title: Proteomic analysis of tumor necrosis factor-alpha resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype.
abstract: BACKGROUND
  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)-alpha. Additionally, the relationship between TNF-alpha 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-alpha resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-alpha 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 (delta-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-alpha 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.
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA.
authors.forename: Changhua
authors.initials: C
authors.lastname: Zhou
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Nitschke
authors.forename: Wei
authors.initials: W
authors.lastname: Xiong
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Tang
authors.forename: Michael
authors.initials: M
authors.lastname: Bloch
authors.forename: Steven
authors.initials: S
authors.lastname: Elliott
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhu
authors.forename: Lindsey
authors.initials: L
authors.lastname: Bazzone
authors.forename: David
authors.initials: D
authors.lastname: Yu
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Weldon
authors.forename: Rachel
authors.initials: R
authors.lastname: Schiff
authors.forename: John A
authors.initials: JA
authors.lastname: McLachlan
authors.forename: Barbara S
authors.initials: BS
authors.lastname: Beckman
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wiese
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Nephew
authors.forename: Bin
authors.initials: B
authors.lastname: Shan
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Burow
authors.forename: Guangdi
authors.initials: G
authors.lastname: Wang
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002853:Chromatography, Liquid; D019008:Drug Resistance, Neoplasm; D015180:Electrophoresis, Gel, Two-Dimensional; D004848:Epithelium / Q000473:pathology*; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006801:Humans; D048671:MAP Kinase Kinase 5 / Q000235:genetics / Q000378:metabolism*; D008648:Mesoderm / Q000473:pathology*; D048054:Mitogen-Activated Protein Kinase 7 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014410:Tumor Stem Cell Assay
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D048054:Mitogen-Activated Protein Kinase 7; D048671:MAP Kinase Kinase 5; C482563:MAP2K5 protein, human