Damage to DNA is an issue for all cells, particularly in cancer, where the mechanisms that repair damage typically fail. The same agents that damage DNA also damage its sister molecule messenger RNA (mRNA), which ferries transcripts of the genes to the tens of thousands of ribosomes in each cell. But little attention has been paid to this damage.

 “There may be cases where messenger RNA is just as important as DNA,” said Carrie Simms, PhD, a postdoctoral associate in Zaher’s lab. “Clearly oxidative damage to RNA is somehow involved in neurodegenerative diseases, such as Alzheimer’s and ALS. It’s not necessarily causing the disease; it may just be some sort of by-product; but it’s in the mix.”

“Under normal conditions only about 1 percent of the cellular mRNAs are oxidized,” Zaher said, “but if you have oxidative stress, for whatever reason, a higher percentage can be damaged.

One of the hallmarks of Alzheimer’s is oxidative stress, and studies have shown that in people with advanced Alzheimer’s, half of the RNA molecules in the neurons may be oxidized.

Zaher, Simms and their colleagues report that when they fed oxidized mRNA to ribosomes, the nanomachines that convert mRNA to protein, the ribosomes jammed and stopped.

A stuck ribosome could be rescued by factors that released it from the mRNA and chewed up the damaged transcipt. But if the factors involved in this quality-control system were absent, damaged mRNA accumulated in the cell, just as it does in Alzheimer’s.

The three cellular processes essential to life—making copies of DNA, copying DNA into mRNA, and translating the mRNA into protein—have been penalized for billions of years by evolution, are astonishingly accurate, because evolution has heavily penalized any sloppiness.

Errors in DNA copying occur only once every billion events. When DNA is transcribed to mRNA, there is a mistake about once every ten thousand events .and when the mRNA is translated to protein, there might be an error once every thousand events.

To test the robustness of translation, the Zaher lab set out to break it, by giving faulty mRNA transcripts to ribosomes. They damaged one letter in a three-letter mRNA coding unit, oxidizing a G (the base guanine), to create what is called 8-oxo-G.

“We chose this oxidized base,” he said,” because we knew that when DNA is copied, an oxidized G causes a mistake. Instead of pairing with a C, as it normally would, 8-oxo-G will pair with an A.”

He thought the ribosome would read the three-letter codon C[8-oxo-G]C not as CGC but rather as CAC and conseqeuntly put the wrong amino acid in the protein chain it was making.

But when 8-oxo-G was added to a soup that contained all the factors needed to translate mRNA into protein, something surprising happened.

“We expected that we might get aberrant proteins,“ Simms said. “But the ribosome didn’t make mistakes.  It just stopped. It couldn’t deal with the mRNA at all”

The scientsts could tell it was stuck because levels of the protein the faulty mRNA encodes plummeted.

To make sure it was the presence rather than the position of the 8-oxo-G that mattered, Simms made mRNAs with the 8-oxo-G in each of the three positions of the three-letter coding unit. Each time the ribosome stalled.

Knowing they had found something interesting, the scientists upped their game. Simms built a longer 300-nucleotide mRNA to use as a probe. And instead of adding the damaged mRNA to a reconstituted bacterial system, she put it in extracts of plant and animal cells.

“We couldn’t look at ribosomes in the extracts,” Simms said, “but we could look at the proteins they made. They made short proteins, exactly the length you’d expect if the ribosome were stopping at the damaged base. “

A single mRNA typically has several ribosomes traveling along it, all simultaneously translating this transcript into protein. When the first ribosome stops, the others pile up behind it.

“You get this small product that is telling you the ribosome cannot go through the 8-oxo-G and then you get even smaller products that are telling you there are multiple ribosomes stuck behind the first ribosome. So the backed up ribosomes make a ladder of peptides,” Zaher said.

“This is a problem,” he said. “Among other things, the ribosome is an expensive machine that the cell has invested a lot of energy in making, and now it’s stuck on an mRNA. You need those ribosomes back.”

Fortunately ribosomes have three quality-control systems that keep watch for errors in the mRNA and rescue the ribosome if spot serious mistakes. One of these systems is “no-go decay.” When ribosomes are stuck and can’t go forward, they recruit factors that come in to pry open the ribosome, chew up the mRNA and add a tag to the defective peptide  that marks it for degradation.”

But no-go decay was originally discovered by throwing artificial roadblocks in the ribosome’s way: mRNAs with large hairpin turns in them that the ribosome could not unwind or plow through.

“Four billion years of evolution has made sure your genome does not have sequences that make hairpins, so these are clearly not the intended targets for no-go decay,” Zaher said.

To find out, the scientists turned to yeast cells. If the yeast’s ribosomes jammed on the oxidized mRNA but were rescued by no-go decay, very little damaged mRNA would accumulate in the cell. This proved to be the case.

Simms then deleted the gene for a factor that releases the ribosome from the mRNA when it jams. In these knockout yeast the level of oxidized mRNA went up. Then she deleted the gene for a factor that is recruited to degrade the mRNA after the ribosome is released, and again the level of oxidized mRNA rose. Without no-go decay, the cells were clearly in trouble.

“The system that translates mRNA into protein is highly conserved, so what’s true for yeast is probably true for people as well,” said Zaher. Washington University in St. Louis.

EKF Diagnostics subsidiary, Selah Genomics, has announced a major, four-way collaboration with Greenville Health System (GHS, South Carolina), DecisionQ Corporation (Virginia), and BD (Becton Dickinson and Company, New Jersey). Expected to last 18 months, the collaboration aims to unite classic clinical annotations with proprietary next generation sequencing (NGS) technology and artificial intelligence-based decision support algorithms in order to improve clinical decision support in the treatment of colon cancer patients. More than eight out of ten US patients are treated in the community, and as many as 60 percent of all patients with solid tumours do not respond to first-line treatment. This results in further treatment cycles, higher cost, elevated toxicity and, perhaps most importantly, lost time. A tool that significantly improves the prognostication for patients by bringing centre of excellence expertise to any clinical setting is therefore highly desirable. Using its PrecisionPath NGS technology, Selah Genomics will first determine the genetic profiles of tumour samples provided by the Institute for Translational Oncology Research, which is part of GHS’s Cancer Institute.  The samples, from colon cancer patients with known outcomes, will be provided with full clinical annotation. DecisionQ will employ its advanced machine-learning platform to integrate genetic profile data with clinical annotations to produce a model that will improve clinical decisions related to the treatment of colon cancer patients. The research project is being funded in part by BD in return for the first opportunity to license the technology should the collaboration be a success. After the initial collaboration, a clinical trial is planned to validate the research and affirm the effectiveness of the new system as a clinical decision support tool for the community-based setting.

www.ekfdiagnostics.com www.selahgenomics.com

Scientists have identified a genetic mutation in about 20 percent of colorectal and endometrial cancers that had been overlooked in recent large, comprehensive gene searches. With this discovery, the altered gene, called RNF43, now ranks as one of the most common mutations in the two cancer types.

The investigators from Dana-Farber Cancer Institute and the Broad Institute of MIT and Harvard said the mutated gene helps control an important cell-signalling pathway, Wnt, that has been implicated in many forms of cancer. They suggest that having a mutation in RNF43 may serve as a biomarker that identifies patients with colorectal and endometrial cancer who could benefit from precision cancer drugs that target the Wnt pathway, although no such drugs have yet been approved.

“Tumours that have this mutation may be telling us that they are dependent on the Wnt signalling pathway, and they will be uniquely sensitive to drugs that inhibit this pathway,” said Charles Fuchs, MD, MPH, an author of the paper and director of the Center for Gastrointestinal Cancer at Dana-Farber. He is also affiliated with Brigham and Women’s Hospital and the Harvard School of Public Health.

In pre-clinical cancer models, tumours with RNF43 mutations have been found to be sensitive to new Wnt pathway inhibitors that are now in clinical trials in humans, according to Marios Giannakis, MD, PhD, who is an attending physician at Dana-Farber and is also a conducting research at the Broad Institute.

The researchers were surprised to find RNF43 mutations in such a significant proportion of colorectal and endometrial cancers because they had not been detected in recent comprehensive searches of tumor DNA conducted by scientists of The Cancer Genome Atlas (TGCA) project.

Authors of the new study believe computer algorithms used by TCGA to parse data from DNA sequencing of tumors may have interpreted the “signal” of the RNF45 mutation as an artifact, and discarded it, much as a legitimate email will sometimes be trapped in a junk filter.

“These mutations occur in repetitive regions of the genome where you often have errors in DNA sequencing, so past algorithms may have been more likely to assume that the RNF43 mutation was an artifact of the sequencing process,” explained Eran Hodis, an MD/PhD student at Harvard Medical School and MIT and also affiliated with the Broad and Dana-Farber. Giannakis and Hodis are co-first authors on the new report.

Other frequently mutated genes in colorectal cancer include APC (75 percent), P53 (50 percent), and KRAS (40 percent).

The new evidence for RNF43 mutations first came from analysis of tumour samples of colorectal cancer that were obtained from two large cohort studies – the Nurses’ Health Study, which has been following 121,000 healthy women since 1976, and the Health Professionals Follow-up Study, which includes 52,000 men enrolled in 1986. About 10 years ago, Fuchs, along with Dana-Farber pathologist Shuji Ogino, MD, PhD, MS, began collecting and studying gastrointestinal tumour samples that had been taken from men and women in the studies who developed cancer. Because these specimens are accompanied by a wealth of data about the patients’ lifestyle, medical history, and other factors, Fuchs calls this collection of tumour samples “a gold mine.”

For the new study, 185 colorectal cancer specimens from this collection were analyzed by whole-exome DNA sequencing at the Broad Institute under the leadership of Levi Garraway, MD, PhD, who is affiliated with Dana-Farber, the Broad, and Brigham and Women’s Hospital, and is corresponding author of the report. The RNF43 mutation was identified in 18.9 percent of the colorectal tumors.

This surprising result prompted the investigators to re-analyse 222 colorectal cancer samples from TCGA project and found the RNF43 mutation in 17.6 percent. The researchers, noting that endometrial cancer is dependent on abnormal Wnt signalling, then re-analysed 248 DNA samples from endometrial cancer that had been previously published by TCGA scientists. They found a strikingly similar proportion – 18.1 percent – of RNF43 mutations in those cancers.

The study authors noted that the discovery of such a significant cancer mutation that hadn’t been picked up in the previous gene hunts shows that carrying out these comprehensive genomic searches continues to have value. Dana-Farber Cancer Institute

After mining the genetic records of thousands of breast cancer patients, researchers from the Johns Hopkins Kimmel Cancer Center have identified a gene whose presence may explain why some breast cancers are resistant to tamoxifen, a widely used hormone treatment generally used after surgery, radiation and other chemotherapy.

The gene, called MACROD2, might also be useful in screening for some aggressive forms of breast cancers, and, someday, offering a new target for therapy, says Ben Ho Park, M.D., Ph.D., an associate professor of oncology in the Kimmel Cancer Center’s Breast Cancer Program and a member of the research team.

The drug tamoxifen is used to treat oestrogen receptor-positive breast cancers. Cells in this type of breast cancer produce protein receptors in their nuclei which bind to and grow in response to the hormone oestrogen. Tamoxifen generally blocks the binding process of the oestrogen-receptor, but some oestrogen receptor-positive cancers are resistant or become resistant to tamoxifen therapy, finding ways to elude its effects. MACROD2 appears to code for a biological path to tamoxifen resistance by diverting the drug from its customary blocking process to a different way of latching onto breast cancer cell receptors, causing cancer cell growth rather than suppression, according to a report by Park and his colleagues.

Specifically, the team’s experiments found that when the gene is overexpressed in breast cancer cells–producing more of its protein product than normal–the cells become resistant to tamoxifen.

One piece of evidence for the gene’s impact was demonstrated when the Johns Hopkins scientists blocked MACROD2’s impact in breast cancer cell cultures by using an RNA molecule that binds to the gene to ‘silence,’ or turn off, the gene’s expression. But the technique only partially restored the cells’ sensitivity to tamoxifen.

To conduct the study, the scientists examined two well-known databases of breast cancer patients’ genetic information, The Cancer Genome Atlas and the Molecular Taxonomy of Breast Cancer International Consortium study. Patients who had MACROD2 overexpressed in primary breast cancers at the original breast cancer site had significantly worse survival rates than those who did not, according to an analysis of the patient databases.

With this in mind, the Johns Hopkins scientists suggest that clinicians may be able to look at MACROD2 activity to help them identify aggressive breast cancers at early stages of growth.

The team’s analysis also found that MACROD2 overexpression was present in the majority of metastases in patients with tamoxifen-resistant tumours and in tumour cells that had spread from their original site in the breast. The latter finding, says Park, suggests that tamoxifen resistance caused by the gene might be a process that develops over time as women take the drug.

Finding a small group of a patient’s cancer cells that overexpress MACROD2, he explained, means those cells are likely to be the ‘survivors’ of early treatment with tamoxifen that go on to multiply and cause metastatic tumours. ‘The resultant cells–or the vast majority of them–are now all overexpressing MACROD2, and are the cells that are aggressive and will cause trouble,’ he adds.

Park and his team cautioned that there may be other genetic factors that control tamoxifen resistance, and that nothing in their study should suggest that tamoxifen use should be avoided. EurekAlert