Chromosome transplantation as a novel approach for correcting complex genomic disorders

Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt gene with a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.     

A combined comparative genomic hybridization and expression microarray analysis of gastric cancer reveals novel molecular subtypes

Comparative genomic hybridization (CGH), microsatellite instability (MSI) assays, and expression microarrays were used to molecularly subclassify a common set of gastric tumor samples. We identified a number of novel genomic aberrations associated with gastric cancer and discovered that gastric tumors could be grouped by their expression profiles into three broad classes: "tumorigenic," "reactive," and "gastric-like." Patients with gastric-like tumors exhibited a significantly better overall survival than patients belonging to the other two classes (P < 0.05). A novel supervised learning methodology for multiclass prediction was used to identify optimal predictor gene sets that accurately predicted the class of an unknown tumor sample. These predictor sets may prove useful in the development of new diagnostic applications for gastric cancer staging and prognostication.     

Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells.

The molecular details of hypoxia-induced cellular responses have been difficult to identify since there is as yet no known oxygen receptor. We used cDNA microarray technology to extend our studies pertaining to these molecular details in human hepatocellular carcinoma (Hep3B) cells that produce erythropoietin (Epo) in response to hypoxia. Of approximately 1200 genes in the array, those associated with integrin-linked kinase (ILK), fibronectin precursor and glycogen synthase kinase-3beta (GSK-3beta) were markedly stimulated after exposure of Hep3B cells to low oxygen (1%) for 6 h. Epo, HIF-1, and von Hippel-Lindau cDNAs were measured in parallel as markers of low oxygen responses in Hep3B cells. ILK is a serine, threonine protein kinase that interacts with the cytoplasmic domains of integrin beta1 and beta3. This interaction localizes ILK to focal adhesion plaques. ILK is stimulated by cell-fibronectin interaction as well as insulin. It is regulated in a phosphatidylinositol 3-kinase dependent manner and can phosphorylate protein kinase B (PKB/AKT) and GSK-3beta. As a result of these and other activities ILK has been shown to affect anchorage-independent cell survival, cell cycle progression and tumorigenesis in nude mice. ILK has also been implicated in the Wnt pathway and as a critical target in PTEN-dependent tumor therapies. To our knowledge this is the first report implicating the ILK pathway in low oxygen responses. Other genes identified as a result of the microarray analysis not previously known to change as a result of low oxygen treatment were elongation factor-1alpha, glycyl-tRNA synthetase, and laminin receptor protein-1. These findings were all corroborated by RT-PCR assays and in some instances Western blot analysis.     

Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators

Although androgen receptor (AR)-mediated signaling is central to prostate cancer, the ability to modulate AR signaling states is limited. Here we establish a chemical genomic approach for discovery and target prediction of modulators of cancer phenotypes, as exemplified by AR signaling. We first identify AR activation inhibitors, including a group of structurally related compounds comprising celastrol, gedunin, and derivatives. To develop an in silico approach for target pathway identification, we apply a gene expression-based analysis that classifies HSP90 inhibitors as having similar activity to celastrol and gedunin. Validating this prediction, we demonstrate that celastrol and gedunin inhibit HSP90 activity and HSP90 clients, including AR. Broadly, this work identifies new modes of HSP90 modulation through a gene expression-based strategy.     

BRCA2 in mitotic exit: a new role in regulating genomic stability

Evaluation of: Daniels MJ, Wang Y, Lee M, Venkitaraman AR: Abnormal cytokinesis in cells deficient in the breast cancer susceptibility protein BRCA2. Science 306, 876-879 (2004). Cytokinesis is the division of the cytoplasm of a parent cell into daughter cells after nuclear division. Cytokinesis failure is often accompanied by the generation of cells with an unstable tetraploidy content, which predisposes the cells to develop aneuploidy and malignancies. A recent study by Venkitaraman's group demonstrates that BRCA2, a breast cancer susceptibility gene product, also functions in mediating normal cytokinesis. Similar to the subcellular localization of Aurora kinase, BRCA2 is present at the cleavage furrow and the midbody during late mitosis. Deficiency in BRCA2 function results in cytokinesis failure, which is associated with abnormal localization of myosin II, a key protein essential for the formation of the cleavage furrow. This study is of significance as it shows for the first time that BRCA2 has a function in controlling mitotic exit, deregulation of which contributes to gross genomic instabilities in daughter cells.     

The role of human papillomavirus in cervical adenocarcinoma carcinogenesis

Human papillomavirus (HPV) is considered the single most important co-factor in the development of cervical squamous cell carcinomas. Adenocarcinomas of the cervix are also related to HPV, but the correlation is reported to be less pronounced. In the present study, 131 cervical adenocarcinomas were identified through the Swedish Cancer Registry, examined morphologically and then analysed with sensitive polymerase chain reaction (PCR)-based HPV methods for a study of age-related prevalence of HPV. HPV was identified in 64% of the tumours after PCR amplification of the HPV L1 gene only and in 71% following PCR amplification of both the L1 and E6 genes of HPV. HPV 18 was the most prevalent (52%), followed by HPV 16 (33%) and other types of HPV (15%). The prevalence of HPV was shown to be age-dependent. In women younger than 40 years, HPV was present in 89%, whereas in women 60 years and older, HPV was observed in only 43%. The difference was statistically significant, P<0.005. The HPV-positive adenocarcinomas were represented by an age distribution similar to that of cervical squamous carcinomas with a maximum age, in the 40-49 year old group, whereas the frequency of HPV-negative adenocarcinomas increased with age, typical of most carcinomas occurring in elderly women.     

A novel human tRNA-dihydrouridine synthase involved in pulmonary carcinogenesis

An increased level of dihydrouridine in tRNA(Phe) was found in human malignant tissues nearly three decades ago, but its biological significance in carcinogenesis has remained unclear. Through analysis of genome-wide gene-expression profiles among non-small cell lung carcinomas (NSCLC), we identified overexpression of a novel human gene, termed hDUS2, encoding a protein that shared structural features with tRNA-dihydrouridine synthases (DUS). The deduced 493-amino-acid sequence showed 39% homology to the dihydrouridine synthase 2 enzyme (Dus2) of Saccharomyces cerevisiae and contained a conserved double-strand RNA-binding motif (DSRM). We found that hDUS2 protein had tRNA-DUS activity and that it physically interacted with EPRS, a glutamyl-prolyl tRNA synthetase, and was likely to enhance translational efficiencies. A small interfering RNA against hDUS2 transfected into NSCLC cells suppressed expression of the gene, reduced the amount of dihydrouridine in tRNA molecules, and suppressed growth. Immunohistochemical analysis showed significant association between higher levels of hDUS2 in tumors and poorer prognosis of lung cancer patients. Our data imply that up-regulation of hDUS2 is a relatively common feature of pulmonary carcinogenesis and that selective suppression of hDUS2 enzyme activity and/or inhibition of formation of the hDUS2-tRNA synthetase complex could be a promising therapeutic strategy for treatment of many lung cancers.     

A novel Src kinase inhibitor reduces tumour formation in a skin carcinogenesis model

The Src family tyrosine kinases are key modulators of cancercell invasion and metastasis and a number of Src kinase inhibitorsare currently in clinical development for the treatment of solidtumours. However, there is growing evidence that Src is alsoupregulated at very early stages of epithelial cancer development.We have investigated the role of Src in mouse skin, which isone of the most tractable models of epithelial homoeostasisand tumorigenesis. We found that Src protein expression andactivity was regulated during the normal hair cycle and wasincreased specifically during the proliferative anagen phaseand also in response to the tumour promoter 12-O-tetradecanoylphorbol-13-acetate(TPA). AZD0530, a selective Src inhibitor, prevented the TPA-inducedproliferation of basal keratinocytes both in vivo and in vitro.Moreover, treatment with AZD0530 reduced papilloma formationfollowing the well-established 7,12-dimethylbenz(a)anthracene/TPAskin carcinogenesis protocol but did not inhibit the subsequentproliferation of the papillomas. Furthermore, AZD0530 did notalter the malignant conversion of papillomas to squamous cellcarcinoma suggesting a role for Src in early tumour developmentin the skin carcinogenesis model, rather than at later stagesof tumour progression. Src expression and activity were alsoseen in human actinic keratoses that are hyperproliferativepre-malignant skin lesions, indicating that Src may also playa role in the early stages of human skin tumour development.Thus, Src inhibitors such as AZD0530 may therefore have chemopreventativeproperties in patients with hyperproliferative epidermal disorders. Abbreviations: DMBA, 7,12-dimethylbenz(a)anthracene; PBS, phosphate-buffered saline; SFK, Src family kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate Received July 2, 2008; revised December 3, 2008; accepted December 3, 2008.     

Whole-exome analysis reveals novel somatic genomic alterations associated with outcome in immunochemotherapy-treated diffuse large B-cell lymphoma

Lack of remission or early relapse remains a major clinical issue in diffuse large B-cell lymphoma (DLBCL), with 30% of patients failing standard of care. Although clinical factors and molecular signatures can partially predict DLBCL outcome, additional information is needed to identify high-risk patients, particularly biologic factors that might ultimately be amenable to intervention. Using whole-exome sequencing data from 51 newly diagnosed and immunochemotherapy-treated DLBCL patients, we evaluated the association of somatic genomic alterations with patient outcome, defined as failure to achieve event-free survival at 24 months after diagnosis (EFS24). We identified 16 genes with mutations, 374 with copy number gains and 151 with copy number losses that were associated with failure to achieve EFS24 (P<0.05). Except for FOXO1 and CIITA, known driver mutations did not correlate with EFS24. Gene losses were localized to 6q21-6q24.2, and gains to 3q13.12-3q29, 11q23.1-11q23.3 and 19q13.12-19q13.43. Globally, the number of gains was highly associated with poor outcome (P=7.4 × 10⁻¹²) and when combined with FOXO1 mutations identified 77% of cases that failed to achieve EFS24. One gene (SLC22A16) at 6q21, a doxorubicin transporter, was lost in 54% of EFS24 failures and our findings suggest it functions as a doxorubicin transporter in DLBCL cells.     

Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes

Genomic analyses aimed at the detection of high-level DNA amplifications were performed on 13 widely used pancreatic cancer cell lines and 6 pancreatic tumor specimens. For these analyses, array-based comparative genomic hybridization (Matrix-CGH) onto dedicated microarrays was used. In comparison with chromosomal CGH (eight amplifications), a >3-fold number of DNA amplifications was detected (n = 29). The most frequent amplifications mapped to 7p12.3 (three pancreatic cancer cell lines and three pancreatic tumor specimens), 8q24 (four pancreatic cancer cell lines and one pancreatic tumor specimen), 11q13 (three pancreatic cancer cell lines and three pancreatic tumor specimens), and 20q13 (four pancreatic cancer cell lines and three pancreatic tumor specimens). Genes contained in the consensus regions were MYC (8q24), EGFR (7p12.3), and FGF3 (11q13). In six of seven pancreatic cancer cell lines and pancreatic tumor specimens with 20q13 amplifications, the novel candidate gene NFAT C2, which plays a role in the activation of cytokines, was amplified. Other amplifications also affected genes for which a pathogenetic role in pancreatic carcinoma has not been described, such as BCL10 and BCL6, two members of the BCL family. A subset of amplified genes was checked for overexpression by means of real-time PCR, revealing the highest expression levels for BCL6 and BCL10. Thus, Matrix-CGH allows the detection of a high number of amplifications, resulting in the identification of novel candidate genes in pancreatic cancer.     

Absence of papillomavirus in skin tumors induced in SENCAR mice by a two-stage carcinogenesis protocol

SENCAR mice are unusually sensitive to induction of papillomas and squamous cell carcinomas by initiation with 7,12-dimethylbenzanthracene (DMBA) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumors induced by this protocol were tested for the presence of papillomavirus by immunohistochemistry, Southern blot, reverse Southern blot and dot blot hybridization techniques. Papillomavirus antigens were not detected in any of 235 tumors or 142 non-tumor-bearing skin samples analyzed. Southern blots and dot blots, using a mixed probe of cloned rodent papillomavirus DNA from the multimammate rat, Mastomys natalensis, and the European harvest mouse, Micromys minutus, did not reveal the presence of either episomal or integrated papillomavirus genomes in total cellular DNA extracts from the tumors or non-tumor-bearing skin. To circumvent the possibility that insufficient cross-homology existed to detect a papillomavirus genome with the mixed probe used, DNAs extracted from six papillomas were labeled and each used to probe reference blots that contained 25 cloned papillomavirus genomes excised from their vectors. No evidence for the presence of a papillomavirus genome was detected by this method. Therefore, it is unlikely that papillomaviruses play a role in the induction of tumors in SENCAR mice by two-stage carcinogenesis protocols.     

Microarray-based comparative genomic hybridization of cancer targets reveals novel, recurrent genetic aberrations in the myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders characterized by ineffective hematopoiesis, cytopenias, and a risk of transformation to acute myeloid leukemia (AML). However, only approximately 50% of primary MDS patients show clonal cytogenetic aberrations. To determine whether high-resolution microarray analysis would reveal new or additional aberrations, we analyzed 35 samples derived from patients with a diagnosis or suspicion of MDS and abnormal karyotypes. We used a whole-genome oligonucleotide microarray with targeted coverage of approximately 1900 genes associated with hematologic and other cancers. Clinically relevant copy number aberrations (CNAs) were identified by microarray-based comparative genomic hybridization (aCGH) in all samples (range 1-31, median 5). In 28 of 35 samples (80%), aCGH revealed new cytogenetic aberrations not seen by karyotype or fluorescence in situ hybridization (FISH). Furthermore, 132 cryptic aberrations (≤5 Mb) were identified in 25 cases (71.4%) including deletions of NF1, RUNX1, RASSF1, CCND1, TET2, DNMT3A, HRAS, PDGFRA and FIP1L1. Additionally, aCGH clarified known complex aberrations in 17 of 35 samples (48.6%). Finally, our results using whole-genome arrays with higher density coverage targeted to cancer features demonstrate the usefulness of arrays to identify rare and cryptic recurring imbalances that may prove to be significant in disease progression or transformation to AML and may improve the suitability or efficacy of molecularly targeted therapy.     

Enhanced colitis-associated colon carcinogenesis in a novel Apc mutant rat

To establish an efficient rat model for colitis-associated colorectal cancer, azoxymethane and dextran sodium sulfate (AOM/DSS)-induced colon carcinogenesis was applied to a novel adenomatous polyposis coli ( Apc ) mutant, the Kyoto Apc Delta (KAD) rat. The KAD rat was derived from ethylnitrosourea mutagenesis and harbors a nonsense mutation in the Apc gene (S2523X). The truncated APC of the KAD rat was deduced to lack part of the basic domain, an EB1-binding domain, and a PDZ domain, but retained an intact β-catenin binding region. KAD rats, homozygous for the Apc mutation on a genetic background of the F344 rat, showed no spontaneous tumors in the gastrointestinal tract. At 5 weeks of age, male KAD rats were given a single subcutaneous administration of AOM (20 mg/kg, bodyweight). One week later, they were given DSS (2% in drinking water) for 1 week. At week 15, the incidence and multiplicity of colon tumors developed in the KAD rat were remarkably severe compared with those in the F344 rat: 100 versus 50% in incidence and 10.7 ± 3.5 versus 0.8 ± 1.0 in multiplicity. KAD tumors were dominantly distributed in the rectum and distal colon, resembling human colorectal cancer. Accumulation of β-catenin protein and frequent β-catenin mutations were prominent features of KAD colon tumors. To our knowledge, AOM/DSS-induced colon carcinogenesis using the KAD rat is the most efficient to induce colon tumors in the rat, and therefore would be available as an excellent model for human colitis-associated CRC.