MOLECULAR AND CELLULAR BIOLOGY, Mar. 2006, p. 2373–2386
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 6
Activation of Transferrin Receptor 1 by c-Myc Enhances Cellular
Proliferation and Tumorigenesis†
Kathryn A. O’Donnell,1,3Duonan Yu,2Karen I. Zeller,3Jung-whan Kim,4Frederick Racke,5
Andrei Thomas-Tikhonenko,2and Chi V. Dang1,3,4,5*
Program in Human Genetics and Molecular Biology,1Department of Medicine, Division of Hematology,3Graduate Program of
Pathobiology,4and Department of Pathology,5The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,
and Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-60512
Received 14 October 2005/Returned for modification 7 December 2005/Accepted 22 December 2005
Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is
a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron
metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and
growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream
target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is
activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved
region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-
mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and
results in G1arrest without affecting cell size. Consistent with these findings, expression profiling reveals that
TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1
expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor
formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors,
illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for
Dysregulated expression of c-Myc is a central event in the
development of a diverse array of human malignancies (1, 10).
c-MYC encodes a helix-loop-helix transcription factor that
dimerizes with its partner Max, binds the consensus sequence
5?-CACGTG-3?, and regulates transcription of target genes.
Through its myriad direct and indirect target genes, the c-Myc
oncoprotein is linked to nearly all cellular processes (1, 12, 20,
40, 43, 48). An emerging view holds that c-Myc regulates 10 to
15% of genes in the human or Drosophila melanogaster ge-
nome, including noncoding microRNAs (18, 30, 38, 39). De-
spite the growing number of target genes being identified, the
precise mechanism by which c-Myc orchestrates cell growth
and division in normal and neoplastic cells is not fully under-
It has become increasingly evident that c-Myc regulates the
expression of several genes required for iron-dependent cellu-
lar processes, such as energy metabolism and mitochondrial
homeostasis (29, 36, 56). The link between c-Myc function and
iron metabolism was directly demonstrated through the iden-
tification of IRP2 and H-ferritin as coordinately regulated tar-
gets of c-Myc (58). Nramp1, which encodes a divalent cation
transporter and removes iron from the cytosol, was recently
shown to be repressed by c-Myc (5, 6). Taken together, these
studies suggest that c-Myc functions to increase the intracel-
lular iron pool.
The transferrin receptor (TFRC1/TFR/p90/CD71) is a key
cell surface molecule that regulates uptake of iron-bound
transferrin by receptor-mediated endocytosis (8). For more
than 20 years, there has been a known correlation between the
number of cell surface transferrin receptors and the rate of cell
proliferation (9, 27, 28, 35, 45). Transferrin receptor expression
is higher in cancer cells than in normal cells (19). Furthermore,
TFRC1 is among a select group of genes that is overexpressed
in a murine Myc-induced prostate cancer model as well as in
primary human prostate cancers (15). Despite these findings,
the mechanism by which transferrin receptor expression is
increased in neoplastic cells remains poorly characterized.
Given that transferrin receptors are highly expressed in can-
cer cells and that TFRC1 has been reported as a c-Myc-respon-
sive gene, we sought to determine if TFRC1 is directly regu-
lated by the c-Myc oncoprotein. In the current study, we
demonstrate that TFRC1 is a direct transcriptional target of
c-Myc in a human B lymphocyte model system. TFRC1 expres-
sion also parallels inducible c-Myc expression in an in vivo
murine model of lymphoma. RNA interference and iron che-
lation with the drug desferrioxamine (DFX) were used to
evaluate the role of TFRC1 in c-Myc-driven cell cycle pro-
gression and cell size control. Here, we report that TFRC1-
depleted lymphocytes undergo G1arrest while maintaining
normal cell size. Conversely, ectopic TFRC1 expression con-
fers a significant growth advantage to cells grown in limiting
serum conditions. We also demonstrate that enforced ex-
pression of TFRC1 significantly enhances the rate of c-Myc-
mediated tumor formation in nude mice. Taken together,
these results support a critical role for TFRC1 in c-Myc-
* Corresponding author. Mailing address: Ross Research Building,
Room 1032, 720 Rutland Avenue, Baltimore, MD 21205. Phone: (410)
955-2773. Fax: (410) 955-0185. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://mcb
MATERIALS AND METHODS
Cell culture. P493-6 cells were the generous gift of D. Eick at the Institute for
Clinical Molecular Biology und Tumor Genetics, GSF-Research Centre, Munich,
Germany. Cells were cultured in RPMI 1640, 10% fetal calf serum, and peni-
cillin-streptomycin (Pen-Strep). To repress c-MYC expression, cells were treated
with RPMI 1640–10% fetal calf serum–Pen-Strep supplemented with 0.1 ?g/ml
tetracycline (Sigma) for 72 h, washed two to three times in 1? phosphate-
buffered saline (PBS), and then restimulated with regular RPMI medium for
various time points. For desferrioxamine treatment, P493-6 cells were placed in
RPMI medium containing 50 or 100 ?M desferrioxamine (Sigma) at the same
time as removal of tetracycline and collected at various time points. TGR-1
(wild-type rat fibroblasts) and HO15.19 (c-MYC knockout rat fibroblasts) cells
(gift of J. Sedivy, Brown University) and c-MYC null cells reconstituted with
W135E (gift of L. Z. Penn, University of Toronto) were cultured in Dulbecco’s
modified Eagle’s medium (Gibco/Life Tech)–10% fetal bovine serum–Pen-Strep.
HO15-MYC cells were made by stable transfection of a MYC expression con-
struct driven by a murine leukemia virus promoter. For growth rate experiments
under limiting conditions, 2.5 ? 104cells were plated and subsequently starved
in 0.1% serum for 48 h. Cell were then washed, grown in 1% serum, and counted
in triplicate wells every 24 h for 6 to 7 days.
Wright staining. Wright staining was performed according to the manufactur-
er’s protocol using the HEMA 3 stain set (Fisher Scientific).
Western blot analysis. For immunoblot analysis, cells were collected and lysed
in incomplete Laemmli buffer at 95°C and protein was quantitated using a
bicinchoninic acid kit (Pierce). Fifteen to twenty micrograms of protein was
loaded on either a 8% or 10% polyacrylamide gel (for TFRC1 or c-MYC,
respectively) and transferred to a nitrocellulose membrane. 9E10 c-Myc and
TFRC1 mouse monoclonal antibodies were obtained from Zymed, Inc. (clone
H68.4). ?-Tubulin mouse monoclonal antibody was obtained from Oncogene
In vivo analysis of TFRC1 in murine B-cell lymphomas. Tumor-derived neo-
plastic lymphocytes were washed twice with PBS and resuspended in fluores-
cence-activated cell sorting (FACS) buffer (0.5% bovine serum albumin in PBS)
at a concentration of 1 ? 106cells/ml. One-hundred-microliter aliquots were
incubated on ice for 45 min with 2 ?l phycoerythrin-labeled anti-mouse CD71
(clone RI7 217.1.4; CALTAG, Burlingame CA). Samples were washed with PBS
and subjected to flow cytometry. Unstained cells were used as a reference.
Chromatin immunoprecipitation. P493-6 cells untreated or treated with 0.1
?g/ml tetracycline for 72 h were used for chromatin immunoprecipitation (ChIP)
assays. Cells were cross-linked with formaldehyde, and chromatin was immuno-
precipitated as described previously (7). The rabbit polyclonal c-Myc (sc-764;
Santa Cruz Biotechnology) and human hepatocyte growth factor (sc-7949; Santa
Cruz) antibodies were used to immunoprecipitate chromatin fragments. Total
input controls were collected from the “no antibody” control supernatant. Mock
control samples lacked chromatin but were treated the same as other samples.
Retroviral production and infection. The TRS1 vector was a gift from Jim
Basilion (Massachusetts General Hospital, Harvard University). The insert was
first cloned into the pSG5 vector using the EcoRV and SalI restriction sites and
then cloned into the pMSCV retroviral vector using the BamHI and BglII
restriction sites. For retroviral transduction, the pMSCV vector containing the
TRS1 insert was transfected into Phoenix amphotropic cells (at 80 to 90%
confluence) using the CaPO4method. Viral supernatant was collected every 24 h
for 3 days. For infection of c-MYC-null cells, approximately 2 ? 105cells were
plated per 10-cm dish and infected with 8 ml viral supernatant and 8 ?l/ml
polybrene 24 h later. Two subsequent rounds of infection were performed, and
cells were then selected with puromycin for 14 days. Cell lysates were then
collected 7 days after removal from puromycin.
RNA interference. Preannealled small interfering RNA (siRNA) duplexes
directed against TFRC1 (and a scrambled control) were purchased from
Dharmacon, Inc., and used according to the manufacturer’s instructions (Option
C). TFRC1 oligonucleotides used were GGAUGGUAACCUCAGAAAGdTdT
(sense) and dTdTCCUACCAUUGGAGUCUUUC (antisense). Scrambled JTV1
control oligonucleotides were CACGCUCGGUCAAAAGGUUdTdT (sense) and
dTdTGUGCGAGCCAGUUUUCCAA (antisense). RNA interference (RNAi)
oligonucleotides were transfected by electroporation. siRNA duplexes were first
added to a 4-mm-gap cuvette (BTX/Cambrige Pharmaceuticals) and then mixed
with 3 ? 106P493-6 cells (in a total volume of 500 ?l). Electroporation settings
used for P493-6 cells were as follows: 240 V and 1500 ?F. Cell viability was
assessed immediately following electroporation by trypan blue exclusion (and
determined to be approximately 30%). Mock-transfected cells and cells trans-
fected with a scrambled siRNA duplex were used as controls. Cells were col-
lected and assayed for gene function approximately 72 h after electroporation.
Transferrin uptake. HO15 and TGR cells were first trypsinized and counted.
Approximately 1 ? 106cells were washed two times in 1? PBS and then
resuspended in 2 ml serum-free Dulbecco’s modified Eagle’s medium. Samples
were then serum starved for 2 h with rotation at 37°C to clear transferrin (present
in culture medium) from the transferrin receptors. Alexa633-labeled transferrin
(Molecular Probes) was then added to each sample in a final concentration of 5
?g/ml, followed by incubation for 30 min, rotating at 37°C. Cells were then
washed three times with 1? PBS, resuspended in chilled 1% paraformaldehyde,
and analyzed using a Beckman Dickinson FACScalibur machine.
Nude mouse experiments. Cells (5 ? 106) in 100 ?l of sterile Hanks balanced
salt solution (Gibco) were injected subcutaneously into the right flank of male
homozygous nude mice at 4 to 6 weeks of age. Tumor volume was measured
using calipers every 3 to 5 days until the tumor mass reached 1,500 mm3. Tumor
volume was calculated using the following formula: [length (mm) ? width
(mm)]2/2. Two independent experiments were performed (n ? 4 to 5 mice per
cell line for experiment 1 and n ? 15 mice per cell line for experiment 2). All
experiments were approved by the Johns Hopkins School of Medicine Animal
Care and Use Committee.
Correlation of TFRC1 and c-MYC mRNA levels using the Atlas Gene Expres-
sion database. TFRC1 and c-MYC mRNA levels were examined in 91 human
tissues and cell lines using the Gene Expression Atlas database (http://expression
.gnf.org/cgi-bin/index.cgi) (52). We first log transformed the expression values
and then performed linear regression analysis (using SPSS version 11.0 for
Windows) on log-transformed mRNA expression values with log TFRC1 expres-
sion levels as the dependent variable and log c-MYC expression values as the
independent variable. An r2value was determined which measured the extent to
which expression of c-MYC and TFRC1 were correlated.
Flow cytometry. For cell size analysis, P493 cells were collected, washed two to
three times, and then resuspended in 1? PBS. For propidium iodide staining,
approximately 1 ? 106cells were washed in 1? PBS, trypsinized for 10 min at
room temperature (RT) (solution A), neutralized with trypsin inhibitor–RNase
A for 10 min at RT (solution B), and then stained with propidium iodide/
spermine tetrahydrochloride (solution C) for 10 min at RT (54). Cells were
filtered and then analyzed using a Becton Dickinson FACScan or FACSCalibur
For anti-human CD71 labeling of P493 lymphocytes, cells were washed in 1?
PBS and then resuspended in staining buffer (1? PBS, 2% fetal bovine serum)
to a concentration of 2 ? 107cells per ml. Twenty microliters fluorescein
isothiocyanate (FITC)-labeled CD71 antibody (BD Biosciences) was added to
1 ? 106cells (in 50-?l aliquots) and incubated on ice in the dark for 30 min. After
washing with cold 1? PBS, cells were resuspended in 500 ?l 1? PBS and
analyzed by flow cytometry.
mRNA quantitation (real-time PCR). Total RNA was extracted from P493-6
cells using Trizol (Invitrogen) or the RNeasy kit (QIAGEN). Quantitative real-
time PCR expression was performed using the ABI 7700 sequence detection
system. TFRC1, p53, and p21 mRNA expression levels were determined using
predeveloped mixtures of specific probe and primers (PE Applied Biosystems)
and the TaqMan One-step RT-PCR Master Mix kit (PE Applied Biosystems). A
predeveloped probe and primers specific to 18S rRNA levels were used for
normalization. All PCRs were performed in triplicate.
Quantitation of ChIP fragments was performed using the SYBR Green core
reagent kit (PE Applied Biosystems) according to the manufacturer’s instruc-
tions. TFRC1-specific primers were designed using the OMIGA program or
Primer Express software (listed in Table S1 in the supplemental material). PCR
parameters were optimized using the Failsafe Real-time PCR PreMix selection
kit (EpiCentre, Madison, WI). Known quantities of 10-fold dilutions of total
input DNA were used to generate standard curves for each primer pair. Relative
amounts of each ChIP sample (expressed as a percentage of total input) were
determined in the linear range according to their CTvalue. For each primer set,
melting curves were used to verify the correct PCR product.
For microarray validation, mRNA samples were first reverse transcribed and
then subjected to SYBR Green quantitative real-time PCR. Primers were de-
signed to cross exon-exon junctions and span less than 400 bp of the target
mRNA. Primer sequences are shown in Table S6 in the supplemental material.
Expression profiling using DNA microarrays. Seventy-two hours after TFRC1
siRNA transfection, P493 cells were stained with the CD71-FITC antibody as
described above and then sorted into TFRC1-negative and -positive populations
using a Becton Dickinson cell sorter. Approximately 40 independent TFRC1
siRNA transfections were pooled for sorting. Samples from two independent
biological replicates were then processed at the JHMI microarray facility and
used to probe an Affymetrix U133 Plus 2.0 array. Total RNA was isolated from
cells using the RNeasy minikit (QIAGEN). Five micrograms of starting total
RNA from control and experimental cell preparations (two independent repli-
2374 O’DONNELL ET AL.MOL. CELL. BIOL.
cates for each) was processed using single-round RNA amplification protocols,
following Affymetrix specifications (Affymetrix GeneChip Expression Analysis
Technical Manual). Briefly, 5 ?g of total RNA was used to synthesize first-strand
cDNA using oligonucleotide probes with 24 oligo(dT) plus T7 promoter as a
primer (Proligo LLC) and the SuperScript Choice system (Invitrogen). Following
double-stranded cDNA synthesis, the product was purified by phenol-chloroform
extraction, and biotinylated antisense cRNA was generated through in vitro
transcription using the BioArray RNA High Yield transcript labeling kit (ENZO
Life Sciences Inc). Fifteen micrograms of the biotinylated labeled cRNA was
fragmented at 94°C for 35 min (100 mM Tris-acetate, pH 8.2, 500 mM potassium
acetate, 150 mM magnesium acetate), and 10 ?g of total fragmented cRNA was
hybridized to the Affymetrix Human Genome GeneChip array U133Plus 2.0 for
16 h at 45°C with constant rotation (60 rpm). An Affymetrix Fluidics Station 450
instrument was then used to wash and stain the chips, removing the nonhybrid-
ized target and incubating with a streptavidin-phycoerythrin conjugate to stain
the biotinylated cRNA. The staining was then amplified using goat immunoglob-
ulin G as a blocking reagent and biotinylated antistreptavidin antibody (goat),
followed by a second staining step with a streptavidin-phycoerythrin conjugate.
Fluorescence was detected using the Affymetrix GeneChip scanner (GS 3000),
and image analysis of each GeneChip was done through the GeneChip operating
system software from Affymetrix (GCOS1.1.1), using the standard default set-
tings. For comparison between different chips, global scaling was used, scaling all
probe sets to a user-defined target intensity of 150.
To ascertain the quality control of the total RNA from the samples, we used
the Agilent Bioanalyzer, Lab on a Chip technology and confirmed that all the
samples had optimal rRNA ratios and clean run patterns. Likewise, this tech-
nology is used to confirm the quality of the RNA in the form of cRNA and
fragmented cRNA. To assess the quality control of the hybridization, GeneChip
image, and comparison between chips, we confirmed the following parameters:
resulting scaling factor values within comparable range, background within
average, significant percentage of present calls, 3?/5? ratios of glyceraldehyde-3-
phosphate dehydrogenase as a representation of housekeeping genes, and pres-
ence of internal spike controls.
The initial analysis of the expression results was based on pairwise compari-
sons among the different experimental conditions represented by the samples.
Any transcript that showed an arbitrary value of at least a twofold change in
expression level between the experimental sample and the control sample was
considered significantly differentially expressed. All analysis was perfomed using
the Li and Wong method. A lower bound of 2.0 was used as the cutoff for a
significant change in gene expression.
Analysis of transferrin receptor 1 expression in response to
c-Myc in P493-6 cells. We used a previously described human
B lymphocyte cell line, P493-6, that expresses a tetracycline-
repressible c-MYC transgene to characterize c-Myc target
genes. In the presence of tetracycline (Tet), P493-6 cells
exhibit low levels of c-Myc protein, as evidenced by Western
blotting (Fig. 1A) (42). P493-6 cells treated with tetracycline
are small with pale blue cytoplasmic staining, whereas c-Myc
induction leads to intense basophilic cytoplasmic staining
and vacuoles that resemble Burkitt’s lymphoma cells (Fig.
1B). In contrast, tetracycline-treated EREB2-5 cells, from
which P493-6 cells were derived and which lack a Tet-re-
pressible c-MYC allele, resemble untreated cells (Fig. 1B).
We sought to characterize c-Myc-induced phenotypes by
examining alterations of cell surface markers typically used in
clinical flow cytometric phenotyping of lymphomas. Because
P493-6 cells serve as a model for Burkitt’s lymphoma, we
examined markers that have prognostic value to determine if
any were indicative of c-Myc status. We analyzed the expres-
sion of 10 cell surface markers in tetracycline-treated (low
c-Myc) or untreated (high c-Myc) cells. In contrast to other
markers, TFRC1 (also known as CD71 or p90) was highly
upregulated (sevenfold) in response to c-Myc (Fig. 1C).
Using the MYC cancer gene database (www.myccancergene
.org), we found that TFRC1 is responsive to c-Myc in several
previously reported microarray screens (11, 37, 47). Comprehen-
sive analysis of gene expression using the Gene Expression
Atlas database (www.expression.gnf.org) revealed that TFRC1
and c-MYC mRNA levels are expressed similarly in 91 human
tissues and cell lines (52). We performed linear regression
analysis (as described in Methods) on log-transformed mRNA
expression values and found that c-MYC expression levels sig-
nificantly correlated with TFRC1 expression values (r2? 0.334;
P value ?0.00023). We also determined that c-Myc alters the
expression of several well-characterized genes that regulate
iron metabolism in three independent microarray analyses (see
Table S1 in the supplemental material). These results are con-
sistent with previous reports that link c-Myc to iron metabo-
lism and provide further support that c-Myc regulates multiple
components of the iron metabolome (5, 6, 58).
During the course of our study, one report using ChIP
showed an area of binding for c-Myc in the TFRC1 gene in
P493-6 cells (18). However, this study did not detect increased
levels of TFRC1 mRNA at 8 h after c-Myc induction. In con-
trast, we discovered that TFRC1 mRNA levels are significantly
increased 24 h after c-Myc induction in these cells (Fig. 1D).
TFRC1 protein levels were also upregulated in response to
c-Myc (Fig. 1E).
Because TFRC1 expression may reflect the proliferative sta-
tus rather than the c-Myc status of P493-6 cells, we determined
whether c-Myc could induce TFRC1 in serum-deprived cells.
Western blotting confirms TFRC1 induction by c-Myc in the
absence of cell proliferation (Fig. 1F). We also determined
that c-Myc induction increased cell size in the absence of
serum (data not shown).
Since TFRC1 expression is tightly coupled to c-Myc expres-
sion, we sought to determine whether TFRC1 is activated by
c-Myc in vivo. We investigated whether c-Myc regulates
TFRC1 expression levels in a murine B-cell lymphoma model,
which is based on retroviral transduction of p53-null bone
marrow cells with a Myc-encoding retrovirus (25, 59, 61). Us-
ing MycER retroviruses, which express a chimeric protein of
c-Myc and the hormone binding domain of the estrogen re-
ceptor, neoplasms were generated in animals whose exponen-
tial growth was contingent upon continuous administration of
4-hydroxytamoxifen (4-OHT) (13, 60). Growing (Myc ON) and
stagnant (Myc OFF) neoplasms from mice were used to pre-
pare single-cell suspensions of neoplastic B-lymphocytes. Via-
ble neoplastic cells were stained with a TFRC1 antibody, and
expression levels were assessed from two different tumors (Fig.
1G). Following inactivation of c-Myc, we observed a sixfold
downregulation of TFRC1. Thus, maintenance of transferrin
receptor expression is dependent on the presence of active
MycER in vivo.
Phylogenetic footprinting and chromatin immunoprecipita-
tion validate TFRC1 as a direct MYC target gene. Although
TFRC1 mRNA and protein levels are increased by c-Myc,
whether TFRC1 is a direct or indirect c-Myc target gene has
not been fully established. We and others use ChIP to deter-
mine where c-Myc binds within target genes (7, 18, 21, 62). We
previously observed that c-Myc binds to phylogenetically con-
served canonical E boxes, 5?-CACGTG-3?, in the promoter
regions or intron 1 of its target genes (22, 63). A detailed
VOL. 26, 2006 c-Myc TARGET TFRC1 ENHANCES TUMORIGENESIS 2375
2376O’DONNELL ET AL.MOL. CELL. BIOL.
analysis of the genomic locus of the human and murine trans-
ferrin receptor 1 gene was performed to identify conserved
canonical E boxes. Sequences were downloaded using the
UCSC and NCBI databases and analyzed using OMIGA soft-
ware (Oxford Molecular Limited, Oxford, United Kingdom)
(22). An alignment of the human and mouse TFRC1 loci, with
two canonical E boxes found flanking exon 1, is shown in Fig. 2A.
While both E boxes are conserved, only the intron 1 E box is
FIG. 1. TFRC1 Expression is responsive to c-Myc in B cells in vitro and in vivo. (A) Immunoblot analysis of c-Myc expression in P493-6. Cells
were untreated or treated with 0.1 ?g/ml tetracycline for 72 h. (B) Wright staining of P493-6 and EREB2-5 cells in the presence or absence of
tetracycline. (C) FACS analysis of P493-6 cells using a panel of leukemia markers. Histograms represent expression of each cell surface marker
in B cells with high c-Myc (red line; untreated) or low c-Myc (black line; Tet treated) expression levels. (D) Real-time PCR analysis of TFRC1
mRNA expression in untreated or Tet-treated P493-6 cells (left) and in P493-6 cells following induction of c-Myc by Tet removal (right). Bar graphs
represent TFRC1 mRNA expression relative to 18S rRNA control. Error bars represent standard deviations derived from three independent
measurements. (E) Immunoblot analysis of TFRC1 after Tet removal. ?-Tubulin is used as a loading control. (F) Immunoblot analysis for TFRC1
and c-Myc expression in the absence of serum. P493-6 cells were treated with tetracycline for 72 h and then deprived of serum (0.1%) at the same
time as tetracycline withdrawal to induce c-Myc expression. (G) Expression levels of transferrin receptor (CD71) on the surfaces of MycER-
induced B-cell tumors. Top panel, flow cytometric analysis of CD71 expression. Cells were either left unstained (gray lines) or stained with the
anti-CD71 antibody. Myc ON 1 and 2 refer to tumors from two different mice continuously treated with 4-OHT. Myc OFF 1 and 2 refer to tumors
from two different mice initially treated with 4-OHT and then deprived of the hormone for 96 h. Bottom panel, quantitative analysis of CD71
expression. Median expression values plotted on the y axis are differences between median fluorescence intensities of stained and unstained cells.
Error bars represent standard deviations derived from two independent measurements in Myc ON and Myc OFF groups.
FIG. 2. Chromatin immunoprecipitation validates TFRC1 as a direct c-Myc target gene. (A) Sequence alignment of the human and mouse
transferrin receptor genomic locus (from 5 kb upstream of the transcriptional start site through intron 1) reveals two canonical E boxes. The E
box flanked by vertical lines falls within a window of 65% nucleotide identity between human and mouse. PCR amplicons that were analyzed for
c-Myc binding are indicated by numbers. (B) Alignment of the human and mouse sequences of the intron 1 E box (boxed). (C) Quantification of
c-Myc binding by real-time PCR analysis (expressed as a percentage of total input DNA). ChIP was performed on untreated P493-6 cells or cells
treated with Tet for 72 h using anti-Myc antibody or anti-hepatocyte growth factor as a control antibody. Error bars represent standard deviations
derived from three independent measurements. A representative experiment is shown.
VOL. 26, 2006c-Myc TARGET TFRC1 ENHANCES TUMORIGENESIS 2377
contained within a 30-bp window of 65% nucleotide identity, a
criterion we have previously used to infer functional conserva-
tion (Fig. 2B) (22).
We localized c-Myc binding to TFRC1 using scanning ChIP
in P493-6 cells (Fig. 2C) (62). Using real-time PCR quantita-
tion, we obtained evidence for in vivo association of c-Myc
with the genomic region containing the conserved intron 1
CACGTG sequence 516 nucleotides downstream of the tran-
scriptional start site (Fig. 2C, amplicon 4). A decrease in signal
intensity was observed upon scanning away from the canonical
E box to amplicon 5 (approximately 106 bp away). Because
amplicon 3, which contained the canonical E box ?2703 to
?2698 bp upstream of the TFRC1 transcriptional start site, was
extraordinarily amplified from total input DNA, we examined
the sequence of this region and found that it lies within a
repetitive short interspersed nucleotide element. We then
scanned approximately 115 bp upstream to a region just out-
side the short interspersed nucleotide element and observed
that amplicon 2 also demonstrated background levels of bind-
ing to c-Myc. (Primer sequences used in our ChIP experiments
are shown in Table S2 in the supplemental material). These
data suggest that c-Myc binds exclusively to the conserved
canonical E-box sequence in intron 1 of the TFRC1 genomic
region and provide strong evidence that TFRC1 is a direct
c-Myc target gene.
Transferrin receptor 1 is required for cellular proliferation
and cell cycle progression in P493-6 cells. Since TFRC1 ex-
pression is elevated in lymphomas and is regulated by c-Myc,
we sought to determine whether TFRC1 is required for cell
cycle proliferation and cell size control. With the P493-6 sys-
tem in place to study these c-Myc-mediated phenotypes, we
utilized RNAi to inhibit TFRC1 expression in these cells (14).
siRNA duplexes were designed to inhibit expression of TFRC1
and introduced into P493-6 cells by electroporation. After 72 h,
TFRC1 protein levels were diminished by approximately 60%
(Fig. 3A). Inhibition of TFRC1 also resulted in decreased rates
of cell proliferation (Fig. 3B).
Staining of P493-6 cells with a FITC-conjugated anti-TFRC1
antibody following electroporation revealed two cell popula-
tions, one with low TFRC1 expression and one with normal
levels of TFRC1 expression (Fig. 3C), reflecting the transfec-
tion efficiency of this cell line. By double labeling cells with
an anti-TFRC1 antibody and the DNA binding dye 7-amino-
actinomycin, we sought to compare cell cycle profiles of the cell
population with low TFRC1 expression to control cell popula-
tions (Fig. 3D). Inhibition of TFRC1 expression resulted in a
significant G1accumulation (Fig. 3E) (79.18% ? 0.27% com-
pared to 55.72% ? 2.4% of cells in G1for TFRC1-negative
and TFRC1-positive populations, respectively). In contrast,
cell size did not change with TFRC1 gene silencing (Fig. 3F).
Taken together, these data demonstrate that TFRC1 is neces-
sary for progression through the G1-S phase transition and cell
To further characterize the cell cycle arrest phenotype fol-
lowing silencing of TFRC1, we examined mRNAs that are
involved in cell cycle regulation. In order to select for a ho-
mogenous population of cells, we sorted for TFRC1-low-ex-
pressing cells. Real-time PCR quantitation detected a twofold
increase in p21 mRNA levels in TFRC1-low-expressing cells
compared to controls, whereas p53 mRNA levels remained the
same (see Fig. S2 in the supplemental material). Immunoblot
analysis further revealed increased levels of the p53 and p21
proteins, whereas p27 levels remained constant (see Fig. S2
in the supplemental material). We also performed TFRC1
knockdown in the human erythroleukemia K562 cells. Despite
the absence of functional p53 in these cells, TFRC1 siRNA-
treated cells maintain a G1phase arrest (our unpublished
As an additional control, we also silenced the c-Myc target
gene JTV1. Diminished JTV1 expression, however, did not af-
fect P493-6 cell proliferation or cell size (data not shown).
These data provide evidence that TFRC1 expression is neces-
sary for c-Myc-mediated cell proliferation but not cell size
Iron chelation inhibits cell proliferation and cell cycle pro-
gression but not cell size increase in P493-6 cells. In order to
determine whether the cell cycle arrest we observed following
TFRC1 knockdown was secondary to depletion of intracellular
iron pools, we directly diminished intracellular iron by chem-
ical chelation. Desferrioxamine (DFX) is a potent iron chela-
tor that has been shown to inhibit the growth of aggressive
tumors, such as neuroblastoma and leukemia (3, 4, 46) and is
currently used to treat iron-overload disorders (45). By 120 h
after tetracycline removal, cells stimulated by c-Myc in the
presence of DFX failed to proliferate, in contrast to control
cells stimulated by c-Myc alone (Fig. 4A). Cell cycle analysis
revealed that Tet-treated cells are arrested in G1, whereas
induction of c-Myc allows progression of cells through the G1-S
transition. In contrast, DFX-treated cells accumulated in G1
phase at early time points (Fig. 4B). With longer exposure to
DFX (72 to 96 h), the lack of cell cycle progression in the
c-Myc-plus-DFX cells was associated with an increase in cells
undergoing apoptosis. These data demonstrate that short-
term iron deprivation mimics TFRC1 knockdown and inhib-
its cellular proliferation in P493-6 cells. Previous studies
have shown that DFX treatment of neuroblastoma cells
inhibits N-Myc expression (16). We thus sought to deter-
mine whether DFX treatment resulted in nonspecific inhi-
bition of c-Myc or TFRC1 expression. Western blot analysis
confirmed that c-Myc and TFRC1 are expressed normally in
the presence of DFX (Fig. 4C).
Since iron is involved in multiple metabolic pathways and
attaining a threshold cell size in G1is required for cell pro-
liferation, it is plausible that iron deprivation could inhibit
c-Myc-mediated cell size increase. Flow cytometric analysis
revealed that DFX treatment had no significant effect on cell
size (data not shown). Thus, these experiments underscore the
requirement of iron for cellular proliferation and clearly dem-
onstrate that disruption of iron metabolism has a profound
effect on cell cycle progression without affecting cell size.
Moreover, these data highlight the importance of TFRC1 in
maintaining sufficient intracellular iron levels for proliferation.
TFRC1 depletion alters cell cycle and apoptosis-promoting
genes. To globally assess alterations in expression of cell cycle
regulatory genes, we examined expression profiles in TFRC1
siRNA-treated B cells. Our oligonucleotide microarray studies
of TFRC1-low cells revealed that the levels of 849 out of ap-
proximately 10,000 expressed transcripts were altered by de-
creased TFRC1 expression. The 202 downregulated transcripts
included cell division cycle genes, cyclins, and other factors
2378 O’DONNELL ET AL.MOL. CELL. BIOL.
FIG. 3. RNAinterferenceofTFRC1expressionabrogatescellproliferationandcellcycleprogression.(A)ImmunoblotanalysisofTFRC1levels72h
after transfection with siRNA oligonucleotides in unsorted cell populations. ?-Tubulin is used as a loading control. (B) Growth rates are diminished after
represent standard deviations from two independent measurements. At least three independently performed experiments yielded similar results.
(C) FACS analysis of anti-TFRC1-FITC labeled cells 72 h after transfection with siRNA oligonucleotides. (D) Cell cycle profiles after knockdown of
TFRC1. Cells were double labeled with 7-amino-actinomycin and an anti-TFRC1-FITC antibody. (E) Bar graphs representing mean values and standard
deviations of each cell cycle phase derived from three independent experiments. (F) Cell size remains unaffected after RNAi-mediated suppression of
TFRC1. Forward light scattering (FSC) histograms obtained by FACS analysis are shown.
VOL. 26, 2006 c-Myc TARGET TFRC1 ENHANCES TUMORIGENESIS2379
FIG. 4. Iron depletion inhibits c-Myc-mediated cell proliferation and cell cycle progression. (A) Growth rates of P493-6 cells grown in the
presence or absence of 100 ?M DFX after tetracycline withdrawal. (B) Cell cycle profiles obtained by propidium iodide labeling of cells stimulated
by c-Myc in the presence or absence of DFX. c-Myc stimulation results in cell cycle progression. In contrast, DFX-treated cells accumulate in G1
at early time points and subsequently undergo apoptosis at later time points. Bar graphs represent the percentage of cells in each phase of the cell
cycle. Results of a representative experiment are shown. One independent experiment yielded similar results. (C) Immunoblot analysis of c-Myc
and TFRC1 after DFX treatment. ?-Tubulin is used as a loading control. Error bars for all panels represent standard deviations derived from at
least two independent experiments.
2380 O’DONNELL ET AL.MOL. CELL. BIOL.
required for DNA replication. The 647 upregulated transcripts
include p53-responsive and apoptosis-promoting genes. A
summary of the various gene categories affected by TFRC1
inhibition is shown in Table 1.
To analyze our microarray data set, we utilized Expression
Analysis Systematic Explorer (EASE), a gene ontology soft-
ware application (24). Each biologic theme is determined from
a list of genes and assigned an EASE score, a measure of
statistical significance that is similar to a one-tailed Fisher
exact probability test. Remarkably, each of the top 15 gene
categories downregulated by inhibition of TFRC1 is associated
with the regulation of the cell cycle (see Table S3 in the sup-
plemental material). Using an EASE score cutoff of 1.84E-07,
the annotated list of down-regulated genes that fall within a
cell cycle regulation category is shown in Table S4 in the
supplemental material. Analysis of upregulated genes using
EASE further revealed that many fall into apoptotic and pro-
grammed cell death categories (see Table S5 in the supple-
mental material). This is consistent with the observed increase
in apoptotic cells after prolonged iron deprivation in P493-6
cells (Fig. 4B).
To validate our microarray results, we performed quantita-
tive real-time PCR analysis to measure expression of diverse
genes that are p53 targets or are involved in cell cycle control
or apoptosis (see Table 1, last column; also see Fig. S3 in the
supplemental material). In summary, 13 out of 15 tested genes
from our microarray experiments showed concordant expres-
sion changes by real-time PCR. (Primer sequences used in our
microarray confirmation are shown in Table S6 in the supple-
To further delineate the role of TFRC1 within the c-Myc
target gene network, we sought to compare our microarray
data set described above with that of all c-Myc-responsive
genes in P493-6 cells. To this end, we performed expression
profiling with P493-6 cells under conditions of high versus low
c-Myc expression (using untreated versus Tet-treated cells). Out
of approximately 10,000 expressed transcripts, 2,679 genes were
c-Myc responsive in P493-6 cells (29). Of these c-Myc-responsive
genes, 175 were also altered by TFRC1 knockdown. Upon
further analysis of these 175 genes, 59 c-Myc-induced genes are
downregulated by TFRC1 inhibition (see Table S7B in the
supplemental material). Consistent with our previous observa-
tion that TFRC1 depletion resulted in a G1arrest but did not
affect cell size, many of the genes that responded to both c-Myc
and TFRC1 levels are involved in cell cycle regulation. In fact,
EASE analysis reveals that the “mitotic cell cycle” is the most
statistically significant gene category (with a score of 3.92E-14)
from this list. The remaining c-Myc-responsive genes, includ-
ing those involved in metabolism and ribosomal biogenesis,
were predominantly unaffected by TFRC1 knockdown. Exam-
ples of these genes are represented in Table S8 in the supple-
Ectopic TFRC1 expression confers a growth advantage in
limiting serum conditions. Having demonstrated that TFRC1
is necessary for cellular proliferation, we next sought to deter-
mine whether TFRC1 overexpression is sufficient to provide a
growth advantage under conditions with limiting growth fac-
tors (including transferrin) that may reflect the physiologic
milieu of cancers (49, 57). We reasoned that cells grown in
limiting 1% serum conditions may mimic the physiologic con-
TABLE 1. Transcripts responsive to TFRC1 knockdowna
Class Gene product (gene name)
Cell cycle Cell division cycle 20 (CDC20)
Cyclin B1 (CCNB1)
Cyclin A2 (CCNA2)
Cell division cycle (CDC2)
Growth arrest and DNA damage-inducible 45 alpha (GADD45A)
Cyclin-dependent kinase inhibitor 1A (CDKN1A)
Inhibitor of DNA binding 2 (ID2)
Apoptosis BCL2 binding component 3 (BBC3/PUMA)
Annexin A4 (ANXA4)
Annexin A1 (ANXA1)
Tumor necrosis factor receptor superfamily, member 6 (TNFRSF6)
p53 regulatedCyclin-dependent kinase inhibitor 1A (CDKN1A)
p53 target zinc finger protein (WIG1)
BCL2 binding component 3 (BBC3/PUMA)
TP53-activated protein 1 (TP53AP1)
Tumor protein p53-inducible nuclear protein 1 (TP53INP)
DNA replicationDNA2 DNA replication helicase 2-like (DNA2L)
Topoisomerase (DNA) II alpha (TOP2A)
Polymerase (DNA directed), epsilon 2 (POLE2)
Chromosome organizationBUB1 budding uninhibited by benzimidazoles 1 homolog (BUB1)
Centromere protein A (CENPA)
Centromere protein F (CENPF)
aFold change represents expression difference between scrambled control siRNA-treated cells and TFRC1 (low)-depleted cells.
VOL. 26, 2006c-Myc TARGET TFRC1 ENHANCES TUMORIGENESIS2381
text of tumor growth, where cells must compete for limiting
growth factors and nutrients. We chose to study wild-type Rat1
TGR fibroblasts, c-Myc null Rat1 cells, and null cells that have
been reconstituted with a hypomorphic mutant MYC W135E
allele (23, 33, 41). We first determined the expression of
TFRC1 in c-MYC null cells (HO15.19), compared to the wild
type (TGR), and null cells reconstituted with murine leukemia
virus-driven wild-type c-MYC (HO15.19-MYC). We found that
TFRC1 expression is greatly diminished in null fibroblasts,
demonstrating that c-Myc is necessary for full physiologic ex-
pression of TFRC1 (Fig. 5A).
To assess whether ectopic expression of TFRC1 affected the
growth of wild-type or knockout cells, we introduced a human
TFRC1-expressing vector or empty vector alone into both cell
lines by retroviral transduction. Western blot analysis confirms
retroviral expression of TFRC1 (Fig. 5B). Anti-TFRC1 stain-
ing by flow cytometry verified functional TFRC1 expression at
the cell surface (data not shown).
Using labeled transferrin (Tf), we found a direct correlation
between TFRC1 expression and the functional uptake of Tf
in c-MYC null and wild-type cells (Fig. 5C). Quantitation of
labeled-transferrin uptake revealed that the magnitude of Tf
uptake is 4.7-fold greater in wild-type cells than in c-MYC
knockout cells. Additionally, we observed that transferrin up-
take increased by 3.3-fold in c-MYC knockout cells expressing
ectopic TFRC1 (Fig. 5C).
It was previously reported that reconstitution of c-MYC null
cells with a hypomorphic mutant MYC W135E allele resulted in
an intermediate growth rate between those of wild-type cells and
c-MYC null cells (41). Intriguingly, TFRC1 expression was dimin-
ished in W135E cells compared to that in wild-type rat fibroblasts
(data not shown). W135E cells expressing a TFRC1 or control
retrovirus were generated as demonstrated by Western blotting
(Fig. 5B, right panel). Despite the ability to increase levels of
transferrin uptake, TFRC1 overexpression was insufficient to res-
cue the slow growth rates of these cells in normal (10%) serum
culture conditions (Fig. 5D, top panel). However, ectopic TFRC1
expression significantly enhanced the growth rates not only of
W135E c-MYC mutant cells but also of wild-type cells when
grown in limiting (1%) serum conditions (Fig. 5D, bottom panel).
These data suggest that upregulation of TFRC1 by c-Myc may be
advantageous to cells growing in limiting nutrients, the typical
environment of tumor cells.
TFRC1 promotes tumorigenicity in Rat1a-Myc cells. Last,
we tested directly whether TFRC1 could enhance the rate of
tumorigenesis in a well-characterized model system, Rat1a fi-
broblasts overexpressing c-Myc. Rat1a cells are immortalized,
nontransformed fibroblasts that do not form tumors when in-
jected subcutaneously into nude mice. Ectopic expression of
select oncogenes, including c-Myc, results in cellular transforma-
tion as indicated by their ability to form tumors in nude mice and
promote the anchorage-independent growth of colonies in soft
agar (50, 51). We observed that TFRC1 is expressed at approxi-
mately twofold-higher levels in Rat1a-Myc cells than in Rat1a
cells (Fig. 6A).
In order to determine whether TFRC1 cooperates with c-
Myc in promoting tumorigenesis, we generated a stable cell
line with enforced TFRC1 expression in Rat1a-Myc cells.
Western blot analysis confirms retroviral expression of TFRC1
in these cells, which already express high levels of endogenous
TFRC1 (Fig. 6B). We examined the growth rates of these cells
under normal (10%) and limiting (1%) serum culture condi-
tions. Consistent with our previous results using W135E mu-
tant and TGR wild-type rat fibroblasts, enforced TFRC1 ex-
pression enhanced the growth rates of Rat1a-Myc cells under
limiting conditions (see Fig. S4A in the supplemental mate-
rial). We then subcutaneously injected Rat1a-Myc (Empty)
and Rat1a-Myc (TFRC1) cells into 4-week-old nude mice and
assessed tumor volume over time. As demonstrated in Fig. 6C,
Rat1a-Myc cells with ectopic TFRC1 expression exhibited a
statistically significant increase in the rate of tumorigenesis
compared to Rat1a-Myc (Empty) cells (P values ? 0.00335,
0.00248, and 0.01472 at days 27, 30, and 35, respectively).
These results provide compelling evidence that TFRC1 en-
hances tumorigenesis in this system.
TFRC1 is a critical gene in the c-Myc target gene network.
c-Myc is one of the most frequently dysregulated proteins in
human malignancies, underscoring the importance of achiev-
ing a more complete understanding of the mechanisms
through which this oncoprotein drives tumorigenesis. The fact
that human or Drosophila c-Myc regulates between 10 and
15% of genes presents a formidable challenge. Thus, it is
critical to identify downstream targets and pathways that are
essential for each of its biologic functions in both normal and
The results reported above lead to three main conclusions
relevant to the role of TFRC1 in the c-Myc target gene net-
work and B-cell lymphoma. First, we have demonstrated that
TFRC1 is a direct transcriptional target of the c-MYC proto-
oncogene. This likely explains why TFRC1 is commonly over-
expressed in tumors. Second, TFRC1 is necessary for B-lympho-
cyte proliferation based on experiments using RNA interference
to inhibit its expression. Enforced TFRC1 expression also confers
a distinct growth advantage to cells grown under limiting serum
conditions, suggesting that upregulation of this pathway by c-
Myc promotes cell proliferation under conditions that are anal-
ogous to the environment of a tumor cell. Third, we present
compelling evidence that TFRC1 promotes tumorigenesis in
Rat1a fibroblasts overexpressing c-Myc.
Our knockdown experiments and microarray studies re-
vealed that many cell cycle regulatory genes and downstream
p53 target genes are affected by reduction of TFRC1 expres-
sion. Since iron depletion is known to affect many cellular
processes, the underlying mechanism for these alterations in
gene expression and the observed cell cycle arrest is likely
complex and dependent on multiple pathways. One possible
mechanism contributing to p53 activation is inhibition of ribo-
nucleotide reductase (RR), an iron-dependent enzyme re-
quired for DNA replication. Treatment with the RR inhibitor
hydroxyurea has been shown to cause a p53-dependent G0/G1
arrest (31). Similarly, knockdown of TFRC1 may render RR
inactive by depriving cells of adequate intracellular iron, thus
depleting deoxyribonucleotide pools and activating p53. Con-
sistent with this model, we observed increased levels of p53
protein but not mRNA, as is known to occur following inhibi-
tion of DNA synthesis.
2382 O’DONNELL ET AL.MOL. CELL. BIOL.
FIG. 5. Ectopic TFRC1 expression in Rat1 fibroblasts confers growth advantage in limiting (1%) serum conditions. (A) Immunoblot analysis
of endogenous TFRC1 expression in c-MYC?/?(HO15.19), c-MYC?/?(TGR), and c-MYC?/?cells reconstituted with human c-Myc (?/? Myc)
Rat1 fibroblasts. ?-Tubulin is used as a loading control. (B) Immunoblot analysis demonstrating TFRC1 overexpression in c-MYC null (HO15.19),
wild-type (TGR), and W135E mutant cells infected with a human TFRC1 retrovirus. ?-Tubulin is used as a loading control. (C) Transferrin uptake
in ?/? (TGR) and ?/? (HO15.19) cells and in ?/? cells infected with an empty vector or a TFRC1 retrovirus. Quantitation was determined by
FACS analysis after incubating cells with fluorescence-labeled transferrin for 30 min. Fold changes shown refer to median Tf uptake levels
compared to those for unlabeled cells. The bar graph represents mean values and standard deviations derived from four independent experiments.
(D) Growth rates under normal (10%) and limiting (1%) serum conditions. Error bars represent standard deviations derived from three
independent measurements. The graph is representative of two independent experiments.
Based on our experiments with K562 lymphocytes which lack
wild-type p53, cell cycle arrest persists in TFRC1 siRNA-
treated cells. These studies reveal that although induced if
present (as in P493-6 cells), wild-type p53 is not required for
the G1phase arrest in response to TFRC1 inhibition. These
data support the idea that iron depletion has pleiotropic effects
on the cell cycle regulatory apparatus. Nevertheless, our find-
ings firmly establish that TFRC1 is a critical downstream target
of c-Myc that is necessary for cellular proliferation and pro-
motes tumorigenesis. Moreover, analysis of gene expression
changes following TFRC1 depletion in the context of all genes
that are responsive to c-Myc has allowed us to pinpoint the role
of TFRC1 within the c-Myc target gene network as a whole.
This type of analysis should also prove useful for identifying
specific pathways affected by other c-Myc target genes.
Our microarray data also revealed that in addition to
TFRC1, c-Myc controls several well-characterized genes
that either regulate iron metabolism or require iron for their
normal function (see Table S1 in the supplemental mate-
rial). These include divalent metal transporter 1 (DMT1 or
Nramp2), which transports iron out of endosomes into the
cytoplasm, and succinate dehydrogenase B, which catalyzes the
oxidation of succinate to fumarate during the citric acid cycle.
Frataxin, which encodes a mitochondrial membrane protein
with putative roles in iron-sulfur cluster synthesis and mito-
chondrial iron metabolism, was also upregulated by c-Myc.
These results provide further support that c-Myc regulates
multiple components of the iron metabolome. Future studies
are necessary to determine the contribution of these additional
factors to c-Myc-mediated proliferation and transformation.
TFRC1 as a clinical marker and therapeutic target for hu-
man cancer. For many years, clinical studies have unknowingly
tumors.67Gallium uptake, now known to be through TFRC1, has
been used clinically to image lymphomas in patients (17, 55).
Since c-Myc is known to play an important role in lym-
FIG. 6. TFRC1 enhances tumorigenesis in Rat1a-Myc cells. (A) Western blot analysis of TFRC1 in Rat1a and Rat1a-Myc cells. ?-Tubulin is
used as a loading control. (B) Western blot analysis demonstrating TFRC1 overexpression in Rat1a-Myc cells. ?-Tubulin is used as a loading
control. (C) Analysis of tumor volume in nude mice. A two-tailed Student t test (type 3, unequal variance) was used to determine whether the
difference between groups was statistically significant. A representative experiment is shown (n ? 15 mice for each group). One independent
tumorigenesis experiment yielded similar results.
2384O’DONNELL ET AL.MOL. CELL. BIOL.
phomagenesis and both c-Myc and TFRC1 have been linked to
the development of aggressive diffuse large B-cell lymphoma
(2, 32), it is not surprising that TFRC1 has served as a useful
Moreover, several lines of evidence demonstrate that TFRC1
expression levels are indicative of c-Myc expression. TFRC1 is
among a select group of genes that are overexpressed in a murine
c-Myc-induced prostate cancer model and in primary human
prostate cancers (15). Numerous expression profiling studies have
lung, colon, and skin (44). A recent study using a correlation-
based model to characterize the gene interaction network of
MycER-stimulated cells identified TFRC1 as one such c-Myc-
activated gene (43a). Our studies, however, not only are cor-
relative but also reveal a direct link between c-MYC expression
and TFRC1 expression. Our experiments also demonstrate for
the first time that TFRC1 can enhance in vivo tumor growth by
cooperating with c-Myc in Rat1a fibroblasts.
The accessibility of TFRC1 as a membrane receptor has
made it an attractive target for cancer therapy. Past therapeu-
tic studies with anti-TFRC1 antibodies have met with limited
clinical success, perhaps due to the lack of appropriate pheno-
typing for TFRC1. More recently a monoclonal TFRC1 anti-
body, A24, was shown to induce apoptosis in T lymphocytes
from patients with CD71-positive T-cell leukemias (26, 34, 53).
In fact, these studies are consistent with our microarray data
where we identified apoptotic genes upregulated in response to
TFRC1 inhibition in P493-6 B lymphocytes. Thus, our studies
provide experimental support for anticancer therapies that tar-
get TFRC1. Future efforts to delineate the molecular mecha-
nisms underlying the cell cycle arrest which occurs following
TFRC1 depletion may allow the development of other thera-
pies that complement the interruption of iron uptake in the
treatment of cancers.
In conclusion, we have demonstrated that the transferrin
receptor is a direct c-Myc target gene that is necessary for
c-Myc-mediated cell cycle progression but not cell size in-
crease. By conferring a significant growth advantage on cells
growing in limiting nutrients, activation of TFRC1 contributes
to the ability of c-Myc to reprogram the metabolic state of the
cell such that growth in the severe environment of a tumor is
promoted. Further definition of the c-Myc transcription factor-
target gene network through the identification of other prin-
cipal tumor-promoting targets will advance our understanding
and perhaps control of the transformation process.
We thank D. Eick, J. Sedivy, L. Penn, and J. Basilion for valuable
cell lines and reagents, D. Warren and Y. Yang for assistance with
electroporation, L. Blosser and A. Tam for flow sorting, D. Arking for
statistical analysis, and F. Martinez Murillo and C. Jie for microarray
hybridization and analysis. We also thank J. Mendell, L. Gardner, M.
McDevitt, R. Osthus, and L. Lee for critical reading of the manuscript.
This work was supported by NCI grants CA57341 (C.V.D.),
CA097932 (A.T.-T.), and CA102709 (A.T.-T.) and NIH-NHLBI grant
T32HL007525 (K.A.O.). C.V.D. is the Johns Hopkins Family Profes-
sor in Oncology Research. J. Kim is a Howard Hughes Predoctoral
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