Different distribution patterns of FACs and F-actin in KO1 and WT1 cells as revealed by vinculin immunostaining (green) and philloidin staining (reddish colored)

Different distribution patterns of FACs and F-actin in KO1 and WT1 cells as revealed by vinculin immunostaining (green) and philloidin staining (reddish colored). cells. These results demonstrate that SRC-1 can promote BC metastasis by directly enhancing expression and thus promoting ITGA5-mediated cell adhesion and migration. Therefore, targeting ITGA5 in SRC-1-positive BCs may result in inhibition of SRC-1-promoted BC metastasis. in the MMTV-polyoma middle T antigen (PyMT) mammary tumor-prone mice dramatically suppresses lung metastasis without affecting primary tumor DPN formation (9). These studies indicate that SRC-1 strongly promotes BC metastasis. SRC-1 upregulates the expression of several key regulators for BC progression. In particular, SRC-1 deficiency in mouse mammary tumors reverses HER2 overexpression and reduces Akt activity (9). Knockout of in these tumors suppresses the expression of colony stimulating factor (CSF-1) (9), a chemoattractant that recruits macrophages to the tumor site. In turn, the macrophages secret EGF to stimulate tumor cell motility. SRC-1 also serves as a coactivator of PEA3 to enhance Twist1 expression in BC cells. Elevated Twist1 promotes breast tumor cell epithelial mesenchymal transition (EMT), invasion and metastasis by recruiting the NuRD protein complex to repress E-cadherin expression (2, 10). Furthermore, SRC-1 works with Ets-2 to induce c-Myc expression and with HOXC11 to induce calcium-binding protein S100beta expression, both of which are positively associated with acquired resistance to endocrine therapy (5, 7). Recently, we discovered that the number of mammary tumor cells in the blood of wild type (WT);PyMT mice is significantly higher than that in the blood of SRC-1 knockout (KO);PyMT mice, suggesting a contribution of SRC-1 to BC cell migration and invasion from the primary tumor to the blood vessels (9). Local migration and invasion of tumor cells are early events partially induced by the tumor microenvironment in metastasis. Resident fibroblasts not only secret TGF to induce tumor cell EMT, but also produce abundant collagen and fibronectin (FN) extracellular matrix (ECM) proteins to provide anchorages for tumor cell adhesion and migration (11C13). Integrins consist of 18 and 8 glycoprotein subunits, which form 24 distinct heterodimeric transmembrane receptors. These receptors bind to ECM proteins such as FN to transport signals bidirectionally across the cell membrane, allowing cells to respond to environmental changes (14). Multiple integrins, including v3, v5, 51, 64, DPN 41 and v6, are detected in cancer cells and their expression levels are associated with tumorigenesis and cancer progression (15). In BC, integrin 4 amplifies HER2 signaling to potentiate mammary tumorigenesis (16). Activation of integrin v3 supports BC cell adhesion to the vascular wall and promotes metastasis (17), while knockout of integrin 1 inhibits mammary tumorigenesis in mice (18). In addition, integrins also regulate Rabbit Polyclonal to CBLN2 tumor cell survival, growth and metastasis in an anchorage-independent manner (15). The mesenchymal integrins 5 (ITGA5) and DPN 1 form heterodimers to mediate cell adhesion to FN (15). Knockout of ITGA5 in mice results in embryonic lethality (19). In human hepatocarcinoma cells, ITGA5 promotes cell adhesion and migration on FN through activating focal adhesion kinase (FAK) (20). In transformed mammary epithelial cells, ITGA5 expression is increased along with the EMT process (21). These findings indicate that expression correlates with cancer progression and plays an important role to enhance cancer cell adhesion to and migration along FN. In this study, we found that SRC-1 works with AP-1 to potentiate expression. The increased ITGA5, in turn, significantly accelerates BC cell adhesion and migration on FN. The identification of as a target gene of SRC-1 DPN and AP-1 in BC cells uncovered a new molecular pathway: SRC-1 regulates expression to promote BC metastasis. Materials and Methods Cell adhesion and migration assays The primary and stable WT;PyMT (WT) and KO;PyMT (KO) mouse mammary tumor cell lines were generated as described previously (22). Adhesion assay was performed on FN or laminin (LN)-coated plates as described previously (23). Individual cell migration was tracked for 18 hours in 96-well plate pre-coated with fluorescent beads and track areas were analyzed using NIH image software as described previously (2, 22). Western blot analysis of human breast tumors A total of 24 human BC specimens were collected from surgically removed tumor tissues at Luzhou Medical College Affiliated Hospital in 2009 2009. All patients were Asian women and aged 33C65 years old. No patient survival data were available at this stage. A portion of the specimen was used for clinical diagnosis.