Oncology Research, Vol. 17, pp. 601–612 0965-0407/09 $90.00 + .00 Printed in the USA. All rights reserved. E-ISSN 1555-3906 Copyright  2009 Cognizant Comm. Corp. www.cognizantcommunication.com Comparative Proteomic Analysis of Mouse Melanoma Cell Line B16, a Metastatic Descendant B16F10, and B16 Overexpressing the Metastasis-Associated Tyrosine Phosphatase PRL-3 Sang Hee Kim,* 1 Yongmo Kim,* 1 Moonil Kim,† Dae Shick Kim,‡ Sang Chul Lee,* Seung-Wook Chi,* Do Hee Lee,* Sung Goo Park,* Byoung Chul Park,* Kwang-Hee Bae,* and Sunghyun Kang* *Medical Proteomics Research Center, KRIBB, Daejeon, 305-806, Korea †BioNanotechnology Research Center, KRIBB, Daejeon 305-806, Korea ‡Department of Pathology, Samsung Medical Center and Sungkyunkwan University School of Medicine, Seoul, 135-710, Korea Metastasis is a complex, multistep process by which a cancer cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary cancer. A deeper understanding of the molecular events underlying metastasis will provide information that will be useful for the development of new diagnostic and therapeutic strategies. The B16 and B16F10 mouse melanoma cell lines are widely used as model system for studying many aspects of cancer biology including metastasis. Compared with B16, which has a low metastatic potential, the highly metastatic cell line B16F10 displayed a higher metastatic ability along with higher expression levels of the metastasis-associated phosphatase of regenerating liver-3 (PRL-3). B16 cells transfected with PRL-3 cDNA (B16-PRL3) had metastatic abilities comparable to those of B16F10 cells. To study the molecular mechanisms that underlie metastasis, the proteomes of the B16, B16F10, and B16-PRL3 cell lines were compared using two-dimensional differential in-gel electrophoresis. Proteins that varied significantly in levels between these cell lines were selected and identified using mass spectrometry. Interestingly, many proteins, especially those present in membrane fractions, were similarly up- or downregulated in both the B16F10 and B16-PRL3 cells lines compared to B16 cell lines. The list of similarly regulated proteins included heat shock protein 70, fascin-1, septin-6, ATP synthase β subunit, and bone morphogenic protein receptor type IB. These proteins may play a causal role in PRL-3-mediated metas- tasis. These investigations open an avenue for the further characterization of the molecular mechanisms that underlie metastasis. Key words: B16 Differential in-gel electrophoresis (DIGE); Melanoma; Metastasis; Proteomics INTRODUCTION gested to play a causal role in tumor metastasis (4). PRLs (PRL-1, -2, and -3) are relatively small proteins of approximately 22 kDa that share at least 75% amino Metastases are the leading cause of death in cancer patients, accounting for more than 90% of all cancer acid sequence similarity. PRLs have an N-terminal cata- lytic domain containing the signature protein tyrosine mortalities (1). Cancer metastasis is a complex, multi- step process (2,3). The process of metastasis begins with phosphatase active site sequence CX 5 R and a C-terminal domain containing a polybasic region and a CAAX se- some tumor cells breaking their adhesions with neigh- boring cells and detaching from the primary tumor. Such quence for prenylation (5). Increasing evidence suggests that PRL-1, -2, and -3 might each take part in the prolif- cells then dissolve the extracellular matrix; migrate and invade surrounding tissues and/or travel via the circula- eration, transformation, and metastasis of cancer cells. PRL-1 is highly expressed in proliferating cells and in a tory system; and invade, survive, and proliferate at dis- tant new sites. However, the detailed molecular and cel- number of human cancer cell lines (6). Recently, Min et. al. demonstrated that PRL-1 caused the degradation lular mechanisms underlying tumor metastasis are not well understood. of p53 and thereby inhibited p53-mediated apoptosis (7). Elevated expression of PRL-2 is observed in prostate Recently, the phosphatase of regenerating liver (PRL) family, a tyrosine phosphatase family, has been sug- cancer cell lines and in tumor tissues (8). Overexpres- 1 Both authors equally contributed to this work. Address correspondence to Sunghyun Kang, Translational Research Center, KRIBB, 52 Eoeun-dong, Yuseong, Daejeon 305-806, Korea. Tel: +82-42-860-4243; Fax: +82-42-860-4593; E-mail: skang@kribb.re.kr or Kwang-Hee Bae, Translational Research Center, KRIBB, 52 Eoeun-dong, Yuseong, Daejeon 305-806, Korea. Tel: +82-42-860-4268; Fax: +82-42-860-4593; E-mail: khbae@kribb.re.kr 601