Regulation of Autophagy via Carbohydrate and Lipid Metabolism in Cancer.

Metabolic changes are an important component of tumor cell progression. Tumor cells adapt to environmental stresses via changes to carbohydrate and lipid metabolism. Autophagy, a physiological process in mammalian cells that digests damaged organelles and misfolded proteins via lysosomal degradation, is closely associated with metabolism in mammalian cells, acting as a meter of cellular ATP levels. In this review, we discuss the changes in glycolytic and lipid biosynthetic pathways in mammalian cells and their impact on carcinogenesis via the autophagy pathway. In addition, we discuss the impact of these metabolic pathways on autophagy in lung cancer.

In vivo evidence supporting a metastasis suppressor role for Stard13 (Dlc2) in ErbB2 (Neu) oncogene induced mouse mammary tumors.

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.

Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9) and Rac1 activation.

The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype.

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.

The role of Dlc1 isoform 2 in K-Ras2(G12D) induced thymic cancer.

The Deleted in liver cancer one (Dlc1) tumor suppressor gene encodes a RhoGTPase activating protein (RhoGAP). The Dlc1 gene has multiple transcriptional isoforms and we have previously established a mouse strain containing a gene trap (gt) insertion, which specifically reduces the expression of the 6.1 kb isoform (isoform 2). This gene trapped allele when homozygous results in embryonic lethality and the heterozygous gene trapped mice do not show an increased incidence of cancers, suggesting that cooperating oncogenic changes may be required for transformation. In the present work, we have studied the in vivo cooperation between oncogenic K-Ras2 and Dlc1 genes in tumourigenesis. We have observed an increase in invasive thymic cancers, including both thymomas and lymphomas, resulting in significantly shortened life spans in mice heterozygous for the gt Dlc1 allele and an inducible LSL-K-Ras2(G12D) allele compared with the LSL-K-Ras2(G12D) only mice. The heterozygous mice showed a high degree of metastasis in the lung. We have found tumour specific selective hypermethylation of the Dlc1 isoform 2 promoter and reduction of the corresponding protein expression in thymic lymphoma (TL) and thymic epithelial carcinoma (TEC) derived from the thymic tumours. The Dlc1 deficient thymic lymphoma cell lines exhibited increased trans-endothelial cell migration. TEC cell lines also exhibited increased stress fiber formation and Rho activity. Introduction of the three Dlc1 isoforms tagged with GFP into these cells resulted in different morphological changes. These results suggest that loss of expression of only isoform 2 may be sufficient for the development of thymic tumors and metastasis.

Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform.

BACKGROUND: The Dlc1 (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. Several transcriptional isoforms have been described but the functional significance and tissue distribution of each form is presently poorly understood. Also, differences in the number of isoforms and splice variants reported still exist between different mammalian species. In order to better understand the number and function of the different variants of the Dlc1 gene in the mouse, we have carried out a detailed analysis. Extensive 3' RACE experiments were carried out in order to identify all possible Dlc1 isoforms and splice variants in the mouse. In addition, we have generated a gene trapped mouse that targets one of these isoforms in order to study its biological function. The effect of this gene trap insertion on the splicing of other isoforms has also been studied. RESULTS: In addition to the known 6.1 and 6.2 Kb transcripts of Dlc1, our study revealed the existence of a novel 7.6 Kb transcriptional isoform in the mouse, which corresponds to the human 7.4 Kb (KIAA1723) cDNA transcript. A gene trapped embryonic cell line, with an insertion between Exon 1 and 2 of the 6.1 Kb transcriptional isoform, was used to generate a transgenic mouse. This line showed a significant reduction in the expression of the trapped isoform. However, reduced expression of the other isoforms was not seen. Mice heterozygous for the gene trapped allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Dlc1gt/gt embryos showed defects in the brain, heart, and placental blood vessels. Cultured serum-free mouse embryo cells from Dlc1 deficient embryos had elevated RhoA activity and displayed alterations in the organization of actin filaments and focal adhesions. The Dlc1 deficient cells also exhibited increased wound closure in an in vitro scratch assay. CONCLUSIONS: The mouse has three major transcriptional isoforms of the Dlc1 gene that are differentially expressed in various tissues. A mouse with exon 1 of the 6.1 Kb transcript gt resulted in hypomorphic expression of Dlc1 protein and an embryonic lethal phenotype in the homozygous condition, which indicates that this isoform plays a major role in mouse development. The Dlc1 deficient cells showed altered cytoskeleton structure, increased RhoA activity and cellular migration.

Duplication of Subcytoband 11E2 of Chromosome 11 Is Regularly Associated with Accelerated Tumor Development in v-abl/myc-Induced Mouse Plasmacytomas.

Chromosome 11 aberrations constitute the second most frequent chromosomal aberration in mouse plasmacytomas (PCTs) in which both the myc and abl oncogenes are constitutively expressed. In these tumors, previous G-banding studies had revealed numerical aberrations including duplication of the entire chromosome 11 or segments of telomeric bands D and E. The trisomy of chromosome 11 was always associated with accelerated pristane + v-abl/myc-induced PCT development. In the present study, PCT development was studied in a unique BALB/c congenic mouse strain, (T38HxBALB/c) F1, carrying a reciprocal translocation between chromosomes X and 11. After v-abl/myc induction, PCTs in this strain had acquired a nonrandom duplication of subcytoband 11E2. This duplication was always associated with accelerated PCT development. Corresponding synteny regions in the human and rat are changed in many tumors and involved in duplication, amplification, or translocation events. Thus, together with these synteny data, our findings strongly suggest a causal involvement of 11E2 in the acceleration of v-abl/myc-induced PCTs.

StARD13(Dlc-2) RhoGap mediates ceramide activation of phosphatidylglycerolphosphate synthase and drug response in Chinese hamster ovary cells.

To identify genes involved in etoposide drug response, we used promoter trap mutagenesis to isolate an etoposide-resistant Chinese hamster ovary (CHO) cell line. This resistant CHO-K1 line, named E91, showed cross-resistance to C(2)-ceramide (N-acetylsphingosine). The promoter trap retrovirus was found integrated into intron 1-2 of the Dlc-2 (Stard13) RhoGap gene. The E91 cells showed elevated guanosine triphosphate (GTP)-bound RhoA levels compared with the parental line, suggesting that retrovirus integration had inactivated one of the Dlc-2 RhoGap alleles. To test whether E91 cells were impaired in an intracellular ceramide-regulated process not directly related to cell killing, we measured mitochondrial phosphatidylglycerolphosphate (PGP) synthase and phospholipase A2 enzyme activities in cells after C(2)-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C(2)-ceramide or tumor necrosis factor alpha, but not the E91 cells. These results suggested that intracellular ceramide signaling was defective in E91 cells due to increased levels of active GTP-bound RhoA. RNA knockdown experiments of the Dlc2 RhoGap resulted in increased GTP-bound RhoA and reduced induction of PGP synthase after C(2)-ceramide addition compared with controls. Expression of a dominant-negative RhoA in the E91 cell line allowed induction of PGP synthase by ceramide. The RNA interference knockdown cell line also showed increased etoposide resistance. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through RhoGap expression.