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Systems Biology analysis of Transcription Networks, Artificial Intelligence, and High-End Computing Coming to Fruition in Personalized Oncology
Curator: Stephen J. Williams, Ph.D.
In the June 2020 issue of the journal Science, writer Roxanne Khamsi has an interesting article “Computing Cancer’s Weak Spots; An algorithm to unmask tumors’ molecular linchpins is tested in patients”[1], describing some early successes in the incorporation of cancer genome sequencing in conjunction with artificial intelligence algorithms toward a personalized clinical treatment decision for various tumor types. In 2016, oncologists Amy Tiersten collaborated with systems biologist Andrea Califano and cell biologist Jose Silva at Mount Sinai Hospital to develop a systems biology approach to determine that the drug ruxolitinib, a STAT3 inhibitor, would be effective for one of her patient’s aggressively recurring, Herceptin-resistant breast tumor. Dr. Califano, instead of defining networks of driver mutations, focused on identifying a few transcription factors that act as ‘linchpins’ or master controllers of transcriptional networks withing tumor cells, and in doing so hoping to, in essence, ‘bottleneck’ the transcriptional machinery of potential oncogenic products. As Dr. Castilano states
“targeting those master regulators and you will stop cancer in its tracks, no matter what mutation initially caused it.”
It is important to note that this approach also relies on the ability to sequence tumors by RNA-seq to determine the underlying mutations which alter which master regulators are pertinent in any one tumor. And given the wide tumor heterogeneity in tumor samples, this sequencing effort may have to involve multiple biopsies (as discussed in earlier posts on tumor heterogeneity in renal cancer).
As stated in the article, Califano co-founded a company called Darwin-Health in 2015 to guide doctors by identifying the key transcription factors in a patient’s tumor and suggesting personalized therapeutics to those identified molecular targets (OncoTarget™). He had collaborated with the Jackson Laboratory and most recently Columbia University to conduct a $15 million 3000 patient clinical trial. This was a bit of a stretch from his initial training as a physicist and, in 1986, IBM hired him for some artificial intelligence projects. He then landed in 2003 at Columbia and has been working on identifying these transcriptional nodes that govern cancer survival and tumorigenicity. Dr. Califano had figured that the number of genetic mutations which potentially could be drivers were too vast:
A 2018 study which analyzed more than 9000 tumor samples reported over 1.5 million mutations[2]
and impossible to develop therapeutics against. He reasoned that you would just have to identify the common connections between these pathways or transcriptional nodes and termed them master regulators.
A Pan-Cancer Analysis of Enhancer Expression in Nearly 9000 Patient Samples
Chen H, Li C, Peng X, et al. Cell. 2018;173(2):386-399.e12.
Abstract
The role of enhancers, a key class of non-coding regulatory DNA elements, in cancer development has increasingly been appreciated. Here, we present the detection and characterization of a large number of expressed enhancers in a genome-wide analysis of 8928 tumor samples across 33 cancer types using TCGA RNA-seq data. Compared with matched normal tissues, global enhancer activation was observed in most cancers. Across cancer types, global enhancer activity was positively associated with aneuploidy, but not mutation load, suggesting a hypothesis centered on “chromatin-state” to explain their interplay. Integrating eQTL, mRNA co-expression, and Hi-C data analysis, we developed a computational method to infer causal enhancer-gene interactions, revealing enhancers of clinically actionable genes. Having identified an enhancer ∼140 kb downstream of PD-L1, a major immunotherapy target, we validated it experimentally. This study provides a systematic view of enhancer activity in diverse tumor contexts and suggests the clinical implications of enhancers.
A diagram of how concentrating on these transcriptional linchpins or nodes may be more therapeutically advantageous as only one pharmacologic agent is needed versus multiple agents to inhibit the various upstream pathways:
VIPER Algorithm (Virtual Inference of Protein activity by Enriched Regulon Analysis)
The algorithm that Califano and DarwinHealth developed is a systems biology approach using a tumor’s RNASeq data to determine controlling nodes of transcription. They have recently used the VIPER algorithm to look at RNA-Seq data from more than 10,000 tumor samples from TCGA and identified 407 transcription factor genes that acted as these linchpins across all tumor types. Only 20 to 25 of them were implicated in just one tumor type so these potential nodes are common in many forms of cancer.
Other institutions like the Cold Spring Harbor Laboratories have been using VIPER in their patient tumor analysis. Linchpins for other tumor types have been found. For instance, VIPER identified transcription factors IKZF1 and IKF3 as linchpins in multiple myeloma. But currently approved therapeutics are hard to come by for targets with are transcription factors, as most pharma has concentrated on inhibiting an easier target like kinases and their associated activity. In general, developing transcription factor inhibitors in more difficult an undertaking for multiple reasons.
Identifying the multiple dysregulated oncoproteins that contribute to tumorigenesis in a given patient is crucial for developing personalized treatment plans. However, accurate inference of aberrant protein activity in biological samples is still challenging as genetic alterations are only partially predictive and direct measurements of protein activity are generally not feasible. To address this problem we introduce and experimentally validate a new algorithm, VIPER (Virtual Inference of Protein-activity by Enriched Regulon analysis), for the accurate assessment of protein activity from gene expression data. We use VIPER to evaluate the functional relevance of genetic alterations in regulatory proteins across all TCGA samples. In addition to accurately inferring aberrant protein activity induced by established mutations, we also identify a significant fraction of tumors with aberrant activity of druggable oncoproteins—despite a lack of mutations, and vice-versa. In vitro assays confirmed that VIPER-inferred protein activity outperforms mutational analysis in predicting sensitivity to targeted inhibitors.
Schematic overview of the VIPER algorithm From: Alvarez MJ, Shen Y, Giorgi FM, Lachmann A, Ding BB, Ye BH, Califano A: Functional characterization of somatic mutations in cancer using network-based inference of protein activity. Nature genetics 2016, 48(8):838-847.
(a) Molecular layers profiled by different technologies. Transcriptomics measures steady-state mRNA levels; Proteomics quantifies protein levels, including some defined post-translational isoforms; VIPER infers protein activity based on the protein’s regulon, reflecting the abundance of the active protein isoform, including post-translational modifications, proper subcellular localization and interaction with co-factors. (b) Representation of VIPER workflow. A regulatory model is generated from ARACNe-inferred context-specific interactome and Mode of Regulation computed from the correlation between regulator and target genes. Single-sample gene expression signatures are computed from genome-wide expression data, and transformed into regulatory protein activity profiles by the aREA algorithm. (c) Three possible scenarios for the aREA analysis, including increased, decreased or no change in protein activity. The gene expression signature and its absolute value (|GES|) are indicated by color scale bars, induced and repressed target genes according to the regulatory model are indicated by blue and red vertical lines. (d) Pleiotropy Correction is performed by evaluating whether the enrichment of a given regulon (R4) is driven by genes co-regulated by a second regulator (R4∩R1). (e) Benchmark results for VIPER analysis based on multiple-samples gene expression signatures (msVIPER) and single-sample gene expression signatures (VIPER). Boxplots show the accuracy (relative rank for the silenced protein), and the specificity (fraction of proteins inferred as differentially active at p < 0.05) for the 6 benchmark experiments (see Table 2). Different colors indicate different implementations of the aREA algorithm, including 2-tail (2T) and 3-tail (3T), Interaction Confidence (IC) and Pleiotropy Correction (PC).
Other articles from Andrea Califano on VIPER algorithm in cancer include:
Echeverria GV, Ge Z, Seth S, Zhang X, Jeter-Jones S, Zhou X, Cai S, Tu Y, McCoy A, Peoples M, Sun Y, Qiu H, Chang Q, Bristow C, Carugo A, Shao J, Ma X, Harris A, Mundi P, Lau R, Ramamoorthy V, Wu Y, Alvarez MJ, Califano A, Moulder SL, Symmans WF, Marszalek JR, Heffernan TP, Chang JT, Piwnica-Worms H.Sci Transl Med. 2019 Apr 17;11(488):eaav0936. doi: 10.1126/scitranslmed.aav0936.PMID: 30996079
Chen H, Li C, Peng X, Zhou Z, Weinstein JN, Liang H: A Pan-Cancer Analysis of Enhancer Expression in Nearly 9000 Patient Samples. Cell 2018, 173(2):386-399 e312.
Alvarez MJ, Shen Y, Giorgi FM, Lachmann A, Ding BB, Ye BH, Califano A: Functional characterization of somatic mutations in cancer using network-based inference of protein activity. Nature genetics 2016, 48(8):838-847.
Other articles of Note on this Open Access Online Journal Include:
As the molecular processes that control mRNA translation and ribosome biogenesis in the eukaryotic cell are extremely complex and multilayered, their deregulation can in principle occur at multiple levels, leading to both disease and cancer pathogenesis. For a long time, it was speculated that disruption of these processes may participate in tumorigenesis, but this notion was, until recently, solely supported by correlative studies. Strong genetic support is now being accrued, while new molecular links between tumor-suppressive and oncogenic pathways and the control of protein synthetic machinery are being unraveled. The importance of aberrant protein synthesis in tumorigenesis is further underscored by the discovery that compounds such as Rapamycin, known to modulate signaling pathways regulatory of this process, are effective anticancer drugs. A number of fundamental questions remain to be addressed and a number of novel ones emerge as this exciting field evolves.
mRNA Translation and Energy Metabolism in Cancer
I. Topisirovic and N. Sonenberg
Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXVI http://dx.doi.org:/10.1101/sqb.2011.76.010785
A prominent feature of cancer cells is the use of aerobic glycolysis under conditions in which oxygen levels are sufficient to support energy production in the mitochondria (Jones and Thompson 2009; Cairns et al. 2010). This phenomenon, named the “Warburg effect,” after its discoverer Otto Warburg, is thought to fuel the biosynthetic requirements of the neoplastic growth (Warburg 1956; Koppenol et al. 2011) and has recently been acknowledged as one of the hallmarks of cancer (Hanahan and Weinberg 2011). mRNA translation is the most energy-demanding process in the cell (Buttgereit and Brand 1995).In mammalian cells it consumes >20% of cellular ATP, not considering the energy that is required for the biosynthesis of the components of the translational machinery (e.g., ribosome biogenesis; Buttgereit and Brand 1995). Control of mRNA translation plays a pivotal role in the regulation of gene expression (Sonenberg and Hinnebusch 2009). In fact, a recent study demonstrated that mammalian proteome is mostly governed at the mRNA translation level (Schwanhausser et al. 2011). Malfunction of mRNA translation critically contributes to human disease, including diabetes, heart disease, blood disorders, and, most notably, cancer (Fig. 1; Crozier et al. 2006; Narla and Ebert 2010; Silvera et al. 2010; Spriggs et al. 2010). The first account of changes in the translational apparatus in cancer dates back to 1896, showing enlarged and irregularly shaped nucleoli that are the site of ribosome biogenesis (Pianese 1896). Rapidly proliferating cancer cells have more ribosomes than normal cells.
Figure 1. Dysregulated mRNA translation plays a pivotal role in cancer. Malignant cells are characterized by enlarged nucleoli and a larger number of ribosomes than their normal counterparts. Mutations and/or altered expression of ribosomal proteins (e.g., RPS19, RPS 24), rRNA-modifying enzymes (e.g., dyskerin), translation initiation factors (e.g., eIF4E), or the initiator tRNA (tRNAiMet) result in malignant transformation. Signaling pathways whose dysfunction is frequent in cancer (e.g., MAPK, PI3K/AKT) affect mRNA translation. Perturbations in the translatome result in aberrant cellular growth, proliferation, and survival characteristic of tumorigenesis.
In stark contrast to normal cells, in cancer cells ribosomal biogenesis is uncoupled from cell proliferation (Stanners et al. 1979). Accordingly, cancer cells exhibit abnormally high rates of protein synthesis (Silvera et al. 2010). That ribosomal dysfunction plays a central role in cancer is further corroborated by the findings that genetic alterations, which encompass the components of the ribosome machinery (i.e., “ribosomopathies”), are characterized by elevated cancer risk (Narla and Ebert 2010).
mRNA translation is the most energy-consuming process in the cell and strongly correlates with cellular metabolic activity. Translation and energy metabolism play important roles in homeostatic cell growth and proliferation, and when dysregulated lead to cancer. eIF4E is a key regulator of translation, which promotes oncogenesis by selectively enhancing translation of a subset of tumor-promoting mRNAs (e.g., cyclins and c-myc). PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways, which are strongly implicated in cancer etiology, exert a number of their biological effects by modulating translation. The PI3K/AKT pathway regulates eIF4E function by inactivating the inhibitory 4E-BPs via mTORC1, whereas MAPKs activate MAP kinase signal-integrating kinases 1 and 2, which phosphorylate eIF4E. In addition, AMP-activated protein kinase, which is a central sensor of the cellular energy balance, impairs translation by inhibiting mTORC1. Thus, eIF4E plays a major role in mediating the effects of PI3K/AKT, MAPK, and cellular energetics on mRNA translation.Figure 2. eIF4E is regulated by multiple mechanisms. The expression of eIF4E is regulated by several transcription factors (e.g., c-myc, hnRNPK, p53) and adenine-uracil-rich element binding proteins (i.e., HuR and AUF1). eIF4E is suppressed by 4E-BPs, which are regulated by mTORC1. MAP kinase signal integrating kinases 1 and 2 (MNKs) phosphorylate eIF4E.
Figure 3. Ras/MAPK and PI3K/AKT/mTORC1 regulate the activity of eIF4E. Various stimuli activate phosphoinositide-3-kinase (PI3K) through the receptor tyrosine kinases (RTKs). Upon activation, PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-triphosphate (PIP3). This reaction is reversed by PTEN. Phosphoinositide-dependent protein kinase 1 (PDK1) and AKT bind to PIP3 via their pleckstrin homology domains, which allows for the phosphorylation and activation of AKT by PDK1. In addition, the mammalian target of rapamycin complex 2 (mTORC2) modulates the activity of AKT by phosphorylating its hydrophobic motif. AKT phosphorylates tuberous sclerosis complex 2 (TSC2) at multiple sites, which results in its inhibition and consequent activation of Ras homolog enriched in brain (Rheb), which is a small GTPase that activates mTORC1. mTORC1 phosphorylates 4E-BPs leading to their dissociation from eIF4E. In addition to the PI3K/AKT pathway, the activity of mTORC1 is regulated by the serine/threonine kinase 11/LKB1/AMP-kinase (LKB1/AMPK) pathway, regulated in development and DNA damage response 1 (REDD1) and Rag GTPases in response to the changes in cellular energy balance, oxygen and amino acid availability, respectively. Ras and the MAPK pathways are activated by various stimuli through receptor tyrosine kinases (RTKs). In addition the MAPK pathway isactivatedthrough theGprotein–coupled receptors(GPCRs) and byproteinkinaseC (PKC;notshown).TheMAPK pathways encompass an initial GTPase-regulated kinase (MAPKKK), which activates an effector kinase (MAPK) via an intermediate kinase (MAPKK). In response to stimuli such as growth factors, hormones, and phorbol-esters, Ras GTPase stimulates Raf kinase (MAPKKK), which activates extracellular signal-regulated kinases 1 and 2 (ERK 1 and 2) via extracellular signal-regulated kinase activator kinases MEK1 and 2 (MAPKK). Cellular stresses, including osmotic shock, inflammatory cytokines, and UV light, activate p38 MAPKs via multiple mechanisms including Rac kinase (MAPKKK) and MKK3 and 6 (MAPKK). p38 MAPK and ERK activate the MAPK signal–integrating kinases 1 and 2 (MNK1/2), which phosphorylate eIF4E. Additional abbreviations are provided in the text.
Breast cancer cells secrete exosomes with specific capacity for cell-independent miRNA biogenesis, while normal cellderivedexosomes lack thisability. Exosomes derivedfrom cancer cellsand serum frompatients withbreast cancer contain the RISC loading complex proteins, Dicer, TRBP, and AGO2, which process pre-miRNAs into mature miRNAs. Cancer exosomes alter the transcriptome of target cells in a Dicer-dependent manner, which stimulate nontumorigenic epithelial cells to form tumors.This study identifies a mechanism whereby cancer cells impart an oncogenic field effect by manipulating the surrounding cells via exosomes. Presence of Dicer in exosomes may serve as biomarker for detection of cancer.
Dicers at RISC. The Mechanism of RNAi
Marcel Tijsterman and Ronald H.A. Plasterk
Cell, Apr 2014; 117:1–4
Figure 1. Model for RNA Silencing in Drosophila In an ordered biochemical pathway, miRNAs (left panel) and siRNAs (right panel) are processed from double-stranded precursor molecules by Dcr-1and Dcr-2, respectively, and stay attached to Dicer-containing complexes, which assemble into RISC. The degree of complementarity between the RNA silencing molecule (in red) and its cognate target determines the fate of the mRNA: blocked translation or immediate destruction.
Fig. 1. Domain organization of RNaseIII gene family. Three classes of RNaseIII genes are shown. The PAZ domain in Dm-Dicer-2 contains mutations in several residues required for RNA binding and may not be functional.
Fig. 2. Model for Dicer catalysis. The PAZ domain binds the 2 nt 30 overhang of a dsRNA terminus. The RNaseIII domains form a pseudo-dimer. Each domain hydrolyzes one strand of the substrate. The binding site of the dsRBD is not defined. The function of the helicase domain is not known.
Fig. 3. Biogenesis pathway of microRNAs. MicroRNA genes are transcribed by RNA polymerase II. The primary transcript is referred to as ‘‘primicroRNA’’. Drosha processing occurs in the nucleus. The resulting precursor, ‘‘pre-microRNA’’, is exported to the cytoplasm for Dicer processing. In a coordinated manner, the mature microRNA is transferred to RISC and unwound by a helicase. mRNA targets that duplex in the Slicer scissile site are cleaved and degraded, if the microRNA is loaded into an Ago2 RISC. Mismatched targets are translationally suppressed. All Ago family members are believed to function in translational suppression.
Fig. 4. Model for Slicer catalysis. The siRNA guide strand is bound at the 50 end by the PIWI domain and at the 30 end by the PAZ domain. The 50 phosphate is coordinated by conserved basic residues. mRNA targets are initially bound by the seed region of the siRNA and pairing is extended to the 30 end. The RNaseH fold hydrolyzes the target in a cation dependent manner. Slicer cleavage is measured from the 50 end of the siRNA. Product is released by an unknown mechanism and the enzyme recycles.
RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing (PTGS), and quelling. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm Caenorhabditis elegans, which they published in 1998.
Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from producing a protein. RNA interference has an important role in defending cells against parasitic nucleotide sequences – viruses and transposons. It also influences development.
The RNAi pathway is found in many eukaryotes, including animals, and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double stranded fragments of ~20 nucleotide siRNAs. Each siRNA is unwound into two single-stranded RNAs (ssRNAs), the passenger strand and the guide strand. The passenger strand is degraded and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. In some organisms, this process spreads systemically, despite the initially limited molar concentrations of siRNA. http://en.wikipedia.org/wiki/RNA_interference
The enzyme dicer trims double stranded RNA, to form small interfering RNA or microRNA. These processed RNAs are incorporated into the RNA-induced silencing.
MiRNA biogenesis and function. (A) The canonical miRNA biogenesis pathway is Drosha- and Dicer-dependent. It begins with RNA Pol II-mediated transcription..
Dicer Promotes Transcription Termination
Dicer Promotes Transcription Termination at Sites of Replication Stress to Maintain Genome Stability
Cell Oct 2014; 159(3): 572–583 http://dx.doi.org/10.1016/j.cell.2014.09.031
18-13 miRNA- protein complex (a) Primary miRNA transcript Translation blocked Hydrogen bond (b) Generation and function of miRNAs Hairpin miRNA miRNA Dicer …
Fig. 1. Small RNA cloning procedure. Outline of the small RNA cloning procedure. RNA is dephosphorylated (step 1) for joining the 30 adapter by T4 RNA ligase 1 in the presence of ATP (step 2). The use of a chemically adenylated adapter and truncated form of T4 RNA ligase 2 (Rnl2) allows eliminating the dephosphorylation step (step 4). If the RNA was dephosphorylated, it is re-phosphorylated (step 3) prior to 50 adapter ligation with T4 RNA ligase 1 and ATP (step 5). After 50 adapter ligation, a standard reverse transcription is performed (step 6). Alternatively, after 30 adapter ligation, the RNA is used directly for reverse transcription simultaneously with 50 adaptor joining (step 7). In this case, the property of reverse transcriptase to add non-templated cytidine residues at the 50 end of synthesized DNA is used to facilitate template switch of the reverse transcriptase to the 30 guanosine residues of the 50 adapter (SMART technology, Invitrogen). Abbreviations: P and OH indicate phosphate and hydroxyl ends of the RNA; App indicates 50 chemically adenylated adapter; L, 30 blocking group; CIP, calf alkaline phosphatase and PNK, polynucleotide kinase.
Fig. 1. Schematic representation of gene silencing by an shRNA-expression vector. The shRNA is processed by Dicer. The processed siRNA enters the RNA-induced silencing complex (RISC), where it targets mRNA for degradation.
Fig. 2. Schematic representation of a transcription system for production of siRNA
Fig. 3. (A) Schematic representation of the proposed siRNA-expression system. Three or four C to U or A to G mutations are introduced into the sense strand. (B) Schematic representation of the discovery of a novel gene using an siRNA library.
Imperfect centered miRNA binding sites are common and can mediate repression of target mRNAs
Martin et al. Genome Biology 2014, 15:R51 http://genomebiology.com/2014/15/3/R51
Table 1 Number of inferred targets for each miRNA tested
miRNA
Probes
Transcripts
Genes
miR-10a
2,206
5,963
1,887
miR-10a-iso
1,648
1,468
4,211
miR-10b
1,588
3,940
1,365
miR-10b-iso
963
2,235
889
miR-17-5p
1,223
2,862
1,137
miR-17-5p-iso
1,656
3,731
1,461
miR-182
2,261
6,423
2,008
miR-182-iso
1,569
4,316
1,444
miR-23b
2,248
5,383
1,990
miR-27a
2,334
5,310
2,069
Probes: number of probes significantly enriched in pull-downs compared to controls (5% FDR). Transcripts: number of transcripts to which those probes map exactly. Genes: number of genes from which those transcripts originate
Figure 2 Biotin pull-downs identify bone fide miRNA targets. (A) Volcano plot showing the significance of the difference in expression between the miR-17-5p pull-down and the mock-transfected control, for all transcripts expressed in HEK293T cells. Both targets predicted by TargetScan or validated previously via luciferase assay were significantly enriched in the pull-down compared to the controls. (B) Results from luciferase assays on previously untested targets predicted using TargetScan and uncovered using the biotin pull-down. The plot indicates mean luciferase activity from either the empty plasmid or from pMIR containing a miRNA binding site in the 3′ UTR, relative to a negative control. Asterisks indicate a significant reduction in luciferase activity (one-sided t-test; P<0.05) and error bars the standard error of the mean over three replicates. (C-E) Targets identified through PAR-CLIP or through miRNA over-expression studies show greater enrichment in the pull-down. Cumulative distribution of log fold-change in the pull-down for transcripts identified as targets by the indicated miRNA over-expression study or not. Red, canonical transcripts found to be miR-17-5p targets in the indicated study (Table S5 in Additional file 1); black, all other canonical transcripts; p, one-sided P-value from Kolmogorov-Smirnov test for a difference in distributions. (F) To confirm that our results were dependent on RISC association, cells were transfected with either single or double-stranded synthetic miRNAs, then subjected to AGO2 immunoprecipitation. The biotin pull-down was performed in the AGO2-enriched and AGO2-depleted fractions. (G-H) Quantitative RT-PCR revealed that, with double-stranded (ds) miRNA (G), four out of five known targets were enriched relative to input mRNA (*P≤0.05, **P<0.01, ***P<0.001) in the AGO2-enriched but not in the AGO2-depleted fractions, but this enrichment was not seen for the cells transfected with a single-stranded (ss) miRNA (H). The numbers on the x-axis correspond to those in Figure 2F. Error bars represent the standard error of mean (sem).
Figure 5 IsomiRs and canonical miRNAs target many of the same transcripts.
Figure 1. Features of hammerhead ribozymes. A generic diagram of a hammerhead ribozyme bound to its target substrate: NUH is the cleavage triplet on target sequence, stems I and III are sites of the specific interactions between ribozyme and target, stem II is the structural element connecting separate parts of the catalytic core. Arrows represent the cleavage site, numbering system according to Hertel et al. [60].
Figure 1 Schematic (A) and ribbon (B) diagrams depicting the crystal structure of the full-length hammerhead ribozyme. The sequence and secondary structure
TABLE 1 Typical examples of successful applications of hammerhead ribozymes. Most of the data are derived from [10] and [11], the others are expressly specified.
Growth factors, receptors, transduction elements
Oncogenes, protoncogenes, fusion genes
Apoptosis, survival factors, drug resistance
Transcription factors
Extracellular matrix, matrix modulating factors
Circulating factors
Viral genome, viral genes
Figure 2.Target–ribozyme interactions. (a) As cheme of ribozyme binding to full substrate. The calculated energy of this binding ensures the formation of a stable complex. At the denaturating temperature, Tm, will allow this complex to survive to biological conditions. Conversely, after cleavage, binding energies calculated on single, (b) and (c), ribozyme arms are very low and no longer stable. These properties will ensure both the efficient release of cleavage fragments and the prevention of binding to unrelated targets. RNAs complementary to one binding arm only will not be bound or cleaved by the hammerhead catalytic sequence.
Figure 3. ‘Chemical omics’ approach. According to this target discovery strategy: (1) a first round of ‘omic’ study (proteomic, genomic, metabolomic, …) will enable the discovery of a set of (2) putative markers. A series of hammerhead ribozymes will then be prepared in order to target each marker. (4) A second ‘omic’ study round will be performed on (3) knocked down samples obtained after ribozymes administration. (5) A new series of markers will then be produced. An expanding analytical process of this type may be further repeated. Finally, a robust bioinformatic algorithm will make it possible to connect the different markers and draw new hypothetical links and pathways.
miRNA
ADAR Enzyme and miRNA Story Sara Tomaselli, Barbara Bonamassa, Anna Alisi, et al.
Int. J. Mol. Sci. 2013, 14, 22796-22816; http://dx.doi.org:/10.3390/ijms141122796
Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.
Figure 1. Structure of ADAR family proteins: ADAR1, ADAR2, and ADAR3. The ADAR enzymes contain a C-terminal conserved catalytic deaminase domain (DM), two or three dsRBDs in the N-terminal portion. ADAR1 full-length protein also contains a N-terminal Zα domain with a nuclear export signal (NES) and a Zβ domain, while ADAR3 has a R-domain. A nuclear localization signal is also indicated.
Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites
Doron Betel, Anjali Koppal, Phaedra Agius, Chris Sander, Christina Leslie
Genome Biology 2010, 11:R90 http://genomebiology.com/2010/11/8/R90
microRNAs are a class of small regulatory RNAs that are involved in post-transcriptional gene silencing. These small (approximately 22 nucleotide) single-strand RNAs guide a gene silencing complex to an mRNA by complementary base pairing, mostly at the 3′ untranslated region (3′ UTR). The association of the RNAinduced silencing complex (RISC) to the conjugate mRNA results in silencing the gene either by translational repression or by degradation of the mRNA. Reliable microRNA target prediction is an important and still unsolved computational challenge, hampered both by insufficient knowledge of microRNA biology as well as the limited number of experimentally validated targets.
mirSVR is a new machine learning method for ranking microRNA target sites by a down-regulation score. The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites. Human RISC – MicroRNA Biogenesis and Posttranscriptional Gene Silencing
Cell 2005; 123:631-640 http://dx.doi.org:/10.1016/j.cell.2005.10.022 Development of microRNA therapeutics
Eva van Rooij & Sakari Kauppinen
EMBO Mol Med (2014) 6: 851–864 http://dx.doi.org:/10.15252/emmm.20110089
MicroRNAs (miRNAs) play key regulatory roles in diverse biological processes and are frequently dysregulated in human diseases. Thus, miRNAs have emerged as a class of promising targets for therapeutic intervention. Here, we describe the current strategies for therapeutic modulation of miRNAs and provide an update on the development of miRNA-based therapeutics for the treatment of cancer, cardiovascular disease and hepatitis C virus (HCV) infection.
Figure 1. miRNA biogenesis and modulation of miRNA activity by miRNA mimics and antimiR oligonucleotides. MiRNA genes are transcribed by RNA polymerase II from intergenic, intronic or polycistronic loci to long primary miRNA transcripts (pri-miRNAs) and processed in the nucleus by the Drosha–DGCR8 complex to approximately 70 nt pre-miRNA hairpin structures. The most common alternative miRNA biogenesis pathway involves short intronic hairpins, termed mirtrons, that are spliced and debranched to form pre-miRNA hairpins. Pre-miRNAs are exported into the cytoplasm and then cleaved by the Dicer–TRBP complex to imperfect miRNA: miRNA* duplexes about 22 nucleotides in length. In the cytoplasm, miRNA duplexes are incorporated into Argonaute-containing miRNA induced silencing complex (miRISC), followed by unwinding of the duplex and retention of the mature miRNA strand in miRISC, while the complementary strand is released and degraded. The mature miRNA functions as a guide molecule for miRISC by directing it to partially complementary sites in the target mRNAs, resulting in translational repression and/or mRNA degradation. Currently, two strategies are employed to modulate miRNA activity: restoring the function of a miRNA using double-stranded miRNA mimics, and inhibition of miRNA function using single-stranded anti-miR oligonucleotides.
Figure 2. Design of chemically modified miRNA modulators. (A) Structures of chemical modifications used in miRNA modulators. A number of different sugar modifications are used to increase the duplex melting temperature (Tm) of anti-miR oligonucleotides. The20-O-methyl(20-O-Me), 20-O-methoxyethyl(20-MOE )and 20-fluoro(20-F) nucleotides are modified at the 20 position of the sugar moiety, whereas locked nucleic acid (LNA) is a bicyclic RNA analogue in which the ribose is locked in a C30-endo conformation by introduction of a 20-O,40-C methylene bridge. To increase nuclease resistance and enhance the pharmacokinetic properties, most anti-miR oligonucleotides harbor phosphorothioate (PS) backbone linkages, in which sulfur replaces one of the non-bridging oxygen atoms in the phosphate group. In morpholino oligomers, a six-membered morpholine ring replaces the sugar moiety. Morpholinos are uncharged and exhibit a slight increase in binding affinity to their cognate miRNAs. PNA oligomers are uncharged oligonucleotide analogues, in which the sugar–phosphate backbone has been replaced by a peptide-like backbone consisting of N-(2-aminoethyl)-glycine units. (B) An example of a synthetic double-stranded miRNA mimic described in this review. One way to therapeutically mimic a miRNA is by using synthetic RNA duplexes that harbor chemical modifications for improved stability and cellular uptake. In such constructs, the antisense (guide) strand is identical to the miRNA of interest, while the sense (passenger) strand is modified and can be linked to a molecule, such as cholesterol, for enhanced cellular uptake. The sense strand contains chemical modifications to prevent mi-RISC loading. Several mismatches can be introduced to prevent this strand from functioning as an anti-miR, while it is further left unmodified to ensure rapid degradation.The20-F modification helps to protect the antisense strand against exonucleases, hence making the guide strand more stable, while it does not interfere with mi-RISC loading. (C) Design of chemically modified anti-miR oligonucleotides described in this review. Antagomirs are30 cholesterol-conjugated,20-O-Me oligonucleotides fully complementary to the mature miRNA sequence with several PS moieties to increase their in vivo stability. The use of unconjugated 20-F/MOE-, 20-MOE- or LNA-modified anti-miR oligonucleotides harboring a complete PS backbone represents another approach for inhibition of miRNA function in vivo. The high duplex melting temperature of LNA-modified oligonucleotides allows efficient miRNA inhibition using truncated, high-affinity 15–16-nucleotide LNA/DNA anti-miR oligonucleotides targeting the 50 region of the mature miRNA. Furthermore, the high binding affinity of fully LNA-modified 8-mer PS oligonucleotides, designated as tiny LNAs, facilitates simultaneous inhibition of entire miRNA seed families by targeting the shared seed sequence.
Human MicroRNA Targets
Bino John, Anton J. Enright, Alexei Aravin, Thomas Tuschl,.., Debora S. Mark
PLoS Biol 2004; 2(11): e363 http://www.plosbiology.org
More than ten years after the discovery of the first miRNA gene, lin-4 (Chalfie et al. 1981; Lee et al. 1993), we know that miRNA genes constitute about 1%–2% of the known genes in eukaryotes. Investigation of miRNA expression combined with genetic and molecular studies in Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana have identified the biological functions of several miRNAs (recent review, Bartel 2004). In C. elegans, lin-4 and let-7 were first discovered as key regulators of developmental timing in early larval developmental transitions (Ambros 2000; Abrahante et al. 2003; Lin et al. 2003; Vella et al. 2004). More recently lsy-6 was shown to determine the left–right asymmetry of chemoreceptor expression (Johnston and Hobert 2003). In D. melanogaster, miR-14 has a role in apoptosis and fat metabolism (Xu et al. 2003) and the bantam miRNA targets the gene hid involved in apoptosis and growth control (Brennecke et al. 2003).
MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. The specific function of most mammalian miRNAs is unknown. We have predicted target sites on the 39 untranslated regions of human gene transcripts for all currently known 218 mammalian miRNAs to facilitate focused experiments. We report about 2,000 human genes with miRNA target sites conserved in mammals and about 250 human genes conserved as targets between mammals and fish. The prediction algorithm optimizes sequence complementarity using position-specific rules and relies on strict requirements of interspecies conservation. Experimental support for the validity of the method comes from known targets and from strong enrichment of predicted targets in mRNAs associated with the fragile X mental retardation protein in mammals. This is consistent with the hypothesis that miRNAs act as sequence-specific adaptors in the interaction of ribonuclear particles with translationally regulated messages. Overrepresented groups of targets include mRNAs coding for transcription factors, components of the miRNA machinery, and other proteins involved in translational regulation, as well as components of the ubiquitin machinery, representing novel feedback loops in gene regulation. Detailed information about target genes, target processes, and open-source software for target prediction (miRanda) is available at http://www.microrna.org. Our analysis suggests that miRNA genes, which are about 1% of all human genes, regulate protein production for 10% or more of all human genes.
Figure 1. Target Prediction Pipeline for miRNA Targets in Vertebrates The mammalian (human, mouse, and rat) and fish (zebra and fugu) 39 UTRs were first scanned for miRNA target sites using position specific rules of sequence complementarity. Next, aligned UTRs of orthologous genes were used to check for conservation of miRNA– target relationships (‘‘target conservation’’) between mammalian genomes and, separately, between fish genomes. The main results (bottom) are the conserved mammalian and conserved fish targets, for each miRNA,as well as a smaller set of super-conserved vertebrate targets. http://dx.doi.org:/10.1371/journal.pbio.0020363.g00
Figure 2. Distribution of Transcripts with Cooperativity of Target Sites and Estimated Number of False Positives Each bar reflects the number of human transcripts with a given number of target sites on their UTR. Estimated rate of false positives(e.g., 39%for2 targets) is given by the number of target sites predicted using shuffled miRNAs processed in a way identical to real miRNAs, including the use of interspecies conservation filter. http://dx.doi.org:/10.1371/journal.pbio.0020363.g002
Conserved Seed Pairing, Often improved an-Flanked by Adenosines, Indicates Thousands of Human Genes are MicroRNA Targets Cell, Jan 2005; 120: 15–20 http://dx.doi.org:/10.1016/j.cell.2004.12.035
Integrated analysis of microRNA and mRNA expression. adding biological significance to microRNA target predictions.
Maarten van Iterson, Sander Bervoets, Emile J. de Meijer, et al.
Nucleic Acids Research, 2013; 41(15), e146 http://dx.doi.org:/10.1093/nar/gkt525
Current microRNA target predictions are based on sequence information and empirically derived rules but do not make use of the expression of microRNAs and their targets. This study aimed to improve microRNA target predictions in a given biological context, using in silico predictions, microRNA and mRNA expression. We used target prediction tools to produce lists of predicted targets and used a gene set test designed to detect consistent effects of microRNAs on the joint expression of multiple targets. In a single test, association between microRNA expression and target gene set expression as well as the contribution of the individual target genes on the association are determined. The strongest negatively associated mRNAs as measured by the test were prioritized. We applied our integration method to a well-defined muscle differentiation model. Validation of our predictions in C2C12 cells confirmed predicted targets of known as well as novel muscle-related microRNAs. We further studied associations between microRNA–mRNA pairs in human prostate cancer, finding some pairs that have been recently experimentally validated by others. Using the same study, we showed the advantages of the global test over Pearson correlation and lasso. We conclude that our integrated approach successfully identifies regulated microRNAs and their targets.
Fig. 1. A ‘‘Domain-centric’’ view of RNAi. (A) The conserved pathways of RNA silencing. The domain structure of each protein in (hypothetical) interaction with its RNA is shown. For clarity, the second column lists domains in order N- to C-terminal. Figures are not to scale. In brief, Drosha, an RNase III enzyme, and its obligate binding partner, Pasha recognize pri-mRNA loops, and cut these into 70 nt hairpin pre-miRNAs. Dicer utilizes a PAZ domain to sense the 30 2-nt overhang created, and further processes these, and dsRNAs into miRNAs and siRNAs. Argonaute binds the 50 end of guide RNAs via its PIWI domain, and the 30 end via a PAZ domain, yielding RISCs that effect RNA silencing through several mechanisms. A Viral protein, VP19 can suppress RNA silencing by sequestering siRNAs. (B) A summary of known siRNA structural biology. Listed by domain are solved structures, their protein/organism of origin, and ligands, where applicable. Also shown are PDB codes.
Fig. 2. Novel modes of RNA recognition. (A) A typical dsRBD: Xenopus binding protein A (1DI2). A RNA helix is modeled pink, and the protein is rendered in transparent electrostatic contours (blue is basic, red acidic). Note the interaction of helices along the major groove, and the position of helix 1. A second dsRBD protein is visible, in the lower right. (B) A dsRBD, Saccharomyces Rnt1P (1T4L), recognizes hairpin loops. A novel third helix (top) pushes helix one into the loop of a hairpin RNA. (C) 30-OH recognition by PAZ. Human Eif2c1 (1SI3) bound to RNA (pink) is shown. PAZ is green, with transparent electrostatic surface plot. The OB-fold (nucleotide binding fold) and the insertion domain are labeled. Note the glove-and-thumb like cleft they form, that the 30-OH is inserted into. A basic groove (blue) the RNA binds along outside the cleft is visible. (D) A close-up view of PAZ, as in C (surface not-transparent, slightly rotated). See white arrows for orientation, and location of 30-OH binding site. RNA is shown red in sticks. The terminal –OH is barely visible, buried in a cleft. It and the carbon it bonds have been colored yellow for clarity. (E) The PIWI domain (2BGG). Note the insertion of the 50P red (labeled) into the binding site. Its complimentary strand (pink) is not annealed to it, and the 30 overhang and first complimentary bases sit on the protein surface. (F) An enlarged view of (E), with protein in slate and RNA modeled as red sticks. The coordinated magnesium is a grey sphere, which is coordinated by the terminal carboxylate of the protein, protein side chains, and RNA phosphate oxygens. The 50 base stacks against a conserved Tyr. Several other sidechain contacts are shown.
Fig. 3. Argonaute/RISC. (A) P. furiosus Argonaute (PDB 1Z26). A color-guided key to the domains is presented. PAZ sits over the PIWI/N/MID bowl and active site. The liganding atoms for the catalytic metal are depicted as yellow balls for clarity. The tungstate binding site (50P surrogate) is shown as tan spheres. (B) A guide strand channel. Looking down from the PAZ domain towards the active site, Z-sections are clipped off. Colors of domains are as in the key in (A). Wrapping down along a basic cleft from the PAZ 30OH binding site (approximate position labeled), a RNA binding groove passes the active site (yellow), and runs down to the 50P binding site (tan balls). A second cleft running perpendicular to this one at its entry may accommodate target strand RNA. For more detail, and models of siRNA placed into the grooves, see [27,29].
Fig. 4. VP19 sequestration of siRNA. (A) CIRV VP19 (1RPU, RNA removed). Two monomers (blue and cyan) form an 8 strand, concave b-sheet with bracketing helices at the ends. (B) Tombus viral VP19 bound to siRNA (1 monomer shown). RNA strands are modeled as sticks, with one strand pink and one red. The bracketing helix places two tryptophans in position to stack over the terminal RNA bases. On the b-sheet surface, and Arg and a Lys interact with the phosphate backbone, and at the center of the RNA binding surface, a number of Ser and Thr mediate an extensive hydrogen bond network. Both the Trp brackets and RNA binding by an extended b-sheet are unique.
Figure 1. Noncoding RNAs Function in Diverse Contexts Noncoding RNAs function in all domains of life, regulating gene expression from transcription to splicing to translation and contributing to genome organization and stability. Self-splicing RNAs, ribosomes, and riboswitches function in both eukaryotes and bacteria. Archaea (not shown) also utilize ncRNA systems including ribosomes, riboswitches, snoRNPs, and CRISPR. Orange strands, ncRNA performing the action indicated; red strands, the RNA acted upon by the ncRNA. Blue strands, DNA. Triangle, small-molecule metabolite bound by a riboswitch. Ovals indicate protein components of an RNP, such as the spliceosome (white oval), ribosome (two purple subunits), or other RNPs (yellow ovals). Because of the importance of RNA structure in these ncRNAs, some structures are shown but they are not meant to be realistic.
miRNAs and cancer targeting
Table 1 of targets
miRNA
Cancer type
reference
NA
GI cancer
Current status of miRNA-targeting therapeutics and preclinical studies against gastroenterological carcinoma
NA
Renal cell
Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis
NA
urothelial
cancer
A microRNA expression ratio defining the invasive phenotype in bladder tumors
miR-31
breast
A Pleiotropically Acting MicroRNA, miR-31, inhibits breast cancer growth
miR-512-3p
NSCLC
Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer
miR-495
gastric
Methylation-associated silencing of miR-495 inhibit the migration and invasion of human gastric cancer cells
microRNA-218
prostate
microRNA-218 inhibits prostate cancer cell growth and promotes apoptosis by repressing TPD52 expression
MicroRNA-373
cervical cancer
MicroRNA-373 functions as an oncogene and targets YOD1 gene in cervical cancer
miR-92a. upregulated in cervical cancer & promotes cell proliferation and invasion by targeting FBXW7
MiR-153
NSCLC
MiR-153 inhibits migration and invasion of human non-small-cell lung cancer by targeting ADAM19
miR-203
melanoma
miR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1
miR-204-5p
Papillary thyroid
miR-204-5p suppresses cell proliferation by inhibiting IGFBP5 in papillary thyroid carcinoma
miR-342-3p
Hepato-cellular
miR-342-3p affects hepatocellular carcinoma cell proliferation via regulating NF-κB pathway
miR-1271
NSCLC
miR-1271 promotes non-small-cell lung cancer cell proliferation and invasion via targeting HOXA5
miR-203
pancreas
Pancreatic cancer derived exosomes regulate the expression of TLR4 in dendritic cells via miR-203
miR-203
metastatic SCC
Rewiring of an Epithelial Differentiation Factor, miR-203, to Inhibit Human SCC Metastasis
miR-204
RCC
TRPM3 and miR-204 Establish a Regulatory Circuit that Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma
NA
urologic
MicroRNAs and cancer. Current and future perspectives in urologic oncology
NA
RCC
MicroRNAs and their target gene networks in renal cell carcinoma
NA
osteoSA
MicroRNAs in osteosarcoma
NA
urologic
MicroRNA in Prostate, Bladder, and Kidney Cancer
NA
urologic
Micro-RNA profiling in kidney and bladder cancers
Current status of miRNA-targeting therapeutics and preclinical studies against gastroenterological carcinoma
Shibata et al. Molecular and Cellular Therapies 2013, 1:5 http://www.molcelltherapies.com/content/1/1/5
Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis
Clinical Biochemistry 43 (2010) 150–158 http://dx.doi.org:/10.1016/j.clinbiochem.2009.07.020
A microRNA expression ratio defining the invasive phenotype in bladder tumors
Urologic Oncology: Seminars and Original Investigations 28 (2010) 39–48 http://dx.doi.org:/10.1016/j.urolonc.2008.06.006
Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer
Intl JBiochem & Cell Biol 2015; 61:103-114 http://dx.doi.org/10.1016/j.biocel.2015.02.005
Methylation-associated silencing of miR-495 inhibit the migration and invasion of human gastric cancer cells by directly targeting PRL-3
Biochem Biochem Res Commun 2014; 456:344-350 http://dx.doi.org/10.1016/j.bbrc.2014.11.083
microRNA-218 inhibits prostate cancer cell growth and promotes apoptosis by repressing TPD52 expression
Biochem Biophys Res Commun 2015; 456:804-809 http://dx.doi.org/10.1016/j.bbrc.2014.12.026
Rewiring of an Epithelial Differentiation Factor, miR-203, to Inhibit Human Squamous Cell Carcinoma Metastasis
Cell Reports 2014; 9:104-117 http://dx.doi.org/10.1016/j.celrep.2014.08.062
TRPM3 and miR-204 Establish a Regulatory Circuit that Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma
Cancer Cell Nov 10, 2014; 26: 738–753 http://dx.doi.org/10.1016/j.ccell.2014.09.015
MicroRNAs and cancer. Current and future perspectives in urologic oncology
Urologic Oncology: Seminars and Original Investigations 2010; 28:4–13 http://dx.doi.org:/10.1016/j.urolonc.2008.10.021
miRNA and mRNA cancer signatures determined by analysis of expression levels in large cohorts of patients
| PNAS | Nov 19, 2013; 110(47): 19160–19165 http://www.pnas.org/cgi/doi/10.1073/pnas.1316991110The study of mRNA and microRNA (miRNA) expression profiles of cells and tissue has become a major tool for therapeutic development. The results of such experiments are expected to change the methods used in the diagnosis and prognosis of disease. We introduce surprisal analysis, an information-theoretic approach grounded in thermodynamics, to compactly transform the information acquired from microarray studies into applicable knowledge about the cancer phenotypic state. The analysis of mRNA and miRNA expression data from ovarian serous carcinoma, prostate adenocarcinoma, breast invasive carcinoma, and lung adenocarcinoma cancer patients and organ specific control patients identifies cancer-specific signatures. We experimentally examine these signatures and their respective networks as possible therapeutic targets for cancer in single cell experiments.
RNA editing is vital to provide the RNA and protein complexity to regulate the gene expression. Correct RNA editing maintains the cell function and organism development. Imbalance of the RNA editing machinery may lead to diseases and cancers. Recently,RNA editing has been recognized as a target for drug discovery although few studies targeting RNA editing for disease and cancer therapy were reported in the field of natural products. Therefore, RNA editing may be a potential target for therapeutic natural products
Aberrant microRNA (miRNA) expression is implicated in tumorigenesis. The underlying mechanisms are unclear because the regulations of each miRNA on potentially hundreds of mRNAs are sample specific.
We describe a novel approach to infer Probabilistic Mi RNA–mRNA Interaction Signature (‘ProMISe’) from a single pair of miRNA–mRNA expression profile. Our model considers mRNA and miRNA competition as a probabilistic function of the expressed seeds (matches). To demonstrate ProMISe, we extensively exploited The Cancer Genome Atlas data. As a target predictor, ProMISe identifies more confidence/validated targets than other methods. Importantly, ProMISe confers higher cancer diagnostic power than using expression profiles alone.
Gene set enrichment analysis on averaged ProMISe uniquely revealed respective target enrichments of oncomirs miR-21 and 145 in glioblastoma and ovarian cancers. Moreover, comparing matched breast (BRCA) and thyroid (THCA) tumor/normal samples uncovered thousands of tumor-related interactions. For example, ProMISe– BRCA network involves miR-155/183/21, which exhibits higher ProMISe coupled with coherently higher miRNA expression and lower target expression; oncomirs miR-221/222 in the ProMISe–THCA network engage with many downregulated target genes. Together, our probabilistic approach of integrating expression and sequence scores establishes a functional link between the aberrant miRNA and mRNA expression, which was previously under-appreciated due to the methodological differences.
This is the final article in a robust series on metabolism, metabolomics, and the “-OMICS-“ biological synthesis that is creating a more holistic and interoperable view of natural sciences, including the biological disciplines, climate science, physics, chemistry, toxicology, pharmacology, and pathophysiology with as yet unforeseen consequences.
There have been impressive advances already in the research into developmental biology, plant sciences, microbiology, mycology, and human diseases, most notably, cancer, metabolic , and infectious, as well as neurodegenerative diseases.
Acknowledgements:
I write this article in honor of my first mentor, Harry Maisel, Professor and Emeritus Chairman of Anatomy, Wayne State University, Detroit, MI and to my stimulating mentors, students, fellows, and associates over many years:
Masahiro Chiga, MD, PhD, Averill A Liebow, MD, Nathan O Kaplan, PhD, Johannes Everse, PhD, Norio Shioura, PhD, Abraham Braude, MD, Percy J Russell, PhD, Debby Peters, Walter D Foster, PhD, Herschel Sidransky, MD, Sherman Bloom, MD, Matthew Grisham, PhD, Christos Tsokos, PhD, IJ Good, PhD, Distinguished Professor, Raool Banagale, MD, Gustavo Reynoso, MD,Gustave Davis, MD, Marguerite M Pinto, MD, Walter Pleban, MD, Marion Feietelson-Winkler, RD, PhD, John Adan,MD, Joseph Babb, MD, Stuart Zarich, MD, Inder Mayall, MD, A Qamar, MD, Yves Ingenbleek, MD, PhD, Emeritus Professor, Bette Seamonds, PhD, Larry Kaplan, PhD, Pauline Y Lau, PhD, Gil David, PhD, Ronald Coifman, PhD, Emeritus Professor, Linda Brugler, RD, MBA, James Rucinski, MD, Gitta Pancer, Ester Engelman, Farhana Hoque, Mohammed Alam, Michael Zions, William Fleischman, MD, Salman Haq, MD, Jerard Kneifati-Hayek, Madeleine Schleffer, John F Heitner, MD, Arun Devakonda,MD, Liziamma George,MD, Suhail Raoof, MD, Charles Oribabor,MD, Anthony Tortolani, MD, Prof and Chairman, JRDS Rosalino, PhD, Aviva Lev Ari, PhD, RN, Rosser Rudolph, MD, PhD, Eugene Rypka, PhD, Jay Magidson, PhD, Izaak Mayzlin, PhD, Maurice Bernstein, PhD, Richard Bing, Eli Kaplan, PhD, Maurice Bernstein, PhD.
This article has EIGHT parts, as follows:
Part 1
Metabolomics Continues Auspicious Climb
Part 2
Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells
Part 3
Neuroscience
Part 4
Cancer Research
Part 5
Metabolic Syndrome
Part 6
Biomarkers
Part 7
Epigenetics and Drug Metabolism
Part 8
Pictorial
genome cartoon
iron metabolism
personalized reference range within population range
Part 1. MetabolomicsSurge
metagraph _OMICS
Metabolomics Continues Auspicious Climb
Jeffery Herman, Ph.D.
GEN May 1, 2012 (Vol. 32, No. 9)
Aberrant biochemical and metabolite signaling plays an important role in
the development and progression of diseased tissue.
This concept has been studied by the science community for decades. However, with relatively
recent advances in analytical technology and bioinformatics as well as
the development of the Human Metabolome Database (HMDB),
metabolomics has become an invaluable field of research.
At the “International Conference and Exhibition on Metabolomics & Systems Biology” held recently in San Francisco, researchers and industry leaders discussed how
the underlying cellular biochemical/metabolite fingerprint in response to
a specific disease state,
toxin exposure, or
pharmaceutical compound
is useful in clinical diagnosis and biomarker discovery and
in understanding disease development and progression.
Developed by BASF, MetaMap® Tox is
a database that helps identify in vivo systemic effects of a tested compound, including
targeted organs,
mechanism of action, and
adverse events.
Based on 28-day systemic rat toxicity studies, MetaMap Tox is composed of
differential plasma metabolite profiles of rats
after exposure to a large variety of chemical toxins and pharmaceutical compounds.
“Using the reference data,
we have developed more than 110 patterns of metabolite changes, which are
specific and predictive for certain toxicological modes of action,”
said Hennicke Kamp, Ph.D., group leader, department of experimental toxicology and ecology at BASF.
With MetaMap Tox, a potential drug candidate
can be compared to a similar reference compound
using statistical correlation algorithms,
which allow for the creation of a toxicity and mechanism of action profile.
“MetaMap Tox, in the context of early pre-clinical safety enablement in pharmaceutical development,” continued Dr. Kamp,
has been independently validated “
by an industry consortium (Drug Safety Executive Council) of 12 leading biopharmaceutical companies.”
Dr. Kamp added that this technology may prove invaluable
allowing for quick and accurate decisions and
for high-throughput drug candidate screening, in evaluation
on the safety and efficacy of compounds
during early and preclinical toxicological studies,
by comparing a lead compound to a variety of molecular derivatives, and
the rapid identification of the most optimal molecular structure
with the best efficacy and safety profiles might be streamlined.
Dynamic Construct of the –Omics
Targeted Tandem Mass Spectrometry
Biocrates Life Sciences focuses on targeted metabolomics, an important approach for
the accurate quantification of known metabolites within a biological sample.
Originally used for the clinical screening of inherent metabolic disorders from dried blood-spots of newborn children, Biocrates has developed
a tandem mass spectrometry (MS/MS) platform, which allows for
the identification,
quantification, and
mapping of more than 800 metabolites to specific cellular pathways.
It is based on flow injection analysis and high-performance liquid chromatography MS/MS.
common drug targets
The MetaDisIDQ® Kit is a
“multiparamatic” diagnostic assay designed for the “comprehensive assessment of a person’s metabolic state” and
the early determination of pathophysiological events with regards to a specific disease.
MetaDisIDQ is designed to quantify
a diverse range of 181 metabolites involved in major metabolic pathways
from a small amount of human serum (10 µL) using isotopically labeled internal standards,
This kit has been demonstrated to detect changes in metabolites that are commonly associated with the development of
metabolic syndrome, type 2 diabetes, and diabetic nephropathy,
Dr. Dallman reports that data generated with the MetaDisIDQ kit correlates strongly with
routine chemical analyses of common metabolites including glucose and creatinine
Biocrates has also developed the MS/MS-based AbsoluteIDQ® kits, which are
an “easy-to-use” biomarker analysis tool for laboratory research.
The kit functions on MS machines from a variety of vendors, and allows for the quantification of 150-180 metabolites.
The SteroIDQ® kit is a high-throughput standardized MS/MS diagnostic assay,
validated in human serum, for the rapid and accurate clinical determination of 16 known steroids.
Initially focusing on the analysis of steroid ranges for use in hormone replacement therapy, the SteroIDQ Kit is expected to have a wide clinical application.
Hormone-Resistant Breast Cancer
Scientists at Georgetown University have shown that
breast cancer cells can functionally coordinate cell-survival and cell-proliferation mechanisms,
while maintaining a certain degree of cellular metabolism.
To grow, cells need energy, and energy is a product of cellular metabolism. For nearly a century, it was thought that
the uncoupling of glycolysis from the mitochondria,
leading to the inefficient but rapid metabolism of glucose and
the formation of lactic acid (the Warburg effect), was
the major and only metabolism driving force for unchecked proliferation and tumorigenesis of cancer cells.
Other aspects of metabolism were often overlooked.
“.. we understand now that
cellular metabolism is a lot more than just metabolizing glucose,”
said Robert Clarke, Ph.D., professor of oncology and physiology and biophysics at Georgetown University. Dr. Clarke, in collaboration with the Waters Center for Innovation at Georgetown University (led by Albert J. Fornace, Jr., M.D.), obtained
the metabolomic profile of hormone-sensitive and -resistant breast cancer cells through the use of UPLC-MS.
They demonstrated that breast cancer cells, through a rather complex and not yet completely understood process,
can functionally coordinate cell-survival and cell-proliferation mechanisms,
while maintaining a certain degree of cellular metabolism.
This is at least partly accomplished through the upregulation of important pro-survival mechanisms; including
the unfolded protein response;
a regulator of endoplasmic reticulum stress and
initiator of autophagy.
Normally, during a stressful situation, a cell may
enter a state of quiescence and undergo autophagy,
a process by which a cell can recycle organelles
in order to maintain enough energy to survive during a stressful situation or,
if the stress is too great,
undergo apoptosis.
By integrating cell-survival mechanisms and cellular metabolism
advanced ER+ hormone-resistant breast cancer cells
can maintain a low level of autophagy
to adapt and resist hormone/chemotherapy treatment.
This adaptation allows cells
to reallocate important metabolites recovered from organelle degradation and
provide enough energy to also promote proliferation.
With further research, we can gain a better understanding of the underlying causes of hormone-resistant breast cancer, with
the overall goal of developing effective diagnostic, prognostic, and therapeutic tools.
NMR
Over the last two decades, NMR has established itself as a major tool for metabolomics analysis. It is especially adept at testing biological fluids. [Bruker BioSpin]
Historically, nuclear magnetic resonance spectroscopy (NMR) has been used for structural elucidation of pure molecular compounds. However, in the last two decades, NMR has established itself as a major tool for metabolomics analysis. Since
the integral of an NMR signal is directly proportional to
the molar concentration throughout the dynamic range of a sample,
“the simultaneous quantification of compounds is possible
without the need for specific reference standards or calibration curves,” according to Lea Heintz of Bruker BioSpin.
NMR is adept at testing biological fluids because of
high reproducibility,
standardized protocols,
low sample manipulation, and
the production of a large subset of data,
Bruker BioSpin is presently involved in a project for the screening of inborn errors of metabolism in newborn children from Turkey, based on their urine NMR profiles. More than 20 clinics are participating to the project that is coordinated by INFAI, a specialist in the transfer of advanced analytical technology into medical diagnostics. The construction of statistical models are being developed
for the detection of deviations from normality, as well as
automatic quantification methods for indicative metabolites
Bruker BioSpin recently installed high-resolution magic angle spinning NMR (HRMAS-NMR) systems that can rapidly analyze tissue biopsies. The main objective for HRMAS-NMR is to establish a rapid and effective clinical method to assess tumor grade and other important aspects of cancer during surgery.
Combined NMR and Mass Spec
There is increasing interest in combining NMR and MS, two of the main analytical assays in metabolomic research, as a means
to improve data sensitivity and to
fully elucidate the complex metabolome within a given biological sample.
to realize a potential for cancer biomarker discovery in the realms of diagnosis, prognosis, and treatment.
.
Using combined NMR and MS to measure the levels of nearly 250 separate metabolites in the patient’s blood, Dr. Weljie and other researchers at the University of Calgary were able to rapidly determine the malignancy of a pancreatic lesion (in 10–15% of the cases, it is difficult to discern between benign and malignant), while avoiding unnecessary surgery in patients with benign lesions.
When performing NMR and MS on a single biological fluid, ultimately “we are,” noted Dr. Weljie,
“splitting up information content, processing, and introducing a lot of background noise and error and
then trying to reintegrate the data…
It’s like taking a complex item, with multiple pieces, out of an IKEA box and trying to repackage it perfectly into another box.”
By improving the workflow between the initial splitting of the sample, they improved endpoint data integration, proving that
a streamlined approach to combined NMR/MS can be achieved,
leading to a very strong, robust and precise metabolomics toolset.
Metabolomics Research Picks Up Speed
Field Advances in Quest to Improve Disease Diagnosis and Predict Drug Response
John Morrow Jr., Ph.D.
GEN May 1, 2011 (Vol. 31, No. 9)
As an important discipline within systems biology, metabolomics is being explored by a number of laboratories for
its potential in pharmaceutical development.
Studying metabolites can offer insights into the relationships between genotype and phenotype, as well as between genotype and environment. In addition, there is plenty to work with—there are estimated to be some 2,900 detectable metabolites in the human body, of which
309 have been identified in cerebrospinal fluid,
1,122 in serum,
458 in urine, and
roughly 300 in other compartments.
Guowang Xu, Ph.D., a researcher at the Dalian Institute of Chemical Physics. is investigating the causes of death in China,
and how they have been changing over the years as the country has become a more industrialized nation.
the increase in the incidence of metabolic disorders such as diabetes has grown to affect 9.7% of the Chinese population.
Dr. Xu, collaborating with Rainer Lehman, Ph.D., of the University of Tübingen, Germany, compared urinary metabolites in samples from healthy individuals with samples taken from prediabetic, insulin-resistant subjects. Using mass spectrometry coupled with electrospray ionization in the positive mode, they observed striking dissimilarities in levels of various metabolites in the two groups.
“When we performed a comprehensive two-dimensional gas chromatography, time-of-flight mass spectrometry analysis of our samples, we observed several metabolites, including
2-hydroxybutyric acid in plasma,
as potential diabetes biomarkers,” Dr. Xu explains.
In other, unrelated studies, Dr. Xu and the German researchers used a metabolomics approach to investigate the changes in plasma metabolite profiles immediately after exercise and following a 3-hour and 24-hour period of recovery. They found that
medium-chain acylcarnitines were the most distinctive exercise biomarkers, and
they are released as intermediates of partial beta oxidation in human myotubes and mouse muscle tissue.
Dr. Xu says. “The traditional approach of assessment based on a singular biomarker is being superseded by the introduction of multiple marker profiles.”
Typical of the studies under way by Dr. Kaddurah-Daouk and her colleaguesat Duke University
is a recently published investigation highlighting the role of an SNP variant in
the glycine dehydrogenase gene on individual response to antidepressants.
patients who do not respond to the selective serotonin uptake inhibitors citalopram and escitalopram
carried a particular single nucleotide polymorphism in the GD gene.
“These results allow us to pinpoint a possible
role for glycine in selective serotonin reuptake inhibitor response and
illustrate the use of pharmacometabolomics to inform pharmacogenomics.
These discoveries give us the tools for prognostics and diagnostics so that
we can predict what conditions will respond to treatment.
“This approach to defining health or disease in terms of metabolic states opens a whole new paradigm.
By screening hundreds of thousands of molecules, we can understand
the relationship between human genetic variability and the metabolome.”
Dr. Kaddurah-Daouk talks about statins as a current
model of metabolomics investigations.
It is now known that the statins have widespread effects, altering a range of metabolites. To sort out these changes and develop recommendations for which individuals should be receiving statins will require substantial investments of energy and resources into defining the complex web of biochemical changes that these drugs initiate.
Furthermore, Dr. Kaddurah-Daouk asserts that,
“genetics only encodes part of the phenotypic response.
One needs to take into account the
net environment contribution in order to determine
how both factors guide the changes in our metabolic state that determine the phenotype.”
Interactive Metabolomics
Researchers at the University of Nottingham use diffusion-edited nuclear magnetic resonance spectroscopy to assess the effects of a biological matrix on metabolites. Diffusion-edited NMR experiments provide a way to
separate the different compounds in a mixture
based on the differing translational diffusion coefficients (which reflect the size and shape of the molecule).
The measurements are carried out by observing
the attenuation of the NMR signals during a pulsed field gradient experiment.
Clare Daykin, Ph.D., is a lecturer at the University of Nottingham, U.K. Her field of investigation encompasses “interactive metabolomics,”which she defines as
“the study of the interactions between low molecular weight biochemicals and macromolecules in biological samples ..
without preselection of the components of interest.
“Blood plasma is a heterogeneous mixture of molecules that
undergo a variety of interactions including metal complexation,
chemical exchange processes,
micellar compartmentation,
enzyme-mediated biotransformations, and
small molecule–macromolecular binding.”
Many low molecular weight compounds can exist
freely in solution,
bound to proteins, or
within organized aggregates such as lipoprotein complexes.
Therefore, quantitative comparison of plasma composition from
diseased individuals compared to matched controls provides an incomplete insight to plasma metabolism.
“It is not simply the concentrations of metabolites that must be investigated,
but their interactions with the proteins and lipoproteins within this complex web.
Rather than targeting specific metabolites of interest, Dr. Daykin’s metabolite–protein binding studies aim to study
the interactions of all detectable metabolites within the macromolecular sample.
Such activities can be studied through the use of diffusion-edited nuclear magnetic resonance (NMR) spectroscopy, in which one can assess
the effects of the biological matrix on the metabolites.
“This can lead to a more relevant and exact interpretation
for systems where metabolite–macromolecule interactions occur.”
Diffusion-edited NMR experiments provide a way to separate the different compounds in a mixture based on
the differing translational diffusion coefficients (which reflect the size and shape of the molecule).
The measurements are carried out by observing
the attenuation of the NMR signals during a pulsed field gradient experiment.
Pushing the Limits
It is widely recognized that many drug candidates fail during development due to ancillary toxicity. Uwe Sauer, Ph.D., professor, and Nicola Zamboni, Ph.D., researcher, both at the Eidgenössische Technische Hochschule, Zürich (ETH Zürich), are applying
high-throughput intracellular metabolomics to understand
the basis of these unfortunate events and
head them off early in the course of drug discovery.
“Since metabolism is at the core of drug toxicity, we developed a platform for
measurement of 50–100 targeted metabolites by
a high-throughput system consisting of flow injection
coupled to tandem mass spectrometry.”
Using this approach, Dr. Sauer’s team focused on
the central metabolism of the yeast Saccharomyces cerevisiae, reasoning that
this core network would be most susceptible to potential drug toxicity.
Screening approximately 41 drugs that were administered at seven concentrations over three orders of magnitude, they observed changes in metabolome patterns at much lower drug concentrations without attendant physiological toxicity.
The group carried out statistical modeling of about
60 metabolite profiles for each drug they evaluated.
This data allowed the construction of a “profile effect map” in which
the influence of each drug on metabolite levels can be followed, including off-target effects, which
provide an indirect measure of the possible side effects of the various drugs.
Dr. Sauer says.“We have found that this approach is
at least 100 times as fast as other omics screening platforms,”
“Some drugs, including many anticancer agents,
disrupt metabolism long before affecting growth.”
killing cancer cells
Furthermore, they used the principle of 13C-based flux analysis, in which
metabolites labeled with 13C are used to follow the utilization of metabolic pathways in the cell.
These 13C-determined intracellular responses of metabolic fluxes to drug treatment demonstrate
the functional performance of the network to be rather robust,
conformational changes leading to substrate efflux.
leading Dr. Sauer to the conclusion that
the phenotypic vigor he observes to drug challenges
is achieved by a flexible make up of the metabolome.
Dr. Sauer is confident that it will be possible to expand the scope of these investigations to hundreds of thousands of samples per study. This will allow answers to the questions of
how cells establish a stable functioning network in the face of inevitable concentration fluctuations.
Is Now the Hour?
There is great enthusiasm and agitation within the biotech community for
metabolomics approaches as a means of reversing the dismal record of drug discovery
that has accumulated in the last decade.
While the concept clearly makes sense and is being widely applied today, there are many reasons why drugs fail in development, and metabolomics will not be a panacea for resolving all of these questions. It is too early at this point to recognize a trend or a track record, and it will take some time to see how this approach can aid in drug discovery and shorten the timeline for the introduction of new pharmaceutical agents.
Degree of binding correlated with function
Diagram_of_a_two-photon_excitation_microscope_
Part 2. Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells
Biologists at UC San Diego have found
the “missing link” in the chemical system that
enables animal cells to produce ribosomes
—the thousands of protein “factories” contained within each cell that
manufacture all of the proteins needed to build tissue and sustain life.
‘Missing Link’
Their discovery, detailed in the June 23 issue of the journal Genes & Development, will not only force
a revision of basic textbooks on molecular biology, but also
provide scientists with a better understanding of
how to limit uncontrolled cell growth, such as cancer,
that might be regulated by controlling the output of ribosomes.
Ribosomes are responsible for the production of the wide variety of proteins that include
enzymes;
structural molecules, such as hair,
skin and bones;
hormones like insulin; and
components of our immune system such as antibodies.
Regarded as life’s most important molecular machine, ribosomes have been intensively studied by scientists (the 2009 Nobel Prize in Chemistry, for example, was awarded for studies of its structure and function). But until now researchers had not uncovered all of the details of how the proteins that are used to construct ribosomes are themselves produced.
In multicellular animals such as humans,
ribosomes are made up of about 80 different proteins
(humans have 79 while some other animals have a slightly different number) as well as
four different kinds of RNA molecules.
In 1969, scientists discovered that
the synthesis of the ribosomal RNAs is carried out by specialized systems using two key enzymes:
RNA polymerase I and RNA polymerase III.
But until now, scientists were unsure if a complementary system was also responsible for
the production of the 80 proteins that make up the ribosome.
That’s essentially what the UC San Diego researchers headed by Jim Kadonaga, a professor of biology, set out to examine. What they found was the missing link—the specialized
system that allows ribosomal proteins themselves to be synthesized by the cell.
Kadonaga says that he and coworkers found that ribosomal proteins are synthesized via
a novel regulatory system with the enzyme RNA polymerase II and
a factor termed TRF2,”
“For the production of most proteins,
RNA polymerase II functions with
a factor termed TBP,
but for the synthesis of ribosomal proteins, it uses TRF2.”
this specialized TRF2-based system for ribosome biogenesis
provides a new avenue for the study of ribosomes and
its control of cell growth, and
“it should lead to a better understanding and potential treatment of diseases such as cancer.”
Coordination of the transcriptome and metabolome
the potential advantages conferred by distal-site protein synthesis
Other authors of the paper were UC San Diego biologists Yuan-Liang Wang, Sascha Duttke and George Kassavetis, and Kai Chen, Jeff Johnston, and Julia Zeitlinger of the Stowers Institute for Medical Research in Kansas City, Missouri. Their research was supported by two grants from the National Institutes of Health (1DP2OD004561-01 and R01 GM041249).
Turning Off a Powerful Cancer Protein
Scientists have discovered how to shut down a master regulatory transcription factor that is
key to the survival of a majority of aggressive lymphomas,
which arise from the B cells of the immune system.
The protein, Bcl6, has long been considered too complex to target with a drug since it is also crucial
to the healthy functioning of many immune cells in the body, not just B cells gone bad.
The researchers at Weill Cornell Medical College report that it is possible
to shut down Bcl6 in diffuse large B-cell lymphoma (DLBCL)
while not affecting its vital function in T cells and macrophages
that are needed to support a healthy immune system.
If Bcl6 is completely inhibited, patients might suffer from systemic inflammation and atherosclerosis. The team conducted this new study to help clarify possible risks, as well as to understand
how Bcl6 controls the various aspects of the immune system.
The findings in this study were inspired from
preclinical testing of two Bcl6-targeting agents that Dr. Melnick and his Weill Cornell colleagues have developed
to treat DLBCLs.
These experimental drugs are
RI-BPI, a peptide mimic, and
the small molecule agent 79-6.
“This means the drugs we have developed against Bcl6 are more likely to be
significantly less toxic and safer for patients with this cancer than we realized,”
says Ari Melnick, M.D., professor of hematology/oncology and a hematologist-oncologist at NewYork-Presbyterian Hospital/Weill Cornell Medical Center.
Dr. Melnick says the discovery that
a master regulatory transcription factor can be targeted
offers implications beyond just treating DLBCL.
Recent studies from Dr. Melnick and others have revealed that
Bcl6 plays a key role in the most aggressive forms of acute leukemia, as well as certain solid tumors.
Bcl6 can control the type of immune cell that develops in the bone marrow—playing many roles
in the development of B cells, T cells, macrophages, and other cells—including a primary and essential role in
enabling B-cells to generate specific antibodies against pathogens.
According to Dr. Melnick, “When cells lose control of Bcl6,
lymphomas develop in the immune system.
Lymphomas are ‘addicted’ to Bcl6, and therefore
Bcl6 inhibitors powerfully and quickly destroy lymphoma cells,” .
The big surprise in the current study is that rather than functioning as a single molecular machine,
Bcl6 functions like a Swiss Army knife,
using different tools to control different cell types.
This multifunction paradigm could represent a general model for the functioning of other master regulatory transcription factors.
“In this analogy, the Swiss Army knife, or transcription factor, keeps most of its tools folded,
opening only the one it needs in any given cell type,”
He makes the following analogy:
“For B cells, it might open and use the knife tool;
for T cells, the cork screw;
for macrophages, the scissors.”
“this means that you only need to prevent the master regulator from using certain tools to treat cancer. You don’t need to eliminate the whole knife,” . “In fact, we show that taking out the whole knife is harmful since
the transcription factor has many other vital functions that other cells in the body need.”
Prior to these study results, it was not known that a master regulator could separate its functions so precisely. Researchers hope this will be a major benefit to the treatment of DLBCL and perhaps other disorders that are influenced by Bcl6 and other master regulatory transcription factors.
The study is published in the journal Nature Immunology, in a paper titled “Lineage-specific functions of Bcl-6 in immunity and inflammation are mediated by distinct biochemical mechanisms”.
Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.
Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.
Tiny vesicles containing protective substances
which they transmit to nerve cells apparently
play an important role in the functioning of neurons.
As cell biologists at Johannes Gutenberg University Mainz (JGU) have discovered,
nerve cells can enlist the aid of mini-vesicles of neighboring glial cells
to defend themselves against stress and other potentially detrimental factors.
These vesicles, called exosomes, appear to stimulate the neurons on various levels:
they influence electrical stimulus conduction,
biochemical signal transfer, and
gene regulation.
Exosomes are thus multifunctional signal emitters
that can have a significant effect in the brain.
Exosome
The researchers in Mainz already observed in a previous study that
oligodendrocytes release exosomes on exposure to neuronal stimuli.
these are absorbed by the neurons and improve neuronal stress tolerance.
Oligodendrocytes, a type of glial cell, form an
insulating myelin sheath around the axons of neurons.
The exosomes transport protective proteins such as
heat shock proteins,
glycolytic enzymes, and
enzymes that reduce oxidative stress from one cell type to another,
but also transmit genetic information in the form of ribonucleic acids.
“As we have now discovered in cell cultures, exosomes seem to have a whole range of functions,” explained Dr. Eva-Maria Krmer-Albers. By means of their transmission activity, the small bubbles that are the vesicles
not only promote electrical activity in the nerve cells, but also
influence them on the biochemical and gene regulatory level.
“The extent of activities of the exosomes is impressive,” added Krmer-Albers. The researchers hope that the understanding of these processes will contribute to the development of new strategies for the treatment of neuronal diseases. Their next aim is to uncover how vesicles actually function in the brains of living organisms.
Neuroscientists use snail research to help explain “chemo brain”
10/08/2014
It is estimated that as many as half of patients taking cancer drugs experience a decrease in mental sharpness. While there have been many theories, what causes “chemo brain” has eluded scientists.
In an effort to solve this mystery, neuroscientists at The University of Texas Health Science Center at Houston (UTHealth) conducted an experiment in an animal memory model and their results point to a possible explanation. Findings appeared in The Journal of Neuroscience.
In the study involving a sea snail that shares many of the same memory mechanisms as humans and a drug used to treat a variety of cancers, the scientists identified
memory mechanisms blocked by the drug.
Then, they were able to counteract or
unblock the mechanisms by administering another agent.
“Our research has implications in the care of people given to cognitive deficits following drug treatment for cancer,” said John H. “Jack” Byrne, Ph.D., senior author, holder of the June and Virgil Waggoner Chair and Chairman of the Department of Neurobiology and Anatomy at the UTHealth Medical School. “There is no satisfactory treatment at this time.”
Byrne’s laboratory is known for its use of a large snail called Aplysia californica to further the understanding of the biochemical signaling among nerve cells (neurons). The snails have large neurons that relay information much like those in humans.
When Byrne’s team compared cell cultures taken from normal snails to
those administered a dose of a cancer drug called doxorubicin,
the investigators pinpointed a neuronal pathway
that was no longer passing along information properly.
With the aid of an experimental drug,
the scientists were able to reopen the pathway.
Unfortunately, this drug would not be appropriate for humans, Byrne said. “We want to identify other drugs that can rescue these memory mechanisms,” he added.
According the American Cancer Society, some of the distressing mental changes cancer patients experience may last a short time or go on for years.
Byrne’s UT Health research team includes co-lead authors Rong-Yu Liu, Ph.D., and Yili Zhang, Ph.D., as well as Brittany Coughlin and Leonard J. Cleary, Ph.D. All are affiliated with the W.M. Keck Center for the Neurobiology of Learning and Memory.
Byrne and Cleary also are on the faculty of The University of Texas Graduate School of Biomedical Sciences at Houston. Coughlin is a student at the school, which is jointly operated by UT Health and The University of Texas MD Anderson Cancer Center.
The study titled “Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase” received support from National Institutes of Health grant (NS019895) and the Zilkha Family Discovery Fellowship.
Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase
Source: Univ. of Texas Health Science Center at Houston
Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase
Rong-Yu Liu*, Yili Zhang*, Brittany L. Coughlin, Leonard J. Cleary, and John H. Byrne +Show Affiliations
The Journal of Neuroscience, 1 Oct 2014, 34(40): 13289-13300; http://dx.doi.org:/10.1523/JNEUROSCI.0538-14.2014
Doxorubicin (DOX) is an anthracycline used widely for cancer chemotherapy. Its primary mode of action appears to be
topoisomerase II inhibition, DNA cleavage, and free radical generation.
However, in non-neuronal cells, DOX also inhibits the expression of
dual-specificity phosphatases (also referred to as MAPK phosphatases) and thereby
inhibits the dephosphorylation of extracellular signal-regulated kinase (ERK) and
p38 mitogen-activated protein kinase (p38 MAPK),
two MAPK isoforms important for long-term memory (LTM) formation.
Activation of these kinases by DOX in neurons, if present,
could have secondary effects on cognitive functions, such as learning and memory.
The present study used cultures of rat cortical neurons and sensory neurons (SNs) of Aplysia
to examine the effects of DOX on levels of phosphorylated ERK (pERK) and
phosphorylated p38 (p-p38) MAPK.
In addition, Aplysia neurons were used to examine the effects of DOX on
long-term enhanced excitability, long-term synaptic facilitation (LTF), and
long-term synaptic depression (LTD).
DOX treatment led to elevated levels of
pERK and p-p38 MAPK in SNs and cortical neurons.
In addition, it increased phosphorylation of
the downstream transcriptional repressor cAMP response element-binding protein 2 in SNs.
DOX treatment blocked serotonin-induced LTF and enhanced LTD induced by the neuropeptide Phe-Met-Arg-Phe-NH2. The block of LTF appeared to be attributable to
overriding inhibitory effects of p-p38 MAPK, because
LTF was rescued in the presence of an inhibitor of p38 MAPK
(SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]) .
These results suggest that acute application of DOX might impair the formation of LTM via the p38 MAPK pathway.
Terms: Aplysia chemotherapy ERK p38 MAPK serotonin synaptic plasticity
Technology that controls brain cells with radio waves earns early BRAIN grant
10/08/2014
bright spots = cells with increased calcium after treatment with radio waves, allows neurons to fire
BRAIN control: The new technology uses radio waves to activate or silence cells remotely. The bright spots above represent cells with increased calcium after treatment with radio waves, a change that would allow neurons to fire.
A proposal to develop a new way to
remotely control brain cells
from Sarah Stanley, a research associate in Rockefeller University’s Laboratory of Molecular Genetics, headed by Jeffrey M. Friedman, is
among the first to receive funding from U.S. President Barack Obama’s BRAIN initiative.
The project will make use of a technique called
radiogenetics that combines the use of radio waves or magnetic fields with
nanoparticles to turn neurons on or off.
The National Institutes of Health is one of four federal agencies involved in the BRAIN (Brain Research through Advancing Innovative Neurotechnologies) initiative. Following in the ambitious footsteps of the Human Genome Project, the BRAIN initiative seeks
to create a dynamic map of the brain in action,
a goal that requires the development of new technologies. The BRAIN initiative working group, which outlined the broad scope of the ambitious project, was co-chaired by Rockefeller’s Cori Bargmann, head of the Laboratory of Neural Circuits and Behavior.
Stanley’s grant, for $1.26 million over three years, is one of 58 projects to get BRAIN grants, the NIH announced. The NIH’s plan for its part of this national project, which has been pitched as “America’s next moonshot,” calls for $4.5 billion in federal funds over 12 years.
The technology Stanley is developing would
enable researchers to manipulate the activity of neurons, as well as other cell types,
in freely moving animals in order to better understand what these cells do.
Other techniques for controlling selected groups of neurons exist, but her new nanoparticle-based technique has a
unique combination of features that may enable new types of experimentation.
it would allow researchers to rapidly activate or silence neurons within a small area of the brain or
dispersed across a larger region, including those in difficult-to-access locations.
Stanley also plans to explore the potential this method has for use treating patients.
“Francis Collins, director of the NIH, has discussed
Why do some cancers spread while others don’t? Scientists have now demonstrated that
metastatic incompetent cancers actually “poison the soil”
by generating a micro-environment that blocks cancer cells
from settling and growing in distant organs.
The “seed and the soil” hypothesis proposed by Stephen Paget in 1889 is now widely accepted to explain how
cancer cells (seeds) are able to generate fertile soil (the micro-environment)
in distant organs that promotes cancer’s spread.
However, this concept had not explained why some tumors do not spread or metastasize.
The researchers, from Weill Cornell Medical College, found that
two key proteins involved in this process work by
dramatically suppressing cancer’s spread.
The study offers hope that a drug based on these
potentially therapeutic proteins, prosaposin and Thrombospondin 1 (Tsp-1),
might help keep human cancer at bay and from metastasizing.
Scientists don’t understand why some tumors wouldn’t “want” to spread. It goes against their “job description,” says the study’s senior investigator, Vivek Mittal, Ph.D., an associate professor of cell and developmental biology in cardiothoracic surgery and director of the Neuberger Berman Foundation Lung Cancer Laboratory at Weill Cornell Medical College. He theorizes that metastasis occurs when
the barriers that the body throws up to protect itself against cancer fail.
But there are some tumors in which some of the barriers may still be intact. “So that suggests
those primary tumors will continue to grow, but that
an innate protective barrier still exists that prevents them from spreading and invading other organs,”
The researchers found that, like typical tumors,
metastasis-incompetent tumors also send out signaling molecules
that establish what is known as the “premetastatic niche” in distant organs.
These niches composed of bone marrow cells and various growth factors have been described previously by others including Dr. Mittal as the fertile “soil” that the disseminated cancer cell “seeds” grow in.
Weill Cornell’s Raúl Catena, Ph.D., a postdoctoral fellow in Dr. Mittal’s laboratory, found an important difference between the tumor types. Metastatic-incompetent tumors
systemically increased expression of Tsp-1, a molecule known to fight cancer growth.
increased Tsp-1 production was found specifically in the bone marrow myeloid cells
that comprise the metastatic niche.
These results were striking, because for the first time Dr. Mittal says
the bone marrow-derived myeloid cells were implicated as
the main producers of Tsp-1,.
In addition, Weill Cornell and Harvard researchers found that
prosaposin secreted predominantly by the metastatic-incompetent tumors
increased expression of Tsp-1 in the premetastatic lungs.
Thus, Dr. Mittal posits that prosaposin works in combination with Tsp-1
to convert pro-metastatic bone marrow myeloid cells in the niche
into cells that are not hospitable to cancer cells that spread from a primary tumor.
“The very same myeloid cells in the niche that we know can promote metastasis
can also be induced under the command of the metastatic incompetent primary tumor to inhibit metastasis,”
The research team found that
the Tsp-1–inducing activity of prosaposin
was contained in only a 5-amino acid peptide region of the protein, and
this peptide alone induced Tsp-1 in the bone marrow cells and
effectively suppressed metastatic spread in the lungs
in mouse models of breast and prostate cancer.
This 5-amino acid peptide with Tsp-1–inducing activity
has the potential to be used as a therapeutic agent against metastatic cancer,
The scientists have begun to test prosaposin in other tumor types or metastatic sites.
Dr. Mittal says that “The clinical implications of the study are:
“Not only is it theoretically possible to design a prosaposin-based drug or drugs
that induce Tsp-1 to block cancer spread, but
you could potentially create noninvasive prognostic tests
to predict whether a cancer will metastasize.”
The study was reported in the April 30 issue of Cancer Discovery, in a paper titled “Bone Marrow-Derived Gr1+ Cells Can Generate a Metastasis-Resistant Microenvironment Via Induced Secretion of Thrombospondin-1”.
Knocking out a single enzyme dramatically cripples the ability of aggressive cancer cells to spread and grow tumors.
The paper, published in the journal Proceedings of the National Academy of Sciences, sheds new light on the importance of lipids, a group of molecules that includes fatty acids and cholesterol, in the development of cancer.
Researchers have long known that cancer cells metabolize lipids differently than normal cells. Levels of ether lipids – a class of lipids that are harder to break down – are particularly elevated in highly malignant tumors.
“Cancer cells make and use a lot of fat and lipids, and that makes sense because cancer cells divide and proliferate at an accelerated rate, and to do that,
they need lipids, which make up the membranes of the cell,”
said study principal investigator Daniel Nomura, assistant professor in UC Berkeley’s Department of Nutritional Sciences and Toxicology. “Lipids have a variety of uses for cellular structure, but what we’re showing with our study is that
lipids can send signals that fuel cancer growth.”
In the study, Nomura and his team tested the effects of reducing ether lipids on human skin cancer cells and primary breast tumors. They targeted an enzyme,
alkylglycerone phosphate synthase, or AGPS,
known to be critical to the formation of ether lipids.
The researchers confirmed that
AGPS expression increased when normal cells turned cancerous.
inactivating AGPS substantially reduced the aggressiveness of the cancer cells.
“The cancer cells were less able to move and invade,” said Nomura.
The researchers also compared the impact of
disabling the AGPS enzyme in mice that had been injected with cancer cells.
Nomura. observes -“Among the mice that had the AGPS enzyme inactivated,
the tumors were nonexistent,”
“The mice that did not have this enzyme
disabled rapidly developed tumors.”
The researchers determined that
inhibiting AGPS expression depleted the cancer cells of ether lipids.
AGPS altered levels of other types of lipids important to the ability of the cancer cells to survive and spread, including
prostaglandins and acyl phospholipids.
“What makes AGPS stand out as a treatment target is that the enzyme seems to simultaneously
regulate multiple aspects of lipid metabolism
important for tumor growth and malignancy.”
Future steps include the
development of AGPS inhibitors for use in cancer therapy,
“This study sheds considerable light on the important role that AGPS plays in ether lipid metabolism in cancer cells, and it suggests that
inhibitors of this enzyme could impair tumor formation,”
said Benjamin Cravatt, Professor and Chair of Chemical Physiology at The Scripps Research Institute, who is not part of the UC.
Agilent Technologies Thought Leader Award Supports Translational Research Program
Published: Mon, March 04, 2013
The award will support Dr DePinho’s research into
metabolic reprogramming in the earliest stages of cancer.
Agilent Technologies Inc. announces that Dr. Ronald A. DePinho, a world-renowned oncologist and researcher, has received an Agilent Thought Leader Award.
DePinho is president of the University of Texas MD Anderson Cancer Center. DePinho and his team hope to discover and characterize
alterations in metabolic flux during tumor initiation and maintenance, and to identify biomarkers for early detection of pancreatic cancer together with
novel therapeutic targets.
Researchers on his team will work with scientists from the university’s newly formed Institute of Applied Cancer Sciences.
The Agilent Thought Leader Award provides funds to support personnel as well as a state-of-the-art Agilent 6550 iFunnel Q-TOF LC/MS system.
“I am extremely pleased to receive this award for metabolomics research, as the survival rates for pancreatic cancer have not significantly improved over the past 20 years,” DePinho said. “This technology will allow us to
rapidly identify new targets that drive the formation, progression and maintenance of pancreatic cancer.
Discoveries from this research will also lead to
the development of effective early detection biomarkers and novel therapeutic interventions.”
“We are proud to support Dr. DePinho’s exciting translational research program, which will make use of
metabolomics and integrated biology workflows and solutions in biomarker discovery,”
said Patrick Kaltenbach, Agilent vice president, general manager of the Liquid Phase Division, and the executive sponsor of this award.
The Agilent Thought Leader Program promotes fundamental scientific advances by support of influential thought leaders in the life sciences and chemical analysis fields.
The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis
Figure 1: Smurf1 expression is elevated in colorectal cancer tissues.
Smurf1 expression is elevated in colorectal cancer tissues.
(a) Smurf1 expression scores are shown as box plots, with the horizontal lines representing the median; the bottom and top of the boxes representing the 25th and 75th percentiles, respectively; and the vertical bars representing the ra
Figure 2: Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer.
Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer
(a) Representative images from immunohistochemical staining of Smurf1, Ubc12, NAE1 and Nedd8 in the same colorectal cancer tumour. Scale bars, 100 μm. (b–d) The expression scores of Nedd8 (b, n=283 ), NAE1 (c, n=281) and Ubc12 (d, n=19…
Figure 3: Smurf1 interacts with Ubc12.
Smurf1 interacts with Ubc12
(a) GST pull-down assay of Smurf1 with Ubc12. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies. Smurf1 interacted with Ubc12 and UbcH5c, but not with Ubc9. (b) Mapping the regions…
Figure 4: Nedd8 is attached to Smurf1through C426-catalysed autoneddylation.
Nedd8 is attached to Smurf1through C426-catalysed autoneddylation
(a) Covalent neddylation of Smurf1 in vitro.Purified His-Smurf1-WT or C699A proteins were incubated with Nedd8 and Nedd8-E1/E2. Reactions were performed as described in the Methods section. Samples were analysed by western blotting wi…
Figure 5: Neddylation of Smurf1 activates its ubiquitin ligase activity.
Neddylation of Smurf1 activates its ubiquitin ligase activity.
(a) In vivo Smurf1 ubiquitylation assay. Nedd8 was co-expressed with Smurf1 WT or C699A in HCT116 cells (left panels). Twenty-four hours post transfection, cells were treated with MG132 (20 μM, 8 h). HCT116 cells were transfected with…
12-LO enzyme promotes the obesity-induced oxidative stress in the pancreatic cells.
An enzyme called 12-LO promotes the obesity-induced oxidative stress in the pancreatic cells that leads
to pre-diabetes, and diabetes.
12-LO’s enzymatic action is the last step in
the production of certain small molecules that harm the cell,
according to a team from Indiana University School of Medicine, Indianapolis.
The findings will enable the development of drugs that can interfere with this enzyme, preventing or even reversing diabetes. The research is published ahead of print in the journal Molecular and Cellular Biology.
In earlier studies, these researchers and their collaborators at Eastern Virginia Medical School showed that
12-LO (which stands for 12-lipoxygenase) is present in these cells
only in people who become overweight.
The harmful small molecules resulting from 12-LO’s enzymatic action are known as HETEs, short for hydroxyeicosatetraenoic acid.
HETEs harm the mitochondria, which then
fail to produce sufficient energy to enable
the pancreatic cells to manufacture the necessary quantities of insulin.
For the study, the investigators genetically engineered mice that
lacked the gene for 12-LO exclusively in their pancreas cells.
Mice were either fed a low-fat or high-fat diet.
Both the control mice and the knockout mice on the high fat diet
developed obesity and insulin resistance.
The investigators also examined the pancreatic beta cells of both knockout and control mice, using both microscopic studies and molecular analysis. Those from the knockout mice were intact and healthy, while
those from the control mice showed oxidative damage,
demonstrating that 12-LO and the resulting HETEs
caused the beta cell failure.
Mirmira notes that fatty diet used in the study was the Western Diet, which comprises mostly saturated-“bad”-fats. Based partly on a recent study of related metabolic pathways, he says that
the unsaturated and mono-unsaturated fats-which comprise most fats in the healthy,
relatively high fat Mediterranean diet-are unlikely to have the same effects.
“Our research is the first to show that 12-LO in the beta cell
is the culprit in the development of pre-diabetes, following high fat diets,” says Mirmira.
“Our work also lends important credence to the notion that
the beta cell is the primary defective cell in virtually all forms of diabetes and pre-diabetes.”
Specially engineered mice gained no weight, and normal counterparts became obese
on the same high-fat, obesity-inducing Western diet.
Specially engineered mice that lacked a particular gene did not gain weight
when fed a typical high-fat, obesity-inducing Western diet.
Yet, these mice ate the same amount as their normal counterparts that became obese.
The mice were engineered with fat cells that lacked a gene called SEL1L,
known to be involved in the clearance of mis-folded proteins
in the cell’s protein making machinery called the endoplasmic reticulum (ER).
When mis-folded proteins are not cleared but accumulate,
they destroy the cell and contribute to such diseases as
mad cow disease,
Type 1 diabetes and
cystic fibrosis.
“The million-dollar question is why don’t these mice gain weight? Is this related to its inability to clear mis-folded proteins in the ER?” said Ling Qi, associate professor of molecular and biochemical nutrition and senior author of the study published online July 24 in Cell Metabolism. Haibo Sha, a research associate in Qi’s lab, is the paper’s lead author.
Interestingly, the experimental mice developed a host of other problems, including
postprandial hypertriglyceridemia,
and fatty livers.
“Although we are yet to find out whether these conditions contribute to the lean phenotype, we found that
there was a lipid partitioning defect in the mice lacking SEL1L in fat cells,
where fat cells cannot store fat [lipids], and consequently
fat goes to the liver.
During the investigation of possible underlying mechanisms, we discovered
a novel function for SEL1L as a regulator of lipid metabolism,” said Qi.
Sha said “We were very excited to find that
SEL1L is required for the intracellular trafficking of
lipoprotein lipase (LPL), acting as a chaperone,” .
and added that “Using several tissue-specific knockout mouse models,
we showed that this is a general phenomenon,”
Without LPL, lipids remain in the circulation;
fat and muscle cells cannot absorb fat molecules for storage and energy combustion,
People with LPL mutations develop
postprandial hypertriglyceridemia similar to
conditions found in fat cell-specific SEL1L-deficient mice, said Qi.
Future work will investigate the
role of SEL1L in human patients carrying LPL mutations and
determine why fat cell-specific SEL1L-deficient mice remain lean under Western diets, said Sha.
Co-authors include researchers from Cedars-Sinai Medical Center in Los Angeles; Wageningen University in the Netherlands; Georgia State University; University of California, Los Angeles; and the Medical College of Soochow University in China.
The study was funded by the U.S. National Institutes of Health, the Netherlands Organization for Health Research and Development National Institutes of Health, the Cedars-Sinai Medical Center, Chinese National Science Foundation, the American Diabetes Association, Cornell’s Center for Vertebrate Genomics and the Howard Hughes Medical Institute.
While work with biomarkers continues to grow, scientists are also grappling with research-related bottlenecks, such as
affinity reagent development,
platform reproducibility, and
sensitivity.
Biomarkers by definition indicate some state or process that generally occurs
at a spatial or temporal distance from the marker itself, and
it would not be an exaggeration to say that biomedicine has become infatuated with them:
where to find them,
when they may appear,
what form they may take, and
how they can be used to diagnose a condition or
predict whether a therapy may be successful.
Biomarkers are on the agenda of many if not most industry gatherings, and in cases such as Oxford Global’s recent “Biomarker Congress” and the GTC “Biomarker Summit”, they hold the naming rights. There, some basic principles were built upon, amended, and sometimes challenged.
In oncology, for example, biomarker discovery is often predicated on the premise that
proteins shed from a tumor will traverse to and persist in, and be detectable in, the circulation.
By quantifying these proteins—singularly or as part of a larger “signature”—the hope is
to garner information about the molecular characteristics of the cancer
that will help with cancer detection and
personalization of the treatment strategy.
Yet this approach has not yet turned into the panacea that was hoped for. Bottlenecks exist in
affinity reagent development,
platform reproducibility, and
sensitivity.
There is also a dearth of understanding of some of the
fundamental principles of biomarker biology that we need to know the answers to,
said Parag Mallick, Ph.D., whose lab at Stanford University is “working on trying to understand where biomarkers come from.”
There are dogmas saying that
circulating biomarkers come solely from secreted proteins.
But Dr. Mallick’s studies indicate that fully
50% of circulating proteins may come from intracellular sources or
proteins that are annotated as such.
“We don’t understand the processes governing
which tumor-derived proteins end up in the blood.”
Other questions include “how does the size of a tumor affect how much of a given protein will be in the blood?”—perhaps
the tumor is necrotic at the center, or
it’s hypervascular or hypovascular.
He points out “The problem is that these are highly nonlinear processes at work, and
there is a large number of factors that might affect the answer to that question,” .
Their research focuses on using
mass spectrometry and
computational analysis
to characterize the biophysical properties of the circulating proteome, and
relate these to measurements made of the tumor itself.
Furthermore, he said – “We’ve observed that the proteins that are likely to
first show up and persist in the circulation, ..
are more stable than proteins that don’t,”
“we can quantify how significant the effect is.”
The goal is ultimately to be able to
build rigorous, formal mathematical models that will allow something measured in the blood
to be tied back to the molecular biology taking place in the tumor.
And conversely, to use those models
to predict from a tumor what will be found in the circulation.
“Ultimately, the models will allow you to connect the dots between
what you measure in the blood and the biology of the tumor.”
Bound for Affinity Arrays
Affinity reagents are the main tools for large-scale protein biomarker discovery. And while this has tended to mean antibodies (or their derivatives), other affinity reagents are demanding a place in the toolbox.
Affimers, a type of affinity reagent being developed by Avacta, consist of
a biologically inert, biophysically stable protein scaffold
containing three variable regions into which
distinct peptides are inserted.
The resulting three-dimensional surface formed by these peptides
interacts and binds to proteins and other molecules in solution,
much like the antigen-binding site of antibodies.
Unlike antibodies, Affimers are relatively small (13 KDa),
non-post-translationally modified proteins
that can readily be expressed in bacterial culture.
They may be made to bind surfaces through unique residues
engineered onto the opposite face of the Affimer,
allowing the binding site to be exposed to the target in solution.
“We don’t seem to see in what we’ve done so far
any real loss of activity or functionality of Affimers when bound to surfaces—
they’re very robust,” said CEO Alastair Smith, Ph.D.
Avacta is taking advantage of this stability and its large libraries of Affimers to develop
very large affinity microarrays for
drug and biomarker discovery.
To date they have printed arrays with around 20–25,000 features, and Dr. Smith is “sure that we can get toward about 50,000 on a slide,” he said. “There’s no real impediment to us doing that other than us expressing the proteins and getting on with it.”
Customers will be provided with these large, complex “naïve” discovery arrays, readable with standard equipment. The plan is for the company to then “support our customers by providing smaller arrays with
the Affimers that are binding targets of interest to them,” Dr. Smith foretold.
And since the intellectual property rights are unencumbered,
Affimers in those arrays can be licensed to the end users
to develop diagnostics that can be validated as time goes on.
Around 20,000-Affimer discovery arrays were recently tested by collaborator Professor Ann Morgan of the University of Leeds with pools of unfractionated serum from patients with symptoms of inflammatory disease. The arrays
“rediscovered” elevated C-reactive protein (CRP, the clinical gold standard marker)
as well as uncovered an additional 22 candidate biomarkers.
other candidates combined with CRP, appear able to distinguish between different diseases such as
rheumatoid arthritis,
psoriatic arthritis,
SLE, or
giant cell arteritis.
Epigenetic Biomarkers
Sometimes biomarkers are used not to find disease but
to distinguish healthy human cell types, with
examples being found in flow cytometry and immunohistochemistry.
These widespread applications, however, are difficult to standardize, being
subject to arbitrary or subjective gating protocols and other imprecise criteria.
Epiontis instead uses an epigenetic approach. “What we need is a unique marker that is
demethylated only in one cell type and
methylated in all the other cell types,”
Each cell of the right cell type will have
two demethylated copies of a certain gene locus,
allowing them to be enumerated by quantitative PCR.
The biggest challenge is finding that unique epigenetic marker. To do so they look through the literature for proteins and genes described as playing a role in the cell type’s biology, and then
look at the methylation patterns to see if one can be used as a marker,
They also “use customized Affymetrix chips to look at the
differential epigenetic status of different cell types on a genomewide scale.”
explained CBO and founder Ulrich Hoffmueller, Ph.D.
The company currently has a panel of 12 assays for 12 immune cell types. Among these is an assay for
regulatory T (Treg) cells that queries the Foxp3 gene—which is uniquely demethylated in Treg
even though it is transiently expressed in activated T cells of other subtypes.
Also assayed are Th17 cells, difficult to detect by flow cytometry because
“the cells have to be stimulated in vitro,” he pointed out.
Developing New Assays for Cancer Biomarkers
Researchers at Myriad RBM and the Cancer Prevention Research Institute of Texas are collaborating to develop
new assays for cancer biomarkers on the Myriad RBM Multi-Analyte Profile (MAP) platform.
The release of OncologyMAP 2.0 expanded Myriad RBM’s biomarker menu to over 250 analytes, which can be measured from a small single sample, according to the company. Using this menu, L. Stephen et al., published a poster, “Analysis of Protein Biomarkers in Prostate and Colorectal Tumor Lysates,” which showed the results of
a survey of proteins relevant to colorectal (CRC) and prostate (PC) tumors
to identify potential proteins of interest for cancer research.
The study looked at CRC and PC tumor lysates and found that 102 of the 115 proteins showed levels above the lower limit of quantification.
Four markers were significantly higher in PC and 10 were greater in CRC.
For most of the analytes, duplicate sections of the tumor were similar, although some analytes did show differences. In four of the CRC analytes, tumor number four showed differences for CEA and tumor number 2 for uPA.
Thirty analytes were shown to be
different in CRC tumor compared to its adjacent tissue.
Ten of the analytes were higher in adjacent tissue compared to CRC.
Eighteen of the markers examined demonstrated —-
significant correlations of CRC tumor concentration to serum levels.
“This suggests.. that the Oncology MAP 2.0 platform “provides a good method for studying changes in tumor levels because many proteins can be assessed with a very small sample.”
Clinical Test Development with MALDI-ToF
While there have been many attempts to translate results from early discovery work on the serum proteome into clinical practice, few of these efforts have progressed past the discovery phase.
Matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry on unfractionated serum/plasma samples offers many practical advantages over alternative techniques, and does not require
a shift from discovery to development and commercialization platforms.
Biodesix claims it has been able to develop the technology into
a reproducible, high-throughput tool to
routinely measure protein abundance from serum/plasma samples.
“.. we improved data-analysis algorithms to
reproducibly obtain quantitative measurements of relative protein abundance from MALDI-ToF mass spectra.
Heinrich Röder, CTO points out that the MALDI-ToF measurements
are combined with clinical outcome data using
modern learning theory techniques
to define specific disease states
based on a patient’s serum protein content,”
The clinical utility of the identification of these disease states can be investigated through a retrospective analysis of differing sample sets. For example, Biodesix clinically validated its first commercialized serum proteomic test, VeriStrat®, in 85 different retrospective sample sets.
Röder adds that “It is becoming increasingly clear that
the patients whose serum is characterized as VeriStrat Poor show
consistently poor outcomes irrespective of
tumor type,
histology, or
molecular tumor characteristics,”
MALDI-ToF mass spectrometry, in its standard implementation,
allows for the observation of around 100 mostly high-abundant serum proteins.
Further, “while this does not limit the usefulness of tests developed from differential expression of these proteins,
the discovery potential would be greatly enhanced
if we could probe deeper into the proteome
while not giving up the advantages of the MALDI-ToF approach,”
Biodesix reports that its new MALDI approach, Deep MALDI™, can perform
simultaneous quantitative measurement of more than 1,000 serum protein features (or peaks) from 10 µL of serum in a high-throughput manner.
it increases the observable signal noise ratio from a few hundred to over 50,000,
resulting in the observation of many lower-abundance serum proteins.
Breast cancer, a disease now considered to be a collection of many complexes of symptoms and signatures—the dominant ones are labeled Luminal A, Luminal B, Her2, and Basal— which suggests different prognose, and
these labels are considered too simplistic for understanding and managing a woman’s cancer.
Studies published in the past year have looked at
somatic mutations,
gene copy number aberrations,
gene expression abnormalities,
protein and miRNA expression, and
DNA methylation,
coming up with a list of significantly mutated genes—hot spots—in different categories of breast cancers. Targeting these will inevitably be the focus of much coming research.
“We’ve been taking these large trials and profiling these on a variety of array or sequence platforms. We think we’ll get
prognostic drivers
predictive markers for taxanes and
monoclonal antibodies and
tamoxifen and aromatase inhibitors,”
explained Brian Leyland-Jones, Ph.D., director of Edith Sanford Breast Cancer Research. “We will end up with 20–40 different diseases, maybe more.”
Edith Sanford Breast Cancer Research is undertaking a pilot study in collaboration with The Scripps Research Institute, using a variety of tests on 25 patients to see how the information they provide complements each other, the overall flow, and the time required to get and compile results.
Laser-captured tumor samples will be subjected to low passage whole-genome, exome, and RNA sequencing (with targeted resequencing done in parallel), and reverse-phase protein and phosphorylation arrays, with circulating nucleic acids and circulating tumor cells being queried as well. “After that we hope to do a 100- or 150-patient trial when we have some idea of the best techniques,” he said.
Dr. Leyland-Jones predicted that ultimately most tumors will be found
to have multiple drivers,
with most patients receiving a combination of two, three, or perhaps four different targeted therapies.
Reduce to Practice
According to Randox, the evidence Investigator is a sophisticated semi-automated biochip system designed for research, clinical, forensic, and veterinary applications.
Once biomarkers that may have an impact on therapy are discovered, it is not always routine to get them into clinical practice. Leaving regulatory and financial, intellectual property and cultural issues aside, developing a diagnostic based on a biomarker often requires expertise or patience that its discoverer may not possess.
Andrew Gribben is a clinical assay and development scientist at Randox Laboratories, based in Northern Ireland, U.K. The company utilizes academic and industrial collaborators together with in-house discovery platforms to identify biomarkers that are
augmented or diminished in a particular pathology
relative to appropriate control populations.
Biomarkers can be developed to be run individually or
combined into panels of immunoassays on its multiplex biochip array technology.
Specificity can also be gained—or lost—by the affinity of reagents in an assay. The diagnostic potential of Heart-type fatty acid binding protein (H-FABP) abundantly expressed in human myocardial cells was recognized by Jan Glatz of Maastricht University, The Netherlands, back in 1988. Levels rise quickly within 30 minutes after a myocardial infarction, peaking at 6–8 hours and return to normal within 24–30 hours. Yet at the time it was not known that H-FABP was a member of a multiprotein family, with which the polyclonal antibodies being used in development of an assay were cross-reacting, Gribben related.
Randox developed monoclonal antibodies specific to H-FABP, funded trials investigating its use alone, and multiplexed with cardiac biomarker assays, and, more than 30 years after the biomarker was identified, in 2011, released a validated assay for H-FABP as a biomarker for early detection of acute myocardial infarction.
Ultrasensitive Immunoassays for Biomarker Development
Research has shown that detection and monitoring of biomarker concentrations can provide
insights into disease risk and progression.
Cytokines have become attractive biomarkers and candidates
for targeted therapies for a number of autoimmune diseases, including rheumatoid arthritis (RA), Crohn’s disease, and psoriasis, among others.
However, due to the low-abundance of circulating cytokines, such as IL-17A, obtaining robust measurements in clinical samples has been difficult.
Singulex reports that its digital single-molecule counting technology provides
increased precision and detection sensitivity over traditional ELISA techniques,
helping to shed light on biomarker verification and validation programs.
The company’s Erenna® immunoassay system, which includes optimized immunoassays, offers LLoQ to femtogram levels per mL resolution—even in healthy populations, at an improvement of 1-3 fold over standard ELISAs or any conventional technology and with a dynamic range of up to 4-logs, according to a Singulex official, who adds that
this sensitivity improvement helps minimize undetectable samples that
could otherwise delay or derail clinical studies.
The official also explains that the Singulex solution includes an array of products and services that are being applied to a number of programs and have enabled the development of clinically relevant biomarkers, allowing translation from discovery to the clinic.
In a poster entitled “Advanced Single Molecule Detection: Accelerating Biomarker Development Utilizing Cytokines through Ultrasensitive Immunoassays,” a case study was presented of work performed by Jeff Greenberg of NYU to show how the use of the Erenna system can provide insights toward
improving the clinical utility of biomarkers and
accelerating the development of novel therapies for treating inflammatory diseases.
A panel of inflammatory biomarkers was examined in DMARD (disease modifying antirheumatic drugs)-naïve RA (rheumatoid arthritis) vs. knee OA (osteoarthritis) patient cohorts. Markers that exhibited significant differences in plasma concentrations between the two cohorts included
CRP, IL-6R alpha, IL-6, IL-1 RA, VEGF, TNF-RII, and IL-17A, IL-17F, and IL-17A/F.
Among the three tested isoforms of IL-17,
the magnitude of elevation for IL-17F in RA patients was the highest.
“Singulex provides high-resolution monitoring of baseline IL-17A concentrations that are present at low levels,” concluded the researchers. “The technology also enabled quantification of other IL-17 isoforms in RA patients, which have not been well characterized before.”
The Singulex Erenna System has also been applied to cardiovascular disease research, for which its
cardiac troponin I (cTnI) digital assay can be used to measure circulating
levels of cTnI undetectable by other commercial assays.
Recently presented data from Brigham and Women’s Hospital and the TIMI-22 study showed that
using the Singulex test to serially monitor cTnI helps
stratify risk in post-acute coronary syndrome patients and
can identify patients with elevated cTnI
who have the most to gain from intensive vs. moderate-dose statin therapy,
according to the scientists involved in the research.
The study poster, “Prognostic Performance of Serial High Sensitivity Cardiac Troponin Determination in Stable Ischemic Heart Disease: Analysis From PROVE IT-TIMI 22,” was presented at the 2013 American College of Cardiology (ACC) Annual Scientific Session & Expo by R. O’Malley et al.
Biomarkers Changing Clinical Medicine
Better Diagnosis, Prognosis, and Drug Targeting Are among Potential Benefits
John Morrow Jr., Ph.D.
Researchers at EMD Chemicals are developing biomarker immunoassays
to monitor drug-induced toxicity including kidney damage.
The pace of biomarker development is accelerating as investigators report new studies on cancer, diabetes, Alzheimer disease, and other conditions in which the evaluation and isolation of workable markers is prominently featured.
Wei Zheng, Ph.D., leader of the R&D immunoassay group at EMD Chemicals, is overseeing a program to develop biomarker immunoassays to
monitor drug-induced toxicity, including kidney damage.
“One of the principle reasons for drugs failing during development is because of organ toxicity,” says Dr. Zheng.
“proteins liberated into the serum and urine can serve as biomarkers of adverse response to drugs, as well as disease states.”
Through collaborative programs with Rules-Based Medicine (RBM), the EMD group has released panels for the profiling of human renal impairment and renal toxicity. These urinary biomarker based products fit the FDA and EMEA guidelines for assessment of drug-induced kidney damage in rats.
The group recently performed a screen for potential protein biomarkers in relation to
kidney toxicity/damage on a set of urine and plasma samples
from patients with documented renal damage.
Additionally, Dr. Zheng is directing efforts to move forward with the multiplexed analysis of
organ and cellular toxicity.
Diseases thought to involve compromised oxidative phosphorylation include
diabetes, Parkinson and Alzheimer diseases, cancer, and the aging process itself.
Good biomarkers allow Dr. Zheng to follow the mantra, “fail early, fail fast.” With robust, multiplexible biomarkers, EMD can detect bad drugs early and kill them before they move into costly large animal studies and clinical trials. “Recognizing the severe liability that toxicity presents, we can modify the structure of the candidate molecule and then rapidly reassess its performance.”
Scientists at Oncogene Science a division of Siemens Healthcare Diagnostics, are also focused on biomarkers. “We are working on a number of antibody-based tests for various cancers, including a test for the Ca-9 CAIX protein, also referred to as carbonic anhydrase,” Walter Carney, Ph.D., head of the division, states.
CAIX is a transmembrane protein that is
overexpressed in a number of cancers, and, like Herceptin and the Her-2 gene,
can serve as an effective and specific marker for both diagnostic and therapeutic purposes.
It is liberated into the circulation in proportion to the tumor burden.
Dr. Carney and his colleagues are evaluating patients after tumor removal for the presence of the Ca-9 CAIX protein. If
the levels of the protein in serum increase over time,
this suggests that not all the tumor cells were removed and the tumor has metastasized.
Dr. Carney and his team have developed both an immuno-histochemistry and an ELISA test that could be used as companion diagnostics in clinical trials of CAIX-targeted drugs.
The ELISA for the Ca-9 CAIX protein will be used in conjunction with Wilex’ Rencarex®, which is currently in a
Phase III trial as an adjuvant therapy for non-metastatic clear cell renal cancer.
Additionally, Oncogene Science has in its portfolio an FDA-approved test for the Her-2 marker. Originally approved for Her-2/Neu-positive breast cancer, its indications have been expanded over time, and was approved
for the treatment of gastric cancer last year.
It is normally present on breast cancer epithelia but
overexpressed in some breast cancer tumors.
“Our products are designed to be used in conjunction with targeted therapies,” says Dr. Carney. “We are working with companies that are developing technology around proteins that are
overexpressed in cancerous tissues and can be both diagnostic and therapeutic targets.”
The long-term goal of these studies is to develop individualized therapies, tailored for the patient. Since the therapies are expensive, accurate diagnostics are critical to avoid wasting resources on patients who clearly will not respond (or could be harmed) by the particular drug.
“At this time the rate of response to antibody-based therapies may be very poor, as
they are often employed late in the course of the disease, and patients are in such a debilitated state
that they lack the capacity to react positively to the treatment,” Dr. Carney explains.
Nanoscale Real-Time Proteomics
Stanford University School of Medicine researchers, working with Cell BioSciences, have developed a
nanofluidic proteomic immunoassay that measures protein charge,
similar to immunoblots, mass spectrometry, or flow cytometry.
unlike these platforms, this approach can measure the amount of individual isoforms,
specifically, phosphorylated molecules.
“We have developed a nanoscale device for protein measurement, which I believe could be useful for clinical analysis,” says Dean W. Felsher, M.D., Ph.D., associate professor at Stanford University School of Medicine.
Critical oncogenic transformations involving
the activation of the signal-related kinases ERK-1 and ERK-2 can now be followed with ease.
“The fact that we measure nanoquantities with accuracy means that
we can interrogate proteomic profiles in clinical patients,
by drawing tiny needle aspirates from tumors over the course of time,” he explains.
“This allows us to observe the evolution of tumor cells and
their response to therapy
from a baseline of the normal tissue as a standard of comparison.”
According to Dr. Felsher, 20 cells is a large enough sample to obtain a detailed description. The technology is easy to automate, which allows
the inclusion of hundreds of assays.
Contrasting this technology platform with proteomic analysis using microarrays, Dr. Felsher notes that the latter is not yet workable for revealing reliable markers.
Dr. Felsher and his group published a description of this technology in Nature Medicine. “We demonstrated that we could take a set of human lymphomas and distinguish them from both normal tissue and other tumor types. We can
quantify changes in total protein, protein activation, and relative abundance of specific phospho-isoforms
from leukemia and lymphoma patients receiving targeted therapy.
Even with very small numbers of cells, we are able to show that the results are consistent, and
our sample is a random profile of the tumor.”
Splice Variant Peptides
“Aberrations in alternative splicing may generate
much of the variation we see in cancer cells,”
says Gilbert Omenn, Ph.D., director of the center for computational medicine and bioinformatics at the University of Michigan School of Medicine. Dr. Omenn and his colleague, Rajasree Menon, are
using this variability as a key to new biomarker identification.
It is becoming evident that splice variants play a significant role in the properties of cancer cells, including
initiation, progression, cell motility, invasiveness, and metastasis.
Alternative splicing occurs through multiple mechanisms
when the exons or coding regions of the DNA transcribe mRNA,
generating initiation sites and connecting exons in protein products.
Their translation into protein can result in numerous protein isoforms, and
these isoforms may reflect a diseased or cancerous state.
Regulatory elements within the DNA are responsible for selecting different alternatives; thus
the splice variants are tempting targets for exploitation as biomarkers.
Analyses of the splice-site mutation
Despite the many questions raised by these observations, splice variation in tumor material has not been widely studied. Cancer cells are known for their tremendous variability, which allows them to
grow rapidly, metastasize, and develop resistance to anticancer drugs.
Dr. Omenn and his collaborators used
mass spec data to interrogate a custom-built database of all potential mRNA sequences
to find alternative splice variants.
When they compared normal and malignant mammary gland tissue from a mouse model of Her2/Neu human breast cancers, they identified a vast number (608) of splice variant proteins, of which
peptides from 216 were found only in the tumor sample.
“These novel and known alternative splice isoforms
are detectable both in tumor specimens and in plasma and
represent potential biomarker candidates,” Dr. Omenn adds.
Dr. Omenn’s observations and those of his colleague Lewis Cantley, Ph.D., have also
shed light on the origins of the classic Warburg effect,
the shift to anaerobic glycolysis in tumor cells.
The novel splice variant M2, of muscle pyruvate kinase,
is observed in embryonic and tumor tissue.
It is associated with this shift, the result of
the expression of a peptide splice variant sequence.
It is remarkable how many different areas of the life sciences are tied into the phenomenon of splice variation. The changes in the genetic material can be much greater than point mutations, which have been traditionally considered to be the prime source of genetic variability.
“We now have powerful methods available to uncover a whole new category of variation,” Dr. Omenn says. “High-throughput RNA sequencing and proteomics will be complementary in discovery studies of splice variants.”
Splice variation may play an important role in rapid evolutionary changes, of the sort discussed by Susumu Ohno and Stephen J. Gould decades ago. They, and other evolutionary biologists, argued that
gene duplication, combined with rapid variability, could fuel major evolutionary jumps.
At the time, the molecular mechanisms of variation were poorly understood, but today
the tools are available to rigorously evaluate the role of
splice variation and other contributors to evolutionary change.
“Biomarkers derived from studies of splice variants, could, in the future, be exploited
both for diagnosis and prognosis and
for drug targeting of biological networks,
in situations such as the Her-2/Neu breast cancers,” Dr. Omenn says.
Aminopeptidase Activities
“By correlating the proteolytic patterns with disease groups and controls, we have shown that
exopeptidase activities contribute to the generation of not only cancer-specific
but also cancer type specific serum peptides.
according to Paul Tempst, Ph.D., professor and director of the Protein Center at the Memorial Sloan-Kettering Cancer Center.
So there is a direct link between peptide marker profiles of disease and differential protease activity.” For this reason Dr. Tempst argues that “the patterns we describe may have value as surrogate markers for detection and classification of cancer.”
To investigate this avenue, Dr. Tempst and his colleagues have followed
the relationship between exopeptidase activities and metastatic disease.
“We monitored controlled, de novo peptide breakdown in large numbers of biological samples using mass spectrometry, with relative quantitation of the metabolites,” Dr. Tempst explains. This entailed the use of magnetic, reverse-phase beads for analyte capture and a MALDI-TOF MS read-out.
“In biomarker discovery programs, functional proteomics is usually not pursued,” says Dr. Tempst. “For putative biomarkers, one may observe no difference in quantitative levels of proteins, while at the same time, there may be substantial differences in enzymatic activity.”
In a preliminary prostate cancer study, the team found a significant difference
in activity levels of exopeptidases in serum from patients with metastatic prostate cancer
as compared to primary tumor-bearing individuals and normal healthy controls.
However, there were no differences in amounts of the target protein, and this potential biomarker would have been missed if quantitative levels of protein had been the only criterion of selection.
It is frequently stated that “practical fusion energy is 30 years in the future and always will be.” The same might be said of functional, practical biomarkers that can pass muster with the FDA. But splice variation represents a new handle on this vexing problem. It appears that we are seeing the emergence of a new approach that may finally yield definitive diagnostic tests, detectable in serum and urine samples.
Part 7. Epigenetics and Drug Metabolism
DNA Methylation Rules: Studying Epigenetics with New Tools
The tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.
New tools may help move the field of epigenetic analysis forward and potentially unveil novel biomarkers for cellular development, differentiation, and disease.
DNA sequencing has had the power of technology behind it as novel platforms to produce more sequencing faster and at lower cost have been introduced. But the tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.
Among these mechanisms, DNA methylation, or the enzymatically mediated addition of a methyl group to cytosine or adenine dinucleotides,
serves as an inherited epigenetic modification that
stably modifies gene expression in dividing cells.
The unique methylomes are largely maintained in differentiated cell types, making them critical to understanding the differentiation potential of the cell.
In the DNA methylation process, cytosine residues in the genome are enzymatically modified to 5-methylcytosine,
which participates in transcriptional repression of genes during development and disease progression.
5-methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. DNA methylation affects gene transcription by physically
interfering with the binding of proteins involved in gene transcription.
Methylated DNA may be bound by methyl-CpG-binding domain proteins (MBDs) that can
then recruit additional proteins. Some of these include histone deacetylases and other chromatin remodeling proteins that modify histones, thereby
forming compact, inactive chromatin, or heterochromatin.
While DNA methylation doesn’t change the genetic code,
it influences chromosomal stability and gene expression.
Epigenetics and Cancer Biomarkers
multistage chemical carcinogenesis
And because of the increasing recognition that DNA methylation changes are involved in human cancers, scientists have suggested that these epigenetic markers may provide biological markers for cancer cells, and eventually point toward new diagnostic and therapeutic targets. Cancer cell genomes display genome-wide abnormalities in DNA methylation patterns,
some of which are oncogenic and contribute to genome instability.
In particular, de novo methylation of tumor suppressor gene promoters
occurs frequently in cancers, thereby silencing them and promoting transformation.
Cytosine hydroxymethylation (5-hydroxymethylcytosine, or 5hmC), the aforementioned DNA modification resulting from the enzymatic conversion of 5mC into 5-hydroxymethylcytosine by the TET family of oxygenases, has been identified
as another key epigenetic modification marking genes important for
pluripotency in embryonic stem cells (ES), as well as in cancer cells.
The base 5-hydroxymethylcytosine was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. In 2011, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2′-deoxycytidine (5hmdC) and 5-methyl-2′-deoxycytidine (5mdC) in genomic DNA, scientists at the Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California investigated
whether levels of 5hmC can distinguish normal tissue from tumor tissue.
They showed that in squamous cell lung cancers, levels of 5hmdC showed
up to five-fold reduction compared with normal lung tissue.
In brain tumors,5hmdC showed an even more drastic reduction
with levels up to more than 30-fold lower than in normal brain,
but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1, the enzyme that converts 5hmC to 5hmdC.
Immunohistochemical analysis indicated that 5hmC is “remarkably depleted” in many types of human cancer.
there was an inverse relationship between 5hmC levels and cell proliferation with lack of 5hmC in proliferating cells.
Their data suggest that 5hmdC is strongly depleted in human malignant tumors,
a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue.
In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis.
Enzymatic Mapping
But according to New England Biolabs’ Sriharsa Pradhan, Ph.D., methods for distinguishing 5mC from 5hmC and analyzing and quantitating the cell’s entire “methylome” and “hydroxymethylome” remain less than optimal.
The protocol for bisulphite conversion to detect methylation remains the “gold standard” for DNA methylation analysis. This method is generally followed by PCR analysis for single nucleotide resolution to determine methylation across the DNA molecule. According to Dr. Pradhan, “.. bisulphite conversion does not distinguish 5mC and 5hmC,”
Recently we found an enzyme, a unique DNA modification-dependent restriction endonuclease, AbaSI, which can
decode the hydryoxmethylome of the mammalian genome.
You easily can find out where the hydroxymethyl regions are.”
AbaSI, recognizes 5-glucosylatedmethylcytosine (5gmC) with high specificity when compared to 5mC and 5hmC, and
cleaves at narrow range of distances away from the recognized modified cytosine.
By mapping the cleaved ends, the exact 5hmC location can, the investigators reported, be determined.
Dr. Pradhan and his colleagues at NEB; the Department of Biochemistry, Emory University School of Medicine, Atlanta; and the New England Biolabs Shanghai R&D Center described use of this technique in a paper published in Cell Reports this month, in which they described high-resolution enzymatic mapping of genomic hydroxymethylcytosine in mouse ES cells.
In the current report, the authors used the enzyme technology for the genome-wide high-resolution hydroxymethylome, describing simple library construction even with a low amount of input DNA (50 ng) and the ability to readily detect 5hmC sites with low occupancy.
As a result of their studies, they propose that
factors affecting the local 5mC accessibility to TET enzymes play important roles in the 5hmC deposition
including include chromatin compaction, nucleosome positioning, or TF binding.
the regularly oscillating 5hmC profile around the CTCF-binding sites, suggests 5hmC ‘‘writers’’ may be sensitive to the nucleosomal environment.
some transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.
“We were able to do complete mapping in mouse embryonic cells and are pleased about what this enzyme can do and how it works,” Dr. Pradhan said.
And the availability of novel tools that make analysis of the methylome and hypomethylome more accessible will move the field of epigenetic analysis forward and potentially novel biomarkers for cellular development, differentiation, and disease.
Patricia Fitzpatrick Dimond, Ph.D. (pdimond@genengnews.com), is technical editor at Genetic Engineering & Biotechnology News.
Epigenetic Regulation of ADME-Related Genes: Focus on Drug Metabolism and Transport
Published: Sep 23, 2013
Epigenetic regulation of gene expression refers to heritable factors that are functionally relevant genomic modifications but that do not involve changes in DNA sequence.
Examples of such modifications include
DNA methylation, histone modifications, noncoding RNAs, and chromatin architecture.
Epigenetic modifications are crucial for
packaging and interpreting the genome, and they have fundamental functions in regulating gene expression and activity under the influence of physiologic and environmental factors.
In this issue of Drug Metabolism and Disposition, a series of articles is presented to demonstrate the role of epigenetic factors in regulating
the expression of genes involved in drug absorption, distribution, metabolism, and excretion in organ development, tissue-specific gene expression, sexual dimorphism, and in the adaptive response to xenobiotic exposure, both therapeutic and toxic.
The articles also demonstrate that, in addition to genetic polymorphisms, epigenetics may also contribute to wide inter-individual variations in drug metabolism and transport. Identification of functionally relevant epigenetic biomarkers in human specimens has the potential to improve prediction of drug responses based on patient’s epigenetic profiles.
Metabolic models can provide a mechanistic framework
to analyze information-rich omics data sets, and are
increasingly being used to investigate metabolic alternations in human diseases.
An expression of the altered metabolic pathway utilization is the selection of metabolites consumed and released by cells. However, methods for the
inference of intracellular metabolic states from extracellular measurements in the context of metabolic models remain underdeveloped compared to methods for other omics data.
Herein, we describe a workflow for such an integrative analysis
emphasizing on extracellular metabolomics data.
We demonstrate,
using the lymphoblastic leukemia cell lines Molt-4 and CCRF-CEM,
how our methods can reveal differences in cell metabolism. Our models explain metabolite uptake and secretion by predicting
a more glycolytic phenotype for the CCRF-CEM model and
a more oxidative phenotype for the Molt-4 model,
which was supported by our experimental data.
Gene expression analysis revealed altered expression of gene products at
key regulatory steps in those central metabolic pathways, and
literature query emphasized the role of these genes in cancer metabolism.
Moreover, in silico gene knock-outs identified unique
control points for each cell line model, e.g., phosphoglycerate dehydrogenase for the Molt-4 model.
Thus, our workflow is well suited to the characterization of cellular metabolic traits based on
-extracellular metabolomic data, and it allows the integration of multiple omics data sets
into a cohesive picture based on a defined model context.
Modern high-throughput techniques have increased the pace of biological data generation. Also referred to as the ‘‘omics avalanche’’, this wealth of data provides great opportunities for metabolic discovery. Omics data sets
contain a snapshot of almost the entire repertoire of mRNA, protein, or metabolites at a given time point or
under a particular set of experimental conditions. Because of the high complexity of the data sets,
computational modeling is essential for their integrative analysis.
Currently, such data analysis is a bottleneck in the research process and methods are needed to facilitate the use of these data sets, e.g., through meta-analysis of data available in public databases [e.g., the human protein atlas (Uhlen et al. 2010) or the gene expression omnibus (Barrett et al. 2011)], and to increase the accessibility of valuable information for the biomedical research community.
Constraint-based modeling and analysis (COBRA) is
a computational approach that has been successfully used to
investigate and engineer microbial metabolism through the prediction of steady-states (Durot et al.2009).
The basis of COBRA is network reconstruction: networks are assembled in a bottom-up fashion based on
genomic data and extensive
organism-specific information from the literature.
Metabolic reconstructions capture information on the
known biochemical transformations taking place in a target organism
to generate a biochemical, genetic and genomic knowledge base (Reed et al. 2006).
Once assembled, a
metabolic reconstruction can be converted into a mathematical model (Thiele and Palsson 2010), and
model properties can be interrogated using a great variety of methods (Schellenberger et al. 2011).
The ability of COBRA models
to represent genotype–phenotype and environment–phenotype relationships arises
through the imposition of constraints, which
limit the system to a subset of possible network states (Lewis et al. 2012).
Currently, COBRA models exist for more than 100 organisms, including humans (Duarte et al. 2007; Thiele et al. 2013).
Since the first human metabolic reconstruction was described [Recon 1 (Duarte et al. 2007)],
biomedical applications of COBRA have increased (Bordbar and Palsson 2012).
One way to contextualize networks is to
define their system boundaries according to the metabolic states of the system, e.g., disease or dietary regimes.
The consequences of the applied constraints can
then be assessed for the entire network (Sahoo and Thiele 2013).
Additionally, omics data sets have frequently been used
to generate cell-type or condition-specific metabolic models.
Models exist for specific cell types, such as
enterocytes (Sahoo and Thiele2013),
macrophages (Bordbar et al. 2010),
adipocytes (Mardinoglu et al. 2013),
even multi-cell assemblies that represent the interactions of brain cells (Lewis et al. 2010).
All of these cell type specific models, except the enterocyte reconstruction
were generated based on omics data sets.
Cell-type-specific models have been used to study
diverse human disease conditions.
For example, an adipocyte model was generated using
transcriptomic, proteomic, and metabolomics data.
This model was subsequently used to investigate metabolic alternations in adipocytes
that would allow for the stratification of obese patients (Mardinoglu et al. 2013).
The biomedical applications of COBRA have been
cancer metabolism (Jerby and Ruppin, 2012).
predicting drug targets (Folger et al. 2011; Jerby et al. 2012).
A cancer model was generated using
multiple gene expression data sets and subsequently used
to predict synthetic lethal gene pairs as potential drug targets
selective for the cancer model, but non-toxic to the global model (Recon 1),
a consequence of the reduced redundancy in the cancer specific model (Folger et al. 2011).
In a follow up study, lethal synergy between FH and enzymes of the heme metabolic pathway
were experimentally validated and resolved the mechanism by which FH deficient cells,
e.g., in renal-cell cancer cells survive a non-functional TCA cycle (Frezza et al. 2011).
Contextualized models, which contain only the subset of reactions active in a particular tissue (or cell-) type,
can be generated in different ways (Becker and Palsson, 2008; Jerby et al. 2010).
However, the existing algorithms mainly consider
gene expression and proteomic data
to define the reaction sets that comprise the contextualized metabolic models.
These subset of reactions are usually defined
based on the expression or absence of expression of the genes or proteins (present and absent calls),
or inferred from expression values or differential gene expression.
Comprehensive reviews of the methods are available (Blazier and Papin, 2012; Hyduke et al. 2013). Only the compilation of a large set of omics data sets
can result in a tissue (or cell-type) specific metabolic model, whereas
the representation of one particular experimental condition is achieved
through the integration of omics data set generated from one experiment only (condition-specific cell line model).
Recently, metabolomic data sets have become more comprehensive and
using these data sets allow direct determination of the metabolic network components (the metabolites).
Additionally, metabolomics has proven to be stable, relatively inexpensive, and highly reproducible (Antonucci et al. 2012). These factors make metabolomic data sets particularly valuable for
interrogation of metabolic phenotypes.
Thus, the integration of these data sets is now an active field of research (Li et al. 2013; Mo et al. 2009; Paglia et al. 2012b; Schmidt et al. 2013).
Generally, metabolomic data can be incorporated into metabolic networks as
qualitative, quantitative, and thermodynamic constraints (Fleming et al. 2009; Mo et al. 2009).
Mo et al. used metabolites detected in the
spent medium of yeast cells to determine intracellular flux states through a sampling analysis (Mo et al. 2009),
which allowed unbiased interrogation of the possible network states (Schellenberger and Palsson 2009) and
prediction of internal pathway use.
Modes of transcriptional regulation during the YMC
Such analyses have also been used to reveal the effects of
enzymopathies on red blood cells (Price et al. 2004),
to study effects of diet on diabetes (Thiele et al. 2005) and
to define macrophage metabolic states (Bordbar et al. 2010).
This type of analysis is available as a function in the COBRA toolbox (Schellenberger et al. 2011).
In this study, we established a workflow
for the generation and analysis of condition-specific metabolic cell line models
that can facilitate the interpretation of metabolomic data.
metabolic differences between two lymphoblastic leukemia cell lines (Fig. 1A).
Fig. 1
metabol leukem cell lines11306_2014_721_Fig1_HTML
A Combined experimental and computational pipeline to study human metabolism.
Experimental work and omics data analysis steps precede computational modeling.
Model predictions are validated based on targeted experimental data.
Metabolomic and transcriptomic data are used for model refinement and submodel extraction.
Functional analysis methods are used to characterize the metabolism of the cell-line models and compare it to additional experimental data.
The validated models are subsequently used for the prediction of drug targets.
B Uptake and secretion pattern of model metabolites. All metabolite uptakes and secretions that were mapped during model generation are shown.
Metabolite uptakes are depicted on the left, and
secreted metabolites are shown on the right.
A number of metabolite exchanges mapped to the model were unique to one cell line.
Differences between cell lines were used to set quantitative constraints for the sampling analysis.
C Statistics about the cell line-specific network generation.
D Quantitative constraints.
For the sampling analysis, an additional set of constraints was imposed on the cell line specific models,
emphasizing the differences in metabolite uptake and secretion between cell lines.
Higher uptake of a metabolite was allowed
in the model of the cell line that consumed more of the metabolite in vitro, whereas
the supply was restricted for the model with lower in vitro uptake.
This was done by establishing the same ratio between the models bounds as detected in vitro.
X denotes the factor (slope ratio) that distinguishes the bounds, and
which was individual for each metabolite.
(a) The uptake of a metabolite could be x times higher in CCRF-CEM cells,
(b) the metabolite uptake could be x times higher in Molt-4,
(c) metabolite secretion could be x times higher in CCRF-CEM, or
(d) metabolite secretion could be x times higher in Molt-4 cells.LOD limit of detection.
The consequence of the adjustment was, in case of uptake, that one model was constrained to a lower metabolite uptake (A, B), and the difference depended on the ratio detected in vitro. In case of secretion, one model
had to secrete more of the metabolite, and again
the difference depended on the experimental difference detected between the cell lines
2 Results
We set up a pipeline that could be used to infer intracellular metabolic states
from semi-quantitative data regarding metabolites exchanged between cells and their environment.
Our pipeline combined the following four steps:
data acquisition,
data analysis,
metabolic modeling and
experimental validation of the model predictions (Fig. 1A).
We demonstrated the pipeline and the predictive potential to predict metabolic alternations in diseases such as cancer based on
^two lymphoblastic leukemia cell lines.
The resulting Molt-4 and CCRF-CEM condition-specific cell line models could explain
^ metabolite uptake and secretion ^ by predicting the distinct utilization of central metabolic pathways by the two cell lines. ^ the CCRF-CEM model resembled more a glycolytic, commonly referred to as ‘Warburg’ phenotype, ^ our model predicted a more respiratory phenotype for the Molt-4 model.
We found these predictions to be in agreement with measured gene expression differences
at key regulatory steps in the central metabolic pathways, and they were also
consistent with additional experimental data regarding the energy and redox states of the cells.
After a brief discussion of the data generation and analysis steps, the results derived from model generation and analysis will be described in detail.
2.1 Pipeline for generation of condition-specific metabolic cell line models
integration of exometabolomic (EM) data
2.1.1 Generation of experimental data
We monitored the growth and viability of lymphoblastic leukemia cell lines in serum-free medium (File S2, Fig. S1). Multiple omics data sets were derived from these cells.Extracellular metabolomics (exo-metabolomic) data,
integration of exometabolomic (EM) data
^ comprising measurements of the metabolites in the spent medium of the cell cultures (Paglia et al. 2012a), ^ were collected along with transcriptomic data, and these data sets were used to construct the models.
2.1.4 Condition-specific models for CCRF-CEM and Molt-4 cells
To determine whether we had obtained two distinct models, we evaluated the reactions, metabolites, and genes of the two models. Both the Molt-4 and CCRF-CEM models contained approximately half of the reactions and metabolites present in the global model (Fig. 1C). They were very similar to each other in terms of their reactions, metabolites, and genes (File S1, Table S5A–C).
(1) The Molt-4 model contained seven reactions that were not present in the CCRF-CEM model (Co-A biosynthesis pathway and exchange reactions).
(2) The CCRF-CEM contained 31 unique reactions (arginine and proline metabolism, vitamin B6 metabolism, fatty acid activation, transport, and exchange reactions).
(3) There were 2 and 15 unique metabolites in the Molt-4 and CCRF-CEM models, respectively (File S1, Table S5B).
(4) Approximately three quarters of the global model genes remained in the condition-specific cell line models (Fig. 1C).
(5) The Molt-4 model contained 15 unique genes, and the CCRF-CEM model had 4 unique genes (File S1, Table S5C).
(6) Both models lacked NADH dehydrogenase (complex I of the electron transport chain—ETC), which was determined by the absence of expression of a mandatory subunit (NDUFB3, Entrez gene ID 4709).
Rather, the ETC was fueled by FADH2 originating from succinate dehydrogenase and from fatty acid oxidation, which through flavoprotein electron transfer
FADH2
could contribute to the same ubiquinone pool as complex I and complex II (succinate dehydrogenase).
Despite their different in vitro growth rates (which differed by 11 %, see File S2, Fig. S1) and
^^^ differences in exo-metabolomic data (Fig. 1B) and transcriptomic data,
^^^ the internal networks were largely conserved in the two condition-specific cell line models.
2.1.5 Condition-specific cell line models predict distinct metabolic strategies
Despite the overall similarity of the metabolic models, differences in their cellular uptake and secretion patterns suggested distinct metabolic states in the two cell lines (Fig. 1B and see “Materials and methods” section for more detail). To interrogate the metabolic differences, we sampled the solution space of each model using an Artificial Centering Hit-and-Run (ACHR) sampler (Thiele et al. 2005). For this analysis, additional constraints were applied, emphasizing the quantitative differences in commonly uptaken and secreted metabolites. The maximum possible uptake and maximum possible secretion flux rates were reduced
^^^ according to the measured relative differences between the cell lines (Fig. 1D, see “Materials and methods” section).
We plotted the number of sample points containing a particular flux rate for each reaction. The resulting binned histograms can be understood as representing the probability that a particular reaction can have a certain flux value.
A comparison of the sample points obtained for the Molt-4 and CCRF-CEM models revealed
a considerable shift in the distributions, suggesting a higher utilization of glycolysis by the CCRF-CEM model
(File S2, Fig. S2).
This result was further supported by differences in medians calculated from sampling points (File S1, Table S6).
The shift persisted throughout all reactions of the pathway and was induced by the higher glucose uptake (34 %) from the extracellular medium in CCRF-CEM cells.
The sampling median for glucose uptake was 34 % higher in the CCRF-CEM model than in Molt-4 model (File S2, Fig. S2).
The usage of the TCA cycle was also distinct in the two condition-specific cell-line models (Fig. 2). Interestingly, the models used succinate dehydrogenase differently (Figs. 2, 3).
TCA_reactions
The Molt-4 model utilized an associated reaction to generate FADH2, whereas
in the CCRF-CEM model, the histogram was shifted in the opposite direction,
toward the generation of succinate.
Additionally, there was a higher efflux of citrate toward amino acid and lipid metabolism in the CCRF-CEM model (Fig. 2). There was higher flux through anaplerotic and cataplerotic reactions in the CCRF-CEM model than in the Molt-4 model (Fig. 2); these reactions include
(1) the efflux of citrate through ATP-citrate lyase,
(2) uptake of glutamine,
(3) generation of glutamate from glutamine,
(4) transamination of pyruvate and glutamate to alanine and to 2-oxoglutarate,
(5) secretion of nitrogen, and
(6) secretion of alanine.
energetics-of-cellular-respiration
The Molt-4 model showed higher utilization of oxidative phosphorylation (Fig. 3), again supported by elevated median flux through ATP synthase (36 %) and other enzymes, which contributed to higher oxidative metabolism. The sampling analysis therefore revealed different usage of central metabolic pathways by the condition-specific models.
Fig. 2
Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).
Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).
The table provides the median values of the sampling results. Negative values in histograms and in the table describe reversible reactions with flux in the reverse direction. There are multiple reversible reactions for the transformation of isocitrate and α-ketoglutarate, malate and fumarate, and succinyl-CoA and succinate. These reactions are unbounded, and therefore histograms are not shown. The details of participating cofactors have been removed.
Figure 3.
Molt-4 has higher median flux through ETC reactions II–IV 11306_2014_721_Fig3_HTML
Atp ATP, cit citrate, adp ADP, pi phosphate, oaa oxaloacetate, accoa acetyl-CoA, coa coenzyme-A, icit isocitrate, αkg α-ketoglutarate, succ-coa succinyl-CoA, succ succinate, fumfumarate, mal malate, oxa oxaloacetate,
pyr pyruvate, lac lactate, ala alanine, gln glutamine, ETC electron transport chain
Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes
metabolic pathways 1476-4598-10-70-1
Metabolic Systems Research Team fig2
Metabolic control analysis of respiration in human cancer tissue. fphys-04-00151-g001