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Purpose: The Wnt/β-catenin (β-cat) signaling cascade is a key regulator of development, and dysregulation of Wnt/β-cat contributes to selected cancers, such as colorectal, breast, and hepatocellular carcinoma, through abnormal activation of Wnt target genes. To identify novel modulators of the Wnt/β-cat pathway that may emerge as therapeutic targets, we did an unbiased high-throughput RNA interference screen.Experimental Design: A synthetic oligonucleotide small interfering RNA library targeting 691 known and predicted human kinases was screened in Wnt3a-stimulated human cells in a live cell luciferase assay for modulation of Wnt/β-cat–dependent transcription. Follow-up studies of a selected high-confidence “hit” were conducted.Results: A robust quartile-based statistical analysis and secondary screen yielded several kinases worthy of further investigation, including Cdc2L1, Lmtk3, Pank2, ErbB3, and, of note, vascular endothelial growth factor receptor (VEGFR)1/Flt1, a receptor tyrosine kinase (TK) with putative weak kinase activity conventionally believed to be a negative regulator of angiogenesis. A series of loss-of-function, genetic null, and VEGFR TK inhibitor assays further revealed that VEGFR1 is a positive regulator of Wnt signaling that functions in a glycogen synthase kinase-3β (GSK3β)–independent manner as a potential synthetic lethal target in Wnt/β-cat–addicted colon carcinoma cells.Conclusions: This unanticipated non-endothelial link between VEGFR1 TK activity and Wnt/β-cat signaling may refine our understanding of aberrant Wnt signaling in colon carcinoma and points to new combinatorial therapeutics targeted to the tumor cell compartment, rather than angiogenesis, in the context of colon cancer. (Clin Cancer Res 2009;15(24):7529–37)

CCR Translation for the Article from Vascular Endothelial Growth Factor Receptor-1 Is Synthetic Lethal to Aberrant β-Catenin Activation in Colon Cancer

Supplementary Data from Vascular Endothelial Growth Factor Receptor-1 Is Synthetic Lethal to Aberrant β-Catenin Activation in Colon Cancer

Development of nanoparticle agents for cancer therapeutics and diagnostics is steadily progressing and was the subject of the inaugural conference entitled, “Nano in Cancer,” held during January 12–15, 2011, in Miami, FL. The meeting program was developed by co-chairs David Piwnica-Worms (Washington University in St. Louis), Jan Schnitzer (Proteogenomics Research Institute for Systems Medicine, San Diego), and Karen Wooley (Texas A&M University). Topics discussed for nanoparticle platforms under development included: nanotechnologies for cancer diagnostics and imaging, overcoming in vivo barriers, therapeutic nanoparticles and clinical prospects, and safety issues for nanotechnologies. Two important concepts emerged from this meeting. The first was the urgent need for uniform standards and protocols for nanoparticle characterization in vitro and in vivo, and the second was the continued need for discovery of definitive targets for tumor-directed nanoparticles across various cancers. Cancer Res; 71(17); 5611–5. ©2011 AACR.

Purpose: The Wnt/β-catenin (β-cat) signaling cascade is a key regulator of development, and dysregulation of Wnt/β-cat contributes to selected cancers, such as colorectal, breast, and hepatocellular carcinoma, through abnormal activation of Wnt target genes. To identify novel modulators of the Wnt/β-cat pathway that may emerge as therapeutic targets, we did an unbiased high-throughput RNA interference screen. Experimental Design: A synthetic oligonucleotide small interfering RNA library targeting 691 known and predicted human kinases was screened in Wnt3a-stimulated human cells in a live cell luciferase assay for modulation of Wnt/β-cat–dependent transcription. Follow-up studies of a selected high-confidence “hit” were conducted. Results: A robust quartile-based statistical analysis and secondary screen yielded several kinases worthy of further investigation, including Cdc2L1, Lmtk3, Pank2, ErbB3, and, of note, vascular endothelial growth factor receptor (VEGFR)1/Flt1, a receptor tyrosine kinase (TK) with putative weak kinase activity conventionally believed to be a negative regulator of angiogenesis. A series of loss-of-function, genetic null, and VEGFR TK inhibitor assays further revealed that VEGFR1 is a positive regulator of Wnt signaling that functions in a glycogen synthase kinase-3β (GSK3β)–independent manner as a potential synthetic lethal target in Wnt/β-cat–addicted colon carcinoma cells. Conclusions: This unanticipated non-endothelial link between VEGFR1 TK activity and Wnt/β-cat signaling may refine our understanding of aberrant Wnt signaling in colon carcinoma and points to new combinatorial therapeutics targeted to the tumor cell compartment, rather than angiogenesis, in the context of colon cancer. (Clin Cancer Res 2009;15(24):7529–37)

Characterization and noninvasive measurement of molecular pathways and biochemistry in living cells, animal models, and humans at the cellular and molecular level is now possible using remote imaging detectors. Positron and single photon emission tomography scanners, highly sensitive cameras for bioluminescence and fluorescence imaging, as well as high-magnetic-field magnetic resonance imaging scanners, can be used to study such diverse processes as signal transduction, receptor density and function, host response to pathogens, cell trafficking, and gene transfer. In many cases, images from more than one modality can be fused, allowing structure-function and multifunction relationships to be studied on a tissue-restricted or regional basis. "Molecular imaging" holds enormous potential for elucidating the molecular mechanisms of pulmonary disease and therapeutic response in intact animal models and humans.

Late-stage malignant melanoma is a cancer that is refractory to current chemotherapeutic treatments. The average survival time for patients with such a diagnosis is 6 months. In general, the vast majority of anticancer drugs operate through induction of cell cycle arrest and cell death in either the DNA synthesis (S) or mitosis (M) phase of the cell cycle. Unfortunately, the same mechanisms that melanocytes possess to protect cells from DNA damage often confer resistance to drugs that derive their toxicity from S or M phase arrest. Described herein is the synthesis of a combinatorial library of potential proapoptotic agents and the subsequent identification of a class of small molecules (triphenylmethylamides, TPMAs) that arrest the growth of melanoma cells in the G1 phase of the cell cycle. Several of these TPMAs are quite potent inducers of apoptotic death in melanoma cell lines (IC(50) approximately 0.5 muM), and importantly, some TPMAs are comparatively nontoxic to normal cells isolated from the bone marrow of healthy donors. Furthermore, the TPMAs were found to dramatically reduce the level of active nuclear factor kappa-B (NFkappaB) in the cell; NFkappaB is known to be constitutively active in melanoma, and this activity is critical for the proliferation of melanoma cells and their evasion of apoptosis. Compounds that reduce the level of NFkappaB and arrest cells in the G1 phase of the cell cycle can provide insights into the biology of melanoma and may be effective antimelanoma agents.

We compare the folding transition state (TS) of ubiquitin previously identified by using ψ analysis to that determined by using φ analysis. Both methods attempt to identify interactions and their relative populations at the rate-limiting step for folding. The TS ensemble derived from ψ analysis has an extensive native-like chain topology, with a four-stranded β-sheet network and a portion of the major helix. According to φ analysis, however, the TS is much smaller and more polarized, with only a local helix/hairpin motif. We find that structured regions can have φ values far from unity, the canonical value for such sites, because of structural relaxation of the TS. Consequently, these sites may be incorrectly interpreted as contributing little to the structure of the TS. These results stress the need for caution when interpreting and drawing conclusions from φ analysis alone and highlight the need for more specific tools for examining the structure and energetics of the TS ensemble.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration ( R G ) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R G of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted ( r 2 = 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.

We quantify the degree to which folding occurs along a complex landscape with structurally distinct pathways using ψ-analysis in combination with a protein engineering method that identifies native, non-covalent polypeptide interactions and their relative populations at the rate-limiting step. By probing the proximity of two specific partners, this method is extremely well-suited for comparison to theoretical simulations. Using ubiquitin as a model system, we detect individual pathways with site-resolved resolution, demonstrating that the protein folds through a native-like transition state ensemble with a common nucleus that contains heterogeneous features on its periphery. The consensus transition state topology has part of the major helix docked against four properly aligned β-strands. However, structural heterogeneity exists in the transition state ensemble, wherein peripheral regions are differentially populated according to their relative stability. Pathway diversity reflects the variable order of formation of these peripheral elements, which radiate outward from the common nucleus. These results, which show only moderate agreement with traditional mutational φ-analysis, provide an extraordinarily detailed and quantitative description of protein folding.

Many of the obligate steps of physiology and disease are dynamic in time and space, and thus, end-point assays do not always provide a full understanding of these processes. Comprehensive understanding of the functional complexity of protein interactions and cell trafficking requires mapping of cellular and molecular function within complex systems over biologically relevant time scales. New approaches to bioluminescence imaging of cell migration, signaling pathways, drug action, and interacting protein partners in vivo allow the study of biology and disease within the context of living animals.

White blood cell cancers constitute a diverse array of neoplasms including leukemia, lymphoma, and myeloma. Leukemias are divided into four major types, acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL). Lymphomas are divided into two major categories, Hodgkin’s and non-Hodgkin’s lymphoma, and multiple myeloma constitutes approximately 90 % of all myeloma cases. The prognosis for patients receiving one of these diagnoses is highly variable. The drug Gleevec(imatinib) has been rec ently developed based on the known chromosomal translocation (the “Philadelphia chromosome”) in patients with CML, a translocation that results in the production of the Bcr-Abl kinase fusion protein. [1] Gleevec has had widely celebrated success as a selective inhibitor of this kinase, and the patient-response rate to this drug in some studies has been a remarkable 90 %. [2] Similarly, Hodgkin’s lymphoma can be cured in > 80 % of cases. [3] In contrast, the survival of patients with CLL (the most common type of leukemia in the western hemisphere) varies widely, with many dying soon after diagnosis. [4] In addition some lymphomas, such as follicular lymphoma, are unresponsive to chemotherapeutic agents. [5] Thus, while great strides have been made in the fight against certain white blood cell cancers, others remain untreatable or are only moderately responsive to even the most potent of chemotherapeuticagents. The vast majority of anticancer agents can be classified as antimetabolites (methotrexate, fluorouracil), DNA-damaging agents (cisplatin, doxorubicin), or antimitotics (Taxol, vincristine). [6] In general, antimetabolites and DNA-damaging agents

The dimensions of a denatured protein, fully reduced ribonuclease A (r-RNase A), have been measured using synchrotron-based small angle X-ray scattering. The radius of gyration, 34-35 A, is unchanged from 0-6 M guanidinium chloride and from 20-90 degrees C at pH 2.5, and agrees with the known scaling behavior for a multitude of chemically denatured states. The polypeptide is behaving as a statistical coil in the non-interacting, high-temperature limit.

We discuss the implementation of Psi-analysis for the structural characterization of protein folding transition states. In Psi-analysis, engineered bi-histidine metal ion binding sites are introduced at surface positions to stabilize secondary and tertiary structures. The addition of metal ions stabilizes the interaction between the two known histidines in a continuous fashion. Measuring the ratio of transition state stabilization to that of the native state provides information about the presence of the metal binding site in the transition state. Psi-Analysis uses noninvasive surface mutations and does not require specialized equipment, so it can be readily applied to characterize the folding of many proteins. As a result, this method can provide a wealth of high-resolution quantitative data for comparison with theoretical folding simulations. Additionally, investigations of other biological processes also may utilize metal binding sites and Psi-analysis to detect conformational events during catalysis, assembly, and function.

We discuss the implementation of Psi-analysis for the structural characterization of protein folding transition states. In Psi-analysis, engineered bi-histidine metal ion binding sites are introduced at surface positions to stabilize secondary and tertiary structures. The addition of metal ions stabilizes the interaction between the two known histidines in a continuous fashion. Measuring the ratio of transition state stabilization to that of the native state provides information about the presence of the metal binding site in the transition state. Psi-Analysis uses noninvasive surface mutations and does not require specialized equipment, so it can be readily applied to characterize the folding of many proteins. As a result, this method can provide a wealth of high-resolution quantitative data for comparison with theoretical folding simulations. Additionally, investigations of other biological processes also may utilize metal binding sites and Psi-analysis to detect conformational events during catalysis, assembly, and function.

The dimensions and secondary structure content of two proteins which fold in a two-state manner are measured within milliseconds of denaturant dilution using synchrotron-based, stopped-flow small-angle X-ray scattering and far-UV circular dichroism spectroscopy. Even upon a jump to strongly native conditions, neither ubiquitin nor common-type acylphosphatase contract prior to the major folding event. Circular dichroism and fluorescence indicate that negligible amounts of secondary and tertiary structures form in the burst phase. Thus, for these two denatured states, collapse and secondary structure formation are not energetically downhill processes even under aqueous, low-denaturant conditions. In addition, water appears to be as good a solvent as that with high concentrations of denaturant, when considering the over-all dimensions of the denatured state. However, the removal of denaturant does subtly alter the distribution of backbone dihedral phi,psi angles, most likely resulting in a shift from the polyproline II region to the helical region of the Ramachandran map. We consider the thermodynamic origins of these behaviors along with implications for folding mechanisms and computer simulations thereof.

Background Positron emission tomography (PET) allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β+)-emitting radionuclides in small animals and humans. Upon β+ decay, the initial velocity of high-energy β+ particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms. Principal Findings To improve optical imaging of Cerenkov radiation in biological systems, we demonstrate that Cerenkov radiation from decay of the PET isotopes 64Cu and 18F can be spectrally coupled by energy transfer to high Stokes-shift quantum nanoparticles (Qtracker705) to produce highly red-shifted photonic emissions. Efficient energy transfer was not detected with 99mTc, a predominantly γ-emitting isotope. Similar to bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), herein we define the Cerenkov radiation energy transfer (CRET) ratio as the normalized quotient of light detected within a spectral window centered on the fluorophore emission divided by light detected within a spectral window of the Cerenkov radiation emission to quantify imaging signals. Optical images of solutions containing Qtracker705 nanoparticles and [18F]FDG showed CRET ratios in vitro as high as 8.8±1.1, while images of mice with subcutaneous pseudotumors impregnated with Qtracker705 following intravenous injection of [18F]FDG showed CRET ratios in vivo as high as 3.5±0.3. Conclusions Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for PET radiopharmaceuticals and other isotopes in biomaterials, tissues and live animals.

Fragment complementation systems based on various luciferase enzymes have allowed for a more complete exploration of the protein interactome within the context of the cellular environment over biologically-relevant timescales. However, this strategy has been plagued by lingering questions regarding the reversibility of the systems that involve luciferase complementation fragments. To further address the need for tools to study the protein interactome, we have developed a novel set of multicolored heteroprotein fragment complementation systems based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Selected heteroprotein fragment complementation systems enabled simultaneous dual-color quantification of multiple protein interactions within live cells in realtime through use of spectral deconvolution analysis. Importantly, we have rigorously characterized the reversibility that is inherent in these systems. Reversibility was noted by the reproduction of association and dissociation constants when probing FK506 blockade of rapamycin-induced mTOR/FKBP interactions. The mean apparent Kd value upon rapamycin induction was 0.25 ± 0.05 nM, irrespective of the composition of the paired luciferases, spectral characteristics, total photon flux, or fold-inducibility. Competitive inhibition by FK506 with all the different pairs, when tested in the presence of 10 nM rapamycin, yielded a mean apparent Ki value of 1.6 ± 0.9 nM, consistent with conventional protein assays. Additionally, using split click-beetle green luciferase fused to the C-termini of EGFR, a more temporally dynamic system, we were able to monitor reversible protein conformational changes associated with the cytoplasmic domains of EGFR upon ligand-induced activation. Addition of EGF resulted in a biphasic response, characterized by an immediate drop in luciferase activity within the first minute after EGF addition followed by a rapid recovery of signal. The initial signal loss correlated with asymmetric movement of the cytosolic C-termini of EGFR dimers and adoption of a constrained conformation after phosphorylation. The subsequent recovery in luciferase activity corresponded to additional conformational changes that placed the relaxed C-terminal tails once again in close proximity. Addition of the MAPK inhibitor U0126 (10 µM) resulted in a blunted recovery of click-beetle green luciferase activity, again demonstrating the requirement for MAPK-mediated phosphorylation to induce post-activation rearrangements of the cytoplamic domain of the receptor. These dual-color reversible protein interaction reporters should enable dynamic analysis of a variety of protein networks in living cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-137.

University of Chicago, Department of Biochemistry; Molecular Biology, 920 E 58th Street, 60637, Chicago IL, USAA revised treatment of j-values is noted but only minimal revision of the identity of the transition state (TS)ensemble is required. On Pg. 465, the f-value should be set to unity in all circumstances, rather than allowedto vary. As a result, a curved Bronsted plot can be fit equally well with a heterogeneous TS model, asinterpreted in the paper, or with a homogenous model with a singular TS having a non-native biHis site.Equations (2)–(4) remain valid for a heterogeneous scenario where one class of TSs has the biHis site presentwith native-like affinity ðK