Epithelial-Mesenchymal Transition Markers and HER3 Expression Are Predictors of Elisidepsin Treatment Response in Breast and Pancreatic Cancer Cell Lines

<div><p>Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734) is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT) and the use of HER family receptors in predicting the <em>in vitro</em> drug response. Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines (MCF-7, HPAC and AsPC-1) and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment.</p> </div

Epithelial-Mesenchymal Transition Markers and HER3 Expression Are Predictors of Elisidepsin Treatment Response in Breast and Pancreatic Cancer Cell Lines

Altres ajuts: This work has been partially funded by Pharmamar Company and by CENIT grant (CEN-2009-1016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734) is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT) and the use of HER family receptors in predicting the in vitro drug response. Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines (MCF-7, HPAC and AsPC-1) and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment

Expression of EMT markers associated with elisidepsin sensitivity in breast cancer cell lines.

<p>Protein expression levels of different EMT markers were evaluated by immunocytochemistry (A), western blot (B) and IHC (C). A) Immunocytochemistry of two epithelial (E-cadherin and β-catenin) and four mesenchymal markers (vimentin, Slug, Snail and Twist). Magnification 100x. B) E-cadherin, β-catenin, Slug, Snail, Twist, vimentin and β-actin (loading control) were detected by western blot analysis using 50 µg of total protein. C) Basal levels of E-cadherin, β-catenin and vimentin were analyzed by IHC. Magnification 20x. Each experiment was performed at least in duplicate.</p

Acquired resistance to elisidepsin induces an EMT phenotype.

<p>A) Cells were lysed, proteins were extracted and western blots were performed with equal amounts of cell lysate (50 µg protein). Expression of epithelial (E-cadherin, β-catenin, γ-catenin)- and mesenchymal (vimentin, Slug, Snail, Twist)-associated proteins differentiates between elisidepsin-sensitive and elisidepsin-resistant cell lines. β-actin was used as an internal control. These western blots were performed in triplicate. B) Expression levels of HER1, HER2, HER3, HER4, pAkt, and pMAPK were analyzed by western blot using 50 µg of protein cell lysate. The membranes were stripped and reprobed with anti-β-actin to verify equal protein loading. C, control; R, resistance.</p

Loss of HER3 expression decreases the sensitivity to elisidepsin treatment.

<p>Cell viability after treatment with various concentrations of elisidepsin for 72 h was determined in SKBR3 (A), MCF-7 (B), MDA-MB-231 (C), MDA-MB-435 (D), BT474 (E), BxPC-3 (F), HPAC (G) and AsPC-1 (H) cells. HER3 expression was downregulated with shRNA (grey squares); LUC shRNA transfected cells were used as the control (black diamonds). Mean, SD, and IC₅₀ values are shown from three independent experiments. Cell viability was measured using a crystal violet assay. Before performing the viability experiments, all cell lines were checked by western blot using 50 µg of protein to confirm their levels of HER3 expression.</p

HER3 expression levels correlate with cell sensitivity to elisidepsin.

<p>A) Cell pellets were fixed in formalin, embedded in paraffin and a HER3 IHC was performed. Cell lines more sensitive to elisidepsin had significant HER3 levels. Magnification 40x. B) Basal expression levels of HER family members were analyzed by western blot; an association between HER3 expression and elisidepsin sensitivity was observed (Mann-Whitney test: p  = 0.0091; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053645#pone.0053645.s003" target="_blank">Fig. S3</a>). Cell lines less sensitive to elisidepsin (MDA-MB-231, PANC-1 and MiaPaCa-2) did not show significant HER3 protein levels, while PANC-1 and MiaPaCa-2 cell lines show levels of other HER family members. No correlation was observed with HER1, HER2 and HER4 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053645#pone.0053645.s003" target="_blank">Fig. S3</a>). These protein expression levels were analyzed in duplicate and 50 µg of protein of cell lysate were loaded in each lane.</p

Upregulation of HER3 increases elisidepsin sensitivity.

<p>Cell viability after treatment with various concentrations of elisidepsin for 72 h was determined in PANC-1 (A), MiaPaCa-2 (B), MDA-MB-435 (C) and MDA-MB-231 (D) cells. Stable cell lines with an upregulation of HER3 expression (with the pIRES HER3) are shown with white circles while black diamonds are used for LUC-transfected control cells (with the pIRES-LUC). Mean, SD, and IC₅₀ values are shown from three independent experiments. Cell viability was measured by a crystal violet assay. Before performing the viability experiments, all cell lines were checked by western blot using 50 µg of protein to confirm their levels of HER3 expression.</p

Elisidepsin sensitivity.

<p>A) Elisidepsin IC₅₀s were determined in a panel of breast (left) and pancreatic (right) cancer cell lines using a crystal violet assay. Cells were exposed to elisidepsin for 72 h. Results are shown as the mean ± SD of at least three independent experiments. B) Cell proliferation in parental and subtoxic elisidepsin-treated cells. Cumulative numbers of cell divisions [shown as population doubling level (PDL)] are shown for MCF-7 and MiaPaCa-2 cells until passage 5. Proliferation of MCF-7 (IC₅₀∶0.4 µM) and MiaPaCa-2 (IC₅₀∶14 µM) cells was suppressed when elisidepsin was added to the culture at subtoxic doses (0.2 and 1 µM, respectively). The number of MiaPaCa-2 and MCF-7 seeded cells were 1.25×10⁵ and 1.4×10⁵, respectively. Each growth curve was performed at least twice with similar results, SDs are shown, and each time point was performed in duplicate. P, passage.</p

A Translational In Vivo and In Vitro Metabolomic Study Reveals Altered Metabolic Pathways in Red Blood Cells of Type 2 Diabetes

Clinical parameters used in type 2 diabetes mellitus (T2D) diagnosis and monitoring such as glycosylated haemoglobin (HbA1c) are often unable to capture important information related to diabetic control and chronic complications. In order to search for additional biomarkers, we performed a pilot study comparing T2D patients with healthy controls matched by age, gender, and weight. By using 1 H-nuclear magnetic resonance (NMR) based metabolomics profiling of red blood cells (RBCs), we found that the metabolic signature of RBCs in T2D subjects differed significantly from non-diabetic controls. Affected metabolites included glutathione, 2,3-bisphophoglycerate, inosinic acid, lactate, 6-phosphogluconate, creatine and adenosine triphosphate (ATP) and several amino acids such as leucine, glycine, alanine, lysine, aspartate, phenylalanine and tyrosine. These results were validated by an independent cohort of T2D and control patients. An analysis of the pathways in which these metabolites were involved showed that energetic and redox metabolism in RBCs were altered in T2D, as well as metabolites transported by RBCs. Taken together, our results revealed that the metabolic profile of RBCs can discriminate healthy controls from T2D patients. Further research is needed to determine whether metabolic fingerprint in RBC could be useful to complement the information obtained from HbA1c and glycemic variability as well as its potential role in the diabetes managemen

Epithelial-Mesenchymal Transition Markers and HER3 Expression Are Predictors of Elisidepsin Treatment Response in Breast and Pancreatic Cancer Cell Lines

<div><p>Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734) is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT) and the use of HER family receptors in predicting the <em>in vitro</em> drug response. Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines (MCF-7, HPAC and AsPC-1) and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment.</p> </div