MEK Inhibition Increases ERα Expression in Human Ovarian Carcinoma Cells.

<p>(A) Expression of ERα protein in human ovarian cancer cell lines. MCF-7, a breast cancer cell line was used as positive control. All cell lines were treated with MEKi at 1 uM for 24 h. (B) The effect of estrogen deprivation on cell cycle. Cells were grown in phenol red-free charcoal stripped RPMI for 48 h to simulate ES-free conditions, and subsequently analyzed for cell cycle distribution and doubling time, as described in Materials and Methods. (C) The effect of MEK inhibition for 24 h on ERα expression and MAPK pathway activation in ovarian cancer cells. DMSO was used as the vehicle-only control. (D) Dose-dependent increase in ERα expression in SKOV3 cells by MEKi (24 h); and densitometric quantification relative to GAPDH. (E) Flow cytometric analysis of cell-cycle distribution at various time points indicates G1 arrest 24 h post MEKi treatment in SKOV3 cells.</p

ERα overexpression is associated with MAPK-dependent phosphorylation, cell-cycle arrest and transactivation of ER-regulated genes.

<p>(A) MEKi treatment for 24 h increases ERα phosphorylation at Serine 118 in ER-immunoprecipitated SKOV3 lysates. (B) The effect of MEKi on <i>ESR1</i> and cell cycle regulatory gene expression, depicting upregulation and suppression, respectively. (C) The effect of MEKi on expression of selected ER-regulated genes in SKOV3 cells. Treatment with MEKi was for 24 h, and mRNA expression was carried out by qRT-PCR as described in Materials and Methods.</p

MEKi-mediated overexpression of ERα is AKT independent.

<p>(A) MEKi-mediated changes in AKT phosphorylation, and not basal phosphorylation, are prognostic of drug sensitivity. Increased phosphorylation of erbB-family receptors in SKOV3 cells also correlate with resistance to MEKi, while S6 dephosphorylation predicts sensitivity to MEKi. Mean IC₅₀’s for MEKi are shown. (B) Temporal dissociation of pAKT and pMEK with ERα overexpression after treatment with 1 µM MEKi. (C) AKT inhibition combined with MEKi does not reverse ERα overexpression in SKOV3 cells. Cells were treated with the specified inhibitors for 24 h.</p

Potentiation of MEKi efficacy by the estrogen receptor antagonist Fulvestrant in ERα-positive cancer cell lines.

<p>The predicted additive effect was determined by applying the Bliss additivity model<b>^§</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054103#pone.0054103-Bliss1" target="_blank">[25]</a>. A greater than additive or synergistic interaction (observed effect exceeds the expected effect) was noted only in the ERα- expressing cell lines, SKOV3 and Ishikawa. Conversely, A2780, ERα-negative ovarian carcinoma cells exhibited antagonism between MEKi and fulvestrant.</p>¶<p>As determined by SRB assay.</p

MEKi-mediated ERα overexpression is independent of erbB activity but MAPK-dependent.

<p>(A) The ERα antagonist fulvestrant prevents MEKi-mediated ERα overexpression, and partially suppresses the phosphorylation of erbB2 and EGFR by MEKi. (B) Overexpression of ERα by MEKi is erbB-independent, since the pan-erbB inhibitor lapatinib (erbBi) does not prevent ERα overexpression by MEKi treatment. Cells were treated with inhibitors for 24 h.</p

The concurrent combination of MEKi and fulvestrant suppresses SKOV3 tumor xenograft growth.

<p>Single agent fulvestrant weakly stimulated tumor growth relative to vehicle, and MEKi had weak anti-tumor activity; however the concurrent combination of fulvestrant and MEKi induced tumor regressions that were statistically significantly different from either MEKi alone (*<i>P</i> = 0.02, unpaired t-test), or fulvestrant (**<i>P</i> = 0.002, unpaired t-test) at day 18. After three weeks, animals in the treatment groups other than combination were euthanized due to tumor burden. Asterisks denote the level of significance. Data are expressed as percent change in initial tumor volume (T₀). The dashed horizontal black line represents initial tumor volume.</p