MYC and RAF: Key Effectors in Cellular Signaling and Major Drivers in Human Cancer.

The prototypes of the human MYC and RAF gene families are orthologs of animal proto-oncogenes that were originally identified as transduced alleles in the genomes of highly oncogenic retroviruses. MYC and RAF genes are now established as key regulatory elements in normal cellular physiology, but also as major cancer driver genes. Although the predominantly nuclear MYC proteins and the cytoplasmic RAF proteins have different biochemical functions, they are functionally linked in pivotal signaling cascades and circuits. The MYC protein is a transcription factor and together with its dimerization partner MAX holds a central position in a regulatory network of bHLH-LZ proteins. MYC regulates transcription conducted by all RNA polymerases and controls virtually the entire transcriptome. Fundamental cellular processes including distinct catabolic and anabolic branches of metabolism, cell cycle regulation, cell growth and proliferation, differentiation, stem cell regulation, and apoptosis are under MYC control. Deregulation of MYC expression by rearrangement or amplification of the MYC locus or by defects in kinase-mediated upstream signaling, accompanied by loss of apoptotic checkpoints, leads to tumorigenesis and is a hallmark of most human cancers. The critically controlled serine/threonine RAF kinases are central nodes of the cytoplasmic MAPK signaling cascade transducing converted extracellular signals to the nucleus for reshaping transcription factor controlled gene expression profiles. Specific mutations of RAF kinases, such as the prevalent BRAF(V600E) mutation in melanoma, or defects in upstream signaling or feedback loops cause decoupled kinase activities which lead to tumorigenesis. Different strategies for pharmacological interference with MYC- or RAF-induced tumorigenesis are being developed and several RAF kinase inhibitors are already in clinical use.

Inhibitor of MYC identified in a Kröhnke pyridine library.

In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The Kd of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.

Expanding the Scope of 2'-SCF3 Modified RNA.

The 2'-trifluoromethylthio (2'-SCF3 ) modification endows ribonucleic acids with exceptional properties and has attracted considerable interest as a reporter group for NMR spectroscopic applications. However, only modified pyrimidine nucleosides have been generated so far. Here, the syntheses of 2'-SCF3 adenosine and guanosine phosphoramidites of which the latter was obtained in highly efficient manner by an unconventional Boc-protecting group strategy, are reported. RNA solid-phase synthesis provided site-specifically 2'-SCF3 -modified oligoribonucleotides that were investigated intensively. Their excellent behavior in (19) F NMR spectroscopic probing of RNA ligand binding was exemplified for a noncovalent small molecule-RNA interaction. Moreover, comparably to the 2'-SCF3 pyrimidine nucleosides, the purine counterparts were also found to cause a significant thermodynamic destabilization when located in double helical regions. This property was considered beneficial for siRNA design under the aspect to minimize off-target effects and their performance in silencing of the BASP1 gene was demonstrated.

Efficient access to 3'-terminal azide-modified RNA for inverse click-labeling patterns.

Labeled RNA becomes increasingly important for molecular diagnostics and biophysical studies on RNA with its diverse interaction partners, which range from small metabolites to large macromolecular assemblies, such as the ribosome. Here, we introduce a fast synthesis path to 3'-terminal 2'-O-(2-azidoethyl) modified oligoribonucleotides for subsequent bioconjugation, as exemplified by fluorescent labeling via Click chemistry for an siRNA targeting the brain acid-soluble protein 1 gene (BASP1). Importantly, the functional group pattern is inverse to commonly encountered alkyne-functionalized "click"-able RNA and offers increased flexibility with respect to multiple and stepwise labeling of the same RNA molecule. Additionally, our route opens up a minimal step synthesis of 2'-O-(2-aminoethyl) modified pyrimidine nucleoside phosphoramidites which are of widespread use to generate amino-modified RNA for N-hydroxysuccinimide (NHS) ester-based conjugations.

Stem cell-specific activation of an ancestral myc protooncogene with conserved basic functions in the early metazoan Hydra.

The c-myc protooncogene encodes a transcription factor (Myc) with oncogenic potential. Myc and its dimerization partner Max are bHLH-Zip DNA binding proteins controlling fundamental cellular processes. Deregulation of c-myc leads to tumorigenesis and is a hallmark of many human cancers. We have identified and extensively characterized ancestral forms of myc and max genes from the early diploblastic cnidarian Hydra, the most primitive metazoan organism employed so far for the structural, functional, and evolutionary analysis of these genes. Hydra myc is specifically activated in all stem cells and nematoblast nests which represent the rapidly proliferating cell types of the interstitial stem cell system and in proliferating gland cells. In terminally differentiated nerve cells, nematocytes, or epithelial cells, myc expression is not detectable by in situ hybridization. Hydra max exhibits a similar expression pattern in interstitial cell clusters. The ancestral Hydra Myc and Max proteins display the principal design of their vertebrate derivatives, with the highest degree of sequence identities confined to the bHLH-Zip domains. Furthermore, the 314-amino acid Hydra Myc protein contains basic forms of the essential Myc boxes I through III. A recombinant Hydra Myc/Max complex binds to the consensus DNA sequence CACGTG with high affinity. Hybrid proteins composed of segments from the retroviral v-Myc oncoprotein and the Hydra Myc protein display oncogenic potential in cell transformation assays. Our results suggest that the principal functions of the Myc master regulator arose very early in metazoan evolution, allowing their dissection in a simple model organism showing regenerative ability but no senescence.

Inhibition of Myc-induced cell transformation by brain acid-soluble protein 1 (BASP1).

Cell transformation by the Myc oncoprotein involves transcriptional activation or suppression of specific target genes with intrinsic oncogenic or tumor-suppressive potential, respectively. We have identified the BASP1 (CAP-23, NAP-22) gene as a novel target suppressed by Myc. The acidic 25-kDa BASP1 protein was originally isolated as a cortical cytoskeleton-associated protein from rat and chicken brain, but has also been found in other tissues and subcellular locations. BASP1 mRNA and protein expression is specifically suppressed in fibroblasts transformed by the v-myc oncogene, but not in cells transformed by other oncogenic agents. The BASP1 gene encompasses 2 exons separated by a 58-kbp intron and a Myc-responsive regulatory region at the 5' boundary of untranslated exon 1. Bicistronic expression of BASP1 and v-myc from a retroviral vector blocks v-myc-induced cell transformation. Furthermore, ectopic expression of BASP1 renders fibroblasts resistant to subsequent cell transformation by v-myc, and exogenous delivery of the BASP1 gene into v-myc-transformed cells leads to significant attenuation of the transformed phenotype. The inhibition of v-myc-induced cell transformation by BASP1 also prevents the transcriptional activation or repression of known Myc target genes. Mutational analysis showed that the basic N-terminal domain containing a myristoylation site, a calmodulin binding domain, and a putative nuclear localization signal is essential for the inhibitory function of BASP1. Our results suggest that down-regulation of the BASP1 gene is a necessary event in myc-induced oncogenesis and define the BASP1 protein as a potential tumor suppressor.

Lipocalin Q83 reveals a dual ligand binding mode with potential implications for the functions of siderocalins.

Siderocalins are particular lipocalins that participate in the innate immune response by interfering with bacterial siderophore-mediated iron uptake. Additionally, siderocalins are involved in several physiological and pathological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. Here we show that siderocalin Q83 displays an unexpected dual ligand binding mode as it can bind enterobactin and unsaturated fatty acids simultaneously. The solution structure of the siderocalin Q83 in complex with arachidonic acid and enterobactin reveals molecular details of this novel dual binding mode and the determinants of fatty acid binding specificity. Our results suggest that Q83 is a metabolic hub linking iron and fatty acid pathways. This unexpected coupling might contribute to the pleiotropic functions of siderocalins.

The metastasis-associated extracellular matrix protein osteopontin forms transient structure in ligand interaction sites.

Osteopontin (OPN) is an acidic hydrophilic glycophosphoprotein that was first identified as a major sialoprotein in bones. It functions as a cell attachment protein displaying a RGD cell adhesion sequence and as a cytokine that signals through integrin and CD44 cell adhesion molecules. OPN is also implicated in human tumor progression and cell invasion. OPN has intrinsic transforming activity, and elevated OPN levels promote metastasis. OPN gene expression is also strongly activated in avian fibroblasts simultaneously transformed by the v-myc and v-mil(raf) oncogenes. Here we have investigated the solution structure of a 220-amino acid recombinant OPN protein by an integrated structural biology approach employing bioinformatic sequence analysis, multidimensional nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism spectroscopy, and small-angle X-ray scattering. These studies suggest that OPN is an intrinsically unstructured protein in solution. Although OPN does not fold into a single defined structure, its conformational flexibility significantly deviates from random coil-like behavior. OPN comprises distinct local secondary structure elements with reduced conformational flexibility and substantially populates a compact subspace displaying distinct tertiary contacts. These compacted regions of OPN encompass the binding sites for α(V)ß(III) integrin and heparin. The conformational flexibility combined with the modular architecture of OPN may represent an important structural prerequisite for its functional diversity.

The v-myc-induced Q83 lipocalin is a siderocalin.

Siderocalins are atypical lipocalins able to capture siderophores with high affinity. They contribute to the innate immune response by interfering with bacterial siderophore-mediated iron uptake but are also involved in numerous physiological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. The Q83 lipocalin was originally identified based on its overexpression in quail embryo fibroblasts transformed by the v-myc oncogene. We show here that Q83 is a siderocalin, binding the siderophore enterobactin with an affinity and mode of binding nearly identical to that of neutrophil gelatinase-associated lipocalin (NGAL), the prototypical siderocalin. This strengthens the role of siderocalins in cancer progression and inflammation. In addition, we also present the solution structure of Q83 in complex with intact enterobactin and a detailed analysis of the Q83 binding mode, including mutagenesis of the critical residues involved in enterobactin binding. These data provide a first insight into the molecular details of siderophore binding and delineate the common molecular properties defining the siderocalin protein family.

Electron detachment dissociation for top-down mass spectrometry of acidic proteins.

Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72% for Melittin, which lacks carboxylic acid functionalities, to 19% for an acidic 147-residue protein, to 12% for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO(2). Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.

MYC Analysis in Cancer and Evolution.

The MYC oncogene was originally identified as a transduced allele (v-myc) in the genome of the highly oncogenic avian retrovirus MC29. The protein product (MYC) of the cellular MYC (c-myc) protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. MYC regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of MYC, leading to increased levels of the MYC protein, is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to quantify the oncogenic potential of MYC alleles, to test MYC inhibitors, and to monitor MYC-specific protein-protein interactions that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of MYC and to analyze structure, function, and regulation of the ancestral MYC proto-oncogenes.

The brain acid-soluble protein 1 (BASP1) interferes with the oncogenic capacity of MYC and its binding to calmodulin.

The MYC protein is a transcription factor with oncogenic potential controlling fundamental cellular processes such as cell proliferation, metabolism, differentiation, and apoptosis. The MYC gene is a major cancer driver, and elevated MYC protein levels are a hallmark of most human cancers. We have previously shown that the brain acid-soluble protein 1 gene (BASP1) is specifically downregulated by the v-myc oncogene and that ectopic BASP1 expression inhibits v-myc-induced cell transformation. The 11-amino acid effector domain of the BASP1 protein interacts with the calcium sensor calmodulin (CaM) and is mainly responsible for this inhibitory function. We also reported recently that CaM interacts with all MYC variant proteins and that ectopic CaM increases the transactivation and transformation potential of the v-Myc protein. Here, we show that the presence of excess BASP1 or of a synthetic BASP1 effector domain peptide leads to displacement of v-Myc from CaM. The protein stability of v-Myc is decreased in cells co-expressing v-Myc and BASP1, which may account for the inhibition of v-Myc. Furthermore, suppression of v-Myc-triggered transcriptional activation and cell transformation is compensated by ectopic CaM, suggesting that BASP1-mediated withdrawal of CaM from v-Myc is a crucial event in the inhibition. In view of the tumor-suppressive role of BASP1 which was recently also reported for human cancer, small compounds or peptides based on the BASP1 effector domain could be used in drug development strategies aimed at tumors with high MYC expression.

Differential regulation of myc homologs by Wnt/ß-Catenin signaling in the early metazoan Hydra.

The c-Myc protein is a transcription factor with oncogenic potential controlling fundamental cellular processes. Homologs of the human c-myc protooncogene have been identified in the early diploblastic cnidarian Hydra (myc1, myc2). The ancestral Myc1 and Myc2 proteins display the principal design and biochemical properties of their vertebrate derivatives, suggesting that important Myc functions arose very early in metazoan evolution. c-Myc is part of a transcription factor network regulated by several upstream pathways implicated in oncogenesis and development. One of these signaling cascades is the Wnt/ß-Catenin pathway driving cell differentiation and developmental patterning, but also tumorigenic processes including aberrant transcriptional activation of c-myc in several human cancers. Here, we show that genetic or pharmacological stimulation of Wnt/ß-Catenin signaling in Hydra is accompanied by specific downregulation of myc1 at mRNA and protein levels. The myc1 and myc2 promoter regions contain consensus binding sites for the transcription factor Tcf, and Hydra Tcf binds to the regulatory regions of both promoters. The myc1 promoter is also specifically repressed in the presence of ectopic Hydra ß-Catenin/Tcf in avian cell culture. We propose that Hydra myc1 is a negative Wnt signaling target, in contrast to vertebrate c-myc, which is one of the best studied genes activated by this pathway. On the contrary, myc2 is not suppressed by ectopic ß-Catenin in Hydra and presumably represents the structural and functional c-myc ortholog. Our data implicate that the connection between ß-Catenin-mediated signaling and myc1 and myc2 gene regulation is an ancestral metazoan feature. Its impact on decision making in Hydra interstitial stem cells is discussed.

Targeting the Architecture of Deregulated Protein Complexes in Cancer.

The architectures of central signaling hubs are precisely organized by static and dynamic protein-protein interactions (PPIs). Upon deregulation, these PPI platforms are capable to propagate or initiate pathophysiological signaling events. This causes the acquisition of molecular features contributing to the etiology or progression of many diseases, including cancer, where deregulated molecular interactions of signaling proteins have been best studied. The reasons for PPI-dependent reprogramming of cancer-initiating cells are manifold; in many cases, mutations perturb PPIs, enzyme activities, protein abundance, or protein localization. Consequently, the pharmaceutical targeting of PPIs promises to be of remarkable therapeutic value. For this review we have selected three key players of oncogenic signaling which are differently affected by PPI deregulation: two (the small G proteins of the RAS family and the transcription factor MYC) are considered "undruggable" using classical drug discovery approaches and in the case of the third protein discussed here, PKA, standard kinase inhibitors, may be unsuitable in the clinic. These circumstances require alternative strategies, which may lie in pharmaceutical drug interference of critical PPIs accountable for oncogenic signaling.

Calcium-dependent binding of Myc to calmodulin.

The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function.

Cell transformation by the v-myc oncogene abrogates c-Myc/Max-mediated suppression of a C/EBP beta-dependent lipocalin gene.

Using differential hybridization techniques, a cDNA clone (Q83) was isolated that corresponds to a highly abundant mRNA in quail embryo fibroblasts transformed by the v-myc oncogene. The deduced 178 amino acid protein product of Q83 contains an N-terminal signal sequence and a lipocalin sequence motif, the hallmark of a family of secretory proteins binding and transporting small hydrophobic molecules of diverse biological function, including retinoids and steroids. The quail Q83 protein displays 87% sequence identity with a developmentally regulated chicken protein, termed p20K or Ch21. Cell transformation specifically by v-myc, but not by other oncogenic agents, induces high-level expression of Q83 mRNA and of the Q83 protein. Nucleotide sequence analysis and transcriptional mapping revealed that the Q83 gene encompasses seven exons with the coding region confined to exons 1 through 6. The promoter region contains consensus binding sites for the transcriptional regulators Myc and C/EBP beta. Transcriptional activation of Q83 is principally dependent on C/EBP beta, but is blocked in normal cells by the endogenous c-Myc/Max/Mad transcription factor network. In v-myc-transformed cells, high-level expression of the v-Myc protein and formation of highly stable v-Myc/Max heterodimers leads to abrogation of Q83 gene suppression and activation by C/EBP beta. A 157 amino acid residue recombinant protein representing the secreted form of Q83 was used for structure determination by nuclear magnetic resonance spectroscopy. Q83 folds into a single globular domain of the lipocalin-type. The central part consists of an eight-stranded up-and-down beta-barrel core flanked by an N-terminal 3(10)-like helix and a C-terminal alpha-helix. The orientation of the C-terminal alpha-helix is partially determined by a disulfide bridge between Cys59 and Cys152. The three-dimensional structure determination of the Q83 protein will facilitate the identification of its authentic ligand and the assessment of its biological function, including the putative role in myc-induced cell transformation.

In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs.

Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.

Hydra myc2, a unique pre-bilaterian member of the myc gene family, is activated in cell proliferation and gametogenesis.

The myc protooncogene encodes the Myc transcription factor which is the essential part of the Myc-Max network controlling fundamental cellular processes. Deregulation of myc leads to tumorigenesis and is a hallmark of many human cancers. We have recently identified homologs of myc (myc1, myc2) and max in the early diploblastic cnidarian Hydra and have characterized myc1 in detail. Here we show that myc2 is transcriptionally activated in the interstitial stem cell system. Furthermore, in contrast to myc1, myc2 expression is also detectable in proliferating epithelial stem cells throughout the gastric region. myc2 but not myc1 is activated in cycling precursor cells during early oogenesis and spermatogenesis, suggesting that the Hydra Myc2 protein has a possible non-redundant function in cell cycle progression. The Myc2 protein displays the principal design and properties of vertebrate Myc proteins. In complex with Max, Myc2 binds to DNA with similar affinity as Myc1-Max heterodimers. Immunoprecipitation of Hydra chromatin revealed that both Myc1 and Myc2 bind to the enhancer region of CAD, a classical Myc target gene in mammals. Luciferase reporter gene assays showed that Myc1 but not Myc2 transcriptionally activates the CAD promoter. Myc2 has oncogenic potential when tested in primary avian fibroblasts but to a lower degree as compared to Myc1. The identification of an additional myc gene in Cnidaria, a phylum that diverged prior to bilaterians, with characteristic expression patterns in tissue homeostasis and developmental processes suggests that principle functions of myc genes have arisen very early in metazoan evolution.