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Leeper A.D.,University of Edinburgh | Farrell J.,MRC Technology | Williams L.J.,University of Edinburgh | Thomas J.S.,University of Edinburgh | And 4 more authors.
Biomaterials | Year: 2012

We developed a three-dimensional assay prepared from primary breast cancer tissue and quantified tumor response to tamoxifen therapy. Freshly harvested breast cancer biopsies obtained at the time of curative surgical resection were fragmented and embedded into collagen I cushions. Changes in proliferation, apoptosis and tumor volume in response to tamoxifen treatment were quantified using image analysis software and optical projection tomography. Individual and collective invasion of epithelial cells into the surrounding collagen I was observed over the course of the experiment using phase contrast light microscopy and histopathological methods. Addition of tamoxifen to preparations derived from ER+ tumors demonstrated a range of response as measured by proliferative and apoptotic markers. In keeping with published data, tamoxifen reduced the percentage of apoptotic cells expressing cleaved caspase-3 (p = 0.02, Poisson regression analysis). Tamoxifen also reduced residual epithelial volume in ER+ tumors (p = 0.001, Mann-Whitney test), but not in ER low/- tumors (p = 0.78). Changes in tumor volume, as measured by optical projection tomography, allowed stratification into responsive and non-responsive tumors. The model mirrors observations of breast cancer response and histopathological changes to tamoxifen in neo-adjuvant trials. This assay provides a method of screening a battery of therapeutics against individual cancers, informing subsequent design of neo-adjuvant trials. © 2011 Elsevier Ltd.


Miller T.C.R.,University of Cambridge | Rutherford T.J.,University of Cambridge | Birchall K.,MRC Technology | Chugh J.,MRC Technology | And 2 more authors.
ACS Chemical Biology | Year: 2014

The Pygo-BCL9 complex is a chromatin reader, facilitating β-catenin-mediated oncogenesis, and is thus emerging as a potential therapeutic target for cancer. Its function relies on two ligand-binding surfaces of Pygos PHD finger that anchor the histone H3 tail methylated at lysine 4 (H3K4me) with assistance from the BCL9 HD1 domain. Here, we report the first use of fragment-based screening by NMR to identify small molecules that block protein-protein interactions by a PHD finger. This led to the discovery of a set of benzothiazoles that bind to a cleft emanating from the PHD-HD1 interface, as defined by X-ray crystallography. Furthermore, we discovered a benzimidazole that docks into the H3K4me specificity pocket and displaces the native H3K4me peptide from the PHD finger. Our study demonstrates the ligandability of the Pygo-BCL9 complex and uncovers a privileged scaffold as a template for future development of lead inhibitors of oncogenesis. © 2014 American Chemical Society.


Leeper A.D.,University of Edinburgh | Farrell J.,MRC Technology | Dixon Michael J.,University of Edinburgh | Wedden S.E.,MRC Technology | And 2 more authors.
Journal of Visualized Experiments | Year: 2011

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account. We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies. We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines. © 2011 Journal of Visualized Experiments.


Smith H.,University of Dundee | Liu X.-Y.,Nanyang Technological University | Dai L.,Nanyang Technological University | Goh E.T.H.,University of Dundee | And 12 more authors.
Biochemical Journal | Year: 2011

Mammalian Pellino isoforms are phosphorylated by IRAK (interleukin receptor associated kinase) 1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. In the present paper we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(I:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-β]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [IκB(inhibitor of nuclear factor κB) kinase]-related protein kinases, TBK1 {TANK [TRAF (tumour-necrosis-factor- receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1} and IKK?. Pellino 1 is not induced in IRF3 (interferon regulatory factor 3) -/- BMDMs, and its induction is only reduced slightly in type 1 interferon receptor -/- BMDMs, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKε as bait, and show that IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser 76, Thr 288 and Ser 293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS- or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by the inhibition of IKKε/TBK1 and is reversed by phosphatase treatment. Thus IKKε/TBK1mediate the activation of Pellino 1's E3 ligase activity, as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists. © The Authors Journal compilation © 2011 Biochemical Society.


Clark K.,University of Dundee | Peggie M.,University of Dundee | Plater L.,University of Dundee | Sorcek R.J.,Boehringer Ingelheim Pharmaceuticals | And 6 more authors.
Biochemical Journal | Year: 2011

Members of the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} family play a central role in innate immunity by inducing NF-κB- and IRF [IFN (interferon) regulatory factor]-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and IFNs. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKKε/TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1) and BI605906, a novel inhibitor of IKKβ, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1 (interleukin-1), TLR (Toll-like receptor) 3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs,which directly phosphorylate and activate IKKε and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFα-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO (NF-κB essential modulator), which is associated with reduced IKKα/β activity and NF-κB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IκBα, p105, RelA (IKKα and IKKβ) and IRF3 (IKKε and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO and TANK) subunits to balance their activities during innate immunity. © The Authors Journal compilation © 2011 Biochemical Society.


De La Roche M.,University of Cambridge | Rutherford T.J.,University of Cambridge | Gupta D.,University of Cambridge | Veprintsev D.B.,University of Cambridge | And 4 more authors.
Nature Communications | Year: 2012

Wnt/β-catenin signalling controls development and tissue homeostasis. Moreover, activated β-catenin can be oncogenic and, notably, drives colorectal cancer. Inhibiting oncogenic β-catenin has proven a formidable challenge. Here we design a screen for small-molecule inhibitors of β-catenin's binding to its cofactor BCL9, and discover five related natural compounds, including carnosic acid from rosemary, which attenuates transcriptional β-catenin outputs in colorectal cancer cells. Evidence from NMR and analytical ultracentrifugation demonstrates that the carnosic acid response requires an intrinsically labile α-helix (H1) amino-terminally abutting the BCL9-binding site in β-catenin. Similarly, in colorectal cancer cells with hyperactive β-catenin signalling, carnosic acid targets predominantly the transcriptionally active ('oncogenic') form of β-catenin for proteasomal degradation in an H1-dependent manner. Hence, H1 is an 'Achilles' Heel' of β-catenin, which can be exploited for destabilization of oncogenic β-catenin by small molecules, providing proof-of-principle for a new strategy for developing direct inhibitors of oncogenic β-catenin. © 2012 Macmillan Publishers Limited. All rights reserved.


Forfar R.,MRC Technology | Lu Z.-L.,Xi'an Jiaotong - Liverpool University
Journal of Biological Chemistry | Year: 2011

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro 174(4.59)-Met 180(4.66)) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln 174(4.60) and Phe 178(4.64) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu 1 and Trp 3 of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe 178(4.64) of the receptor and Trp 3 of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu 175(4.61), Ile 177(4.63), and Met 180(4.66) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe 177(4.63) and Phe 178(4.64) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an -helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.


News Article | December 7, 2016
Site: www.latimes.com

Researchers in Spain have taken a key step in unraveling one of nature’s most malignant mysteries: How do cancerous tumor cells that establish a beachhead in one organ strike out in search of new territory to colonize? And more important, how might they be stopped? Some answers to those questions came Wednesday in a study published in the journal Nature. In a series of experiments using human oral cancer cells implanted in mice, scientists at Barcelona's Institute for Research in Biomedicine focused on a single protein that sits on the surface of some cancer cells. Known only as CD36, this protein helps usher a key energy source — fatty acids — into the cell for use as fuel. The presence of CD36 on a cell’s surface was revealing. Amid millions of cells that make up a tumor, only a very small number are equipped with this telltale fatty acid receptor. Within groups of oral cancer cells transferred from humans to mice, these were among the first to break free of their beachhead in search of new territory, the scientists found. They functioned as cancer’s expeditionary forces, plying the body’s bloodstream or lymphatic tissue to find new sites to conquer. And they were not limited to oral cancers:  The same fat-metabolizing proteins appear to play a key role in spurring metastasis in ovarian, bladder and lung cancers, and possibly others. The researchers, led by stem cell biologist Salvador Aznar Benitah, used this finding to develop two experimental antibody treatments capable of disarming CD36. Ideally, these armies of cloned immune cells would knock out CD36 and deny the expansionist cancer cells the means, or perhaps just the impetus, to wander beyond their place of origin. It worked. Among the mice implanted with human cancer cells, those that got an infusion of cloned antibodies every three days showed no metastases, even though the cancer cells had established themselves firmly inside the lab animals’ mouths. When mice whose cancer had already metastasized got daily infusions of the antibodies, their lymph node metastases shrunk by 80% to 90%. And in 15% to 20% of these mice, the antibodies brought about complete remission. The new findings suggest that fatty acids — dietary fats that are plentiful in all modern human diets and ubiquitous in Western diets — play an outsize role in promoting cancer’s spread. When mice were fed a high-fat diet and seeded with human cancer cells, they developed more and larger lymph node metastases. And researchers found one type of fat that encouraged metastasis with particular effectiveness. The researchers bathed human oral cancer cells in palmitic acid — a type of fat widely added to modern processed food — and injected the treated cells into the tongues of mice. The animals’ original tumors did not change in size. But the size and frequency of lymph node metastases increased markedly. Indeed, 10% of the affected mice saw their cancer spread to the lung, a very rare metastasis for this type of cancer. The finding that dietary fats and large stores of excess fat contribute to cancer’s spread is not new. University of Chicago cancer researcher Ernst Lengyel and his colleagues discovered in 2011 that dietary fat was a key promoter of metastasis in ovarian cancer. Lengyel, who wasn’t involved in the new research, called the Nature paper “fantastic.” But he cautioned that it is far too early for cancer patients to halt their consumption of fat in a bid to prevent their disease from spreading. “You can take away from this paper that cancer cells depend on the nutrients in their environment,” Lengyel said. But, he added, “fatty acids have very important roles for normal cells and also help immune cells to protect the body from the tumor.” So cutting consumption of all fats could be harmful, he said. The findings are striking, given that 90% of the world’s 7.6 million cancer deaths each year come after a cancer has metastasized. Benitah said his team is working with a London-based medical research charity, MRC Technology, to identify human versions of the mouse antibodies that have shown such promise in blocking cancer’s spread. The work of identifying those human antibodies, and of beginning to test them in humans, is expected to take up to 10 years, Benitah said. However, with a few lucky breaks, antibodies capable of knocking out CD36 in human cells could be found in just three or four years, he said. “Science is much tougher than many people might think. We constantly have negative results and failure,” Benitah said. But his lab’s four-year quest to find a way to block the spread of cancer has given him some “magical moments,” he said, and “this is what we’re here for.” Follow me on Twitter @LATMelissaHealy and "like" Los Angeles Times Science & Health on Facebook. Click here for a Spanish version of this story States with background checks of those who buy guns and bullets also have fewer school shootings Explosives detector works 16 times better when it can ‘sniff’ like a dog Here's something Americans disagree about that has nothing to do with partisan politics: food


News Article | December 7, 2016
Site: www.eurekalert.org

A study headed by Salvador Aznar Benitah, ICREA researcher at the Institute for Research in Biomedicine (IRB Barcelona), and published today in Nature identifies metastasis-initiating cells through a specific marker, namely the protein CD36. This protein, which is found in the membranes of tumour cells, is responsible for taking up fatty acids. CD36 activity and dependence on lipid (fat) metabolism distinguish metastasis-initiating cells from other tumour cells. The researchers discovered the metastatic CD36+ cells in samples from patients with oral cancer with different degrees of aggressiveness, provided as part of a collaboration with the Hospital Vall d'Hebrón de Barcelona. In the mouth tumours analysed, very few cells were found to have metastasis-initiating capacity. The addition of CD36 expression to tumours that did not cause metastasis made them become metastatic. Furthermore, the researchers have demonstrated that the effect exerted by CD36 on metastasis is the same for melanoma cells and luminal breast cancer cells. Likewise, statistical analyses of samples from patients reveal that the metastasis of ovarian, bladder and lung cancer are also dependent on CD36. "Although we have not yet tested this in all tumour types, we can state that CD36 is a general marker of metastatic cells, the first marker I know of that is generally specific to metastasis," says Salvador Aznar Benitah, head of the Stem Cell and Cancer Lab at IRB Barcelona. "We can now obtain metastatic cells in the laboratory. This will allow us to trace them and to study, for example, their distribution in the tumour, where they anchor when they leave it, or why they are so sensitive to fat, among other questions," adds the first author of the study Gloria Pascual. Dr. Aznar-Benitah pointed that "We expect this study to have a big impact on the scientific community and to further advances in metastasis research, and we hope to be able to validate the potential of CD36 as an anti-metastasis treatment. Things like this don't happen every day." Given the involvement of lipid metabolism in metastasis and the function of CD36 in this process, the next logical question for the researchers was: does fat intake have a direct effect on metastasis? The researchers provided mice with a high-fat diet (15% more fat that normal, equivalent to what a so-called "cafeteria diet"). They then inoculated them with a type of oral cancer, which in standard dietary conditions would lead to 30% of the animals developing metastasis. Strikingly, under the high-fat diet and thus with a greater amount of lipids in blood, around 80% of the mice developed many more and larger metastases. They also tested the effect of a specific fatty acid, palmitic acid, on metastasis. This plant-derived fatty acid is the main component of palm oil, and in lower proportions, of coconut and other oils, and is used in many kinds of processed food. The researchers treated the oral tumours with palmitic acid for two days and then injected them into mice on a standard diet,.An increase from 50 to 100% was observed in the frequency of the metastatic tumour. That is to say, all the mice developed metastasis in a CD36-dependent manner. "In mice inoculated with human tumour cells, there appears to be a direct link between fat intake and an increase in metastatic potential through CD36. More studies are needed to unravel this intriguing relationship between diet and metastasis, above all because industrialised countries are registering an alarming increase in the consumption of saturated fats and sugar," warns Aznar Benitah. "Fat is necessary for the function of the body, but uncontrolled intake can have an effect on health, as already shown for some tumours such as colon cancer, and in metastasis, as we demonstrate here," explains the researcher. The study demonstrates the anti-metastatic effect of blocking the CD36 protein, both in immunodepressed mice and in mice with intact immune systems. Figures were similar for all tests. The inhibition of CD36 when the animals were inoculated with the tumour cells completely eliminated their metastatic potential. In addition, the administration of CD36-blocking antibodies to mice with already established metastases led to total removal of the metastases in 20% of the animals, while for the others it brought about a dramatic reduction of 80-90% in the number of metastatic foci and their size. The mice tolerated the treatment in the therapeutic window required to achieve an anti-metastasis effect and no intolerable side-effects were observed in autopsies, and blood and tissue analyses. IRB Barcelona has applied for IP protection of the results, and the researchers are working with MRC Technology in the UK to co-develop new antibody-based therapeutics against CD36 that are suitable for treatment of patients in a range of cancers. If the development of the novel therapeutic was successful, a new product could be available in 5-10 years. The study has been possible thanks to the collaboration of the Service of Oral and Maxillofacial Surgery, the Service of Pathology Anatomy and the biobank of the Hospital Vall d'Hebrón de Barcelona, with the participation of Coro Bescós and Juan Antonio Hueto from the Vall d'Hebron Institut de Recerca (VHIR). The study is thus the result of collaboration between basic and clinical research and it aims to transform research results into useful treatments for patients. This work has been supported by the Fundació La Marató de TV3, two project grants from the World Wide Cancer Research of the UK, and Fundación Botín and Banco Santander, through Santander Universidades. Furthermore, the laboratory is supported by funding from the European Research Council, the Ministry of Science and Innovation, and the Generalitat de Catalunya (Government of Catalonia).

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