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Pfeffer U.,Functional Genomics | Amaro A.,Functional Genomics | Bachmeier B.,Ludwig Maximilians University of Munich | Angelini G.,Functional Genomics
New Horizons in Translational Medicine | Year: 2014

Everybody is at risk for cancer yet environmental factors, life style and diet as well as genetic factors influence the individual cancer risk. Targeted or personalized cancer prevention is based on the knowledge of the molecular characteristics of the tumor to be prevented, the molecular mechanisms of action of the compounds to be used and the genetic make-up of the person who opts for prevention medicine. Genetic factors are to a certain extent specific for cancer types or even subtypes as it has been shown for breast cancer. The growing knowledge of such genotype cancer risk associations will allow for the definition of personalized prevention strategies. Prevention in intermediate risk populations requires non-toxic, well tolerated and cheap compounds, such as Curcumin. Its main activity is the inhibition of nuclear factor kappa B (NFkB) activation. NFkB is involved in many cancers where it acts through the generation of chronic inflammation that can be contrasted with anti-inflammatory drugs such as Curcumin. Targeted prevention of cancer also increases the possibility to conduct serious clinical experimentation with target based patient selection. © 2014 European Society for Translational Medicine.

News Article
Site: www.biosciencetechnology.com

National Institutes of Health researchers have identified a striking signature in tumor DNA that occurs in five different types of cancer. They also found evidence that this methylation signature may be present in many more types of cancer. The specific signature results from a chemical modification of DNA called methylation, which can control the expression of genes like a dimmer on a light switch. Higher amounts of DNA methylation (hypermethylation), like that found by the researchers in some tumor DNA, decreases a gene's activity. Based on this advance, the researchers hope to spur development of a blood test that can be used to diagnose a variety of cancers at early stages, when treatments can be most effective. The study appeared February 5, 2016, in The Journal of Molecular Diagnostics. "Finding a distinctive methylation-based signature is like looking for a spruce tree in a pine forest," said Laura Elnitski, Ph.D., a computational biologist in the Intramural Research Program at NIH's National Human Genome Research Institute (NHGRI). "It's a technical challenge to identify, but we found an elevated methylation signature around the gene known as ZNF154 that is unique to tumors." Dr. Elnitski is head of the Genomic Functional Analysis Section and senior investigator in the Translational and Functional Genomics Branch at NHGRI. In 2013, her research group discovered a methylation mark (or signature) around ZNF154 in 15 tumor types in 13 different organs and deemed it a possible universal cancer biomarker. Biomarkers are biological molecules that indicate the presence of disease. Dr. Elnitski's group identified the methylation mark using DNA taken from solid tumors. "No one in my group slept the night after that discovery," Dr. Elnitski said. "We were so excited when we found this candidate biomarker. It's the first of its kind to apply to so many types of cancer." In this new study, they developed a series of steps that uncovered telltale methylation marks in colon, lung, breast, stomach and endometrial cancers. They showed that all the tumor types and subtypes consistently produced the same methylation mark around ZNF154. "Finding the methylation signature was an incredibly arduous and valuable process," said NHGRI Scientific Director Dan Kastner, M.D., Ph.D. "These findings could be an important step in developing a test to identify early cancers through a blood test." The NIH Intramural Sequencing Center sequenced the tumor DNA that had been amplified using a technique called polymerase chain reaction (PCR). Dr. Elnitski and her group then analyzed the results, finding elevated levels of methylation at ZNF154 across the different tumor types. To verify the connection between increased methylation and cancer, Dr. Elnitski's group developed a computer program that looked at the methylation marks in the DNA of people with and without cancer. By feeding this information into the program, they were able to predict a threshold for detecting tumor DNA. Even when they reduced the amount of methylated molecules by 99 percent, the computer could still detect the cancer-related methylation marks in the mixture. Knowing that tumors often shed DNA into the bloodstream, they calculated the proportions of circulating tumor DNA that could be found in the blood. Dr. Elnitski will next begin screening blood samples from patients with bladder, breast, colon, pancreatic and prostate cancers to determine the accuracy of detection at low levels of circulating DNA. Tumor DNA in a person with cancer typically comprises between 1 and 10 percent of all DNA circulating in the bloodstream. The group noted that when 10 percent of the circulating DNA contains the tumor signature, their detection rate is quite good. Because the methylation could be detected at such low levels, it should be adequate to detect advanced cancer as well as some intermediate and early tumors, depending on the type. Dr. Elnitski's group will also collaborate with Christina Annunziata, M.D., Ph.D., an investigator in the Women's Malignancies Branch and head of the Translational Genomics Section at NIH's National Cancer Institute (NCI). They will test blood samples from women with ovarian cancer to validate the process over the course of treatment and to determine if this type of analysis leads to improved detection of a recurrence and, ultimately, improved outcomes. "Ovarian cancer is difficult to detect in its early stages, and there are no proven early detection methods," said Dr. Annunziata. "We need a reliable biomarker for detecting the disease when a cure is more likely. We are looking forward to testing Dr. Elnitski's novel approach using DNA methylation signatures." Current blood tests are specific to a known tumor type. In other words, clinicians must first find the tumor, remove a sample of it and determine its genome sequence. Once the tumor-specific mutations are known, they can be tracked for appearance in the blood. The potential of the new approach is that no prior knowledge of cancer is required, it would be less intrusive than other screening approaches like colonoscopies and mammograms and it could be used to follow individuals at high risk for cancer or to monitor the activity of a tumor during treatment. Once the blood test is developed, the scientific community must conduct studies to ensure that it does not indicate the presence of cancer when it is not there or miss cancer when it is there. Dr. Elnitski does not yet understand the connection between tumors and elevated DNA methylation. It may represent derailment of normal processes in the cell, or it may have something to do with the fact that tumors consume a lot of energy and circumvent the cellular processes that keep growth in check. Researchers also don't know exactly what the gene ZNF154 does. "We have laid the groundwork for developing a diagnostic test, which offers the hope of catching cancer earlier and dramatically improving the survival rate of people with many types of cancer," Dr. Elnitski said.

Amaro A.,Functional Genomics | Esposito A.I.,Functional Genomics | Gallina A.,Functional Genomics | Nees M.,VTT Technical Research Center of Finland | And 3 more authors.
Cancer and Metastasis Reviews | Year: 2014

Biomarkers are important for early detection of cancer, prognosis, response prediction, and detection of residual or relapsing disease. Special attention has been given to diagnostic markers for prostate cancer since it is thought that early detection and surgery might reduce prostate cancer-specific mortality. The use of prostate-specific antigen, PSA (KLK3), has been debated on the base of cohort studies that show that its use in preventive screenings only marginally influences mortality from prostate cancer. Many groups have identified alternative or additional markers, among which PCA3, in order to detect early prostate cancer through screening, to distinguish potentially lethal from indolent prostate cancers, and to guide the treatment decision. The large number of markers proposed has led us to the present study in which we analyze these indicators for their diagnostic and prognostic potential using publicly available genomic data. We identified 380 markers from literature analysis on 20,000 articles on prostate cancer markers. The most interesting ones appeared to be claudin 3 (CLDN3) and alpha-methysacyl-CoA racemase highly expressed in prostate cancer and filamin C (FLNC) and keratin 5 with highest expression in normal prostate tissue. None of the markers proposed can compete with PSA for tissue specificity. The indicators proposed generally show a great variability of expression in normal and tumor tissue or are expressed at similar levels in other tissues. Those proposed as prognostic markers distinguish cases with marginally different risk of progression and appear to have a clinically limited use. We used data sets sampling 152 prostate tissues, data sets with 281 prostate cancers analyzed by microarray analysis and a study of integrated genomics on 218 cases to develop a multigene score. A multivariate model that combines several indicators increases the discrimination power but does not add impressively to the information obtained from Gleason scoring. This analysis of 10 years of marker research suggests that diagnostic and prognostic testing is more difficult in prostate cancer than in other neoplasms and that we must continue to search for better candidates. © 2014 The Author(s).

Bianchi G.,Laboratorio Of Oncologia Istituto slini | Martella R.,Laboratorio Of Oncologia Istituto slini | Ravera S.,University of Genoa | Marini C.,CNR Institute of Neuroscience | And 12 more authors.
Oncotarget | Year: 2015

Tumor chemoresistance is associated with high aerobic glycolysis rates and reduced oxidative phosphorylation, a phenomenon called "Warburg effect" whose reversal could impair the ability of a wide range of cancer cells to survive in the presence or absence of chemotherapy. In previous studies, Short-term-starvation (STS) was shown to protect normal cells and organs but to sensitize different cancer cell types to chemotherapy but the mechanisms responsible for these effects are poorly understood. We tested the cytotoxicity of Oxaliplatin (OXP) combined with a 48hour STS on the progression of CT26 colorectal tumors. STS potentiated the effects of OXP on the suppression of colon carcinoma growth and glucose uptake in both in vitro and in vivo models. In CT26 cells, STS down-regulated aerobic glycolysis, and glutaminolysis, while increasing oxidative phosphorylation. The STS-dependent increase in both Complex I and Complex II-dependent O2 consumption was associated with increased oxidative stress and reduced ATP synthesis. Chemotherapy caused additional toxicity, which was associated with increased succinate/Complex IIdependent O2 consumption, elevated oxidative stress and apoptosis . These findings indicate that the glucose and amino acid deficiency conditions imposed by STS promote an anti-Warburg effect characterized by increased oxygen consumption but failure to generate ATP, resulting in oxidative damage and apoptosis.

"CRISPR technology allows researchers to easily knock out or remove specific genes from a host cell," said Sandia researcher Oscar Negrete, who is spearheading the project for Sandia to develop the libraries that will be used to screen Zika and many other viruses. "CRISPR libraries are built to target large sets of genes simultaneously." Using these libraries, researchers can rapidly understand how removal of these genes affects virus infection. By discovering host genes essential for infection through CRISPR library screening, researchers can begin to design anti-viral treatments using those identified gene or gene products as targets. Negrete is co-leading the project with Robert Damoiseaux, director of the Molecular Screening Shared Resource at UCLA. Sandia researchers Joe Schoeniger, Sara Bird and Edwin Saada also are working on the yearlong project, "Development of Arrayed CRISPR-based Libraries for Functional Genomics Screening." The work is part of the first-ever Cooperative Research and Development Agreement (CRADA) between Sandia and UCLA. This work is conducted under Sandia's Laboratory Directed Research and Development program, which invests in high-risk, potentially high-payoff science, technology and engineering challenges with high potential to make a significant impact on national security. "The purpose of our CRADA is using an alternative method of library assembly to produce arrayed CRISPR libraries at a fraction of the cost of standard methods," Negrete said. "Instead of producing individual constructs one-by-one which by standard methods are relatively expensive and labor intensive, we will start out with a mixture of CRISPR constructs called a pooled library that is easy and inexpensive to produce, then separate or array the mixture into individual constructs using high-throughput robotic equipment. Using next-generation sequencing, we can then decode the constructs to build the arrayed formatted library." "Like a jar of jellybeans, a pooled CRISPR library is a complex mixture," Negrete explained. "An arrayed library on the other hand, is more like individually wrapped and labeled jellybeans. When you identify a particular CRISPR of interest from a pooled screen, you still have to run a deconvolution experiment to know what you have. In arrayed libraries, the CRISPR of interest is already labeled, therefore hit identification is much easier, quicker and cheaper." Researchers aim to produce a genome-scale arrayed CRISPR library, but will initially focus on building a sub-library that targets the membrane proteins involved in cell communication and that act as receptors for pathogen entry. CRISPR evolved in nature as a bacterial defense system and now serves as the basis of a powerful genome editing technology. Unlike other gene-editing methods, CRISPR is cheap, quick and easy to use. Negrete said CRISPR has created a biotechnology revolution similar to the discovery of the polymerase chain reaction (PCR) technique used to make millions of copies of a segment of DNA. "This technology is moving medicine to a whole new direction where researchers are trying to cure any disease that has a genetic component," Negrete said. "With CRISPR, it is theoretically possible to modify any genome including the human genome. It could one day hold the cure to any number of genetic diseases." This CRADA effort will develop efficient routes toward arrayed libraries using high-throughput screening and sequencing methods. "As a research tool, CRISPR is an ideal genome engineering system for functional genomic screening," Negrete said. "CRISPR-based genetic screens are particularly useful for studying diseases or phenotypes for which the underlying genetic cause is not known." Before CRISPR, scientists relied heavily on RNA interference-based genetic screens, which are prone to off-target effects and may result in false negatives due to incomplete knock-down of target genes. The CRISPR system can make highly specific, permanent genetic modifications in target genes and already has been used to screen for novel genes that regulate disease-related phenotypes. The most common method for conducting genome-wide screens using CRISPR involves the use of "pooled" libraries. A pooled CRISPR library is a complex mixture of thousands of unique sequences, which is then delivered to a single group of cells in bulk for screening disease phenotypes. Since the libraries are delivered in bulk, analyses of phenotypes are limited. By contrast, arrayed CRISPR libraries are generated in multiwell plates, where each well contains a vector preparation targeting an individual gene. Using this type of library, it is possible to explore complex phenotypes arising from a vast number of distinct cell perturbations. In addition, arrayed libraries provide access to rapid secondary follow-up screens. Access to screening of this novel CRISPR library will be made available to a wider scientific audience on a case-by-case basis. Sandia also plans to use the libraries for screening of viral mechanisms and drug target discovery. The Molecular Screening Shared Resource (MSSR) offers a comprehensive range of high throughput screening services including chemical genomics, functional genomics and drug discovery. The MSSR is an open environment and welcomes academic investigators from UCLA and from all over the globe. "This is just the beginning for the applications of this type of technology," Negrete said. "If we can make an impact on Ebola and Zika, then we have accomplished our goal."

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