identification of actively translated mrna transcripts in a rat model of early-stage colon...

Identification of Actively Translated mRNA Transcripts in a RatModel of Early Stage Colon Carcinogenesis

Laurie A. Davidson1,2, Naisyin Wang3, Ivan Ivanov4, Jennifer Goldsby2, Joanne R.Lupton1,2, and Robert S. Chapkin1,21Program in Integrative Nutrition & Complex Diseases, Texas A&M University, College Station, TX778432Center for Environmental and Rural Health, Texas A&M University, College Station, TX 778433Department of Statistics, Texas A&M University, College Station, TX 778434Department of Veterinary Physiology & Pharmacology, Texas A&M University, College Station, TX77843

AbstractWith respect to functional mapping of gene expression signatures, the steady-state mRNA expressionlevel does not always accurately reflect the status of critical signaling proteins. In these cases, controlis exerted at the epigenetic level of recruitment of mRNAs to polysomes, the factories of ribosomesthat mediate efficient translation of many cellular messages. However, to date, a genome-wideperspective of the effect of carcinogen and chemoprotective bioactive diets on actively translated(polysomal) mRNA populations has not been performed. Therefore, we utilized an established coloncancer model, i.e., the azoxymethane (AOM)-treated rat, in combination with a chemoprotective dietextensively studied in our laboratory, i.e., n-3 polyunsaturated fatty acids (PUFA), to characterizethe molecular processes underlying the transformation of normal colonic epithelium. The number ofgenes affected by AOM treatment 10 wk after carcinogen injection was significantly greater in thepolysome RNA fraction compared with the total RNA fraction as determined using a high-densitymicroarray platform. In particular, polysomal loading patterns of mRNAs associated with the Wnt-beta catenin, phospholipase A2-eicosanoid and the MAP kinase signaling axes were significantly up-regulated at a very early period of tumor development in the colon. These data indicate thattranslational alterations are far more extensive relative to transcriptional alterations in mediatingmalignant transformation. In contrast, transcriptional alterations were found to be more extensiverelative to translational alterations in mediating the effects of diet. Therefore, during early stagecolonic neoplasia, diet and carcinogen appear to predominantly regulate gene expression at multiplelevels via unique mechanisms.

KeywordsPolysomes; Translation alterations; Gene expression profiling; Azoxymethane

Address Correspondence to: Dr. Robert S. Chapkin, Center for Environmental and Rural Health, Kleberg Biotechnology Center, MS2253, Texas A&M University, College Station, TX 77843-2253, USA. Phone: 979-845-0419, Fax: 979-862-2378, [email protected] disclosures: Laurie A. Davidson, Naisyin Wang, Ivan Ivanov, Jennifer Goldsby, Joanne R. Lupton and Robert S. Chapkin, noconflict of interest.

NIH Public AccessAuthor ManuscriptCancer Prev Res (Phila). Author manuscript; available in PMC 2010 November 1.

Published in final edited form as:Cancer Prev Res (Phila). 2009 November ; 2(11): 984–994. doi:10.1158/1940-6207.CAPR-09-0144.

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IntroductionTotal steady-state messenger RNA (mRNA) levels have utility in the prediction of theexpression levels of an array of proteins. However, in many cases the steady-state mRNAexpression level does not accurately reflect the expression rate of proteins. This is especiallyrelevant in the case of Ras transformed cells (1-4). Because prolonged activation of p21 Rasdrives colonic tumor development (5), it is likely that translational regulation plays animportant role in the control of colonic gene expression during malignant transformation.

It has been recently reported that several tumor suppressors and proto-oncogenes can influencethe formation of the mature ribosome or regulate the activity of translation factors (6,7).Traditionally, the differential mobilization of mRNAs onto polyribosomes has been utilizedto identify genes whose transcripts are translationally controlled (8). Translational control hasbeen verified by demonstrating the redistribution of mRNAs on polysome gradients at variousstages of colon tumor cell line transformation (3). Recently, Provenzani and colleagues (9),using SW480 colon carcinoma cells and their relative, lymph node mestastasis SW620 cells,demonstrated that the colorectal cancer genome acquires changes impacting by far thetranslational more than the transcriptional control of gene expression. These changes wouldhave been completely missed in a conventional transcriptosome analysis detecting cellularmRNAs irrespective of their degree of polysomal loading.

Although the analysis of cancer cells in tissue culture is informative, it is possible that thismodel system may not accurately represent the state of cancer cells in vivo. Therefore, we haveapplied a systems biology approach to the characterization of gene expression changesoccurring during carcinogen-induced colon tumorigenesis. Specifically, to take full advantageof the polysomal isolation method, we have combined ribosome-free and polysome-boundmRNAs with an open, high throughput quantitative mRNA analysis detection platform thatsimultaneously measures and identifies existing mRNA species in order to provide a panoramicoverview of gene expression at both the total transcript and post-transcriptional levels. To ourknowledge, the effect of a colon-specific carcinogen on the pattern of existing mRNAs thatare recruited to polysomes has never been attempted in vivo.

With respect to environmental risk factors, there is growing evidence that long-chain n-3PUFA, e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), suppress coloncancer risk in humans (10-11). Recently, we have demonstrated that chemoprotective n-3PUFA reprogram genetic signatures during colon cancer initiation and progression and reducecolon tumor formation (12). Therefore, we further investigated whether n-3 PUFA modulatethe levels of actively translated mRNAs in colonic mucosa following carcinogen exposure.

Material and MethodsAnimals

Sixty-four 8 wk old male Sprague Dawley rats (Harlan, Houston, TX) were acclimated for twoweeks in a temperature and humidity controlled facility on a 12-hour light/dark cycle. Theanimal use protocol was approved by the University Animal Care Committee of Texas A&MUniversity. The study was a 2 × 2 × 2 factorial design with two types of dietary fat (n-6 PUFAor n-3 PUFA), two types of dietary fiber (cellulose or pectin) and two treatments (injectionwith the colon carcinogen, AOM, or with saline). Animals (n=6-9 per group) were stratifiedby body weight after the acclimation period so that mean initial body weights were not differentbetween groups. Body weight was monitored throughout the study.

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DietsAfter a 1-wk acclimation on standard pelleted diet, rats were assigned to one of four diet groupswhich differed in the type of fat and fiber as previously described (13). Diets contained (g/100g diet): dextrose, 51.00; casein, 22.40; D,L-methionine, 0.34; AIN-76 salt mix, 3.91; AIN-76vitamin mix, 1.12; choline chloride, 0.13; pectin or cellulose, 6.00. The total fat content of eachdiet was 15% by weight with the n-6 PUFA diet containing 15.0 g corn oil/100 g diet and then-3 PUFA diet containing 11.5 g fish oil/100g diet plus 3.5 g corn oil/100 g diet as previouslydescribed (12).

Carcinogen treatmentAfter 2 wk on the experimental diets, 9 rats per diet group received injections of saline (control)and 9 rats per diet group received injections of AOM (Sigma, St. Louis, MO) s.c. at 15 mg/kgbody weight. Rats received a second AOM or saline injection one week later and wereterminated 10 wk after the first injection.

Polysome and total RNA isolationUpon termination, each colon was cut open longitudinally, flushed clean with PBS, 1 cm fromthe distal colon was collected for fixation and embedding for immunohistochemical assays, anadjacent cm was taken for total RNA isolation and the remainder of the colon was used forpolysome RNA isolation. For total RNA isolation, epithelial cells were scraped from theunderlying muscle layer with a glass microscope slide, homogenized on ice in lysis buffer(mirVana miRNA Isolation Kit, Ambion, Austin, TX), and frozen at -80°C until RNA wasisolated. Using the mirVana kit, total RNA enriched with microRNA was isolated followed byDNase treatment. For polysome RNA isolation, colons were incubated for 5 min at roomtemperature in PBS containing 100 μg/ml cycloheximide, an inhibitor of translation whichlocks the mRNA/ribosome complex, facilitating its isolation and preventing RNA degradation.Following incubation, epithelial cells were scraped with a microscope slide on a chilled glassplate and immediately processed according to the procedure of Ju et al. (14). Briefly, epithelialcells were allowed to swell in LSB (20 mM Tris pH 7.5, 10 mM NaCl and 3 mM MgCl2)containing 1 mM dithiothreitol and 50 U RNase inhibitor for 2 min followed by lysis in LSBcontaining 0.2 M sucrose and 1.2% Triton X-100. After removal of nuclei by centrifugation,the supernatant was layered over a 15-50% linear sucrose gradient (in LSB) and centrifugedat 247,000 × g for 2 h at 4°C in a swinging bucket rotor. Gradients were fractionated using apipette, an aliquot was taken for absorbance at 254 nm and 3 vol denaturation solution (AmbionTotally RNA kit) was immediately added to the remainder of each fraction. Samples werefrozen at -80°C until RNA was isolated using the Totally RNA kit as per manufacturer'sinstructions followed by DNase treatment. Both total RNA and polysome RNA were analyzedon an Agilent Bioanalyzer to assess RNA integrity. In select experiments, 10 mM EDTA wasadded to the post nuclear supernatant in order to examine the shift in elution due to disruptionof polyribosome formation by EDTA.

MicroarrayCodeLink™ rat whole genome bioarrays (Applied Microarray, Tempe, AZ) were used to assessgene expressionp. For total RNA arrays, the Ambion MessageAmp II-Biotin Enhanced aRNAamplification kit was used to prepare labeled cRNA. For polysome arrays, the AmbionMessageAmp II aRNA amplification kit followed by the MessageAmp II-Biotin EnhancedaRNA amplification kit were used with two rounds of in vitro transcription (IVT) to generatelabeled cRNA. For both total RNA and polysome RNA, 10 μg of biotin-labeled cRNA washybridized to the CodeLink genome array following manufacturer's instructions (12). Slideswere scanned with an Axon GenePix 4000B scanner.

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ImmunohistochemistryAt necropsy, a one cm section of distal colon was removed, fixed in 4% paraformaldehyde for4 h, and paraffin embedded. Following antigen retrieval in 1X TE containing 0.05% Tween-20brought to a boil and maintained at sub-boiling temperature for 10 min in a microwave,followed by cooling on the bench for 30 min, sections were treated with 3% hydrogen peroxidein methanol for 15 min to quench endogenous peroxidase activity. Sections were then processedusing rabbit eIF4E (Cell Signaling Technology, Danvers, MA) at 1:100 or rabbit phopho-eIF4E(Cell Signaling) at 1:7 to quantify in situ eIF4E and phosphorylated eIF4E staining aspreviously described (13). Images were quantified using NIS Elements software (Nikon) bycollecting staining intensity at the base, middle and top of at least 20 crypts per animal, 8animals per treatment group (AOM or saline).

Statistics and pathway analysisPolysome and total RNA expression data were analyzed using GenMapp(www.genmapp.org) and GO (www.geneontology.org) and the approaches described belowusing the R computation platform (http://www.R-project.org). Raw data were first normalizedthrough a robust procedure that eliminates the intensity-based chip-by-chip bias and reducesthe between-chip variation and the potential outlier-effects. Robust local median estimationwas applied to all observations from chips of the same treatment and potential outlyingobservations identified (15). A within-treatment quantile-normalization was then applied to allnon-outlying observations (16). An alternative of replacing the within-treatment targetdistribution in quantile-normalization by the cross-all-chip method was also investigated;testing outcomes remained unchanged between the two alternatives. Mixed-effect ANOVAwas applied to normalized logarithm based 2 transformed data to obtain p-values, q-values,and positive false discovery rate (17). Significant genes for the AOM vs saline as well as dietcomparisons were identified. Genes that met the criteria of q-value <0.05 were submitted toGenMapp and GO for pathway profiling.

ResultsWhole genome polysomal profiles

Colonic mucosa polysome isolation was monitored by examining sucrose gradient fractionson an Agilent Bioanalyzer. As shown in Figure 1, the top of the gradient contained smallribonuclear proteins (region A). Through the remainder of the gradient, the progression toheavier particles was seen first with the elution of the 40S (B) then 60S (C) ribosomal subunits,followed by 80S ribosomes (D) and finally polysomes (E), which eluted as a broad area dueto mRNA transcripts loaded with varying numbers of ribosomes. The entire broad region inthe second half of the gradient, excluding the final fraction containing RNA granules (18), wascollected as the polysome fraction. Addition of EDTA to the sample prior to gradientfractionation resulted in the expected shift away from the bottom of the gradient due to thedisruption of polysome and 80S ribosome formation (Supplemental Figure 1).

Carcinogen-induced changes in gene expression differentially affect recruitment of mRNAsto polysomes

Polysome analysis and microarray technology was utilized to assess the impact of a colon-specific carcinogen (azoxymethane) on global mRNA polysome recruitment. Total andpolysomal mRNA from scraped colonic mucosa were isolated, processed and hybridized usingthe Codelink™ (Applied Microarray) microarray platform. The number of genes affected byAOM treatment 10 wk after carcinogen treatment was significantly (p<0.05) greater in thepolysome RNA fraction compared with the total RNA fraction. As shown in Figure 2A, 3,239genes were significantly altered (p<0.05) by AOM vs saline treatment in the polysome RNA

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http://www.genmapp.org

http://www.geneontology.org

http://www.R-project.org

fraction. In contrast, the total RNA fraction resulted in only 69 unique genes affected by theAOM treatment. There were 233 genes significantly (p<0.05) affected by the carcinogentreatment identified in both the polysome and total RNA fractions. These data indicate thattranslational alterations were more extensive (11.5-fold higher) relative to transcriptionalalterations in mediating malignant transformation.

Analysis of translationally activated genes in colonic mucosaSince an increase in polysomal RNA can reflect changes in mRNA abundance and/ortranslatability, the relative translatability of each differentially (AOM vs saline) expressedmRNA was calculated. Specifically, data were normalized to determine the change inabundance in polysomal RNA relative to the change in abundance in total RNA for each mRNA(polysome/total) (19). Table 1A shows that 35 genes were translationally enhanced followingcarcinogen exposure, i.e., polysome-associated mRNAs were enriched in AOM vs salinetreated animals. We have defined translationally enhanced mRNAs as those that aresignificantly increased in AOM polysomes by 30% or greater (poly/total ratio >1.3, p<0.05).These mRNAs are present on polysomes to a greater extent than can be explained by increasesin total mRNA abundance alone. Notable genes maintained on polysomes included CD47,involved in regulating neutrophil transepithelial migration (20); phosphatidylinositol-glycanbiosynthesis class S protein (PIGS), essential for the transfer of GPI to proteins (21); cyclinG1, associated with G2/M phase arrest in response to DNA damage (22); and SMAD4, a tumorsuppressor gene implicated in intestinal cell cycle regulation (23). Our analysis also identified8 genes (Table 1B) that were reduced by 30% or greater (poly/total ratio <0.7, p<0.05) in AOMvs saline-injected rats. Of interest is the observation that several of these genes are associatedwith immune response. Among these were C-C motif chemokine 17 (CCL17) (24), Best5(25), and CXC chemokine ligand (CXCL9) (26).

Microarray chips from a total of 6 rats per treatment were processed and data mining wasperformed as described in the Methods. Functional analysis of differentially expressedpolysome fraction genes was performed by differential pathway analysis (GSEA) (27). Thesystematic profiling approach shown in Figure 2B, revealed that carcinogen affected gene setsclassified into 26 well-defined biological process categories. Interestingly, the top biologicalprocesses affected by AOM exposure were related to electron transport, coenzyme andprosthetic group metabolism.

The canonical Wnt cascade has emerged as a critical regulator of stem cells (28). There is alsocompelling evidence that oncogenic K-ras stimulates Wnt signaling in colon cancer and thatMyc is required for the majority of Wnt target gene activation (29,30). Therefore, we examinedthe effect of carcinogen (AOM) on gene activation of the Wnt pathway. Colonic mucosalpolysome and total RNA were isolated from rats terminated at 10 wk post-injection (saline orAOM). Only significantly (p<0.05) altered genes are shown, n=6-9 rats per group (Figure 3).The data clearly demonstrate the impact of carcinogen on the mobilization of mRNAs ontopolyribosomes for genes associated with the Wnt pathway (Figure 3A). Interestingly, at this“early” promotional stage (tumors typically do not appear until 30 wk after carcinogeninjection), almost no changes were observed in the total mRNA transcriptosome (Figure 3B),indicating minimal perturbation at the transcriptional level. Furthermore, becausecyclooxygenase-2 (COX-2)-derived PGE2 can promote colonic tumor initiation andprogression by enhancing cell proliferation, angiogenesis, cell migration and invasion, whileinhibiting apoptosis (31), we also investigated the impact of carcinogen exposure on the patternof eicosanoid-related mRNAs that are recruited to polysomes. Overall, 8 enzymes weresignificantly (p<0.05) up-regulated at the polysome mRNA level as opposed to only 2 in thetotal RNA fraction (Figure 4).

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Microarray analysis also revealed that a number of genes associated with Ras and Mitogen-activated protein kinase (MAPK) cascades were significantly (p<0.05) up-regulated (expressedas fold increase - AOM vs saline; e.g., Map2k1/MEK1 − 2.0) predominantly in polysomes(Supplemental Figures 2 & 3). This is noteworthy, because blockade of the MAPK pathwaysuppresses growth of tumors in vivo (5, 32).

Diet preferentially influences global transcriptional profilesThe number of genes affected by diet (n-3 vs n-6 PUFA) 10 wk following placebo (saline)treatment was much greater in the total RNA fraction compared with the polysome RNAfraction. As shown in Supplemental Figure 4, only 63 genes were significantly altered (p<0.05)by PUFA treatment in the polysome RNA fraction. In contrast, the total RNA fraction contained328 unique genes affected by diet. In addition, there were 2 genes significantly (p<0.05)affected by diet treatment identified in both the polysome and total RNA fractions.Interestingly, a similar trend was observed in n-3 vs n-6 PUFA fed animals injected withcarcinogen (Supplemental Figure 5). These data indicate that transcriptional alterations weremore extensive relative to translational alterations in mediating the effects of diet.

In previous experiments, we have shown that diets containing both a fermentable fiber source,e.g., pectin, and n-3 PUFA as the lipid component, e.g., fish oil, are maximally protectiveagainst experimentally-induced colon cancer compared to cellulose and corn oil (33,34).Therefore, we examined the effect of dietary combination chemotherapy on the mucosaltranslatability index. As shown in Supplemental Table 1, 13 genes were modulatedtranslationally following carcinogen exposure and corn oil + cellulose (CC) relative to fish oil+ pectin (FP) feeding. Notably, the tumor suppressor retinoblastoma-associated protein (RB1)gene was translationally suppressed in CC relative to FP-fed animals (translatability index =0.57). A similar trend was also noted with respect to epoxide hydrolase 2 (Ephx2), a xenobioticmetabolizing phase I enzyme (translatability index = 0.15).

Effect of carcinogen on expression levels of colonocyte eIF4E and phospho-eIF4ESince elevated eukaryotic translation initiation factor 4E (eIF4E) function can inducemalignancy by selectively enhancing translation of key malignancy-related transcripts (35),eIF4E expression in tissue sections was quantified. Expression of eIF4E was higher (p<0.01)in the carcinogen (AOM)-treated compared to saline-control animals (Figure 5). Tissuepolysomal mRNA and protein eIF4E levels (AOM/saline ratio) were similar, 1.22 and 1.19,respectively. Interestingly, eIF4E was higher (p<0.01) at the base of the crypt (proliferativezone) and progressively decreased toward the luminal surface (Supplemental Figure 6). Wealso evaluated whether the phosphorylation status of eIF4E, which typically correlates withthe translation rate and growth status of the cell, was affected by carcinogen exposure. Similarto eIF4E, phospho-eIF4E levels were significantly (p<0.01) elevated in carcinogen treatedanimals (Figure 5).

DiscussionIn this study we examined ribosome-free and polysome-bound mRNAs using a high throughputmicroarray platform in order to provide a panoramic overview of gene expression at both thetotal transcript and post-transcriptional levels during the early stages of colon carcinogenesis.To our knowledge, this has never been attempted in the context of a highly relevant in vivocolon cancer model. The AOM-induced rat colon tumor model was selected because it providesa clear distinction between tumor initiation and promotion (36). Similar to humans, tumorsfrom AOM-injected animals exhibit the presence of mutated/nuclear β-catenin (37) and theoverexpression of cyclooxygenase-2 (COX-2) (31,32), consistent with the dramatic up-regulation of the Wnt signaling pathway (32). In addition, rat colonic tumors closely parallel

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human colonic neoplasia in pathologic features (38). Therefore, in the absence ofcomprehensive human data, the AOM chemical carcinogenesis model serves as a highlyrelevant means of assessing human colon cancer risk.

The examination of global alterations in gene expression in colonic mucosa at a very earlystage (10 wk post AOM injection) of colorectal cancer development revealed that the colonacquires changes predominantly impacting the translational control of gene expression. Thisfinding is supported by the elevation in eIF4E and phospho-eIF4E in colonic sections, inaddition to a previous study in which colon carcinoma cell lines of cancer progression tometastasis was examined (9). Collectively, these data support a model whereby oncogenicsignaling leads to cellular transformation by altering the transcriptome and producing a radicalshift in the composition of mRNAs associated with actively translating polysomes (2). Weargue, therefore, that studies employing polysome purification and microarray analysis areneeded in order to fully elucidate the mechanisms of translational deregulation associated withcolon tumor development. Indeed, major advancements in genome-wide techniques forsystematically monitoring protein translation in response to environmental factors haverecently been made (39).

By determining mRNA relative translatability (polysome/total mRNA ratio), we cataloguedwhich mRNAs are translated efficiently under malignant transformation conditions and arethus refractory to translational repression. Our data indicated a profound alteration intranslational control following AOM treatment, suggesting that signaling pathways cancooperate to differentially translate existing pools of mRNA. The shift of existing mRNAs intoand out of the polysome fraction appears to be more rapid and extensive than the change in thetotal mRNA pool. Since conditions such as cell stress and malignant transformation often resultin a poor correlation between mRNA levels and protein production (9,40,41), it is evident thatcomplementary experiments investigating the causal relationship between translational controland oncogenesis are needed. In addition, having determined the level of concordance betweensteady-state and translated mRNA populations at a very early stage of malignanttransformation, future analyses will need to focus on how the expression of translated mRNAis altered in (late stage) tumor samples compared to uninvolved mucosa. This will involverunning separate analyses on uninvolved mucosa and tumors, since there is growing evidencethat epigenetic perturbations in cancer are not confined to tumor cells but also involve adjacentcells (7).

It is well known that the APC gene is mutated, truncated or deleted in the majority of humancolon tumors. This is significant because mutant forms of the APC protein cause activation ofWnt-beta catenin-dependent signaling (42), COX-2 expression/prostaglandin/leukotrienesynthesis (43), MAPK signaling (32) and crypt stem cell survival (44). Similarly, colon tumorsinduced in rodents by chemical alkylating carcinogens, e.g., AOM, exhibit deregulation of theWnt and MAPK signaling pathways (45). However, from the perspective of the developmentof a lethal cancer, it is not clear precisely at which point in the evolution of a colonic tumordoes Wnt, COX-2 or MAPK-dependent signaling become chronically perturbed. Our datademonstrate that polysomal loading patterns of mRNAs associated with the Wnt-beta catenin,phospholipase A2-eicosanoid and the MAPK signaling axes are significantly up-regulated ata very early period of tumor development in the colon. Interestingly, previous reports indicatethat MAPKs are highly activated during late progression of colorectal carcinoma. Indeed, incolorectal cancer, at least 21 pathways have been identified to be enriched in mutations fromdifferent pathway databases (46). We have applied a systems biology approach involvingsimultaneous microarray analysis of rat colonic total (steady-state) mRNA and activelytranslated (polysomal) mRNA populations to show that 2 pathways (Wnt and eicosanoid)previously implicated in colorectal tumorigenesis, are affected at the level of translation at avery early stage of tumor development. It appears that transcriptional differences seen with

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AOM-induced signaling alterations are generated secondary to translational effects onmRNAs. It is likely, therefore, that differential recruitment into polysomes of a given pool ofmRNA may be sufficient to promote tumor formation. This interpretation is consistent withprevious reports indicating that as cancer cells become more transformed they acquireresistance to translation repression (47,48). To the best of our knowledge, this is the first reportdemonstrating a profound alteration of translational control in early colorectal progression invivo. These data complement previous studies using sequencing and transcriptome profilingapproaches to examine early stage tumor evolution (49,50).

Our data also offer insight into the effects of chemoprotective n-3 PUFA on colonic polysomalmRNA and total mRNA profiles. In striking contrast to the effects of AOM, transcriptionalalterations were found to be more extensive relative to translational alterations in mediatingthe effects of diet. This implies that dietary intervention does not simply counteract the effectsof carcinogen. Although very little is known regarding the role of diet on post-transcriptionalcontrol, the n-3 PUFA, eicosapentaenoic acid (EPA, 20:5n-3) has been shown to inhibit proteinsynthesis at the level of protein initiation (40). Collectively, therefore, these findings arenoteworthy because they provide further inroads into how diet regulates global gene expressionduring the early stages of colorectal tumorigenesis.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Evelyn Callaway, Chandra Emani and Carl Raetzsch for technical assistance.

This work was supported in part by NIH grants DK71707, CA59034, CA129444, CA74552, and P30ES09106.

Abbreviations used

AOM azoxymethane

MAPK mitogen activated protein kinase

PUFA polyunsaturated fatty acids

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34. Crim KC, Sanders L, Hong MY, et al. Upregulation of p21waf1/cip1 expression in vivo by butyrateadministration can be chemoprotective or chemopromotive depending on the lipid component of thediet. Carcinogenesis 2008;29:1415–20. [PubMed: 18567619]

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37. Yamada Y, Yoshimi N, Hirose Y, et al. Frequent β-catenin gene mutations and accumulations of theprotein in the putative preneoplastic lesions lacking macroscopic aberrant crypt foci appearance, inrat colon carcinogenesis. Cancer Res 2000;60:3323–27. [PubMed: 10910031]

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40. Palakurthi SS, Fluckiger R, Aktas H, et al. Inhibition of translation initiation mediates the anticancereffect of n-3 polyunsaturated fatty acid eicosapentaenoic acid. Cancer Res 2000;60:2919–25.[PubMed: 10850438]

41. Lu X, de la Pena L, Barker C, Camphausen K, Tofilon PJ. Radiation-induced changes in geneexpression involve recruitment of existing messenger RNAs to and away from polysomes. CancerRes 2006;66:1052–61. [PubMed: 16424041]

42. Gregorieff A, Clevers H. Wnt signaling in the intestinal epithelium: form endoderm to cancer. Genes& Dev 2005;19:877–90. [PubMed: 15833914]

43. Araki Y, Okamura S, Hussain SP, et al. Regulation of cyclooxygenase-2 expression by the Wnt andRas pathways. Cancer Res 2003;63:728–34. [PubMed: 12566320]

44. Barker N, Clevers H. Tracking down the stem cells of the intestine: Strategies to identify adult stemcells. Gastroenterology 2007;133:1755–60. [PubMed: 18054544]

45. Femia AP, Dolara P, Giannini A, Salvadori M, Biggeri A, Caderni G. Frequent mutation of Apc genein rat colon tumors and mucin-depleted foci, preneoplastic lesions in experimental coloncarcinogenesis. Cancer Res 2007;67:445–9. [PubMed: 17234750]

46. Lin J, Gan CM, Zhang X, et al. A multidimensional analysis of genes mutated in breast and coloncolorectal cancers. Genome Res 2007;17:1304–18. [PubMed: 17693572]

47. Connolly E, Braunstein S, Formenti S, Schneider RJ. Hypoxia inhibits protein synthesis through 4E-BP1 and elongation factor 2 kinase pathway controlled by mTOR and uncoupled in breast cancercells. Mol Cell Biol 2006;26:3955–65. [PubMed: 16648488]

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Davidson et al.


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Figure 1. Isolation of polysomes by sucrose gradient centrifugationColonic mucosal cells were lysed and post-nuclear supernatants layered onto 15-50% linearsucrose gradients. Following centrifugation, fractions were analyzed by absorbance at 254 nmand RNA was isolated. Select fractions were analyzed by Agilent Bioanalyzer and identifiedas (A) small ribonuclear protein complexes, (B) 40S ribosomal subunit, (C) 60S ribosomalsubunit, (D) 80S ribosomes, and (E) polysomes.

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Figure 2.(A) Number of genes significantly affected by treatment with AOM compared to saline (control)in polysome and total RNA fractions. Polysomes were isolated from colonic mucosa by sucrosegradient centrifugation followed by RNA isolation from the polysome fraction. Total RNAwas also isolated from the same animals (n=6-9). Following microarray analysis, genesexpressed differentially (p<0.05) between the AOM and saline treated rats were compared inthe polysome and total RNA fractions. (B) Effect of carcinogen on various biological processgene categories. Data are expressed as the percent of AOM affected genes (p<0.05) in abiological process category for the polysome fraction divided by the percent of genes for thetotal RNA fraction.

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Figure 3. Regulated genes in the Wnt signaling pathway following carcinogen treatment at 10 weeksSee legend to Figure 2 for details. Gene expression in AOM and saline treated rats wascompared using GenMAPP pathway analysis. Colors indicate significantly (p < 0.05) up-regulated genes. Numbers show the fold changes. (A) Polysome RNA fraction, (B) Total RNAfraction.

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Figure 4. Regulated genes in the eicosanoid biosynthesis pathway following carcinogen treatmentat 10 weeksColors indicate significantly (p < 0.05) up-regulated genes. Numbers show the fold changes.(A) Polysome RNA fraction, (B) Total RNA fraction.

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Figure 5. Carcinogen effect on in situ expression of eIF4E and phosphorylated eIF4E in coloniccryptsA, Immunhistochemistry of eIF4E and phosphorylated eIF4E (ph-eIF4E) in colon of rats 10wk after AOM or saline treatment. Negative control, primary antibody was omitted.Magnification, 100×. B, Expression level (staining intensity minus local background) wasquantified with image analysis software by taking measurements at the base, middle and topof at least 20 crypts per animal, n=8 rats per treatment group.

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Davidson et al.

Tabl

e 1A

Tra

nsla

tiona

lly u

p-re

gula

ted

gene

s sig

nific

antly

affe

cted

by

carc

inog

en e

xpos

ure

Abu

ndan

ce o

f mR

NA

s in

poly

som

e an

d to

tal R

NA

frac

tions

in A

OM

vs s

alin

e tre

atm

ent w

as q

uant

ified

by

mic

roar

ray

anal

ysis

. Tra

nsla

tabi

lity

inde

x w

asca

lcul

ated

as t

he fo

ld c

hang

e in

pol

ysom

e R

NA

ratio

(AO

M/s

alin

e) o

ver f

old

chan

ge in

tota

l RN

A ra

tio. G

enes

show

n ha

ve a

tran

slat

abili

ty in

dex

of 1

.3 o

rgr

eate

r and

a fa

lse

disc

over

y ra

te <

0.0

5.

NC

BI A

cces

sion

#Po

lyso

me

RN

A A

OM

/sal

ine

Tot

al R

NA

AO

M/s

alin

eT

rans

lata

bilit

y In

dex

Poly

som

e R

NA

p-v

alue

Tot

al R

NA

p-v

alue

Gen

e D

escr

iptio

n

NM

_019

195

2.09

1.26

1.66

1.78

E-03

4.88

E-02

CD

47 a

ntig

en (R

h-re

late

d an

tigen

,in

tegr

in-a

ssoc

iate

d si

gnal

tran

sduc

er)

(Cd4

7)

NM

_012

571

1.95

1.18

1.65

4.76

E-04

2.10

E-03

glut

amat

e ox

aloa

ceta

te tr

ansa

min

ase

1(G

ot1)

NM

_001

0066

021.

781.

111.

607.

27E-

061.

68E-

02ph

osph

atid

ylin

osito

l gly

can,

cla

ss S

(Pig

s)

NM

_012

923

1.81

1.17

1.55

3.29

E-03

2.39

E-02

cycl

in G

1 (C

cng1

)

NM

_019

275

1.76

1.14

1.54

1.14

E-03

4.75

E-02

MA

D h

omol

og 4

(Dro

soph

ila) (

Mad

h4)

NM

_080

907

1.75

1.15

1.52

1.72

E-04

4.47

E-02

prot

ein

phos

phat

ase

4, re

gula

tory

subu

nit 1

(Ppp

4r1)

NM

_001

0798

891.

731.

141.

516.

91E-

053.

36E-

02se

leno

phos

phat

e sy

nthe

tase

2 (S

ephs

2)

NM

_001

0069

701.

671.

121.

497.

73E-

044.

03E-

02ub

iqui

nol-c

ytoc

hrom

e c

redu

ctas

e co

repr

otei

n II

(Uqc

rc2)

NM

_017

030

1.81

1.22

1.48

1.74

E-04

4.12

E-02

prop

iony

l coe

nzym

e A

car

boxy

lase

,be

ta p

olyp

eptid

e (P

ccb)

NM

_022

536

1.59

1.09

1.46

4.34

E-04

2.23

E-02

pept

idyl

prol

yl is

omer

ase

B (P

pib)

NM

_175

597

1.58

1.10

1.44

1.96

E-04

1.28

E-02

syno

vial

sarc

oma,

X b

reak

poin

t 2in

tera

ctin

g pr

otei

n (S

sx2i

p)

NM

_022

390

1.62

1.13

1.43

1.50

E-04

1.46

E-02

quin

oid

dihy

drop

terid

ine

redu

ctas

e(Q

dpr)

NM

_001

0069

891.

561.

111.

413.

33E-

034.

88E-

02sc

otin

(MG

C94

600)

NM

_001

0131

971.

611.

151.

401.

84E-

032.

78E-

02se

rine/

thre

onin

e ki

nase

19

(Stk

19)

NM

_022

512

1.58

1.12

1.40

2.45

E-03

5.95

E-03

acyl

-coe

nzym

e A

deh

ydro

gena

se, s

hort

chai

n (A

cads

)

NM

_001

0093

991.

601.

141.

401.

78E-

033.

86E-

03N

AD

(P) d

epen

dent

ster

oid

dehy

drog

enas

e-lik

e (N

sdhl

)

NM

_207

591

1.72

1.23

1.39

1.23

E-03

2.39

E-02

glio

ma

tum

or su

ppre

ssor

can

dida

tere

gion

gen

e 2

(Glts

cr2)

NM

_024

392

1.55

1.12

1.38

2.27

E-04

1.03

E-02

hydr

oxys

tero

id (1

7-be

ta)

dehy

drog

enas

e 4

(Hsd

17b4

)


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Davidson et al.

NC

BI A

cces

sion

#Po

lyso

me

RN

A A

OM

/sal

ine

Tot

al R

NA

AO

M/s

alin

eT

rans

lata

bilit

y In

dex

Poly

som

e R

NA

p-v

alue

Tot

al R

NA

p-v

alue

Gen

e D

escr

iptio

n

NM

_012

914

1.55

1.12

1.38

4.08

E-04

2.20

E-02

ATP

ase,

Ca+

+ tra

nspo

rting

, ubi

quito

us(A

tp2a

3)

NM

_031

328

1.62

1.18

1.38

1.48

E-02

4.08

E-02

B-c

ell C

LL/ly

mph

oma

10 (B

cl10

)

NM

_054

004

1.53

1.11

1.37

1.56

E-03

3.17

E-02

TBP-

inte

ract

ing

prot

ein

120A

(Tip

120A

)

NM

_001

0130

461.

501.

091.

371.

83E-

059.

17E-

03R

AB

35, m

embe

r RA

S on

coge

ne fa

mily

(Rab

35)

NM

_019

163

1.67

1.22

1.37

1.63

E-03

2.52

E-02

pres

enili

n 1

(Pse

n1)

NM

_017

050

1.63

1.20

1.36

5.32

E-03

4.31

E-02

supe

roxi

de d

ism

utas

e 1

(Sod

1)

NM

_001

0121

521.

491.

111.

341.

60E-

034.

83E-

02TB

C1

dom

ain

fam

ily, m

embe

r 14

(Tbc

1d14

)

NM

_053

556

1.50

1.12

1.34

3.43

E-03

3.68

E-04

mat

erna

l G10

tran

scrip

t (G

10)

NM

_001

0083

281.

601.

201.

348.

00E-

041.

36E-

02po

ly (A

DP-

ribos

e) p

olym

eras

e fa

mily

,m

embe

r 3 (P

arp3

)

NM

_031

146

1.57

1.18

1.33

5.28

E-04

2.14

E-02

actin

rela

ted

prot

ein

2/3

com

plex

,su

buni

t 1A

(Arp

c1a)

NM

_053

527

1.46

1.10

1.33

1.74

E-04

3.08

E-03

cell

divi

sion

cyc

le 5

-like

(S p

ombe

)(C

dc5l

)

NM

_001

0077

311.

611.

211.

338.

80E-

032.

33E-

02go

lgi a

utoa

ntig

en, g

olgi

n su

bfam

ily a

, 7(G

olga

7)

NM

_022

281

1.62

1.22

1.33

7.27

E-04

1.54

E-02

ATP

-bin

ding

cas

sette

, sub

-fam

ily C

(CFT

R/M

RP)

, mem

ber 1

(Abc

c1)

NM

_001

0085

191.

541.

171.

317.

45E-

031.

36E-

02le

ucin

e-ric

h PP

R-m

otif

cont

aini

ng(L

rppr

c)

NM

_024

374

1.64

1.25

1.31

6.81

E-03

4.16

E-02

myo

troph

in (M

tpn)

NM

_138

896

1.54

1.18

1.31

8.24

E-04

3.00

E-02

praj

a 2,

RIN

G-H

2 m

otif

cont

aini

ng(P

ja2)

NM

_001

0130

971.

491.

141.

303.

78E-

044.

88E-

02rib

onuc

leas

e H

1 (R

nase

h1)


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Davidson et al.

Tabl

e 1B

Tra

nsla

tiona

lly d

own-

regu

late

d ge

nes s

igni

fican

tly a

ffect

ed b

y ca

rcin

ogen

exp

osur

e

Abu

ndan

ce o

f mR

NA

s in

poly

som

e an

d to

tal R

NA

frac

tions

in A

OM

vs s

alin

e tre

atm

ent w

ere

quan

tifie

d by

mic

roar

ray

anal

ysis

. Tra

nsla

tabi

lity

inde

x w

asca

lcul

ated

as t

he fo

ld c

hang

e in

pol

ysom

e R

NA

ratio

(AO

M/s

alin

e) o

ver f

old

chan

ge in

tota

l RN

A ra

tio. G

enes

show

n ha

ve a

tran

slat

abili

ty in

dex

of 0

.7 o

rle

ss a

nd a

fals

e di

scov

ery

rate

< 0

.05.

NC

BI A

cces

sion

#Po

lyso

me

RN

A A

OM

/sal

ine

Tot

al R

NA

AO

M/s

alin

eT

rans

lata

bilit

y In

dex

Poly

som

e p-

valu

eT

otal

RN

A p

-val

ueG

ene

Des

crip

tion

NM

_013

069

1.22

1.76

0.70

2.01

E-02

7.95

E-03

CD

74 a

ntig

en (i

nvar

iant

pol

pype

ptid

e of

maj

or h

isto

com

patib

ility

cla

ss II

ant

igen

-as

soci

ated

) (C

d74)

NM

_001

0042

731.

171.

710.

684.

82E-

021.

14E-

02zi

nc fi

nger

pro

tein

403

(Znf

403)

NM

_173

840

1.22

1.83

0.67

3.70

E-02

2.31

E-02

Will

iam

s-B

eure

n sy

ndro

me

chro

mos

ome

regi

on 5

hom

olog

(hum

an) (

Wbs

cr5)

NM

_138

881

1.54

2.35

0.66

3.16

E-03

1.81

E-02

Bes

t5 p

rote

in (B

est5

)

NM

_138

913

1.56

2.68

0.58

8.72

E-06

0.00

E+00

2′,5′-o

ligoa

deny

late

synt

heta

se 1

, 40/

46kD

a(O

as1)

NM

_057

151

1.19

2.30

0.52

2.89

E-02

2.76

E-02

smal

l ind

ucib

le c

ytok

ine

subf

amily

A (C

ys-

Cys

), m

embe

r 17

(Ccl

17)

NM

_145

672

1.52

2.97

0.51

1.44

E-05

4.62

E-02

chem

okin

e (C

-X-C

mot

if) li

gand

9 (C

xcl9

)

NM

_053

299

1.51

3.06

0.49

7.94

E-05

4.88

E-02

ubiq

uitin

D (U

bd)


identification of actively translated mrna transcripts in a rat model of early-stage colon...

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