Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer.
BACKGROUND
The molecular mechanisms involved in breast cancer metastasis still remain unclear to date. In our previous study, differential expression of peroxiredoxin 6 was found between the highly metastatic MDA-MB-435HM cells and their parental counterparts, MDA-MB-435 cells. In this study, we investigated the effects of peroxiredoxin 6 on the proliferation and metastatic potential of human breast cancer cells and their potential mechanism.


METHODS
Expression of peroxiredoxin 6 in the highly metastatic MDA-MB-231HM cells was investigated by RT-PCR, real-time PCR and western blot. A recombinant expression plasmid of the human peroxiredoxin 6 gene was constructed and transfected into MDA-MB-231 and MDA-MB-435 cells. The effects of peroxiredoxin 6 on the proliferation and invasion of MDA-MB-231 and MDA-MB-435 cells were investigated by the Cell Counting Kit-8 method, colony-formation assay, adhesion assay, flow cytometry and invasion assay in vitro. miRNA was used to downregulate the expression of peroxiredoxin 6. Genes related to the invasion and metastasis of cancer were determined by RT-PCR, real-time PCR and western blot. The tumorigenicity and spontaneously metastatic capability regulated by peroxiredoxin 6 were determined using an orthotopic xenograft tumor model in athymic mice.


RESULTS
Overexpression of peroxiredoxin 6 in MDA-MB-231HM cells compared with their parental counterparts was confirmed. Upregulation of peroxiredoxin 6 enhanced the in vitro proliferation and invasion of breast cancer cells. The enhancement was associated with decreasing levels of tissue inhibitor of matrix metalloproteinase (TIMP)-2 and increasing levels of the urokinase-type plasminogen activator receptor (uPAR), Ets-1 (E26 transformation-specific-1), matrix metalloproteinase (MMP)-9 and RhoC (ras homolog gene family, member C) expression. The results were further demonstrated by RNA interference experiments in vitro. In an in vivo study, we also demonstrated that peroxiredoxin 6-transfected breast cancer cells grew much faster and had more pulmonary metastases than control cells. By contrast, peroxiredoxin 6 knockdown breast cancer cells grew more slowly and had fewer pulmonary metastases. Effects similar to those of peroxiredoxin 6 on the uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression observed in in vitro studies were found in the in vivo study.


CONCLUSIONS
Overexpression of peroxiredoxin 6 leads to a more invasive phenotype and metastatic potential in human breast cancer, at least in part, through regulation of the levels of uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression.
Chang
Xin-Zhong
XZ
Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical Science, Fudan University, Shanghai, 200032, People's Republic of China.
Li
Da-Qiang
DQ
Hou
Yi-Feng
YF
Wu
Jiong
J
Lu
Jin-Song
JS
Di
Gen-Hong
GH
Jin
Wei
W
Ou
Zhou-Luo
ZL
Shen
Zhen-Zhou
ZZ
Shao
Zhi-Ming
ZM
D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018450:Disease Progression; D015536:Down-Regulation; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D066298:In Vitro Techniques; D008175:Lung Neoplasms / Q000378:metabolism / Q000556:secondary; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000378:metabolism; D009361:Neoplasm Invasiveness; D054465:Peroxiredoxin VI / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D050784:Proto-Oncogene Protein c-ets-1 / Q000378:metabolism; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous; D015854:Up-Regulation; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D000075024:rhoC GTP-Binding Protein
C494041:ETS1 protein, human; D035683:MicroRNAs; C524898:PLAUR protein, human; C524899:Plaur protein, mouse; D050784:Proto-Oncogene Protein c-ets-1; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D019716:Tissue Inhibitor of Metalloproteinase-2; C515977:PRDX6 protein, human; D054465:Peroxiredoxin VI; C515983:Prdx6 protein, mouse; D020780:Matrix Metalloproteinase 9; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D000075024:rhoC GTP-Binding Protein