Finding Keys to Glioblastoma Therapeutic Resistance
Researchers at the University of California, San Diego School of Medicine have found one of the keys to why certain glioblastomas – the primary form of a deadly brain cancer – are resistant to drug therapy. The answer lies not in the DNA sequence of the tumor, but in its epigenetic signature. These findings have been published online as a priority report in the journal Oncotarget.
“There is a growing interest to guide cancer therapy by sequencing the DNA of the cancer cell,” said Clark Chen, MD, PhD, vice-chairman of Research and Academic Development, UC San Diego Division of Neurosurgery and the principal investigator of the study. “Our study demonstrates that the sensitivity of glioblastoma to a drug is influenced not only by the content of its DNA sequences, but also by how the DNA sequences are organized and interpreted by the cell.”
The team of scientists, led by Chen, used a method called comparative gene signature analysis to study the genetic profiles of tumor specimens collected from approximately 900 glioblastoma patients. The method allows investigators to discriminate whether specific cellular processes are “turned on” or “turned off” in glioblastomas. “Our study showed that not all glioblastomas are the same. We were able to classify glioblastomas based on the type of cellular processes that the cancer cells used to drive tumor growth,” said Jie Li, PhD, senior postdoctoral researcher in the Center for Theoretical and Applied Neuro-Oncology at UC San Diego and co-first author of the paper.
One of these cellular processes involves Epidermal Growth Factor Receptor (EGFR). The study revealed that EGFR signaling is suppressed in a subset of glioblastomas. Importantly, this suppression is not the result of altered DNA sequences or mutations. Instead, EGFR is turned off as a result of how the DNA encoding the EGFR gene is organized in the cancer cell. This form of regulation is termed “epigenetic.” Because EGFR is turned off in these glioblastomas, they become insensitive to drugs designed to inhibit EGFR signaling.
“Our research suggests that the selection of appropriate therapies for our brain tumor patients will require a meaningful synthesis of genetic and epigenetic information derived from the cancer cell,” said co-first author Zachary J. Taich.
The epigenetics of Parkinson’s disease
Parkinson’s disease (PD) is a neurodegenerative disorder with high incidence in the elderly, caused by a combination of environmental and genetic factors. In addition, epigenetic changes – changes in gene expression that are caused by non-genetic factors – such as DNA methylation have recently been associated with PD.
In the August 1 online edition of the Landes Biosciences journal Epigenetics, researchers from the University of California, San Diego School of Medicine investigated genome-wide methylation in brain and blood samples from PD patients. They observed a distinctive pattern of methylation involving many genes previously associated with the disease, supporting the role of epigenetic alterations as a molecular mechanism in neurodegeneration.
DNA methylation, a biochemical process that is one of the methods used to regulate the expression of genes, represents a highly promising biomarker for neurodegenerative disorders. Emerging techniques allow monitoring DNA methylation from blood – a method that could replace post-mortem brain tissue as a way to detect PD.
“Since early identification of pathological changes is crucial to enable therapeutic intervention in Parkinson’s disease before major neurologic damage occurs, these findings could prove important in monitoring disease progression and the effectiveness of treatment,” said lead author Paula Desplats, PhD, project scientist with the Department of Neurosciences at UC San Diego School of Medicine.
In this study, Desplats and UC San Diego colleagues Eliezer Masliah, MD, Wilmar Dumaop and Douglas Galasko, MD, report differential methylation for several genes, and identified concordant methylation alterations in a subset of genes in both the brain and blood samples of patients with PD.
Earlier studies by the UC San Diego researchers reported a significant decrease in DNA methylation in the frontal cortex of patients with PD and the related disorder called Dementia with Lewy bodies. Here, they further investigated the extent of epigenetic deregulation by analyzing genome-wide DNA methylation profiles in both postmortem frontal cortex samples and peripheral blood leukocytes (or PBLs) from the same individuals in a cohort of PD patients. They then compared these profiles to those of age-matched, healthy control subjects. Individual methylation profiles obtained from blood distinguished control subjects from those with PD.
“We observed similar overall methylation patterns in the brain and blood, and that the same functional groups were affected – genes involved with cell communication, as well as with cellular and metabolic processes, such as genes related to cell death,” Desplats said.
The scientists’ analyses suggest that a number of methylation changes are shared between brain and blood, positioning 124 genes (which co-varied among blood and brain tissues) as candidates for biomarker discovery.
Since accurate diagnosis of PD cannot currently be achieved until clear motor features have developed (which occurs when half of more of neurons in a particular region of the brain called the substantia nigra), increasing efforts are being dedicated to identifying early, non-motor symptoms. These may include depression and sleep disorders as well as olfactory dysfunction or reduced sense of smell.
A false color scanning electron micrograph of chromosomes X and Y.
Finding the “epigenetic mark”
The centromere (Greek for center and part) is the anchor and focal point of cell division and, ultimately, of the heritability of life. It is here in the structure of a chromosome that DNA unspools and performs the act of mitosis, dividing to create two identical daughter cells.
This act occurs uncountable times every moment in species ranging in diversity and complexity from single-cell organisms to human beings. It is fundamental to life, and centromeres are fundamental to it.
Without centromeres, cells do not divide properly: Chromosomes do not align and their sister strands of DNA called chromatids do not separate correctly. The wrong number of chromosomes is passed to daughter cells. Inherited conditions like Down syndrome, which occurs when an individual has a full or partial extra copy of chromosome 21, can result.
But while the role and importance of centromeres has long been appreciated, a basic mystery has endured, said Don W. Cleveland, Distinguished Professor and Chair of the Department of Cellular and Molecular Medicine in the UC San Diego School of Medicine and head of the Laboratory for Cell Biology at the Ludwig Institute for Cancer Research at UC San Diego:
Why, if centromeres are responsible for insuring proper inheritance of chromosomal DNA, are they apparently not defined or determined by that very DNA? Rather, something else directs and maintains them, something researchers called “epigenetic mark.” The precise identity and nature of this mark was not known.
Writing in this week’s issue of Nature Cell Biology, Cleveland, with first author Daniele Fachinetti, and colleagues describe a two-step mechanism for defining centromere identity and function.
The first step, said Cleveland, is marking centromere DNA with a specific histone called Centromere Protein A or CENP-A, which provides a stable structure for attached proteins and other machinery to begin the second step of chromatid separation and mitosis.
The findings go beyond boosting our understanding of basic biology, said Cleveland.
“Discovery of the epigenetic mark of centromere identity adds a key insight into how chromosome inheritance is normally achieved with high fidelity,” he said. “Understanding how normal centromere function prevents errors in chromosome delivery is important to understanding, treating and preventing human disease. Many conditions, including cancers, are the result of an abnormal number of chromosomes.”
An x-ray micrograph of a yeast cell, Saccharomyces cerevisiae, as it buds before dividing. Courtesy of Carolyn Larabell, UC San Francisco, Lawrence Berkeley National Laboratory and the National Institute of General Medical Sciences.
In Epigenomics, Location is Everything
Researchers exploit gene position to test “histone code”
In a novel use of gene knockout technology, researchers at the University of California, San Diego School of Medicine tested the same gene inserted into 90 different locations in a yeast chromosome – and discovered that while the inserted gene never altered its surrounding chromatin landscape, differences in that immediate landscape measurably affected gene activity.
The findings, published online in the Jan. 3 issue of Cell Reports, demonstrate that regulation of chromatin – the combination of DNA and proteins that comprise a cell’s nucleus – is not governed by a uniform “histone code” but by specific interactions between chromatin and genetic factors.
“One of the main challenges of epigenetics has been to get a handle on how the position of a gene in chromatin affects its expression,” said senior author Trey Ideker, PhD, chief of the Division of Genetics in the School of Medicine and professor of bioengineering in UC San Diego’s Jacobs School of Engineering. “And one of the major elements of that research has been to look for a histone code, a general set of rules by which histones (proteins that fold and structure DNA inside the nucleus) bind to and affect genes.”
The Cell Reports findings indicate that there is no singular universal code, according to Ideker. Rather, the effect of epigenetics on gene expression or activity depends not only on the particular mix of histones and other epigenetic material, but also on the identity of the gene being expressed.
To show this, the researchers exploited an overlooked feature of an existing resource. The widely-used gene knockout library for yeast, originally created to see what happens when a particular gene is missing, was built by systematically inserting the same reporter gene into different locations. Ideker and colleagues focused on this reporter gene and observed what happens to gene expression at different locations along yeast chromosome 1.
“If epigenetics didn’t matter – the state of histones and DNA surrounding the gene – the expression of a gene would be the same regardless of where on the chromosome that gene is positioned,” said Ideker. But in every case, gene expression was measurably influenced by interaction with nearby epigenetic players.
Epigenetics Alters Genes in Rheumatoid Arthritis
It’s not just our DNA that makes us susceptible to disease and influences its impact and outcome. Scientists are beginning to realize more and more that important changes in genes that are unrelated to changes in the DNA sequence itself – a field of study known as epigenetics – are equally influential.
A research team at the University of California, San Diego – led by Gary S. Firestein, MD, professor in the Division of Rheumatology, Allergy and Immunology at UC San Diego School of Medicine – investigated a mechanism usually implicated in cancer and in fetal development, called DNA methylation, in the progression of rheumatoid arthritis (RA). They found that epigenetic changes due to methylation play a key role in altering genes that could potentially contribute to inflammation and joint damage. Their study is currently published in the online edition of the Annals of the Rheumatic Diseases.
“Genomics has rapidly advanced our understanding of susceptibility and severity of rheumatoid arthritis,” said Firestein. “While many genetic associations have been described in this disease, we also know that if one identical twin develops RA that the other twin only has a 12 to 15 percent chance of also getting the disease. This suggests that other factors are at play – epigenetic influences.”
DNA methylation is one example of epigenetic change, in which a strand of DNA is modified after it is duplicated by adding a methyl to any cytosine molecule (C) – one of the 4 main bases of DNA. This is one of the methods used to regulate gene expression, and is often abnormal in cancers and plays a role in organ development.
While DNA methylation of individual genes has been explored in autoimmune diseases, this study represents a genome-wide evaluation of the process in fibroblast-like synoviocytes (FLS), isolated from the site of the disease in RA. FLS are cells that interact with the immune cells in RA, an inflammatory disease in the joints that damages cartilage, bone and soft tissues of the joint.
“Birth of DNA (Epigenetics)” by Zdenko Herceg
Deciphering DNA’s hidden code
Reading the genetic “Book of Life” is not easy, an observation scientists learn all of the time. Consider the well-known nucleobases that comprise DNA. There are only four: adenine, thymine, guanine and cytosine (plus uracil, which is found in RNA). It turns out, however, that cytosine comes in two modified forms: 5-methylcytosine (5-mc) and 5-hydroxymethlcytosine (5-hmC). The versions look almost alike, but affect genes in very different ways.
In a paper published in the journal Cell today, researchers at the University of Chicago, the Ludwig Institute for Cancer Research at UC San Diego and Emory University describe a new technique for reading the particular differences in cytosine, an achievement that has ramifications for better understanding fundamental life processes.
These two modifications of cytosine “regulate gene expression that has broad impact on stem cell development, various human diseases such as cancer, and potentially neurodegenerative disease,” said Chuan He, a professor of chemistry at the University of Chicago. “They may even shape the development of the human brain.”
He, with Bing Ren, PhD, head of the Laboratory of Gene Regulation at the Ludwig Institute for Cancer Research at UC San Diego, and colleagues developed a method called TAB-Seq that directly measures 5-hmC and produced the first map of the entire genome of 5-hmC at single-base resolution. Ren applied TAB-Seq to human embryonic stem cells; Peng Jin of Emory applied the method to mouse embryonic stem cells.
The work is expected to have a significant impact upon the field of epigenetics, which looks at changes in gene expression caused by factors other than alterations in the actual DNA. 5-mC and 5-hmC appear to be major epigenetic players. 5-mC is generally found on genes that are turned off; it helps silence genes that aren’t supposed to be turned on. Conversely, 5-hmC appears to be abundant on active genes, especially in brain cells.
“This is a major breakthrough in that TAB-Seq allows precise mapping of all 5-hydroxymethylcytosine sites in a mammalian genome using well-established, next-generation DNA sequencing methods,” said Joseph Ecker, a professor at the Salk Institute for Biological Studies, who was not involved in the Cell study. “The study showed very clearly that deriving useful knowledge about this poorly understood epigenetic regulator requires determination of the exact locations of 5-hmC with base-level accuracy. I expect that their new method will immediately become widely adopted.”
A five-day-old human blastocyst.
Express Yourself: How Zygotes Sort Out Imprinted Genes
Researchers at the Ludwig Institute for Cancer Research, the University of California, San Diego School of Medicine and the Toronto Western Research Institute peel away some of the enduring mystery of how zygotes or fertilized eggs determine which copies of parental genes will be used or ignored.
In developing humans and other mammals, not all genes are created equal – or equally used. The expression of certain genes, known as imprinted genes, is determined by just one copy of the parents’ genetic contribution. In humans, there are at least 80 known imprinted genes. If a copy of an imprinted gene fails to function correctly – or if both copies are expressed – the result can be a variety of heritable conditions, such as Prader-Willi and Angelman syndromes, or diseases like cancer.
In the Cell paper, a team of scientists, led by Bing Ren, PhD, head of the Laboratory of Gene Regulation at the Ludwig Institute for Cancer Research at UC San Diego, describe in greater detail how differential DNA methylation in the two parental genomes set the stage for selective expression of imprinted genes in the mouse. Differential DNA methylation is essential to normal development in humans and other higher organisms. It involves the addition of hydrocarbon compounds called methyls to cytosine, one of the four bases or building blocks of DNA. Such addition alters the expression of different genes, boosting or suppressing them to help direct embryonic growth and development.
The process is sometimes called epigenetic regulation. Epigenetics is the study of factors influencing inheritance beyond the genes themselves. “DNA is just half the story,” said Ren, who also heads the San Diego Epigenome Center, one of four centers established by the National Institutes of Health to focus on epigenetics research.
“Understanding how these limited imprinted regions control regulation can help us better understand how certain diseases happen,” said Ren, a professor of cellular and molecular medicine in the UC San Diego School of Medicine. “That can help us develop better diagnostic tools for detecting genetic abnormalities and perhaps learn how to predict whether something bad will happen.”