Proteomic analysis of tumor necrosis factor-alpha resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype.
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.
Zhou
Changhua
C
Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA.
Nitschke
Ashley M
AM
Xiong
Wei
W
Zhang
Qiang
Q
Tang
Yan
Y
Bloch
Michael
M
Elliott
Steven
S
Zhu
Yun
Y
Bazzone
Lindsey
L
Yu
David
D
Weldon
Christopher B
CB
Schiff
Rachel
R
McLachlan
John A
JA
Beckman
Barbara S
BS
Wiese
Thomas E
TE
Nephew
Kenneth P
KP
Shan
Bin
B
Burow
Matthew E
ME
Wang
Guangdi
G
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
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