Proteomic Profiling of Proteins Associated With Lymph Node Metastasis in Colorectal Cancer Yiming Ma, 1 Mei Zhao, 1 Jialing Zhong, 1 Lan Shi, 1 Qing Luo, 1 Jian Liu, 1 Jia Wang, 1 Xinghua Yuan, 2 ** and Changzhi Huang 1 * 1 Key Laboratory of Molecular Oncology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China 2 Department of Abdomen Surgery, Cancer Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China ABSTRACT Lymph node metastasis (LNM) is associated with poor prognosis in colorectal cancer (CRC). The presence or absence of lymph node metastases is a strong independent prognostic factor for CRC survival. Investigation of proteins associated with the process of lymph node metastasis (LNM) is crucial for understanding of the molecular mechanisms underlying the LNM process and for predicting the CRC prognosis. In the present study, proteins from CRC tissues and adjacent normal mucosa (NMC) were examined using two-dimensional gel electrophoresis coupled with MALDI-TOF-MS. The expression levels of Ferritin Heavy Chain (FHC) were decreased in LNM CRC as compared to those in non- LNM CRC, while the expression of Cathepsin D and Ubiquitin C-terminal hydrolase-L1 (UCH-L1) were increased in LNM CRC. The results were confirmed by Western blotting and immunohistochemical staining. Furthermore, in vitro cell invasion assay showed that the overexpression of UCH-L1 through gene transfection increased the invasive ability of HCT8 cells, suggesting that UCH-L1 is not only a biomarker for LNM in CRC, but also a functional protein that may play a significant role in cell migration. The proteins identified in the present study should further our understanding of the LNM process of CRC and may become useful markers for diagnosis and targets for therapeutic interventions. J. Cell. Biochem. 110: 1512–1519, 2010. ß 2010 Wiley-Liss, Inc. KEY WORDS: PROTEOMICS; LYMPH NODE METASTASIS; COLORECTAL CANCER; UCH-L1 C olorectal cancer (CRC) is one of the most common malignant tumors in the world. It causes significant morbidity and mortality, especially in industrialized nations. The morbidity of CRC in China has also risen rapidly in recent years and is the third most common cancers. In 2008, the morbidity rate of CRC grew by 4.2% in China and the estimated number of new CRC cases was up to 400,000. It has been suggested by earlier reports [Newcomb et al., 1992; Selby et al., 1992] that the survival rate decreases while progressing through the stages. A major proportion of patients in China were being diagnosed in the advanced stages and only 10% of patients were diagnosed in T1 stage. As a result, the 5-year survival rate was between 20% and 50%. One of the most important prognostic factor for predicting the outcome of long-term survival of cancer is whether there is positive lymph nodes or distant metastases. The lymph node metastasis rate in T 3 –T 4 stages was much higher than that in T 1 –T 2 stages in CRC. The lymph node ratio (ratio of positive over excised lymph nodes) is a highly significant prognostic factor in stage III CRC. Studies by Derwinger et al. [2008] showed that with the increase of lymph node ratios, the 3-year disease-free survival (DFS) dropped sharply from 80% to 30%. However, previous studies has suggested that substantial variations in nodal staging is closely associated with surgical, pathological, and patients factors [Baxter et al., 2005; Chang et al., 2007]. Inadequate lymph node sampling can lead to positive lymph node being missed and thus to patients being misclassified and not being appropriately treated. Therefore, additional markers are much needed to detect lymph node metastasis (LNM) in CRC and to improve systemic treatments. In recent years, the focus of cancer research has expanded from genomic to proteomic. Proteins are responsible for the functional execution in a diversity of cells. Many regulatory processes and disease processes occur at the protein level, and most drug targets Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 110:1512–1519 (2010) 1512 Grant sponsor: National Key Basic Research Program of China; Grant number: 2007CB914700. *Correspondence to: Changzhi Huang, Cancer Institute, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China. E-mail: huangcz8849@126.com **Correspondence to: Xinghua Yuan, Department of Abdomen Surgery, Cancer Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China. E-mail: haroldyuan@sohu.com Received 15 March 2010; Accepted 27 May 2010  DOI 10.1002/jcb.22726  ß 2009 Wiley-Liss, Inc. Published online 11 June 2010 in Wiley InterScience (www.interscience.wiley.com).