Reversible Kinetic Analysis of Myc Targets In vivo Provides Novel Insights into Myc-Mediated Tumorigenesis

Reversible Kinetic Analysis of Myc Targets In vivo Provides Novel

Insights into Myc-Mediated Tumorigenesis

Elizabeth R. Lawlor, Laura Soucek, Lamorna Brown-Swigart, Ksenya Shchors,C. Uli Bialucha, and Gerard I. Evan

Cancer Research Institute, University of California San Francisco Comprehensive Cancer Center, San Francisco, California

Abstract

Deregulated expression of the Myc transcription factor is afrequent causal mutation in human cancer. Thousands ofputative Myc target genes have been identified in in vitrostudies, indicating that Myc exerts highly pleiotropic effectswithin cells and tissues. However, the complexity and diversityof Myc gene targets has confounded attempts at identifyingwhich of these genes are the critical targets mediating Myc-driven tumorigenesis in vivo . Acute activation of Myc in areversibly switchable transgenic model of Myc-mediated B celltumorigenesis induces rapid tumor onset, whereas subsequentMyc deactivation triggers equally rapid tumor regression.Thus, sustained Myc activity is required for tumor mainte-nance. We have used this reversibly switchable kinetic tumormodel in combination with high-density oligonucleotidemicroarrays to develop an unbiased strategy for identifyingcandidate Myc-regulated genes responsible for maintenanceof Myc-dependent tumors. Consistent with known Mycfunctions, some Myc-regulated genes are involved in cellgrowth, cycle, and proliferation. In addition, however, manyMyc-regulated genes are specific to B cells, indicating that asignificant component of Myc action is cell type specific.Finally, we identify a very restricted cadre of genes withexpression that is inversely regulated upon Myc activation-induced tumor progression and deactivation-induced tumorregression. By definition, such genes are candidates for tumormaintenance functions. Combining reversibly switchable,transgenic models of tumor formation and regression withgenomic profiling offers a novel strategy with which todeconvolute the complexities of oncogenic signaling pathwaysin vivo . (Cancer Res 2006; 66(9): 4591-601)

Introduction

The Myc transcription factor regulates many genes implicated indiverse cellular functions, including proliferation, differentiation,death, and tissue reorganization. Disruption of the control of theseprocesses is a mandatory feature of cancer and, consistent with

this, genetic lesions in the Myc locus occur frequently in humanmalignancy. In general, such lesions result in the overexpressedand/or deregulated expression of Myc. However, the molecularmechanisms by which Myc promotes cellular transformation andoncogenesis remain unclear.

The pleiotropic effects of Myc function are thought to arisethrough its action as a transcription factor, positively or negativelyregulating target effector genes. Induction of gene targets by Mycrequires its dimerization with its partner protein, Max, andsubsequent heterodimeric binding to canonical (CACGTG) andnoncanonical DNA E-box sequences (1–4). The mechanisms bywhich Myc represses gene targets are less clear but likely involvefunctional interference via binding to and inhibiting the activity ofpositive regulators, such as Miz-1 (5). In attempts to define thetranscriptional target genes of Myc, several genome-wide screenshave been conducted using different cell types and experimentalconditions (6–9). These studies have indicated the great diversity ofMyc target gene function and have, cumulatively, identified wellover 1,000 putative transcriptional targets. Unfortunately, thedegree of overlap among these in vitro studies has been small,alluding to the critical potential role of cell type and context indetermining the biological consequences of Myc action. Withrespect to its specific role in cancer, it has not been possible tosubstantiate which of these Myc targets are mission critical fortumorigenesis and which are functionally inconsequential. Impor-tantly, to date, no systematic analysis of Myc transcriptional targetsin any orthotopic tissue setting in vivo has been conducted.

MycERTAM is a regulatable fusion protein that exhibits Mycactivity only in the presence of the ligand 4-hydroxytamoxifen(4-OHT). In pIns-MycERTAM transgenic mice, MycERTAM expressionis targeted to pancreatic h cells via the insulin promoter (10). Theh cells of pIns-MycERTAM mice exhibit uniform cell cycle entryupon systemic administration of 4-OHT; however, the net biolo-gical outcome of such acute Myc activation depends on the extentto which Myc-induced apoptosis is opposed. By itself, activation ofMyc triggers massive h-cell apoptosis that overwhelms prolifera-tion and leads to islet involution (10). In contrast, coexpression ofBcl-xL in h cells inhibits apoptosis, allowing Myc-inducedproliferation to supersede death and resulting in rapid andprogressive h-cell expansion (10). Upon sustained activation ofMycERTAM, hyperplastic islets progressively acquire the definingcharacteristics of cancer, including dysplasia and dedifferentiation,angiogenesis, local invasion, and metastasis. Moreover, thesefeatures are completely dependent on continuous Myc action asevidenced by the uniform regression of tumors upon 4-OHTwithdrawal (10).

Given the consistent, synchronous, and reproducible pattern ofphenotypic changes that Myc elicits and maintains in this model,the pIns-MycERTAM x RIP7-Bcl-xL double-transgenic mouse is anideal system with which to study the genetic processes that

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Current address for E.R. Lawlor: Division of Hematology-Oncology, Children’sHospital Los Angeles, Keck School of Medicine, University of Southern California, LosAngeles, California.

Current address for C.U. Bialucha: Medical Research Council Laboratory forMolecular Cell Biology, University College London, London, United Kingdom.Requests for reprints: Gerard I. Evan, Cancer Research Institute, University of

California San Francisco, Comprehensive Cancer Center, 2340 Sutter Street, SanFrancisco, CA 94143-0875. E-mail: [email protected] or Elizabeth R. Lawlor, Children’sHospital Los Angeles, 4650 Sunset Boulevard MS57, Los Angeles, CA 90027. Phone:323-644-8579; E-mail: [email protected].

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-3826

www.aacrjournals.org 4591 Cancer Res 2006; 66: (9). May 1, 2006

Research Article

Research. on February 16, 2016. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

underlie Myc-induced tumorigenesis. We have, therefore, used thismodel to track the transcriptional changes that follow acuteactivation of Myc in pancreatic h cells in their orthotopicenvironment. Using genomic profiling of laser capture micro-dissected islets, we show that acute activation of Myc drives aprogressively more complex pattern of expression changes overtime as direct Myc targets engage secondary transcriptionalprograms. Interestingly, although we identified the anticipated cellcycle and metabolic genes, most of the Myc-regulated genes havenot been previously reported as such in in vitro studies.Furthermore, numerous genes implicated in pancreatic isletdevelopment and h-cell differentiation were profoundly modulatedby Myc, demonstrating that cell type is an integral determinant ofthe biological outcome of Myc action. Finally, by combining thisactivation analysis with analogous kinetic analysis of thetranscriptional changes that accompany deactivation of Myc inestablished h-cell tumors, we have defined a discrete set of Myctarget genes whose functions are implicated in the maintenance ofMyc-dependent tumors.

Materials and Methods

Mice, tissue sample generation, and preparation. All mice were

housed and treated in accordance with protocols approved by thecommittee for animal research at the University of California, San

Francisco. Transgenic mice expressing switchable pIns-MycERTAM and

constitutive RIP7-Bcl-XL in their pancreatic h cells (M+XL+) have been

previously described and characterized (10).For expression microarray studies, 8- to 12-week-old M+XL+ mice were

injected i.p. every 24 hours with 1 mg 4-OHT (early activation) or 1 mg

tamoxifen (sustained activation and regression cohorts) to activateMycERTAM. Tamoxifen is metabolized in vivo to 4-OHT and studies in our

laboratory confirm their equivalent effects when administered to M+XL+

mice (data not shown). For the activation cohort, mice were sacrificed by

cervical dislocation at 2, 4, 8, and 24 hours, or 21 days postinjection.Regression cohort mice were sacrificed 2, 4, or 6 days after completing 21

days of treatment. Pancreata were immediately dissected and frozen on dry

ice in optimum cutting temperature medium and stored at �80jC. Organs

from untreated (0 hours) M+XL+ littermates were obtained to act asreference controls. Pancreata from 0- and 24-hour 4-OHT-treated single

transgenic RIP7-Bcl-XL littermates (M�XL+) served as additional 4-OHT-

only controls. Three mice were obtained for each experimental conditionand mice of both sexes were randomly included across all conditions with

the proviso that each triplet included at least one male and one female

mouse.

For studies of explanted whole islets, pancreata were obtained fromuntreated or 4-OHT-treated M+XL+ mice 4 hours after injection and islets

isolated from surrounding exocrine pancreas using a collagenase-based

enzymatic digestion protocol. In brief, the pancreas was inflated with 4 mL

of a 0.7 mg/mL collagenase P solution (Sigma, St. Louis, MO) in 1� HBSS.Isolated pancreas was then cut into small pieces and transferred to

siliconized glass vials where it was incubated with gentle shaking for a

further 15 to 20 minutes at 37jC in 2 mL collagenase solution. Digestedpancreas was washed twice in ice-cold h-cell purification buffer [123 mmol/L

NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl2, 4.2 mmol/L NaHCO3, 0.8 mmol/L

MgSO4, 10 mmol/L HEPES, 5.6 mmol/L glucose, 0.25% bovine serum

albumin (pH 7.4)] and intact islets were handpicked from surroundingexocrine tissue under a stereomicroscope. Total RNA was then isolated from

the islets in preparation for reverse transcription (see below).

Laser capture microdissection and RNA amplification. Fresh-frozen

pancreatic sections (10-14 Am) were fixed in ice-cold 70% ethanol beforelaser capture microdissection. A modified H&E staining protocol was used

to preserve RNA integrity while allowing microscopic visualization of islets

(11, 12). Using an Arcturus PixCell II laser capture microscope system

(Arcturus Engineering, Mountain View, CA), islets of Langerhans were

isolated from surrounding exocrine pancreas. For each independent sample,6 to 10 islets were captured from four to eight adjacent tissue sections and

islets were pooled for RNA isolation. Total RNA was isolated and DNase I

treated using the Arcturus PicoPure RNA Isolation kit and subsequently

quantified (Ribogreen, Molecular Probes, Carlsbad, CA). Two rounds ofT7-RNA polymerase–driven linear amplification was done on each sample

using established protocols (13–15) and biotin-labeled nucleotides were

incorporated during the second round in vitro transcription reaction

(Bioarray High Yield RNA transcript Labeling kit, Enzo, Farmingdale, NY),thus yielding biotinylated, twice-amplified cRNA target (2a-cRNA).

Affymetrix GeneChip arrays, data analysis, and promoter studies.Affymetrix MG-U74Av2 arrays were hybridized and processed following the

Affymetrix GeneChip Expression Analysis Technical Manual. In brief, 20 Ag2a-cRNA was combined with prelabeled hybridization controls (Affymetrix,

Santa Clara, CA) and then hybridized to the arrays. Scaling of scanned

images was done using Affymetrix Microarray Suite 5.0 software and datafrom all chips were normalized using robust multichip averaging before

further statistical analysis (16).

Normalized data were filtered to exclude probe sets with raw signal

intensities of <50 in all samples (log2 <5.7) and statistical analysis ofmicroarrays was then used to identify statistically significant differences in

gene expression between different groups of mice.1 Probe sets with a median

false discovery rate of <5% were called significant (17). Hierarchical clustering

of significantly regulated genes was done using Cluster 3.0 software2 andclusters visualized using TreeView (18).3 Fold changes for each probe set were

calculated relative to the average signal intensity of that probe set in the

corresponding control mice (i.e., activation samples were compared with0-hour mice whereas regression samples were compared with established

islet tumors isolated from 21-day mice). Probe sets with a false discovery

rate V5% or a 2-fold change in expression between untreated and 24-hour

4-OHT-treated M�XL+ islets were eliminated to exclude Myc-independent4-OHT effects. Gene ontology classifications were taken from the MG-U74Av2

annotation file provided on the Affymetrix web site (19).

Immunocytochemical studies of proliferation and differentiation.To evaluate the kinetics of proliferation, transgenic mice were treated with1 mg 4-OHT i.p. 2, 4, 8, 12, 16, 20, or 24 hours before sacrifice. Mice were also

injected with 100 AL of 10 mmol/L stock 5-bromo-2V-deoxyuridine (BrdUrd)

3 hours before sacrifice. Dissected pancreata were fixed overnight inneutral-buffered formalin, embedded in paraffin, and 10 Am sections were

cut for staining. BrdUrd was detected using a mouse monoclonal anti-

BrdUrd primary antibody (Roche, Indianapolis, IN). Studies of h-cell

differentiation in activation samples were done on paraffin-embeddedsections obtained from transgenic mice treated daily with 1 mg tamoxifen

i.p. for 0 to 7 days. Immunohistologic studies of regression samples were

done using fresh-frozen tissue sections obtained from the pancreata used

for gene expression studies. Primary antibodies against Ki-67 (Lab Vision,Fremont, CA), insulin (Linco Research, Inc., St. Charles, MO), Glut2

(Chemicon, Temecula, CA), and Isl-1 (Developmental Studies, Iowa City, IA)

were used along with species-appropriate secondary Alexa Fluor 488 dye–

conjugated antibodies (Amersham, Piscataway, NJ). Stained images werevisualized using a Zeiss Axiovert s100 fluorescent microscope and captured

using Open lab 3.5.1 software.

Quantitative real-time PCR. To validate microarray data, Taqmanquantitative reverse-transcriptase PCR (qRT-PCR) was done on cDNA

generated from laser capture microdissection islets as well as from whole

explanted transgenic islets and murine embryo fibroblasts (MEF). For laser

capture microdissection samples, tissue sections were cut from the samefrozen tissue blocks as those used for microarray analysis, stained with

hematoxylin, and processed as described above. For studies of in vitro

fibroblasts, wild-type MEFs stably transduced with pBabePuro-MycERTAM

were treated with 100 nmol/L 4-OHT or ethanol control for 2 hours beforeRNA isolation. MEF experiments were repeated using cells that had been

1 http://www-stat.Stanford.edu/%7Etibs/SAM/index.html.2 http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/index.html.3 http://rana.lbl.gov/EisenSoftwareSource.htm.

Cancer Research

Cancer Res 2006; 66: (9). May 1, 2006 4592 www.aacrjournals.org



exposed to cycloheximide (10 Amol/L) for 30 minutes before 4-OHT/ethanoltreatment to determine if gene regulation was dependent on functional

protein synthesis. Total RNA (1 Ag) from all islet and MEF samples was

reverse transcribed to first strand cDNA (iScript Reverse Transcriptase kit,

Bio-Rad, Hercules, CA), and gene expression levels were detected usingstandardized TaqMan Assays-on-Demand (Applied Biosystems, Foster City,

CA) and an ABI Prism 7700 PCR sequence detector (Applied Biosystems).

Samples were run in triplicate and the level of expression of each assayed

gene was averaged and then quantified relative to the level of expression ofh-glucuronidase.

Results

Kinetic profiling of Myc effects in vivo is feasible using thecombined technologies of laser capture microdissection, RNAamplification, and expression microarrays. Using linearlyamplified RNA from laser-captured transgenic islets and high-density oligonucleotide microarrays, we profiled the transcription-al consequences of Myc activation and subsequent deactivation inthe h cells of M+XL+ mice in vivo . cMycERTAM was activatedin vivo by injection of 4-OHT and islets were laser captured frompancreatic frozen sections. All samples were treated independentlyyielding 20 to 50 ng total islet RNA per sample. Two rounds oflinear amplification generated 20 to 50 Ag of high-quality, biotin-labeled 2a-cRNA/sample. The overall quality of microarray dataobtained for 33 independently amplified and hybridized RNAsamples was excellent, with an average ‘‘present’’ call of 46 F 4%,which is consistent with published results (20). Followingnormalization of the raw data, 9,813 probe sets were identifiedas being reproducibly detectable in pIns-MycERTAM–positivepancreatic islets.4 We then analyzed these probe sets for evidenceof modulation by Myc.Global gene expression profiling in pancreatic islets

following Myc activation in vivo reveals complex and dynamicchanges. To define immediate early transcriptional responses toMyc, we first identified those genes modulated within 24 hours ofacute Myc activation. Statistical analysis of microarray software(17) defined 293 probe sets as significantly altered in this earlyactivation cohort. Expression of eight probe sets was also alteredfollowing 4-OHT treatment of control MycERTAM transgene–negative M�XL+ mice, indicating that they represent genes thatcan be directly modulated by 4-OHT. These eight probe sets wereexcluded from further analysis, leaving 285 probe sets whoseexpression in pancreatic h cells in vivo was significantly alteredwithin 24 hours of acute Myc activation (Supplementary Table S1).Supervised hierarchical clustering of the 285 significantly regulatedprobe sets shows the dynamic nature of Myc-mediated generegulation, even over such a short time period (Fig. 1A).

To define Myc-dependent effects in established h cell tumors, weexpanded our data analysis to include samples obtained from miceexposed to tamoxifen for 21 days. M+XL+ transgenic islets exposedto sustained Myc activity exhibit all the phenotypic characteristicsof malignant h-cell tumors (10). Consistent with our publishedresults, histologic sections confirmed that the islets isolated fromthese mice were massive, proliferative, and angiogenic (data notshown). Kinetic expression profiling of this sustained activationcohort, revealed that 2,532 probe sets were significantly modulatedby Myc over the course of 21 days (Supplementary Table S2).

Supervised hierarchical clustering of the 2,532 probe sets showssignificant elaboration and evolution of the dynamic changesobserved in the early activation cohort (Fig. 1B). Many of the probesets that were initially induced within 24 hours of Myc activationwere repressed in established islet tumors and vice versa. Thedramatic transcriptional differences apparent in 24-hour and 21-day islets underscore the profound effect exerted by sustained Mycactivation.Myc activation in vivo induces synchronous cell cycle entry

and modulates previously identified in vitro targets. Consistentwith its potent mitogenic action, cell cycle genes figure promi-nently among in vitro Myc targets (21). We monitored S-phaseentry of h cells in vivo by BrdUrd incorporation following acuteMycERTAM activation. This showed that onset of h cell cycle entryis extremely synchronous, with S-phase entry of h cells evident inall islets within 16 to 24 hours (Fig. 2A).

Of the 285 significant early activation probe sets, 36 (represent-ing 34 unique genes) are designated ‘‘cell cycle genes’’ by geneontology classification of biological function (Fig. 2B ; ref. 22). Thesynchrony of cell cycle entry we observe upon MycERTAM activationis reflected in the kinetics of expression of key cell cycle regulatory

4 The data discussed in this article have been deposited in NCBIs Gene ExpressionOmnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEOSeries accession number GSE4356.

Figure 1. Myc activation in pancreatic h cells in vivo rapidly and dynamicallyalters the islet transcriptome. Global genomic profiling of transgenic isletsfollowing acute activation of MycERTAM in h cells identifies 285 probe sets asbeing significantly regulated by Myc in the first 24 hours and 2,532 probes sets asbeing modulated following a 21-day period of sustained Myc activity. Hierarchicalgene clustering of these early (A) and sustained (B) activation cohorts shows therapid kinetics of gene induction and repression in vivo , as well as the dynamicnature of the gene expression levels over time. Significance was defined as afalse detection rate of V5%. For gene clustering, expression levels werecalculated relative to the average signal intensity of the probe set in the threeuntreated samples.

Kinetic Analysis of Myc Function In vivo




genes. Cyclin D1 gene (Ccnd1), whose product is pivotal for G1

phase progression, was induced within 2 hours of Myc activation,whereas expression of the S-phase cyclin A (Ccna2) and mitoticcyclin B1 (Ccnb1) and cyclin B2 (Ccnb2) genes peaked subsequently(Fig. 2B). Induction of genes encoding the G2-M transitionregulatory phosphatase Cdc25c and the mitotic regulator kinaseCdk1 (Cdc2) paralleled induction of the mitotic cyclins. Thissequential pattern of cyclin induction was confirmed by qRT-PCR(Fig. 2C).

Next, we used an established database (21) to determine whichof our significant in vivo targets had been previously labeled as Myctargets in vitro .5 Perhaps surprisingly, only 50 of 285 early activationtarget probe sets (equivalent to 47 unique genes) have beenpreviously identified as Myc targets (Supplementary Fig. S1). In thesustained activation cohort, we identified 303 designated Myctarget genes among our list of 2,532 regulated probe sets(Supplementary Table S3). Thus, kinetic profiling of Myc targetgenes in pancreatic h cells in vivo corroborates in vitro studies butalso identifies multiple novel targets with diverse biological roles.Myc represses mature B-cell genes and results in global islet

dedifferentiation. Activation of Myc in many normal andneoplastic cell types is associated with an undifferentiatedphenotype. It remains unclear whether this inverse relationshipbetween proliferation and differentiation is merely correlative orrepresents some obligate exclusivity in the two biologicalprograms. In h cells, it has been noted that ectopic expression ofMyc represses insulin expression (10, 23, 24). To assess whetherMyc activation more globally suppresses terminal h-cell differen-tiation, we analyzed our microarray database to determine theeffect of Myc activation on the expression of mature h-cell-specificgenes.

The transcriptional profile of murine pancreas during develop-ment has been characterized, providing a suite of geneticfingerprints that define the developing organ at various stages ofdifferentiation (25). Adult islets can be distinguished from lessdifferentiated pancreas by the differential expression of 217 matureislet-specific genes that are, in the main, markers of fullydifferentiated h cells (25). One hundred eighty-nine of 217 matureislet genes are represented in our M+XL+ islet data set by 218 probesets and expression of 89 (47%) of these was altered as aconsequence of Myc activation (Supplementary Table S4). Thevast majority (73 of 89) of these islet genes were down-regulated byMyc and, in the case of 25 genes, sustained Myc activity repressedexpression by z2-fold (Fig. 3A). Interestingly, several islet-specificgenes, including Gad1, Ptprn, Slc2a2 , and the key transcriptionalregulator of h-cell-specific gene expression Ipf1 (Pdx1), wererepressed within the first 2 to 4 hours of Myc activation, intimatingthat they may be direct targets of Myc (Supplementary Table S4).qRT-PCR analysis of three well-characterized h cell genes, Th,Slc2a2 , and Ins2 , which encode tyrosine hydroxylase, facilitatedglucose-transporter 2 (glut2), and insulin, respectively, corroborat-ed the microarray data (Fig. 3B). To establish whether this generepression leads to loss of the differentiated h-cell phenotype, weassessed the influence of Myc activation on expression of themature h-cell proteins insulin, Glut2, and Isl-1. Expression of allthree h-cell markers was suppressed upon Myc activation, whereasnone was affected by 4-OHT treatment of MycERTAM-negativeM�XL+ mice (Fig. 3C). Finally, to show that h-cell gene repressionand dedifferentiation were a consequence of Myc activity, weassessed expression in established h-cell tumors 2, 4, and 6 daysfollowing acute deactivation of Myc. Consistent with our previouslypublished observations (10), acute deactivation of Myc in theseh-cell tumors resulted in tumor regression and reappearance ofmature h-cell markers insulin, Glut2, and Isl1 (data not shown).Genomic profiling of these regressing islet tumors showed thatreappearance of the mature h-cell phenotype was heralded by the5 http://www.myccancergene.org/site/mycTargetDB.asp.

Figure 2. Myc activation in h cells in vivo inducesrapid and synchronous cell cycle entry andsustained proliferation. A, activation of Myc indouble-transgenic (M+XL+) pancreatic h cells bysystemic administration of 4-OHT inducedsynchronous S-phase entry in h cells in all isletsof the pancreas by f16 to 24 hours, asevidenced by BrdUrd-positive staining (greenimmunofluorescence ). Analogous 4-OHTtreatment of MycERTAM-negative, M�XL+ miceinduced no S-phase transition. B, hierarchical genecluster of 41 probe sets (38 genes) with geneontology functions of cell cycle or proliferation,which were regulated in the first 24 hours ofMycERTAM activation. C, qRT-PCR analysisof cyclin D1 (Ccnd1 ) and cyclin B2 (Ccnb2 )expression in laser-captured islets confirmed thesequential modulation observed in the microarraydata. Columns, fold change; bars, SD.

Cancer Research




reexpression of h-cell-specific genes (Fig. 3D). Thus, sustained Mycactivity in pancreatic h cells represses the expression of mature h-cell genes, leading to loss of differentiation, whereas subsequentdeactivation of Myc rapidly restores h-cell gene expression. Theseresults indicate that the process of Myc-mediated tumorigenesisinvolves profound changes in differentiation status, many of whicharise from modulation of tissue-specific genes.Comparison of genomic profiles during B-cell tumor

formation and regression identifies genes whose modulationis exquisitely dependent on continual Myc activity. Myc-induced h-cell tumorigenesis arises through induction of h-cellproliferation, dedifferentiation, and angiogenesis. Subsequent Mycdeactivation induces proliferative arrest, redifferentiation, andcollapse of vascular integrity, indicating that tumor regression isessentially a reversal of the processes that drive tumor formation(10). Comparison of the regression cohort expression profiles withthose of stable tumors identified 1,431 probe sets that weresignificantly altered during the onset of tumor regression(Supplementary Table S5). Hierarchical clustering of these genesidentifies two distinct categories: 1,000 probe sets that were rapidly

down-regulated upon Myc deactivation and 431 probe sets thatwere induced (Supplementary Fig. S2). To identify which Myceffectors may be involved in maintaining established h-cell tumors,we compared the 2,532 probe sets that were significantly regulatedupon Myc activation with the 1,431 regression probe setsmodulated by Myc deactivation. This comparison revealed 428probe sets common to both cohorts, 257 of which were regulated inopposing directions during tumor initiation (Myc on) andregression (Myc off; Fig. 4; Table 1). Thus, regulation of thesegenes (either positive or negative) was, like maintenance of theh-cell tumors, dependent on sustained Myc activity. Of the 257‘‘candidate tumor maintenance’’ probe sets, 154 (equivalent to 132genes) were both induced upon Myc activation and repressed upondeactivation (type A genes), whereas 103 probe sets (representing101 genes) were repressed by Myc activation and then reexpressedwhen Myc was deactivated (type B genes; Table 1). The diversebiological functions of these candidate tumor maintenance genesare apparent from their gene ontology designations (Table 1A).

To identify which of the 257 probe sets are likely to be direct Myctargets, we determined which were regulated immediately

Figure 3. Myc activation in h cells represses mature islet-specific genes and results in h-cell dedifferentiation. A, list of significantly regulated (false discoveryrate <5%) adult islet-associated genes that were down-regulated at least 2-fold following 21 days of sustained Myc activity in h cells. B, Taqman analysis oflaser-captured islets confirms Myc-induced repression of three known genetic markers of h cell differentiation. Columns, fold change; bars, SD. C, down-regulationof islet-specific genes in M+XL+ h cells is accompanied by suppression of insulin, Glut2, and Isl-1 proteins. Insulin and Glut2 are cytoplasmic, whereas Isl-1 is visibleas a nuclear protein. All positive immunofluorescent staining is represented in green and, in the case of Isl-1, autofluorescent RBC appear white. The differentiationstate of control M�XL+ h cells is unaffected by 4-OHT treatment. Representative of islets from three different mice for each time point and genotype (�400).D, h-cell genes repressed z2-fold at 21 days were largely reexpressed following Myc deactivation. Fold change in signal intensity is relative to the average of21-day Myc-on samples. Columns, fold change; bars, SD. Representative for three replicate mice at each time point. FC, fold change.





following MycERTAM activation. Thirty maintenance probe setswere modulated within 24 hours of Myc activation (Fig. 5A) and 10were altered >1.5-fold within 2 to 4 hours (Fig. 5B). Of these, sixwere induced (BC037006, Eif4ebp1, Fzd9, Gmip, St6galnac4 , andZbtb16), whereas four were repressed (5730592L21, Dap, Derl2 , andSec23b ; Fig. 5B ; Table 1B). Although only 3 of these 10 genes havebeen previously reported to be Myc targets (Gmip, St6galnac4 , andSec23b), by sequence analysis all six type A genes contain canonicalMyc:Max binding sites in their promoters (data not shown),supporting the idea that these are direct Myc targets. To furthervalidate these targets, we selected three type A genes (Fzd9,Eif4ebp1 , and Gmip) and one type B gene (Dap) for analysis by qRT-PCR. These studies confirmed Myc-mediated regulation of all fourgenes in h cells in vivo (Fig. 5C).

Finally, to determine if Myc-dependent regulation of keymaintenance genes is dependent on cell type, we assessed theeffects of acute MycERTAM activation in primary MEFs in vitro .Evaluation of the aforementioned three type A and one type Bgenes revealed that Eif4ebp1 was not measurably expressed inMEFs, Gmip was unaffected by Myc status, and Dap was repressedto an insignificant degree (data not shown). However, Fzd9 wassignificantly induced following Myc activation in MEFs and thisoccurred even in the presence of cycloheximide, suggesting thatFzd9 is likely to be a direct transcriptional target of Myc (Fig. 5D).Such data are consistent with the emerging notion that Myc targets

comprise both genes that are universally regulated and genes thatare specific to certain cell lineages and/or environmental circum-stances.

Discussion

Understanding how genes implicated in cancer conspire todisrupt normal cell and tissue biology is one of the most intractableproblems in contemporary biology. The Myc oncoprotein exem-plifies this problem: Originally identified through its role inpromoting cell proliferation, its mechanism of action involves thecoordinated regulation of a multitude of biological propertiesmediated through a myriad of target genes (7–9, 21, 26–31). Thismakes determining the extent to which these various genescontribute to Myc oncogenic potential virtually impossible todetermine. It is also apparent that cell type, genetic background,and experimental context can all influence the downstreamconsequences of Myc activation, leading some to propose thatMyc may act as a global transcriptional regulator (6).

Notwithstanding such a ‘‘global’’ role in biology, the mechanismsby which Myc drives and maintains tumorigenesis are likely to bemore restricted. However, the problem remains that as Myc exertsits inexorable tumorigenic effect, a legion of secondary geneticprograms evolve. Dissecting which of these is cause or conse-quence of Myc action has proven very difficult, as has identifying

Figure 4. Genomic profiling of islet tumorsfollowing deactivation of Myc identifiescandidate Myc-regulated genes witha role in tumor maintenance. Supervisedhierarchical gene clustering is shownof 257 probe sets that were oppositelyinfluenced by Myc activation anddeactivation. One hundred fifty-four probesets (representing 132 genes) wereinduced when Myc was activated andthen down-regulated when Myc wasdeactivated, whereas 103 probe sets(representing 101 genes) were repressedupon Myc activation and reexpressed uponsubsequent deactivation. A close-upof three clusters is shown here and acomplete listing of all represented genesis shown in Table 1A .

Cancer Research




which, if any, of its direct gene targets act as linchpins of Myconcogenic action. In this article, we have addressed the problem ofobjectively defining what attributes of Myc are required for themaintenance of tumors by combining a switchable in vivo geneticmodel with a global genomic analysis of Myc-dependent transcrip-tion. In this way, we have, for the first time, correlated Myconcogenic action with the underlying transcriptional changes thataccompany tumorigenesis. In particular, the reversibility of ourswitchable model has enabled us to define a highly restricted cadre

of Myc target genes that are candidates for mediating tumormaintenance function of Myc in vivo .

Not surprisingly, cell cycle genes figure prominently among ourMyc targets. Ccnd1 , the gene encoding cyclin D1, has beenpreviously identified as a Myc target in vitro and our in vivo datasupport the induction of Ccnd1 by Myc. Because cell growth mustaccompany cell cycle progression, it is also noteworthy that genesencoding several members of the AKT-mTOR nutrient use andresponse pathway are also modulated by Myc. These include

Table 1. Putative tumor maintenance genes

A. Maintenance genes sorted by gene ontology biological process

Type A genes induced when

Myc is activated and repressed

when Myc is deactivated

Type B genes repressed upon Myc activation and

reexpressed when Myc is deactivated

Angiogenesis Crhr2, Epas1 —

Apoptosis Accn1, Casp2, Fadd, Foxo3 Dap, Nfkb1, Sgpl1, Siah2Cell cycle/proliferation Ak1, Ccng2, Cdc25c, Ect2, Jun, Lzf D17Wsu104e, Pard6a, Siah2

Cell adhesion Cspg2, Vcam1 —

Cell growth and/or maintenance Ect2, Foxo3, Jun, Socs3 —Cytoskeleton or microtubule Kifc5a, Krt1-18, Mtap6 6720463E02Rik, Map1lc3, Nde1

Development or organogenesis/

morphogenesis

Epas1, Fzd9, Jun, Zbtb16 Arpc1a, Bmp1, Dcx, Nfkb1, Siah2, Th

Endocytosis Dnm, Cubn —Glycolysis Eno1, Pklr —

Glycosylation St6galnac4, St6gal1 Rpn1

Metabolism Ak1, Ccs, Galm, Gmip, Gsr, Gyk, Mvk, Sms, Uox 2410012M04Rik, Atp2a2, Cryl1, Fh1,

Mcfd2, Psap, Sgpl1, ThmRNA processing — A1462438, Cpsf3

Phosphorylation/

dephosphorylation

Cdc25c, Dyrk1a, Ltk, Matk, Mvk, Pdk4, Ptk2b Ppm1b, Pxk, Slk, Stk16

Protein biosynthesis or

translation

2410005K20Rik, Cubn, Eif4ebp1, Sars2 Eef2, Eif4b, Mrpl4, Mrps18a, Mrps18b, Tars, Wars

Protein folding Ccs Cct3, Cct4, Dnajb1, Dnajb10, Dnajb11, Tra1

Proteolysis Adprtl2, Blmh, Casp2, Erf Bmp1, Xpnpep1Signaling/signal transduction Crhr2, Ect2, Eif4ebp1, Epas1, Fadd, Fzd9,

Gabrg3, Gcgr, Gmip, Ltk, Matk, Olfr16,

Pde1b, Racgap1, Rasal1, Socs3, Stmn4

Arfrp1, Arl4, Crcp, Dcx, Nfkb1, Pdzk11, Pxk

Transcription D1Ertd161e, Epas1, Erf, Fhl2, Jun, Mnt,Myt1, Rpo1-2, Sox4, Tead4, Tieg1, Zfp28

Nfkb1, Rnf14, Xbp1

Transport Accn1, Atp1b1, Ccs, Clcn1, Gabrg3,

Gsr, Kif20a, Slc6a8, Txndc9, Txn12

1300013B2Rik, Aass, Abcc8, Arl4,

Atp2a2, Gdi1, Mcfd2,

Sec22l1, Sec23b, Sytl4, Txndc7Ubiquitin cycle 2700084L22Rik, Usp2 Pxmp3, Rnf14, Siah2

Other 2010016F14Rik, C1qtnf1, Dbn1, Mpz,

Serpina1a, Serpina1d, Txnip

Anxa5, Cmas, Gch1, H2-Q7, H2-D1,

LOC280487, Nme2Not specified 1500010J02Rik, 1500034J01Rik, 1810030O07Rik,

1810044D09Rik, 2410018L13Rik, 2900054D09Rik,

6230416J20Rik, AI464131, AI591476, BC037006,

Bub1, C330027C09Rik, C79407, C80638, Cck,Ceacam11, Cep2, Col5a1, Crlf1, Csnk, D10627,

D430019H16Rik, D4Ertd196e, D5Ertd363e,

D930048N14Rik, Dlk1, Dnajc11, Egln2, Eif4ebp2,

Fez1, Gtse1, H1fx, Hrc, Ide, Iqcf4, Itgb1bp1,Jarid1b, Lpin1, Lpp, Midn, Pcdha10, Phlda3,

Pspc1, Riok3, Sct, Sez6, Slc35b1, Slc35e4, Ssbp4,

Surf2, Tmem5, Wiz, Zdhhc9, Zfp259, Zfp277

0610010K14Rik, 1110008P14Rik, 1110014C03Rik,

1700023O11Rik, 1810010N17Rik, 1810046J19Rik,

2310004K06Rik, 2410022L05Rik, 2610208E05Rik,

2610507B11Rik, 2900010J23Rik, 833439L19Rik,5730592L21Rik, 6330442E10Rik, Armet,

B230114J08Rik, C130052I12Rik, Cdk5rap3,

Cenpc1, Commd7, D030028O16Rik, D19Ertd144e,

Derl2, Dnajc3, Fut8, Gas5, Gng12, Ik,Mapbpip, Mrpl54, Ndg2, Pik3c3, Pprc1,

Pscd3, Rad17, Sec23ip, Tbrg1, Tgtp, Vasp

(Continued on the following page)





Table 1. Putative tumor maintenance genes (Cont’d)

B. Ten early target genes implicated in tumor maintenance in pIns-MycERTAM/RIP7-Bcl-xL transgenic mice

Probeset ID Gene name Gene

symbol

Ref Seq EBOX* Present

in databasec

(a) GO biological process

(b) Published role in cancer

96518_at cDNA sequence BC037006 BC037006 XM_109956 Yes No (a) —

(b) No

100636_at Eukaryotic translation

initiation factor 4E

binding protein 1

Eif4ebp1 NM_007918 Yes No (a) Insulin receptor signaling pathway,

negative regulation of protein

biosynthesis, negative regulationof translational initiation,

regulation of protein biosynthesis,

regulation of translation, andregulation of translational

initiation

(b) Yes (45)

99844_at Frizzled homologue 9

(Drosophila)

Fzd9 XM_284144 Yes No (a) G-protein coupled receptor protein

signaling pathway, Wnt receptorsignaling pathway, cell surface

receptor linked signal transduction,

development, and frizzledsignaling pathway

(b) Yes (46)

93647_at Gem-interacting protein Gmip NM_198101 Yes Yes (a) Intracellular signaling cascade

and metabolism(b) No

96682_at ST6 (a-N-acetyl-neuraminyl-2,

3-b-galactosyl-1,3)-N-acetylgalactosaminide

a-2,6-sialyltransferase 4

St6galnac4 NM_011373 Yes Yes (a) Protein amino acid glycosylation

(b) Yes (44)92202_g_at Zinc finger and BTB domain

containing 16

Zbtb16 XM_134826 Yes No (a) Skeletal development, negative

regulation of cell proliferation,

embryonic pattern specification,

anterior, posterior patternformation, embryonic limb

morphogenesis, forelimb

morphogenesis, positive

regulation of apoptosis, regulationof transcription, negative

regulation of transcription, and

DNA-dependent, male germ-linestem cell division

(b) Yes (43)

160119_at RIKEN cDNA5730592L21 gene

5730592L21Rik NM_029720 Yes No (a) —

(b) No

93842_at Death-associated protein Dap NM_146057 No No (a) Apoptosis and induction of apoptosisby extracellular signal

(b) No

160769_at Der1-like domain family,

member 2

Derl2 NM_033562 No No (a) —

(b) Yes (48)

98944_at SEC23B

(Saccharomyces cerevisiae)

Sec23b NM_019787 No Yes (a) Endoplasmic reticulum to Golgi

transport, intracellular protein

transport, protein transport,and transport

(b) No

*MYC:MAX binding site determined by promoter sequence analysis using Matinspector Software (http://www.genomatix.de).chttp://www.myccancergene.org/site/mycTargetDB.asp.

Cancer Research




Eif4ebp1 , which encodes the eukaryotic translation initiation factor4 binding protein 1, and is one of only 10 early Myc targets that weidentify as candidate mediators of tumor maintenance. In addition,the inhibitor of mTOR, tuberin (Tsc2), is rapidly and continuouslydown-regulated following MycERTAM activation whereas expressionof the S6 kinase (Stk6) gene is induced 5-fold at 24 hours(Supplementary Table S1). Our data lend support to recent in vitrostudies implicating Myc in the regulation of the AKT-mTor pathway(32) and confirm that the effects of Myc on cell cycle andproliferation are intimately linked to cell growth and survival.

Despite substantial evidence for direct Myc-mediated inductionof the ARF-p53 pathway both in vitro and in vivo (33, 34), we foundno evidence for this relationship in M+XL+ islets in vivo . NeitherCdkn2a , which encodes both p19ARF and p16Ink4a , nor key p53target genes Cdkn1a (p21) or Mdm2 were induced during the first24 hours of Myc activation. We suspect that this lack of p53activation may be due to the influence of Bcl-xL overexpression inour model. It has been proposed that overexpression of Bcl-xL maylimit Myc induction of p19ARF, thereby suppressing p53-mediated

cell cycle arrest and apoptosis (35). Consistent with this, we findthat p19ARF is induced as expected following in vivo activation ofMycERTAM in h cells lacking overexpression of Bcl-xL.6 Thus, inaddition to suppressing apoptosis, Bcl-xL may cooperate oncogeni-cally with Myc by abrogating Myc-induced activation of the ARF-p53 pathway. Ongoing work in our laboratory is directed towardelucidating this issue.

Myc status has a profound influence on embryonic development(36, 37), in part through its ability to disrupt differentiationprograms. In this study, acute activation of Myc triggered awidespread change in h-cell phenotype, suppressing expression ofmany genes that are signatures of terminally differentiated h cells.The observed association between Myc-ERTAM activation and lossof h-cell differentiation was substantiated by the rapid reappear-ance of such markers upon deactivation of Myc. Whether these

Figure 5. Tumor maintenance genes includea subset of novel, putative direct targets ofMyc. A, hierarchical gene cluster of 30maintenance gene probe sets that weresignificantly modulated within the first 24 hoursof acute Myc activation in vivo and oppositelyregulated following Myc deactivation. B, 10 ofthe 30 maintenance gene probe sets wereinduced or repressed within 4 hours of acuteMyc activation, suggesting that they may bedirect transcriptional targets of Myc. All sixinduced genes have a putative Myc:Maxbinding site in their promoter region and haveknown functions relevant to cancer (seeTable 1B). C, Taqman analysis of explantedwhole islets isolated from M+XL+ mice 4 hoursafter 4-OHT treatment confirmed Mycmodulation of four putative target maintenancegenes. Columns, fold change relative to theaverage of untreated mice for replicate mice ateach time point; bars, SD. D, acute activationof MycERTAM in primary MEFs causeda 2-fold induction of Fzd9 relative to ethanolcontrol–treated cells 2 hours after 4-OHTtreatment. Pretreatment of cells withcycloheximide (CHX ) did not abrogate theFzd9 response, confirming that it is a directtranscriptional target of Myc. NS, notstatistically significant.

6 Unpublished data.





differentiation genes are direct targets of the Myc:Max complex willneed to be determined by further studies. These findings parallelrecent observations of Myc-induced hepatic tumors that undergodifferentiation upon oncogene deactivation (38). Aberrant orderegulated expression of intrinsic developmental pathways may,therefore, be an integral mechanistic component of Myc-inducedtumorigenesis. It is noteworthy that several of the genes rapidlyrepressed by Myc in our study are tissue-specific and cannot,therefore, be universal Myc targets. However, other genes notrestricted in their expression to pancreas (e.g., Eif4ebp1, Dap , andGmip) were nonetheless regulated by Myc in h cells in vivo yet notin fibroblasts in vitro . Defining the precise role of Myc targets, bethey direct or indirect, in tumorigenesis will thus require carefulstudy in biologically relevant cell types and environments in vivo .

By directly comparing the transcriptional fingerprints of isletsduring tumor formation and regression, we were able to identify257 probe sets (233 genes) whose expression was inverselyregulated during Myc activation and subsequent deactivation.Because Myc is required to maintain the tumors it induces, thiscadre of genes must include those whose functions are required formaintenance of h-cell tumors. Functional classification of geneswithin this ‘‘tumor maintenance’’ cohort identifies many targets ofpotential interest. Among the type A genes, whose sustainedexpression requires sustained Myc activity, are the cytokinesisregulator Ect2 and the activator protein transcription factormember c-Jun , both of which have been implicated in tumorigen-esis (39, 40). Another type A gene is Epas1 , implicated in hypoxia-induced angiogenesis (41) and potentially involved in maintainingh-cell tumor vasculature. The designated biological functions ofthese type A genes are extremely diverse and underscore the ideathat tumor maintenance involves multiple processes that evolveover time. For example, whereas cell cycle and proliferation genesmake up over 30% of early activation gene targets, they representonly 10% of the identified maintenance gene cohort.

The kinetics of regulation of the 233 candidate maintenancegenes indicates that the great majority are indirect consequences ofMyc action rather than direct target genes. By confining our searchto those genes that were rapidly modulated by Myc, we identified asubset of 10 genes that share the correct dynamics to be putativedirect transcriptional targets. Most of these genes (i.e., Dap, Derl2,Eif4ebp1, Fzd9, St6galnac4 , and Zbtb16) possess both requisite

regulatory Myc recognition elements in their promoters and havealready been implicated elsewhere in various aspects of cancer(42–48). Of note, the Wnt receptor–encoding gene, Fzd9 , was alsofound to be regulated by Myc in cultured MEFs. Wnt signaling iscritical to normal development and is frequently deregulated inhuman cancer, largely through constitutive activation of h-catenin(49). Fzd9 knockout mice show significant abnormalities inhematologic development, principally secondary to a failure ofB-cell self-renewal (50). Definitive proof that Fzd9 is a directtranscriptional target of Myc and the extent to which it mediatesessential aspects of Myc function will need to be resolved by directexperimentation. To that end, we have begun studies to determinethe effect of acute MycERTAM activation on the islets of Fzd9knockout mice. Demonstration of impaired tumor formation inthese mice will confirm the critical role of this Wnt receptor inMyc-induced transformation of pancreatic h cells in vivo .

In summary, we have, for the first time, defined, in real time, theMyc-induced transcriptional changes that occur during tumorinitiation and regression in an orthotopic tissue in vivo . We arecurrently extending this approach to other inducible tumormodel systems to elucidate which of the identified Myc targetsare h cell-specific and which are more universal. By unraveling thecomplexities of the tumorigenic process in vivo , we hope to identifygenes and pathways that will be strategic targets for the focuseddevelopment of novel, targeted anticancer strategies.

Acknowledgments

Received 10/24/2005; revised 2/24/2006; accepted 3/2/2006.Grant support: Juvenile Diabetes Research Foundation grants 4-2004-372 and 4-

1999-841, National Cancer Institute grant R01 CA98018 (G.I. Evan), and CanadianInstitutes of Health Research Senior Research Fellowship (E.R. Lawlor).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank The J. David Gladstone Genomics Core Laboratory at San FranciscoGeneral Hospital (where part of this work was carried out) and the University ofCalifornia San Francisco Genome Core, Mt. Zion Cancer Center (where qRT-PCR wasdone), for invaluable assistance; their respective directors, Dr. Chris Barker andDr. David Ginzinger, and personnel; Dr. Chris Haqq and his laboratory at theUniversity of California San Francisco Cancer Center for their instruction andguidance in the techniques of laser capture microdissection and RNA amplification;Dr. Tobias Dansen for help with fluorescence microscopy; Mike Hagen for technicalsupport with islet preparations; and Dr. Matthias Hebrok (Diabetes Center,University of California San Francisco, San Francisco, CA) for anti-Glut2 and anti-Isl-1 antibodies.

References

1. Blackwood EM, Eisenman RN. Max: a helix-loop-helixzipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 1991;251:1211–7.2. Littlewood TD, Amati B, Land H, Evan GI. Max and c-

Myc/Max DNA-binding activities in cell extracts.Oncogene 1992;7:1783–92.3. Kretzner L, Blackwood EM, Eisenman RN. Myc and

Max proteins possess distinct transcriptional activities.Nature 1992;359:426–9.4. Solomon DL, Amati B, Land H. Distinct DNA binding

preferences for the c-Myc/Max and Max/Max dimers.Nucleic Acids Res 1993;21:5372–6.5. Staller P, Peukert K, Kiermaier A, et al. Repression of

p15INK4b expression by Myc through association withMiz-1. Nat Cell Biol 2001;3:392–9.6. Li Z, Van Calcar S, Qu C, Cavenee WK, Zhang MQ, Ren

B. A global transcriptional regulatory role for c-Myc inBurkitt’s lymphoma cells. Proc Natl Acad Sci U S A 2003;100:8164–9.7. Mao DY, Watson JD, Yan PS, et al. Analysis of Myc

bound loci identified by CpG island arrays shows thatMax is essential for Myc-dependent repression. CurrBiol 2003;13:882–6.8. O’Connell BC, Cheung AF, Simkevich CP, et al. A large

scale genetic analysis of c-Myc-regulated gene expres-sion patterns. J Biol Chem 2003;278:12563–73.9. Fernandez PC, Frank SR, Wang L, et al. Genomic

targets of the human c-Myc protein. Genes Dev 2003;17:1115–29.10. Pelengaris S, Khan M, Evan GI. Suppression of Myc-

induced apoptosis in h cells exposes multiple oncogenicproperties of Myc and triggers carcinogenic progression.Cell 2002;109:321–34.11. Goldsworthy SM, Stockton PS, Trempus CS, Foley JF,

Maronpot RR. Effects of fixation on RNA extraction andamplification from laser capture microdissected tissue.Mol Carcinog 1999;25:86–91.12. Luzzi V, Holtschlag V, Watson MA. Expression

profiling of ductal carcinoma in situ by laser capturemicrodissection and high-density oligonucleotidearrays. Am J Pathol 2001;158:2005–10.13. Eberwine J. Amplification of mRNA populations

using aRNA generated from immobilized oligo(dT)-T7primed cDNA. Biotechniques 1996;20:584–91.14. Phillips J, Eberwine JH. Antisense RNA amplification:

a linear amplification method for analyzing the mRNApopulation from single living cells. Methods 1996;10:283–8.15. Baugh LR, Hill AA, Brown EL, Hunter CP. Quantita-

tive analysis of mRNA amplification by in vitrotranscription. Nucleic Acids Res 2001;29:E29.16. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B,

Speed TP. Summaries of Affymetrix GeneChip probelevel data. Nucleic Acids Res 2003;31:e15.17. Tusher VG, Tibshirani R, Chu G. Significance analysis

of microarrays applied to the ionizing radiationresponse. Proc Natl Acad Sci U S A 2001;98:5116–21.18. Eisen MB, Spellman PT, Brown PO, Botstein D.

Cluster analysis and display of genome-wide expressionpatterns. Proc Natl Acad Sci U S A 1998;95:14863–8.19. Liu G, Loraine AE, Shigeta R, et al. NetAffx:

Affymetrix probesets and annotations. Nucleic AcidsRes 2003;31:82–6.20. Luzzi V, Mahadevappa M, Raja R, Warrington JA,

Cancer Research






Watson MA. Accurate and reproducible gene expressionprofiles from laser capture microdissection, transcriptamplification, and high density oligonucleotide micro-array analysis. J Mol Diagn 2003;5:9–14.21. Zeller KI, Jegga AG, Aronow BJ, O’Donnell KA, Dang

CV. An integrated database of genes responsive to theMyc oncogenic transcription factor: identification ofdirect genomic targets. Genome Biol 2003;4:R69.22. Ashburner M, Ball CA, Blake JA, et al. Gene ontology:

tool for the unification of biology. The Gene OntologyConsortium. Nat Genet 2000;25:25–9.23. Laybutt DR, Weir GC, Kaneto H, et al. Overexpression

of c-Myc in h-cells of transgenic mice causes prolifer-ation and apoptosis, downregulation of insulin geneexpression, and diabetes. Diabetes 2002;51:1793–804.24. Kaneto H, Sharma A, Suzuma K, et al. Induction of

c-Myc expression suppresses insulin gene transcriptionby inhibiting NeuroD/BETA2-mediated transcriptionalactivation. J Biol Chem 2002;277:12998–3006.25. Gu G, Wells JM, Dombkowski D, Preffer F, Aronow B,

Melton DA. Global expression analysis of gene regula-tory pathways during endocrine pancreatic develop-ment. Development 2004;131:165–79.26. Menssen A, Hermeking H. Characterization of the

c-MYC-regulated transcriptome by SAGE: identificationand analysis of c-MYC target genes. Proc Natl Acad SciU S A 2002;99:6274–9.27. Orian A, van Steensel B, Delrow J, et al. Genomic

binding by the Drosophila Myc, Max, Mad/Mnt tran-scription factor network. Genes Dev 2003;17:1101–14.28. Haggerty TJ, Zeller KI, Osthus RC, Wonsey DR, Dang

CV. A strategy for identifying transcription factorbinding sites reveals two classes of genomic c-Myctarget sites. Proc Natl Acad Sci U S A 2003;100:5313–8.29. Godfried MB, Veenstra M, v Sluis P, et al. The N-myc

and c-myc downstream pathways include the chromo-

some 17q genes nm23-1 and nm23-2. Oncogene 2002;21:2097–101.30. Guo QM, Malek RL, Kim S, et al. Identification of

c-myc responsive genes using rat cDNA microarray.Cancer Res 2000;60:5922–8.31. Coller HA, Grandori C, Tamayo P, et al. Expression

analysis with oligonucleotide microarrays reveals thatMYC regulates genes involved in growth, cell cycle,signaling, and adhesion. Proc Natl Acad Sci U S A 2000;97:3260–5.32. Ruggero D, Montanaro L, Ma L, et al. The translation

factor eIF-4E promotes tumor formation and cooperateswith c-Myc in lymphomagenesis. Nat Med 2004;10:484–6.33. Zindy F, Eischen CM, Randle DH, et al. Myc signaling

via the ARF tumor suppressor regulates p53-dependentapoptosis and immortalization. Genes Dev 1998;12:2424–33.34. Zindy F, Williams RT, Baudino TA, et al. Arf tumor

suppressor promoter monitors latent oncogenic signalsin vivo. Proc Natl Acad Sci U S A 2003;100:15930–5.35. Nilsson JA, Cleveland JL. Myc pathways provoking

cell suicide and cancer. Oncogene 2003;22:9007–21.36. Davis AC, Wims M, Spotts GD, Hann SR, Bradley A.

A null c-myc mutation causes lethality before 10.5 daysof gestation in homozygotes and reduced fertility inheterozygous female mice. Genes Dev 1993;7:671–82.37. Levens DL. Reconstructing MYC. Genes Dev 2003;17:

1071–7.38. Shachaf CM, Kopelman AM, Arvanitis C, et al. MYC

inactivation uncovers pluripotent differentiation andtumour dormancy in hepatocellular cancer. Nature2004;431:1112–7.39. Jochum W, Passegue E, Wagner EF. AP-1 in mouse

development and tumorigenesis. Oncogene 2001;20:2401–12.

40. Saito S, Liu XF, Kamijo K, et al. Deregulation andmislocalization of the cytokinesis regulator ECT2activate the Rho signaling pathways leading to malig-nant transformation. J Biol Chem 2004;279:7169–79.41. Takeda N, Maemura K, Imai Y, et al. Endothelial PAS

domain protein 1 gene promotes angiogenesis throughthe transactivation of both vascular endothelial growthfactor and its receptor, Flt-1. Circ Res 2004;95:146–53.42. Avdulov S, Li S, Michalek V, et al. Activation of

translation complex eIF4F is essential for the genesisand maintenance of the malignant phenotype in humanmammary epithelial cells. Cancer Cell 2004;5:553–63.43. Costoya JA, Pandolfi PP. The role of promyelocytic

leukemia zinc finger and promyelocytic leukemia inleukemogenesis and development. Curr Opin Hematol2001;8:212–7.44. Dall’Olio F, Chiricolo M. Sialyltransferases in cancer.

Glycoconj J 2001;18:841–50.45. Holland EC. Regulation of translation and cancer.

Cell Cycle 2004;3:452–5.46. Kirikoshi H, Sekihara H, Katoh M. Expression profiles

of 10 members of Frizzled gene family in human gastriccancer. Int J Oncol 2001;19:767–71.47. Li S, Takasu T, Perlman DM, et al. Translation factor

eIF4E rescues cells from Myc-dependent apoptosis byinhibiting cytochrome c release. J Biol Chem 2003;278:3015–22.48. Ying H, Yu Y, Xu Y. Cloning and characterization of

F-LANa, upregulated in human liver cancer. BiochemBiophys Res Commun 2001;286:394–400.49. Karim R, Tse G, Putti T, Scolyer R, Lee S. The

significance of the Wnt pathway in the pathology ofhuman cancers. Pathology 2004;36:120–8.50. Ranheim EA, Kwan HC, Reya T, Wang YK, Weissman

IL, Francke U. Frizzled 9 knock-out mice have abnormalB-cell development. Blood 2005;105:2487–94.



2006;66:4591-4601. Cancer Res Elizabeth R. Lawlor, Laura Soucek, Lamorna Brown-Swigart, et al. Novel Insights into Myc-Mediated Tumorigenesis

ProvidesIn vivoReversible Kinetic Analysis of Myc Targets

Updated version

http://cancerres.aacrjournals.org/content/66/9/4591

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2006/05/05/66.9.4591.DC1.html

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/66/9/4591.full.html#ref-list-1

This article cites 49 articles, 25 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/66/9/4591.full.html#related-urls

This article has been cited by 14 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

[email protected] at

To request permission to re-use all or part of this article, contact the AACR Publications


http://cancerres.aacrjournals.org/content/66/9/4591

http://cancerres.aacrjournals.org/content/suppl/2006/05/05/66.9.4591.DC1.html

http://cancerres.aacrjournals.org/content/66/9/4591.full.html#ref-list-1

http://cancerres.aacrjournals.org/content/66/9/4591.full.html#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]


Documents

Reversible Kinetic Analysis of Myc Targets In vivo Provides Novel Insights into Myc-Mediated Tumorigenesis