The Deleted in Liver Cancer 1 (Dlc1) tumor suppressor is haploinsufficient for mammary gland development and epithelial cell polarity.
BACKGROUND
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. 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.


METHODS
Mammary gland whole mount preparations from 10-week virgin heterozygous Dlc1(gt/+) 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 Dlc1(gt/+) 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.


RESULTS
Dlc1(gt/+) 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 Dlc1(gt/+) mice. Dlc1(gt/+) 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 Dlc1(gt/+) mice showed increased RhoA activity compared with WT cells.


CONCLUSIONS
The 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.
Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, MB, R3E 0V9, Canada. onlyprat@gmail.com.|Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, MB, Canada. onlyprat@gmail.com.|Department of Immunology, University of Manitoba, Winnipeg, MB, Canada. onlyprat@gmail.com.|Regenerative Medicine Program, University of Manitoba, Winnipeg, MB, Canada. onlyprat@gmail.com.
Pratima
P
Basak
Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, MB, R3E 0V9, Canada. rachelle.dillon@gmail.com.
Rachelle
R
Dillon
Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, MB, R3E 0V9, Canada. Heather.Leslie@umanitoba.ca.
Heather
H
Leslie
Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, MB, R3E 0V9, Canada. Afshin.Raouf@umanitoba.ca.|Department of Immunology, University of Manitoba, Winnipeg, MB, Canada. Afshin.Raouf@umanitoba.ca.|Regenerative Medicine Program, University of Manitoba, Winnipeg, MB, Canada. Afshin.Raouf@umanitoba.ca.
Afshin
A
Raouf
Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, MB, R3E 0V9, Canada. Michael.Mowat@umanitoba.ca.|Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, MB, Canada. Michael.Mowat@umanitoba.ca.
Michael R A
MR
Mowat
D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics; D016764:Cell Polarity / Q000502:physiology*; D004847:Epithelial Cells / Q000502:physiology*; D005260:Female; D005455:Fluorescent Antibody Technique; D020690:GTPase-Activating Proteins / Q000502:physiology*; D057895:Haploinsufficiency / Q000502:physiology*; D008321:Mammary Glands, Animal / Q000254:growth & development*; D051379:Mice; D018613:Microscopy, Confocal; D060888:Real-Time Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000502:physiology*
C430864:DLC-1 (deleted in liver cancer) protein, mouse; D020690:GTPase-Activating Proteins; D025521:Tumor Suppressor Proteins