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.
Overexpression of dominant oncogenes and the loss of tumor suppressor genes are basic genetic events in the acquisition of the malignant phenotype. The erb-b2 receptor tyrosine kinase 2 (ERBB-2) proto-oncogene is overexpressed in 20-30% of human breast cancers. The StAR related lipid transfer domain containing 13 gene (STARD13), also known as Deleted in Liver Cancer-2 (DLC-2), maps to chromosome band 13q12.3 and is frequently downregulated in human cancers, including 72% of breast cancers. It encodes a RhoGAP protein with sterile α motif (SAM) and StAR-related lipid transfer (START) domains. The objective of this study was to determine if loss of Stard13 plays a role in mammary tumor progression using transgenic mice expressing the activated ErbB-2 (Neu) oncogene and Cre recombinase (NIC) in mammary epithelium under transcriptional control of the murine mammary tumor virus (MMTV) promoter (MMTV-NIC). These mice were crossed with a conditional Stard13 knockout mouse (floxed exon 3), resulting in simultaneous Neu expression and Stard13 deletion, specifically in the mammary epithelium. We found that loss of Stard13 did not alter tumor growth nor significantly modify overall survival and tumor free survival. However, there was an increase in the total number of lung metastases in the Stard13 heterozygous or homozygous mice compared with the parental MMTV-NIC strain. Altogether our results indicate that Stard13 acts as a metastasis suppressor rather than a tumor suppressor gene, in Neu oncogene induced mammary tumorigenesis.
Introduction: Overexpression of dominant oncogenes and the loss of tumor suppressor genes are basic genetic events in acquisition of the malignant phenotype. The ERBB-2 proto-oncogene is overexpressed in 20% - 30% of human breast cancers. The tumor suppressor gene Deleted in liver cancer 2 (DLC-2, also known as STARD13) maps to chromosome 13q12.3 and is frequently downregulated by 72% in human breast cancer. It encodes a RhoGAP protein containing a START lipid binding domain. It is not known what role DLC-2 silencing plays in breast carcinogenesis. The purpose of our study is to assess the biological significance of inactivation of DLC-2 in ERBB2 induced mammary tumorigenesis mouse model. For this, we used MMTV-NIC mice, which carry an activated NEU oncogene (ERBB2) under control of the MMTV-LTR promoter that also co-expresses Cre recombinase. These mice were crossed to a conditional DLC-2 knockout mouse (floxed exon 3). Objectives: To determine if heterozygous or homozygous DLC-2 loss plays a role in mouse mammary tumor progression in MMTV-NIC mice. Results: Our preliminary results showed that heterozygous deletion of DLC-2 in the mammary epithelium of the MMTV-NIC mouse model resulted in increased tumor burden with accelerated tumor growth. Metastases to the lungs were quantified by scanning H&E stained step sections (4X150uM). We observed a significant increase in the incidence of lung metastases in heterozygous DLC-2 exon3floxed/+ MMTV-NIC mice. The lung metastases showed a homogenous morphology and histopathologically similar phenotype to the MMTV-NIC strain. DLC-2 heterozygous tumor cells showed increased tumorosphere formation under low-attachment conditions with minimal growth factor supplementation. These tumor cells also showed increased expression of mesenchymal cell markers, as determined by quantitative real time RT-PCR, suggesting an Epithelial to Mesenchymal transition is occurring. Experiments with homozygous floxed DLC-2 mice are ongoing and will be presented. Conclusion: These results indicate that loss of one allele of DLC-2 is sufficient to accelerate NEU oncogene induced tumorigenesis. Citation Format: Pratima Basak, Pratima Basak, Heather Leslie, Heather Leslie, Afshin Raouf, Afshin Raouf, Michael RA Mowat, Michael RA Mowat. Role of the tumor suppressor gene DLC-2 in ERBB2 induced mouse mammary tumorigenesis and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B18.
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