First analysis of tumor-suppressor interactions with whole genome in normal human cells reveals key differences with cancer cells

ScienceDaily (Nov. 30, 2011) — Scientists investigating the interactions, or binding patterns, of a major tumor-suppressor protein known as p53 with the entire genome in normal human cells have turned up key differences from those observed in cancer cells. The distinct binding patterns reflect differences in the chromatin (the way DNA is packed with proteins), which may be important for understanding the function of the tumor suppressor protein in cancer cells.

The study was conducted by scientists at the U.S. Department of Energy's (DOE) Brookhaven National Laboratory and collaborators at Cold Spring Harbor Laboratory, and is published in the December 15 issue of the journal Cell Cycle.

"No other study has shown such a dramatic difference in a tumor suppressor protein binding to DNA between normal and cancer-derived cells," said Brookhaven biologist Krassimira Botcheva, lead author on the paper. "This research makes it clear that it is essential to study p53 functions in both types of cells in the context of chromatin to gain a correct understanding of how p53 tumor suppression is affected by global epigenetic changes -- modifications to DNA or chromatin -- associated with cancer development."

Because of its key role in tumor suppression, p53 is the most studied human protein. It modulates a cell's response to a variety of stresses (nutrient starvation, oxygen level changes, DNA damage caused by chemicals or radiation) by binding to DNA and regulating the expression of an extensive network of genes. Depending on the level of DNA damage, it can activate DNA repair, stop the cells from multiplying, or cause them to self-destruct -- all of which can potentially prevent or stop tumor development. Malfunctioning p53 is a hallmark of human cancers.

Most early studies of p53 binding explored its interactions with isolated individual genes, and all whole-genome studies to date have been conducted in cancer-derived cells. This is the first study to present a high-resolution genome-wide p53-binding map for normal human cells, and to correlate those findings with the "epigenetic landscape" of the genome.

"We analyzed the p53 binding in the context of the human epigenome, by correlating the p53 binding profile we obtained in normal human cells with a published high-resolution map of DNA methylation -- a type of chemical modification that is one of the most important epigenetic modifications to DNA -- that had been generated for the same cells," Botcheva said.

Key findings

In the normal human cells, the scientists found p53 binding sites located in close proximity to genes and particularly at the sites in the genome, known as transcriptions start sites, which represent "start" signals for transcribing the genes. Though this association of binding sites with genes and transcription start sites was previously observed in studies of functional, individually analyzed binding sites, it was not seen in high-throughput whole-genome studies of cancer-derived cell lines. In those earlier studies, the identified p53 binding sites were found not close to genes, and not close to the sites in the human genome where transcription starts.

Additionally, nearly half of the newly identified p53 binding sites in the normal cells (in contrast to about five percent of the sites reported in cancer cells) reside in so-called CpG islands. These are short DNA sequences with unusually high numbers of cytosine and guanine bases (the C and G of the four-letter genetic code alphabet, consisting of A, T, C, and G). CpG islands tend to be hypo- (or under-) methylated relative to the heavily methylated mammalian genome.

"This association of binding sites with CpG islands in the normal cells is what prompted us to investigate a possible genome-wide correlation between the identified sites and the CpG methylation status," Botcheva said.

The scientists found that p53 binding sites were enriched at hypomethylated regions of the human genome, both in and outside CpG islands.

"This is an important finding because, during cancer development, many CpG islands are subjected to extensive methylation while the bulk of the genomic DNA becomes hypomethylated," Botcheva said. "These major epigenetic changes may contribute to the differences observed in the p53-binding-sites' distribution in normal and cancer cells."

The scientists say this study clearly illustrates that the genomic landscape -- the DNA modifications and the associated chromatin changes -- have a significant effect on p53 binding. Furthermore, it greatly extends the list of experimentally defined p53 binding sites and provides a general framework for investigating the interplay between transcription factor binding, tumor suppression, and epigenetic changes associated with cancer development.

This research, which was funded by the DOE Office of Science, lays groundwork for further advancing the detailed understanding of radiation effects, including low-dose radiation effects, on the human genome.

The research team also includes John Dunn and Carl Anderson of Brookhaven Lab, and Richard McCombie of Cold Spring Harbor Laboratory, where the high-throughput Illumina sequencing was done.

Methodology

The p53 binding sites were identified by a method called ChIP-seq: for chromatin immunoprecipitation (ChIP), which produces a library of DNA fragments bound by a protein of interest using immunochemistry tools, followed by massively parallel DNA sequencing (seq) for determining simultaneously millions of sequences (the order of the nucleotide bases A, T, C and G in DNA) for these fragments.

"The experiment is challenging, the data require independent experimental validation and extensive bioinformatics analysis, but it is indispensable for high-throughput genomic analyses," Botcheva said. Establishing such capability at BNL is directly related to the efforts for development of profiling technologies for evaluating the role of epigenetic modifications in modulating low-dose ionizing radiation responses and also applicable for plant epigenetic studies.

The analysis required custom-designed software developed by Brookhaven bioinformatics specialist Sean McCorkle.

"Mapping the locations of nearly 20 million sequences in the 3-billion-base human genome, identifying binding sites, and performing comparative analysis with other data sets required new programming approaches as well as parallel processing on many CPUs," McCorkle said. "The sheer volume of this data required extensive computing, a situation expected to become increasingly commonplace in biology. While this work was a sequence data-processing milestone for Brookhaven, we expect data volumes only to increase in the future, and the computing challenges to continue."

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Journal References:

Krassimira Botcheva, Sean R. McCorkle, W.R. McCombie, John J. Dunn, Carl W. Anderson. Distinct p53 genomic binding patterns in normal and cancer-derived human cells. Cell Cycle, 2011; 10 (24) [link]William A. Freed-Pastor, Carol Prives. Dissimilar DNA binding by p53 in normal and tumor-derived cells. Cell Cycle, 2011; 10 (24) [link]

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Cancer cells' DNA repair disrupted to increase radiation sensitivity

ScienceDaily (Dec. 1, 2011) — Shortening end caps on chromosomes in human cervical cancer cells disrupts DNA repair signaling, increases the cells' sensitivity to radiation treatment and kills them more quickly, according to a study in Cancer Prevention Research.

Researchers would to like see their laboratory findings -- published in the journal's Dec. 5 print edition -- lead to safer, more effective combination therapies for hard-to-treat pediatric brain cancers like medulloblastoma and high-grade gliomas. To this end, they are starting laboratory tests on brain cancer cells.

"Children with pediatric brain cancers don't have very many options because progress to find new treatments has been limited the last 30 years," said Rachid Drissi, PhD, principal investigator on the study and a researcher in the Division of Oncology at Cincinnati Children's. "The ability to make cancer cells more sensitive to radiation could allow physicians to use lower radiation doses to lessen side effects. Too many children with brain cancer can develop disabilities or die from treatment."

Before treating cells with ionizing radiation, the researchers blocked an enzyme called telomerase, found in over 90 percent of cancer cells but barely detectable in most normal human cells. In cancer cells, telomerase helps maintain the length of caps on the ends of chromosomes called telomeres. This helps cancer cells replicate indefinitely, grow and spread, Drissi said.

Unraveling DNA stability

Found on chromosomes in both cancerous and normal cells, telomeres are analogous to plastic caps that keep shoestring ends from unraveling. Telomeres help preserve DNA stability in cells by containing genetic miscues. This helps explain why cells with maintained or long telomeres appear to be more resistant to radiation.

In normal cells lacking the telomerase enzyme, telomeres get shorter each time cells divide. They continue doing so until normal cells stop dividing, reaching a condition called senescence. If this first cell-cycle "stop sign" is bypassed, cells continue dividing until telomeres become critically short and reach a second stopping point, when most cells die. In rare instances, cells bypass this second "stop sign" and survive. This survival is often associated with telomerase activation and the onset of cancer.

This was the basis for experiments Drissi and his colleagues conducted to compare the radiation sensitivity and survivability of cells based on telomere length. They also monitored DNA repair responses in the cells by looking for specific biochemical signs that indicate whether the repair systems are working.

The tests involved normal human foreskin cells -- called fibroblasts -- and human cervical carcinoma cells. They exposed the cells to ionizing radiation and analyzed DNA repair responses as telomeres became progressively shorter. In the cervical cancer cells, researchers blocked the telomerase enzyme before radiation treatment to induce progressively shorter telomeres.

Both late-stage noncancerous cells with shorter telomeres, and cancer cells with induced shorter telomeres, were more radiosensitive and died more quickly, according to the study.

Among cancer cells with maintained telomere length, close to 10 percent receiving the maximum dose of ionizing radiation used in the study (8 Gy, or Gray Units) survived the treatment. None of the cancer cells with the shortest telomeres survived that exposure.

Researchers said the cancer cells became more radiosensitive because material inside the chromosomes -- called chromatin -- compacted as telomeres became shorter. Compacted chromatin then disrupted the biochemical signaling of a protein called ATM (ataxiatelangeietasia mutated).

ATM is a master regulator of DNA repair and cell division. It sends signals to activate other biochemical targets (H2AX, SMC1, NBS1 and p53) that help direct DNA repair and preserve genetic stability. In telomere-shortened cancer cells, the compacted chromatin inhibited ATM signaling to all of the chromatin-bound targets tested in the study. This disrupted DNA repair responses and increased radiation sensitivity.

Testing brain cancer cells

The researchers are now testing their findings in cells from hard-to-treat pediatric brain tumors. These tests begin as Drissi's laboratory also leads correlative cancer biology studies of tumor samples from a current clinical trial. The trial is evaluating telomere shortening as a stand-alone therapy for pediatric cancer.

Managed through the National Institutes of Health's Children's Oncology Group (COG), the multi-institutional Phase 1 trial is testing the safety and tumor response capabilities of the drug Imetelstat, which blocks telomerase in cancer cells. Drissi serves on the clinical trial committee along with Maryam Fouladi, MD, MSc, and medical director of Neuro-Oncology at Cincinnati Children's. She leads the medical center's clinical participation in the trial.

Drissi and Fouladi are starting preparatory work to develop, and seek approvals for, a possible clinical trial to test telomere shortening and radiation treatment as a safer, more effective treatment for pediatric brain tumors.

Funding support for the current study in Cancer Prevention Research -- published by the American Society for Cancer Research -- came from the National Institutes of Health, the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital and Cincinnati Children's Hospital Medical Center. Also collaborating were researchers from Children's National Medical Center in Washington, D.C., and from St. Jude. Funding support for the Drissi lab's correlative studies on the COG clinical trial comes from CancerFree Kids Pediatric Cancer Research Alliance and from Children's Cancer Research Fund.

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Journal Reference:

R. Drissi, J. Wu, Y. Hu, C. A. Bockhold, J. S. Dome. Telomere shortening alters the kinetics of the DNA damage response after ionizing radiation in human cells. Cancer Prevention Research, 2011; DOI: 10.1158/1940-6207.CAPR-11-0069

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High tech detection of breast cancer using nanoprobes and SQUID

ScienceDaily (Oct. 28, 2011) — Mammography saves lives by detecting very small tumors. However, it fails to find 10-25% of tumors and is unable to distinguish between benign and malignant disease. New research published in BioMed Central's open access journal Breast Cancer Research provides a new and potentially more sensitive method using tumor-targeted magnetic nanoprobes and superconducting quantum interference device (SQUID) sensors.

A team of researchers from University of New Mexico School of Medicine and Cancer Research and Treatment Center, Senior Scientific, LLC, and the Center for Integrated Nanotechnologies facility at Sandia National Laboratories created nanoprobes by attaching iron-oxide magnetic particles to antibodies against HER-2, a protein overexpressed in 30% of breast cancer cases. Using these tiny protein-iron particles the team was able to distinguish between cells with HER-2 and those without, and were able to find HER-2 cancer cells in biopsies from mice. In their final test the team used a synthetic breast to determine the potential sensitivity of their system.

Dr Helen Hathaway explained, "We were able to accurately pinpoint 1 million cells at a depth of 4.5 cm. This is about 1000x fewer cells than the size at which a tumor can be felt in the breast and 100x more sensitive than mammographic x-ray imaging. While we do not expect the same level of nanoparticle uptake in the clinic, our system has an advantage in that dense breast tissue, which can mask traditional mammography results, is transparent to the low-frequency magnetic fields detected by the SQUID sensors."

Future refining of the system could allow not only tumor to be found but to be classified according to protein expression (rather than waiting for biopsy results). This in turn could be used to predict disease progression and refine treatment plans and so improve patient survival.

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Helen J Hathaway, Kimberly S Butler, Natalie L Adolphi, Debbie M Lovato, Robert Belfon, Danielle L Fegan, Todd C Monson, Jason E Trujillo, Trace E Tessier, Howard C Bryant, Dale L Huber, Richard S Larson and Edward R Flynn. Detection of breast cancer cells using targeted magnetic nanoparticles and ultra-sensitive magnetic field sensors. Breast Cancer Research, 2011; 13: R108 DOI: 10.1186/bcr3050

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More clues to causes of breast cancer: Hyperactivation of Akt and overexpression of IKBKE observed in 50 percent of human cancers

ScienceDaily (Oct. 27, 2011) — Publishing in the current issue of The Journal of Biological Chemistry, researchers at Moffitt Cancer Center in Tampa, Fla., have discovered additional mechanisms of "Akt" activation and suggest a component of that activation mechanism -- inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE) -- could be targeted as a therapeutic intervention for treating cancer.

Akt, also known as protein kinase B, is one of about 500 protein kinases in the human genome. Kinases are known to regulate the majority of cellular pathways. Akt modifies other proteins chemically and regulates cell proliferation.

"Recent evidence suggests that IKBKE is an oncogenic kinase that participates in malignant transformation and tumor development," said Moffitt senior researcher and lead author Jin Q. Cheng, Ph.D., M.D. "Our study identified Akt as a bona fide substrate of IKBKE and IKBKE direct activation of Akt independent PI3K and revealed a functional link between IKBKE and Akt activation in breast cancer."

Cheng's lab studies a variety of genetic alterations and their molecular mechanisms in both ovarian and breast cancer, particularly on their effect on the molecules that are regulated by Akt and the small molecule inhibitors of Akt.

"We found that inhibition of Akt suppresses IKBKE's oncogenic transformation," said Cheng. "This is significant because overexpression of IKBKE and activation of Akt has been observed in more than 50 percent of human cancers. Akt inhibitors targeting PH domain do not have inhibitory effect on IKBKE-induced Akt."

The researchers experimented with a variety of inhibitors currently being used in clinical trials.

The laboratory study utilized breast cancer cell lines from received from patient donors at Moffitt and cell lines received from Harvard University and Johns Hopkins University. The work was supported by a National Institutes of Health grant and a grant from the James and Esther King Biomedical Research Program.

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The above story is reprinted from materials provided by H. Lee Moffitt Cancer Center & Research Institute.

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J.-P. Guo, D. Coppola, J. Q. Cheng. IKBKE Protein Activates Akt Independent of Phosphatidylinositol 3-Kinase/PDK1/mTORC2 and the Pleckstrin Homology Domain to Sustain Malignant Transformation. Journal of Biological Chemistry, 2011; 286 (43): 37389 DOI: 10.1074/jbc.M111.287433

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Regular aspirin intake halves cancer risk, study finds

ScienceDaily (Oct. 28, 2011) — Scientists including those from Queen's University have discovered that taking regular aspirin halves the risk of developing hereditary cancers.

Hereditary cancers are those which develop as a result of a gene fault inherited from a parent. Bowel and womb cancers are the most common forms of hereditary cancers. Fifty thousand people in the UK are diagnosed with bowel and womb cancers every year; 10 per cent of these cancers are thought to be hereditary.

The decade-long study, which involved scientists and clinicians from 43 centres in 16 countries and was funded by Cancer Research UK, followed nearly 1,000 patients, in some cases for over 10 years. The study found that those who had been taking a regular dose of aspirin had 50 per cent fewer incidents of hereditary cancer compared with those who were not taking aspirin.

The research focused on people with Lynch syndrome which is an inherited genetic disorder that causes cancer by affecting genes responsible for detecting and repairing damage in the DNA. Around 50 per cent of those with Lynch syndrome develop cancer, mainly in the bowel and womb. The study looked at all cancers related to the syndrome, and found that almost 30 per cent of the patients not taking aspirin had developed a cancer compared to around 15 per cent of those taking the aspirin.

Those who had taken aspirin still developed the same number of polyps, which are thought to be precursors of cancer, as those who did not take aspirin but they did not go on to develop cancer. It suggests that aspirin could possibly be causing these cells to destruct before they turn cancerous.

Over 1,000 people were diagnosed with bowel cancer in Northern Ireland last year; 400 of these died from the disease. Ten per cent of bowel cancer cases are hereditary and by taking aspirin regularly the number of those dying from the hereditary form of the disease could be halved.

Professor Patrick Morrison from Queen's University in Belfast, who led the Northern Ireland part of the study, said: "The results of this study, which has been ongoing for over a decade, proves that the regular intake of aspirin over a prolonged period halves the risk of developing hereditary cancers. The effects of aspirin in the first five years of the study were not clear but in those who took aspirin for between five and ten years the results were very clear."

"This is a huge breakthrough in terms of cancer prevention. For those who have a history of hereditary cancers in their family, like bowel and womb cancers, this will be welcome news. Not only does it show we can reduce cancer rates and ultimately deaths, it opens up other avenues for further cancer prevention research. We aim now to go forward with another trial to assess the most effective dosage of aspirin for hereditary cancer prevention and to look at the use of aspirin in the general population as a way of reducing the risk of bowel cancer.

"For anyone considering taking aspirin I would recommend discussing this with your GP first as aspirin is known to bring with it a risk of stomach complaints, including ulcers."

The research was published online Oct. 28 in The Lancet.

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Sir John Burn, Anne-Marie Gerdes, Finlay Macrae, Jukka-Pekka Mecklin, Gabriela Moeslein, Sylviane Olschwang, Diane Eccles, Gareth Evans, Eamonn R. Maher, Lucio Bertario, Marie-Luise Bisgaard, Malcolm G. Dunlop, Judy W.C. Ho, Shirley V. Hodgson, Annika Lindblom, Jan Lubinski, Patrick J. Morrison, Victoria Murday, Raj Ramesar, Lucy Side, Rodney J. Scott, Huw J.W. Thomas, Hans F. Vasen, Gail Barker, Gillian Crawford, Faye Elliott, Mohammad Movahedi, Kirsi Pylvanainen, Juul T. Wijnen, Riccardo Fodde, Henry T. Lynch, John C. Mathers, D. Timothy Bishop, on behalf of the CAPP2 Investigators. Long-term effect of aspirin on cancer risk in carriers of hereditary colorectal cancer: an analysis from the CAPP2 randomised controlled trial. The Lancet, Pubished online Oct. 28, 2011; DOI: 10.1016/S0140-6736(11)61049-0

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