2007. then oxidized PTEN and protein tyrosine phosphatase 1B (PTP1B) followed by activation of the Akt pathway. This contributed to directional cell migration. The expression level of AQP3 Columbianadin in breast malignancy cells was related to their migration ability both and through CXCL12/CXCR4- or H2O2-dependent pathways. Coincidentally, spontaneous metastasis of orthotopic xenografts to the lung was reduced upon AQP3 knockdown. These findings underscore the importance of AQP3-transported H2O2 in CXCL12/CXCR4-dependent signaling and migration in breast malignancy cells and suggest that AQP3 has potential as a therapeutic target for breast cancer. INTRODUCTION Breast cancer mortality remains high owing to clinical relapse associated with metastases, primarily to the lungs, brain, and bones (1). Metastases are the result of several sequential processes, including cell migration and invasion (2). Organ-specific metastasis requires chemokine-dependent cancer cell migration toward destination sites (3). In particular, the CXCL12/CXCR4 axis is usually a key step in breast malignancy cell migration toward the lungs (4, 5). The binding of CXCL12 to CXCR4 stimulates downstream G protein signaling, leading to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt or mitogen-activated protein kinase (MAPK) pathway. These effects regulate a variety of cellular functions, such as cell proliferation and migration, thereby contributing to cancer metastasis and progression (6). However, the underlying mechanism by which the pathways downstream of CXCL12/CXCR4 result in breast malignancy cell migration and metastasis remain to be fully elucidated. Aquaporin-3 (AQP3), a member of the aquaporin water channel family (AQP0 to -12), has the primary function of transporting water and glycerol (7, 8). AQP3 is usually expressed in various cancer cells derived from diverse types of cancer tissues, including breast, colon, and lung (9, 10). Recent results from clinical studies have suggested the relevance of AQP3 expression in Columbianadin tumor progression and the prognosis of several malignant cancers (11,C14). studies using cancer cell lines have implicated AQP3 expression in cancer cell proliferation and migration (15,C17). However, the mechanism by which AQP3 participates as a biological pore channel in cancer progression remains unknown. AQP3-facilitated cellular uptake of hydrogen peroxide (H2O2) was discovered recently (18). We subsequently demonstrated that this uptake of extracellular H2O2 by T cells, generated in response to chemokines, including CXCL12, was facilitated by AQP3 and was required for the chemotaxis necessary to produce a sufficient immune response (19). The cellular redox state appears to play a role in the pathology of cancer (20). Columbianadin Many studies have proposed the involvement of reactive oxygen species (ROS), including H2O2, in cell growth, survival, motility, and metastasis during cancer progression (21). Furthermore, H2O2 is usually emerging as an important second messenger in cell signaling (22, 23). We hypothesized that H2O2, taken up through an AQP3-facilitated process, might act as a second messenger and be involved in a variety of cellular PML functions, including the focus of the current studybreast cancer cell migration and metastasis. In this study, we utilized the breast malignancy cell lines MDA-MB-231 and DU4475 and their transplantation into immune-deficient mice, since both cells express CXCR4 but do not express Columbianadin epidermal growth factor receptor 2 (EGFR2) (HER2) and are well characterized by their metastatic potentials and properties (4, 5, 24). The results demonstrate that AQP3 is required for CXCL12-induced breast malignancy cell signaling and directional migration by a mechanism involving the CXCL12-induced generation of extracellular H2O2 and subsequent intracellular transport by AQP3. An spontaneous-metastasis model using AQP3 knockdown (KD) cells consistently showed markedly reduced breast malignancy cell metastasis to the lungs. MATERIALS AND METHODS Cell lines. Breast malignancy cell lines, MDA-MB-231 and DU4475, were obtained from the American Type Culture Collection (ATCC). The cells were maintained in Dulbecco’s altered Eagle medium (DMEM) or RPMI 1640 made up of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a CO2 incubator (5% CO2; 37C). RNA interference (RNAi) and plasmid DNAs. For AQP3, NADPH.