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Long interspersed nuclear elements 1 (LINE1) is repeated half a million times in the human genome, making up nearly a fifth of the DNA in every cell. But nobody cared to study it and may be the reason to call it junk DNA. LINE1, like other transposons (or “jumping genes”), has the unusual ability to copy and insert itself in random places in the genome. Many other research groups uncovered possible roles in early mouse embryos and in brain cells. But nobody quite established a proper report about the functions of LINE1.
Geneticists gave attention to LINE1 when it was found to cause cancer or genetic disorders like hemophilia. But researchers at University of California at San Francisco suspected there was more characteristics of LINE1. They suspected that if it can be most harmless then it can be worst harmful also.
Many reports showed that LINE1 is especially active inside developing embryos, which suggests that the segment actually plays a key role in coordinating the development of cells in an embryo. Researchers at University of California at San Francisco figured out how to turn LINE1 off in mouse embryos by blocking LINE1 RNA. As a result the embryos got stuck in the two-cell stage, right after a fertilized egg has first split. Without LINE1, embryos essentially stopped developing.
The researchers thought that LINE1 RNA particles act as molecular “glue,” bringing together a suite of molecules that switch off the two-cell stage and kick it into the next phase of development. In particular, it turns off a gene called Dux, which is active in the two-cell stage.
LINE1’s ability to copy itself, however, seems to have nothing to do with its role in embryonic development. When LINE1 was blocked from inserting itself into the genome, the embryonic stem cells remained unaffected. It’s possible that cells in embryos have a way of making LINE1 RNA while also preventing its potentially harmful “jumping” around in the genome. But it’s unlikely that every one of the thousands of copies of LINE1 is actually being used to regulate embryonic development.
LINE1 is abundant in the genomes of almost all mammals. Other transposons, also once considered junk DNA, have turned out to have critical roles in development in human cells too. There are differences between mice and humans, so, the next obvious step is to study LINE1 in human cells, where it makes up 17 percent of the genome.
Sequencing of the human genome via massive programs such as the Cancer Genome Atlas Program (CGAP) and the Encyclopedia of DNA Elements (ENCODE) consortium in conjunction with considerable bioinformatics efforts led by the National Center for Biotechnology Information (NCBI) have unlocked a myriad of yet unclassified genes (for good review see (2). The project encompasses 32 institutions worldwide which, so far, have generated 1640 data sets, initially depending on microarray platforms but now moving to the more cost effective new sequencing technology. Initially the ENCODE project focused on three types of cells: an immature white blood cell line GM12878, leukemic line K562, and an approved human embryonic cell line H1-hESC. The analysis was rapidly expanded to another 140 cell types. DNA sequencing had revealed 20,687 known coding regions with hints of 50 more coding regions. Another 11,224 DNA stretches were classified as pseudogenes. The ENCODE project reveals that many genes encode for an RNA, not protein product, so called regulatory RNAs.
However some of the most recent and interesting results focus on the noncoding regions of the human genome, previously discarded as uninteresting or “junk” DNA . Only 2% of the human genome contains coding regions while 98% of this noncoding part of the genome is actually found to be highly active “with about 4 million constantly communicating switches” (3). Some of these “switches” in the noncoding portion contain small, repetitive elements which are mobile throughout the genome, and can control gene expression and/or predispose to disease such as cancer. These mobile elements, found in almost all organisms, are classified as transposable elements (TE), inserting themselves into far-reaching regions of the genome. Retro-transposons are capable of generating new insertions through RNA intermediates. These transposable elements are normally kept immobile by epigenetic mechanisms(4-6) however some TEs can escape epigenetic repression and insert in areas of the genome, a process described as insertional mutagenesis as the process can lead to gene alterations seen in disease(7). In addition, this insertional mutagenesis can lead to the transformation of cells and, as described in Post 2, act as a model system to determine drivers of oncogenesis. This insertional mutagenesis is a different mechanism of genetic alteration and rearrangement seen in cancer like recombination and fusion of gene fragments as seen with the Philadelphia chromosome and BCR/ABL fusion protein (8). The mechanism of transposition and putative effects leading to mutagenesis are described in the following figure:
Figure. Insertional mutagenesis based on transposon-mediated mechanism. A) Basic structure of transposon contains gene/sequence flanked by two inverted repeats (IR) and/or direct repeats (DR). An enzyme, the transposase (red hexagon) binds and cuts at the IR/DR and transposon is pasted at another site in DNA, containing an insertion site. B) Multiple transpositions may results in oncogenic events by inserting in promoters leading to altered expression of genes driving oncogenesis or inserting within coding regions and inactivating tumor suppressors or activating oncogenes. Deep sequencing of the resultant tumor genomes ( based on nested PCR from IR/DRs) may reveal common insertion sites (CIS) and oncogenic mutations could be identified.
In a bioinformatics study Eunjung Lee et al.(1), in collaboration with the Cancer Genome Atlas Research Network, the authors had analyzed 43 high-coverage whole-genome sequencing datasets from five cancer types to determine transposable element insertion sites. Using a novel computational method, the authors had identified 194 high-confidence somatic TE insertion sites present in cancers of epithelial origin such as colorectal, prostate and ovarian, but not in brain or blood cancers. Sixty four of the 194 detected somatic TE insertions were located within 62 annotated genes. Genes with TE insertion in colon cancers have commonly high mutation rates and enriched genes were associated with cell adhesion functions (CDH12, ROBO2,NRXN3, FPR2, COL1A1, NEGR1, NTM and CTNNA2) or tumor suppressor functions (NELL1m ROBO2, DBC1, and PARK2). None of the somatic events were located within coding regions, with the TE sequences being detected in untranslated regions (UTR) or intronic regions. Previous studies had shown insertion in these regions (UTR or intronic) can disrupts gene expression (9). Interestingly, most of the genes with insertion sites were down-regulated, suggested by a recent paper showing that local changes in methylation status of transposable elements can drive retro-transposition (10,11). Indeed, the authors found that somatic insertions are biased toward the hypomethylated regions in cancer cell DNA. The authors also confirmed that the insertion sites were unique to cancer and were somatic insertions, not germline (germline: arising during embryonic development) in origin by analyzing 44 normal genomes (41 normal blood samples from cancer patients and three healthy individuals).
The authors conclude:
“that some TE insertions provide a selective advantage during tumorigenesis,
rather than being merely passenger events that precede clonal expansion(1).”
The authors also suggest that more bioinformatics studies, which utilize the expansive genomic and epigenetic databases, could determine functional consequences of such transposable elements in cancer. The following Post will describe how use of transposon-mediated insertional mutagenesis is leading to discoveries of the drivers (main genetic events) leading to oncogenesis.
1. Lee, E., Iskow, R., Yang, L., Gokcumen, O., Haseley, P., Luquette, L. J., 3rd, Lohr, J. G., Harris, C. C., Ding, L., Wilson, R. K., Wheeler, D. A., Gibbs, R. A., Kucherlapati, R., Lee, C., Kharchenko, P. V., and Park, P. J. (2012) Science337, 967-971
Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer death in the United States with a median survival of <6 mo and a dismal 5-yr survival rate of 3%–5%. The cancer’s lethal nature stems from its propensity to rapidly disseminate to the lymphatic system and distant organs. This aggressive biology and resistance to conventional and targeted therapeutic agents leads to a typical clinical presentation of incurable disease at the time of diagnosis.
Also, it has been well documented that despite much progress in its molecular characterization, Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy.
Recent article published in the journal Nature talks about discovering the link between a gene and the prognosis of Pancreatic Ductal Adenocarcenoma (PDA). The discovery might have therapeutic relevance in PDA.
Although previous work had attributed a pro-survival role to USP9X in human neoplasia, the researchers found instead that loss of Usp9x protects pancreatic cancer cells from death. Thus, the study proposed USP9X to be a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.
Study points to potential new treatment for deadly pancreatic cancer
In a study published in Nature (Sunday 29 April), researchers have identified a potential new therapeutic target for pancreatic cancer.
The team found that when a gene involved in protein degradation is switched-off through chemical tags on the DNA’s surface, pancreatic cancer cells are protected from the bodies’ natural cell death processes, become more aggressive, and can rapidly spread.
Pancreatic cancer kills around 8,000 people every year in the UK and, although survival rates are gradually improving, fewer than 1 in 5 patients survive for a year or more following their diagnosis.
Co-lead author Professor David Tuveson, from Cancer Research UK’s Cambridge Research Institute, said: “The genetics of pancreatic cancer has already been studied in some detail, so we were surprised to find that this gene hadn’t been picked up before. We suspected that the fault wasn’t in the genetic code at all, but in the chemical tags on the surface of the DNA that switch genes on and off, and by running more lab tests we were able to confirm this.”
The team expects this gene, USP9X, could be faulty in up to 15 per cent of pancreatic cancers, raising the prospect that existing drugs, which strip away these chemical tags, could be an effective way of treating some pancreatic cancers.
” This study strengthens our emerging understanding that we must also look into the biology of cells to identify all the genes that play a role in cancer. ” Dr David Adams
“Drugs which strip away these tags are already showing promise in lung cancer and this study suggests they could also be effective in treating up to 15 per cent of pancreatic cancers,” continues Professor Tuveson.
The researchers used a mouse model of pancreatic cancer to screen for genes that speed up pancreatic cancer growth using a technique called ‘Sleeping Beauty transposon mutagenesis’. This system uses mobile genetic elements that hop around the cell’s DNA from one location to the next. Cells that acquire mutations in genes that contribute to cancer development will grow out and ‘driver’ cancer genes may be identified.
By introducing the Sleeping Beauty transposon into mice pre-disposed to develop pancreatic cancer, the researchers were able to screen for a class of genes called a tumour suppressor that, under normal circumstances, would protect against cancer. These genes are a bit like the cell’s ‘brakes’, so when they become faulty there is little to stop the cell from multiplying out of control.
This approach uncovered many genes already linked to pancreatic cancer. But unexpectedly, USP9X, was identified.
Co-lead author Dr David Adams, from the Wellcome Trust Sanger Institute, said: “The human genome sequence has delivered many promising new leads and transformed our understanding of cancer. Without it, we would have only a small, shattered glimpse into the causes of this disease. This study strengthens our emerging understanding that we must also look into the biology of cells to identify all the genes that play a role in cancer.”
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