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B-Raf Is Dispensable for K-Ras-Mediated Oncogenesis in Human Cancer Cells¹

¹ Department of Oncology and ² Tumor Biology Training Program, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, DC

	ABSTRACT

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Oncogenic mutations in B-Raf and Kirsten-Ras (K-Ras) are mutually exclusive during human cancer pathogenesis. In an effort to study the biological basis of this epistasis, gene targeting was used to create isogenic sets of human cancer cells differing only in presence or absence of endogenous oncogenic K-Ras or wild-type B-Raf. Whereas cells lacking the K-Ras oncogene were unable to efficiently form xenograft tumors, isogenic cells retaining activated K-Ras but deleted for B-Raf remained highly tumorigenic. Deletion of oncogenic K-Ras failed to reduce the activation state of B-Raf or ERK1/2, despite the requirement of oncogenic K-Ras for tumorigenesis. Genechip analysis revealed numerous genes in which the regulation by oncogenic K-Ras did not require B-Raf. These studies suggest that despite the mutual exclusivity of K-Ras and B-Raf mutations in human cancer and the well-described role for Raf proteins as Ras effectors, B-Raf is dispensable for K-Ras-mediated oncogenesis in a human cancer cell line. Additional studies are required to demonstrate the generalizability of these unexpected findings.

	Introduction

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Oncogenic mutations in the Ras signaling pathway are found in the majority of human cancers. Activation of Kirsten-Ras (K-Ras) leads to signaling through at least three different pathways: the RAF/MEK/ERK signaling pathway; the phosphatidylinositol 3-kinase signaling pathway; and the Ral guanine-nucleotide exchange factor signaling pathway (1) . In a recent genomic scale mutational analysis of genes encoding elements of Ras signal transduction pathways, activating mutations of B-Raf were identified in several common human cancers, including melanoma, colon cancer, and others (2) . Interestingly, mutations of K-Ras and B-Raf were mutually exclusive in tumor types in which both occur, suggesting that K-Ras and B-Raf provide an equivalent or at least redundant oncogenic stimulus in cancer pathogenesis (3) . B-Raf is one of three Raf family members and functions as a serine-threonine kinase (4) . Activation of Raf proteins via Ras binding leads to phosphorylation and activation of MAP kinase/ERK kinase (MEK)1/2, which in turn phosphorylates extracellular signal-regulated kinase 1/2 (ERK1/2).

The fact that K-Ras and B-Raf mutations are mutually exclusive, combined with the fact that Raf proteins are well-known effectors of Ras signal transduction pathways, has led to the hypothesis that B-Raf in particular is a critical effector of K-Ras mediated oncogenesis. To test this, human somatic cell gene targeting was used to create targeted deletions of either oncogenic K-Ras or wild-type B-Raf in a human cancer cell line. Phenotypic and biochemical analysis of the resultant isogenic sets of gene-targeted cell lines showed that B-Raf is dispensable for K-Ras mediated oncogenesis.

	Materials and Methods

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Creation of a Human K-Ras Targeting Vector.
A promoterless human K-Ras targeting vector was created using homologous recombination in Saccharomyces cerevisiae. The targeting vector was designed to delete exon I, which encodes the NH₂ terminus of K-Ras including commonly mutated codons 12 and 13. To do this, a 6.5-kb EcoRI fragment containing exon I of K-Ras was subcloned from BAC 66P21 into yeast shuttle vector pRS426 and sequenced. Next, a PCR product containing a neo^R gene flanked by a priming site for PCR-based identification of knockouts, restriction sites for Southern blot-based identification of knockouts, and 50 nucleotides of homology to the subcloned K-Ras genomic fragment was cotransformed into S. cerevisiae with the linearized recombinant yeast shuttle vector. Successful recombinants were identified by whole-cell PCR. Recombinant plasmids were then shuttled into Escherichia coli, and their integrity confirmed via restriction analysis and DNA sequencing. Additional technical details of this approach to human somatic cell gene targeting are discussed elsewhere (5) .

Creation of a Human B-Raf Targeting Vector.
A 6.0-kb SpeI fragment containing exons VI and VII of B-Raf was cloned from P1 Artificial Chromosome (PAC) RP4–726N20 into yeast shuttle vector YEp24 and sequenced. Next, a PCR product containing an IRES-neo^R gene flanked by LoxP sites, a priming site for PCR-based identification of knockouts, restriction sites for Southern blot-based identification of knockouts, and 50 nucleotides of homology to the subcloned B-Raf genomic fragment was cotransformed into S. cerevisiae with the linearized recombinant yeast shuttle vector. Recombinants were identified and prepared as described for the K-Ras targeting vector. The specific details of construction and the sequences of all PCR primers used are available from the authors upon request.

Tissue Culture and Transfection.
HEC1A cells (American Type Culture Collection, Manassas, VA) were grown in McCoy’s 5A media (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Mediatech, Herndon, VA) and 1% penicillin-streptomycin (Invitrogen) at 37°C in 5% CO₂. To obtain stably transfected clones in HEC1A cells, 20 25-cm² flasks were transfected with ClaI-linearized K-Ras or NotI-linearized B-Raf targeting vectors using Lipofectamine (Invitrogen), following the manufacturer’s protocol. After 3 weeks of growth and selection (0.75 mg/ml G418) in 96-well plates, individual colonies were transferred first to wells in a 24-well plate, then to a 25-cm² flask. Cells in confluent flasks were trypsinized and approximately one-third of the cells used for preparation of genomic DNA and two-thirds used for cryopreservation. For homozygous B-Raf targeting, heterozygous targeted clones were infected with a cre-expressing adenovirus to excise the IRES-neo^R gene. G418-sensitive clones were then retransfected with the linearized targeting vector and homozygous knockout cells identified by PCR and Southern blot.

Genomic PCR, Southern Blots, and DNA Sequencing.
Preparation of genomic DNA, PCR, Southern blots, and automated sequencing were all performed using standard techniques. Taq Platinum (Invitrogen) was used for PCR, according to the manufacturer’s instructions. For Southern blots, 5 µg of digested genomic DNA were separated on a 1% agarose gel and transferred to a Zeta-Probe nylon membrane (Bio-Rad, Hercules, CA). Membranes were prehybridized overnight at 60°C, then hybridized for 24 h at 60°C using radiolabelled, PCR-generated probes for K-Ras or B-Raf. Blots were then washed and imaged using a PhosphorImager 445 SI (Amersham Biosciences, Piscataway, NJ).

Western Blot Analysis.
Cell lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), probed with primary and horseradish peroxidase-coupled secondary antibodies, and visualized by enhanced chemiluminescence (Amersham Biosciences). Antibodies were obtained from Upstate Biotechnology (C-Raf), Cell Signaling Technologies (pan-ERK1/2, phospho-ERK1/2), and Santa Cruz Biotechnology (A-Raf, B-Raf).

Xenograft Growth Assay.
Tumors were established by s.c. injection of 6 x 10⁶ cells suspended in McCoy’s 5A media in the flanks of immunodeficient mice. Tumor growth rate was determined by measuring three orthogonal diameters of each tumor twice a week, and the tumor volume was estimated at /6 (D₁D₂D₃).

B-Raf Kinase Assay.
After serum starvation (24 h), cells were stimulated with 100 ng/ml epidermal growth factor (EGF; Cell Sciences, Canton, MA) for 30 min, solubilized in lysis buffer containing Triton X-100 (Cell Signaling), and immunoprecipitated with 4 µg of anti-B-Raf antibodies (Upstate Biotechnology) overnight. The ability of immunoprecipitated B-Raf to phosphorylate MEK1 was measured in vitro using the B-Raf kinase cascade assay, following the manufacturer’s protocol (Upstate Biotechnology).

Microarrays.
Total RNA from HEC1A cells and gene-targeted derivatives lacking K-Ras or B-Raf was prepared with TRIzol Reagent (Invitrogen) using standard techniques. cRNA probes were prepared and hybridizations to U133A arrays performed as described in the Genechip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). Data analysis was performed using Microarray Suite 5.0 software (Affymetrix). Hierarchical clustering was performed using Genecluster 3.0. Expression levels in HEC1A parental cells were used as the baseline to generate increase and decrease calls. A gene was designated as regulated by oncogenic K-Ras if it met the following stringent criteria: increased (or decreased) in both of two independently derived HEC1A-derivative cell lines with targeted deletion of oncogenic K-Ras but unchanged in HEC1A neo^R control cells and in both of two independently derived cell lines with targeted deletion of wild-type K-Ras. Genes meeting these criteria were then further stratified based on their level of expression in B-Raf^-/- cells. A gene was designated as being regulated by B-Raf if it was increased (or decreased) in both of two independent expression measurements of B-Raf^-/- cells. Primary data are available from the authors upon request.

	Results

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Heterozygous Deletion of Oncogenic K-Ras in HEC1A Cells.
HEC1A was identified in a comprehensive search for new cell lines appropriate for human somatic cell gene targeting. Originally derived from a human endometrial carcinoma, HEC1A is near-diploid, grows rapidly and clonally, and transfects with high efficiency (6) . Sequencing of K-Ras revealed a heterozygous Gly¹² (GGT)Asp¹²(GAT) mutation. Of note, 20% of endometrial carcinomas harbor activating mutations in K-Ras (7) . A human promoterless K-Ras targeting vector was constructed for creating heterozygous deletions of K-Ras in HEC1A cells, as described in "Materials and Methods" (Fig. 1A) . After transfection of the linearized K-Ras targeting vector, individual G418-resistant colonies were expanded, cryopreserved, and tested by PCR to identify cells with heterozygous deletion of K-Ras (data not shown). Putative knockouts were confirmed by Southern blot (Fig. 1B) . In heterozygous knockout clones, the remaining untargeted allele was PCR amplified and sequenced to determine whether the targeting vector had disrupted the wild-type or the mutant allele of K-Ras (Fig. 1C) . The wild-type allele was deleted in 9 of 15 heterozygous knockout clones, and the oncogenic allele was deleted in 6 of 15 heterozygous knockout clones.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. K-Ras gene targeting. A, homologous recombination between the genomic locus and the targeting vector deletes exon I and replaces it with a promoterless neoR gene. PCR primers used for identification of knockouts are indicated, as are the restriction enzyme cleavage sites and probe used for Southern blot-based confirmation of knockouts. B, confirmation of K-Ras targeting by Southern blot analysis. Fragments corresponding to the wild-type allele and the targeted allele are shown. C, PCR products from the untargeted allele were sequenced to identify mutant and wild-type alleles of K-Ras. D, xenograft growth of HEC1A cells and derivatives with targeted deletion of oncogenic K-Ras.

View larger version (62K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Cytological and biochemical features of gene-targeted cells. A, light microscopy of HEC1A parental cells (a) and derivatives with wild-type K-Ras deleted (b), oncogenic K-Ras deleted (c), and homozygous deletion of B-Raf (d). Scale bar represents 100 µm. B, B-Raf kinase assay. Lane 1 is a negative control (lysis buffer), and Lane 2 is a positive control (recombinant B-Raf protein). The data shown are representative of three independent experiments. C, levels of active and total ERK1/2 were measured in HEC1A parental cells and two independently derived K-Ras gene-targeted derivatives of each genotype after serum starvation (-), or stimulation with 100 ng/ml epidermal growth factor (EGF; +). D, levels of active and total ERK1/2 were measured in B-Raf+/+ and B-Raf-/- cells after serum starvation (-) or stimulation with EGF (+).

Homozygous Deletion of B-Raf in HEC1A Cells.
The studies described above demonstrated that oncogenic K-Ras is required to maintain the tumorigenic properties of HEC1A cells. If B-Raf was a required effector of K-Ras oncogenic activity, B-Raf should be similarly required to maintain the tumorigenic properties of HEC1A cells. To test this, we created and studied HEC1A cells with homozygous deletion of the B-Raf gene. Of note, sequencing of B-Raf in HEC1A cells revealed no mutations (although one allele harbors a polymorphism; Ref. 2 ). The frequency of B-Raf oncogenic mutations in endometrial cancer in general has not yet been reported in the literature. A human promoterless B-Raf targeting vector was constructed as described in "Materials and Methods" (Fig. 2A) . After transfection of the linearized B-Raf targeting vector, individual G418-resistant colonies were expanded, cryopreserved, and tested by PCR to identify cells with heterozygous deletion of B-Raf (data not shown). Heterozygous knockouts were then infected with a cre-expressing adenovirus to remove the integrated IRES-neo^R gene via cre-lox recombination. G418-sensitive B-Raf^+/-clones were then retransfected with the targeting vector, and B-Raf^-/- cells were identified by PCR (data not shown) and confirmed by Southern blot (Fig. 2B) . The absence of B-Raf protein was confirmed via Western blot (Fig. 2C) . Deletion of B-Raf did not lead to a measurable compensatory increase in expression or kinase activity of either A-Raf or C-Raf (Fig. 2C and data not shown).

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. B-Raf gene targeting. A, homologous recombination between the genomic locus and the targeting vector deletes exons VI and VII, replacing them with a promoterless IRES-neoR gene flanked by LoxP sites. PCR primers used for identification of knockouts are indicated, as are the restriction enzyme cleavage sites and probe used for Southern blot-based confirmation of knockouts. B, confirmation of B-Raf targeting by Southern blot analysis. Fragments corresponding to the wild-type allele and the targeted allele before and after cre-mediated excision of IRES-neoR are shown. C, Western blot analysis of HEC1A isogenic B-Raf+/+, B-Raf+/-, and B-Raf-/- cells. Immunoblotting was performed with the antibodies indicated. D, xenograft growth of HEC1A cells and derivatives with targeted homozygous deletion of B-Raf.

Maintenance of Tumorigenicity in B-Raf^-/- Cells.
Xenograft growth assays were performed to determine whether B-Raf was necessary for the tumorigenic potential of HEC1A cells (Fig. 2D) . HEC1A cells retaining oncogenic K-Ras but lacking wild-type B-Raf retained their ability to efficiently form xenograft tumors (in fact, B-Raf^-/- tumors grew slightly faster than B-Raf^+/+ tumors). The fact that deletion of oncogenic K-Ras virtually eliminated the tumorigenicity of HEC1A cells but that deletion of B-Raf did not reduce tumorigenicity led us to conclude that B-Raf is dispensable for K-Ras-mediated oncogenesis in HEC1A cells.

B-Raf Kinase Activity.
In light of the tumorigenicity data described above, we decided to test whether endogenous oncogenic Ras modulates the activation state of endogenous B-Raf. To date, this has been demonstrated exclusively in overexpression systems (11) . B-Raf kinase assays were performed in parental HEC1A cells and derivatives with targeted deletions of either oncogenic K-Ras or wild-type K-Ras (Fig. 3B) . Deletion of oncogenic (or wild-type) K-Ras failed to reduce B-Raf kinase activity in either serum-starved or EGF-stimulated cells. The absence of kinase activity in B-Raf^-/- cells demonstrated the specificity of the assay. These data suggest that endogenous oncogenic K-Ras does not control B-Raf kinase activity in HEC1A human cancer cells.

ERK1/2 Phosphorylation.
It is well established that ectopic overexpression of oncogenic Ras can lead to ERK1/2 activation. To test whether endogenous oncogenic K-Ras modulates ERK1/2 activation in HEC1A cells, Western blots with phospho-specific antibodies were performed to assess the activation state of ERK1/2 in HEC1A cells and gene-targeted derivatives (Fig. 3C) . After both serum starvation and EGF stimulation, there was more phosphorylated ERK1/2 in cells harboring a wild-type allele of K-Ras (i.e., HEC1A cells and derivatives with targeted deletion of oncogenic K-Ras) than in isogenic cells harboring only an oncogenic allele of K-Ras (i.e., HEC1A derivatives with targeted deletion of wild-type K-Ras). Similar findings have been previously reported for DLD1 colon cancer cells and K-Ras gene-targeted derivatives (12) . These results suggest that wild-type K-Ras more effectively transduces EGF-induced signals to ERK1/2 than does oncogenic K-Ras. Furthermore, they paradoxically suggest that the activity of wild-type K-Ras is dominant to oncogenic K-Ras in growth factor-induced signal transduction. Next, we tested whether endogenous B-Raf was required for ERK1/2 activation in HEC1A cells. To do this, Western blots with phospho-specific antibodies were performed to assess the activation state of ERK1/2 in isogenic B-Raf^+/+ and B-Raf^-/- cells (Fig. 3D) . EGF stimulation induced a significant increase in ERK1/2 phosphorylation that was similar in B-Raf^+/+ and B-Raf^-/- cells. These data demonstrate that B-Raf is not required to transduce EGF-induced signals to ERK1/2 in HEC1A cells.

K-Ras and B-Raf Transcriptomes.
Affymetrix U133A Genechips were used to measure the expression levels of 14,500 well-characterized human genes in HEC1A cells and isogenic gene-targeted derivatives. To do this, gene expression profiles were obtained for the following isogenic cell lines: HEC1A parental cells; a G418-resistant control cell line with random integration of the K-Ras targeting vector; two independently derived cell lines with deletion of oncogenic K-Ras; two independently derived cell lines with deletion of wild-type K-Ras; and duplicate preparations of HEC1A B-Raf^-/- cells. Probe preparation, hybridization, and data analysis were performed as described in "Materials and Methods."

A total of 143 genes (0.98%) was specifically down-regulated after deletion of oncogenic but not wild-type K-Ras (Fig. 4A–C) , and 143 genes (0.98%) were specifically up-regulated after deletion of oncogenic but not wild-type K-Ras (Fig. 4D–F) . Of the down-regulated genes, 34 (24%) were similarly down-regulated after deletion of B-Raf (Fig. 4A) , 7 (5%) were up-regulated after deletion of B-Raf (Fig. 4B) , and 102 (71%) were unchanged after deletion of B-Raf (Fig. 4C) . Of the up-regulated genes, 15 (10%) were similarly up-regulated after deletion of B-Raf (Fig. 4D) , 4 (3%) were down-regulated after deletion of B-Raf (Fig. 4E) , and 124 (87%) were unchanged after deletion of B-Raf (Fig. 4F) . Of note, the stringent criteria used to define up-regulation and down-regulation (see "Materials and Methods") has likely caused us to overestimate the number of genes regulated by oncogenic K-Ras but not by wild-type B-Raf (note the green boxes in columns 6 and 7 in Fig. 4C and the red boxes in columns 6 and 7 in Fig. 4F ). However, our data clearly demonstrate that a significant fraction of the genes regulated by oncogenic K-Ras do not require B-Raf for this modulation.

View larger version (87K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Expression analysis. Hierarchical clustering of genes specifically down-regulated (A–C) and up-regulated (D–F) after deletion of oncogenic K-Ras but not wild-type K-Ras. These genes were additionally stratified based on whether their expression change was concordant in oncogenic K-Ras and B-Raf knockout cells (A and D), discordant (B and E), or relatively unchanged in B-Raf knockout cells (C and F). Cluster diagram columns correspond to a HEC1A G418-resistant control cell line with random integration of the K-Ras targeting vector (column 1), two independently derived cell lines with deletion of wild-type K-Ras (columns 2 and 3), two independently derived cell lines with deletion of oncogenic K-Ras (columns 4 and 5), and duplicate preparations of HEC1A B-Raf-/- cells (columns 6 and 7). HEC1A parental cells are not depicted in the cluster diagram because they serve as the baseline for all comparisons. The color intensity reflects the magnitude of down-regulation (green) or up-regulation (red).

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Here, we demonstrate that the oncogenic properties of K-Ras do not require either B-Raf or activation of ERK1/2 in a human cancer cell line. Three types of data have led us to this conclusion. First, we have shown that although oncogenic K-Ras is required to maintain the tumorigenic potential of HEC1A cells, B-Raf is not. Second, we have demonstrated that deletion of oncogenic K-Ras attenuates the tumorigenic properties of HEC1A cells without diminishing B-Raf kinase or ERK1/2 activity. Third, we have identified a significant number of genes in which the regulation by oncogenic K-Ras does not require B-Raf.

These conclusions are consistent with a number of recent studies demonstrating that the Ral guanine-nucleotide exchange factor pathway, not the Raf/MEK/ERK pathway or the phosphatidylinositol 3-kinase pathway, is the critical effector of Ras-induced transformation in human cells (16) ; deletion of oncogenic K-Ras reduces the tumorigenic potential of DLD1 colon cancer cells without simultaneously reducing the activation state of ERK1/2 (8 , 12) ; and A-Raf and C-Raf are dispensable for Ras-induced transformation in mouse embryo fibroblasts (although such a study using B-Raf^-/- mouse embryo fibroblasts has not been reported; Refs. 4 , 17 ).

These data have potential implications for anticancer drug discovery efforts. A recent study convincingly demonstrated that specific siRNA-mediated inhibition of oncogenic B-Raf can revert the transformed properties of human cancer cells (18) . This suggested that pharmacological inhibition of B-Raf may be an effective anticancer strategy in tumors harboring oncogenic mutations of B-Raf. Our data suggest that pharmacological inhibition of B-Raf may be less efficacious in the significantly larger number of human tumors harboring oncogenic mutations of K-Ras instead.

Finally, the B-Raf^-/- cells described herein are, to our knowledge, the only human cell line currently available completely lacking expression of B-Raf. As such, they will likely prove useful for future studies addressing the role of B-Raf in cancer pathogenesis and for B-Raf targeted drug discovery efforts. Furthermore, the K-Ras and B-Raf targeting vectors we describe should make it possible to create and study additional isogenic sets of K-Ras and B-Raf-deficient human cancer cells to demonstrate the generalizability of these findings.

	ACKNOWLEDGMENTS

 
We thank Heather Crooks for characterizing the biological properties of HEC1A cells; Bert Vogelstein and Ben Ho Park for the cre-expressing adenovirus; John Sedivy for the IRES-neo construct; Carlos Benitez and Syid Abdullah for assistance with animal husbandry; Jianguo Yang and Xiaojun Zhou for assistance with microarrays; Henry Yang for DNA sequencing; Chip Dye for assistance with microscopy; and Tagvor G. Nishanian, Challice Bonifant, and Matthew Anderson for comments on the manuscript.

	FOOTNOTES

 
Grant support: NIH Grant K01 CA87828 (T. Waldman) and the Lombardi Cancer Center Support Grant (P30 CA51008). T. Waldman is a V Foundation Scholar and the recipient of a Career Development Award from the American Society of Clinical Oncology. C. Lee is an Umeko Strauss Scholar.

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: Todd Waldman, Lombardi Cancer Center, Georgetown School of Medicine, Research Building, Room E304, 3970 Reservoir Road, N.W., Washington, DC 20057. Phone: (202) 687-1340; Fax: (202) 687-7505; E-mail: waldmant{at}georgetown.edu

Received 12/10/03. Revised 1/23/04. Accepted 1/23/04.

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