The microphthalmia-associated transcription factor (MITF) promotes melanocyte differentiation and cell cycle arrest. Paradoxically, MITF also promotes melanoma survival and proliferation, acting like a lineage survival oncogene. Thus, it is critically important to understand the mechanisms that regulate MITF activity in melanoma cells. SWI/SNF chromatin remodeling enzymes are multiprotein complexes composed of one of two related ATPases, BRG1 or BRM, and 9-12 associated factors (BAFs). We previously determined that BRG1 interacts with MITF to promote melanocyte differentiation. However, it was unclear whether SWI/SNF enzymes regulate the expression of different classes of MITF target genes in melanoma. In this study, we characterized SWI/SNF subunit expression in melanoma cells and observed down-regulation of BRG1 or BRM, but not concomitant loss of both ATPases. Re-introduction of BRG1 in BRG1 deficient SK-MEL5 cells enhanced expression of differentiation specific MITF target genes and resistance to cisplatin. Down-regulation of the single ATPase, BRM, in SK-MEL5 cells inhibited expression of both differentiation specific and pro-proliferative MITF target genes and inhibited tumorigenicity in vitro. Our data suggest that heterogeneous SWI/SNF complexes composed of either the BRG1 or BRM subunit promote expression of distinct and overlapping MITF target genes and that at least one ATPase is required for melanoma tumorigenicity.

No abstract

The pCMV4 plasmid containing the cancer-promoting gene, c-erbB2/neu, was cotransfected into the human hepatocarcinoma cell line 7721 with the pcDNA3 vector, which contains the`neo' selectable marker. Several clones showing stable expression of c-erbB2/neu were established and characterized by determination of c-erbB2/neu mRNA and its encoded protein p185. Expression of Lewis antigens and a1,3-fucosyltransferases and the biological behavior of 7721 cells after c-erbB2/neu transfection were studied using mock cells transfected with the vectors pCMV4 and pcDNA3 as controls. SLe x expression on the surface of mock cells was high, whereas expression of SDLe x , Le x and SLe a was absent or negligible. This is compatible with the abundant expression of a1,3-fucosyltransferase VII, very low expression of afucosyltransferase III/VI, and almost absent expression of a1,3-fucosyltransferase IV in the mock cells. After transfection of c-erbB2/neu, expression of SLe x and a1,3-fucosyltransferase VII were simultaneously elevated, but that of afucosyltransferase III/VI was not altered. The expression of both SLe x and a1,3-fucosyltransferase VII correlated positively with the expression of c-erbB2/neu in different clones, being highest in clone 13, medium in clone 6, and lowest in clone 7. In addition, the adhesion of 7721 cells to human umbilical vein endothelial cells (HUVECs) or P-selectin, as well as cell migration and invasion, were increased in c-erbB2/neu-transfected cells.These increases also correlated positively with the expression intensities of c-erbB2/neu, SLe x and a1,3-fucosyltransferase VII in the different clones, whereas cell adhesion to fibronectin correlated negatively with these variables. mAbs to SLe x (KM93) and SDLe x (FH6) significantly and slightly, respectively, abolished cell adhesion to HUVECs or P-selectin and cell migration and invasion. mAbs to SDLe x and SLe a did not suppress cell adhesion to HUVECs nor inhibit cell migration and invasion. Transfection of a1,3-fucosyltransferase VII cDNA into 7721 cells showed similar results to transfection of c-erbB2/neu, and the increased adhesion to HUVECs, cell migration, and invasion were also inhibited significantly by KM93 and slightly by FH6. These results indicate that expression of a1,3-fucosyltransferase VII and its specific product, SLe x , and their capacity for cell adhesion, migration and invasion are closely related. Therefore, the cerbB2/neu gene is proposed to be a metastasis-promoting gene, and its effects are at least partially mediated by the increased expression of a1,3-fucosyltransferase VII and SLe x .Keywords: c-erbB2/ neu; hepatocarcinoma cells; metastasisassociated phenotype; sialyl Lewis X; a1, 3-fucosyltransferase.The c-erbB2 gene (also known as neu, HER-2 or NGL) encodes a transmembrane glycoprotein of molecular mass 185 kDa (p185), which shares extensive sequence homology with the receptor of epidermal growth factor (EGF-R) [1,2]. Amplification or overexpression of the c-erbB2/neu gene was first found in human breast cancer [3] ...

BackgroundMetastatic melanoma is an aggressive malignancy that is resistant to therapy and has a poor prognosis. The progression of primary melanoma to metastatic disease is a multi-step process that requires dynamic regulation of gene expression through currently uncharacterized epigenetic mechanisms. Epigenetic regulation of gene expression often involves changes in chromatin structure that are catalyzed by chromatin remodeling enzymes. Understanding the mechanisms involved in the regulation of gene expression during metastasis is important for developing an effective strategy to treat metastatic melanoma. SWI/SNF enzymes are multisubunit complexes that contain either BRG1 or BRM as the catalytic subunit. We previously demonstrated that heterogeneous SWI/SNF complexes containing either BRG1 or BRM are epigenetic modulators that regulate important aspects of the melanoma phenotype and are required for melanoma tumorigenicity in vitro.ResultsTo characterize BRG1 expression during melanoma progression, we assayed expression of BRG1 in patient derived normal skin and in melanoma specimen. BRG1 mRNA levels were significantly higher in stage IV melanomas compared to stage III tumors and to normal skin. To determine the role of BRG1 in regulating the expression of genes involved in melanoma metastasis, we expressed BRG1 in a melanoma cell line that lacks BRG1 expression and examined changes in extracellular matrix and adhesion molecule expression. We found that BRG1 modulated the expression of a subset of extracellular matrix remodeling enzymes and adhesion proteins. Furthermore, BRG1 altered melanoma adhesion to different extracellular matrix components. Expression of BRG1 in melanoma cells that lack BRG1 increased invasive ability while down-regulation of BRG1 inhibited invasive ability in vitro. Activation of metalloproteinase (MMP) 2 expression greatly contributed to the BRG1 induced increase in melanoma invasiveness. We found that BRG1 is recruited to the MMP2 promoter and directly activates expression of this metastasis associated gene.ConclusionsWe provide evidence that BRG1 expression increases during melanoma progression. Our study has identified BRG1 target genes that play an important role in melanoma metastasis and we show that BRG1 promotes melanoma invasive ability in vitro. These results suggest that increased BRG1 levels promote the epigenetic changes in gene expression required for melanoma metastasis to proceed.

SummaryThe expressions of Lewis (Le) antigens, α-1,3/1,4 fucosyltransferases (α-1,3/1,4 FuTs), and metastatic potential after the treatment of 2 differentiation inducers, all-trans retinoic acid (ATRA), 8-bromo-cyclic 3′,5′adenosine monophosphate (8-Br-cAMP); and 2 proliferation inducers, epidermal growth factor (EGF) and phobol-12-myristate-13-acetate (PMA), on 7721 human hepatocarcinoma cell line were studied. Cell adhesion to human umbilical vein endothelial cells (HUVEC), cell migration through transwell and invasion through matrigel were selected as the indexes of metastatic potential-related phenotypes. Using fluorescence-labelled antibodies and flow-cytometric analysis, it was found that 7721 cells mainly expressed sialyl Lewis X (SLe x ) and a less amount of sialyl dimeric Lewis X (SDLe x ) antigens on the cell surface. Their expressions were down-regulated by ATRA, and up-regulated by EGF. SLe x antigen was also decreased and increased by the treatment of 8-Br-cAMP and PMA respectively. With Northern blot to detect the mRNAs of α-1,3/1,4 FuTs, the main enzymatic basis for the change in SLe x expression was found to be the alteration of the expression of α-1,3 FuT-VII. It was evidenced by the observations that α-1,3 FuT-VII was the main α-1,3/1,4 FuT in 7721 cells, while α-1,3/1,4 FuT-III and α-1,3 FuT-VI were expressed rather low. The changes in the expressions of SLe x antigen and α-1,3 FuT-VII resulted in the altered cell adhesion to tumour necrosis factor-α stimulated HUVEC, since only the monoclonal antibody of the SLe