Marian E. Durkin

Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties

Growing evidence indicates that microRNAs have a significant role in tumor development and may constitute robust biomarkers for cancer diagnosis and prognosis. In this study, we evaluated the clinical and functional relevance of microRNA-122 (miR-122) expression in human hepatocellular carcinoma (HCC). We report that miR-122 is specifically repressed in a subset of primary tumors that are characterized by poor prognosis. We further show that the loss of miR-122 expression in tumor cells segregates with specific gene expression profiles linked to cancer progression, namely the suppression of hepatic phenotype and the acquisition of invasive properties. We identify liver-enriched transcription factors as central regulatory molecules in the gene networks associated with loss of miR-122, and provide evidence suggesting that miR-122 is under the transcriptional control of HNF1A, HNF3A and HNF3B. We further show that loss of miR-122 results in an increase of cell migration and invasion and that restoration of miR-122 reverses this phenotype. In conclusion, miR-122 is a marker of hepatocyte-specific differentiation and an important determinant in the control of cell migration and invasion. From a clinical point of view, our study emphasizes miR-122 as a diagnostic and prognostic marker for HCC progression.

DLC‐1:a Rho GTPase‐activating protein and tumour suppressor

•  Introduction ‐  The deleted in liver cancer family of RhoGAP domain proteins ‐  DLC‐1 ‐  DLC‐2 ‐  DLC‐3 ‐  Invertebrate DLC‐1‐like proteins •  Expression of DLC family proteins •  Features of DLC family protein domains ‐  SAM domain ‐  RhoGAP domain ‐  START domain ‐  Serine‐rich, unstructured middle region •  Biological functions of DLC‐1 ‐  Cytoskeletal organization ‐  DLC‐1 localizes to focal adhesions via binding to tensin family proteins ‐  Interaction of DLC‐1 with caveolin‐1 ‐  DLC‐1 and phosphoinositide signalling ‐  Biological activities of DLC‐2 and DLC‐3 •  Genetic analysis of DLC‐1 function ‐  Mouse DLC‐1 gene knockout ‐  RhoGAP88 C in Drosophila •  The DLC family proteins in cancer ‐  Decreased expression of DLC‐1 in human cancers ‐  Deletions of DLC1 in tumours ‐  Epigenetic inactivation of DLC‐1 expression ‐  DLC‐1 sequence variants in human cancers ‐  DLC‐1 suppresses tumour cell growth ‐  DLC‐1 as a metastasis suppressor gene ‐  Regulation of DLC‐1 by anti‐oncogenic factors ‐  Animal models of DLC‐1 in cancer ‐  Evidence for roles of DLC‐2 and DLC‐3 in neoplasia •  Conclusions and future directions

Tissue-specific expression of the human laminin α5-chain, and mapping of the gene to human chromosome 20q13.2-13.3 and to distal mouse chromosome 2 near the locus for the ragged (Ra) mutation

To investigate the function of the laminin α5‐chain, previously identified in mice, cDNA clones encoding the 953‐amino‐acid carboxy terminal G‐domain of the human laminin α5‐chain were characterized. Northern blot analysis showed that the laminin α5‐chain is expressed in human placenta, heart, lung, skeletal muscle, kidney, and pancreas. The human laminin α5‐chain gene (LAMA5) was assigned to chromosome 20q13.2‐q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra) mutation.

Mice with a Targeted Deletion of the Tetranectin Gene Exhibit a Spinal Deformity

ABSTRACT Tetranectin is a plasminogen-binding, homotrimeric protein belonging to the C-type lectin family of proteins. Tetranectin has been suggested to play a role in tissue remodeling, due to its ability to stimulate plasminogen activation and its expression in developing tissues such as developing bone and muscle. To test the functional role of tetranectin directly, we have generated mice with a targeted disruption of the gene. We report that the tetranectin-deficient mice exhibit kyphosis, a type of spinal deformity characterized by an increased curvature of the thoracic spine. The kyphotic angles were measured on radiographs. In 6-month-old normal mice (n= 27), the thoracic angle was 73° ± 2°, while in tetranectin-deficient 6-month-old mice (n = 35), it was 93° ± 2° (P < 0.0001). In approximately one-third of the mutant mice, X-ray analysis revealed structural changes in the morphology of the vertebrae. Histological analysis of the spines of these mice revealed an apparently asymmetric development of the growth plate and of the intervertebral disks of the vertebrae. In the most advanced cases, the growth plates appeared disorganized and irregular, with the disk material protruding through the growth plate. Tetranectin-null mice had a normal peak bone mass density and were not more susceptible to ovariectomy-induced osteoporosis than were their littermates as determined by dual-emission X-ray absorptiometry scanning. These results demonstrate that tetranectin plays a role in tissue growth and remodeling. The tetranectin-deficient mouse is the first mouse model that resembles common human kyphotic disorders, which affect up to 8% of the population.

MYC activates stem-like cell potential in hepatocarcinoma by a p53-dependent mechanism

Activation of c-MYC is an oncogenic hallmark of many cancers, including liver cancer, and is associated with a variety of adverse prognostic characteristics. Despite a causative role during malignant transformation and progression in hepatocarcinogenesis, consequences of c-MYC activation for the biology of hepatic cancer stem cells (CSC) are undefined. Here, distinct levels of c-MYC overexpression were established by using two dose-dependent tetracycline-inducible systems in four hepatoma cell lines with different p53 mutational status. The CSCs were evaluated using side population (SP) approach as well as standard in vitro and in vivo assays. Functional repression of p53 was achieved by lentiviral shRNA transduction. The results show that c-MYC expression levels have a differential impact on liver CSC characteristics. At low levels, c-MYC activation led to increased proliferation and enhanced CSC properties including activation of reprogramming transcription factors and CSC marker expression (e.g., NANOG, OCT4, and EpCAM), expansion of SP, and acceleration of tumor growth upon subcutaneous transplantation into immunocompromised mice. However, when exceeding a threshold level, c-MYC induced a proapoptotic program and loss of CSC potential both in vitro and in vivo. Mechanistically, c-MYC-induced self-renewal capacity of liver cancer cells was exerted in a p53-dependent manner. Low c-MYC activation increased spheroid formation in p53-deficient tumor cells, whereas p53-dependent effects were blunted in the absence of c-MYC overexpression. Together, our results confirm the role of c-MYC as a master regulator during hepatocarcinogenesis and establish a new gatekeeper role for p53 in repressing c-MYC-induced CSC phenotype in liver cancer cells.

Epigenetic reprogramming modulates malignant properties of human liver cancer

Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of hepatocellular carcinoma (HCC) cells. Our strategy employed a 3‐day treatment of established and primary human HCC‐derived cell lines grown as a monolayer at various cell densities with the DNMT1 inhibitor zebularine (ZEB) followed by a 3D culture to identify cells endowed with self‐renewal potential. Differences in self‐renewal, gene expression, tumorigenicity, and metastatic potential of spheres at generations G1‐G5 were examined. Transient ZEB exposure produced differential cell density‐dependent responses. In cells grown at low density, ZEB caused a remarkable increase in self‐renewal and tumorigenicity associated with long‐lasting gene expression changes characterized by a stable overexpression of cancer stem cell‐related and key epithelial‐mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast, at high cell density, ZEB caused a gradual decrease in self‐renewal and tumorigenicty, and up‐regulation of apoptosis‐ and differentiation‐related genes. A permanent reduction of DNMT1 protein using short hairpin RNA (shRNA)‐mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, WRL68 and HepG2 hepatoblastoma cells expressing low DNMT1 basal levels also possessed a high self‐renewal, irrespective of cell density or ZEB exposure. Spheres formed by low‐density cells treated with ZEB or shDNMT1 displayed a high molecular similarity which was sustained through consecutive generations, confirming the essential role of DNMT1 depletion in the enhancement of cancer stem cell properties. Conclusion: These results identify DNA methylation as a key epigenetic regulatory mechanism determining the pool of cancer stem cells in liver cancer and possibly other solid tumors. (Hepatology 2014;59:2251–2262)

Structural Organization of the Human and Mouse Laminin β2 Chain Genes, and Alternative Splicing at the 5′ End of the Human Transcript

We have determined the structural organization of the human and mouse genes that encode the laminin β2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon-intron organization and are the smallest laminin chain genes characterized to date, due to the unusually small size of their introns. The laminin β2 chain genes of both species consist of 33 exons that span ≤12 kilobase pairs of genomic DNA. The exon-intron pattern of the laminin β2 chain gene is also highly similar to that of the human genes encoding the homologous laminin β1 and β3 chains. The putative promoter regions of the human and mouse laminin β2 chain genes have features characteristic of the promoters of genes that have a limited tissue expression. Considerable conservation of the intron sequences of the mouse and human genes was observed. The first intron of the human gene, located 1 base pair upstream of the translation start codon, contains a non-consensus 5′ splice site. This intron was shown to be inefficiently spliced in humans, suggesting that post-transcriptional mechanisms may be involved in the regulation of laminin β2 chain gene expression.

Assignment of the gene for human tetranectin (TNA) to chromosome 3p22→p21.3 by somatic cell hybrid mapping

Tetranectin is a plasminogen-binding protein that is induced during the mineralization phase of osteogenesis. By screening a human chromosome 3 somatic cell hybrid mapping panel, we have localized the human tetranectin gene (TNA) to 3p22-->p21.3, which is distinct from the loci of two human connective tissue disorders that map to the short arm of chromosome 3, MFS2 and LRS1.

DLC1 is the principal biologically-relevant down-regulated DLC family member in several cancers

The RHO family of RAS-related GTPases in tumors may be activated by reduced levels of RHO GTPase accelerating proteins (GAPs). One common mechanism is decreased expression of one or more members of the Deleted in Liver Cancer (DLC) family of Rho-GAPs, which comprises three closely related genes (DLC1, DLC2, and DLC3) that are down-regulated in a wide range of malignancies. Here we have studied their comparative biological activity in cultured cells and used publicly available datasets to examine their mRNA expression patterns in normal and cancer tissues, and to explore their relationship to cancer phenotypes and survival outcomes. In The Cancer Genome Atlas (TCGA) database, DLC1 expression predominated in normal lung, breast, and liver, but not in colorectum. Conversely, reduced DLC1 expression predominated in lung squamous cell carcinoma (LSC), lung adenocarcinoma (LAD), breast cancer, and hepatocellular carcinoma (HCC), but not in colorectal cancer. Reduced DLC1 expression was frequently associated with promoter methylation in LSC and LAD, while DLC1 copy number loss was frequent in HCC. DLC1 expression was higher in TCGA LAD patients who remained cancer-free, while low DLC1 had a poorer prognosis than low DLC2 or low DLC3 in a more completely annotated database. The poorest prognosis was associated with low expression of both DLC1 and DLC2 (P < 0.0001). In cultured cells, the three genes induced a similar reduction of Rho-GTP and cell migration. We conclude that DLC1 is the predominant family member expressed in several normal tissues, and its expression is preferentially reduced in common cancers at these sites.

Characterization of the human laminin β2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS)

The laminin β2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin β2 chain gene (LAMB2) on chromosome 3p21.3→p21.2, a region paralogous with the chromosome 7q22→q31 region that contains the laminin β1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3′ end of LAMB2 lies 16 kb from the 5′ end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3′ end of LAMB2L was similar to exons 8–12 of the laminin β2 chain gene. The LAMB2L–LAMB2–QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).