Targeting dopamine receptor D2 as a novel therapeutic strategy in endometrial cancer

Abstract

Background: ONC201 is a dopamine receptor D2 (DRD2) antagonist that inhibits tumor growth in preclinical models through ClpP activation to induce integrated stress response pathway and mitochondrial events related to inhibition of cell growth, which is being explored in clinical trials for solid tumors and hematological malignancies. In this study, we investigated the anti-tumorigenic effect of ONC201 in endometrial cancer cell lines and a genetically engineered mouse model of endometrial cancer.

Methods: Cell proliferation was assessed by MTT and colony formation assays. Cell cycle and apoptosis were evaluated by Cellometer. Invasion capacity was tested using adhesion, transwell and wound healing assays. LKB1^fl/flp53^fl/fl mouse model of endometrial cancer were fed a control low fat diet versus a high fat diet to mimic diet-induced obesity. Following tumor onset, mice were treated with placebo or ONC201. Metabolomics and lipidomics were used to identify the obesity-dependent effects of ONC201 in the mouse endometrial tumors. DRD2 expression was analyzed by immunohistochemistry in human endometrioid and serous carcinoma specimens. DRD2 mRNA expression from the Cancer Genome Atlas (TCGA) database was compared between the four molecular subtypes of endometrial cancer.

Results: Increasing DRD2 expression in endometrial cancer was significantly associated with grade, serous histology and stage, as well as worse progression free survival and overall survival. Higher expression of DRD2 mRNA was found for the Copy Number High (CNH) subtype when compared to the other subtypes. ONC201 inhibited cell proliferation, induced cell cycle G1 arrest, caused cellular stress and apoptosis and reduced invasion in endometrial cancer cells. Diet-induced obesity promoted endometrial tumor growth while ONC201 exhibited anti-tumorigenic efficacy in the obese and lean LKB1^fl/fl/p53^fl/fl mice. Metabolomic analysis demonstrated that ONC201 reversed the obesity-driven upregulation of lipid biosynthesis and reduced protein biosynthesis in obese and lean mice.

Conclusion: ONC201 has anti-tumorigenic effects in endometrial cancer cells and a transgenic mouse model of endometrial cancer, and DRD2 expression was documented in both human serous and endometrioid endometrial cancer. These studies support DRD2 antagonism via ONC201 as a promising therapeutic strategy for endometrial cancer that has already demonstrated pharmacodynamic activity and clinical benefit in both serous and endometrioid endometrial cancer patients.

Keywords: DRD2; Endometrial cancer; Invasion; ONC201; Proliferation.

Fig. 1

The expression of DRD2 in human ECs. The expression of DRD2 was detected by immunohistochemistry in 118 human tissues of serous and endometrioid ECs (a). Using median composite H-scores, DRD2 expression demonstrated statistically significant positive association with grade, stage, histologic types, PFS and OS in ECs (b and c). Identification of DRD2 mRNA expression from TCGA database in 371 human endometrial tumors based on histology (d) and molecular subtypes (e). High expression of DRD2 was found in serous-like copy-number high subtype when compared to the other subtypes. CN Low: Copy number low. CN high: Copy number high. MSI: Microsatellite Instability

Fig. 2

ONC201 induced dose-dependent growth inhibition in EC cell lines. Four endometrial EC cell lines, Ishikawa, ECC-1, HEC1A and KLE, were treated with various doses of ONC201 for 72 h. Cell proliferation was determined by MTT assay. Relative survival was determined by dividing the number of remaining ONC201 treated cells by the number of remaining viable DMSO (control). Representative dose-response curves and IC50 values are shown (a). The effect of ONC201 on the colony forming ability in ECC-1 and KLE cells was assessed through a colony-formation assay (b). The ECC-1 and KLE cells displayed distinct morphological alterations after 48 h treatment with ONC201 (c). Western blot analysis was performed to detect the expression of DRD2 in the four EC cell lines (d) and alternations of DRD2 and DRD5 after ONC201 treatment for 24 h in ECC-1 and KLE cell lines (e). The ECC-1 and KLE cells were cultured with various concentrations of ONC201 for 24 h followed by Western blot analyses to determine the expression of phosphorylation of AKT and S6. The results demonstrate that ONC201 inhibited the mTOR pathway in both cell lines (f). The levels of β-actin or α-tubulin served as protein loading controls. Data are representative of one of three independent experiments

Fig. 3

ONC201 induced cell cycle G1 arrest and apoptosis in EC cells. The ECC-1 and KLE cells were treated with various doses of ONC201 for 36 h. Cellometer analysis showed ONC201 induced cell cycle arrest in G1 phase in both cell lines (a). Cell lysates from the ECC-1 and KLE cell lines were subjected to Western blotting analysis for CDK4 and Cyclin D1 after treatment ONC201 for 24 h (b). Apoptosis was determined by Annexin V/ PI analyses after 24 h of treatment with ONC201 in ECC-1 and KLE cells. The profiles of Annexin V-FITC/PI staining are presented. The percentage of early apoptotic cells is located in the lower right quadrant (c). ECC-1 and KLE cells were cultured with different concentrations of ONC201 for 24 h, and then subjected to Western blot analysis on the levels of anti-apoptotic proteins. ONC201 reduced BCL-2 and MCL-1 expression in both cell lines (d). Data are representative of one of three repeats

Fig. 4

ONC201 induced cellular stress in EC cells. The ECC-1 and KLE cells were treated with ONC201 at indicated doses for 24 h and ROS production was assessed by DCFH-DA assay. ONC201 increased ROS production in a dose dependent manner in both cell lines (a). The cell lysates underwent Western blotting analysis for PERK, Bip, PD-1, Erol-1 and IRE1-a in both cells after 24 h treatment with ONC201. The results showed that ONC201 increased the expression of PERK, Bip, PD-1, Erol-1 and IRE1-a (b). The levels of α-tubulin served as protein loading controls. Data are representative of three or more independent experiments. *p < 0.05 and **p < 0.01

Fig. 5

ONC201 reduced adhesion and invasion in EC cells. The ECC-1 and KLE cells were cultured with different doses of ONC201 for 90 min. Adhesion was determined by laminin-1 assay (a). Invasion was assessed by transwell assay in both cell lines after treatment with ONC201 for 6 h (b). Migration was measured by wound healing assay after treatment with ONC201 for 48 h (c). ONC201 significantly reduced the ability of adhesion, migration and invasion in both cell lines. Cell lysates from ECC-1 and KLE were subjected to Western blotting analysis for VEGF, Slug and E-cadherin after 24 h of treatment with ONC201 (d). ONC201 reduced the expression of VEGF and Slug and increased E-cadherin expression in both cell lines. The levels of α-tubulin served as protein loading controls. Results shown are representative of four independent experiment. *p < 0.05 and **p < 0.01

Fig. 6

ONC201 inhibited tumor growth in the LKB1fl/fl/p53fl/fl mouse model of EC. The LKB1^fl/fl p53^fl/fl mouse genetically engineered mouse model of EC was used to evaluate the effect of ONC201 in vivo. The obese (HFD) or lean (LFD) mice were injected Ad Cre into left side of uterus at 6–8 weeks of age to induce ECs. After 8 weeks of injections, the mice were treated with ONC201 (130 mg/kg, oral, weekly) or vehicle for 4 weeks. The mean tumor weight was reduced in the ONC201 treatment groups in both obese and lean mice (a). Serum VEGF from each group was measured by ELISA assay (b). ONC201 decreased serum VEGF level in obese and lean mice treated with ONC201. The expression of KI67, VEGF, phosphorylated-S6 and BCL-2 was assessed using immunohistochemistry following ONC201 or placebo treatment in endometrial tumors under obese and lean conditions (c). ONC201 significantly reduced the expression of Ki67, VEGF, BCL-2 and phosphorylated-S6 in endometrial tumors under obese and lean conditions. *p < 0.05 and **p < 0.01

Fig. 7

The effect of ONC201 on metabolites in the LKB1^fl/fl/p53^fl/fl mouse model of EC. Metabolomic profiling were analyzed from the four groups (N = 5/group) by Metabolon. Endometrial tumors were found that lipid and protein biosynthesis were upregulated in the ECs of obese mice compared to lean mice. ONC201 increased lipid degradation and oxidation (a) and eicosanoid production in the ECs of obese mice but not lean mice (b). In contrast, ONC201 reduced glycolysis in the ECs of lean mice but not in the ECs of obese mice (b). ONC201 decreased protein biosynthesis in the ECs of both obese and lean mice (c). Red indicate metabolites with higher mean levels at a p ≤ 0.05 significance. Green cells indicate lower levels relative to control group at a p ≤ 0.05 significance. Data are presented as relative measures of “scaled intensity” and median scaling to 1