REPORTS Collapsin Response Mediator Protein-1 and the Invasion and Metastasis of Cancer Cells Jin-Yuan Shih, Shuenn-Chen Yang, Tse-Ming Hong, Ang Yuan, Jeremy J. W. Chen, Chong-Jen Yu, Yih-Leong Chang, Yung-Chie Lee, Konan Peck, Cheng-Wen Wu, Pan-Chyr Yang
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Metastasis, the spread of tumor cells from a primary tumor to secondary sites within the body, is a complicated process involving degradation of the basement membrane, invasion of the stroma, adhesion, angiogenesis, cell proliferation, and migration (1). Molecular aspects of metastasis are not clearly understood, and a variety of positive and negative factors may be involved (2), as well as numerous genetic changes. In addition, current clinical methods cannot accurately identify which patients will develop metastatic disease. Thus, improved methods to predict and to diagnose metastasis and improved treatments of metastasis are needed. One approach to solving this problem is to identify the genes controlling metastasis (3). Cancers are a heterogeneous mass of neoplastic cells with various properties, including different metastatic activity (4). During carcinogenesis, some tumor cells probably acquire new phenotypes by the increased expression of metastasispromoting genes or the decreased expression of metastasis-suppressor genes. Studies of differential gene expression in poorly metastatic cancer cells and highly metastatic cancer cells should identify genes associated with metastasis and, in fact, several metastasis-suppressor genes, such as NM23, KAI1, and KiSS-1, have been so identified (5–7). New highthroughput genomic technologies, such as DNA microarray technology, may facilitate gene-expression profiling of cancers. DNA microarray technology can analyze the expression of many genes simultaneously and has identified numerous differentially expressed genes (8–11). For
example, Clark et al. (12) found that the small guanosine 5⬘-triphosphatase RhoC, when overexpressed, enhanced metastasis. To identify genes associated with invasion, we used an established panel of human lung adenocarcinoma cell lines (13,14) with different invasive activities (CL1–0 cells and its sublines CL1–1, CL1–5, and CL1–5-F4 cells, in ascending order of activity), a complementary DNA (cDNA) microarray, and a colorimetric detection system (14,15). We selected a differentially expressed gene, collapsin response mediator protein-1 (CRMP-1), and determined whether its expression was associated with the invasive activity and metastasis of cancer cells.
MATERIALS
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METHODS
Cell Lines and Culture Conditions The panel of human lung adenocarcinoma cell lines, CL1–0, CL1–1, CL1–5, and CL1–5-F4, in ascending order of invasiveness, was established in our laboratory (13,14). Cells were grown in RPMI-1640 medium (Life Technologies, Inc. [GIBCO BRL], Rockville, MD) with 10% fetal bovine serum (FBS) (Life Technologies, Inc.) and 2 mM L-glutamine (Life Technologies, Inc.) at 37 °C in a humidified atmosphere of 5% CO2–95% air. CL1–0 is the parent cell line; CL1–1 and CL1–5 are sublines that were selected from CL1–0 cultures with a polycarbonate membrane coated with Matrigel (Collaborative Biomedical, Becton Dickinson Labware, Bedford, MA) in a Transwell invasion chamber (13). The cells that migrated through the membrane were harvested. The sublines from the first and fifth rounds of selection were designed CL1–1 and CL1–5, respectively (13). In the next selection step, CL1–5 cells were
Affiliations of authors: J.-Y. Shih, C.-J. Yu (Department of Internal Medicine), A. Yuan (Department of Emergency Medicine), J. J. W. Chen (Department of Clinical Research), Y.-L. Chang (Department of Pathology), Y.-C. Lee (Department of Surgery), National Taiwan University Hospital, Taipei; S.-C. Yang, T.-M. Hong, K. Peck, Institute of Biomedical Sciences, Academia Sinica, Taipei; C.-W. Wu, National Health Research Institutes, Taipei; P.-C. Yang, Department of Internal Medicine, National Taiwan University Hospital, National Health Research Institutes, and Institute of Biomedical Sciences, Taipei. Correspondence to: Pan-Chyr Yang, M.D., Ph.D., Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Rd., Taipei 100, Taiwan (e-mail:
[email protected]. edu.tw). See “Notes” following “References.” © Oxford University Press
Journal of the National Cancer Institute, Vol. 93, No. 18, September 19, 2001
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Background: Numerous genetic changes are associated with metastasis and invasion of cancer cells. To identify differentially expressed invasionassociated genes, we screened a panel of lung cancer cell lines (CL1–0, CL1–1, CL1–5, and CL1–5-F4 in order of increasing invasive activity) for such genes and selected one gene, collapsin response mediator protein-1 (CRMP1), to characterize. Methods: We used a microarray containing 9600 gene sequences to assess gene expression in the cell panel and selected the differentially expressed CRMP-1 gene for further study. We confirmed the differential expression of CRMP-1 with northern and western blot analyses. After transfecting and overexpressing CRMP-1 in highly invasive CL1–5 cells, the cells were assessed morphologically and with an in vitro invasion assay. We used enhanced green fluorescent proteintagged CRMP-1 and fluorescence microscopy to localize CRMP-1 intracellularly. CRMP-1 expression in 80 lung cancer specimens was determined by real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR). All statistical tests were two-sided. Results: Expression of CRMP-1 was inversely associated with invasive activity in the cell panel, an observation confirmed by northern and western blot analyses. CRMP-1-transfected CL1–5 cells became rounded and had fewer filopodia and statistically significantly lower in vitro invasive activity than untransfected cells (all P
