Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype

Changhua Zhou, Ashley M. Nitschke, Wei Xiong, Qiang Zhang, Yan Tang, Michael Bloch, Steven Elliott, Yun Zhu, Lindsey Bazzone, David Yu, Christopher B. Weldon, Rachel Schiff, John A. McLachlan, Barbara S. Beckman, Thomas E. Wiese, Kenneth Nephew, Bin Shan, Matthew E. Burow, Guangdi Wang

Research output: Contribution to journalArticle

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Abstract

Introduction: Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.Methods: To compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.Results: Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.Conclusions: This study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.

Original languageEnglish
Article numberR105
JournalBreast Cancer Research
Volume10
Issue number6
DOIs
StatePublished - Dec 16 2008

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Epithelial-Mesenchymal Transition
Proteomics
MCF-7 Cells
Glutathione Transferase
Breast Neoplasms
Phenotype
Tumor Necrosis Factor-alpha
Vimentin
Cadherins
Keratin-8
Keratin-19
Down-Regulation
Creatine Kinase
Fluorescent Antibody Technique
Up-Regulation
Staining and Labeling
Gene Expression
Cell Line
Polymerase Chain Reaction
Gastropoda

ASJC Scopus subject areas

  • Cancer Research
  • Oncology
  • Medicine(all)

Cite this

Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype. / Zhou, Changhua; Nitschke, Ashley M.; Xiong, Wei; Zhang, Qiang; Tang, Yan; Bloch, Michael; Elliott, Steven; Zhu, Yun; Bazzone, Lindsey; Yu, David; Weldon, Christopher B.; Schiff, Rachel; McLachlan, John A.; Beckman, Barbara S.; Wiese, Thomas E.; Nephew, Kenneth; Shan, Bin; Burow, Matthew E.; Wang, Guangdi.

In: Breast Cancer Research, Vol. 10, No. 6, R105, 16.12.2008.

Research output: Contribution to journalArticle

Zhou, C, Nitschke, AM, Xiong, W, Zhang, Q, Tang, Y, Bloch, M, Elliott, S, Zhu, Y, Bazzone, L, Yu, D, Weldon, CB, Schiff, R, McLachlan, JA, Beckman, BS, Wiese, TE, Nephew, K, Shan, B, Burow, ME & Wang, G 2008, 'Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype', Breast Cancer Research, vol. 10, no. 6, R105. https://doi.org/10.1186/bcr2210
Zhou, Changhua ; Nitschke, Ashley M. ; Xiong, Wei ; Zhang, Qiang ; Tang, Yan ; Bloch, Michael ; Elliott, Steven ; Zhu, Yun ; Bazzone, Lindsey ; Yu, David ; Weldon, Christopher B. ; Schiff, Rachel ; McLachlan, John A. ; Beckman, Barbara S. ; Wiese, Thomas E. ; Nephew, Kenneth ; Shan, Bin ; Burow, Matthew E. ; Wang, Guangdi. / Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype. In: Breast Cancer Research. 2008 ; Vol. 10, No. 6.
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title = "Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype",
abstract = "Introduction: Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.Methods: To compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.Results: Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.Conclusions: This study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.",
author = "Changhua Zhou and Nitschke, {Ashley M.} and Wei Xiong and Qiang Zhang and Yan Tang and Michael Bloch and Steven Elliott and Yun Zhu and Lindsey Bazzone and David Yu and Weldon, {Christopher B.} and Rachel Schiff and McLachlan, {John A.} and Beckman, {Barbara S.} and Wiese, {Thomas E.} and Kenneth Nephew and Bin Shan and Burow, {Matthew E.} and Guangdi Wang",
year = "2008",
month = "12",
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doi = "10.1186/bcr2210",
language = "English",
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TY - JOUR

T1 - Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype

AU - Zhou, Changhua

AU - Nitschke, Ashley M.

AU - Xiong, Wei

AU - Zhang, Qiang

AU - Tang, Yan

AU - Bloch, Michael

AU - Elliott, Steven

AU - Zhu, Yun

AU - Bazzone, Lindsey

AU - Yu, David

AU - Weldon, Christopher B.

AU - Schiff, Rachel

AU - McLachlan, John A.

AU - Beckman, Barbara S.

AU - Wiese, Thomas E.

AU - Nephew, Kenneth

AU - Shan, Bin

AU - Burow, Matthew E.

AU - Wang, Guangdi

PY - 2008/12/16

Y1 - 2008/12/16

N2 - Introduction: Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.Methods: To compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.Results: Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.Conclusions: This study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.

AB - Introduction: Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.Methods: To compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.Results: Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.Conclusions: This study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.

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DO - 10.1186/bcr2210

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