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[Cancer Research 64, 7248-7255, October 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

The Nuclear Factor {kappa}B Subunits RelA/p65 and c-Rel Potentiate but Are Not Required for Ras-Induced Cellular Transformation

Julie L. Hanson1,2, Noel A. Hawke1, David Kashatus1,2 and Albert S. Baldwin1,2,3

1 Lineberger Comprehensive Cancer Center, 2 Curriculum in Genetics and Molecular Biology, and 3 Department of Biology, University of North Carolina, Chapel Hill, North Carolina


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {kappa}B (NF-{kappa}B). Supportive of the importance of NF-{kappa}B in transformation, Ras-induced cellular transformation can be suppressed by expression of I{kappa}B{alpha}, an inhibitor of NF-{kappa}B, or by dominant-negative forms of the upstream activator I{kappa}B kinase (IKK). However, conclusive evidence for a requirement for NF-{kappa}B subunits in oncogenic transformation has not been reported. Furthermore, there is little understanding of the gene targets controlled by NF-{kappa}B that might support oncogenic conversion. The data presented here demonstrate that, although both p65 and c-Rel enhance the frequency of Ras-induced cellular transformation, these NF-{kappa}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-{kappa}B for their expression and that likely play contributory roles in promoting Ras-induced oncogenic transformation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {kappa}B (NF-{kappa}B) is activated by oncogenic Ras. Expression of interfering proteins such as the inhibitor of NF-{kappa}B (I{kappa}B{alpha}) or dominant-negative forms of I{kappa}B kinase (IKK) suppresses Ras-induced transformation in vitro (3, 4, 5, 6) , suggesting the involvement of NF-{kappa}B in the transformation process induced by oncogenic Ras expression.

NF-{kappa}B represents a family of dimeric transcription factors that are characterized by a 300-amino-acid region called the Rel homology domain (7, 8, 9) . The five mammalian NF-{kappa}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{kappa}B proteins localize NF-{kappa}B dimers to the cytoplasmic compartment through masking of nuclear localization sequences of NF-{kappa}B subunits. Activation of NF-{kappa}B can occur through various stimuli, including inflammatory cytokines and bacterial and viral infection, as well as oncogenic signals (7, 8, 9) . Activation of NF-{kappa}B, 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{kappa}B kinase (IKK) complex. The IKK complex is composed of three subunits, IKK{alpha}, IKKß and IKK{gamma} (7, 8, 9) . Both IKK{alpha} and IKKß have inducible catalytic activity, whereas IKK{gamma} is a regulatory subunit. Following its activation, the IKK complex phosphorylates I{kappa}B{alpha} and I{kappa}Bß at specific serine residues, which targets I{kappa}B for ubiquitination and degradation by a proteasome-dependent pathway. After I{kappa}B degradation, the unmasked nuclear localization sequence of NF-{kappa}B allows nuclear accumulation that, in turn, promotes sequence-specific DNA binding and transcriptional activation of target genes (9) .

Although nuclear accumulation of NF-{kappa}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-{kappa}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-{kappa}B target genes (10 , 11) . Additional sites of phosphorylation have also been described that appear to contribute to the inherent transcriptional activity of NF-{kappa}B (12, 13, 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-{kappa}B (15 , 16) . Consistent with this, we have shown that both oncogenic Ras and oncogenic Raf activate an NF-{kappa}B-dependent reporter in mouse fibroblasts without inducing DNA binding of NF-{kappa}B as measured by gel mobility shift assay (4 , 6) . It is noted that human epithelial cells, transformed with oncogenic Ras, exhibit enhanced NF-{kappa}B DNA-binding activity in association with I{kappa}B{alpha} degradation (3) .

Certain transcription factors, including Ets and NF-{kappa}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 factor-regulated genes are induced during transformation. Recently, we demonstrated that oncogenic Ras, although stimulating a NF-{kappa}B-dependent reporter, suppressed the ability of TNF to activate NF-{kappa}B in murine fibroblasts (19) . The mechanism of suppression of NF-{kappa}B involved Ras-controlled inhibition of TNF-induced IKK activation and I{kappa}B degradation. These studies revealed that Ras suppressed the ability of TNF to activate known NF-{kappa}B-regulated genes such as iNOS, indicating that, although Ras can stimulate NF-{kappa}B functional activity, it does so in a selective manner.

Although it is suggested that NF-{kappa}B plays a role in Ras-induced transformation, direct genetic evidence is missing. We previously reported that Ras stimulates the activity of the NF-{kappa}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 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 Ras-induced transformation of immortalized murine fibroblasts. Despite this, each of these NF-{kappa}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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 H-RasV12 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 µg/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 x 105 target cells with 1 mL of virus, 1 mL of complete medium with 8 µg/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 x 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 x 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 µg 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 bands were visualized with enhanced chemiluminescence detection (Amersham Life Science).

Northern Blot Analysis.
Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s protocol. Ten µg of RNA were then electrophoresed on a 1.5% formaldehyde-agarose gel and were transferred to a nylon membrane according to standard procedures. The RNA was then cross-linked to the membrane by UV irradiation (Stratagene) and were probed with a randomly labeled gene specific probe. Hybridization and washing were preformed with ExpressHyb (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.

Reverse Transcription-PCR.
Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer’s protocol. Four µg total RNA were then treated with RNase-free DNase (Invitrogen) to eliminate any DNA 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 ß-actin. In a total volume of 25 µL, 0.5 µg of total RNA was used to analyze H-Ras expression, and 0.01 µg was used to determine the level of ß-actin expression.

Real-time PCR.
Total RNA was isolated with the Promega SV total RNA kit. Two µg 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 mRNA-based PCR products. Each PCR reaction contained 2 µL of cDNA, a 2 µmol/L concentration of each primer, and Qiagen QuantiTect Syber Green PCR master mix in a 20-µL 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-{kappa}B-Regulated Genes.
Seven µg 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 [5x 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 µg of fragmented cRNA were then added to a hybridization cocktail (0.05 µg/µL 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 µg 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-{kappa}B dependent (correlation coefficient of 0.95).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
H-RasV12 Induces Transformation of p65–/– and c-Rel–/– MEFs.
Previous studies examining the involvement of NF-{kappa}B in Ras-induced transformation used dominant-negative forms of IKK or the I{kappa}B-super repressor (SR), the trans-dominant-negative inhibitor of NF-{kappa}B (3 , 4) . These interfering proteins either inhibit upstream signaling to a variety of NF-{kappa}B dimers or block activation of any NF-{kappa}B subunit that will associate with I{kappa}B family members. Use of dominant-negative IKK or I{kappa}B-SR does not allow for the determination of a potential role of specific NF-{kappa}B subunits in the transformation process. Additionally, overexpression of I{kappa}B{alpha} or I{kappa}B-SR can have effects on cells that are independent of NF-{kappa}B (22 , 23) . Therefore, to determine whether NF-{kappa}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-{kappa}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-H-RasV12), 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)Citation . At 14 days posttransfection, the two remaining plates were stained and were analyzed for focus formation. As shown in Fig. 1A and BCitation , 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.



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  		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 µg 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 ß-actin (loading control) expression. The data shown are representative of three independent experiments.

 
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)Citation . 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)Citation . 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. 1ACitation 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 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)Citation , indicating that p65 and c-Rel are not essential for Ras-induced focus formation in MEFs. Importantly, p65 and c-Rel expression alone, or in combination, substantially enhance Ras-induced focus formation (Fig. 2ACitation and 2BCitation ) without affecting levels of exogenous oncogenic Ras expression (Figs. 2C)Citation , again indicating that p65 and c-Rel contribute to the potential for Ras-induced transformation.



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  		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 ß-actin (loading control) expression (lower panel). The data shown are representative of three independent experiments.

 
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 µg/mL). Drug-resistant colonies were then pooled and examined for Ras expression and morphologic transformation. As observed in Fig. 3ACitation , 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.



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  		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.

 
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 form colonies in soft agar, whereas the vector control cells were unable to grow (Fig. 3B)Citation . 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 Ras-induced 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-{kappa}B subunits substantially enhance transformation. Genes that are known to be regulated by NF-{kappa}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)Citation , 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 Ras-induced expression of c-IAP1/2 or TRAF1/2 (see Fig. 5BCitation ). These data are consistent with our recent report that Ras does not regulate certain NF-{kappa}B-dependent genes (19) .



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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.

 


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  		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.]

 
To determine which, if any, Ras-induced genes are NF-{kappa}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-or-greater 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, Ras-inducible genes (Table 1Citation ; 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/c-Rel-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 identified 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).


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  		Table 1 Ras-inducible, NF-{kappa}B-regulated genes

 
A list of those Ras-induced genes clearly dependent on p65 and/or c-Rel and their relative expression is presented in Table 1Citation . Among the NF-{kappa}B-dependent genes are: (a) plasminogen activator inhibitor type 2 (Serpin B2) known to be regulated by NF-{kappa}B and by oncogenic Ras and to suppress TNF-induced apoptosis (26, 27, 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/ß-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-{kappa}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-{kappa}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-{kappa}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-{kappa}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. 5Citation , SerpinB2 and Bsf3 (both identified as NF-{kappa}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)Citation . This analysis demonstrated that c-IAP2 and TRAF2, although known to be regulated by NF-{kappa}B downstream of TNF-induced signaling (25) , were not induced by oncogenic H-Ras (Fig. 5B)Citation . 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-{kappa}B. These NF-{kappa}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-{kappa}B and whether knockdown of expression of any of these genes would exhibit a measurable effect on Ras-induced transformation efficiency.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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-{kappa}B is activated downstream of Ras-induced as well as Raf-induced signaling and that inhibition of NF-{kappa}B suppresses the ability of Ras to induce oncogenic conversion (3, 4, 5, 6) . In this regard, recent reviews have covered the potential involvement of NF-{kappa}B in promoting oncogenic conversion and progression (39 , 40) . Importantly, our data with murine fibroblasts for analysis of Ras-induced responses indicate that the activation of NF-{kappa}B in this process does not involve the traditional I{kappa}B degradation pathway that is associated with cytokine-induced signaling (19) . However, it is noted that oncogenic Ras induces IKK activity and I{kappa}B degradation in human epithelial cells (3) .

The results in this article indicate that the two major transactivating NF-{kappa}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. 1Citation and 2Citation ). These findings help to clarify previous results with expression of I{kappa}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)Citation . The proposed functions of these gene products are generally consistent with promotion of cell growth and oncogenic transformation. Of the genes identified as NF-{kappa}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-{kappa}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-{kappa}B-regulated genes contribute to the enhancement of Ras-induced cellular transformation.

The function of NF-{kappa}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 1Citation and Fig. 5Citation and with the proposed functions of several of the genes identified as Ras inducible and NF-{kappa}B dependent. However, based on the known antiapoptotic function of NF-{kappa}B, we cannot rule out the possibility that the loss of NF-{kappa}B and certain NF-{kappa}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-{kappa}B-dependent manner. Although the NF-{kappa}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-{kappa}B in suppressing Ras-induced apoptosis has been previously described by our group (5) . Another potential role for NF-{kappa}B in promoting Ras-induced oncogenic transformation is the control of Ras-induced signaling. Thus, NF-{kappa}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-{kappa}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-{kappa}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-{kappa}B function (44) . However, clearly, Ras can use NF-{kappa}B in a functional manner in the null MEFs restored for NF-{kappa}B expression, indicating that Arf does not suppress NF-{kappa}B activity directed toward certain genes.

Regarding the genes induced by Ras in a manner dependent on NF-{kappa}B, it is noteworthy that the majority of these genes have not been identified as NF-{kappa}B regulated downstream of cytokine-induced signaling. We did not find any of the well-characterized NF-{kappa}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-{kappa}B pathway while suppressing the ability of TNF to activate NF-{kappa}B and NF-{kappa}B-dependent gene expression in murine fibroblasts (19) . Thus, a remaining challenge is to understand how Ras-induced signals converge on NF-{kappa}B to activate certain genes while simultaneously suppressing the ability of cytokines to activate the more traditional, and better characterized, NF-{kappa}B-response. It is likely that this key process controlled by oncogenic Ras, selectively activating NF-{kappa}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-{kappa}B-null MEFs.


   FOOTNOTES
 
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: jhall{at}med.unc.edu

4 Internet address: http://www.silicongenetics.com/cgi/Products/GeneSpring?index.smf. Back

Received 12/16/03. Revised 7/15/04. Accepted 8/10/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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