Researchers from the University of Wisconsin School of Medicine and Public Health and Carbone Cancer Center have better defined a pro-growth signalling pathway common to many cancers that, when blocked, kills cancer cells but leaves healthy cells comparatively unharmed.

The study could establish new avenues of therapeutic treatments for many types of solid tumours.

Growth signals typically come in the form of chemical agonists outside of cells that bind to protein receptors on cells. Activated receptors are responsible for transmitting the signal to the inside of the cell, ultimately generating a growth messenger called PIP3.

Two years ago, research out of UW–Madison professor Richard Anderson’s lab found that some of these agonist-stimulated receptors continue to transmit the signal even after they have been pulled into the cell, sequestered in vesicles called endosomes and presumably on their way to being degraded.

“According to dogma in the literature, receptors shouldn’t make PIP3 at these internal sites, but they were,” Anderson says. “We set out to ask, ‘Why is that?’”

In this new study, a postdoctoral fellow in Anderson’s lab, Suyong Choi, showed that the proteins known to be in this signal transmission cascade were all present on endosomes inside the cell, supporting the idea that the key growth message was being signalled from these internal compartments.

However, there was one fact which they could not biologically explain: In a typical signalling cascade, each step amplifies the signal, suggesting there should be more and more of the messenger molecules; but here, levels of PIP3 and other intermediary messengers were too low to be detected in endosomes.

“A scaffold completely solves this issue, because it acts like an assembly line, bringing together all of the proteins and passing one messenger molecule to the next protein in the cascade until the last protein, PI3K, is activated and generates PIP3,” Anderson says. “Suyong Choi found that the scaffolding protein IQGAP1 brings all of these proteins together like a happy family on the endosome. It’s an incredibly efficient mechanism.”

Choi discovered that the IQGAP1 complex pulls together all of the signalling components in the PI3K pathway. Remarkably, this assembly happens in response to nearly all agonists that switch on growth and cell survival signals in cells. Once Choi had established how the proteins in the complex interacted, he was able to block scaffold formation in cells by adding a small, competing fragment of the IQGAP1 protein.

“It worked beautifully to block assembly of IQGAP1 and PI3K complex,” Anderson says. “The really cool thing was, when we treated different cells with these inhibitory fragments, the disruption of IQGAP1 and PI3K complex formation had almost no effect on normal cells but it killed cancer cells very efficiently.”

PI3K is an essential protein, and cells (and whole organisms) die if they do not have any functional PI3K because the protein is involved in multiple signalling pathways. However, it is specifically this pathway, mediated through IQGAP1, that is required for the growth and survival of cancer cells but not normal cells. In fact, mice lacking IQGAP1 develop normally but are resistant to developing solid tumours.

“Pharmaceutical companies have developed PI3K inhibitors, but many of these have failed, likely because they’re hitting all PI3Ks and the different pathways,” Anderson said. “If you can specifically disrupt this agonist-activated PI3K pathway, the one that has a specific role in cancer, then you can effectively treat

University of Wisconsin School of Medicine and Public Health www.med.wisc.edu/news-events/cancer-signaling-pathway-could-lead-to-new-cancer-therapies/49720

A multi-institutional group of researchers, led by investigators at Children’s Hospital Los Angeles and the University of Michigan, have identified a simple and inexpensive tool for assessing the prognosis of paediatric brain tumours called ependymomas. Their study, which demonstrates the epigenetic mechanism behind these tumours, may offer future opportunities for novel therapeutic options.

Childhood posterior fossa ependymomas (PF) are tumours found largely in the hind brain (consisting of the cerebellum, pons and the brainstem) of children. Routine assessment of tumour grade and other markers in PF ependymomas do not correlate well with outcomes in these tumours, highlighting the need for new prognostic markers. Genomic sequencing efforts have not identified mutations in these tumours, and the origin of PF ependymomas remains obscure.

While lacking recurrent genetic mutations, a subset of these tumours exhibit alterations in DNA methylation. In this study, the researchers looked at modification of histones – protein components of the chromatin around which DNA winds, and which play a role in gene regulation – in particular, histone H3.

Co-lead investigator, Sriram Venneti, MD, PhD, of the Department of Pathology at the University of Michigan, observed that histone H3 is modified differently in paediatric posterior fossa ependymoma. Specifically, 80 percent of these tumours exhibited loss of the H3K27me3 a repressive mark, while 20 percent of tumours retained H3K27me3.  Researchers went back and looked at MRIs and outcomes of children treated for these tumours and identified that tumours with loss of H3K27me3 tumours behaved more aggressively and showed poor overall survival.  This suggests that reduced H3K27me3 may be a prognostic indicator in PF ependymomas.

“Detection of H3K27me3 by immunohistochemical staining is a widely available and cost effective surrogate molecular marker.  This test can be readily implemented in most departments of pathology and provides a much-needed tool to risk stratify and identify ependymoma patients who would potentially benefit from epigenetic therapies,” said co-lead investigator Alexander R. Judkins, MD, of the Department of Pathology and Laboratory Medicine at CHLA and Keck School of Medicine of the University of Southern California.

This loss in H3K27me3, along with other epigenetic changes, was similar to that observed in another type of paediatric brain tumour of the hind brain region termed diffuse intrinsic pontine gliomas (DIPGs).  This suggests that both of these tumours arise from similar epigenetic states. Intriguingly, researchers found that certain progenitor cells in this part of the brain also showed low H3K27me3, suggesting – as both tumours share epigenetic similarities – that low methylation of H3K27me3 is important to the development of tumours in this region of the brain.

Children’s Hospital of Los Angeles www.chla.org/press-release/biomarker-identified-aid-prognosis-pediatric-ependymomas

Bleeding disorders could one day be diagnosed by putting platelets through strength tests, researchers have proposed. Biomedical engineers from Emory University and the Georgia Institute of Technology have devised a microfluidic testing ground where platelets can demonstrate their strength by squeezing two protein dots together. Imagine rows and rows of strength testing machines from a carnival, but very tiny. A platelet is capable of exerting forces that are several times larger, in relation to its size, than a muscle cells.
After a blood clot forms, it contracts, promoting wound closure and restoration of normal blood flow. This process can be deficient in a variety of blood clotting disorders. Previously, it was difficult to measure an individual platelet’s contributions to contraction, because clots’ various components got in the way.
“We discovered that platelets from some patients with bleeding disorders are ‘wimpier’ than platelets from healthy people,” says Wilbur Lam, an assistant professor in the Department of Pediatrics at Emory University School of Medicine and in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University. “Our device may function as a new physics-based method to test for bleeding disorders, complementary to current methods.”
The first author of the paper is David Myers, an instructor at Emory’s medical school. Lam is also a physician in the Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta.
The scientists infer how strong or wimpy someone’s platelets are by measuring how far the protein dots move, taking a picture of the rows of dots, and then analysing the picture on a computer.
The dots are made of fibrinogen, a sticky protein that is the precursor for fibrin, which forms a mesh of insoluble strands in a blood clot.
In addition to detecting problems with platelet contraction in patients with known inherited disorders such as Wiskott Aldrich syndrome, Myers, Lam and colleagues could also see differences in some patients who had bleeding symptoms, but who performed normally on standard diagnostic tests.
The researchers also used chemical tools to dissect the process of platelet contraction. They showed that inhibitors of Rho/ ROCK enzymes shut down platelet contraction, but inhibitors of a related pathway, MLCK (myosin light chain kinase), did not. Individual platelet contraction could become an assay for development or refinement of blood thinning drugs, Lam says.

Georgia Techhttp://tinyurl.com/j8byzzg

Loss of a key protein leads to defects in skeletal development including reduced bone density and a shortening of the fingers and toes — a condition known as brachydactyly. The discovery was made by researchers at Penn State University who knocked out the Speckle-type POZ Protein (Spop) in the mouse and characterized the impact on bone development. The research redefines the role of Spop during bone development and provides a new potential target for the diagnosis and treatment of bone diseases such as osteoporosis.
“The Spop protein is involved in Hedgehog signalling — a well-studied cell-tocell communication pathway that plays multiple roles during development,” said Aimin Liu, associate professor of biology at Penn State and the corresponding author of the study. “Previous studies done in cell culture suggested that Spop negatively regulates or ‘turns down’ Hedgehog signalling. However, in our study, we show that Spop positively regulates the pathway downstream of a member of the Hedgehog family, a protein called Indian Hedgehog, during bone development. This new understanding adds to our knowledge of the genetic basis of bone development and could open new avenues to study bone disease.”
Indian Hedgehog (Ihh) plays an essential role in bone development. It is near the top of a hierarchical cascade of genes that program cells to produce cartilage and bone. Ihh controls gene expression by regulating the activity of the transcription factors — proteins that control the expression of other genes — Gli2 and Gli3. Gli2 acts mainly as an activator of gene expression and Gli3 acts mainly to repress gene expression. The Spop protein tags the Gli proteins to be degraded in the cell. “Previous studies led to a hypothesis that a loss of Spop function would increase Hedgehog signalling because the Gli activators were no longer being degraded,” said Hongchen Cai, a graduate student at Penn State and an author of the paper. “We were surprised to see in our study the repressor of gene expression, Gli3, built up in developing bone, but not the activator of gene expression, Gli2. This imbalance led to an overall decrease in Hedgehog signalling.”
In order to study the role of Spop in bone development more closely, the researchers knocked the gene out specifically in the limb. Limbs that lacked Spop had less dense bone, mimicking osteopenia — a human condition characterized by low bone density, but not as severe as osteoporosis. The limbs also had shorter than normal fingers and toes. The researchers also showed that the effects of losing Spop could be mitigated by simultaneously reducing the amount of Gli3 in the limbs.

Penn State http://tinyurl.com/jx3y6nj

Researchers at the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB in Stuttgart are developing a test which rapidly and cost-effectively identifies bacteria, fungi or viruses. It can be carried out directly in situ without laboratory equipment and specialist knowledge. “The ImmuStick can even detect pathogens outside the body – on medical devices or in hospital rooms for example. However, the technology would certainly also be of interest for testing human blood for germs or allergies“, says Dr. Anke Burger-Kentischer.
The method works as simply as a pregnancy test. The ImmuStick is a test strip onto which a few drops of fluid are applied. If the fluid contains pyrogens, fragments of pathogens, this is shown by a coloured strip in a viewing window. First of all, human immune receptors sensitive to certain pyrogens are applied to the surface of the stick. These are laboratory-produced immune receptors which are synthesized on the basis of the biological model. During production, at the docking point of the immune receptors to which the pyrogens normally bind, a type of placeholder is mounted which is marked with a dye. When drops of a fluid containing pyrogens are then applied to the test strip, the pyrogens rush to the docking point on the immune receptor. The placeholders marked with the dye migrate with the fluid through the test strip until they are visible in the viewing window. The colour signal thus indicates that pyrogens that have docked on the immune receptors are present.
The ImmuStick project was financed with money from the Discover programme. In this way the Fraunhofer-Gesellschaft is supporting projects for the duration of one year in order to demonstrate the feasibility of a technology. The ImmuStick has passed this test. “We were able to show that it works very well for the bacterial pyrogen LPS. Together with industrial partners, we now want to develop it into a product“, says project manager Burger-Kentischer. “We are currently testing further immune receptors that are specific for other pyrogens.“
Currently envisaged are applications in the food and pharmaceuticals sector or in medical technology, as a complete absence of germs or pyrogens is required there. In principle, the ImmuStick would also be of interest for blood analysis. Pyrogens in the blood often lead to blood poisoning, sepsis, from which many people still die today, especially weakened intensive care patients. “However, blood is a special challenge as it is complex and contains many constituent parts. But in the medium term we are aiming at blood analysis“, says Burger-Kentischer.
As pyrogens also include certain allergy trigger factors, an application here would also be conceivable. In the food and pharmaceutical industries, for example, it is important that products are free of allergens. With the ImmuStick these could be detected quickly, cost-effectively and simply. Costly and laborious laboratory tests would therefore no longer be needed or could be supplemented. At present the IGB researchers are seeking cooperation partners who want to further develop the ImmuStick to make it ready for the market.
Pyrogens become a problem when hygiene is of particular importance – in the food and pharmaceutical industries for example, or on intensive care wards in hospitals. Especially people with weakened immune systems can become severely ill. For this reason, tests are frequently carried out and the surfaces of machines or medical devices are tested for pyrogens using swabs. However, to date these tests have been costly and laborious as pyrogens can only be detected with laboratory equipment. A widely used standard test is the detection of LPS, a structure that is present in the membrane of certain bacteria. At present this test takes up around two hours. Other pyrogens can even only be detected in animal experiment.

Fraunhofer Institute for Interfacial Engineering and Biotechnology IGBhttp://tinyurl.com/jyrlqct