Skip to main content
ArticleOPEN ACCESS

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

PLoS ONE
DOI: 10.1371/journal.pone.0053645
15Citations
Citations of this article
32Readers
Mendeley users who have this article in their library.

Abstract

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. © 2013 Teixidó et al.

Figures

  • Figure 1. Elisidepsin sensitivity. 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 6 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 mM) and MiaPaCa-2 (IC50:14 mM) cells was suppressed when elisidepsin was added to the culture at subtoxic doses (0.2 and 1 mM, respectively). The number of MiaPaCa-2 and MCF-7 seeded cells were 1.256105 and 1.46105, 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. doi:10.1371/journal.pone.0053645.g001
  • Figure 2. Expression of EMT markers associated with elisidepsin sensitivity in breast cancer cell lines. Protein expression levels of different EMT markers were evaluated by immunocytochemistry (A), western blot (B) and IHC (C). A) Immunocytochemistry of two epithelial (Ecadherin and b-catenin) and four mesenchymal markers (vimentin, Slug, Snail and Twist). Magnification 100x. B) E-cadherin, b-catenin, Slug, Snail, Twist, vimentin and b-actin (loading control) were detected by western blot analysis using 50 mg of total protein. C) Basal levels of E-cadherin, bcatenin and vimentin were analyzed by IHC. Magnification 20x. Each experiment was performed at least in duplicate. doi:10.1371/journal.pone.0053645.g002
  • Figure 3. Expression of EMT markers associated with elisidepsin sensitivity in pancreatic cancer cell lines. E-cadherin, b-catenin, vimentin, Slug, Snail and Twist basal expression levels were evaluated by immunocytochemistry (A) and western blot (50 mg of protein/lane) (B). Magnification 100x. Membranes were stripped and reprobed with anti-b-actin as an internal control. C) E-cadherin, b-catenin and vimentin protein expression levels were evaluated by IHC. Magnification 20x. These analyses were performed in duplicate. doi:10.1371/journal.pone.0053645.g003
  • Figure 4. HER3 expression levels correlate with cell sensitivity to elisidepsin. 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; Fig. S3). 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 (Fig. S3). These protein expression levels were analyzed in duplicate and 50 mg of protein of cell lysate were loaded in each lane. doi:10.1371/journal.pone.0053645.g004
  • Figure 5. Acquired resistance to elisidepsin induces an EMT phenotype. A) Cells were lysed, proteins were extracted and western blots were performed with equal amounts of cell lysate (50 mg protein). Expression of epithelial (E-cadherin, b-catenin, c-catenin)- and mesenchymal (vimentin, Slug, Snail, Twist)-associated proteins differentiates between elisidepsin-sensitive and elisidepsin-resistant cell lines. b-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 mg of protein cell lysate. The membranes were stripped and reprobed with anti-b-actin to verify equal protein loading. C, control; R, resistance. doi:10.1371/journal.pone.0053645.g005
  • Figure 6. Loss of HER3 expression decreases the sensitivity to elisidepsin treatment. 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 mg of protein to confirm their levels of HER3 expression. doi:10.1371/journal.pone.0053645.g006
  • Figure 7. Upregulation of HER3 increases elisidepsin sensitivity. 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 mg of protein to confirm their levels of HER3 expression. doi:10.1371/journal.pone.0053645.g007

References Powered by Scopus

This article is free to access.

Epithelial-mesenchymal transitions in tumor progression

Nature Reviews Cancer
5819Citations
2961Readers
Get full text

Complex networks orchestrate epithelial-mesenchymal transitions

Nature Reviews Molecular Cell Biology
3434Citations
2137Readers
Get full text
View more at Scopus

Cited by Powered by Scopus

Therapeutic properties and biological benefits of marine-derived anticancer peptides

International Journal of Molecular Sciences
69Citations
116Readers

This article is free to access.

View more at Scopus

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Cite

CITATION STYLE

APA

Teixidó, C., Marés, R., Aracil, M., Ramón y Cajal, S., & Hernández-Losa, J. (2013, January 8). Epithelial-Mesenchymal Transition Markers and HER3 Expression Are Predictors of Elisidepsin Treatment Response in Breast and Pancreatic Cancer Cell Lines. PLoS ONE. Public Library of Science. https://doi.org/10.1371/journal.pone.0053645

Readers over time

‘13‘14‘15‘16‘17‘18‘19‘20‘21‘24036912

Readers' Seniority

Tooltip

PhD / Post grad / Masters / Doc 11

48%

Researcher 8

35%

Professor / Associate Prof. 3

13%

Lecturer / Post doc 1

4%

Readers' Discipline

Tooltip

Agricultural and Biological Sciences 13

57%

Medicine and Dentistry 5

22%

Biochemistry, Genetics and Molecular Bi... 3

13%

Pharmacology, Toxicology and Pharmaceut... 2

9%

Save time finding and organizing research with Mendeley

Sign up for free
0