, HK2, PDHA1, and p21 in ONC206-treated ARK1, ARK2, and HEC50 cells compared with control cells without treatment. β-actin served as a loading control. Relative normalized protein levels with respect to the corresponding control are presented. Three independent experiments were performed. (D) ATP production as measured by the ATP production kit, (E) lactate secretion as measured by the Lactate-Glo assay kit, and (F) cytochrome c oxidase activity as measured by the cytochrome c oxidase assay kit were decreased in ARK1, ARK2, and HEC50 cells after treatment with ONC206. Results were averaged from three independent experiments and are shown as mean ± standard deviation. ** p < 0.01, * p < 0.05, two-tailed Student t test. (G) Reactive oxygen species (ROS) productions was increased in ARK1, ARK2, and HEC50 cells after 24 h of treatment with ONC206 as measured using the ROS detection assay kit. Results were averaged from three independent experiments and are shown as mean ± standard deviation. ** p < 0.01, * p < 0.05, two-tailed Student t test.

Figure 4

Role of DRD2 in mediating the effect of ONC206 on uterine serous cancer (USC) cells. (A) DRD2 knockout (DRD2-KO) ARK2 cells are more resistant to ONC206 than control knockout (Control-KO) ARK2 cells, as measured by the MTT assay. Three independent experiments were performed (mean ± standard deviation). (B) Decrease in ATP production in DRD2-KO ARK2 cells after 48 h of treatment with ONC206 as measured by the ATP determination kit. Results were averaged from three independent experiments and are shown as mean ± standard deviation. *** p < 0.001, ** p < 0.01, two-tailed Student t test. (C) Decrease in lactate secretion in DRD2-KO ARK2 cells after 24 h of treatment with ONC206 as measured by the Lactate-Glo kit. Results were averaged from three independent experiments and are shown as mean ± standard deviation. *** p < 0.001, ** p < 0.01, two-tailed Student t test. (D) Representative microscopic images of paraffinized sections of different stages of gynecological tissues including normal endometrium, endometrioid endometrial cancer (EEC; grades 1 to 3) and uterine serous cancer (USC). An increase in DRD2 expression from normal endometrium to USC is shown. Bar = 100 μm. Arrows indicate the uterine gland structures in normal endometrium. (E) Quantification of DRD2 staining intensity for normal endometrium (n = 5), grade 1 EEC (n = 10), grade 2 EEC (n = 10), grade 3 EEC (n = 5) and USC (n = 15) is shown in the box plot. (F) Box plot shows a significant decrease in DRD2 expression in long-term USC survivors (>5 years; n = 6) compared with short-term survivors (<2 years; n = 9; p = 0.012, Mann–Whitney U test).

Figure 5

Effect of ONC206 in combination with paclitaxel on uterine serous cancer cell viability. ARK1, ARK2 and HEC50 cells were treated with ONC206 alone (0–10 μM) or in combination with paclitaxel (0–25 nM) for 72 h. ONC206 synergized with paclitaxel in reducing cell viability of USC cells. Three independent experiments were performed (mean ± standard deviation). The combination index was calculated using CompuSyn (combination index <1 indicates a synergistic effect).