ABSTRACT
BACKGROUND: Treatment failure for breast cancer is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate invasion- and metastasis-related genes in breast cancer cells in vitro and in vivo. Identification of new targets will facilitate the developmental pace of new techniques in screening and early diagnosis. Improved abilities to predict progression and metastasis, therapeutic response and toxicity will help to increase survival of breast cancer patients. METHODS: Differential protein expression in two breast cancer cell lines, one with high and the other with low metastatic potential, was analyzed using two-dimensional liquid phase chromatographic fractionation (Proteome Lab PF 2D system) followed by matrix-assisted laser desorption/time-of-flight mass spectrometry (MALDI-TOF/MS). RESULTS: Up regulation of α-subunit of ATP synthase was identified in high metastatic cells compared with low metastatic cells. Immunohistochemical analysis of 168 human breast cancer specimens on tissue microarrays revealed a high frequency of ATP synthase α-subunit expression in breast cancer (94.6%) compared to normal (21.2%) and atypical hyperplasia (23%) breast tissues. Levels of ATP synthase expression levels strongly correlated with large tumor size, poor tumor differentiation and advanced tumor stages (P < 0.05). ATP synthase α-subunit over-expression was detected on the surface of a highly invasive breast cancer cell line. An antibody against the ATP synthase α-subunit inhibited proliferation, migration and invasion in these breast cancer cells but not that of a non-tumor derived breast cell line. CONCLUSIONS: Over-expression of ATP synthase α-subunit may be involved in the progression and metastasis of breast cancer, perhaps representing a potential biomarker for diagnosis, prognosis and a therapeutic target for breast cancer. This finding of this study will help us to better understand the molecular mechanism of tumor metastasis and to improve the screening, diagnosis, as well as prognosis and/or prediction of responses to therapy for breast cancer.
Subject(s)
Breast Neoplasms/enzymology , Breast Neoplasms/therapy , Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors , Molecular Targeted Therapy , Adult , Aged , Aged, 80 and over , Antibodies/pharmacology , Apoptosis/drug effects , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Chromatography, Liquid , Female , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hyperplasia , Immunohistochemistry , Middle Aged , Mitochondrial Proton-Translocating ATPases/chemistry , Mitochondrial Proton-Translocating ATPases/metabolism , Neoplasm Invasiveness , Protein Subunits/antagonists & inhibitors , Protein Subunits/chemistry , Protein Subunits/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue Array AnalysisABSTRACT
BACKGROUND: Treatment failure for esophageal carcinoma is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate invasion- and metastasis-related genes in esophageal carcinoma cells in vitro and in vivo. METHODS: A metastasis model using a Matrigel invasion clonal selection approach was employed to establish a highly invasive subline EC9706-P4 from the esophageal carcinoma cell (ESCC) line EC9706. The differentially expressed genes of the subline and the parental cells determined by gene microarrays were further analyzed by RT-PCR and Western blotting. RESULTS: We identified sphingosine kinase 1 (SPHK1) as an invasion and metastasis-related gene of esophageal cancer. SPHK1 was overexpressed in the EC9706-P4 subline with high invasive capacity. Among six ESCC lines tested, KYSE2 and KYSE30 cells showed the highest SPHK1 mRNA and protein expressions as well as the most invasive phenotype. By Western blotting, in 7/12 cases (58%), SPHK1 expression was higher in esophageal carcinomas than in the companion normal tissue. In 23/30 cases (76%), SPHK1 protein expression was upregulated in the tumors compared to matched normal tissue by immunohistochemistry (IHC). Esophageal carcinoma tissue microarray analysis indicated that SPHK1 expression correlated with the depth of tumor invasion (P < 0.0001) and lymph node metastasis (P = 0.016). By Kaplan-Meier analysis, strong SPHK1 expression was significantly associated with clinical failure (P < 0.01), suggesting the involvement of SPHK1 in aggressiveness of human esophageal carcinoma. SPHK1 overexpression significantly increased the invasiveness of EC9706 cells in vitro and also increased EC9706 cell growth and spontaneous metastasis in vivo, promoting significant increases in tumor growth, tumor burden and spontaneous lung metastasis in nude mice. SPHK1 expression significantly correlated with the expression of many EGFR pathway genes associated with invasion of cancer cells. SPHK1 protein expression also significantly correlated with the phosphorylation of EGFR. CONCLUSION: In summary, our data implicate SPHK1 in the metastasis of esophageal cancer. Our study also identified downstream mediators of SPHK1 in esophageal cancer cells that may mediate enhanced malignant behavior, and several of these mediators may be useful as therapeutic targets.
