Holiday 6/9.qxd

Breast cancer (BrCa) is one of the most frequently diagnosed cancers and the second leading cause of cancerrelated deaths in North American women. Most deaths are caused by metastasis, and BrCa is characterized by a distinct metastatic pattern involving lymph nodes, bone marrow, lung, liver and brain. Migration of metastatic cells share many similarities with leukocyte trafficking, which are regulated by chemokines and their receptors. The current study evaluates the expression and functional role of CCR9, and its only known ligand, CCL25, in BrCa cell migration and invasion. Quantitative immunohistochemical analysis showed that both moderately and poorly differentiated BrCa tissue expressed significantly more (P<0.0001) CCR9 compared to non-neoplastic breast tissue. Interestingly, poorly differentiated BrCa tissue expressed significantly more (P<0.0001) CCR9 compared to moderately differentiated BrCa tissue. Similarly, CCR9 was highly expressed by the aggressive breast cancer cell line (MDA-MD-231) compared to the less aggressive MCF-7. Migration as well as invasion assays were used to evaluate the functional interaction between CCR9 and CCL25 in BrCa cell lines (MDA-MB-231 and MCF-7). Neutralizing CCR9-CCL25 interactions significantly impaired the migration and invasion of BrCa cells. Furthermore, CCL25 enhanced the expression of MMP-1, -9, -11 and -13 active proteins by BrCa cells in a CCR9dependent fashion. These studies show CCR9 is functionally and significantly expressed by BrCa (poorly > moderately differentiated) tissue and cells as well as that CCL25 activation of this receptor promotes breast tumor cell migration, invasion and MMP expression, which are key components of BrCa metastasis. Introduction In the United States, BrCa is the most common non-skin cancer and the second leading cause of cancer deaths in women, behind lung cancer (1). Metastasis is responsible for the majority of BrCa-related deaths. The development of metastasis consists of a complex series of sequential steps (2). These processes include invasion through extracellular matrix, intravasation, survival in the circulation, extravasation into a distant site, and progressive growth at the site of metastasis (3). BrCa progression is characterized by a distinct metastatic pattern involving regional lymph nodes, bone marrow, lung, brain and liver (4). Many factors have been implicated in the process of metastasis, but the precise mechanisms for the directional migration of malignant cells to different organs are incompletely known. This process shares many similarities with leukocyte trafficking, which is largely mediated by chemokines. In this regard, chemokines are a super family of small, cytokine-like proteins that induce, through their interaction with G protein-coupled receptors, cytoskeletal rearrangement, firm adhesion to endothelial cells, and directional migration. These secreted proteins act in a coordinated fashion with cell-surface proteins, including integrins, to direct the specific homing of various subsets of cells. It has been shown that CXCL12-CXCR4 interactions play a role in the metastasis of BrCa cells to regional lymph nodes and lung (5). It has also been demonstrated that neoplastic breast tissues express higher levels of CXCR4 than normal breast tissue. CXCR4/CXCL12 interactions alone do not fully explain the pattern of BrCa metastasis or the potential of BrCa cells to migrate and invade other tissues. In this study, we have tested the hypothesis that breast carcinomas partially use CCR9-dependent mechanisms for migration, invasion and matrix metalloproteinase (MMP) expression. To test this hypothesis, we evaluated the levels of CCR9 mRNA and surface protein expression by BrCa cell lines and tissue. The functional relevance of this expression was demonstrated using migration, extracellular matrix (ECM) invasion and MMP expression assays. These studies suggest the expression of CCR9 and its interaction with CCL25 aid in BrCa cell migration, invasion and MMP expression to support the INTERNATIONAL JOURNAL OF ONCOLOGY 38: 1279-1285, 2011 CCL25 mediates migration, invasion and matrix metalloproteinase expression by breast cancer cells in a CCR9-dependent fashion CRYSTAL JOHNSON-HOLIDAY1, RAJESH SINGH1, ERICA JOHNSON1, SHAILESH SINGH1, CECIL R. STOCKARD2, WILLIAM E. GRIZZLE2 and JAMES W. LILLARD Jr1 1Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA; 2Department of Pathology, University of Alabama at Birmingham, School of Medicine, Birmingham, AL, USA Received September 6, 2010; Accepted October 4, 2010 DOI: 10.3892/ijo.2011.953 _________________________________________ Correspondence to: Dr James W. Lillard Jr, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310-1495, USA E-mail: [email protected]