Reversible Kinetic Analysis of Myc Targets in Vivo Provides Novel Insights Into Myc-Mediated Tumorigenesis

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Research Article

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 this, genetic lesions in the Myc locus occur frequently in human Deregulated expression of the Myc transcription factor is a malignancy. In general, such lesions result in the overexpressed frequent causal mutation in human cancer. Thousands of and/or deregulated expression of Myc. However, the molecular mechanisms by which Myc promotes cellular transformation and putative Myc target genes have been identified in in vitro studies, indicating that Myc exerts highly pleiotropic effects oncogenesis remain unclear. within cells and tissues. However, the complexity and diversity The pleiotropic effects of Myc function are thought to arise of Myc gene targets has confounded attempts at identifying through its action as a transcription factor, positively or negatively which of these genes are the critical targets mediating Myc- regulating target effector genes. Induction of gene targets by Myc driven tumorigenesis in vivo. Acute activation of Myc in a requires its dimerization with its partner protein, Max, and reversibly switchable transgenic model of Myc-mediated B cell subsequent heterodimeric binding to canonical (CACGTG) and tumorigenesis induces rapid tumor onset, whereas subsequent noncanonical DNA E-box sequences (1–4). The mechanisms by Myc deactivation triggers equally rapid tumor regression. which Myc represses gene targets are less clear but likely involve Thus, sustained Myc activity is required for tumor mainte- functional interference via binding to and inhibiting the activity of nance. We have used this reversibly switchable kinetic tumor positive regulators, such as Miz-1 (5). In attempts to define the model in combination with high-density oligonucleotide transcriptional target genes of Myc, several genome-wide screens microarrays to develop an unbiased strategy for identifying have been conducted using different cell types and experimental candidate Myc-regulated genes responsible for maintenance conditions (6–9). These studies have indicated the great diversity of of Myc-dependent tumors. Consistent with known Myc Myc target gene function and have, cumulatively, identified well functions, some Myc-regulated genes are involved in cell over 1,000 putative transcriptional targets. Unfortunately, the growth, cycle, and proliferation. In addition, however, many degree of overlap among these in vitro studies has been small, Myc-regulated genes are specific to B cells, indicating that a alluding to the critical potential role of cell type and context in significant component of Myc action is cell type specific. determining the biological consequences of Myc action. With Finally, we identify a very restricted cadre of genes with respect to its specific role in cancer, it has not been possible to expression that is inversely regulated upon Myc activation- substantiate which of these Myc targets are mission critical for induced tumor progression and deactivation-induced tumor tumorigenesis and which are functionally inconsequential. Impor- regression. By definition, such genes are candidates for tumor tantly, to date, no systematic analysis of Myc transcriptional targets maintenance functions. Combining reversibly switchable, in any orthotopic tissue setting in vivo has been conducted. transgenic models of tumor formation and regression with MycERTAM is a regulatable fusion protein that exhibits Myc genomic profiling offers a novel strategy with which to activity only in the presence of the ligand 4-hydroxytamoxifen deconvolute the complexities of oncogenic signaling pathways (4-OHT). In pIns-MycERTAM transgenic mice, MycERTAM expression in vivo. (Cancer Res 2006; 66(9):4591-601) is targeted to pancreatic h cells via the insulin promoter (10). The h cells of pIns-MycERTAM mice exhibit uniform cell cycle entry Introduction upon systemic administration of 4-OHT; however, the net biological outcome of such acute Myc activation depends on the extent The Myc transcription factor regulates many genes implicated in to which Myc-induced apoptosis is opposed. By itself, activation of diverse cellular functions, including proliferation, differentiation, Myc triggers massive h-cell apoptosis that overwhelms prolifera- death, and tissue reorganization. Disruption of the control of these tion and leads to islet involution (10). In contrast, coexpression of processes is a mandatory feature of cancer and, consistent with h Bcl-xL in cells inhibits apoptosis, allowing Myc-induced proliferation to supersede death and resulting in rapid and progressive h-cell expansion (10). Upon sustained activation of Note: Supplementary data for this article are available at Cancer Research Online TAM (http://cancerres.aacrjournals.org/). MycER , hyperplastic islets progressively acquire the defining Current address for E.R. Lawlor:Division of Hematology-Oncology, Children’s characteristics of cancer, including dysplasia and dedifferentiation, Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los angiogenesis, local invasion, and metastasis. Moreover, these Angeles, California. Current address for C.U. Bialucha:Medical Research Council Laboratory for features are completely dependent on continuous Myc action as Molecular Cell Biology, University College London, London, United Kingdom. evidenced by the uniform regression of tumors upon 4-OHT Requests for reprints: Gerard I. Evan, Cancer Research Institute, University of California San Francisco, Comprehensive Cancer Center, 2340 Sutter Street, San withdrawal (10). Francisco, CA 94143-0875. E-mail:[email protected] or Elizabeth R. Lawlor, Children’s Given the consistent, synchronous, and reproducible pattern of Hospital Los Angeles, 4650 Sunset Boulevard MS57, Los Angeles, CA 90027. Phone: phenotypic changes that Myc elicits and maintains in this model, 323-644-8579; E-mail:[email protected]. TAM I2006 American Association for Cancer Research. the pIns-MycER x RIP7-Bcl-xL double-transgenic mouse is an doi:10.1158/0008-5472.CAN-05-3826 ideal system with which to study the genetic processes that www.aacrjournals.org 4591 Cancer Res 2006; 66: (9). May 1, 2006

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research underlie Myc-induced tumorigenesis. We have, therefore, used this isolated from surrounding exocrine pancreas. For each independent sample, model to track the transcriptional changes that follow acute 6 to 10 islets were captured from four to eight adjacent tissue sections and activation of Myc in pancreatic h cells in their orthotopic islets were pooled for RNA isolation. Total RNA was isolated and DNase I environment. Using genomic profiling of laser capture micro- treated using the Arcturus PicoPure RNA Isolation kit and subsequently quantified (Ribogreen, Molecular Probes, Carlsbad, CA). Two rounds of dissected islets, we show that acute activation of Myc drives a T7-RNA polymerase–driven linear amplification was done on each sample progressively more complex pattern of expression changes over using established protocols (13–15) and biotin-labeled nucleotides were time as direct Myc targets engage secondary transcriptional incorporated during the second round in vitro transcription reaction programs. Interestingly, although we identified the anticipated cell (Bioarray High Yield RNA transcript Labeling kit, Enzo, Farmingdale, NY), cycle and metabolic genes, most of the Myc-regulated genes have thus yielding biotinylated, twice-amplified cRNA target (2a-cRNA). not been previously reported as such in in vitro studies. Affymetrix GeneChip arrays, data analysis, and promoter studies. Furthermore, numerous genes implicated in pancreatic islet Affymetrix MG-U74Av2 arrays were hybridized and processed following the development and h-cell differentiation were profoundly modulated Affymetrix GeneChip Expression Analysis Technical Manual. In brief, 20 Ag by Myc, demonstrating that cell type is an integral determinant of 2a-cRNA was combined with prelabeled hybridization controls (Affymetrix, the biological outcome of Myc action. Finally, by combining this Santa Clara, CA) and then hybridized to the arrays. Scaling of scanned images was done using Affymetrix Microarray Suite 5.0 software and data activation analysis with analogous kinetic analysis of the from all chips were normalized using robust multichip averaging before transcriptional changes that accompany deactivation of Myc in further statistical analysis (16). h established -cell tumors, we have defined a discrete set of Myc Normalized data were filtered to exclude probe sets with raw signal target genes whose functions are implicated in the maintenance of intensities of <50 in all samples (log2 <5.7) and statistical analysis of Myc-dependent tumors. microarrays 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 Materials and Methods of significantly regulated genes was done using Cluster 3.0 software2 and Mice, tissue sample generation, and preparation. All mice were clusters visualized using TreeView (18).3 Fold changes for each probe set were housed and treated in accordance with protocols approved by the calculated relative to the average signal intensity of that probe set in the committee for animal research at the University of California, San corresponding control mice (i.e., activation samples were compared with Francisco. Transgenic mice expressing switchable pIns-MycERTAM and 0-hour mice whereas regression samples were compared with established constitutive RIP7-Bcl-XL in their pancreatic h cells (M+XL+) have been islet tumors isolated from 21-day mice). Probe sets with a false discovery previously described and characterized (10). rate V5% or a 2-fold change in expression between untreated and 24-hour À For expression microarray studies, 8- to 12-week-old M+XL+ mice were 4-OHT-treated M XL+ islets were eliminated to exclude Myc-independent injected i.p. every 24 hours with 1 mg 4-OHT (early activation) or 1 mg 4-OHTeffects. Gene ontology classifications were taken from the MG-U74Av2 tamoxifen (sustained activation and regression cohorts) to activate annotation file provided on the Affymetrix web site (19). MycERTAM. Tamoxifen is metabolized in vivo to 4-OHT and studies in our Immunocytochemical studies of proliferation and differentiation. laboratory confirm their equivalent effects when administered to M+XL+ To evaluate the kinetics of proliferation, transgenic mice were treated with mice (data not shown). For the activation cohort, mice were sacrificed by 1 mg 4-OHT i.p. 2, 4, 8, 12, 16, 20, or 24 hours before sacrifice. Mice were also cervical dislocation at 2, 4, 8, and 24 hours, or 21 days postinjection. injected with 100 AL of 10 mmol/L stock 5-bromo-2V-deoxyuridine (BrdUrd) Regression cohort mice were sacrificed 2, 4, or 6 days after completing 21 3 hours before sacrifice. Dissected pancreata were fixed overnight in days of treatment. Pancreata were immediately dissected and frozen on dry neutral-buffered formalin, embedded in paraffin, and 10 Am sections were ice in optimum cutting temperature medium and stored at À80jC. Organs cut for staining. BrdUrd was detected using a mouse monoclonal anti- h from untreated (0 hours) M+XL+ littermates were obtained to act as BrdUrd primary antibody (Roche, Indianapolis, IN). Studies of -cell reference controls. Pancreata from 0- and 24-hour 4-OHT-treated single differentiation in activation samples were done on paraffin-embedded transgenic RIP7-Bcl-XL littermates (MÀXL+) served as additional 4-OHT- sections obtained from transgenic mice treated daily with 1 mg tamoxifen only controls. Three mice were obtained for each experimental condition i.p. for 0 to 7 days. Immunohistologic studies of regression samples were and mice of both sexes were randomly included across all conditions with done using fresh-frozen tissue sections obtained from the pancreata used the proviso that each triplet included at least one male and one female for gene expression studies. Primary antibodies against Ki-67 (Lab Vision, mouse. Fremont, CA), insulin (Linco Research, Inc., St. Charles, MO), Glut2 For studies of explanted whole islets, pancreata were obtained from (Chemicon, Temecula, CA), and Isl-1 (Developmental Studies, Iowa City, IA) untreated or 4-OHT-treated M+XL+ mice 4 hours after injection and islets were used along with species-appropriate secondary Alexa Fluor 488 dye– isolated from surrounding exocrine pancreas using a collagenase-based conjugated antibodies (Amersham, Piscataway, NJ). Stained images were enzymatic digestion protocol. In brief, the pancreas was inflated with 4 mL visualized using a Zeiss Axiovert s100 fluorescent microscope and captured of a 0.7 mg/mL collagenase P solution (Sigma, St. Louis, MO) in 1Â HBSS. using Open lab 3.5.1 software. Isolated pancreas was then cut into small pieces and transferred to Quantitative real-time PCR. To validate microarray data, Taqman siliconized glass vials where it was incubated with gentle shaking for a quantitative reverse-transcriptase PCR (qRT-PCR) was done on cDNA further 15 to 20 minutes at 37jC in 2 mL collagenase solution. Digested generated from laser capture microdissection islets as well as from whole pancreas was washed twice in ice-cold h-cell purification buffer [123 mmol/L explanted transgenic islets and murine embryo fibroblasts (MEF). For laser

NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl2,4.2mmol/LNaHCO3,0.8mmol/L capture microdissection samples, tissue sections were cut from the same MgSO4, 10 mmol/L HEPES, 5.6 mmol/L glucose, 0.25% bovine serum frozen tissue blocks as those used for microarray analysis, stained with albumin (pH 7.4)] and intact islets were handpicked from surrounding hematoxylin, and processed as described above. For studies of in vitro exocrine tissue under a stereomicroscope. Total RNA was then isolated from fibroblasts, wild-type MEFs stably transduced with pBabePuro-MycERTAM the islets in preparation for reverse transcription (see below). were treated with 100 nmol/L 4-OHT or ethanol control for 2 hours before Laser capture microdissection and RNA amplification. Fresh-frozen RNA isolation. MEF experiments were repeated using cells that had been pancreatic sections (10-14 Am) were fixed in ice-cold 70% ethanol before laser capture microdissection. A modified H&E staining protocol was used to preserve RNA integrity while allowing microscopic visualization of islets 1 http://www-stat.Stanford.edu/%7Etibs/SAM/index.html. (11, 12). Using an Arcturus PixCell II laser capture microscope system 2 http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/index.html. (Arcturus Engineering, Mountain View, CA), islets of Langerhans were 3 http://rana.lbl.gov/EisenSoftwareSource.htm.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Kinetic Analysis of Myc Function In vivo exposed to cycloheximide (10 Amol/L) for 30 minutes before 4-OHT/ethanol Supervised hierarchical clustering of the 2,532 probe sets shows treatment to determine if gene regulation was dependent on functional significant elaboration and evolution of the dynamic changes A protein synthesis. Total RNA (1 g) from all islet and MEF samples was observed in the early activation cohort (Fig. 1B). Many of the probe reverse transcribed to first strand cDNA (iScript Reverse Transcriptase kit, sets that were initially induced within 24 hours of Myc activation Bio-Rad, Hercules, CA), and gene expression levels were detected using were repressed in established islet tumors and vice versa. The standardized TaqMan Assays-on-Demand (Applied Biosystems, Foster City, CA) and an ABI Prism 7700 PCR sequence detector (Applied Biosystems). dramatic transcriptional differences apparent in 24-hour and 21- Samples were run in triplicate and the level of expression of each assayed day islets underscore the profound effect exerted by sustained Myc gene was averaged and then quantified relative to the level of expression of activation. h-glucuronidase. Myc activation in vivo induces synchronous cell cycle entry and modulates previously identified in vitro targets. Consistent Results with its potent mitogenic action, cell cycle genes figure prominently among in vitro Myc targets (21). We monitored S-phase Kinetic profiling of Myc effects in vivo is feasible using the entry of h cells in vivo by BrdUrd incorporation following acute combined technologies of laser capture microdissection, RNA MycERTAM activation. This showed that onset of h cell cycle entry amplification, and expression microarrays. Using linearly is extremely synchronous, with S-phase entry of h cells evident in amplified RNA from laser-captured transgenic islets and high- all islets within 16 to 24 hours (Fig. 2A). density oligonucleotide microarrays, we profiled the transcription- Of the 285 significant early activation probe sets, 36 (represent- al consequences of Myc activation and subsequent deactivation in ing 34 unique genes) are designated ‘‘cell cycle genes’’ by gene the h cells of M+X + mice in vivo. cMycERTAM was activated L ontology classification of biological function (Fig. 2B; ref. 22). The in vivo by injection of 4-OHT and islets were laser captured from synchrony of cell cycle entry we observe upon MycERTAM activation pancreatic frozen sections. All samples were treated independently is reflected in the kinetics of expression of key cell cycle regulatory yielding 20 to 50 ng total islet RNA per sample. Two rounds of linear amplification generated 20 to 50 Ag of high-quality, biotin- labeled 2a-cRNA/sample. The overall quality of microarray data obtained for 33 independently amplified and hybridized RNA samples was excellent, with an average ‘‘present’’ call of 46 F 4%, which is consistent with published results (20). Following normalization of the raw data, 9,813 probe sets were identified as being reproducibly detectable in pIns-MycERTAM–positive pancreatic islets.4 We then analyzed these probe sets for evidence of modulation by Myc. Global gene expression profiling in pancreatic islets following Myc activation in vivo reveals complex and dynamic changes. To define immediate early transcriptional responses to Myc, we first identified those genes modulated within 24 hours of acute Myc activation. Statistical analysis of microarray software (17) defined 293 probe sets as significantly altered in this early activation cohort. Expression of eight probe sets was also altered following 4-OHT treatment of control MycERTAM transgene– À negative M XL+ mice, indicating that they represent genes that can be directly modulated by 4-OHT. These eight probe sets were excluded from further analysis, leaving 285 probe sets whose expression in pancreatic h cells in vivo was significantly altered within 24 hours of acute Myc activation (Supplementary Table S1). Supervised hierarchical clustering of the 285 significantly regulated probe sets shows the dynamic nature of Myc-mediated gene regulation, even over such a short time period (Fig. 1A). To define Myc-dependent effects in established h cell tumors, we expanded our data analysis to include samples obtained from mice exposed to tamoxifen for 21 days. M+XL+ transgenic islets exposed to sustained Myc activity exhibit all the phenotypic characteristics of malignant h-cell tumors (10). Consistent with our published results, histologic sections confirmed that the islets isolated from these mice were massive, proliferative, and angiogenic (data not Figure 1. Myc activation in pancreatic h cells in vivo rapidly and dynamically shown). Kinetic expression profiling of this sustained activation alters the islet transcriptome. Global genomic profiling of transgenic islets following acute activation of MycERTAM in h cells identifies 285 probe sets as cohort, revealed that 2,532 probe sets were significantly modulated being significantly regulated by Myc in the first 24hours and 2,532 probes sets as by Myc over the course of 21 days (Supplementary Table S2). being modulated following a 21-day period of sustained Myc activity. Hierarchical gene clustering of these early (A) and sustained (B) activation cohorts shows the rapid kinetics of gene induction and repression in vivo, as well as the dynamic nature of the gene expression levels over time. Significance was defined as a 4 The data discussed in this article have been deposited in NCBIs Gene Expression false detection rate of V5%. For gene clustering, expression levels were Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO calculated relative to the average signal intensity of the probe set in the three Series accession number GSE4356. untreated samples. www.aacrjournals.org 4593 Cancer Res 2006; 66: (9). May 1, 2006

Figure 2. Myc activation in h cells in vivo induces rapid and synchronous cell cycle entry and sustained proliferation. A, activation of Myc in double-transgenic (M+XL +) pancreatic h cells by systemic administration of 4-OHT induced synchronous S-phase entry in h cells in all islets of the pancreas by f16 to 24hours, as evidenced by BrdUrd-positive staining (green immunofluorescence). Analogous 4-OHT TAM treatment of MycER -negative, MÀXL + mice induced no S-phase transition. B, hierarchical gene cluster of 41 probe sets (38 genes) with gene ontology functions of cell cycle or proliferation, which were regulated in the first 24hours of MycERTAM activation. C, qRT-PCR analysis of cyclin D1 (Ccnd1) and cyclin B2 (Ccnb2) expression in laser-captured islets confirmed the sequential modulation observed in the microarray data. Columns, fold change; bars, SD.

genes. Cyclin D1 gene (Ccnd1), whose product is pivotal for G1 The transcriptional profile of murine pancreas during develop- phase progression, was induced within 2 hours of Myc activation, ment has been characterized, providing a suite of genetic whereas expression of the S-phase cyclin A (Ccna2) and mitotic fingerprints that define the developing organ at various stages of cyclin B1 (Ccnb1) and cyclin B2 (Ccnb2) genes peaked subsequently differentiation (25). Adult islets can be distinguished from less (Fig. 2B). Induction of genes encoding the G2-M transition differentiated pancreas by the differential expression of 217 mature regulatory phosphatase Cdc25c and the mitotic regulator kinase islet-specific genes that are, in the main, markers of fully Cdk1 (Cdc2) paralleled induction of the mitotic cyclins. This differentiated h cells (25). One hundred eighty-nine of 217 mature sequential pattern of cyclin induction was confirmed by qRT-PCR islet genes are represented in our M+XL+ islet data set by 218 probe (Fig. 2C). sets and expression of 89 (47%) of these was altered as a Next, we used an established database (21) to determine which consequence of Myc activation (Supplementary Table S4). The of our significant in vivo targets had been previously labeled as Myc vast majority (73 of 89) of these islet genes were down-regulated by targets in vitro.5 Perhaps surprisingly, only 50 of 285 early activation Myc and, in the case of 25 genes, sustained Myc activity repressed target probe sets (equivalent to 47 unique genes) have been expression by z2-fold (Fig. 3A). Interestingly, several islet-specific previously identified as Myc targets (Supplementary Fig. S1). In the genes, including Gad1, Ptprn, Slc2a2, and the key transcriptional sustained activation cohort, we identified 303 designated Myc regulator of h-cell-specific gene expression Ipf1 (Pdx1), were target genes among our list of 2,532 regulated probe sets repressed within the first 2 to 4 hours of Myc activation, intimating (Supplementary Table S3). Thus, kinetic profiling of Myc target that they may be direct targets of Myc (Supplementary Table S4). genes in pancreatic h cells in vivo corroborates in vitro studies but qRT-PCR analysis of three well-characterized h cell genes, Th, also identifies multiple novel targets with diverse biological roles. Slc2a2, and Ins2, which encode tyrosine hydroxylase, facilitated Myc represses mature B-cell genes and results in global islet glucose-transporter 2 (glut2), and insulin, respectively, corroborat- dedifferentiation. Activation of Myc in many normal and ed the microarray data (Fig. 3B). To establish whether this gene neoplastic cell types is associated with an undifferentiated repression leads to loss of the differentiated h-cell phenotype, we phenotype. It remains unclear whether this inverse relationship assessed the influence of Myc activation on expression of the between proliferation and differentiation is merely correlative or mature h-cell proteins insulin, Glut2, and Isl-1. Expression of all represents some obligate exclusivity in the two biological three h-cell markers was suppressed upon Myc activation, whereas programs. In h cells, it has been noted that ectopic expression of none was affected by 4-OHT treatment of MycERTAM-negative Myc represses insulin expression (10, 23, 24). To assess whether MÀXL+ mice (Fig. 3C). Finally, to show that h-cell gene repression Myc activation more globally suppresses terminal h-cell differen- and dedifferentiation were a consequence of Myc activity, we tiation, we analyzed our microarray database to determine the assessed expression in established h-cell tumors 2, 4, and 6 days effect of Myc activation on the expression of mature h-cell-specific following acute deactivation of Myc. Consistent with our previously genes. published observations (10), acute deactivation of Myc in these h-cell tumors resulted in tumor regression and reappearance of mature h-cell markers insulin, Glut2, and Isl1 (data not shown). Genomic profiling of these regressing islet tumors showed that 5 http://www.myccancergene.org/site/mycTargetDB.asp. reappearance of the mature h-cell phenotype was heralded by the

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Kinetic Analysis of Myc Function In vivo reexpression of h-cell-specific genes (Fig. 3D). Thus, sustained Myc down-regulated upon Myc deactivation and 431 probe sets that activity in pancreatic h cells represses the expression of mature h- were induced (Supplementary Fig. S2). To identify which Myc cell genes, leading to loss of differentiation, whereas subsequent effectors may be involved in maintaining established h-cell tumors, deactivation of Myc rapidly restores h-cell gene expression. These we compared the 2,532 probe sets that were significantly regulated results indicate that the process of Myc-mediated tumorigenesis upon Myc activation with the 1,431 regression probe sets involves profound changes in differentiation status, many of which modulated by Myc deactivation. This comparison revealed 428 arise from modulation of tissue-specific genes. probe sets common to both cohorts, 257 of which were regulated in Comparison of genomic profiles during B-cell tumor opposing directions during tumor initiation (Myc on) and formation and regression identifies genes whose modulation regression (Myc off; Fig. 4; Table 1). Thus, regulation of these is exquisitely dependent on continual Myc activity. Myc- genes (either positive or negative) was, like maintenance of the induced h-cell tumorigenesis arises through induction of h-cell h-cell tumors, dependent on sustained Myc activity. Of the 257 proliferation, dedifferentiation, and angiogenesis. Subsequent Myc ‘‘candidate tumor maintenance’’ probe sets, 154 (equivalent to 132 deactivation induces proliferative arrest, redifferentiation, and genes) were both induced upon Myc activation and repressed upon collapse of vascular integrity, indicating that tumor regression is deactivation (type A genes), whereas 103 probe sets (representing essentially a reversal of the processes that drive tumor formation 101 genes) were repressed by Myc activation and then reexpressed (10). Comparison of the regression cohort expression profiles with when Myc was deactivated (type B genes; Table 1). The diverse those of stable tumors identified 1,431 probe sets that were biological functions of these candidate tumor maintenance genes significantly altered during the onset of tumor regression are apparent from their gene ontology designations (Table 1A). (Supplementary Table S5). Hierarchical clustering of these genes To identify which of the 257 probe sets are likely to be direct Myc identifies two distinct categories:1,000 probe sets that were rapidly targets, 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 discovery rate <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 of laser-captured islets confirms Myc-induced repression of three known genetic markers of h cell differentiation. Columns, fold change; bars, SD. C, down-regulation of 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 visible as a nuclear protein. All positive immunofluorescent staining is represented in green and, in the case of Isl-1, autofluorescent RBC appear white. The differentiation Â state of control MÀXL + 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 of 21-day Myc-on samples. Columns, fold change; bars, SD. Representative for three replicate mice at each time point. FC, fold change. www.aacrjournals.org 4595 Cancer Res 2006; 66: (9). May 1, 2006

Figure 4. Genomic profiling of islet tumors following deactivation of Myc identifies candidate Myc-regulated genes with a role in tumor maintenance. Supervised hierarchical gene clustering is shown of 257 probe sets that were oppositely influenced by Myc activation and deactivation. One hundred fifty-four probe sets (representing 132 genes) were induced when Myc was activated and then down-regulated when Myc was deactivated, whereas 103 probe sets (representing 101 genes) were repressed upon Myc activation and reexpressed upon subsequent deactivation. A close-up of three clusters is shown here and a complete listing of all represented genes is shown in Table 1A.

following MycERTAM activation. Thirty maintenance probe sets comprise both genes that are universally regulated and genes that were modulated within 24 hours of Myc activation (Fig. 5A) and 10 are specific to certain cell lineages and/or environmental circum- were altered >1.5-fold within 2 to 4 hours (Fig. 5B). Of these, six stances. were induced (BC037006, Eif4ebp1, Fzd9, Gmip, St6galnac4, and Zbtb16), whereas four were repressed (5730592L21, Dap, Derl2, and Sec23b;Fig.5B; Table 1B). Although only 3 of these 10 genes have Discussion been previously reported to be Myc targets (Gmip, St6galnac4, and Understanding how genes implicated in cancer conspire to Sec23b), by sequence analysis all six type A genes contain canonical disrupt normal cell and tissue biology is one of the most intractable Myc:Max binding sites in their promoters (data not shown), problems in contemporary biology. The Myc oncoprotein exem- supporting the idea that these are direct Myc targets. To further plifies this problem:Originally identified through its role in validate these targets, we selected three type A genes (Fzd9, promoting cell proliferation, its mechanism of action involves the Eif4ebp1, and Gmip) and one type B gene (Dap) for analysis by qRT- coordinated regulation of a multitude of biological properties PCR. These studies confirmed Myc-mediated regulation of all four mediated through a myriad of target genes (7–9, 21, 26–31). This genes in h cells in vivo (Fig. 5C). makes determining the extent to which these various genes Finally, to determine if Myc-dependent regulation of key contribute to Myc oncogenic potential virtually impossible to maintenance genes is dependent on cell type, we assessed the determine. It is also apparent that cell type, genetic background, effects of acute MycERTAM activation in primary MEFs in vitro. and experimental context can all influence the downstream Evaluation of the aforementioned three type A and one type B consequences of Myc activation, leading some to propose that genes revealed that Eif4ebp1 was not measurably expressed in Myc may act as a global transcriptional regulator (6). MEFs, Gmip was unaffected by Myc status, and Dap was repressed Notwithstanding such a ‘‘global’’ role in biology, the mechanisms to an insignificant degree (data not shown). However, Fzd9 was by which Myc drives and maintains tumorigenesis are likely to be significantly induced following Myc activation in MEFs and this more restricted. However, the problem remains that as Myc exerts occurred even in the presence of cycloheximide, suggesting that its inexorable tumorigenic effect, a legion of secondary genetic Fzd9 is likely to be a direct transcriptional target of Myc (Fig. 5D). programs evolve. Dissecting which of these is cause or conse- Such data are consistent with the emerging notion that Myc targets quence of Myc action has proven very difficult, as has identifying

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Table 1. Putative tumor maintenance genes

A. Maintenance genes sorted by gene ontology biological process

Type A genes induced when Type B genes repressed upon Myc activation and Myc is activated and repressed reexpressed when Myc is deactivated when Myc is deactivated

Angiogenesis Crhr2, Epas1 — Apoptosis Accn1, Casp2, Fadd, Foxo3 Dap, Nfkb1, Sgpl1, Siah2 Cell 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/ Epas1, Fzd9, Jun, Zbtb16 Arpc1a, Bmp1, Dcx, Nfkb1, Siah2, Th morphogenesis 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, Th mRNA processing — A1462438, Cpsf3 Phosphorylation/ Cdc25c, Dyrk1a, Ltk, Matk, Mvk, Pdk4, Ptk2bPpm1b,Pxk, Slk, Stk16 dephosphorylation Protein biosynthesis or 2410005K20Rik, Cubn, Eif4ebp1, Sars2 Eef2, Eif4b, Mrpl4, Mrps18a, Mrps18b, Tars, Wars translation Protein folding Ccs Cct3, Cct4, Dnajb1, Dnajb10, Dnajb11, Tra1 Proteolysis Adprtl2, Blmh, Casp2, Erf Bmp1, Xpnpep1 Signaling/signal transduction Crhr2, Ect2, Eif4ebp1, Epas1, Fadd, Fzd9, Arfrp1, Arl4, Crcp, Dcx, Nfkb1, Pdzk11, Pxk Gabrg3, Gcgr, Gmip, Ltk, Matk, Olfr16, Pde1b, Racgap1, Rasal1, Socs3, Stmn4 Transcription D1Ertd161e, Epas1, Erf, Fhl2, Jun, Mnt, Nfkb1, Rnf14, Xbp1 Myt1, Rpo1-2, Sox4, Tead4, Tieg1, Zfp28 Transport Accn1, Atp1b1, Ccs, Clcn1, Gabrg3, 1300013B2Rik, Aass, Abcc8, Arl4, Gsr, Kif20a, Slc6a8, Txndc9, Txn12 Atp2a2, Gdi1, Mcfd2, Sec22l1, Sec23b, Sytl4, Txndc7 Ubiquitin cycle 2700084L22Rik, Usp2 Pxmp3, Rnf14, Siah2 Other 2010016F14Rik, C1qtnf1, Dbn1, Mpz, Anxa5, Cmas, Gch1, H2-Q7, H2-D1, Serpina1a, Serpina1d, Txnip LOC280487, Nme2 Not specified 1500010J02Rik, 1500034J01Rik, 1810030O07Rik, 0610010K14Rik, 1110008P14Rik, 1110014C03Rik, 1810044D09Rik, 2410018L13Rik, 2900054D09Rik, 1700023O11Rik, 1810010N17Rik, 1810046J19Rik, 6230416J20Rik, AI464131, AI591476, BC037006, 2310004K06Rik, 2410022L05Rik, 2610208E05Rik, Bub1, C330027C09Rik, C79407, C80638, Cck, 2610507B11Rik, 2900010J23Rik, 833439L19Rik, Ceacam11, Cep2, Col5a1, Crlf1, Csnk, D10627, 5730592L21Rik, 6330442E10Rik, Armet, D430019H16Rik, D4Ertd196e, D5Ertd363e, B230114J08Rik, C130052I12Rik, Cdk5rap3, D930048N14Rik, Dlk1, Dnajc11, Egln2, Eif4ebp2, Cenpc1, Commd7, D030028O16Rik, D19Ertd144e, Fez1, Gtse1, H1fx, Hrc, Ide, Iqcf4, Itgb1bp1, Derl2, Dnajc3, Fut8, Gas5, Gng12, Ik, Jarid1b, Lpin1, Lpp, Midn, Pcdha10, Phlda3, Mapbpip, Mrpl54, Ndg2, Pik3c3, Pprc1, Pspc1, Riok3, Sct, Sez6, Slc35b1, Slc35e4, Ssbp4, Pscd3, Rad17, Sec23ip, Tbrg1, Tgtp, Vasp Surf2, Tmem5, Wiz, Zdhhc9, Zfp259, Zfp277

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which, if any, of its direct gene targets act as linchpins of Myc of Myc target genes that are candidates for mediating tumor oncogenic action. In this article, we have addressed the problem of maintenance function of Myc in vivo. objectively defining what attributes of Myc are required for the Not surprisingly, cell cycle genes figure prominently among our maintenance of tumors by combining a switchable in vivo genetic Myc targets. Ccnd1, the gene encoding cyclin D1, has been model with a global genomic analysis of Myc-dependent transcrip- previously identified as a Myc target in vitro and our in vivo data tion. In this way, we have, for the first time, correlated Myc support the induction of Ccnd1 by Myc. Because cell growth must oncogenic action with the underlying transcriptional changes that accompany cell cycle progression, it is also noteworthy that genes accompany tumorigenesis. In particular, the reversibility of our encoding several members of the AKT-mTOR nutrient use and switchable model has enabled us to define a highly restricted cadre response pathway are also modulated by Myc. These include www.aacrjournals.org 4597 Cancer Res 2006; 66: (9). May 1, 2006

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

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

Probeset ID Gene name Gene Ref Seq EBOX* Present (a) GO biological process c symbol in database (b) Published role in cancer

96518_at cDNA sequence BC037006 BC037006 XM_109956 Yes No (a)— (b)No 100636_at Eukaryotic translation Eif4ebp1 NM_007918 Yes No (a) Insulin receptor signaling pathway, initiation factor 4E negative regulation of protein binding protein 1 biosynthesis, negative regulation of translational initiation, regulation of protein biosynthesis, regulation of translation, and regulation of translational initiation (b) Yes (45) 99844_at Frizzled homologue 9 Fzd9 XM_284144 Yes No (a) G-protein coupled receptor protein (Drosophila) signaling pathway, Wnt receptor signaling pathway, cell surface receptor linked signal transduction, development, and frizzled signaling 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, St6galnac4 NM_011373 Yes Yes (a) Protein amino acid glycosylation 3-b-galactosyl-1,3)- N-acetylgalactosaminide a-2,6-sialyltransferase 4 (b) Yes (44) 92202_g_at Zinc finger and BTB domain Zbtb16 XM_134826 Yes No (a) Skeletal development, negative containing 16 regulation of cell proliferation, embryonic pattern specification, anterior, posterior pattern formation, embryonic limb morphogenesis, forelimb morphogenesis, positive regulation of apoptosis, regulation of transcription, negative regulation of transcription, and DNA-dependent, male germ-line stem cell division (b) Yes (43) 160119_at RIKEN cDNA 5730592L21Rik NM_029720 Yes No (a)— 5730592L21 gene (b)No 93842_at Death-associated protein Dap NM_146057 No No (a) Apoptosis and induction of apoptosis by extracellular signal (b)No 160769_at Der1-like domain family, Derl2 NM_033562 No No (a)— member 2 (b) Yes (48) 98944_at SEC23B Sec23b NM_019787 No Yes (a) Endoplasmic reticulum to Golgi (Saccharomyces cerevisiae) 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.

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Figure 5. Tumor maintenance genes include a subset of novel, putative direct targets of Myc. A, hierarchical gene cluster of 30 maintenance gene probe sets that were significantly modulated within the first 24hours of acute Myc activation in vivo and oppositely regulated following Myc deactivation. B, 10 of the 30 maintenance gene probe sets were induced or repressed within 4hours of acute Myc activation, suggesting that they may be direct transcriptional targets of Myc. All six induced genes have a putative Myc:Max binding site in their promoter region and have known functions relevant to cancer (see Table 1B). C, Taqman analysis of explanted whole islets isolated from M+XL + mice 4hours after 4-OHT treatment confirmed Myc modulation of four putative target maintenance genes. Columns, fold change relative to the average of untreated mice for replicate mice at each time point; bars, SD. D, acute activation of MycERTAM in primary MEFs caused a 2-fold induction of Fzd9 relative to ethanol control–treated cells 2 hours after 4-OHT treatment. Pretreatment of cells with cycloheximide (CHX) did not abrogate the Fzd9 response, confirming that it is a direct transcriptional target of Myc. NS, not statistically significant.

Eif4ebp1, which encodes the eukaryotic translation initiation factor cell cycle arrest and apoptosis (35). Consistent with this, we find 4 binding protein 1, and is one of only 10 early Myc targets that we that p19ARF is induced as expected following in vivo activation of TAM 6 identify as candidate mediators of tumor maintenance. In addition, MycER in h cells lacking overexpression of Bcl-xL. Thus, in the inhibitor of mTOR, tuberin (Tsc2), is rapidly and continuously addition to suppressing apoptosis, Bcl-xL may cooperate oncogeni- down-regulated following MycERTAM activation whereas expression cally with Myc by abrogating Myc-induced activation of the ARF- of the S6 kinase (Stk6) gene is induced 5-fold at 24 hours p53 pathway. Ongoing work in our laboratory is directed toward (Supplementary Table S1). Our data lend support to recent in vitro elucidating this issue. studies implicating Myc in the regulation of the AKT-mTor pathway Myc status has a profound influence on embryonic development (32) and confirm that the effects of Myc on cell cycle and (36, 37), in part through its ability to disrupt differentiation proliferation are intimately linked to cell growth and survival. programs. In this study, acute activation of Myc triggered a Despite substantial evidence for direct Myc-mediated induction widespread change in h-cell phenotype, suppressing expression of of the ARF-p53 pathway both in vitro and in vivo (33, 34), we found many genes that are signatures of terminally differentiated h cells. TAM no evidence for this relationship in M+XL+ islets in vivo. Neither The observed association between Myc-ER activation and loss Cdkn2a, which encodes both p19ARF and p16Ink4a, nor key p53 of h-cell differentiation was substantiated by the rapid reappear- target genes Cdkn1a (p21) or Mdm2 were induced during the first ance of such markers upon deactivation of Myc. Whether these 24 hours of Myc activation. We suspect that this lack of p53 activation may be due to the influence of Bcl-xL overexpression in our model. It has been proposed that overexpression of Bcl-xL may ARF limit Myc induction of p19 , thereby suppressing p53-mediated 6 Unpublished data. www.aacrjournals.org 4599 Cancer Res 2006; 66: (9). May 1, 2006

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research differentiation genes are direct targets of the Myc:Max complex will regulatory Myc recognition elements in their promoters and have need to be determined by further studies. These findings parallel already been implicated elsewhere in various aspects of cancer recent observations of Myc-induced hepatic tumors that undergo (42–48). Of note, the Wnt receptor–encoding gene, Fzd9, was also differentiation upon oncogene deactivation (38). Aberrant or found to be regulated by Myc in cultured MEFs. Wnt signaling is deregulated expression of intrinsic developmental pathways may, critical to normal development and is frequently deregulated in therefore, be an integral mechanistic component of Myc-induced human cancer, largely through constitutive activation of h-catenin tumorigenesis. It is noteworthy that several of the genes rapidly (49). Fzd9 knockout mice show significant abnormalities in repressed by Myc in our study are tissue-specific and cannot, hematologic development, principally secondary to a failure of therefore, be universal Myc targets. However, other genes not B-cell self-renewal (50). Definitive proof that Fzd9 is a direct restricted in their expression to pancreas (e.g., Eif4ebp1, Dap, and transcriptional target of Myc and the extent to which it mediates Gmip) were nonetheless regulated by Myc in h cells in vivo yet not essential aspects of Myc function will need to be resolved by direct in fibroblasts in vitro. Defining the precise role of Myc targets, be experimentation. To that end, we have begun studies to determine they direct or indirect, in tumorigenesis will thus require careful the effect of acute MycERTAM activation on the islets of Fzd9 study in biologically relevant cell types and environments in vivo. knockout mice. Demonstration of impaired tumor formation in By directly comparing the transcriptional fingerprints of islets these mice will confirm the critical role of this Wnt receptor in during tumor formation and regression, we were able to identify Myc-induced transformation of pancreatic h cells in vivo. 257 probe sets (233 genes) whose expression was inversely In summary, we have, for the first time, defined, in real time, the regulated during Myc activation and subsequent deactivation. Myc-induced transcriptional changes that occur during tumor Because Myc is required to maintain the tumors it induces, this initiation and regression in an orthotopic tissue in vivo. We are cadre of genes must include those whose functions are required for currently extending this approach to other inducible tumor maintenance of h-cell tumors. Functional classification of genes model systems to elucidate which of the identified Myc targets within this ‘‘tumor maintenance’’ cohort identifies many targets of are h cell-specific and which are more universal. By unraveling the potential interest. Among the type A genes, whose sustained complexities of the tumorigenic process in vivo, we hope to identify expression requires sustained Myc activity, are the cytokinesis genes and pathways that will be strategic targets for the focused regulator Ect2 and the activator protein transcription factor development of novel, targeted anticancer strategies. member c-Jun, both of which have been implicated in tumorigenesis (39, 40). Another type A gene is Epas1, implicated in hypoxia- Acknowledgments induced angiogenesis (41) and potentially involved in maintaining h Received 10/24/2005; revised 2/24/2006; accepted 3/2/2006. -cell tumor vasculature. The designated biological functions of Grant support: Juvenile Diabetes Research Foundation grants 4-2004-372 and 4- these type A genes are extremely diverse and underscore the idea 1999-841, National Cancer Institute grant R01 CA98018 (G.I. Evan), and Canadian that tumor maintenance involves multiple processes that evolve Institutes of Health Research Senior Research Fellowship (E.R. Lawlor). The costs of publication of this article were defrayed in part by the payment of page over time. For example, whereas cell cycle and proliferation genes charges. This article must therefore be hereby marked advertisement in accordance make up over 30% of early activation gene targets, they represent with 18 U.S.C. Section 1734 solely to indicate this fact. only 10% of the identified maintenance gene cohort. We thank The J. David Gladstone Genomics Core Laboratory at San Francisco General Hospital (where part of this work was carried out) and the University of The kinetics of regulation of the 233 candidate maintenance California San Francisco Genome Core, Mt. Zion Cancer Center (where qRT-PCR was genes indicates that the great majority are indirect consequences of done), for invaluable assistance; their respective directors, Dr. Chris Barker and Myc action rather than direct target genes. By confining our search Dr. David Ginzinger, and personnel; Dr. Chris Haqq and his laboratory at the University of California San Francisco Cancer Center for their instruction and to those genes that were rapidly modulated by Myc, we identified a guidance in the techniques of laser capture microdissection and RNA amplification; subset of 10 genes that share the correct dynamics to be putative Dr. Tobias Dansen for help with fluorescence microscopy; Mike Hagen for technical support with islet preparations; and Dr. Matthias Hebrok (Diabetes Center, direct transcriptional targets. Most of these genes (i.e., Dap, Derl2, University of California San Francisco, San Francisco, CA) for anti-Glut2 and anti- Eif4ebp1, Fzd9, St6galnac4, and Zbtb16) possess both requisite Isl-1 antibodies.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Reversible Kinetic Analysis of Myc Targets In vivo Provides Novel Insights into Myc-Mediated Tumorigenesis

Elizabeth R. Lawlor, Laura Soucek, Lamorna Brown-Swigart, et al.

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