Statins Impair Glucose Uptake in Tumor Cells1

Statins, HMG-CoA reductase inhibitors, are used in the prevention and treatment of cardiovascular diseases owing to their lipid-lowering effects. Previous studies revealed that, by modulating membrane cholesterol content, statins could induce conformational changes in cluster of differentiation 20 (CD20) tetraspanin. The aim of the presented study was to investigate the influence of statins on glucose transporter 1 (GLUT1)-mediated glucose uptake in tumor cells. We observed a significant concentration- and time-dependent decrease in glucose analogs' uptake in several tumor cell lines incubated with statins. This effect was reversible with restitution of cholesterol synthesis pathway with mevalonic acid as well as with supplementation of plasma membrane with exogenous cholesterol. Statins did not change overall GLUT1 expression at either transcriptional or protein levels. An exploratory clinical trial revealed that statin treatment decreased glucose uptake in peripheral blood leukocytes and lowered 18F-fluorodeoxyglucose (18F-FDG) uptake by tumor masses in a mantle cell lymphoma patient. A bioinformatics analysis was used to predict the structure of human GLUT1 and to identify putative cholesterol-binding motifs in its juxtamembrane fragment. Altogether, the influence of statins on glucose uptake seems to be of clinical significance. By inhibiting 18F-FDG uptake, statins can negatively affect the sensitivity of positron emission tomography, a diagnostic procedure frequently used in oncology

MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy

<div><p>Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased <i>in vitro</i> dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.</p></div

Peroxiredoxin-1 protects estrogen receptor alpha from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer

Introduction: Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H2O2) scavenger, molecular chaperone and immune modulator. Although differential PRDX1 expression has been described in many tumors, the potential role of PRDX1 in breast cancer remains highly ambiguous. Using a comprehensive antibody-based proteomics approach, we interrogated PRDX1 protein as a putative biomarker in estrogen receptor (ER)-positive breast cancer. Methods: An anti-PRDX1 antibody was validated in breast cancer cell lines using immunoblotting, immunohistochemistry and reverse phase protein array (RPPA) technology. PRDX1 protein expression was evaluated in two independent breast cancer cohorts, represented on a screening RPPA (n = 712) and a validation tissue microarray (n = 498). In vitro assays were performed exploring the functional contribution of PRDX1, with oxidative stress conditions mimicked via treatment with H2O2, peroxynitrite, or adenanthin, a PRDX1/2 inhibitor. Results: In ER-positive cases, high PRDX1 protein expression is a biomarker of improved prognosis across both cohorts. In the validation cohort, high PRDX1 expression was an independent predictor of improved relapse-free survival (hazard ratio (HR) = 0.62, 95% confidence interval (CI) = 0.40 to 0.96, P = 0.032), breast cancer-specific survival (HR = 0.44, 95% CI = 0.24 to 0.79, P = 0.006) and overall survival (HR = 0.61, 95% CI = 0.44 to 0.85, P = 0.004). RPPA screening of cancer signaling proteins showed that ER alpha protein was upregulated in PRDX1 high tumors. Exogenous H2O2 treatment decreased ER alpha protein levels in ER-positive cells. PRDX1 knockdown further sensitized cells to H2O2- and peroxynitrite-mediated effects, whilst PRDX1 overexpression protected against this response. Inhibition of PRDX1/2 antioxidant activity with adenanthin dramatically reduced ER alpha levels in breast cancer cells. Conclusions: PRDX1 is shown to be an independent predictor of improved outcomes in ER-positive breast cancer. Through its antioxidant function, PRDX1 may prevent oxidative stress-mediated ER alpha loss, thereby potentially contributing to maintenance of an ER-positive phenotype in mammary tumors. These results for the first time imply a close connection between biological activity of PRDX1 and regulation of estrogen-mediated signaling in breast cancer

Overexpression of a constitutively active MEK1 mutant (MEK-Q56P) in GC-sensitive RS4;11 cells induces resistance to DEX.

<p>(A) RS4;11 cells were retrovirally transduced with MEK-Q56P or empty control. Cells were lysed and ERK1/2 phoshorylation status was assessed by immunoblotting. (B-C) Control cells and MEK-Q56P—transduced cells were incubated with DEX (0.05, 2 or 30 μg/ml) for 72h. Thereafter, cell death was assessed by annexinV/PI staining and flow cytometry analysis. Absolute, averaged numbers of apoptotic cells in two independent experiments are indicated in (C). Error bars represent SD. P value was calculated using Student’s t-test.</p

Primer sequences used for MEK1 mutagenesis.

<p>Primer sequences used for MEK1 mutagenesis.</p

BCN1 knockdown reduces SEL-mediated sensitization to DEX.

<p>(A) SEMK2 cells were retrovirally transduced with BCN1-specific shRNA or scrambled control (SCR). After selection of stable transfectants, BCN1 knockdown was confirmed by western blot. (B,C) Cells with BCN1 knockdown or control cells were incubated with DEX, (0.05 μg/ml or 2 μg/ml) in the presence or absence of MEK1/2 inhibitor, selumetinib (SEL, 200 nM) for 72h. Apoptosis was assessed by annexinV/PI staining followed by flow cytometry analysis. Representative dot-plots from FACS analysis are shown in B; averaged results from 3 independent experiments with SD are indicated in (C) Statistical difference in responses between mock- and shBCN1-transduced cells were determined using 2-sided Student’s t-test. (D) Beclin-1 expression in cells treated with DEX, SEL or their combination. SEMK2 cells were incubated for 24 h with DEX (0.05 μg/ml), SEL (200 nM) or combination of DEX+SEL and BCN1 expression level was assessed by western blot.</p

MEK1/2 inhibitor, selumetinib, intensifies DEX induced LC3 conversion, MDC staining and GFP-LC3 relocalization in GC-resistant SEMK2 ALL cells.

<p>(A) GC-sensitive (RS4;11) and GC-resistant (SEMK2) cells were incubated with DEX (0.05 μg/mL) in the presence or absence of MEK1/2 inhibitor, selumetinib (SEL, 200 nM) for 24h. When indicated, cells were pretreated for 3 h with 50 μM or 100 μM of chloroquine (CQ). Thereafter, LC3 processing was assessed by immunoblotting. Densitometric analyses of LC3II/I are indicated below the blots. (B) SEMK2 and RS4;11 cells were cultured as described above for 24h, stained with MDC (50 μM) and analyzed by fluorescence microscopy. (C) SEMK2 cells were stably transduced with GFP-LC3 and incubated with DEX, SEL or combination of DEX+SEL for 24h. GFP-LC3 relocalization from diffuse cytoplasmic in control cells to a massive dotty pattern in DEX+SEL treated cells indicates LC3 recruitment to autophagosome membranes. In the lower panel, the percentage of cells with GFP-LC3 dots was quantified by counting the number of cells with > 3 dots and divided by a total number of GFP positive cells in 5 random non-overlapping fields. Pictures were taken at 630 × magnification. P value was calculated using Student’s t-test. (D) Induction of autophagy markers by the DEX and SEL co-treatment involves mTOR suppression. SEMK2 and RS4;11 cells were incubated with DEX in the presence or absence of SEL and lysed. 4E-BP1 phoshorylation status was assessed by immunoblotting. (E) GC-resistant (SEMK2) and—sensitive (RS4;11) cells were incubated with mTOR inhibitor rapamycin (100nM) in the presence or absence of DEX (0.05 μg/mL) for 24h. Thereafter, LC3 processing was assessed by immunoblotting and quantified. Densitometric analyses of LC3II/I are indicated below the blots. (F) Cells were treated as in (E) for 72h. Thereafter, cell numbers were assessed by counting 6 independent fields in Burker’s chamber. Data represent two independent experiments. P-values were calculated using 2-sided Student’s t-test.</p

Viability of ALL cell lines incubated with SEL.

<p>ALL cell lines were incubated with SEL at the indicated doses for 72h and cell death was assessed by annexinV/PI staining followed by flow cytometry analysis. Error bars indicate SD from at least 3 independent repeats.</p

MEK1/2 inhibitor, selumetinib sensitizes GC-resistant ALL cells to dexamethasone.

<p>(A) GC-resistant cells exhibit coordinate upregulation of MAPK/ERK pathway components. GSEA plots show relative upregulation of MAPK/ERK cascade components in GC-resistant ALL cells in two independent datasets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155893#pone.0155893.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155893#pone.0155893.ref026" target="_blank">26</a>]. Relative expression of pathway components is visualized by the heat map. FDR- false discovery rate. (B) ALL cell lines with active MAPK/ERK pathway (SEMK2, 697, CCRF-CEM) and ALL cells with undetectable expression of MAPK/ERK pathway (RS4;11) were incubated for 4h with MEK1/2 inhibitor, selumetinib (SEL, 200 nM). Thereafter, phosphorylation status of ERK1/2 and its substrate p90RSK were assessed by immunoblotting. (C) ALL cell lines were incubated with DEX (0.05 μg/ml 2 μg/ml and 30 μg/ml), SEL (200 nM) or combination of DEX+SEL for 72h and cell death was assessed by annexinV/PI staining followed by flow cytometry analysis. * p<0.05, ** p<0.01, ns- not significant. Error bars represent SD of three independent experiment. P values were calculated using Student’s t-test. (D) Cells were incubated as in (C) and used to determine the phosphorylation status of ERK1/2 by immunoblotting.</p