Researchers silence leading cancer-causing gene

Using bioluminescence, researchers showed that the novel molecule “KRAS silencing RNA” or "KRAS siRNA” (right) reduced the size of a tumor in mice. Researchers used a “non-KRAS silencing” molecule as the control (left) as a comparison. Credit: Image courtesy of University of North Carolina Health Care

Researchers from the UNC School of Medicine and colleagues at The University of Texas MD Anderson Cancer Center have developed a new approach to block the KRAS oncogene, one of the most frequently mutated genes in human cancer. The approach, led by Chad Pecot, MD, an assistant professor of medicine at UNC, offers another route to attack KRAS, which has proven to be an elusive and frustrating target for drug developers.

The new method relies upon a specifically sequenced type of small interfering RNA -- or siRNA. The findings, published in the journal Molecular Cancer Therapeutics, show that using a form of siRNA to halt KRAS not only dramatically stunted the growth of lung and colon cancers in cultured cells and mice but also stopped metastasis -- the main cause of cancer deaths.

"KRAS has been widely regarded as an undruggable protein, but we show that that's simply not the case," said Pecot, the study lead author and member of the UNC Lineberger Comprehensive Cancer Center.

KRAS is a signaling molecule -- a protein switch that triggers a cascade of molecular events that tell cells to grow and survive. Mutations in the KRAS gene create a switch that is perpetually "on," causing cells to divide uncontrollably. KRAS mutations are present in roughly 30 percent of human cancers, particularly lung, colon, pancreatic, and thyroid.

"It is the elephant in the room," Pecot said. "KRAS was one of the first cancer-causing genes ever discovered, and it was the obvious target to go after. People have been trying for decades to hit it, but they haven't had much luck."

Inhibiting KRAS signaling has been tricky because it lacks good pockets or crevices for small molecules and drugs to bind to. Some researchers have tried instead to target the proteins downstream in the KRAS signaling cascade, but those attempts have also had limited success.

Rather than try another conventional approach, Pecot decided to use a new genetic tool known as RNA interference -- or RNAi -- to destroy the KRAS protein before it fully forms. RNAi uses bits of synthetically engineered RNA -- the single-stranded molecule transcribed from DNA -- to silence specific genes. These bits of RNA bind to specific genetic messages called mRNA in the cell and direct enzymes to recognize the messages as enemies. In this context, the enzymes destroyed the genetic messages of KRAS mRNA so that KRAS can't be made. As a result, the cells don't grow, replicate, or move nearly as well.

RNAi has shown great promise in the treatment of liver diseases, viral infections, and cancers. To see if this approach could thwart the KRAS oncogene, Pecot and his colleagues first had to test different sequences of RNA to determine which one most effectively tagged KRAS for destruction. Of five RNA sequences, the researchers identified two candidates worthy to take into cancer models.

When they delivered these sequences into tissue culture cells, they found that the siRNAs destroyed more than 90 percent of the KRAS gene messages, significantly impairing the growth of cancer cell lines. The technique also led to marked reduction of two signaling molecules called pERK and pMEK, which lie downstream of KRAS and have been implicated in cancer cell proliferation and tumor growth.

Next, Pecot and his colleagues tested the siRNAs in mouse models of lung and colon cancer. They wrapped the sequences in protective lipid nanoparticles and delivered the siRNA solution into the mice. The researchers found that this treatment significantly slowed the growth of primary tumors. For example, tumors from colon cancer models that had been treated with the KRAS siRNAs were 69 percent smaller than tumors treated with control RNA sequences.

In addition, the researchers discovered that silencing KRAS stemmed the spread of cancer cells to other organs. The siRNA treatment reduced the number of these secondary malignant growths by about 80 percent in mice with lung cancer and to a similar degree in colon cancer models.

Pecot's findings come on the heels of two other papers using siRNAs to target KRAS, one from Frank McCormick's laboratory at the University of California at San Francisco and the other from Tyler Jacks' laboratory at the Massachusetts Institute of Technology. What sets the UNC study apart is that it demonstrates that this approach can be used to control the development of metastatic disease.

"Having all three papers come out at about the same time is encouraging because it means that KRAS is druggable if you use outside-the-box methods," Pecot said. "Now, we essentially have three platforms for targeting KRAS with siRNAs that may get to the clinic."

Pecot said the results, while promising, are just a first step in combating this cancer-causing gene. Ultimately, the siRNA sequences will have to be designed to specifically target the mutant form of KRAS without disrupting the normal form of the gene, which is necessary for maintaining normal growth in healthy cells.

Source: University of North Carolina Health Care

How to sell the drugs of the future

Drugs
Credit: Getty Images

Only a decade ago, basing medical treatment on your DNA seemed like science fiction. Not any more. Thanks in part to the sequencing of the human genome, personalized medicine (PM), a specific course of treatment developed for the individual patient, is now science fact.

PM has already shown its effectiveness in the treatment of cancer, and medical professionals are eager to expand it to treat other chronic diseases. But first patients need to understand how PM can work for them.

Will they buy into it? "Yes -- but only if patients are armed with knowledge about their own disease and understand the relative advantages of PM," says Concordia University marketing professor Lea Prevel Katsanis, the co-author of a new study on the subject, published in the International Journal of Pharmaceutical and Healthcare Marketing. She adds that if patients are going to accept PM, doctor-patient communication is vital.

For the study, Katsanis and her co-author, Anja Hitz, a former John Molson School of Business MBA student and current head of medical compliance and prevention at the Military Hospital in Hamburg, Germany, polled 307 consumers through an online survey. 
They found that knowledge and the relative advantages of PM have the most significant influence on patient acceptance of PM.

"The more a patient knows about how she is being treated, the more likely she is to accept that treatment," says Katsanis. "So it's important to educate consumers on potential benefits and risks associated with PM."

Indeed, patient understanding is a key factor in getting healthcare professionals, governments and insurance companies to adopt and pay for PM, particularly when these targeted treatments are often more costly than traditional medical methods.

With PM, the same drug isn't given to millions of people. It's a targeted treatment regime. While that reduced patient pool means an increased cost, there can be long-term benefits. Increased efficiency and prevention may result in fewer drugs being prescribed. And PM may also result in the reduction of secondary costs as a result of overdosing, incorrect prescriptions and adverse drug reactions.

"If PM can be successfully integrated into the healthcare system at a reasonable cost, it represent a significant improvement in the treatment of chronic disease," says Katsanis.

But she warns that marketers need to proceed with caution: "The promotion of personalized medications will increasingly focus on the healthy patient with a genetic disposition for a particular illness. While this might lead to new and potentially greater opportunities for marketers, it might also result in the targeting of healthy patients who don't actually need treatment for an active disease. Ultimately, this could increase healthcare costs and cause unnecessary patient treatment."

Source: Concordia University

Software models more detailed evolutionary networks from genetic data

Phylogenetic networks depict the movement of genetic sequences from one species to another as a means of showing where horizontal gene transfer may have taken place. Software by scientists at Rice University aims to reveal far more about species’ evolutionary histories than traditional tree models are able to. Credit: Luay Nakhleh/Rice University

The tree has been an effective model of evolution for 150 years, but a Rice University computer scientist believes it's far too simple to illustrate the breadth of current knowledge.

Rice researcher Luay Nakhleh and his group have developed PhyloNet, an open-source software package that accounts for horizontal as well as vertical inheritance of genetic material among genomes. His "maximum likelihood" method, detailed this month in the Proceedings of the National Academy of Sciences, allows PhyloNet to infer network models that better describe the evolution of certain groups of species than do tree models.

"Inferring" in this case means analyzing genes to determine their evolutionary history with the highest probability -- the maximum likelihood -- of connections between species. Nakhleh and Rice colleague Christopher Jermaine recently won a $1.1 million National Science Foundation grant to analyze evolutionary patterns using Bayesian inference, a statistics-based technique to estimate probabilities based on a data set.

To build networks that account for all of the genetic connections between species, the software infers the probability of variations that phylogenetic trees can't illustrate, such as horizontal gene transfers. These transfers circumvent simple parent-to-offspring evolution and allow genetic variations to move from one species to another by means other than reproduction.

Biologists want to know when and how these transfers happened, but tree structures conceal such information. "When horizontal transfer occurs, as with the hybridization of two species, the tree model becomes inadequate to describe the evolutionary history, and networks that incorporate horizontal gene transfer become the more appropriate model," Nakhleh said.

Nakhleh's Java-based software accounts for incomplete lineage sorting, in which clues to gene evolution that don't match the established lineage of species appear in the genetic record.

"We are the first group to develop a general model that will allow biologists to estimate hybridization while accounting for all these complexities in evolution," Nakhleh said.

Most existing programs for phylogenetics (the study of evolutionary relationships) ignore such complexities. "They end up overestimating the amount of hybridization," Nakhleh said. 

"They start seeing lots of complexities in the data and say, 'Oh, it's complex here; it must be hybridization,' and end up inferring too much. Our method acknowledges that part of the complexity has nothing to do with hybridization; it has to do with other random processes that happened during evolution."

The Rice researchers used two data sets to test the new program. One, a computer-generated set of data that mimics a realistic model of evolution, allowed them to evaluate the accuracy of the program. The second involved multiple genomes of mice found across Europe and Asia. "There have been stories about mice hybridizing," Nakhleh said. "Now that we have the first method to allow for systematic analysis, we ran it on a very large amount of data from five mouse samples and we detected hybridization" -- most notably in the presence of a genetic signal from a mouse in Kazakhstan that found its way to mice in France and Germany, he said.

Nakhleh hopes evolutionary biologists will use PhyloNet to take a fresh look at the massive amount of genomic data collected over the past few decades. "The exciting thing for me about this is that biologists can now systematically go through lots of data they have generated and check to see if there has been hybridization."

Source: Rice University

Body weight heavily influenced by gut microbes: Genes shape body weight by affecting gut microbes

Body sizes (stock image). New research shows that our genetic makeup influences whether we are fat or thin by shaping which types of microbes thrive in our body. Credit: © olly / Fotolia

Our genetic makeup influences whether we are fat or thin by shaping which types of microbes thrive in our body, according to a study by researchers at King's College London and Cornell University.

By studying pairs of twins at King's Department of Twin Research, researchers identified a specific, little known bacterial family that is highly heritable and more common in individuals with low body weight. This microbe also protected against weight gain when transplanted into mice.

The results, published today in the journal Cell, could pave the way for personalised probiotic therapies that are optimised to reduce the risk of obesity-related diseases based on an individual's genetic make-up.

Previous research has linked both genetic variation and the composition of gut microbes to metabolic disease and obesity. Despite these shared effects, the relationship between human genetic variation and the diversity of gut microbes was presumed to be negligible.

In the study, funded by National Institutes of Health (NIH), researchers sequenced the genes of microbes found in more than 1,000 fecal samples from 416 pairs of twins. The abundances of specific types of microbes were found to be more similar in identical twins, who share 100 per cent of their genes, than in non-identical twins, who share on average only half of the genes that vary between people. These findings demonstrate that genes influence the composition of gut microbes.

The type of bacteria whose abundance was most heavily influenced by host genetics was a recently identified family called 'Christensenellaceae'. Members of this health-promoting bacterial family were more abundant in individuals with a low body weight than in obese individuals. Moreover, mice that were treated with this microbe gained less weight than untreated mice, suggesting that increasing the amounts of this microbe may help to prevent or reduce obesity.

Professor Tim Spector, Head of the Department of Twin Research and Genetic Epidemiology at King's College London, said: 'Our findings show that specific groups of microbes living in our gut could be protective against obesity -- and that their abundance is influenced by our genes. The human microbiome represents an exciting new target for dietary changes and treatments aimed at combating obesity.

'Twins have been incredibly valuable in uncovering these links -- but we now want to promote the use of microbiome testing more widely in the UK through the British Gut Project. This is a crowd-sourcing experiment that allows anyone with an interest in their diet and health to have their personal microbes tested genetically using a simple postal kit and a small donation via our website (www.britishgut.org). We want thousands to join up so we can continue to make major discoveries about the links between our gut and our health.'

Ruth Ley, Associate Professor at Cornell University in the United States, said: 'Up until now, variation in the abundances of gut microbes has been explained by diet, the environment, lifestyle, and health. This is the first study to firmly establish that certain types of gut microbes are heritable -- that their variation across a population is in part due to host genotype variation, not just environmental influences. These results will also help us find new predictors of disease and aid prevention.'

Source: King's College London

Ancient human genome from southern Africa throws light on our origins

Professor Vanessa Hayes in the field.

The skeleton of a man who lived 2,330 years ago in the southernmost tip of Africa tells us about ourselves as humans, and throws some light on our earliest common genetic ancestry.

What can DNA from the skeleton of a man who lived 2,330 years ago in the southernmost tip of Africa tell us about ourselves as humans? A great deal when his DNA profile is one of the 'earliest diverged' -- oldest in genetic terms -- found to-date in a region where modern humans are believed to have originated roughly 200,000 years ago.

The man's maternal DNA, or 'mitochondrial DNA', was sequenced to provide clues to early modern human prehistory and evolution. Mitochondrial DNA provided the first evidence that we all come from Africa, and helps us map a figurative genetic tree, all branches deriving from a common 'Mitochondrial Eve'.
When archaeologist Professor Andrew Smith from the University of Cape Town discovered the skeleton at St. Helena Bay in 2010, very close to the site where 117,000 year old human footprints had been found -- dubbed "Eve's footprints" -- he contacted Professor Vanessa Hayes, an expert in African genomes.

At the time, Hayes was Professor of Genomic Medicine at the J. Craig Venter Institute in San Diego, California. She now heads the Laboratory for Human Comparative and Prostate Cancer Genomics at Sydney's Garvan Institute of Medical Research.

The complete 1.5 metre tall skeleton was examined by Professor Alan Morris, from the University of Cape Town. A biological anthropologist, Morris showed that the man was a 'marine forager'. A bony growth in his ear canal, known as 'surfer's ear', suggested that he spent some time diving for food in the cold coastal waters, while shells carbon-dated to the same period, and found near his grave, confirmed his seafood diet. Osteoarthritis and tooth wear placed him in his fifties.

Due to the acidity of the soil within the region, acquiring DNA from skeletons has proven problematic. The Hayes team therefore worked with the world's leading laboratory in ancient DNA research, namely that of paleogeneticist Professor Svante Pääbo at the Max Planck Institute for Evolutionary Anthropolgy in Leipzig, Germany, who successfully sequenced a Neanderthal.

The team generated a complete mitochondrial genome, using DNA extracted from a tooth and a rib. The findings provided genomic evidence that this man, from a lineage now presumed extinct, as well as other indigenous coastal dwellers like him, were the most closely related to 'Mitochondrial Eve'.

The study underlines the significance of southern African archaeological remains in defining human origins, and is published in the journal Genome Biology and Evolution, now online.

"We were thrilled that archaeologist Andrew Smith understood the importance of not touching the skeleton when he found it, and so did not contaminate its DNA with modern human DNA," said Professor Hayes.

"I approached Svante Pääbo because his lab is the best in the world at DNA extraction from ancient bones. This skeleton was very precious and we needed 
to make sure the sample was in safe hands."

"Alan Morris undertook some incredible detective work. He used his skills in forensics and murder cases to assemble a profile of the man behind the St Helena skeleton."

"Alan helped establish that this man was a marine hunter-gatherer -- in contrast to the contemporary inland hunter-gatherers from the Kalahari dessert. We were very curious to know how this man related to them."

"We also know that this man pre-dates migration into the region, which took place around 2,000 years ago when pastoralists made their way down the coast from Angola, bringing herds of sheep. We could demonstrate that our marine hunter-gatherer carried a different maternal lineage to these early migrants -- containing a DNA variant that we have never seen before."

"Because of this, the study gives a baseline against which historic herders at the Cape can now be compared."

While interested in African lineages, and how they interact with each other, Professor Hayes is especially keen for Africa to inform genomic research and medicine worldwide.

"One of the biggest issues at present is that no-one is assembling genomes from scratch -- in other words, when someone is sequenced, their genome is not pieced together as is," she said.

"Instead, sections of the sequenced genome are mapped to a reference genome. Largely biased by European contribution, the current reference is poorly representative of indigenous peoples globally."

"If we want a good reference, we have to go back to our early human origins."
"None of us that walk on this planet now are pure anything -- we are all mixtures. For example 1-4% of Eurasians even carry Neanderthal DNA"

"We need more genomes that don't have extensive admixture. In other words, we need to reduce the noise."

"In this study, I believe we may have found an individual from a lineage that broke off early in modern human evolution and remained geographically isolated. That would contribute significantly to refining the human reference genome."

Source: Garvan Institute of Medical Research

Uncovering one of humankind’s most ancient lineages

A Khoisan hunter/gatherer with his bow and arrows. Credit: Image courtesy of Nanyang Technological University

Scientists at Nanyang Technological University (NTU Singapore) and Penn State University in the United States have successfully discovered one of modern humans' ancient lineages through the sequencing of genes.

A geneticist from NTU, Professor Stephan Christoph Schuster, who led an international research team from Singapore, United States and Brazil, said this is the first time that the history of humankind populations has been analysed and matched to Earth's climatic conditions over the last 200,000 years.
Their breakthrough findings are published today (4 Dec) in Nature Communications.

The team has sequenced the genome of five living individuals from a hunter/gatherer tribe in Southern Africa, and compared them with 420,000 genetic variants across 1,462 genomes from 48 ethnic groups of the global population.

Through advanced computation analysis, the team found that these Southern African Khoisan tribespeople are genetically distinct not only from Europeans and Asians, but also from all other Africans.

The team also found that there are individuals of the Khoisan population whose ancestors did not interbreed with any of the other ethnic groups for the last 150,000 years and that Khoisan was the majority group of living humans for most of that time until about 20,000 years ago.

Their findings mean it is now possible to use genetic sequencing to reveal the ancestral lineage of any ethnic group even up to 200,000 years ago, if non-admixed individuals are found, like in the case of the Khoisan. This will show when in history there have been important genetic changes to an ancestral lineage due to intermarriages or geographical migrations that may have occurred over the centuries.

"Khoisan hunter/gatherers in Southern Africa have always perceived themselves as the oldest people," said Prof Schuster, an NTU scientist at the Singapore Centre on Environmental Life Sciences Engineering (SCELSE) and a former Penn State University professor.

"Our study proves that they truly belong to one of mankind's most ancient lineages, and these high quality genome sequences obtained from the tribesmen will help us better understand human population history, especially the understudied branch of mankind such as the Khoisan.

"The new data gathered will also enable scientists to better understand how the human genome has evolved and hopefully lead to more effective treatment options for certain genetic diseases and illnesses."

Of the five tribesmen who were the oldest members of the Ju/'hoansi tribe and other tribes living in protected areas of northwest Namibia, two individuals were found to have a genome which had not admixed with other ethnic groups.

The Ju/'hoansi tribe was made famous in the 80s and 90s by the box-office hit movie series "The Gods Must Be Crazy." The main character of the series was a hunter/gatherer tribesman, played by Nǃxau, a bushman.

The research paper's first author, Dr Hie Lim Kim, a SCELSE senior research fellow, said "it was very surprising that this group apparently did not intermarry with non-Khoisan neighbours for thousands of years." This is because the Khoisan peoples and the rest of modern humanity shared their most recent common ancestor around 150,000 years ago.

The current Khoisan culture and tradition, where marriage occurs either among Khoisan groups or results in female members leaving their tribes after marrying non-Khoisan men, appears to be long-standing.

"A key finding from this study is that even today after 150,000 years, single non-admixed individuals or descendants of those who did not interbreed with separate populations can be identified within the Ju/'hoansi population, which means there might be more of such unique individuals in other parts of the world," added Dr Kim.

The Khoisan tribespeople participating in this study had parts of their genomes sequenced in an earlier study by the same team in 2010. The new study generated complete genome sequences at high quality, which enabled the analysis of admixture and population history. The availability of such high quality Southern African genomes will allow further investigation of the population history of this largely understudied branch of humankind at high resolution.

This research project involving six investigators was led by NTU and Penn State University. Other institutions participating in the study include the Ohio State University and Sao Paulo State University, Brazil.

Moving forward, Prof Schuster added that they will be looking to find more non-admixed individuals who are in the other parts of the world, such as in South Asia and South America, where uncontacted tribes still exist. The team will also be seeking more funding to further their research which will have large impact on the study of life sciences.

Source: Nanyang Technological University

Poisonous cure: Toxic fungi may hold secrets to tackling deadly diseases

Take two poisonous mushrooms, and call me in the morning, said no doctor ever. Credit: Photo by G.L. Kohuth

Take two poisonous mushrooms, and call me in the morning. While no doctor would ever write this prescription, toxic fungi may hold the secrets to tackling deadly diseases.

A team of Michigan State University scientists has discovered an enzyme that is the key to the lethal potency of poisonous mushrooms. The results, published in the current issue of the journal Chemistry and Biology, reveal the enzyme's ability to create the mushroom's molecules that harbor missile-like proficiency in attacking and annihilating a single vulnerable target in the human liver.

The team revealed how the enzyme contributes to the manufacture of chemical compounds known as cyclic peptides, a favorite type of molecule that pharmaceutical companies use to create new drugs. These findings could lead to single-minded medicines with zero side effects, said Jonathan Walton, professor of plant biology and co-lead author.

"Mushrooms are prolific chemical factories, yet only a few of their peptides are poisonous," he said. "These toxins survive the high temperatures of cooking and the acids of digestion, and yet they're readily absorbed by the bloodstream and go directly to their intended target. These are the exact qualities needed for an effective medicine."

Walton published the paper with fellow MSU scientists Hong Luo, Sung-Yong Hong, R. Michael Sgambelluri and Evan Angelos. Working with the mushroom species Amanita, Walton and his teammates disassembled one of its poisonous peptides, which can be compared to a laser-guided missile with a nuclear warhead.

By removing the molecular equivalent of the deadly warhead, they now have a sturdy, precise delivery system that can supply medicine -- rather than poison -- to a single target. By taking a laser, rather than a shotgun approach, scientists could develop medicines capable of curing disease without the patient suffering any side effects.

The enzyme the team discovered is called POPB, and it converts toxins from their initial linear shape into cyclic peptides, fortress-like molecular circles comprising eight amino acids.

Harnessing the distinct properties of POPB will allow scientist to create billions of variant molecules, which can be tested against many different medical targets such as pathogenic bacteria and cancer.

"We've found some variables that are key," said Walton, an AgBioResearch scientist. "By making more variants, we can add or replace molecules that may or may not work. To date we've created a library of a hundred or so, and we eventually plan to create millions."

The challenge of the next stage of research, though, is testing the variants against diseases such as cancer. The bottleneck lies in the screening process. While Walton's team has discovered a missile capable of carrying a million different potential medicines, as of yet the payload that will be effective remains a mystery.

Source: Michigan State University