Analysis of dystroglycan regulation and functions in mouse mammary epithelial cells and implications for mammary tumorigenesis

Abstract

Abnormalities in the interactions of cells with the extracellular matrix (ECM) play an important role in the development and progression of many types of cancer and are a hallmark of malignant transformation. The dystroglycan (DG) complex is a transmembrane glycoprotein that forms a continuous link from the ECM to the actin cytoskeleton, providing structural integrity and perhaps transducing signal, in a manner similar to integrins. Deregulated expression of DG has been reported in a variety of human malignancies and related to tumor differentiation and aggressiveness. In breast cancer, reduced DG expression has been associated with patient survival and with loss of differentiation of tumor cells. Limited data are available on DG physiology in epithelial cells. In this study, we used the HC11 spontaneously immortalized murine mammary epithelial cells to study DG function(s) and regulation in normal cells. We found that expression of DG protein and mRNA is cell-cycle and cell-density regulated in these cells. Moreover, expression of both DG subunits increased upon lactogenic differentiation of the HC11 cells. The turnover of cell-surface-expressed DG was evaluated in the same cells and half-life of DG subunits was evaluated to be about 12 h. DG-specific small inhibitory RNAs were used to analyze the effects of a reduced expression of DG in these cells. Cells in which DG expression was suppressed were growth inhibited, accumulated in the S-phase of the cell cycle, failed to undergo lactogenic differentiation, and displayed an increase in the percentage of apoptotic cells. Moreover, changes were observed in the expression and/or activity of several molecules involved in cell growth control. These results demonstrate that DG expression is tightly regulated in normal mammary epithelial cells and support the hypothesis that DG is involved in several functions other than structural integrity in these cells. This finding provides new insight into the roles played by DG in epithelial cell physiology and will contribute to our understanding of its involvement in the process of epithelial cell transformation.

Dystroglycan expression is reduced during prostate tumorigenesis and is regulated by androgens in prostate cancer cells

“…Cell cultures were collected by cell scraping and cell pellets were added to 3-5 volumes of sonication buffer containing proteases and phosphatase inhibitors (20 mM Tris-HCl pH 7.4, 2 mM EGTA, 6 mM b-mercaptoethanol, 1% NP40, 0.1% SDS, 50 mM NaF, 15 mg/ ml benzamidine, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and sonicated at 48C, as previously described (Sgambato et al, 2004(Sgambato et al, , 2006a. Homogenates were incubated in ice for 30 min and then centrifuged at 14,000 rpm for 15 min at 48C.…”

“…Densitometric analysis was performed four times for each sample (two runs for each protein extract) and, after correction for the internal control, mean values were arbitrarily considered identical when the difference between them was lower than 10%. The monoclonal antibodies to a-DG (clone VIA4-1) and to b-DG (clone 43DAG/8D5) were obtained from Upstate Biotechnology (Lake Placid, NY) and from Novocastra (Newcastle, UK), respectively and were used as previously described (Sgambato et al, 2004(Sgambato et al, , 2006a.…”

“…All assays were performed in triplicate and all experiments were repeated three times. For cell cycle analysis, exponentially growing cells were collected, washed with PBS, fixed in 70% ethanol and stored at 48C (Sgambato et al, 2004(Sgambato et al, , 2006a. For the analysis, cells were collected by centrifugation, stained with propidium iodide and analyzed for DNA content on a Coulter EPICS 753 flow cytometer, as previously described.…”

“…Moreover, DG has been shown to be essential for epithelial development in vivo and for epithelial morphogenesis in vitro (Durbeej et al, 2001). Recently, DG has been implicated in several cell functions (i.e., growth control, differentiation, shape change, and movement) which are all relevant in the process of tumor development and metastasis although the mechanisms by which the DG complex exerts these effects remain unknown (Winder, 2001;Muschler et al, 2002;Sgambato et al, 2006a).…”

Altered expression of natively glycosylated α dystroglycan in pediatric solid tumors

“…Altered dystroglycan expression has also been reported in oral epithelial cancers [30]. In addition, aberrant dystroglycan expression and dystroglycan cleavage have been reported in cell lines representative of adult tumor types including breast and colon [29][30][31][32][33]. Here, we expand such analyses of dystroglycan expression and glycosylation to pediatric solid tumors and demonstrate marked alterations of α dystroglycan in pediatric rhabdomyosarcoma (RMS), neuroblastoma (NBL), and medulloblastoma.…”