Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry.
To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression.
Li
Da-Qiang
DQ
Department of Breast Surgery, Breast Cancer Institute, Cancer Hospital/Cancer Institute, Fudan University, Shanghai, P. R. China.
Wang
Lei
L
Fei
Fei
F
Hou
Yi-Feng
YF
Luo
Jian-Min
JM
Zeng
Rong
R
Wu
Jiong
J
Lu
Jin-Song
JS
Di
Gen-Hong
GH
Ou
Zhou-Luo
ZL
Xia
Qi-Chang
QC
Shen
Zhen-Zhou
ZZ
Shao
Zhi-Min
ZM
D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000556:secondary*; D015674:Mammary Neoplasms, Animal / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D020543:Proteome / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D014185:Transplantation, Isogeneic
D014408:Biomarkers, Tumor; D020543:Proteome