Published OnlineFirst October 20, 2009; DOI: 10.1158/1940-6207.CAPR-09-0144
Cancer Prevention Research
Identification of Actively Translated mRNA Transcripts in a Rat Model of
Early-Stage Colon Carcinogenesis
Laurie A. Davidson,1,2 Naisyin Wang,3 Ivan Ivanov,4 Jennifer Goldsby,2
Joanne R. Lupton1,2 and Robert S. Chapkin1,2
Abstract
With respect to functional mapping of gene expression signatures, the steady-state
mRNA expression level does not always accurately reflect the status of critical signaling proteins. In these cases, control is exerted at the epigenetic level of recruitment of mRNAs to
polysomes, the factories of ribosomes that mediate efficient translation of many cellular
messages. However, to date, a genome-wide perspective of the effect of carcinogen and
chemoprotective bioactive diets on actively translated (polysomal) mRNA populations has
not been done. Therefore, we used an established colon cancer model, i.e., the azoxymethane (AOM)-treated rat, in combination with a chemoprotective diet extensively studied
in our laboratory, i.e., n-3 polyunsaturated fatty acids, to characterize the molecular processes underlying the transformation of normal colonic epithelium. The number of genes affected
by AOM treatment 10 weeks after carcinogen injection was significantly greater in the polysome RNA fraction compared with the total RNA fraction as determined using a high-density
microarray platform. In particular, polysomal loading patterns of mRNAs associated with the
Wnt-β catenin, phospholipase A2-eicosanoid and the mitogen-activated protein kinase signaling axes were significantly upregulated at a very early period of tumor development in the
colon. These data indicate that translational alterations are far more extensive relative to
transcriptional alterations in mediating malignant transformation. In contrast, transcriptional
alterations were found to be more extensive relative to translational alterations in mediating
the effects of diet. Therefore, during early stage colonic neoplasia, diet and carcinogen seem
to predominantly regulate gene expression at multiple levels via unique mechanisms.
Total steady-state mRNA levels have utility in the prediction
of the expression levels of an array of proteins. However, in
many cases, the steady-state mRNA expression level does
not accurately reflect the expression rate of proteins. This is
especially relevant in the case of Ras-transformed cells (1–4).
Because prolonged activation of p21 Ras drives colonic tumor
development (5), it is likely that translational regulation plays
an important role in the control of colonic gene expression
during malignant transformation.
It has been recently reported that several tumor suppressors
and proto-oncogenes can influence the formation of the mature
ribosome or regulate the activity of translation factors (6, 7).
Traditionally, the differential mobilization of mRNAs onto
polyribosomes has been used to identify genes whose transcripts are translationally controlled (8). Translational control
has been verified by demonstrating the redistribution of
mRNAs on polysome gradients at various stages 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, showed that
the colorectal cancer genome acquires changes impacting by
far the translational more than the transcriptional control of
gene expression. These changes would have been completely
missed in a conventional transcriptosome analysis detecting
cellular mRNAs irrespective of their degree of polysomal loading.
Although the analysis of cancer cells in tissue culture is informative, it is possible that this model system may not accurately
represent the state of cancer cells in vivo. Therefore, we have
applied a systems biology approach to the characterization of
gene expression changes occurring during carcinogen-induced
colon tumorigenesis. Specifically, to take full advantage of the
polysomal isolation method, we have combined ribosome-free
and polysome-bound mRNAs with an open, high throughput
quantitative mRNA analysis detection platform that simultaneously measures and identifies existing mRNA species to provide a panoramic overview of gene expression at both the total
Authors' Affiliations: 1Program in Integrative Nutrition and Complex Diseases,
2
Center for Environmental and Rural Health, and Departments of 3Statistics and
4
Veterinary Physiology and Pharmacology, Texas A&M University, College
Station, Texas
Received 9/30/08; revised 7/2/09; accepted 7/27/09; published OnlineFirst
10/20/09.
Grant support: NIH grants DK71707, CA59034, CA129444, CA74552, and
P30ES09106.
Note: Supplementary data for this article are available at Cancer Prevention
Research Online (http://cancerprevres.aacrjournals.org/).
Requests for reprints: Robert S. Chapkin, Center for Environmental and
Rural Health, Kleberg Biotechnology Center, MS 2253, Texas A&M University,
College Station, TX 77843-2253. Phone: 979-845-0419; Fax: 979-862-2378;
E-mail: [email protected].
©2009 American Association for Cancer Research.
doi:10.1158/1940-6207.CAPR-09-0144
Cancer Prev Res 2009;2(11) November 2009
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Global Alterations in Colonic Polysomal mRNA
Fig. 1. Isolation of polysomes by sucrose gradient centrifugation. Colonic mucosal cells were lysed and postnuclear supernatants layered onto 15% to 50% linear
sucrose gradients. Following centrifugation, fractions were analyzed by absorbance at 254 nm and RNA was isolated. Select fractions were analyzed by Agilent
Bioanalyzer and identified as (A) small ribonuclear protein complexes, (B) 40S ribosomal subunit, (C) 60S ribosomal subunit, (D) 80S ribosomes, and (E) polysomes.
Diets
transcript and posttranscriptional levels. To our knowledge,
the effect of a colon-specific carcinogen on the pattern of existing mRNAs that are recruited to polysomes has never been
attempted in vivo.
With respect to environmental risk factors, there is growing evidence that long-chain n-3 polyunsaturated fatty acids (PUFA),
e.g., eicosapentaenoic acid and docosahexaenoic acid, suppress
colon cancer risk in humans (10, 11). Recently, we have shown
that chemoprotective n-3 PUFA reprogram genetic signatures
during colon cancer initiation and progression and reduce colon tumor formation (12). Therefore, we further investigated
whether n-3 PUFA modulate the levels of actively translated
mRNAs in colonic mucosa following carcinogen exposure.
After a 1-wk acclimation on standard pelleted diet, rats were assigned to one of four diet groups that differed in the type of fat and
fiber as previously described (13). Diets contained (grams/100 grams
diet) the following: dextrose, 51.00; casein, 22.40; D,L-methionine, 0.34;
AIN-76 salt mix, 3.91; AIN-76 vitamin mix, 1.12; choline chloride, 0.13;
and pectin or cellulose, 6.00. The total fat content of each diet was 15%
by weight with the n-6 PUFA diet containing 15.0 grams of corn oil/
100 grams diet, and the n-3 PUFA diet containing 11.5 grams of fish
oil/100 grams diet plus 3.5 grams of corn oil/100 grams diet as previously described (12).
Carcinogen treatment
After 2 wk on the experimental diets, nine rats per diet group received injections of saline (control) and nine rats per diet group received injections of AOM (Sigma) s.c. at 15 mg/kg body weight.
Rats received a second AOM or saline injection 1 week later and were
terminated 10 wk after the first injection.
Materials and Methods
Animals
Sixty-four 8-wk-old male Sprague-Dawley rats (Harlan) were acclimated for 2 wk in a temperature and humidity controlled facility on a
12-h light/dark cycle. The animal use protocol was approved by the
University Animal Care Committee of Texas A&M University. The
study was a 2 × 2 × 2 factorial design with two types of dietary fat
(n-6 PUFA or n-3 PUFA), two types of dietary fiber (cellulose or pectin), and two treatments [injection with the colon carcinogen, azoxymethane (AOM), or with saline]. Animals (n = 6-9 per group) were
stratified by body weight after the acclimation period so that mean
initial body weights were not different between groups. Body weight
was monitored throughout the study.
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Polysome and total RNA isolation
Upon termination, each colon was cut open longitudinally, was
flushed clean with PBS, 1-cm from the distal colon was collected for
fixation and embedding for immunohistochemical assays, an adjacent
centimeter was taken for total RNA isolation, and the remainder of the
colon was used for polysome RNA isolation. For total RNA isolation,
epithelial cells were scraped from the underlying muscle layer with a
glass microscope slide, were homogenized on ice in lysis buffer (mirVana miRNA Isolation kit, Ambion), and were frozen at −80°C until
RNA was isolated. Using the mirVana kit, total RNA enriched with
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Cancer Prevention Research
Fig. 2. A, number of genes significantly affected by treatment with AOM compared with saline (control) in polysome and total RNA fractions. Polysomes were
isolated from colonic mucosa by sucrose gradient centrifugation followed by RNA isolation from the polysome fraction. Total RNA was also isolated from the
same animals (n = 6-9). Following microarray analysis, genes expressed differentially (P < 0.05) between the AOM- and saline-treated rats were compared in the
polysome and total RNA fractions. B, effect of carcinogen on various biological process gene categories. Data are expressed as the percent of AOM affected genes
(P < 0.05) in a biological process category for the polysome fraction divided by the percent of genes for the total RNA fraction.
microRNA was isolated followed by DNase treatment. For polysome
RNA isolation, colons were incubated for 5 min at room temperature
in PBS containing 100 μg/mL cycloheximide, an inhibitor of translation that locks the mRNA/ribosome complex, facilitating its isolation
and preventing RNA degradation. Following incubation, epithelial
cells were scraped with a microscope slide on a chilled glass plate
and immediately processed according to the procedure of Ju et al.
(14). Briefly, epithelial cells were allowed to swell in LSB [20 mmol/L
Cancer Prev Res 2009;2(11) November 2009
Tris (pH 7.5), 10 mmol/L NaCl, and 3 mmol/L MgCl2] containing
1 mmol/L DTT and 50 units RNase inhibitor for 2 min followed
by lysis in LSB containing 0.2 mol/L sucrose and 1.2% Triton
X-100. After removal of nuclei by centrifugation, the supernatant
was layered over a 15% to 50% linear sucrose gradient (in LSB)
and centrifuged at 247,000 × g for 2 h at 4°C in a swinging bucket
rotor. Gradients were fractionated using a pipette, an aliquot was
taken for absorbance at 254 nm and 3-volume denaturation solution
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Global Alterations in Colonic Polysomal mRNA
Fig. 3. Regulated genes in the Wnt signaling pathway following carcinogen treatment at 10 wk. See legend of Fig. 2 for details. Gene expression in AOM- and
saline-treated rats was compared using GenMAPP pathway analysis. Colors indicate significantly (P < 0.05) up- or down-regulated genes. Numbers show the fold
changes. A, polysome RNA fraction; B, total RNA fraction.
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Cancer Prevention Research
Table 1. Carcinogen-induced changes in actively translated mRNA transcripts
A. Translationally upregulated genes significantly affected by carcinogen exposure*
NCBI
accession #
Polysome RNA
AOM/saline
Total RNA
AOM/saline
Translatability
index
Polysome
RNA P
Total
RNA P
Gene description
NM_019195
2.09
1.26
1.66
1.78E-03
4.88E-02
NM_012571
1.95
1.18
1.65
4.76E-04
2.10E-03
NM_001006602
1.78
1.11
1.60
7.27E-06
1.68E-02
NM_012923
NM_019275
1.81
1.76
1.17
1.14
1.55
1.54
3.29E-03
1.14E-03
2.39E-02
4.75E-02
NM_080907
1.75
1.15
1.52
1.72E-04
4.47E-02
NM_001079889
1.73
1.14
1.51
6.91E-05
3.36E-02
NM_001006970
1.67
1.12
1.49
7.73E-04
4.03E-02
NM_017030
1.81
1.22
1.48
1.74E-04
4.12E-02
NM_022536
NM_175597
1.59
1.58
1.09
1.10
1.46
1.44
4.34E-04
1.96E-04
2.23E-02
1.28E-02
NM_022390
1.62
1.13
1.43
1.50E-04
1.46E-02
NM_001006989
NM_001013197
NM_022512
1.56
1.61
1.58
1.11
1.15
1.12
1.41
1.40
1.40
3.33E-03
1.84E-03
2.45E-03
4.88E-02
2.78E-02
5.95E-03
NM_001009399
1.60
1.14
1.40
1.78E-03
3.86E-03
NM_207591
1.72
1.23
1.39
1.23E-03
2.39E-02
NM_024392
1.55
1.12
1.38
2.27E-04
1.03E-02
NM_012914
1.55
1.12
1.38
4.08E-04
2.20E-02
NM_031328
NM_054004
1.62
1.53
1.18
1.11
1.38
1.37
1.48E-02
1.56E-03
4.08E-02
3.17E-02
NM_001013046
1.50
1.09
1.37
1.83E-05
9.17E-03
NM_019163
NM_017050
NM_001012152
1.67
1.63
1.49
1.22
1.20
1.11
1.37
1.36
1.34
1.63E-03
5.32E-03
1.60E-03
2.52E-02
4.31E-02
4.83E-02
NM_053556
NM_001008328
1.50
1.60
1.12
1.20
1.34
1.34
3.43E-03
8.00E-04
3.68E-04
1.36E-02
NM_031146
1.57
1.18
1.33
5.28E-04
2.14E-02
NM_053527
1.46
1.10
1.33
1.74E-04
3.08E-03
CD47 antigen (Rh-related antigen,
integrin-associated signal
transducer) (Cd47)
Glutamate oxaloacetate
transaminase 1 (Got1)
Phosphatidylinositol glycan,
class S (PIGS)
Cyclin G1 (Ccng1)
MAD homologue 4 (Drosophila)
(Madh4)
Protein phosphatase 4, regulatory
subunit 1 (Ppp4r1)
Selenophosphate synthetase 2
(Sephs2)
Ubiquinol-cytochrome c reductase
core protein II (Uqcrc2)
Propionyl CoA carboxylase,
β polypeptide (Pccb)
Peptidylprolyl isomerase B (Ppib)
Synovial sarcoma, X breakpoint 2
interacting protein (Ssx2ip)
Quinoid dihydropteridine
reductase (Qdpr)
Scotin (MGC94600)
Serine/threonine kinase 19 (Stk19)
Acyl-CoA dehydrogenase,
short chain (Acads)
NAD(P)-dependent steroid
dehydrogenase-like (Nsdhl)
Glioma tumor suppressor
candidate region gene 2 (Gltscr2)
Hydroxysteroid (17-β)
dehydrogenase 4 (Hsd17b4)
ATPase, Ca2+ transporting,
ubiquitous (Atp2a3)
B-cell CLL/lymphoma 10 (Bcl10)
TBP-interacting protein
120A (Tip120A)
RAB35, member RAS oncogene
family (Rab35)
Presenilin 1 (Psen1)
Superoxide dismutase 1 (Sod1)
TBC1 domain family,
member 14 (Tbc1d14)
Maternal G10 transcript (G10)
Poly (ADP-ribose) polymerase
family, member 3 (Parp3)
Actin-related protein 2/3 complex,
subunit 1A (Arpc1a)
Cell division cycle 5–like
(S pombe) (Cdc5l)
(Continued on the following page)
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Global Alterations in Colonic Polysomal mRNA
Table 1. Carcinogen-induced changes in actively translated mRNA transcripts (Cont'd)
A. Translationally upregulated genes significantly affected by carcinogen exposure*
NCBI
accession #
Polysome RNA
AOM/saline
Total RNA
AOM/saline
Translatability
index
Polysome
RNA P
Total
RNA P
Gene description
NM_001007731
1.61
1.21
1.33
8.80E-03
2.33E-02
NM_022281
1.62
1.22
1.33
7.27E-04
1.54E-02
NM_001008519
1.54
1.17
1.31
7.45E-03
1.36E-02
NM_024374
NM_138896
1.64
1.54
1.25
1.18
1.31
1.31
6.81E-03
8.24E-04
4.16E-02
3.00E-02
NM_001013097
1.49
1.14
1.30
3.78E-04
4.88E-02
Golgi autoantigen, golgin
subfamily a, 7 (Golga7)
ATP-binding cassette, sub-family C
(CFTR/MRP), member 1 (Abcc1)
Leucine-rich PPR-motif
containing (Lrpprc)
Myotrophin (Mtpn)
Praja 2, RING-H2 motif
containing (Pja2)
RNase H1 (Rnaseh1)
B. Translationally downregulated genes significantly affected by carcinogen exposure†
NM_013069
1.22
1.76
0.70
2.01E-02
7.95E-03
NM_001004273
NM_173840
1.17
1.22
1.71
1.83
0.68
0.67
4.82E-02
3.70E-02
1.14E-02
2.31E-02
NM_138881
NM_138913
1.54
1.56
2.35
2.68
0.66
0.58
3.16E-03
8.72E-06
1.81E-02
0.00E+00
NM_057151
1.19
2.30
0.52
2.89E-02
2.76E-02
NM_145672
1.52
2.97
0.51
1.44E-05
4.62E-02
NM_053299
1.51
3.06
0.49
7.94E-05
4.88E-02
CD74 antigen (invariant polpypeptide
of major histocompatibility class II
antigen-associated) (Cd74)
Zinc finger protein 403 (Znf403)
Williams-Beuren syndrome
chromosome region 5
homologue (human) (Wbscr5)
Best5 protein (Best5)
2′,5′-oligoadenylate synthetase 1,
(Oas1)
Small inducible cytokine subfamily A
(Cys-Cys), member 17 (Ccl17)
Chemokine (C-X-C motif)
ligand 9 (Cxcl9)
Ubiquitin D (Ubd)
Abbreviation: NCBI, National Center for Biotechnology Information.
*Abundance of mRNAs in polysome and total RNA fractions in AOM vs saline treatment was quantified by microarray analysis. Translatability index was calculated as the fold change in polysome RNA ratio (AOM/saline) over fold change in total RNA ratio. Genes shown have a
translatability index of 1.3 or greater and a false discovery rate of <0.05.
†
Abundance of mRNAs in polysome and total RNA fractions in AOM vs saline treatment were quantified by microarray analysis. Translatability index was calculated as the fold change in polysome RNA ratio (AOM/saline) over fold change in total RNA ratio. Genes shown have a
translatability index of 0.7 or less and a false discovery rate of <0.05.
For both total RNA and polysome RNA, 10 μg of biotin-labeled
cRNA was hybridized to the CodeLink genome array following
manufacturer's instructions (12). Slides were scanned with an Axon
GenePix 4000B scanner.
(Ambion Totally RNA kit) was immediately added to the remainder of
each fraction. Samples were frozen at −80°C until RNA was isolated
using the Totally RNA kit as per manufacturer's instructions followed
by DNase treatment. Both total RNA and polysome RNA were analyzed on an Agilent Bioanalyzer to assess RNA integrity. In select experiments, 10 mmol/L EDTA was added to the post–nuclear
supernatant to examine the shift in elution due to disruption of polyribosome formation by EDTA.
Immunohistochemistry
At necropsy, a 1-cm section of distal colon was removed, fixed in
4% paraformaldehyde for 4 h, and paraffin embedded. Following antigen retrieval in 1× TE containing 0.05% Tween 20 brought 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 peroxide in methanol for 15 min to quench endogenous peroxidase activity. Sections were then processed using rabbit
eukaryotic translation initiation factor 4E (eIF4E; Cell Signaling Technology) at 1:100 or rabbit phospho-eIF4E (Cell Signaling) at 1:7 to
quantify in situ eIF4E and phosphorylated eIF4E staining as previously described (13). Images were quantified using NIS Elements
Microarray
CodeLink rat whole genome bioarrays (Applied Microarray)
were used to assess gene expression. For total RNA arrays, the
Ambion MessageAmp II-Biotin Enhanced aRNA amplification kit
was used to prepare labeled cRNA. For polysome arrays, the
Ambion MessageAmp II aRNA amplification kit followed by the
MessageAmp II-Biotin Enhanced aRNA amplification kit were used
with two rounds of in vitro transcription to generate labeled cRNA.
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software (Nikon) by collecting staining intensity at the base, middle,
and top of at least 20 crypts per animal, eight animals per treatment
group (AOM or saline).
ed by the AOM treatment. There were 233 genes significantly
(P < 0.05) affected by the carcinogen treatment identified in
both the polysome and total RNA fractions. These data indicate that translational alterations were more extensive (11.5fold higher) relative to transcriptional alterations in mediating
malignant transformation.
Statistics and pathway analysis
Polysome and total RNA expression data were analyzed using
GenMapp5 and GO6 and the approaches were described below using
the R computation platform. 7 Raw data were first normalized
through a robust procedure that eliminates the intensity-based
chip-by-chip bias and reduces the between-chip variation and the
potential outlier effects. Robust local median estimation was applied
to all observations from chips of the same treatment and potential
outlying observations identified (15). A within-treatment quantile
normalization was then applied to all nonoutlying observations
(16). An alternative of replacing the within-treatment target distribution in quantile normalization by the cross-all-chip method was also
investigated, testing outcomes remained unchanged between the two
alternatives. Mixed-effect ANOVA was 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 versus saline as well as diet comparisons were identified. Genes that
met the criteria of q value of <0.05 were submitted to GenMapp
and GO for pathway profiling.
Analysis of translationally activated genes in colonic
mucosa
Because an increase in polysomal RNA can reflect changes
in mRNA abundance and/or translatability, the relative translatability of each differentially (AOM versus saline) expressed
mRNA was calculated. Specifically, data were normalized to
determine the change in abundance in polysomal RNA relative to the change in abundance in total RNA for each mRNA
(polysome/total; ref. 19). Table 1A shows that 35 genes were
translationally enhanced following carcinogen exposure, i.e.,
polysome-associated mRNAs were enriched in AOM versus
saline-treated animals. We have defined translationally enhanced mRNAs as those that are significantly 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 increases in total mRNA
abundance alone. Notable genes maintained on polysomes included CD47, involved in regulating neutrophil transepithelial
migration (20); phosphatidylinositol-glycan biosynthesis class
S protein (PIGS), essential for the transfer of GPI to proteins
(21); cyclin G1, associated with G2-M phase arrest in response
to DNA damage (22); and SMAD4, a tumor suppressor gene
implicated in intestinal cell cycle regulation (23). Our analysis
also identified eight genes (Table 1B) that were reduced by
30% or greater (poly/total ratio, <0.7; P < 0.05) in AOM versus
saline-injected rats. Of interest is the observation that several
of these genes are associated with immune response. Among
these were C-C motif chemokine 17 (CCL17; ref. 24), Best5
(25), and CXC chemokine ligand (CXCL9; ref. 26).
Microarray chips from a total of six rats per treatment were
processed and data mining was done as described in the
Materials and Methods. Functional analysis of differentially
expressed polysome fraction genes was done by differential
pathway analysis (GSEA; ref. 27). The systematic profiling approach shown in Fig. 2B revealed that carcinogen affected
gene sets classified into 26 well-defined biological process
categories. Interestingly, the top biological processes affected
by AOM exposure were related to electron transport and
coenzyme and prosthetic group metabolism.
The canonical Wnt cascade has emerged as a critical regulator of stem cells (28). There is also compelling evidence that
oncogenic K-ras stimulates Wnt signaling in colon cancer
and that Myc is required for the majority of Wnt target gene
activation (29, 30). Therefore, we examined the effect of carcinogen (AOM) on gene activation of the Wnt pathway. Colonic
mucosal polysome and total RNA were isolated from rats
terminated at 10 weeks postinjection (saline or AOM). Only
significantly (P < 0.05) altered genes are shown (n = 6-9 rats
per group; Fig. 3). The data clearly show the impact of carcinogen on the mobilization of mRNAs onto polyribosomes for
genes associated with the Wnt pathway (Fig. 3A). Interestingly, at this “early” promotional stage (tumors typically do not
appear until 30 weeks after carcinogen injection), almost no
changes were observed in the total mRNA transcriptosome
Results
Whole genome polysomal profiles
Colonic mucosa polysome isolation was monitored by examining sucrose gradient fractions on an Agilent Bioanalyzer.
As shown in Fig. 1, the top of the gradient contained small
ribonuclear proteins (region A). Through the remainder of
the gradient, the progression to heavier 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 due to mRNA transcripts loaded
with varying numbers of ribosomes. The entire broad region
in the second half of the gradient, excluding the final fraction
containing RNA granules (18), was collected as the polysome
fraction. Addition of EDTA to the sample before gradient fractionation resulted in the expected shift away from the bottom
of the gradient due to the disruption of polysome and 80S ribosome formation (Supplementary Fig. S1).
Carcinogen-induced changes in gene expression
differentially affect recruitment of mRNAs to
polysomes
Polysome analysis and microarray technology was used to
assess the impact of a colon-specific carcinogen (AOM) on
global mRNA polysome recruitment. Total and polysomal
mRNA from scraped colonic mucosa were isolated, processed,
and hybridized using the Codelink (Applied Microarray) microarray platform. The number of genes affected by AOM
treatment 10 wk after carcinogen treatment was significantly
(P < 0.05) greater in the polysome RNA fraction compared
with the total RNA fraction. As shown in Fig. 2A, 3,239 genes
were significantly altered (P < 0.05) by AOM versus saline
treatment in the polysome RNA fraction only. In contrast,
the total RNA fraction resulted in only 69 unique genes affect-
5
6
7
http://www.genmapp.org
http://www.geneontology.org
http://www.R-project.org
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Global Alterations in Colonic Polysomal mRNA
Fig. 4. Regulated genes in the eicosanoid biosynthesis pathway following carcinogen treatment at 10 wk. Colors indicate significantly (P < 0.05) upregulated genes.
Numbers show the fold changes. A, polysome RNA fraction; B, total RNA fraction.
cell migration, and invasion, while inhibiting apoptosis (31), we
also investigated the impact of carcinogen exposure on the pattern of eicosanoid-related mRNAs that are recruited to polysomes. Overall, eight enzymes were significantly (P < 0.05)
(Fig. 3B), indicating minimal perturbation at the transcriptional level. Furthermore, because cyclooxygenase-2 (COX-2)–
derived prostaglandin E 2 can promote colonic tumor initiation
and progression by enhancing cell proliferation, angiogenesis,
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Cancer Prevention Research
Fig. 5. Carcinogen effect on in situ expression of eIF4E and phosphorylated eIF4E in colonic crypts. A, immunhistochemistry of eIF4E and phosphorylated eIF4E
(phospho-eIF4E) in colon of rats 10 wk after AOM or saline treatment. Negative control, primary antibody was omitted. Magnification, ×100. B, expression level
(staining intensity minus local background) was quantified with image analysis software by taking measurements at the base, middle, and top of at least 20 crypts per
animal; n = 8 rats per treatment group.
13 genes were modulated translationally following carcinogen exposure and corn oil + cellulose relative to fish oil +
pectin feeding. Notably, the tumor suppressor retinoblastomaassociated protein (RB1) gene was translationally suppressed
in corn oil + cellulose relative to fish oil + pectin–fed animals
(translatability index, 0.57). A similar trend was also noted
with respect to epoxide hydrolase 2, a xenobiotic metabolizing phase I enzyme (translatability index, 0.15).
upregulated at the polysome mRNA level as opposed to only
two in the total RNA fraction (Fig. 4).
Microarray analysis also revealed that a number of genes
associated with Ras and mitogen-activated protein kinase
(MAPK) cascades were significantly (P < 0.05) upregulated
(expressed as fold increase—AOM versus saline; e.g.,
Map2k1/MAP/ERK kinase 1-2.0) predominantly in polysomes (Supplementary Figs. S2 and S3). This is noteworthy,
because blockade of the MAPK pathway suppresses growth
of tumors in vivo (5, 32).
Effect of carcinogen on expression levels of colonocyte
eIF4E and phospho-eIF4E
Because elevated eIF4E function can induce malignancy 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 with saline-control
animals (Fig. 5). Tissue polysomal 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 (proliferative zone) and progressively decreased toward the luminal surface (Supplementary Fig. S6).
We also evaluated whether the phosphorylation status of
eIF4E, which typically correlates with the translation rate
and growth status of the cell, was affected by carcinogen
exposure. Similar to eIF4E, phospho-eIF4E levels were significantly (P < 0.01) elevated in carcinogen treated animals
(Fig. 5).
Diet preferentially influences global transcriptional
profiles
The number of genes affected by diet (n-3 versus n-6 PUFA)
10 weeks following placebo (saline) treatment was much
greater in the total RNA fraction compared with the polysome
RNA fraction. As shown in Supplementary Fig. S4, only 63
genes were significantly altered (P < 0.05) by PUFA treatment
in the polysome RNA fraction. In contrast, the total RNA fraction contained 328 unique genes affected by diet. In addition,
there were two 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– versus n-6 PUFA–fed animals injected with carcinogen
(Supplementary Fig. S5). These data indicate that transcriptional alterations were more 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 protective
against experimentally induced colon cancer compared with
cellulose and corn oil (33, 34). Therefore, we examined the effect of dietary combination chemotherapy on the mucosal
translatability index. As shown in Supplementary Table S1,
Cancer Prev Res 2009;2(11) November 2009
Discussion
In this study, we examined ribosome-free and polysomebound mRNAs using a high throughput microarray platform
to provide a panoramic overview of gene expression at both
the total transcript and posttranscriptional levels during the
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Global Alterations in Colonic Polysomal mRNA
activation of Wnt-β catenin–dependent signaling (42), COX2 expression/prostaglandin/leukotriene synthesis (43),
MAPK signaling (32), and crypt stem cell survival (44). Similarly, colon tumors induced in rodents by chemical alkylating carcinogens, e.g., AOM, exhibit deregulation of the Wnt
and MAPK signaling pathways (45). However, from the perspective of the development of a lethal cancer, it is not clear
precisely at which point in the evolution of a colonic tumor
does Wnt, COX-2, or MAPK-dependent signaling become
chronically perturbed. Our data show that polysomal loading patterns of mRNAs associated with the Wnt-β catenin,
phospholipase A2-eicosanoid, and the MAPK signaling axes
are significantly upregulated at a very early period of tumor
development in the colon. Interestingly, previous reports indicate that MAPKs are highly activated during late progression of colorectal carcinoma. Indeed, in colorectal cancer, at
least 21 pathways have been identified to be enriched in mutations from different pathway databases (46). We have applied a systems biology approach involving simultaneous
microarray analysis of rat colonic total (steady-state) mRNA
and actively translated (polysomal) mRNA populations to
show that two pathways (Wnt and eicosanoid), previously
implicated in colorectal tumorigenesis, are affected at the
level of translation at a very early stage of tumor development. It seems that transcriptional differences seen with
AOM-induced signaling alterations are generated secondary to translational effects on mRNAs. It is likely, therefore, that differential recruitment into polysomes of a
given pool of mRNA may be sufficient to promote tumor
formation. This interpretation is consistent with previous
reports indicating that as cancer cells become more transformed, they acquire resistance to translation repression
(47, 48). To the best of our knowledge, this is the first report demonstrating a profound alteration of translational
control in early colorectal progression in vivo. These data
complement previous studies using sequencing and transcriptome profiling approaches to examine early stage tumor evolution (49, 50).
Our data also offer insight into the effects of chemoprotective n-3 PUFA on colonic polysomal mRNA and total
mRNA profiles. In striking contrast to the effects of AOM,
transcriptional alterations were found to be more extensive
relative to translational alterations in mediating the effects
of diet. This implies that dietary intervention does not simply counteract the effects of carcinogen. Although very little
is known regarding the role of diet on posttranscriptional
control, the n-3 PUFA, eicosapentaenoic acid (20:5n-3) has
been shown to inhibit protein synthesis at the level of protein initiation (40). Collectively, therefore, these findings are
noteworthy because they provide further inroads into how
diet regulates global gene expression during the early stages
of colorectal tumorigenesis.
early stages of colon carcinogenesis. To our knowledge, this
has never been attempted in the context of a highly relevant
in vivo colon cancer model. The AOM-induced rat colon tumor model was selected because it provides a clear distinction between tumor initiation and promotion (36). Similar to
humans, tumors from AOM-injected animals exhibit the
presence of mutated/nuclear β-catenin (37) and the overexpression of COX-2 (31, 32), consistent with the dramatic
upregulation of the Wnt signaling pathway (32). In addition,
rat colonic tumors closely parallel human colonic neoplasia
in pathologic features (38). Therefore, in the absence of comprehensive human data, the AOM chemical carcinogenesis
model serves as a highly relevant means of assessing human
colon cancer risk.
The examination of global alterations in gene expression
in colonic mucosa at a very early stage (10 weeks post–
AOM injection) of colorectal cancer development revealed
that the colon acquires changes predominantly impacting
the translational control of gene expression. This finding
is supported by the elevation in eIF4E and phospho-eIF4E
in colonic sections, in addition to a previous study in
which colon carcinoma cell lines of cancer progression to
metastasis was examined (9). Collectively, these data support a model whereby oncogenic signaling leads to cellular
transformation by altering the transcriptome and producing
a radical shift in the composition of mRNAs associated
with actively translating polysomes (2). We argue, therefore, that studies using polysome purification and microarray analysis are needed to fully elucidate the mechanisms
of translational deregulation associated with colon tumor
development. Indeed, major advancements in genome-wide
techniques for systematically monitoring protein translation in response to environmental factors have recently
been made (39).
By determining mRNA relative translatability (polysome/
total mRNA ratio), we catalogued which mRNAs are translated efficiently under malignant transformation conditions and
are thus refractory to translational repression. Our data indicated a profound alteration in translational control following
AOM treatment, suggesting that signaling pathways can cooperate to differentially translate existing pools of mRNA.
The shift of existing mRNAs into and out of the polysome
fraction seems to be more rapid and extensive than the change
in the total mRNA pool. Because conditions such as cell stress
and malignant transformation often result in a poor correlation between mRNA levels and protein production (9, 40,
41), it is evident that complementary experiments investigating the causal relationship between translational control and
oncogenesis are needed. In addition, having determined the
level of concordance between steady-state and translated
mRNA populations at a very early stage of malignant transformation, future analyses will need to focus on how the expression of translated mRNA is altered in (late stage) tumor
samples compared with uninvolved mucosa. This will involve
running separate analyses on uninvolved mucosa and tumors,
because there is growing evidence that epigenetic perturbations in cancer are not confined to tumor cells but also involve
adjacent cells (7).
It is well known that the APC gene is mutated, truncated,
or deleted in the majority of human colon tumors. This is
significant because mutant forms of the APC protein cause
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Disclosure of Potential Conflicts of Interest
Laurie A. Davidson, Naisyin Wang, Ivan Ivanov, Jennifer Goldsby, Joanne R.
Lupton, and Robert S. Chapkin, no conflict of interest.
Acknowledgments
We thank Evelyn Callaway, Chandra Emani, and Carl Raetzsch for technical
assistance.
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Cancer Prevention Research
References
1. Prendergast GC. Signal transduction: putting
translation before transcription. Cancer Cell 2003;
4:244–5.
2. Rajasekhar VK, Viale A, Socci ND, Wiedmann M,
Hu X, Holland EC. Oncogenic ras and akt signaling
contribute to glioblastoma formation by differential
recruitment of existing mRNAs to polysomes. Mol
Cell 2003;12:889–901.
3. Spence J, Duggan BM, Eckhardt C, McClelland M,
Mercola D. Messenger RNAs under differential
translational control in Ki-ras-transformed cells.
Mol Cancer Res 2006;4:47–60.
4. Xi Y, Shalgi R, Fodstad O, Pilpel Y, Ju Jingfang J.
Differentially regulated micro-RNAs and actively
translated messenger RNA transcripts by tumor
suppressor p53 in colon cancer. Clin Cancer Res
2006;12:2014–24.
5. Sebolt-Leopold JS, Dudley DT, Herrera R, et al.
Blockade of MAP kinase pathway suppresses
growth of colon tumors in vivo. Nature Med 1999;
5:810–6.
6. Ruggero D, Pandolfi PP. Does the ribosome translate cancer? Nature Rev Cancer 2003;3:179–92.
7. Bilanges B, Stokoe D. Mechanisms of translational deregulation in human tumors and therapeutic intervention strategies. Oncogene 2007;26:
5973–90.
8. Mikulits W, Pradet-Balade B, Habermann B, Beug
H, Garcia-Sanz JA, Mullner EW. Isolation of translationally controlled mRNAs by differential screening. FASEB J 2000;14:1641–52.
9. Provenzani A, Fronza R, Loreni F, Pascale A,
Amadio M, Quattrone A. Global alterations in mRNA
polysomal recruitment in a cell model of colorectal
cancer progression to metastasis. Carcinogenesis
2006;27:1323–33.
10. Anti M, Armelao F, Marra G, et al. Effects of different doses of fish oil on rectal cell proliferation in
patients with sporadic colonic adenomas. Gastroenterology 1994;107:1709–18.
11. Bartram HP, Gostner A, Scheppach W, et al. Effects of fish oil on rectal cell proliferation, mucosal
fatty acids, and prostaglandin E2 release in healthy
subjects. Gastroenterology 1993;105:1317–22.
12. Davidson LA, Nguyen DV, Hokanson RM, et al.
Chemopreventive n-3 polyunsaturated fatty acids
reprogram genetic signatures during colon cancer
initiation and progression in the rat. Cancer Res
2004;64:6797–804.
13. Chang WC, Chapkin RS, Lupton JR. Predictive
value of proliferation, differentiation and apoptosis
as intermediate markers for colon tumorigenesis.
Carcinogenesis 1997;18:721–30.
14. Ju J, Huang C, Minskoff SA, Mayotte JE, Taillon
BE, Simons JF. Simultaneous gene expression
analysis of steady-state and actively translated
mRNA populations from osteosarcoma MG-63
cells in response to IL-1α via an open expression
analysis platform. Nucleic Acids Res 2003;31:
5157–66.
15. Koenker R, Hallock K. Quantile Regression.
J Econ Perspect 2001;15:143–56
16. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A
comparison of normalization methods for high density oligonucleotide array data based on variance
and bias. Bioinformatics 2003;19:185.
17. Storey D, Tibshirani R. Statistical significance for
genome-wide studies. Proc Natl Acad Sci U S A
2003;100:9440–5.
18. Krichevsky AM, Kosik KS. Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation. Neuron 2001;32:
683–96.
19. Thomas JD, Johannes GJ. Identification of
mRNAs that continue to associate with polysomes
during hypoxia. RNA 2007;13:1–16.
20. Parkos CA, Colgan SP, Liang TW, et al. CD47
mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia.
J Cell Biol 1996;132:437–50.
21. Nicolas F, Tiveron MC, Davoust J, Reggio H. GPI
membrane anchor is determinant in intracellular accumulation of apical plasma membrane proteins in
the non-polarized human colon cancer cell HT-29
18. J Cell Sci 1994;107:2679–89.
22. Perez R, Wu N, Klipfel AA, Beart BW. A better cell
cycle target for gene therapy of colorectal cancer:
cyclin G. J Gastrointest Surg 2003;7:884–9.
23. Chiao PJ, Hunt KK, Grau AM, et al. Tumor suppressor gene SMAD4/DPC4, its downstream target
genes, and regulation of cell cycle. Ann NY Acad
Sci 1999;880:31–7.
24. Kanagawa N, Niwa M, Hatanaka Y, et al. CCchemokine ligand 17 gene therapy induces tumor
regression through augmentation of tumor-infiltrating
immune cells in a murine model of preexisting
CT26 colon carcinoma. Int J Cancer 2007;121:
2013–22.
25. Grewal TS, Genever PG, Brabbs AC, Birch M,
Skerry TM. Best5: a novel interferon-inducible gene
expressed during bone formation. FASEB J 2000;
14:523–31.
26. Zhang R, Tian L, Chen LJ, et al. Combination of
MIG (CXCL9) chemokine gene therapy with lowdose cisplastin improves therapeutic efficacy
against murine carcinoma. Gene Ther 2006;13:
1263–71.
27. Subramanian A, Tamayo P, Mootha VK, et al.
Gene set enrichment analysis: a knowledge-based
approach for interpreting genome-wide expression
profiles. Proc Natl Acad Sci U S A 2005;102:
15545–50.
28. D'Errico I, Moschetta A. Nuclear receptors, intestinal architecture and colon cancer: an intriguing
link. Cell Mol Life Sci 2008;65:1523–43.
29. Nandan MO, McConnell BB, Ghaleb AM, et al.
Kruppel-like factor 5 mediates cellular transformation during oncogenic KRAS-induced intestinal tumorigenesis. Gastroenterology 2008;134:120–30.
30. Haigis KM, Kendall KR, Wang Y, et al. Differential
effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the
colon. Nat Genet 2008;40:600–8.
31. Buchanan FG, DuBois RN. Connecting Cox-2
and Wnt in cancer. Cancer Cell 2006;9:6–8.
32. Komatsu K, Buchanan G, Katkuri S, et al. Oncogenic potential of MEK1 in rat intestinal epithelial
cells in mediated via cyclooxygenase-2. Gastroenterology 2005;129:577–90.
33. Chang WC, Chapkin RS, Lupton JR. Fish oil
blocks azoxymethane-induced tumorigenesis by
increased cell Differentiation and apoptosis rather
than decreased cell proliferation. J Nutr 1998;18:
351–7.
Cancer Prev Res 2009;2(11) November 2009
994
34. Crim KC, Sanders L, Hong MY, et al. Upregulation of p21waf1/cip1 expression in vivo by butyrate
administration can be chemoprotective or chemopromotive depending on the lipid component of the
diet. Carcinogenesis 2008;29:1415–20.
35. Mamane Y, Petroulakis E, Martineau Y, et al. Epigenetic activation of a subset of mRNAs by eIF4E
explains its effects on cell proliferation. PLoS ONE
2007;2:e242.
36. Reddy BS, Burill C, Rigotty J. Effect of diets high
in ω-3 and ω-6 fatty acids on initiation and postinitiation stages of colon carcinogenesis. Cancer Res
1991;51:487–91.
37. Yamada Y, Yoshimi N, Hirose Y, et al. Frequent
β-catenin gene mutations and accumulations of the
protein in the putative preneoplastic lesions lacking
macroscopic aberrant crypt foci appearance, in
rat colon carcinogenesis. Cancer Res 2000;60:
3323–7.
38. Ahnen DJ. Are animal models of colon cancer
relevant to human disease. Dig Dis Sci 1985;30:
103–6S.
39. Ingolia NT, Ghaemmaghami S, Newman JRS,
Weissman JS. Genome-wide analysis in vivo of
translation with nucleotide resolution using ribosome profiling. Science 2009;324:218–23.
40. Palakurthi SS, Fluckiger R, Aktas H, et al. Inhibition of translation initiation mediates the anticancer
effect of n-3 polyunsaturated fatty acid eicosapentaenoic acid. Cancer Res 2000;60:2919–25.
41. Lu X, de la Pena L, Barker C, Camphausen K,
Tofilon PJ. Radiation-induced changes in gene expression involve recruitment of existing messenger
RNAs to and away from polysomes. Cancer Res
2006;66:1052–61.
42. Gregorieff A, Clevers H. Wnt signaling in the
intestinal epithelium: form endoderm to cancer.
Genes Dev 2005;19:877–90.
43. Araki Y, Okamura S, Hussain SP, et al. Regulation
of cyclooxygenase-2 expression by the Wnt and
Ras pathways. Cancer Res 2003;63:728–34.
44. Barker N, Clevers H. Tracking down the stem
cells of the intestine: strategies to identify adult
stem cells. Gastroenterology 2007;133:1755–60.
45. Femia AP, Dolara P, Giannini A, Salvadori M,
Biggeri A, Caderni G. Frequent mutation of Apc
gene in rat colon tumors and mucin-depleted foci,
preneoplastic lesions in experimental colon carcinogenesis. Cancer Res 2007;67:445–9.
46. Lin J, Gan CM, Zhang X, et al. A multidimensional
analysis of genes mutated in breast and colon colorectal cancers. Genome Res 2007;17:1304–18.
47. Connolly E, Braunstein S, Formenti S, Schneider
RJ. Hypoxia inhibits protein synthesis through 4EBP1 and elongation factor 2 kinase pathway controlled by mTOR and uncoupled in breast cancer
cells. Mol Cell Biol 2006;26:3955–65.
48. Shaw RJ, Cantley LC. Ras, PI([3])K and mTOR
signaling controls tumour cell growth. Nature
2006;441:424–30.
49. Sabates-Beliver J, Van der Flier LG, de Palo M,
et al. Transcriptome profile of human colorectal
adenomas. Mol Cancer Res 2007;5:1263–75.
50. Jones S, Chen WD, Parmigiani G, et al. Comparative lesion sequencing provides insights into tumor evolution. Proc Natl Acad Sci U S A 2008;
105:4283–8.
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Identification of Actively Translated mRNA Transcripts in a Rat
Model of Early-Stage Colon Carcinogenesis
Laurie A. Davidson, Naisyin Wang, Ivan Ivanov, et al.
Cancer Prev Res 2009;2:984-994. Published OnlineFirst October 20, 2009.
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