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. 2018 Feb;17(2):464-473.
doi: 10.1158/1535-7163.MCT-17-0006. Epub 2017 Dec 13.

Inhibiting Nuclear Phospho-Progesterone Receptor Enhances Antitumor Activity of Onapristone in Uterine Cancer

Yan Huang #  1 , Wei Hu #  1 , Jie Huang  1 , Fangrong Shen  1 , Yunjie Sun  1 , Cristina Ivan  2 , Sunila Pradeep  1 , Robert Dood  1 , Monika Haemmerle  1 , Dahai Jiang  2 , Lingegowda S Mangala  1 , Kyunghee Noh  1 , Jean M Hansen  1 , Heather J Dalton  1 , Rebecca A Previs  1 , Archana S Nagaraja  1 , Michael McGuire  1 , Nicholas B Jennings  1 , Russell Broaddus  3 , Robert L Coleman  1 , Anil K Sood  4   2   5
Affiliations

Inhibiting Nuclear Phospho-Progesterone Receptor Enhances Antitumor Activity of Onapristone in Uterine Cancer

Yan Huang et al. Mol Cancer Ther. 2018 Feb.

Abstract

Although progesterone receptor (PR)-targeted therapies are modestly active in patients with uterine cancer, their underlying molecular mechanisms are not well understood. The clinical use of such therapies is limited because of the lack of biomarkers that predict response to PR agonists (progestins) or PR antagonists (onapristone). Thus, understanding the underlying molecular mechanisms of action will provide an advance in developing novel combination therapies for cancer patients. Nuclear translocation of PR has been reported to be ligand-dependent or -independent. Here, we identified that onapristone, a PR antagonist, inhibited nuclear translocation of ligand-dependent or -independent (EGF) phospho-PR (S294), whereas trametinib inhibited nuclear translocation of EGF-induced phospho-PR (S294). Using orthotopic mouse models of uterine cancer, we demonstrated that the combination of onapristone and trametinib results in superior antitumor effects in uterine cancer models compared with either monotherapy. These synergistic effects are, in part, mediated through inhibiting the nuclear translocation of EGF-induced PR phosphorylation in uterine cancer cells. Targeting MAPK-dependent PR activation with onapristone and trametinib significantly inhibited tumor growth in preclinical uterine cancer models and is worthy of further clinical investigation. Mol Cancer Ther; 17(2); 464-73. ©2017 AACR.

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Conflict of interest statement

Disclosures: WH received research support from Arno Therapeutics to support some of the preclinical experiments. All other authors report no conflicts of interest.

Figures

Figure 1
Figure 1. In vitro effects of onapristone in uterine cancer cells
(A) Real time-PCR and (B) Western blot analyses of PR expression in a panel of uterine cancer cell lines. (C) Cell viability after treatment with the indicated concentrations of onapristone for 72 hours. Cell viability was defined as the percentage of viable cells in the treatment group relative to viable cells in the control group. Data represent the means of triplicate measurements with error bars to represent the standard error of the mean. **p<0.01 compared with the control group. In vitro functional studies of (D) apoptosis for 72 hours, (E) proliferation for 96 hours, and (F) cell cycle for 48 hours in ISHIKAWA and SKUT2 cells treated with 20 μM onapristone. Data represent the means of triplicate measurements with error bars to represent the standard error of the mean. ** p<0.01 (G) Western blot analysis of phospho-PR (S345) and phospho-p44/42 MAPK expression in ISHIKAWA cells treated with the indicated concentrations of onapristone for 24 hours. Quantification of band intensity relative to phospho-PR intensity is shown graphically. *p<0.05, **p<0.01 compared with the control group. Quantification of band intensity relative to p44/42 MAPK intensity is shown graphically. NS indicates p>0.05, compared with the control group. (H) Western blot analysis of p21 expression in ISHIKAWA and SKUT2 cells treated with 10 μM onapristone for 24, 48, and 72 hours. Quantification of band intensity relative to β-actin intensity is shown graphically.
Figure 2
Figure 2. In vitro effects of onapristone and trametinib in uterine cancer cell lines
(A, B) Cell viability after treatment with onapristone and trametinib at the indicated doses in ISHIKAWA cells for 72 hours. Data represent the means of triplicate measurements with error bars to represent the standard error of the mean. *p<0.05, **p<0.01 compared with the control group. (C) Isobologram analysis of the 2-drug interaction in ISHIKAWA cells. (D-F) In vitro functional studies of (D) apoptosis for 72 hours, (E) cell proliferation for 96 hours, and (F) cell cycle at 48 hours in ISHIKAWA cells treated with 20 μM onapristone combined with 100 nM trametinib. **p<0.01 compared with the single drug.
Figure 3
Figure 3. In vivo effects of onapristone and trametinib in orthotopic mouse model of uterine cancer
In vivo effect of onapristone, trametinib, and the combination of both drugs on (A) tumor weight, (B) number of tumor nodules, and (C) body weight. Error bars indicate the standard error of the mean. *p<0.05. (D) Immunohistochemical staining showing the effects of onapristone, trametinib, and the combination on apoptosis (cleaved caspase 3), cell proliferation (Ki67), and MVD (CD31) in the ISHIKAWA model. Predictive markers (phospho-PR (S345) and p21) are shown for the ISHIKAWA model. * p<0.05, ** p<0.01, compared to the single drug. Original magnification ×200.
Figure 4
Figure 4. Effects of onapristone or trametinib on the nuclear translocation of phospho-PR (S345) in uterine cancer cells
(A) Western blot and semi-quantitative analysis of phospho-PR (S345) and phospho-p42/44 MAPK expression in ISHIKAWA cells treated with trametinib at the indicated doses for 24 hours. (B) Western blot and (C) semi-quantitative analysis of phospho-PR(S345) and phospho-p42/44 MAPK expression in ISHIKAWA and SKUT2 cells treated with 10 μM onapristone, 100nM trametinib, or a combination for 24 hours. * p<0.05, compared to control. P21 was assessed using 10 μM onapristone, 100nM trametinib, or a combination for 48 hours (p>0.05, compared to control). (D) IF and (E) semi-quantitative analysis of the nuclear phospho-PR (S345) in ISHIKAWA cells treated with 10 μM onapristone, 100nM trametinib, or a combination for 24 hours. Data represent means of 6 random field measurements with error bars to represent SEM. * p<0.05, ** p<0.01, compared to the control.
Figure 5
Figure 5. Effects of onapristone, trametinib, and combination treatment on the nuclear translocation of R5020 or EGF-induced phospho-PR (S294) in uterine cancer cells
(A) Western blot and semi-quantitative analysis of R5020-induced phospho-PR expression in ISHIKAWA cells treated with 10 μM onapristone, 100 nM trametinib, or both for 2 hours, followed by 25 nM R5020 for 1 hour. (B) IF and (C) semi-quantitative analysis of R5020-induced phospho-PR expression and location in ISHIKAWA cells treated with 10 μM onapristone, 100 nM trametinib, or both for 2 hours followed by 25 nM R5020 for 1 hour. Data represent means of 6 random field measurements with error bars to represent SEM. *p<0.05, compared to the R5020 treatment. (D) Western blot and semi-quantitative analysis of EGF-induced phospho-PR expression and location in ISHIKAWA cells treated with 10 μM onapristone, 100 nM trametinib, or both for 2 hours, followed by treatment with 30 ng/mL EGF for 30 minutes. ** p<0.01, compared to EGF treatment or control. (E) IF and (F) semi-quantitative analysis of EGF-induced phospho-PR expression and location in ISHIKAWA cells treated with 10 μM onapristone, 100 nM trametinib, or both for 2 hours, followed by treatment with 30 ng/mL EGF for 30 minutes. Data represent means of 6 random field measurements with error bars to represent SEM. * p<0.05, compared to EGF treatment.
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