A model-based scan statistic for identifying extreme chromosomal regions of gene expression in human tumors

MOTIVATION: The analysis of gene expression data in its chromosomal context has been a recent development in cancer research. However, currently available methods fail to account for variation in the distance between genes, gene density and genomic features (e.g. GC content) in identifying increased or decreased chromosomal regions of gene expression. RESULTS: We have developed a model-based scan statistic that accounts for these aspects of the complex landscape of the human genome in the identification of extreme chromosomal regions of gene expression. This method may be applied to gene expression data regardless of the microarray platform used to generate it. To demonstrate the accuracy and utility of this method, we applied it to a breast cancer gene expression dataset and tested its ability to predict regions containing medium-to-high level DNA amplification (DNA ratio values >2). A classifier was developed from the scan statistic results that had a 10-fold cross-validated classification rate of 93% and a positive predictive value of 88%. This result strongly suggests that the model-based scan statistic and the expression characteristics of an increased chromosomal region of gene expression can be used to accurately predict chromosomal regions containing amplified genes. AVAILABILITY: Functions in the R-language are available from the author upon request. CONTACT: fcouples@umich.edu.     

Epigenetic events in malignant melanoma

Irreversible changes in the DNA sequence, including chromosomal deletions or amplification, activating or inactivating mutations in genes, have been implicated in the development and progression of melanoma. However, increasing attention is being turned towards the participation of 'epigenetic' events in melanoma progression that do not affect DNA sequence, but which nevertheless may lead to stable inherited changes in gene expression. Epigenetic events including histone modifications and DNA methylation play a key role in normal development and are crucial to establishing the correct program of gene expression. In contrast, mistargeting of such epigenetic modifications can lead to aberrant patterns of gene expression and loss of anti-cancer checkpoints. Thus, to date at least 50 genes have been reported to be dysregulated in melanoma by aberrant DNA methylation and accumulating evidence also suggests that mistargetting of histone modifications and altered chromatin remodeling activities will play a key role in melanoma. This review gives an overview of the many different types of epigenetic modifications and their involvement in cancer and especially in melanoma development and progression.     

A comparison study reveals important features of agreement and disagreement between summarized DNA and RNA data obtained from renal cell carcinoma

Cytogenetic abnormalities, such as DNA amplifications and deletions, often lead to significant changes in gene expression levels within a chromosomal region. Instead of generating additional DNA copy number data, one method to identify DNA copy number abnormalities has been to search existing gene expression data for regional perturbations in gene expression. However, it is not clear how well this surrogate method performs in the examination of individual tumors and how we can use both DNA and RNA data to identify candidate genes that may be mutated. Here we report a comparison study using summarized DNA and RNA data to identify chromosomal abnormalities in human samples. Forty-four tissue samples from patients diagnosed as having renal cell carcinoma (RCC) were collected, together with 15 normal kidney samples as controls, and for each sample the genome-wide DNA and RNA data were obtained for comparison using Affymetrix 100K SNP and HGU133plus2 gene expression chips, respectively. The DNA and RNA data was summarized by both chromosome arm and cytogenetic banding patterns and compared. The result of this analysis revealed that the two summarized data sets used to identify cytogenetic changes agreed well. However, some differences between the two were also identified. These differences of large-scale gene expression deregulation without evidence of the comparable DNA copy number alterations may be the result of known mechanisms, such as large-scale methylation or chromosome inactivation, or may be the result of some new mechanism of DNA-RNA translation. The usefulness of the combined data set for identifying regions of mutated genes is also discussed.     

Coupled analysis of gene expression and chromosomal location

Microarray technology can be used to assess simultaneously global changes in expression of mRNA or genomic DNA copy number among thousands of genes in different biological states. In many cases, it is desirable to determine if altered patterns of gene expression correlate with chromosomal abnormalities or assess expression of genes that are contiguous in the genome. We describe a method, differential gene locus mapping (DIGMAP), which aligns the known chromosomal location of a gene to its expression value deduced by microarray analysis. The method partitions microarray data into subsets by chromosomal location for each gene interrogated by an array. Microarray data in an individual subset can then be clustered by physical location of genes at a subchromosomal level based upon ordered alignment in genome sequence. A graphical display is generated by representing each genomic locus with a colored cell that quantitatively reflects its differential expression value. The clustered patterns can be viewed and compared based on their expression signatures as defined by differential values between control and experimental samples. In this study, DIGMAP was tested using previously published studies of breast cancer analyzed by comparative genomic hybridization (CGH) and prostate cancer gene expression profiles assessed by cDNA microarray experiments. Analysis of the breast cancer CGH data demonstrated the ability of DIGMAP to deduce gene amplifications and deletions. Application of the DIGMAP method to the prostate data revealed several carcinoma-related loci, including one at 16q13 with marked differential expression encompassing 19 known genes including 9 encoding metallothionein proteins. We conclude that DIGMAP is a powerful computational tool enabling the coupled analysis of microarray data with genome location.     

A Mechanism of Gene Amplification Driven by Small DNA Fragments

DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s). Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA) occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB) external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation in nature.     

Evidence for a DNA inversion system in Bordetella pertussis

The expression of virulence-associated genes in Bordetella pertussis can be lost in three ways: phase variation, antigenic modulation, or serotype conversion. The mechanism(s) of these alterations in gene expression is unclear. B. pertussis chromosomal DNA was probed with cloned pin genes from Escherichia coli and cloned hin genes from Salmonella typhimurium. DNA duplex melting temperature experiments indicated significant homology between B. Pertussis chromosomal DNA and both DNA inversion genes. Southern blots using the hin gene probe showed homology with a 15 kb EcoRI fragment of B. pertussis chromosomal DNA. We postulate here that B. pertussis contains a DNA inversion system which may be responsible for serotype conversion or virulence phase change in this organism.     

Radiation-induced epigenetic alterations after low and high LET irradiations

Epigenetics, including DNA methylation and microRNA (miRNA) expression, could be the missing link in understanding radiation-induced genomic instability (RIGI). This study tests the hypothesis that irradiation induces epigenetic aberrations, which could eventually lead to RIGI, and that the epigenetic aberrations induced by low linear energy transfer (LET) irradiation are different than those induced by high LET irradiations. GM10115 cells were irradiated with low LET X-rays and high LET iron (Fe) ions and evaluated for DNA damage, cell survival and chromosomal instability. The cells were also evaluated for specific locus methylation of nuclear factor-kappa B (NFκB), tumor suppressor in lung cancer 1 (TSLC1) and cadherin 1 (CDH1) gene promoter regions, long interspersed nuclear element 1 (LINE-1) and Alu repeat element methylation, CpG and non-CpG global methylation and miRNA expression levels. Irradiated cells showed increased micronucleus induction and cell killing immediately following exposure, but were chromosomally stable at delayed times post-irradiation. At this same delayed time, alterations in repeat element and global DNA methylation and miRNA expression were observed. Analyses of DNA methylation predominantly showed hypomethylation, however hypermethylation was also observed. We demonstrate that miRNA expression levels can be altered after X-ray irradiation and that these miRNA are involved in chromatin remodeling and DNA methylation. A higher incidence of epigenetic changes was observed after exposure to X-rays than Fe ions even though Fe ions elicited more chromosomal damage and cell killing. This distinction is apparent at miRNA analyses at which only three miRNA involved in two major pathways were altered after high LET irradiations while six miRNA involved in five major pathways were altered after low LET irradiations. This study also shows that the irradiated cells acquire epigenetic changes suggesting that epigenetic aberrations may arise in the cell without initiating chromosomal instability.     

Accurate detection of aneuploidies in array CGH and gene expression microarray data

MOTIVATION: Chromosomal copy number changes (aneuploidies) are common in cell populations that undergo multiple cell divisions including yeast strains, cell lines and tumor cells. Identification of aneuploidies is critical in evolutionary studies, where changes in copy number serve an adaptive purpose, as well as in cancer studies, where amplifications and deletions of chromosomal regions have been identified as a major pathogenetic mechanism. Aneuploidies can be studied on whole-genome level using array CGH (a microarray-based method that measures the DNA content), but their presence also affects gene expression. In gene expression microarray analysis, identification of copy number changes is especially important in preventing aberrant biological conclusions based on spurious gene expression correlation or masked phenotypes that arise due to aneuploidies. Previously suggested approaches for aneuploidy detection from microarray data mostly focus on array CGH, address only whole-chromosome or whole-arm copy number changes, and rely on thresholds or other heuristics, making them unsuitable for fully automated general application to gene expression datasets. There is a need for a general and robust method for identification of aneuploidies of any size from both array CGH and gene expression microarray data. RESULTS: We present ChARM (Chromosomal Aberration Region Miner), a robust and accurate expectation-maximization based method for identification of segmental aneuploidies (partial chromosome changes) from gene expression and array CGH microarray data. Systematic evaluation of the algorithm on synthetic and biological data shows that the method is robust to noise, aneuploidal segment size and P-value cutoff. Using our approach, we identify known chromosomal changes and predict novel potential segmental aneuploidies in commonly used yeast deletion strains and in breast cancer. ChARM can be routinely used to identify aneuploidies in array CGH datasets and to screen gene expression data for aneuploidies or array biases. Our methodology is sensitive enough to detect statistically significant and biologically relevant aneuploidies even when expression or DNA content changes are subtle as in mixed populations of cells. AVAILABILITY: Code available by request from the authors and on Web supplement at http://function.cs.princeton.edu/ChARM/     

Cause and Consequences of Genetic and Epigenetic Alterations in Human Cancer

Both genetic and epigenetic changes contribute to development of human cancer. Oncogenomics has primarily focused on understanding the genetic basis of neoplasia, with less emphasis being placed on the role of epigenetics in tumourigenesis. Genomic alterations in cancer vary between the different types and stages, tissues and individuals. Moreover, genomic change ranges from single nucleotide mutations to gross chromosomal aneuploidy; which may or may not be associated with underlying genomic instability. Collectively, genomic alterations result in widespread deregulation of gene expression profiles and the disruption of signalling networks that control proliferation and cellular functions. In addition to changes in DNA and chromosomes, it has become evident that oncogenomic processes can be profoundly influenced by epigenetic mechanisms. DNA methylation is one of the key epigenetic factors involved in regulation of gene expression and genomic stability, and is biologically necessary for the maintenance of many cellular functions. While there has been considerable progress in understanding the impact of genetic and epigenetic mechanisms in tumourigenesis, there has been little consideration of the importance of the interplay between these two processes. In this review we summarize current understanding of the role of genetic and epigenetic alterations in human cancer. In addition we consider the associated interactions of genetic and epigenetic processes in tumour onset and progression. Furthermore, we provide a model of tumourigenesis that addresses the combined impact of both epigenetic and genetic alterations in cancer cells.     

From amplification to function: the case of the MDR1 gene.

This review describes the features of gene amplification associated with the selection of multidrug-resistant cell lines. Some of these lines carry multiple copies of the MDR1 gene that encodes P-glycoprotein, a broad specificity efflux pump. The MDR1 gene was initially identified as the common component of the amplicons found in multidrug-resistant cell lines selected with different drugs. Subsequent studies have established that increased MDR1 expression is sufficient for the multidrug-resistant phenotype. MDR1-containing amplicons may include a number of additional transcribed genes that do not appear to contribute to multidrug resistance. MDR1 amplification is associated with specific chromosomal changes and apparently non-random recombinational events. Increased expression of the MDR1 gene, however, does not necessarily require gene amplification. Although amplification of the MDR1 gene has not been found in clinical tumor samples, increased expression of this gene is commonly observed in different types of cancer and appears to be a significant marker of clinical drug resistance.     

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.     

The role of epigenetic inactivation of 14-3-3σ in human cancer

Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specific promoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. The p53-regulated gene 14-3-3σ undergoes frequent epigenetic silencing in several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of 14-3-3σ expression may be causally involved in tumor progression. Functional studies demonstrated that 14-3-3σ is involved in cell-cycle control and prevents the accumulation of chromosomal damage. The recent identification of novel 14-3-3if-associated proteins by a targeted proteomics approach implies that 14-3-3σ regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3σ expression in cancer cells.     

Borealin/Dasra B is a cell cycle-regulated chromosomal passenger protein and its nuclear accumulation is linked to poor prognosis for human gastric cancer

Chromosomal passenger proteins including Aurora B, Survivin, and Borealin/Dasra B, also called CDCA8/FLJ10468, are known to play crucial roles during mitosis and cell division. Inappropriate chromosomal segregation and cell division may cause auneuploidy leading to cancer. However, it is still unclear how the expression of chromosomal passenger proteins may be linked to cancer. In this study, we demonstrated that Borealin is a cell cycle-regulated gene and is upregulated at G2-M phases of the cell cycle. We showed that Borealin interacts with Survivin but not with Aurora B. The interaction domain of Survivin in Borealin was mapped to the N-terminal 92 amino-acid residues of Borealin. To examine the linkage between expression of Borealin and cancer, we performed immunohistochemistry analysis using anti-Borealin specific antibody on the paraffin-embedded gastric cancer tissues. Our results showed that Borealin expression is significantly correlated with Survivin (P = 0.003) and Ki67 (P = 0.007) in gastric cancer. Interestingly, an increased nuclear Borealin level reveals borderline association with a poor survival rate (P = 0.047). Taken together, our results demonstrated that Borealin is a cell cycle-regulated chromosomal passenger protein and its aberrant expression is linked to a poor prognosis for gastric cancer.     

Viral infection-oxidative stress/DNA damage-aberrant DNA methylation: separate or interrelated events responsible for genetic instability and childhood ALL development?

Acute lymphoblastic leukemia (ALL) is a malignant disorder that originates in a single B- or T-lymphocyte progenitor and is characterized by a range of numeric and structural chromosomal aberrations. Although, so far no clear cause can be found for ALL the most commonly recognized and strongest causal factor is infection. However, an interesting question is how viral infection may be responsible for genetic changes that lead to lymphoid cell transformation. A plausible mechanism by which infection might impact the process of leukemogenesis via genetic alteration is through: oxidative stress/DNA damage which is closely linked with inflammation, aberrant expression of AID/ABOBEC family enzymes which may be responsible for massive mutation introduction and alteration of DNA methylation, leading to changes in the expression of hematopoietic genes. In this review we propose several specific molecular mechanisms which link infection with all the above-mentioned processes. The most likely event which links common virus infection with ALL pathogenesis is aberrant expression of AID/APOBEC. This event may be directly responsible for the introduction of point mutations (as the result of cytosine or 5-methylcytosine deamination and formation of G:U or G:T misspairs) as well as changes in DNA methylation status.