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

Gut microbiota influences blood-brain barrier permeability

Uptake of the substance Raclopride in the brain of germ-free versus conventional mice.
Credit: Miklos Toth

A new study in mice, conducted by researchers at Sweden's Karolinska Institutet together with colleagues in Singapore and the United States, shows that our natural gut-residing microbes can influence the integrity of the blood-brain barrier, which protects the brain from harmful substances in the blood. According to the authors, the findings provide experimental evidence that our indigenous microbes contribute to the mechanism that closes the blood-brain barrier before birth. The results also support previous observations that gut microbiota can impact brain development and function.

The blood-brain barrier is a highly selective barrier that prevents unwanted molecules and cells from entering the brain from the bloodstream. In the current study, being published in the journal Science Translational Medicine, the international interdisciplinary research team demonstrates that the transport of molecules across the blood-brain barrier can be modulated by gut microbes -- which therefore play an important role in the protection of the brain.

The investigators reached this conclusion by comparing the integrity and development of the blood-brain barrier between two groups of mice: the first group was raised in an environment where they were exposed to normal bacteria, and the second (called germ-free mice) was kept in a sterile environment without any bacteria.

"We showed that the presence of the maternal gut microbiota during late pregnancy blocked the passage of labeled antibodies from the circulation into the brain parenchyma of the growing fetus," says first author Dr. Viorica Braniste at the Department of Microbiology, Tumor and Cell Biology at Karolinska Institutet. "In contrast, in age-matched fetuses from germ-free mothers, these labeled antibodies easily crossed the blood-brain barrier and was detected within the brain parenchyma."

The team also showed that the increased 'leakiness' of the blood-brain barrier, observed in germ-free mice from early life, was maintained into adulthood. Interestingly, this 'leakiness' could be abrogated if the mice were exposed to fecal transplantation of normal gut microbes. 

The precise molecular mechanisms remain to be identified. However, the team was able to show that so-called tight junction proteins, which are known to be important for the blood-brain barrier permeability, did undergo structural changes and had altered levels of expression in the absence of bacteria.

According to the researchers, the findings provide experimental evidence that alterations of our indigenous microbiota may have far-reaching consequences for the blood-brain barrier function throughout life.

"These findings further underscore the importance of the maternal microbes during early life and that our bacteria are an integrated component of our body physiology," says Professor Sven Pettersson, the principal investigator at the Department of Microbiology, Tumor and Cell Biology. "Given that the microbiome composition and diversity change over time, it is tempting to speculate that the blood-brain barrier integrity also may fluctuate depending on the microbiome. This knowledge may be used to develop new ways for opening the blood-brain-barrier to increase the efficacy of the brain cancer drugs and for the design of treatment regimes that strengthens the integrity of the blood-brain barrier."

Source: Karolinska Institutet

Transplant drug could boost power of brain tumor treatments, study finds

Drs. Maria Castro and Pedro Lowenstein, both of the U-M Department of Neurosurgery, co-led the research. Credit: Image courtesy of University of Michigan Health System

Every day, organ transplant patients around the world take a drug called rapamycin to keep their immune systems from rejecting their new kidneys and hearts. New research suggests that the same drug could help brain tumor patients by boosting the effect of new immune-based therapies.

In experiments in animals, researchers from the University of Michigan Medical School showed that adding rapamycin to an immunotherapy approach strengthened the immune response against brain tumor cells.

What's more, the drug also increased the immune system's "memory" cells so that they could attack the tumor if it ever reared its head again. The mice and rats in the study that received rapamycin lived longer than those that didn't.

Now, the U-M team plans to add rapamycin to clinical gene therapy and immunotherapy trials to improve the treatment of brain tumors. They currently have a trial under way at the U-M Health System which tests a two-part gene therapy approach in patients with brain tumors called gliomas in an effort to get the immune system to attack the tumor. In future clinical trials, adding rapamycin could increase the therapeutic response.

The new findings, published online in the journal Molecular Cancer Therapeutics, show that combining rapamycin with a gene therapy approach enhanced the animals' ability to summon immune cells called CD8+ T cells to kill tumor cells directly. Due to this cytotoxic effect, the tumors shrank and the animals lived longer.

But the addition of rapamycin to immunotherapy even for a short while also allowed the rodents to develop tumor-specific memory CD8+ T cells that remember the specific "signature" of the glioma tumor cells and attacked them swiftly when a tumor was introduced into the brain again.

"We had some indication that rapamycin would enhance the cytotoxic T cell effect, from previous experiments in both animals and humans showing that the drug produced modest effects by itself," says Maria Castro, Ph.D., senior author of the new paper. Past clinical trials of rapamycin in brain tumors have failed.

"But in combination with immunotherapy, it became a dramatic effect, and enhanced the efficacy of memory T cells too. This highlights the versatility of the immunotherapy approach to glioma." Castro is the R.C. Schneider Collegiate Professor of neurosurgery and a professor of cell and developmental biology at U-M.

Rapamycin is an FDA-approved drug that produces few side effects in transplant patients and others who take it to modify their immune response. So in the future, Castro and her colleagues plan to propose new clinical trials that will add rapamycin to immune gene therapy trials like those already ongoing at UMHS.

She notes that other researchers currently studying immunotherapies for glioma and other brain tumors should also consider doing the same. "This could be a universal mechanism for enhancing efficacy of immunotherapies in glioma," she says.

Rapamycin inhibits a specific molecule in cells, called mTOR. As part of the research, Castro and her colleagues determined that brain tumor cells use the mTOR pathway to hamper the immune response of patients.

This allows the tumor to trick the immune system, so it can continue growing without alerting the body's T cells that a foreign entity is present. Inhibiting mTOR with rapamycin, then, uncloaks the cells and makes them vulnerable to attack.

Castro notes that if the drug proves useful in human patients, it could also be used for long-term prevention of recurrence in patients who have had the bulk of their tumor removed. "This tumor always comes back," she says.

Source: University of Michigan Health System

Handheld scanner could make brain tumor removal more complete, reducing recurrence

A handheld device that resembles a laser pointer could someday help surgeons remove all of the cells in a brain tumor. Credit: Moritz Kircher

Cancerous brain tumors are notorious for growing back despite surgical attempts to remove them -- and for leading to a dire prognosis for patients. But scientists are developing a new way to try to root out malignant cells during surgery so fewer or none get left behind to form new tumors. The method, reported in the journal ACS Nano, could someday vastly improve the outlook for patients.

Moritz F. Kircher and colleagues at Memorial Sloan Kettering Cancer Center point out that malignant brain tumors, particularly the kind known as glioblastoma multiforme (GBM), are among the toughest to beat. Although relatively rare, GBM is highly aggressive, and its cells multiply rapidly. Surgical removal is one of the main weapons doctors have to treat brain tumors. The problem is that currently, there's no way to know if they have taken out all of the cancerous cells. And removing extra material "just in case" isn't a good option in the brain, which controls so many critical processes. The techniques surgeons have at their disposal today are not accurate enough to identify all the cells that need to be excised. So Kircher's team decided to develop a new method to fill that gap.

The researchers used a handheld device resembling a laser pointer that can detect "Raman nanoprobes" with very high accuracy. These nanoprobes are injected the day prior to the operation and go specifically to tumor cells, and not to normal brain cells. Using a handheld Raman scanner in a mouse model that mimics human GBM, the researchers successfully identified and removed all malignant cells in the rodents' brains. Also, because the technique involves steps that have already made it to human testing for other purposes, the researchers conclude that it has the potential to move readily into clinical trials. Surgeons might be able to use the device in the future to treat other types of brain cancer, they say.

The authors acknowledge funding from the National Institutes of Health.

Source: American Chemical Society

What makes pancreatic cancer so aggressive? New study sheds light

“We know that patients with the earliest stage of pancreatic cancer have a survival rate of only 30 percent. This suggests that even in that very early stage of invasive cancer there are already cells that have spread to distant parts of the body,” says study author Diane M. Simeone, M.D. Credit: Image courtesy of University of Michigan Health System

New research from the University of Michigan Comprehensive Cancer Center helps explain why pancreatic cancer is so lethal, with fewer than a third of patients surviving even early stage disease.

The researchers found a gene known to be involved in nearly 90 percent of pancreatic cancers promotes cancer growth and spread. The gene, ATDC, plays a key role in how a tumor progresses from a preinvasive state to an invasive cancer to metastatic cancer.

"We know that patients with the earliest stage of pancreatic cancer have a survival rate of only 30 percent. This suggests that even in that very early stage of invasive cancer there are already cells that have spread to distant parts of the body," says study author Diane M. Simeone, M.D., director of the Pancreatic Cancer Center at the University of Michigan Comprehensive Cancer Center.

"This study sheds important light on what it is about pancreatic cancer that makes it so aggressive early in the game," she adds. The study appears Jan. 15 in Genes and Development.

Researchers used a mouse model to replicate pancreatic cancer as it appears in humans. They also studied pancreatic cancer tissue samples and samples of pre-invasive pancreatic lesions. They found ATDC was expressed in a subset of the pre-invasive cells and played a role in the development of pancreatic cancer stem cells, the small number of cells in a tumor that fuel its growth and spread. This suggests that ATDC promotes a tumor's invasiveness and spread early in the course of disease.

The researchers suspect that ATDC may be a potent drug target. No drugs currently exist to target this pathway in part because researchers do not understand the crystal structure of the protein. Simeone's team, working with the University of Michigan Center for Structural Biology has made crystals of the protein and begun to create a three-dimensional structure that they can use as a model for drug development.

Preliminary data suggests that ATDC may also play a role in other cancer types, including bladder, ovarian, colorectal and lung cancers and multiple myeloma. But, Simeone notes, it's particularly critical to find new treatment options for pancreatic cancer. About 46,400 Americans will be diagnosed with pancreatic cancer this year, and more than 39,000 will die of the disease. Pancreatic cancer is expected to become the second-leading cause of cancer death in the United States by 2030.

Source: University of Michigan Health System

3-D culture system for pancreatic cancer has potential to change therapeutic approaches

A team of researchers has developed a method to grow pancreatic tissue in a three-dimensional culture system, called organoids. The scientists are able to use tissue not only from laboratory mouse models, but also from human patients. The technology promises to change the way pancreatic cancer research is done, offering a path to personalized treatment approaches in the future. Credit: D. Tuveson/ Cold Spring Harbor Laboratory

Cold Spring Harbor and Bethpage, N.Y. -- Pancreatic cancer is one of the most deadly forms of cancer, with only 6 percent of patients surviving five years after diagnosis. Today, Cold Spring Harbor Laboratory (CSHL) and The Lustgarten Foundation jointly announce the development of a new model system to grow both normal and cancerous pancreatic cells in the laboratory. Their work offers the potential to change the way pancreatic cancer research is done, allowing scientists to interrogate the pathways driving this devastating disease while searching for new drug targets.

In work published in Cell, the research team describes a three-dimensional "organoid" culture system for pancreatic cancer. Co-led by David Tuveson, CSHL Professor and Director of Research for The Lustgarten Foundation, and Hans Clevers, Professor and Director of the Hubrecht Institute and President of the Royal Netherlands Academy of Arts and Sciences, the team developed a method to grow pancreatic tissue not only from laboratory mouse models, but also from human patient tissue, offering a path to personalized treatment approaches in the future.

All cancer research relies on a steady supply of cells -- both normal and cancerous -- that can be grown in the laboratory. By comparing normal cells to cancer cells, scientists can then identify the changes that lead to disease. However, both types of pancreatic cells have been extremely difficult to culture in the laboratory.

Furthermore, the normal ductal cells that are able to develop into pancreatic cancer represent about 10 percent of the cells in the pancreas, complicating efforts to pinpoint the changes that occur as the tumor develops. Until now, scientists have been entirely unable to culture human normal ductal pancreatic cells under standard laboratory conditions. 

Because of these limitations, most pancreatic cancer research relies on genetically engineered mouse models of the disease, which can take up to one year to generate. "With this development, we are now able to culture both mouse and human organoids, providing a very powerful tool in our fight against pancreatic cancer," explains Tuveson.

The organoids are entirely made up of ductal cells, eliminating the surrounding cell types that often contaminate samples from the pancreas. They grow as hollow spheres within a complex gel-like substance filled with growth-inducing factors and connecting fibers. Once they have grown to a sufficient size, the organoids can be transplanted back into mice, where they fully recapitulate pancreatic cancer. "We now have a model for each stage in the progression of the disease," says Chang-Il Hwang, Ph.D., one of the lead authors working in The Lustgarten Foundation's Pancreatic Cancer Research Lab at CSHL directed by Dr. Tuveson.

Traditionally, cancer cells are isolated during surgery or autopsies. Unfortunately, approximately 85 percent of cancer patients are ineligible for surgery at the time of diagnosis, either because the tumor is entwined in critical vasculature or the disease has progressed too far. Researchers therefore have had limited access to patient samples. The new research provides a way for scientists to grow organoids from biopsy material, which is comparatively easy to obtain. "Biopsies are the standard for diagnosis," says Dannielle Engle, Ph.D., also a lead author on the paper. "We can now rapidly generate organoids from any patient, which offers us the potential to study the disease in a much wider population."

The team is now working to create a repository of pancreatic tumor samples, coordinating with the National Cancer Institute. "We hope to make this available to the entire pancreatic cancer research community," says Tuveson. Additionally, Lindsey Baker, Ph.D., another lead author of the paper, has started holding an "organoid school" for other researchers, and has already taught six laboratories from around the world this technique.

Source: Cold Spring Harbor Laboratory

First steps in formation of pancreatic cancer identified

Shown is a region of a pancreas with preneoplastic lesions. Red labeling indicates macrophages, green labeling indicates pancreatic acinar cells that dedifferentiate, and grey labeling indicates further progressed pancreatic lesions. Credit: Image courtesy of Mayo Clinic

Researchers at Mayo Clinic's campus in Jacksonville say they have identified first steps in the origin of pancreatic cancer and that their findings suggest preventive strategies to explore.

In an online issue of Cancer Discovery, the scientists described the molecular steps necessary for acinar cells in the pancreas -- the cells that release digestive enzymes -- to become precancerous lesions. Some of these lesions can then morph into cancer.

"Pancreatic cancer develops from these lesions, so if we understand how these lesions come about, we may be able to stop the cancer train altogether," says the study's lead investigator, Peter Storz, Ph.D., a cancer biologist.

The need for new treatment and prevention strategies is pressing, Dr. Storz says. Pancreatic cancer is one of the most aggressive human cancers -- symptoms do not occur until the cancer is well advanced. One-year survival after diagnosis is only 20 percent. It is the fourth leading cause of cancer death in this country.

The scientists studied pancreatic cells with Kras genetic mutations. Kras produces a protein 

that regulates cell division, and the gene is often mutated in many cancers. More than 95 percent of pancreatic cancer cases have a Kras mutation.

The researchers detailed the steps that led acinar cells with Kras mutations to transform into duct-like cells with stem cell-like properties. Stem cells, which can divide at will, are also often implicated in cancer.

They found that Kras proteins in the acinar cells induce the expression of a molecule, ICAM-1, which attracts macrophages, a specific kind of immune cells. These inflammatory macrophages release a variety of proteins, including some that loosen the structure of the cells, allowing acinar cells to morph into different types of cells. These steps produced the precancerous pancreatic lesions.

"We show a direct link between Kras mutations and the inflammatory environment that drive the initiation of pancreatic cancer," Dr. Storz says.

But the process can be halted in laboratory mice, he adds. "We could do this two ways -- by depleting the macrophages or by treating the transformed cells with a blocking antibody that shuts down ICAM-1," says Dr. Storz. "Doing either one reduced the number of precancerous lesions."

Dr. Storz noted that a neutralizing antibody that blocks ICAM-1has already been developed. It is being tested for a wide variety of disorders, including stroke and rheumatoid arthritis.

"Understanding the crosstalk between acinar cells with Kras mutations and the microenvironment of those cells is key to developing targeted strategies to prevent and treat this cancer," he says.

Source: Mayo Clinic

Powerful new system for classifying tumors revealed

This diagram illustrates how tumors with different tissues of origin were reclassified on the basis of molecular analyses. Credit: Zhong Chen, NIH/NIDCD

Cancers are classified primarily on the basis of where in the body the disease originates, as in lung cancer or breast cancer. According to a new study, however, one in ten cancer patients would be classified differently using a new classification system based on molecular subtypes instead of the current tissue-of-origin system. This reclassification could lead to different therapeutic options for those patients, scientists reported in a paper published August 7 in Cell.

"It's only ten percent that were classified differently, but it matters a lot if you're one of those patients," said senior author Josh Stuart, a professor of biomolecular engineering at UC Santa Cruz.

Stuart helped organize the study as part of the Pan-Cancer Initiative of the Cancer Genome Atlas (TCGA) project. A large team of researchers from multiple institutions performed a comprehensive analysis of molecular data from thousands of patients representing 12 different types of cancer. This was the most comprehensive and diverse collection of tumors ever analyzed by systematic genomic methods. Each tumor type was characterized using six different "platforms" or methods of molecular analysis--mostly genomic platforms such as DNA and RNA sequencing, plus a protein expression analysis.

The research team used statistical analyses of the molecular data to divide the tumors into groups or "clusters," first analyzing the data from each platform separately and then combining them in an integrated cross-platform analysis developed by co-first author Katherine Hoadley of the University of North Carolina. All six platforms as well as the integrated analysis converged on the same divisions of the cancers into 11 major subtypes. 

Five of those subtypes were nearly identical to their tissue-of-origin counterparts. But some tissue-of-origin categories split into several different molecular subtypes, and some subtypes encompass tumors with several different tissues of origin.

Bladder cancer was a particularly interesting group, because it split into seven different clusters, with most samples falling into one of three subtypes. One subtype was bladder cancer only, but some bladder cancers clustered with lung adenocarcinomas, and others with a subtype called 'squamous-like' that includes some lung cancers, some head-and-neck cancers, and some bladder cancers.

"If you look at survival rates, the bladder cancers that clustered with other tumor types had a worse prognosis. So this is not just an academic exercise," Stuart said.

Other findings from the study reconfirmed cancer subtypes that were already recognized, such as the different subtypes of breast cancer based on well-characterized biomarkers. The findings provide a more refined, quantitative picture of the differences between breast cancer subtypes, Stuart said. For example, the results reinforce the idea that 'basal-like' breast cancers are a unique tumor type. "Basal-like breast cancers are as different from luminal breast cancers as they are from lung cancers," he said.

The fact that all six platforms for molecular analysis identified the same set of subtypes, both individually and in multi-platform analyses, is an important result, Stuart noted. Not only does it give the researchers confidence in the subtypes they identified, it also means that different kinds of data can be used to classify a tumor.

"We can now say what the telltale signatures of the subtypes are, so you can classify a patient's tumor just based on the gene expression data, or just based on mutation data, if that's what you have," Stuart said. "Having a molecular map like this could help get a patient into the right clinical trial."

Although follow-up studies are needed to validate the findings, this new analysis lays the groundwork for classifying tumors into molecularly defined subtypes. The new classification scheme could be used to enroll patients in clinical trials and could lead to different treatment options based on molecular subtypes.

According to Stuart, the percentage of tumors that are reclassified based on molecular signatures is likely to grow as more samples and tumor types are included in the analysis (the next major Pan-Cancer analysis will include 21 tumor types). Coauthor Christopher Benz, an oncologist at the Buck Institute for Research on Aging and UC San Francisco, noted that the 10 percent reclassification rate in the current study is likely an underestimate due to the unequal representation of different tumors. "If our study had included as many bladder cancers as breast cancers, for example, we would have reclassified 30 percent," Benz said.

The researchers reported that each molecular subtype may reflect tumors arising from distinct cell types. For example, the data showed a marked difference between cancers of epithelial and non-epithelial origins. "We think the subtypes reflect primarily the cell of origin. Another factor is the nature of the genomic lesion, and third is the microenvironment of the cell and how surrounding cells influence it," Stuart said. "We are disentangling the signals from these different factors so we can gauge each one for its prognostic power."

The study involved an enormous amount of molecular and clinical data, which was managed by data coordinator Kyle Ellrott, a software developer in Stuart's lab at UC Santa Cruz. The data sets and results have been made available to other researchers through the Synapse web site (http://www.synapse.org). Stuart worked with the bioinformatics company Sage Bionetworks to create Synapse as a data repository for the Pan-Cancer Initiative.

"It's a huge amount of information, and all the data is available as programmable data sets that other researchers can use to do further analysis," Stuart said. "The scale of this project is hard to imagine. All of the data that the TCGA project has been churning out got funneled into this paper, and it's giving us an unbiased look at what the data have to tell us about cancer."

Source: University of California - Santa Cruz

New approach aims to silence cancer 'survival genes'

Silencing the SIRT1 gene: Cancer cells before and after treatment in vitro. Non-cancerous cells (not shown) are unaffected. Credit: Image courtesy of University of York

Scientists at the University of York are working on a promising new approach for tackling colorectal cancer, the second most common cause of cancer-related death.

The new method works by silencing cancer 'survival genes' and could potentially combat cancer through the selective killing of colorectal cancer cells without adverse effects on normal, non-cancer cells.

Funded by York's Centre for Chronic Diseases and Disorders (C2D2), the project led by Professor Jo Milner from York's Department of Biology involved preliminary studies to establish the suitability of an ex vivo model for the future development of anti-cancer therapies for colorectal cancer using a technique called RNA interference.

The new approach builds on ground-breaking research by Professor Milner and her team at York more than a decade ago. This early work, funded by Yorkshire Cancer Research (YCR), used the newly-developed technique of RNA interference to successfully kill human cervical cancer cells grown in culture without causing damage to healthy cells.

Professor Milner explained: "When a mammalian cell elects to die it does so with great precision and without harming its neighbours. This process of 'programmed cell death' enables the continuous replacement of aging cells and also the sculpting of tissues and neuronal pathways.

"However, when this normal process of programmed cell death fails the continued abnormal growth of affected cells can lead to cancer. Some cancers develop following infection with a virus, such as human papilloma virus which causes human cervical cancer. Here the virus expresses specific viral genes that disrupt normal cellular control mechanisms resulting in abnormal cell proliferation and survival.

"Using RNA interference (RNAi) we first identified the viral gene responsible for the continued survival of cervical cancer cells. Then we established the feasibility of RNAi-based therapeutics for the selective killing of human cervical cancer cells growing in vitro."

Professor Milner and her team next studied cells from other cancer types, including colorectal cancer and breast cancer. Such cancers develop when the cell's internal control system fails due to damage to one or more of the regulatory genes.

Professor Milner said "We discovered that other genes, belonging to a group called stress-response genes, acquire a new pro-survival function during the process of cancerous transformation. Importantly, this acquired cancer-specific survival function operates under normal, physiological conditions. Silencing these cancer-specific survival genes by RNA interference causes the cancer cells to die while the survival of non-cancerous cells appears normal. This is in contrast to treating cancer by radiotherapy and/or genotoxic drugs -- these agents cause genotoxic stress and damage both cancer and normal cells and tissues in the body, resulting in unwanted adverse side effects for the patient."

For the work on colorectal cancer therapies to progress towards the clinic, the team has had to meet the challenge of modifying the agent siRNA. siRNA is the synthetic RNA molecule which is designed to silence a chosen gene by inducing RNA interference and selectively suppressing expression of that gene. However, siRNA is very unstable and is rapidly degraded when in contact with human tissues.

As reported in the journal Molecular Therapy, the team has now successfully met this challenge and converted the unstable siRNA molecule into a stable form without losing its ability and very high efficacy for targeted gene silencing. A novel siRNA/DNA has been shown to be resistant to degradation while retaining high efficacy and selectivity for target gene silencing when tested on human cancer cells grown in culture.

The next step will involve testing this novel therapeutic agent for cancer-specific cell killing using human tissue maintained ex vivo, using an experimental model which was validated in the course of the C2D2-funded research.

Professor Paul Kaye, Director of C2D2, said: "Professor Milner's team has now shown that ex vivo cultures of colorectal tumour material, derived from human patients, maintain cancer-related biochemistry over several days, and of sufficient time known to produce a killing effect with the novel siRNA/DNA in vitro. It is marvelous that C2D2 has been able to support this ground breaking research that has validated an ex vivo model that can be used to progress this novel therapeutic towards the clinic, and without the need for animal research."

Source: University of York

Shape of things to come in platelet mimicry

By mimicking the shape, size, flexibility and surface chemistry of real platelets, artificial platelets are pushed out of the main blood flow to vessel walls. There, the surface chemistry enables them to anchor on damaged cells and induce faster clotting at the site. Credit: Anirban Sen Gupta

Artificial platelet mimics developed by a research team from Case Western Reserve University and University of California, Santa Barbara, are able to halt bleeding in mouse models 65 percent faster than nature can on its own.

For the first time, the researchers have been able to integratively mimic the shape, size, flexibility and surface chemistry of real blood platelets on albumin-based particle platforms. The researchers believe these four design factors together are important in inducing clots to form faster selectively at vascular injury sites while preventing harmful clots from forming indiscriminately elsewhere in the body.

The new technology, reported in the journal ACS Nano, is aimed at stemming bleeding in patients suffering from traumatic injury, undergoing surgeries or suffering clotting disorders from platelet defects or a lack of platelets. Further, the technology may be used to deliver drugs to target sites in patients suffering atherosclerosis, thrombosis or other platelet-involved pathologic conditions.

Anirban Sen Gupta, an associate professor of biomedical engineering at Case Western Reserve, previously designed peptide-based surface chemistries that mimic the clot-relevant activities of real platelets. Building on this work, Sen Gupta now focuses on incorporating morphological and mechanical cues that are naturally present in platelets to further refine the design.

"Morphological and mechanical factors influence the margination of natural platelets to the blood vessel wall, and only when they are near the wall can the critical clot-promoting chemical interactions take place," he said.

These natural cues motivated Sen Gupta to team up with Samir Mitragotri, a professor of chemical engineering at UC Santa Barbara, whose laboratory has recently developed albumin-based technologies to make particles that mimic the geometry and mechanical properties of red blood cells and platelets.

Together, the team has developed artificial platelet-like nanoparticles (PLNs) that combine morphological, mechanical and surface chemical properties of natural platelets.

The researchers believe this refined design will be able to simulate natural platelet's ability to collide effectively with larger and softer red blood cells in systemic blood flow. The collisions cause margination -- pushing the platelets out of the main flow and closer to the blood vessel wall -- increasing the probability of interacting with an injury site.

The surface coatings enable the artificial platelets to anchor to injury-site-specific proteins, von Willebrand Factor and collagen, while inducing the natural and artificial platelets to aggregate faster at the injury site.

Testing in mouse models showed that intravenous injection of these artificial platelets formed clots at the site of injury three times faster than natural platelets alone in control mice.

The ability to interact selectively with injury site proteins, as well as the ability to remain mechanically flexible like natural platelets, enables these artificial platelets to safely ride through the smallest of blood vessels without causing unwanted clots.

Albumin, a protein found in blood serum and eggs, is already used with cancer drugs and considered a safe material. Artificial platelets that don't become involved in a clot and continue to circulate are metabolized within one to two days.

The researchers believe the new artificial platelet design may be even more effective in larger volume blood flows where margination to the blood vessel wall is more prominent. They expect to soon begin testing those capabilities.

This research was previously funded by American Heart Association and is currently funded by National Institutes of Health.

In addition to stemming bleeding, Sen Gupta believes the technology could also be useful in delivering clot-busting medicines directly to clots, to treat heart attack or stroke without having to systemically suspend the body's coagulation mechanism. The artificial platelets may also be used to deliver cancer medicines to metastatic tumors that have high platelet interactions. Sen Gupta is seeking grants to pursue that work.

Source: Case Western Reserve University

'Microlesions' in epilepsy discovered by novel technique

Clusters of differentially expressed genes predict cellular abnormalities. Credit: Jeffrey Loeb

Using an innovative technique combining genetic analysis and mathematical modeling with some basic sleuthing, researchers have identified previously undescribed microlesions in brain tissue from epileptic patients. The millimeter-sized abnormalities may explain why areas of the brain that appear normal can produce severe seizures in many children and adults with epilepsy.

The findings, by researchers at the University of Illinois at Chicago College of Medicine, Wayne State University and Montana State University, are reported in the journal Brain.

Epilepsy affects about 1 percent of people worldwide. Its hallmark is unpredictable seizures that occur when groups of neurons in the brain abnormally fire in unison. Sometimes epilepsy can be traced back to visible abnormalities in the brain where seizures start, but in many cases, there are no clear abnormalities or scaring that would account for the epileptic activity.

"Understanding what is wrong in human brain tissues that produce seizures is critical for the development of new treatments because roughly one third of patients with epilepsy don't respond to our currently available medications," said Dr. Jeffrey Loeb, professor and head of neurology and rehabilitation in the UIC College of Medicine and corresponding author on the study. "Knowing these microlesions exist is as huge step forward in our understanding of human epilepsy and present new targets for treating this disease."

Loeb and colleagues searched for cellular changes associated with epilepsy by analyzing thousands genes in tissues from 15 patients who underwent surgery to treat their epilepsy. They used a mathematical modeling technique called cluster analysis to sort through huge amounts of genetic data.

Using the model, they were able to predict and then confirm the presence of tiny regions of cellular abnormalities -- the microlesions -- in human brain tissue with high levels of epileptic electrical activity, or 'high-spiking' areas where seizures begin.

"Using cluster analysis is like using a metal detector to find a needle in a haystack," said Loeb. The model, he said, revealed 11 gene clusters that "jumped right out at us" and were either up-regulated or down-regulated in tissue with high levels of epileptic electrical activity compared to tissue with less epileptic activity from the same patient.

When they matched the genes to the types of cells they came from, the results predicted that there would be reductions of certain types of neurons and increases in blood vessels and inflammatory cells in brain tissue with high epileptic activity.

When Fabien Dachet, an expert in bioinformatics research at UIC and first author of the study, went back to the tissue samples and stained for these cells, he found that all of the prediction were correct- there was a marked increase in blood vessels, inflammatory cells, and there were focal microlesions made up of neurons that had lost most of their normal connections that allow them to communicate with one another. "We think that these newly found microlesions lead to spontaneous, abnormal electrical currents in the brain that lead to epileptic seizures," said Loeb.

Loeb and his colleagues at UIC are using the same approach to look for the clusters of differentially expressed genes associated with ALS, a neurodegenerative disease, and in brain tumors. "We now have a way to predict cellular changes by simply measuring the genetic composition, with some fairly simple calculations, between more- and less-affected epileptic human tissues," explained Loeb.

"This technique gives us the ability to discover previously unknown cellular abnormalities in almost any disease where we have access to human tissues," Loeb said. He is currently developing at UIC a national 'neurorepository' of electrically mapped and genetically analyzed brain tissue for such studies.

Source: University of Illinois at Chicago

Slow to mature, quick to distract: ADHD brain study finds slower development of key connections

By examining hundreds of fMRI brain scans of children with ADHD and those without, the researchers identified key connections between brain networks that matured more slowly in ADHD brains.
Credit: Sripada lab, University of Michigan

A peek inside the brains of more than 750 children and teens reveals a key difference in brain architecture between those with attention deficit hyperactivity disorder and those without.

Kids and teens with ADHD, a new study finds, lag behind others of the same age in how quickly their brains form connections within, and between, key brain networks.

The result: less-mature connections between a brain network that controls internally-directed thought (such as daydreaming) and networks that allow a person to focus on externally-directed tasks. That lag in connection development may help explain why people with ADHD get easily distracted or struggle to stay focused.

What's more, the new findings, and the methods used to make them, may one day allow doctors to use brain scans to diagnose ADHD -- and track how well someone responds to treatment. This kind of neuroimaging "biomarker" doesn't yet exist for ADHD, or any psychiatric condition for that matter.

The new findings come from a team in the University of Michigan Medical School's Department of Psychiatry. They used highly advanced computing techniques to analyze a large pool of detailed brain scans that were publicly shared for scientists to study. Their results are published in the Proceedings of the National Academy of Sciences.

Lead author Chandra Sripada, M.D., Ph.D., and colleagues looked at the brain scans of 275 kids and teens with ADHD, and 481others without it, using "connectomic" methods that can map interconnectivity between networks in the brain.

The scans, made using function magnetic resonance imaging (fMRI) scanners, show brain activity during a resting state. This allows researchers to see how a number of different brain networks, each specialized for certain types of functions, were "talking" within and amongst themselves.

The researchers found lags in development of connection within the internally-focused network, called the default mode network or DMN, and in development of connections between DMN and two networks that process externally-focused tasks, often called task-positive networks, or TPNs. They could even see that the lags in connection development with the two task-related networks -- the frontoparietal and ventral attention networks -- were located primarily in two specific areas of the brain.

The new findings mesh well with what other researchers have found by examining the physical structure of the brains of people with and without ADHD in other ways.
Such research has already shown alterations in regions within DMN and TPNs. So, the new findings build on that understanding and add to it.

The findings are also relevant to thinking about the longitudinal course of ADHD from childhood to adulthood. For instance, some children and teens "grow out" of the disorder, while for others the disorder persists throughout adulthood. Future studies of brain network maturation in ADHD could shed light into the neural basis for this difference.

"We and others are interested in understanding the neural mechanisms of ADHD in hopes that we can contribute to better diagnosis and treatment," says Sripada, an assistant professor and psychiatrist who holds a joint appointment in the U-M Philosophy department and is a member of the U-M Center for Computational Medicine and Bioinformatics. "But without the database of fMRI images, and the spirit of collaboration that allowed them to be compiled and shared, we would never have reached this point."

Sripada explains that in the last decade, functional medical imaging has revealed that the human brain is functionally organized into large-scale connectivity networks. These networks, and the connections between them, mature throughout early childhood all the way to young adulthood. "It is particularly noteworthy that the networks we found to have lagging maturation in ADHD are linked to the very behaviors that are the symptoms of ADHD," he says.

Studying the vast array of connections in the brain, a field called connectomics, requires scientists to be able to parse through not just the one-to-one communications between two specific brain regions, but the patterns of communication among thousands of nodes within the brain. This requires major computing power and access to massive amounts of data -- which makes the open sharing of fMRI images so important.

"The results of this study set the stage for the next phase of this research, which is to examine individual components of the networks that have the maturational lag," he says. 

"This study provides a coarse-grained understanding, and now we want to examine this phenomenon in a more fine-grained way that might lead us to a true biological marker, or neuromarker, for ADHD."

Sripada also notes that connectomics could be used to examine other disorders with roots in brain connectivity -- including autism, which some evidence has suggested stems from over-maturation of some brain networks, and schizophrenia, which may arise from abnormal connections. Pooling more fMRI data from people with these conditions, and depression, anxiety, bipolar disorder and more could boost connectomics studies in those fields.
Volunteers needed for research:

To develop such a neuromarker, Sripada has embarked on follow-up research. One study is enrolling children between the ages of 7 and 17 who have ADHD and a comparison group of those without it; information is at http://umhealth.me/adhdchild. Another study is enrolling adults between the ages of 18 and 35 who have ADHD and a comparison group of those without it; information is at http://umhealth.me/adhdadult. Of note, fMRI scans do not expose a person to radiation. Anyone interested in these studies can email Psych-study@med.umich.edu or call (734) 232-0353; for the study of children, parents should make the contact and consent to research on behalf of their children.

Source: University of Michigan Health System

New way to diagnose brain damage from concussions, strokes, and dementia

A new tool to assess cerebrovascular health: Coherent Hemodynamics Spectroscopy (CHS).
Credit: Tufts University Professor of Biomedical Engineering Sergio Fantini

New optical diagnostic technology developed at Tufts University School of Engineering promises new ways to identify and monitor brain damage resulting from traumatic injury, stroke or vascular dementia -- in real time and without invasive procedures.

Coherent hemodynamics spectroscopy (CHS), developed and published by Tufts Professor of Biomedical Engineering Sergio Fantini, measures blood flow, blood volume, and oxygen consumption in the brain. It uses non-invasive near infrared (NIR) light technology to scan brain tissue, and then applies mathematical algorithms to interpret that information.

"CHS is based on measurements of brain hemodynamics that are interpreted according to unique algorithms that generate measures of cerebral blood flow, blood volume and oxygen consumption," says Fantini. "This technique can be used not only to assess brain diseases but also to study the blood flow and how it is regulated in the healthy brain."

Tufts has licensed CHS on a non-exclusive basis to ISS, a Champaign, Ill.-based company that specializes in technology to measure hemoglobin concentration and oxygenation in brain and muscle tissue.

"Potentially the market for CHS is large as it encompasses several applications from the monitoring of cerebrovascular disorders to assessing neurological disorders," says Beniamino Barbieri, president of ISS. "It reminds me of the introduction of ultrasound technology at beginning of the seventies; nobody back then knew how to utilize the new technology and of course, nowadays, its applications are ubiquitous in any medical center."

How It Works

CHS uses laser diodes which emit NIR light that is delivered to the scalp by fiber optics. Light waves are absorbed by the blood vessels in the brain. Remaining light is reflected back to sensors, resulting in optical signals that oscillate with time as a result of the heartbeat, respiration, or other sources of variations in the blood pressure.

By analyzing the light signals with algorithms developed for this purpose, Fantini's model is able to evaluate blood flow and the way the brain regulates it--which is one marker for brain health.

CHS technology has been tested among patients undergoing hemodialysis at Tufts Medical Center. Published research reported a lower cerebral blood flow in dialysis patients compared with healthy patients.

"Non-invasive ways to measure local changes in cerebral blood flow, particularly during periods of stress such as hemodialysis, surgeries, and in the setting of stroke, could have major implications for maintaining healthy brain function," says Daniel Weiner, M.D., a nephrologist at Tufts Medical Center (Tufts MC) and associate professor of medicine at Tufts University School of Medicine (TUSM), who is a member of the research team.

Josh Kornbluth, M.D., a neurologist at Tufts MC and associate professor of medicine at TUSM, is also working with Fantini to explore CHS's potential to assess the cerebrovascular state of patients who suffer traumatic brain injury or stroke. They hope to test CHS further among neurological critical care patients.

"Having data about local cerebral blood flow and whether it is properly regulated can allow us to more accurately develop individualized therapy and interventions instead of choosing a 'one size fits all' approach to traumatic brain injury, stroke, or subarachnoid hemorrhage," Kornbluth says.

Source: Tufts University

Football players found to have brain damage from mild 'unreported' concussions

The images from the Ben-Gurion University of the Negev JAMA Neurology study represent Blood-Brain Barrier (BBB) Permeability in Football Players (A) vs. a control group (B). The players in the pathological-BBB group (B) presented focal BBB lesions in different cortical regions including the temporal (player 4), frontal (player 5), and parietal (player 6) lobes. Both gray and white matter were involved. Credit: Image courtesy of American Associates, Ben-Gurion University of the Negev

A new, enhanced MRI diagnostic approach was, for the first time, able to identify significant damage to the blood-brain barrier (BBB) of professional football players following "unreported" trauma or mild concussions. Published in the current issue of JAMA Neurology, this study could improve decision making on when an athlete should "return to play."

According to Prof. Alon Friedman, from the Ben-Gurion University Brain Imaging Research Center and discoverer of the new diagnostic, "until now, there wasn't a diagnostic capability to identify mild brain injury early after the trauma. In the NFL, other professional sports and especially school sports, concern has grown about the long-term neuropsychiatric consequences of repeated mild Traumatic Brain Injury (mTBI) and specifically sports-related concussive and sub-concussive head impacts."

The paper, published by researchers at Ben-Gurion University of the Negev (BGU) and Soroka University Medical Center, describes a new diagnostic approach using Magnetic Resonance Imaging (MRI) for detection and localization of vascular pathology and blood-brain barrier breakdown in football players.

The images from the Ben-Gurion University of the Negev JAMA Neurology study represent Blood-Brain Barrier (BBB) Permeability in Football Players (A) vs. a control group (B). The players in the pathological-BBB group (B) presented focal BBB lesions in different cortical regions including the temporal (player 4), frontal (player 5), and parietal (player 6) lobes. Both gray and white matter were involved.

"The goal of our study was to use our new method to visualize the extent and location of BBB dysfunction in football players using Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI) on a Phillips 3-T Ingenia. Specifically, it generates more detailed brain maps showing brain regions with abnormal vasculature, or a 'leaky BBB.' "

Study participants included 16 football players from Israel's professional football team, Black Swarm, as well as 13 track and field athletes from Ben-Gurion University who served as controls. All underwent the newly developed MRI-based diagnostic.

The DCE-MRIs were given between games during the season and revealed significant damage.

Forty percent of the examined football players with unreported concussions had evidence of "leaky BBB" compared to 8.3 percent of the control athletes.

"The group of 29 volunteers was clearly differentiated into an intact-BBB group and a pathological-BBB group," Friedman explains. "This showed a clear association between football and increased risk for BBB pathology that we couldn't see before. In addition, high-BBB permeability was found in six players and in only one athlete from the control group."

Friedman also explains that not all the players showed pathology. This indicates that repeated, mild concussive events might impact some players differently than others. This level of diagnosis of individual players can provide the basis of more rational decision making on "return to play" for professionals as well amateurs of any age.

"Generally, players return to the game long before the brain's physical healing is complete, which could exacerbate the possibility of brain damage later in life," says Friedman.

A decade of research in the BGU Laboratory for Experimental Neurosurgery has shown that vascular pathology, and specifically dysfunction of the blood-brain barrier (BBB), plays a key role in brain dysfunction and degeneration, and may be an underlying cause of neurodegenerative complications after brain injuries.

The BBB is a highly selective permeable membrane that separates circulating blood from extracellular fluid. It protects the brain by preventing many dangerous substances from penetrating, and therefore is not meant to be damaged.

Medical researchers, including Friedman's group at BGU, are working to find ways to find drugs that will target the BBB and facilitate its repair, allowing for the prevention of Alzheimer's disease and other brain-related disease.

"Prof. Friedman has been able to conduct this breakthrough brain research using the state-of-the-art MRI machine donated as a result of contributions from American Associates, Ben-Gurion University of the Negev (AABGU)," explains Doron Krakow, AABGU executive vice president. "We believe that with continued support, Prof. Friedman and the DCE-MRI can help render more accurate and informed decisions by athletes and others exposed to mild concussions about when to resume activities."

Source: American Associates, Ben-Gurion University of the Negev