MEK1/2 inhibitor selumetinib (AZD6244) inhibits growth of ovarian clear cell carcinoma in a PEA-15-dependent manner in a mouse xenograft model

Abstract

Clear cell carcinoma (CCC) of the ovary tends to show resistance to standard chemotherapy, which results in poor survival for patients with CCC. Developing a novel therapeutic strategy is imperative to improve patient prognosis. Epidermal growth factor receptor (EGFR) is frequently expressed in epithelial ovarian cancer. One of the major downstream targets of the EGFR signaling cascade is extracellular signal-related kinase (ERK). PEA-15, a 15-kDa phosphoprotein, can sequester ERK in the cytoplasm. MEK1/2 plays a central role in integrating mitogenic signals into the ERK pathway. We tested the hypothesis that inhibition of the EGFR-ERK pathway suppresses tumorigenicity in CCC, and we investigated the role of PEA-15 in ERK-targeted therapy in CCC. We screened a panel of 4 CCC cell lines (RMG-I, SMOV-2, OVTOKO, and KOC-7c) and observed that the EGFR tyrosine kinase inhibitor erlotinib inhibited cell proliferation of EGFR-overexpressing CCC cell lines through partial dependence on the MEK/ERK pathway. Furthermore, erlotinib-sensitive cell lines were also sensitive to the MEK inhibitor selumetinib (AZD6244), which is under clinical development. Knockdown of PEA-15 expression resulted in reversal of selumetinib-sensitive cells to resistant cells, implying that PEA-15 contributes to selumetinib sensitivity. Both selumetinib and erlotinib significantly suppressed tumor growth (P < 0.0001) in a CCC xenograft model. However, selumetinib was better tolerated; erlotinib-treated mice exhibited significant toxic effects (marked weight loss and severe skin peeling) at high doses. Our findings indicate that the MEK-ERK pathway is a potential target for EGFR-overexpressing CCC and indicate that selumetinib and erlotinib are worth exploring as therapeutic agents for CCC.

Figure 1

Downregulation of EGFR suppresses proliferation of CCC cells. A, Four CCC cell lines were cultured in serum-free medium for 24 h and then exposed to EGF in culture medium at a concentration of 100 ng/ml for 15 min. Western blot analysis was performed and β-actin was used as a loading control. B and C, RMG-I cells were treated with siRNA-control or siRNA-EGFR for 6 h, medium was changed, and 96 h after addition of siRNAs, (B) Western blot analysis was performed and (C) cell viability was measured by trypan blue exclusion test.

Figure 2

Erlotinib suppresses proliferation of CCC cells via pERK downregulation and G₁ arrest. A, Four CCC cell lines were plated in 96-well plates and treated with 0.1 and 1 μM erlotinib for 72 h. WST-1 assay was performed. B, RMG-I cells were cultured in serum-free medium for 24 h and treated with 0.1 or 1 μM erlotinib for 3 h. Protein samples were collected after exposure to EGF in culture medium at a concentration of 100 ng/ml for 15 min. Western blot analysis was performed. C, RMG-I cells were treated with 0.1 or 1 μM erlotinib for 48 h, and FACScan analysis was performed to determine cell cycle distribution. D, RMG-I cells stably expressing MEK1^CA were treated with erlotinib (1, 2, 5, or 10 μM), and WST-1 assay was performed at 72 h. Cells were also treated with erlotinib (2 μM), and trypan blue exclusion test was performed at 48 h to assess cell viability.

Figure 3

Selumetinib suppresses proliferation of CCC cells via pERK downregulation and G₁ arrest. A, Four CCC cell lines were plated in 96-well plates and treated with 0.1 and 1 μM selumetinib for 72 h. WST-1 assay was performed to quantify the activity of erlotinib.B, RMG-I cells were treated with 0.01 or 0.1 μM selumetinib for 24 h. Western blot analysis was performed (α-tubulin was used as a loading control). C, RMG-I cells were treated with 0.01 or 0.1 μM selumetinib for 24 h, and FACScan analysis was performed. D, RMG-I and OVTOKO cells were treated with selumetinib (0.1 μM for RMG-I, 1 μM for OVTOKO) for 72 h and divided into cytoplasmic and nuclear fractions, and western blot analysis was performed.

Figure 4

Phosphorylated PEA-15 sensitizes CCC cells to selumetinib. A, RMG-I and OVTOKO cells were treated with selumetinib (0.01 and 0.1 μM for RMG-I, 1 and 10 μM for OVTOKO) for 72 h. Western blot analysis was performed to detect pERK1/2, PEA-15, pPEA-15 (S116), and pPEA-15 (S104) expression. α-tubulin was used as a loading control. B, RMG-I cells were transfected with siRNA-PEA-15. Forty-eight hours later, cells were treated with 1 μM selumetinib for 48 h. Cell viability was examined by trypan blue exclusion test. C, OVTOKO cells were transfected with vector, nonphosphorylated mutant PEA-15-AA, or mutant PEA-15-AD. Twenty-four hours later, cells were treated with 10 μM selumetinib for 44 h. Cell viability was examined by trypan blue exclusion test.

Figure 5

Selumetinib inhibits tumorigenicity in a CCC xenograft model without producing toxic effects. Female athymic nude mice were injected intraperitoneally with 4 × 10⁶ luciferase-transfected RMG-I cells (RMG-I/luc). The experimental groups were treated with vehicle control (0.5% hydroxypropyl methyl cellulose and 0.1% Tween 80), 50 mg/kg/day erlotinib, or 50 or 100 mg/kg/day selumetinib for 5 weeks beginning 5 days after cell injection. A, Mean body weight in each treatment group. Data from an initial experiment in which mice were treated with 100 mg/kg/day erlotinib followed by 50 mg/kg/day erlotinib are included for reference. B, The photon counts per area (monitored by IVIS) of each treatment group were quantitatively analyzed on days 0, 7, 14, 21, 28, and 35 after inoculation. C, Immunohistochemical stains and quantification of ERK, pERK, PEA-15, pPEA-15 (S116D), and Ki-67 in representative tumor tissue samples from each treatment group.