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

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

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

Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods.

<p>(A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.</p

Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods.

<p>Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.</p

MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods.

<p>The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC_t (δC_t target miRNA - δC_t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.</p

DNA/RNA extractions using archived human specimens.

<p>Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.</p