Abstract
BackgroundDeleted in Liver Cancer 1 (Dlc1) is a tumor suppressor gene, which maps to human chromosome 8p21-22 and is found frequently deleted in many cancers including breast cancer. The promoter of the remaining allele is often found methylated. The Dlc1 gene encodes a RhoGAP protein that regulates cell proliferation, migration and inhibits cell growth and invasion when restored in Dlc1 deficient tumor cell lines. This study focuses on determining the role of Dlc1 in normal mammary gland development and epithelial cell polarity in a Dlc1 gene trapped (gt) mouse.MethodsMammary gland whole mount preparations from 10-week virgin heterozygous Dlc1gt/+ gene-trapped mice were compared with age-matched wild type (WT) controls. Hematoxylin-Eosin (H&E) and Masson’s Trichrome staining of histological sections were carried out. Mammary glands from Dlc1gt/+ mice and WT controls were enzymatically digested with collagenase and dispase and then cultured overnight to deplete hematopoietic and endothelial cells. The single cell suspensions were then cultured in Matrigel for 12 days. To knockdown Dlc1 expression, primary WT mammary epithelial cells were infected with short hairpin (sh) RNA expressing lentivirus or with a scrambled shRNA control.ResultsDlc1gt/+ mice showed anomalies in the mammary gland that included increased ductal branching and deformities in terminal end buds and branch points. Compared to the WT controls, Masson’s Trichrome staining showed a thickened stromal layer with increased collagen deposition in mammary glands from Dlc1gt/+ mice. Dlc1gt/+ primary mammary epithelial cells formed increased solid acinar spheres in contrast with WT and scrambled shRNA control cells, which mostly formed hollow acinar structures when plated in 3D Matrigel cultures. These solid acinar structures were similar to the acinar structures formed when Dlc1 gene expression was knocked down in WT mammary cells by shRNA lentiviral transduction. The solid acinar structures were not due to a defect in apoptosis as determined by a lack of detectible cleaved caspase 3 antibody staining. Primary mammary cells from Dlc1gt/+ mice showed increased RhoA activity compared with WT cells.ConclusionsThe results illustrate that decreased Dlc1 expression can disrupt the normal cell polarization and mammary ductal branching. Altogether this study suggests that Dlc1 plays a role in maintaining normal mammary epithelial cell polarity and that Dlc1 is haploinsufficient.
Highlights
Deleted in Liver Cancer 1 (Dlc1) is a tumor suppressor gene, which maps to human chromosome 8p21-22 and is found frequently deleted in many cancers including breast cancer
Scale bar 50 μm Heterozygous Dlc1 loss affects mammary gland branching Previous results had suggested that the chromosome region 8p22, where Dlc1 maps, may contain a haploinsufficient tumor suppressor gene in breast cancer [1]
On comparing the mammary glands from the age matched wild type (WT) and heterozygous Dlc1gt/+ gene-trapped mice, we found that the WT mammary gland showed regular ductal branching as is observed in virgin females of the C57BL background [Fig. 1a–d]
Summary
Deleted in Liver Cancer 1 (Dlc1) is a tumor suppressor gene, which maps to human chromosome 8p21-22 and is found frequently deleted in many cancers including breast cancer. Xue et al showed that heterozygous deletion of Dlc occurred in approximately 50 % of breast, liver, pancreatic and lung tumors and more than 70 % of colon cancers [5]. These deletions could be up to five Mbps (~20 genes), they always included the Dlc locus (ibid.). Another study, using matched malignant and nonmalignant human breast cancer cell lines, showed that the nonmalignant line had Dlc transcript levels 3-fold greater than the malignant clone [11] Overall these results suggest that Dlc may be an important tumor suppressor in breast cancer
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