Download The Nuclear Factor B Subunits RelA/p65 and c

[CANCER RESEARCH 64, 7248 –7255, October 15, 2004]
The Nuclear Factor ␬B Subunits RelA/p65 and c-Rel Potentiate but Are Not
Required for Ras-Induced Cellular Transformation
Julie L. Hanson,1,2 Noel A. Hawke,1 David Kashatus,1,2 and Albert S. Baldwin1,2,3
1
Lineberger Comprehensive Cancer Center, 2Curriculum in Genetics and Molecular Biology, and 3Department of Biology, University of North Carolina, Chapel Hill, North
Carolina
ABSTRACT
Extensive data indicate that oncoproteins, such as oncogenic H-Ras,
initiate signal transduction cascades that ultimately lead to the activation
of specific transcription factors. We and others have previously demonstrated that Ras activates the inherent transcriptional activation function
of the transcription factor nuclear factor ␬B (NF-␬B). Supportive of the
importance of NF-␬B in transformation, Ras-induced cellular transformation can be suppressed by expression of I␬B␣, an inhibitor of NF-␬B,
or by dominant-negative forms of the upstream activator I␬B kinase
(IKK). However, conclusive evidence for a requirement for NF-␬B subunits in oncogenic transformation has not been reported. Furthermore,
there is little understanding of the gene targets controlled by NF-␬B that
might support oncogenic conversion. The data presented here demonstrate that, although both p65 and c-Rel enhance the frequency of Rasinduced cellular transformation, these NF-␬B subunits are not essential
for Ras to transform spontaneously immortalized murine fibroblasts.
Microarray analysis identified a set of genes induced by Ras that is
dependent on NF-␬B for their expression and that likely play contributory
roles in promoting Ras-induced oncogenic transformation.
INTRODUCTION
Proteins of the Ras family function as important signaling molecules in a variety of cellular processes, including growth, differentiation, and survival (1). Approximately 30% of human tumors have
mutations in Ras alleles that result in constitutively active proteins (2).
The oncogenic activation of Ras ultimately leads to the chronic
stimulation of signaling cascades that promote activation of transcription factors involved in controlling proliferation, differentiation, and
apoptosis (1). As an example, experiments have demonstrated that the
transcription factor nuclear factor ␬B (NF-␬B) is activated by oncogenic Ras. Expression of interfering proteins such as the inhibitor of
NF-␬B (I␬B␣) or dominant-negative forms of I␬B kinase (IKK)
suppresses Ras-induced transformation in vitro (3– 6), suggesting the
involvement of NF-␬B in the transformation process induced by
oncogenic Ras expression.
NF-␬B represents a family of dimeric transcription factors that are
characterized by a 300-amino-acid region called the Rel homology
domain (7–9). The five mammalian NF-␬B family members are
RelA/p65, RelB, c-Rel, p50/p105, and p52/p100, with the RelA/p65
and c-Rel subunits exhibiting strong transactivation potential. In unstimulated cells, I␬B proteins localize NF-␬B dimers to the cytoplasmic compartment through masking of nuclear localization sequences
of NF-␬B subunits. Activation of NF-␬B can occur through various
stimuli, including inflammatory cytokines and bacterial and viral
infection, as well as oncogenic signals (7–9). Activation of NF-␬B,
Received 12/16/03; revised 7/15/04; accepted 8/10/04.
Grant support: Research support was provided by NIH grants RO1 CA73756 and
RO1 CA75080 (A. Baldwin), and CA096418 (N. Hawke). We also gratefully acknowledge the Waxman Cancer Research Institute for research support.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Albert Baldwin, Lineberger Comprehensive Cancer Center,
CB 7295, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 966-3652;
Fax: (919) 966-0444; E-mail: [email protected].
©2004 American Association for Cancer Research.
through receptors such as tumor necrosis factor receptor 1 (TNFR1),
interleukin 1 receptor (IL-1R), or various Toll-like receptors, initiates
signal transduction cascades ultimately leading to activation of the
I␬B kinase (IKK) complex. The IKK complex is composed of three
subunits, IKK␣, IKK␤ and IKK␥ (7–9). Both IKK␣ and IKK␤ have
inducible catalytic activity, whereas IKK␥ is a regulatory subunit.
Following its activation, the IKK complex phosphorylates I␬B␣ and
I␬B␤ at specific serine residues, which targets I␬B for ubiquitination
and degradation by a proteasome-dependent pathway. After I␬B degradation, the unmasked nuclear localization sequence of NF-␬B allows nuclear accumulation that, in turn, promotes sequence-specific
DNA binding and transcriptional activation of target genes (9).
Although nuclear accumulation of NF-␬B is a key step in the
overall activation process controlled by receptor-dependent activation,
posttranslational modifications on RelA/p65 also appear to be necessary for the transcriptional competence of NF-␬B. One such modification, phosphorylation on serine 276, has been shown to be required
for stable interactions between RelA/p65 and the transcriptional coactivator CBP and for transcriptional activation of certain NF-␬B
target genes (10, 11). Additional sites of phosphorylation have also
been described that appear to contribute to the inherent transcriptional
activity of NF-␬B (12–14). Additionally, Akt, which functions downstream of Ras and phosphatidylinositol 3⬘-kinase (PI3K), can control
transcriptional activation function through a mechanism dependent on
IKK function and on RelA/p65 phosphorylation, but in a manner that
does not promote enhanced nuclear accumulation of NF-␬B (15, 16).
Consistent with this, we have shown that both oncogenic Ras and
oncogenic Raf activate an NF-␬B-dependent reporter in mouse fibroblasts without inducing DNA binding of NF-␬B as measured by gel
mobility shift assay (4, 6). It is noted that human epithelial cells,
transformed with oncogenic Ras, exhibit enhanced NF-␬B DNAbinding activity in association with I␬B␣ degradation (3).
Certain transcription factors, including Ets and NF-␬B proteins,
have been shown to be important for transformation in response to
oncoprotein expression (4, 17, 18); however, it was unclear whether
transforming proteins elicit a full range of transcription factor-dependent gene expression or whether a limited set of transcription factorregulated genes are induced during transformation. Recently, we
demonstrated that oncogenic Ras, although stimulating a NF-␬Bdependent reporter, suppressed the ability of TNF to activate NF-␬B
in murine fibroblasts (19). The mechanism of suppression of NF-␬B
involved Ras-controlled inhibition of TNF-induced IKK activation
and I␬B degradation. These studies revealed that Ras suppressed the
ability of TNF to activate known NF-␬B-regulated genes such as
iNOS, indicating that, although Ras can stimulate NF-␬B functional
activity, it does so in a selective manner.
Although it is suggested that NF-␬B plays a role in Ras-induced
transformation, direct genetic evidence is missing. We previously
reported that Ras stimulates the activity of the NF-␬B RelA/p65
transcriptional activation domain (5, 6). Although a specific role for
c-Rel in Ras transformation has not been established, c-Rel does have
documented transforming potential. Thus, c-Rel has been shown to
transform chicken lymphoid cells (20) and recently mouse mammary
tumor virus (MMTV)-c-Rel transgenic mice were shown to develop
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NF-␬B SUBUNITS POTENTIATE RAS TRANSFORMATION
mammary tumors with a high frequency (21). In the present study, we
have addressed the roles of the RelA/p65 and c-Rel subunits in
controlling Ras-induced cellular transformation. Somewhat surprisingly, neither RelA/p65 nor c-Rel is absolutely required for Rasinduced transformation of immortalized murine fibroblasts. Despite
this, each of these NF-␬B subunits potently enhances the efficacy of
oncogenic transformation induced by Ras. We have extended these
studies to identify a set of genes induced by Ras that require either p65
or c-Rel for their expression and presumably encode proteins that
potentiate the Ras-induced transformation response.
MATERIALS AND METHODS
contamination. Reverse transcription-PCR (RT-PCR) was then preformed with
the Qiagen One-Step RT-PCR kit with gene-specific primers for H-Ras and
␤-actin. In a total volume of 25 ␮L, 0.5 ␮g of total RNA was used to analyze
H-Ras expression, and 0.01 ␮g was used to determine the level of ␤-actin
expression.
Real-time PCR. Total RNA was isolated with the Promega SV total RNA
kit. Two ␮g of RNA was reverse transcribed with random hexamers and
MMLV-RT (Invitrogen) according to the manufacturer’s protocol. The cDNA
was then diluted 1:5 and was used for quantitative PCR. Primers were designed
for each gene with consideration of intron/exon borders to favor only mRNAbased PCR products. Each PCR reaction contained 2 ␮L of cDNA, a 2 ␮mol/L
concentration of each primer, and Qiagen QuantiTect Syber Green PCR master
mix in a 20-␮L total volume. PCR was preformed in an ABI Prism 7900
sequence detection system (Applied Biosystems, Foster City, CA). All of the
reactions were preformed in triplicate and were normalized to the number of
copies of 18S detected in each sample.
Affymetrix Microarray and Identification of NF-␬B-Regulated Genes.
Seven ␮g of total RNA were used to synthesize cDNA. A custom cDNA kit
from Invitrogen was used with a T7-(dT)24 primer for this reaction. Biotinylated cRNA was then generated from the cDNA reaction with the BioArray
High Yield RNA Transcript kit. The cRNA was then fragmented in fragmentation buffer [5⫻ fragmentation buffer: 200 mmol/L Tris-acetate (pH 8.1), 500
mmol/L KOAc, 150 mmol/L MgOAc] at 94°C for 35 minutes before the chip
hybridization. Fifteen ␮g of fragmented cRNA were then added to a hybridization cocktail (0.05 ␮g/␮L fragmented cRNA, 50 pmol/L control oligonucleotide B2, BioB, BioC, BioD, and cre hybridization controls, 0.1 mg/ml
herring sperm DNA, 0.5 mg/mL acetylated bovine serum albumin, 100
mmol/L 2-(N-morpholino)ethanesulfonic acid (MES), 1 mol/L Na⫹, 20
mmol/L EDTA, 0.01% Tween 20). Ten ␮g of cRNA were used for hybridization. Arrays were hybridized for 16 hours at 45°C in the GeneChip Hybridization Oven 640. The arrays were washed and stained with R-phycoerythrin streptavidin in the GeneChip Fluidics Station 400. After this, the
arrays were scanned with the Hewlett Packard GeneArray Scanner. Affymetrix
GeneChip Microarray Suite 5.0 software was used for washing, scanning, and
basic analysis. The results of these analyses were input into GeneSpring
(Silicon Genetics, Redwood City, CA, http://www.silicongenetics.com/cgi/
Products/GeneSpring?index.smf)4 for comparative analysis of expression. The
data were “double polished,” i.e., each chip was normalized with a distribution
of all of the genes around the 50th percentile (arbitrarily a value of 1), and each
individual oligonucleotide, or spot, was normalized across the eight samples by
dividing the individual value at each spot by the average of the eight samples.
The polished data set was reduced to only those genes that indicated at least
four present or marginal calls of the eight samples. The data set was also
filtered for absolute, nonnormalized values, of at least 10 (note 0.1 equals 0 or
less signal detected) to 21,523 provide an indication of the range of abundances. Subsequent filters for errors (accepting ⬍1 SD between the replicates
in two of four conditions) and confidence (accepting only probe sets with t test
P values ⬍ 0.5 in two of four conditions) yielded the experimental data set.
Applying a final filter for at least a 3-fold change in expression between any
two conditions resulted in the final set of dynamically regulated genes, which
was then used for cluster analysis with the GeneSpring software. Various
algorithms were used to define clusters of genes with similar expression
patterns. The cluster of genes reported was identified as Ras induced and
NF-␬B dependent (correlation coefficient of 0.95).
Cell Culture and Retroviral Infections. Spontaneously immortalized
mouse embryo fibroblasts (MEFs)—wild-type (MEF⫹/⫹), p65⫺/⫺, cRel⫺/⫺, or
p65⫺/⫺/cRel⫺/⫺ [p65/cRel double knockout (DKO)]—were maintained in
DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% donor calf serum
(Invitrogen) and penicillin-streptomycin. MEF lines, stably expressing HRasV12 or vector control, were obtained by infecting various MEF lines with
a retrovirus-encoding oncogenic H-Ras or with vector control virus and then
selecting with 2 ␮g/mL puromycin. Retrovirus was prepared by cotransfecting
293T cells with pVPack-Eco, pVPack GP (Stratagene) and either pBabe-Puro
or pBabe H-RasV12. Twenty-four hours posttransfection, the medium on the
293T cells was changed to DMEM containing 2% fetal bovine serum, and the
cells were transferred to a 32°C, 5% CO2 environment. Twenty-four and
forty-eight hours later, the supernatant was collected, filter sterilized, and used
to infect target cells. Retroviral infection of target cells was performed by
incubating 1 ⫻ 105 target cells with 1 mL of virus, 1 mL of complete medium
with 8 ␮g/mL Polybrene for 2 to 3 hours and then adding 3 mL of complete
medium and incubating the cells for 20 hours. Incubation of the target cells
with retrovirus was then repeated approximately 24 hours after the initial
infection. Forty-eight hours after the second infection, the cells were either
split into puromycin selection for stable cell line or were harvested for RNA.
Focus Formation Assays. All MEF cell lines were plated at 4 ⫻ 105
cells/60-mm plate 24 hours before transfection. The MEFs were then transfected with the indicated amounts of DNA with GenePorter2 (Gene Therapy
Systems) according to the manufacturer’s protocol. Twenty-four hours posttransfection, the medium was changed and then was changed again every 3 to
4 days for two weeks. After 2 weeks, the cells were fixed with 10% acetic acid
and 10% methanol and then were stained with a 4% crystal violet-10%
methanol solution.
Soft Agar Assays. MEF lines, stably maintaining the vector control or
expressing H-RasV12, were suspended in a 0.33% bactoagar/DMEM layer at
a concentration of 2 ⫻ 104 cells/mL and were plated over a 0.5% bactoagar/
DMEM layer in 6-well plates. Two weeks after plating the cells, the wells were
analyzed for soft agar growth.
Western Blot Analysis. Western blot analysis was performed by preparing
whole cell extracts and then separating 30 to 50 ␮g of total protein by
SDS-PAGE. After transferring the separated proteins to nitrocellulose, blots
were blocked in Tris-buffered saline-Tween (TBST) with 5% milk and then
were incubated in primary antibody [p65 (Rockland), c-Rel, tubulin (Santa
Cruz Biotechnology, Santa Cruz, CA)] for either 1 to 2 hours at room
temperature or overnight at 4°C. The membranes were then washed in TBST
and were incubated for 1 hour in antirabbit horseradish peroxidase-conjugated
secondary antibody (Promega, Madison, WI) and were washed again. Protein RESULTS
bands were visualized with enhanced chemiluminescence detection (Amersham Life Science).
H-RasV12 Induces Transformation of p65ⴚ/ⴚ and c-Relⴚ/ⴚ
Northern Blot Analysis. Total RNA was isolated with Trizol (Invitrogen) MEFs. Previous studies examining the involvement of NF-␬B in
according to the manufacturer’s protocol. Ten ␮g of RNA were then electro- Ras-induced transformation used dominant-negative forms of IKK or
phoresed on a 1.5% formaldehyde-agarose gel and were transferred to a nylon
the I␬B-super repressor (SR), the trans-dominant-negative inhibitor of
membrane according to standard procedures. The RNA was then cross-linked
NF␬B (3, 4). These interfering proteins either inhibit upstream sigto the membrane by UV irradiation (Stratagene) and were probed with a
randomly labeled gene specific probe. Hybridization and washing were pre- naling to a variety of NF-␬B dimers or block activation of any NF-␬B
formed with ExpressHyb (Stratagene, La Jolla, CA) according to the manu- subunit that will associate with I␬B family members. Use of dominant-negative IKK or I␬B-SR does not allow for the determination of
facturer’s protocol.
Reverse Transcription-PCR. Total RNA was isolated with Trizol (In4
vitrogen) according to the manufacturer’s protocol. Four ␮g total RNA were
Internet address: http://www.silicongenetics.com/cgi/Products/GeneSpring?index.
then treated with RNase-free DNase (Invitrogen) to eliminate any DNA smf.
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NF-␬B SUBUNITS POTENTIATE RAS TRANSFORMATION
a potential role of specific NF-␬B subunits in the transformation
process. Additionally, overexpression of I␬B␣ or I␬B-SR can have
effects on cells that are independent of NF-␬B (22, 23). Therefore, to
determine whether NF-␬B subunits, specifically p65 and c-Rel, have
essential roles in Ras-induced transformation, we used genetic knockout-derived MEFs. Spontaneously immortalized MEFs that lacked the
p65 or c-Rel subunit of NF-␬B and that expressed oncogenic Ras were
analyzed for the loss of contact inhibition through the use of focus
formation assays. MEFs, either p65⫺/⫺ or c-Rel⫺/⫺, were transfected
with vector control plasmid (pZip-neo), oncogenic H-Ras (pZip-HRasV12), or oncogenic H-Ras with an expression vector for either p65
or c-Rel. All of the transfections were done in quadruplicate for each
condition. At 48 hours posttransfection, one plate was harvested for
whole cell protein extraction and another was harvested for total
RNA. These extracts were used as controls for protein expression of
p65 and c-Rel and for expression of H-Ras RNA (Fig. 1C). At 14 days
posttransfection, the two remaining plates were stained and were
analyzed for focus formation. As shown in Fig. 1A and B, Ras alone
was able to induce a low level of focus formation in both the p65⫺/⫺
and the c-Rel⫺/⫺ MEFs. This suggests that neither of these subunits
alone is absolutely required for Ras-induced focus formation, but
these experiments do not allow us to determine whether p65 and c-Rel
are able to compensate for each other in facilitating Ras-induced
transformation.
Cotransfection of p65 and Ras into the p65⫺/⫺ MEFs, or c-Rel and
Ras into the c-Rel⫺/⫺ MEFs resulted in an increase in focus formation
over that of Ras alone (comparable with levels attained with wild-type
cells; see ref. 4), indicating that both p65 and c-Rel can enhance
Ras-induced cellular transformation (Fig. 1A and B). p65⫺/⫺ and
c-Rel⫺/⫺ MEFs transfected with p65 or c-Rel, respectively, without
Ras do not display any focus-forming activity (data not shown).
Western blot analysis of whole cell extracts for p65 and c-Rel and
RT-PCR analysis for H-Ras confirmed the concentration-dependent
expression of the transfected proteins and also shows, importantly,
that exogenous expression of p65 and c-Rel does not have an effect on
expression of exogenous H-Ras (Fig. 1C). Thus, enhanced focus
formation after p65 or c-Rel expression cannot be explained by simply
inducing higher levels of H-RasV12 expression and must, instead, be
derived from inherent p65 or c-Rel activity.
The raw data presented in Fig. 1A suggest that p65⫺/⫺ cells have a
higher transforming potential than do the c-Rel⫺/⫺ cells. This observation could be interpreted to indicate that c-Rel plays a more important role in Ras-induced focus formation than does p65. However, we
have analyzed at least two independent spontaneously immortalized
cell lines for each genotype and have found that not all cell lines have
the same transforming potential as measured by focus formation
assay. Some MEF cell lines (p65⫺/⫺ and c-Rel⫺/⫺) will not transform
with or without cotransfection of either p65 or c-Rel, which indicates
that the absence of p65 or c-Rel is not the reason for the lack of focus
formation (data not shown). Furthermore, all MEF lines do not have
equal transfection efficiency, presumably because of genetic alterations associated with immortalization. Therefore, it is not appropriate
to directly compare the transforming potential of two different spontaneously immortalized cell lines. Thus, our analysis is limited to the
analysis of p65-null, c-Rel-null, and double-null MEFs as well as the
restored isogenic cell lines.
H-RasV12 Induces Transformation of p65/c-Rel DKO MEFs.
To further examine the role of p65 and c-Rel in Ras-induced transformation and to analyze the possibility that p65 and c-Rel serve
redundant functions in transformation, we performed focus formation
assays in p65/c-Rel DKO MEFs. Immunoblotting analysis reveals that
Arf and p53 are inducible (by oncogenic Ras expression and by UV
exposure, respectively; data not shown), which indicates that these
Fig. 1. p65 and c-Rel enhance Ras-induced transformation in p65⫺/⫺ and cRel⫺/⫺
MEFs, respectively. A, focus formation assay of p65⫺/⫺ (top panel) and cRel⫺/⫺ (bottom
panel) MEFs transfected with pBabe-Puro (Control) or H-RasV12 alone or with 1 or 2 ␮g
of pBabe-p65 (top panel) or pBabe-cRel (bottom panel). B, graphical representation of the
number of foci/plate (in duplicate) in the focus formation assay shown in A. C, RT-PCR
analysis for H-Ras and ␤-actin (loading control) expression. The data shown are representative of three independent experiments.
tumor suppressor proteins are wild-type in these cells (see Discussion). As shown above for the p65 and c-Rel knockout MEFs, oncogenic H-Ras alone was able to induce focus formation in the p65/cRel
DKO MEFs (Figs. 2A and B), indicating that p65 and c-Rel are not
essential for Ras-induced focus formation in MEFs. Importantly, p65
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NF-␬B SUBUNITS POTENTIATE RAS TRANSFORMATION
Fig. 2. p65 and c-Rel enhance Ras-induced transformation in p65/c-Rel DKO MEFs.
A, focus formation assays in p65/cRel DKO MEFs transfected with pBabe-Puro (Control)
or pBabe-H-RasV12 alone or in combination with pBabe-p65, pBabe-c-Rel, or both. B,
graphical representation of the number of foci/plate (in duplicate) from the focus formation assays shown in A. C, Immunoblotting for p65 and c-Rel (upper panel). RT-PCR
analysis for H-Ras and ␤-actin (loading control) expression (lower panel). The data shown
are representative of three independent experiments.
and c-Rel expression alone, or in combination, substantially enhance
Ras-induced focus formation (Fig. 2A and 2B) without affecting levels
of exogenous oncogenic Ras expression (Figs. 2C), again indicating
that p65 and c-Rel contribute to the potential for Ras-induced transformation.
MEFs Stably Expressing Oncogenic H-Ras Are Morphologically Transformed and Grow in Soft Agar. The results observed in
the focus formation assays demonstrate that in the presence of oncogenic H-Ras, p65⫺/⫺, c-Rel⫺/⫺, and p65/cRel DKO MEFs display a
loss of normal contact-inhibited growth, one measure of cellular
transformation. Two other phenotypes associated with oncogenic
transformation are altered cellular morphology and anchorage-independent growth. To determine whether p65 and/or c-Rel are required
for morphologic transformation and anchorage-independent growth,
we created MEF⫹/⫹ and p65⫺/⫺, c-Rel⫺/⫺, and p65/c-Rel DKO
MEFs stably expressing either vector control or H-RasV12 by infecting the cells with a control retrovirus, pBabe-Puro, or a retrovirus
expressing pBabe-HRasV12. At 48 hours postinfection, infected cells
were selected for 14 days in growth medium supplemented with
puromycin (1 ␮g/mL). Drug-resistant colonies were then pooled and
examined for Ras expression and morphologic transformation. As
observed in Fig. 3A, MEF⫹/⫹, p65⫺/⫺, c-Rel⫺/⫺, and p65/c-Rel DKO
cells expressing RasV12, each display a spindly and refractile phenotype characteristic of Ras-transformed cells, which demonstrates
that p65 and c-Rel are not required for Ras to induce morphologic
transformation of MEFs.
A hallmark of cellular transformation is the ability of cells to grow
in an anchorage-independent manner. Therefore, soft agar assays were
performed to determine whether p65 and/or c-Rel are required for
Ras-induced anchorage-independent growth. All MEF lines (⫹/⫹,
p65⫺/⫺, c-Rel⫺/⫺, and p65/cRel DKO), stably expressing either vector control or H-RasV12, were suspended in 0.33% agar/medium and
were analyzed for colony formation 14 days later. All of the cell lines
(including the DKO MEFs) that expressed oncogenic Ras were able to
Fig. 3. p65-, cRel-, and p65/cRel-null MEFs
stably expressing H-RasV12 display morphologic
transformation and anchorage-independent growth.
A, morphologic transformation observed in ⫹/⫹,
p65⫺/⫺, c-Rel⫺/⫺, and p65/cRel DKO MEFs stably expressing pBabe-H-RasV12 (bottom panel)
compared with pBabe-Puro control cells (top
panel). B, soft agar assay with MEFs described in A
were suspended in 0.33% bactoagar growth medium and were incubated for 14 days at 37°C, 5%
CO2. Fourteen days later, the cells were examined
for colony formation indicative of anchorage-independent growth. The data shown are representative
of three independent experiments, each performed
in triplicate.
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Fig. 4. p65 and c-Rel do not contribute to Ras-induced expression of Cyclin D1 or
c-myc. A, RNase protection assay for Cyclin D1 RNA levels. Total RNA isolated from
⫹/⫹ or p65/c-Rel DKO MEFs, infected with either control or HRasV12-expressing
retrovirus, was incubated with probes for cyclin D1 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (as an internal control) as described under Materials and
Methods. B, total RNA (as described for A) was separated by electrophoresis in a
formaldehyde-agarose gel and was analyzed by Northern blot with a 32P-labeled c-myc
DNA probe. 18S RNA is included as a control for RNA loading. Data shown are
representative of three independent experiments.
form colonies in soft agar, whereas the vector control cells were
unable to grow (Fig. 3B). Furthermore, the p65⫺/⫺ MEFs did not
show transforming potential on their own as suggested in another
study (24), although cell origin may explain the difference. Together
these data indicated that p65 and c-Rel are not required for Rasinduced loss of contact-inhibited growth, morphologic transformation,
or anchorage-independent growth.
Determination of Ras-Induced Genes Controlled by p65 and/or
c-Rel. The data thus provided indicated that, although p65 and c-Rel
are not required for Ras-induced transformation, these NF-␬B subunits substantially enhance transformation. Genes that are known to
be regulated by NF-␬B (at least in response to certain stimuli) include
c-IAP1/2, TRAF1 and -2, cyclin D1, and c-myc (25). To determine
whether p65 and c-Rel regulate cyclin D1 or c-myc expression in
response to oncogenic Ras signaling, we infected MEFs⫹/⫹ or p65/
c-Rel DKO MEFs with either control or H-RasV12 retrovirus. At 48
hours postinfection, total RNA was isolated and Northern blot analysis was performed. As expected, oncogenic Ras induced the expression of cyclin D1 and c-myc in MEFs⫹/⫹. Interestingly, inducible
expression of cyclin D1 and c-myc was largely unaffected in the
p65/cRel DKO MEFs (Fig. 4), indicating that p65 and c-Rel do not
contribute substantially to Ras-induced expression of these genes.
Furthermore, at least in murine fibroblasts, we did not observe Rasinduced expression of c-IAP1/2 or TRAF1/2 (see Fig. 5B). These data
are consistent with our recent report that Ras does not regulate certain
NF-␬B-dependent genes (19).
To determine which, if any, Ras-induced genes are NF-␬B (p65
and/or c-Rel)-dependent, we performed Affymetrix microarray analysis. Total RNA from ⫹/⫹ or p65/c-Rel DKO MEFs was isolated 48
hours post-retroviral infection with control or H-RasV12 virus. The
RNA was then amplified and hybridized to the Affymetrix mouse
gene array 430A, as described in Materials and Methods. Affymetrix
analysis identified 899 differentially regulated genes with the criteria
for inclusion (see Materials and Methods) defined by a 2-fold-orgreater change in expression among any two of the four samples. Of
these genes, 135 were considered to be specifically Ras-inducible
based on a 3-fold increase in Ras-expressing wild-type MEFs over
control/wild-type MEFs irrespective of their regulation by RelA/p65
and c-Rel. Clustering algorithms also identified a single subset of 25
similarly regulated genes that represent p65/c-Rel-dependent, Rasinducible genes (Table 1; see below). This collective expression
profile demonstrates a requirement for p65/c-Rel to exhibit induced
gene expression on oncogenic Ras expression. The largest single
cluster identified 88 genes, including c-myc, cyclin D1, HMGCI, and
HMGY that were induced by Ras in both the wild-type and p65/cRel-null cells. A more refined clustering of these 88 genes demonstrated that the basal expression levels of 44 genes were unaffected by
the loss of p65/c-Rel. The remaining 44 genes segregated into two
additional profiles based on repressed or elevated basal expression in
the DKO cells versus the wild-type MEFs. Therefore, we have iden-
Table 1 Ras-inducible, NF-␬B-regulated genes
Normalized fluorescent intensity†
Gene name
gb identifier
Fold*
DKO–H-ras
DKO-PURO
p65⫹/⫹ PURO
p65⫹/⫹ H-ras
P value‡
Serpinb2
Tslp
Adra2a
Tmod2
PCA-1§
Galactin7
Posh
PI4P5K-I
Or10
Psgl-1/Selp1
Aeg1
Pkp1
Tage4
EST
Nppb
Stk10
Col18a1
Bsf3
AldH1a3§
Klf3
Adra2c
Cgref1
Sox5
RN19§
Oas16
NM_011111
NM_021367
NM_0074717
AK018223
BB560177
AF038562
NM_021506
BC003763
NM_020513
NM_009151
NM_009638
NM_019645
NM_009310
AV302409
NM_008726
NM_009288
NM_009929
NM_019952
BC026667
NM_008453
NM_007418
BC023116
AK015212
AK015966
AB067530
33.32
11.92
6.339
5.895
5.695
5.586
5.315
4.896
4.022
3.925
3.862
3.786
3.712
3.684
3.515
3.228
3.045
2.999
2.809
2.626
2.597
2.501
2.466
2.256
2.079
1.036
0.491
1.063
1.613
0.884
0.921
0.751
1.307
0.843
0.95
0.725
1.151
1.07
0.998
0.995
0.809
0.998
1.112
0.897
0.803
0.965
1.064
0.831
0.885
0.929
0.511
0.79
0.897
0.904
0.94
0.777
0.743
0.926
0.655
0.965
1.004
0.84
0.86
0.905
0.998
0.796
0.767
0.949
0.847
0.821
0.778
0.83
1.023
1.021
1.116
0.989
1.224
0.823
0.838
1.058
0.972
0.856
0.76
1.043
0.877
0.956
0.754
0.768
0.775
0.831
1.074
0.925
0.925
0.958
1.111
1.044
0.887
0.993
0.951
0.856
32.95
14.59
5.217
4.94
6.025
5.43
4.55
3.721
4.195
3.442
3.692
2.855
2.851
2.855
2.921
3.467
2.817
2.774
2.691
2.917
2.711
2.218
2.449
2.145
1.78
0.00936
0.078
0.00518
0.028
0.179
0.0437
0.0231
0.207
0.00204
0.00351
0.251
0.242
0.13
0.0662
0.054
0.021
0.179
0.0256
0.227
0.15
0.0211
0.0112
0.14
0.0129
0.248
Abbreviations: gb, GenBank; DKO, double knockout; PURO, (supplemented with) puromycin.
* Fold induction on response to H-ras expression in p65⫹/⫹ MEFs.
† Normalization procedure is described in Materials and Methods.
‡ t test P values for significance across two independent experiments involving independent Affymetrix chips.
§ Gene name based on ⬎80% nucleotide identity to Hs homolog.
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NF-␬B SUBUNITS POTENTIATE RAS TRANSFORMATION
Fig. 5. Ras-induced expression BSF3 is dependent on p65 and c-Rel. A, quantitative real-time PCR
for expression of BSF3, Serpinb2 (A), c-IAP1 and
TRAF2 (B) from ⫹/⫹ and p65/c-Rel DKO MEFs
infected with either control or HRasV12-expressing virus. Data shown are representative of at least
three independent experiments, each performed in
triplicate. [Data represented as molecules (mol.) of
a transcript versus attomole of 18S.]
tified four distinct populations of Ras-inducible genes: (a) p65/c-Rel
independent (unaffected basally in DKO cells); (b) p65/c-Rel basally
activated (basal levels repressed in DKO cells); (c) p65/c-Rel basally
repressed (i.e., basal levels up-regulated in DKO cells); and (d)
p65/c-Rel-dependent (unaffected basally in DKO cells).
A list of those Ras-induced genes clearly dependent on p65 and/or
c-Rel and their relative expression is presented in Table 1. Among the
NF-␬B-dependent genes are: (a) plasminogen activator inhibitor type
2 (Serpin B2) known to be regulated by NF-␬B and by oncogenic Ras
and to suppress TNF-induced apoptosis (26 –28); (b) TSLP (thymic
stromal lymphopoietin) which can promote the growth of pre-B cells
(29, 30); (c) plakophilin-1 (a member of the armadillo/␤-catenin
family, which is known to be up-regulated in certain cancers (31); (d)
Tage4/nectain-like molecule 5 [associated with cell migration (32)],
(e) neurotrophin/B-cell stimulating factor 3 [a member of the IL-6
family (33)]; (f) Posh-like molecule [posh is a Rac-regulated scaffold
protein associated with JNK and NF-␬B signaling pathways (34)]; (g)
galectin 7, expression of which is associated with a more aggressive
phenotype in lymphoma cells (35); (h) Stk10 [a recently described
polo-like kinase expressed in hematopoietic tissues (36)]; and (i)
PCA-1, a cell surface marker for multiple myeloma (37).
In agreement with a recent study (29), we found several genes,
including phosholipase A2 group VII, connexin 43, transferrin receptor, ERK3, and Mdm2 to be induced by H-RasV12 in ⫹/⫹ MEFs.
Expression of Tage4, shown here to be NF-␬B-dependent, was also
found increased in K-RasV12 transformed NIH3T3 cells by Ikeda et
al. (32). Additionally, other Ras-induced genes were identified for
which NF-␬B played a measurable, but less substantial, role in regulating gene expression. These genes and those induced by Ras in a
manner independent of NF-␬B will be reported/deposited elsewhere.
To confirm results derived from the microarray studies, we further
analyzed the expression of several of the inducible genes with quantitative PCR. Quantitative real-time PCR was performed on cDNA
generated from ⫹/⫹ and p65/cRel DKO infected with control or
H-RasV12 virus with primers specific to Serpinb2, Bsf3, c-IAP2, and
TRAF2. As shown in Fig. 5, SerpinB2 and Bsf3 (both identified as
NF-␬B regulated in the array studies) were found to be induced by
Ras in a manner highly dependent on the presence of p65/RelA and/or
c-Rel (Fig. 5A). This analysis demonstrated that c-IAP2 and TRAF2,
although known to be regulated by NF-␬B downstream of TNFinduced signaling (25), were not induced by oncogenic H-Ras (Fig.
5B). In fact, Ras repressed c-IAP2 expression. These results, therefore, have identified a gene set induced by oncogenic Ras in a manner
highly dependent on the p65 and/or c-Rel subunits of NF-␬B. These
NF-␬B-dependent genes are likely to encode proteins that promote the
efficacy of transformation of cells in response to oncogenic Ras
expression. Longer-term experimentation would be required to determine how many of these genes are direct targets for NF-␬B and
whether knockdown of expression of any of these genes would exhibit
a measurable effect on Ras-induced transformation efficiency.
DISCUSSION
Ras-induced cellular transformation is mediated by interactions
between Ras and downstream effector proteins such as PI3K, Raf, and
RalGEFs (1). Raf is considered to be the most potent of this group in
controlling transformation in murine cells, whereas RalGDS appears
to play a more important role in Ras-induced transformation of human
cells (1, 38). In support of the key role of Raf in transformation of
murine fibroblasts, it has been observed that constitutively active Raf
or mitogen-activated protein/extracellular signal-regulated kinase
[MAP/ERK kinase (MEK)] can cause tumorigenic conversion of
immortalized 3T3 cells and that inhibitors of this pathway can block
this process (1, 38). Activation of the ERK pathway presumably
targets certain transcription factors that promote the changes in gene
expression that stimulate oncogenic transformation. Our group and
others have shown that NF-␬B is activated downstream of Rasinduced as well as Raf-induced signaling and that inhibition of NF-␬B
suppresses the ability of Ras to induce oncogenic conversion (3– 6). In
this regard, recent reviews have covered the potential involvement of
NF-␬B in promoting oncogenic conversion and progression (39, 40).
Importantly, our data with murine fibroblasts for analysis of Rasinduced responses indicate that the activation of NF-␬B in this process
does not involve the traditional I␬B degradation pathway that is
associated with cytokine-induced signaling (19). However, it is noted
that oncogenic Ras induces IKK activity and I␬B degradation in
human epithelial cells (3).
The results in this article indicate that the two major transactivating
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NF-␬B SUBUNITS POTENTIATE RAS TRANSFORMATION
NF-␬B subunits, p65 and c-Rel, are not essential for Ras to induce
cellular transformation of murine fibroblasts. However, re-expression
of these subunits in p65- or c-Rel-null cells strongly promotes the
ability of Ras to induce focus formation (Figs. 1 and 2). These
findings help to clarify previous results with expression of I␬B-SR in
which inhibition suppressed, but was unable to completely block,
Ras-induced cellular transformation. Furthermore, we identify a gene
set that is activated by oncogenic Ras (V12) in murine fibroblasts, the
expression of which is highly dependent on either p65 or c-Rel (Table
1). The proposed functions of these gene products are generally
consistent with promotion of cell growth and oncogenic transformation. Of the genes identified as NF-␬B regulated, Bsf3, TSLP, and
Stk10, all have documented effects on cellular proliferation. For
example, BSF3 and TSLP have both been shown to stimulate the
growth of myeloid leukemia cells (29, 33). Although the precise
function of serine/threonine kinase Stk10 is unknown, the only known
substrate of Stk10, Plk1, is a protein that is involved in cellular
proliferation (36).
A number of the other genes identified as Ras inducible and NF-␬B
dependent have also been found to exhibit increased expression in
cancer. Tage4, a nectin-like protein, was originally found up-regulated
in rat colon carcinomas and Min mouse intestinal adenomas (41).
More recently, it has been determined that Tage4 interacts with
nectin-3 and enhances cell migration (32). Additionally, Plakophilin-1, a component of desmosomes, has been found overexpressed in
head and neck squamous cell carcinomas (42, 43). At this point, it is
unclear which of the NF-␬B-regulated genes contribute to the enhancement of Ras-induced cellular transformation.
The function of NF-␬B in potentiating Ras-induced cell transformation could be manifested at several levels, some of which may be
independent of Ras-signaling. The simplest hypothesis is that the
induction of certain genes by Ras in a manner dependent on p65 and
c-Rel promotes the efficacy or likelihood of cellular transformation.
This hypothesis is consistent with the microarray data presented in
Table 1 and Fig. 5 and with the proposed functions of several of the
genes identified as Ras inducible and NF-␬B dependent. However,
based on the known antiapoptotic function of NF-␬B, we cannot rule
out the possibility that the loss of NF-␬B and certain NF-␬B–regulated (but potentially not Ras-inducible) genes provides a proapoptotic
background for Ras. Thus, oncogenic Ras expression may induce a
higher level of apoptosis in p65- and c-Rel-null cells leading to less
efficient oncogenic conversion. Additionally, Ras may induce certain
antiapoptotic genes in an NF-␬B-dependent manner. Although the
NF-␬B–regulated antiapoptotic gene c-IAP2 was not found to be
inducible by Ras in murine fibroblasts, Serpinb2 has been shown to
protect cells from TNF-induced apoptosis and may serve an antiapoptotic function in Ras transformation as well (26). A role for NF-␬B in
suppressing Ras-induced apoptosis has been previously described by
our group (5). Another potential role for NF-␬B in promoting Rasinduced oncogenic transformation is the control of Ras-induced signaling. Thus, NF-␬B may be required for more potent or persistent
activation of Raf-induced signaling, e.g., promoting higher levels of
Ras-induced cellular transformation. Additionally, we cannot rule out
the possibility that Ras represses a set of genes in an NF-␬B– dependent manner and that this process also promotes transformation. Combinations of any of these possibilities remain possible and additional
studies in these areas are currently underway. Additionally, it must be
noted that the genetic phenotype of an immortalized cell may control
or modulate the ability of NF-␬B to regulate specific gene expression
and, correspondingly, to contribute to oncogenic transformation. In
this regard, we have analyzed the status of the tumor suppressors Arf
and p53 in the p65/c-Rel-null MEFs used in our studies. Both proteins
appear to be wild-type, because they are inducible by Ras and UV
exposure, respectively (data not shown). On the basis of earlier
studies, induction of Arf by Ras in these cells would be expected to
block NF-␬B function (44). However, clearly, Ras can use NF-␬B in
a functional manner in the null MEFs restored for NF-␬B expression,
indicating that Arf does not suppress NF-␬B activity directed toward
certain genes.
Regarding the genes induced by Ras in a manner dependent on
NF-␬B, it is noteworthy that the majority of these genes have not been
identified as NF-␬B regulated downstream of cytokine-induced signaling. We did not find any of the well-characterized NF-␬B–regulated cytokine, cytokine receptors, cell adhesion genes, and so forth,
associated with immune and inflammatory function as Ras-inducible
genes. These results are consistent with our previous hypothesis that
Ras induces a selective arm of the NF-␬B pathway while suppressing
the ability of TNF to activate NF-␬B and NF-␬B– dependent gene
expression in murine fibroblasts (19). Thus, a remaining challenge is
to understand how Ras-induced signals converge on NF-␬B to activate certain genes while simultaneously suppressing the ability of
cytokines to activate the more traditional, and better characterized,
NF-␬B-response. It is likely that this key process controlled by oncogenic Ras, selectively activating NF-␬B in a manner leading to
up-regulation of a distinct subset of genes, is an important component
in the ability of Ras and potentially other oncoproteins to efficiently
transform cells.
ACKNOWLEDGMENTS
We thank Dr. Alexander Hoffmann (University of California San Diego, La
Jolla, CA) for the generous gift of NF-␬B-null MEFs.
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The Nuclear Factor κB Subunits RelA/p65 and c-Rel
Potentiate but Are Not Required for Ras-Induced Cellular
Transformation
Julie L. Hanson, Noel A. Hawke, David Kashatus, et al.
Cancer Res 2004;64:7248-7255.
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