Endometrial Cancers With Activating KRas Mutations Have Activated Estrogen Signaling and Paradoxical Response to MEK Inhibition

Abstract

Objectives: The aims of this study were to determine if activating KRas mutation alters estrogen signaling in endometrial cancer (EC) and to explore the potential therapeutic impact of these alterations.

Methods: The Cancer Genome Atlas was queried for changes in estrogen-regulated genes in EC based on KRas mutation status. In vitro studies were conducted to evaluate estrogen receptor α (ERα) phosphorylation changes and related kinase changes in KRas mutant EC cells. The resulting effect on response to MEK inhibition, using trametinib, was evaluated. Immunohistochemistry was performed on KRas mutant and wild-type EC tumors to test estrogen signaling differences.

Results: KRas mutant tumors in The Cancer Genome Atlas showed decreased progesterone receptor expression (P = 0.047). Protein analysis in KRas mutant EC cells also showed decreased expression of ERα (P < 0.001) and progesterone receptor (P = 0.001). Although total ERα is decreased in KRas mutant cells, phospho-ERα S118 was increased compared with wild type. Treatment with trametinib in KRas mutant cells increased phospho-ERα S167 and increased expression of estrogen-regulated genes. While MEK inhibition blocked estradiol-stimulated phosphorylation of ERK1/2 and p90RSK in wild-type cells, phospho-ERK1/2 and phospho-p90RSK were substantially increased in KRas mutants. KRas mutant EC tumor specimens showed similar changes, with increased phospho-ERα S118 and phospho-ERα S167 compared with wild-type EC tumors.

Conclusions: MEK inhibition in KRas mutant cells results in activation of ER signaling and prevents the abrogation of signaling through ERK1/2 and p90RSK that is achieved in KRas wild-type EC cells. Combination therapy with MEK inhibition plus antiestrogen therapy may be necessary to improve response rates in patients with KRas mutant EC.

Figure 1

Analysis of gene expression changes between KRas mutant and wild-type uterine cancers. A) Sample selection from Uterine Cancer TCGA data to include endometrioid tumors for analysis of gene expression changes and KRas mutations. Of 185 endometrioid tumors with PI3K pathway aberrations, 26.5% had KRas mutations and 73.5% had normal KRas. B) A significant decrease in progesterone receptor (p=0.047) and IGF1 (p=0.044) expression was found between KRas mutant (red) and KRas wild-type (green) tumors.

Figure 2

Analysis of protein expression levels after estradiol stimulation in Ishikawa cells using Reverse Phase Protein Array (RPPA) indicates that cells transfected to express mutant KRas G12V have reduced expression of both Progesterone Receptor (PGR) and Estrogen Receptor alpha (ESR1) compared to cells expressing wild-type (WT) KRas. Cells were stimulated with 0.01μM estradiol and cell lysates were harvested at 30 minutes. Experiments were performed in triplicate. Graphs represent mean ± standard deviation.

Figure 3

A) Total levels of ERα are decreased in KRas mutant cells compared to wild-type (WT) and this is maintained following treatment with trametinib (MEK inhibitor, MEKi). Differences in phosphorylation of S118 and S167 in response to estradiol stimulation were observed based on KRas mutation status (vehicle versus 30 minute time point). A. Phosphorylation at S118 was increased by estradiol stimulation at 30 minutes in both wild-type and KRasG12V. However, the magnitude of induction is reduced in KRasG12V. Baseline phosphorylation at S167 is increased by estradiol stimulation only in wild-type cells. We then evaluated phosphorylation of S167 and S118 at the 30 minute time point to evaluate differences in phosphorylated states between KRas wild type and mutant cells. KRas wild type cells had increased phosphorylation at S167 compared to KRas mutant cells. Cells with mutant KRas had increased phosphorylation at S118 after estradiol stimulation compared to cells with wild-type KRas. Following treatment with a MEK inhibitor, KRas mutant cells have increased phosphorylation at S167. The triangle at left indicates the band of interest for phosphorylation of S167. B) Graphical depiction of immunoblot results at the 30 minute time point expressed as normalized relative density. Phospho-ERs are normalized to total ERα; ERα is normalized to β-actin control. V, vehicle. Cells were stimulated with 0.01μM estradiol and cell lysates were harvested at 10minutes, 30 minutes, 1 hour, and 4 hours. All experiments were performed in triplicate.

Figure 4

No differences in levels of phospho-AKT or phospho-p70S6K were seen in response to estradiol stimulation. Both had a slight decrease in expression following treatment with trametinib (MEKi). Wild-type (WT) KRas cells had increased phosphorylation of ERK1/2 and p90RSK in response to estradiol stimulation compared to mutant KRas cells. Following treatment with trametinib, wild-type cells exhibited decreased phosphorylation at ERK1/2 and p90RSK, while KRas mutant cells had increased phosphorylation of ERK1/2 and p90RSK. Cells were stimulated with 0.01μM estradiol and cell lysates were harvested at 10minutes, 30 minutes, 1 hour, and 4 hours. All experiments were performed in triplicate.

Figure 5

Ishikawa cells expressing mutant KRas had increased expression of estrogen-induced genes following treatment with trametinib (MEKi), while cells with wild-type KRas had stable or slightly decreased expression. Gene expression changes are presented as fold-change compared to the vehicle control. Specifically, wild-type with trametinib treatment are normalized to wild-type with vehicle control and KRas mutant cells treated with trametinib are normalized to KRas mutant cells with vehicle control. *p<0.05 comparing the difference between genotypes. All experiments were performed in triplicate. Graphs represent mean ± standard error.

Figure 6

Immunohistochemical staining was conducted on endometrioid endometrial tumor specimens to compare ER and PR expression in KRas wild-type and KRas mutant tumors. PR expression was lower in KRas mutants (p=0.044). KRas mutants showed increased expression of phospho- ERα S118 (p=0.033) and phospho- ERα S167 (P<0.001).