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Proc Natl Acad Sci U S A. Jul 15, 2008; 105(28): 9697–9702.
Published online Jul 10, 2008. doi:  10.1073/pnas.0804709105
PMCID: PMC2453074

A genome-wide RNAi screen for Wnt/β-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer


The Wnt family of secreted proteins coordinate cell fate decision-making in a broad range of developmental and homeostatic contexts. Corruption of Wnt signal transduction pathways frequently results in degenerative diseases and cancer. We have used an iterative genome-wide screening strategy that employs multiple nonredundant RNAi reagents to identify mammalian genes that participate in Wnt/β-catenin pathway response. Among the genes that were assigned high confidence scores are two members of the TCF/LEF family of DNA-binding proteins that control the transcriptional output of the pathway. Surprisingly, we found that the presumed cancer-promoting gene TCF7L2 functions instead as a transcriptional repressor that restricts colorectal cancer (CRC) cell growth. Mutations in TCF7L2 identified from cancer genome sequencing efforts abolish its ability to function as a transcriptional regulator and result in increased CRC cell growth. We describe a growth-promoting transcriptional program that is likely activated in CRC tumors with compromised TCF7L2 function. Taken together, the results from our screen and studies focused on members of the TCF/LEF gene family refine our understanding of how aberrant Wnt pathway activation sustains CRC growth.

Keywords: cancer genome sequencing, colorectal cancer, functional genomics, TCF/LEF transcription factors, Wnt signal transduction

The Wnt/β-catenin signal transduction pathway is a global regulator of embryonic development (1, 2). In postembryonic animals, Wnt/β-catenin pathway activity sustains homeostatic tissue renewal but can be exploited in cancer to promote deviant cell growth (13). For example, pathway activity is necessary for maintenance of stem cells in the intestinal crypts (4) and, when misactivated, gives rise to familial and sporadic CRC (5). Indeed, >90% of CRCs are induced by loss-of-function mutations in the adenomatosis polyposis coli (APC) gene, which encodes a scaffolding protein that mediates constitutive destruction of the β-catenin transcriptional coactivator in the absence of Wnt ligand (6). Loss of APC function results in the accumulation of β-catenin and subsequent activation of a member of the T cell factor/lymphoid enhancer-binding factor (TCF/LEF) family of transcriptional effectors, presumably Tcf7l2 [formerly Tcf4 (79)].

Despite its importance to cancer, much of our genetic understanding of the Wnt/β-catenin pathway is based on developmental studies in model organisms. The relevance of these findings to human diseases often varies because of differences in gene function in developing and adult animals and the species-dependent sensitivity of tissues to pathway perturbation. For example, mice that have decreased APC function develop tumors primarily in the small intestines, whereas, in humans, loss of APC frequently results in cancer of the colon (1). Also complicating our understanding of human diseases from model organisms are differences in the redundancy of gene function observed between humans and other animals. For example, a single TCF family member (Pangolin) controls both transcriptional repression and activation of Wnt pathway target genes in Drosophila, whereas, in mammals, the TCF/LEF gene family consists of four members that are subject to alternative splicing and whose transcriptional roles are isoform dependent (10).

The advent of RNA-mediated interference (RNAi) has enabled rapid testing of gene function, using loss-of-function strategies in human cells derived from diseased tissues, and may allow us to bypass many of the issues associated with the use of model organisms to study human disease processes. Using a screening strategy designed to limit off-targeting effects that are commonly associated with the use of RNAi technology and genome-wide short interfering RNA (siRNA) libraries, we identified novel genes that contribute to mammalian Wnt/β-catenin pathway response and assigned new functions to previously described pathway components in CRC cells. Most importantly, our study reveals a tumor suppressor role for Tcf7l2 in CRC and a novel mechanism for Tcf7l2 action in normal and cancerous Wnt pathway response.


A High-Throughput RNAi Screen for Wnt Pathway Components Using Human Cells.

After testing several human cell lines for their responsiveness to Wnt ligand, using an extensively tested Wnt-inducible luciferase reporter construct (SuperTopFlash/STF reporter; Materials and Methods), we found that HeLa cells exhibited a robust and faithful response to Wnt3A [supporting information (SI) Fig. S1 A–C]. We incorporated this assay into a screening strategy that enabled us to monitor Wnt/β-catenin pathway response and aspects of general cellular functions in cells transiently transfected with chemically synthesized siRNAs and several reporter constructs (Fig. 1A). Using this approach, we first screened a human genome-scale siRNA library (Dharmacon) consisting of ≈21,000 pools of siRNAs, each containing four distinct siRNAs targeting a single gene (Fig. 1B and Fig. S1 D and E), an approach shown to be useful in gene discovery studies (11).

Fig. 1.
Overview of the RNAi-based screening strategy to identify components of the Wnt/β-catenin pathway. (A) The screening strategy. HeLa cells were cotransfected with siRNA pools targeting a single gene, and various reporter and expression constructs ...

Confounding the effective use of genome-scale RNAi screens has been the inability to discriminate between siRNAs that induce off-targeting effects or otherwise grossly affect general cellular functions, from those that target genes-of-interest (12). Directly relevant to this study is the prevalence of false positives resulting from off-targeting observed in a similar screen performed in Drosophila to identify Wnt pathway components (12, 13). To limit the influence of off-targeting in our screen, we retested 530 siRNAs of interest identified from the primary screen (Fig. 1B and Fig. S2; see Table S1 for screening data), using gene-specific siRNA pools from human and mouse genome-scale siRNA libraries developed by Qiagen in either HeLa or NIH 3T3 cells (Fig. 1B, Fig. S1F, Table S2, Table S3, and Table S4). Because these libraries were engineered by using a different algorithm for identifying targeting sequences than that used in the primary screen siRNA library, an observed effect on pathway by at least two siRNA pools are less likely to be due to off-targeting events (Fig. S3). Ultimately, seven known pathway components [Apc, Axin2, Tcf7, Tcf7l2, Ctnnb1 (encoding β-catenin), Bcl9l, and Nlk] were included among the highest scoring genes, thus confirming the utility of our screening strategy (Table 1).

Table 1.
The role of known and candidate Wnt/β-catenin pathway components in supporting CRC cell growth

To assign confidence values to candidate pathway components, we established a scoring system that takes into account the number of different human and mouse siRNA pools targeting the same gene that induce the same effect on pathway response (Fig. 1B). Additionally, the “exogenous Wnt test” eliminated those components that likely affected Wnt production. Thus, a maximal score of 3 (N in Table 1 and Table S2, Table S3, and Table S4) could be assigned to a gene if it was identified by using three different siRNA pools. All genes assigned a score are listed in Table S2, Table S3, and Table S4, whereas only the highest scoring hits from each screen are listed in Table 1. Because some known pathway components were not identified among the highest confidence hits (Fig. S4), genes with n < 3 may also be relevant to pathway response but failed to retest either as a result of differences in the effectiveness of siRNA pools or redundancy of gene function in the cells tested.

We performed epistasis-type experiments using overexpression of a Wnt receptor (Lrp6) or the transcriptional activator β-catenin to gain insight into the role of candidate genes within the pathway (Table 1 and Table S5). For example, loss of the novel secreted protein Tmem43 was fully rescued by expression of Lrp6, which suggests that Tmem43 functions in Wnt response at the cell membrane (Table 1, see also Table S5). We also tested whether the actions of some of our candidate positive regulators within the pathway are limiting by using overexpression studies in our cell-based reporter assay (Table 1, Table S2, and Table S3).

Identification of Wnt/β-catenin pathway components relevant to CRC.

Aberrant activation of the Wnt/β-catenin pathway resulting from inactivating mutations in APC is present in >90% of sporadic and familial CRCs (6). Less frequently, CRCs are associated with activating mutations in CTNNB1 (8). We tested the relevance of positive regulators of the Wnt pathway that are predicted to function at the level or downstream of Ctnnb1 in aberrant pathway response using STF reporter assays in two CRC cell lines that either harbor a mutation in APC (DLD-1 cells) or an activating mutation in CTNNB1 (HCT116 cells; Table 1 and Fig. S5). These genes include Ctnnb1, Bcl9l, Tcf7 (which encodes Tcf1), components of the Mediator complex (Surb7/Med21 and Pcqap/Pc2/Med15) that regulate RNA polymerase II activity, and the nuclear import factor karyopherin alpha 3 (Kpna3). Components of the Mediator complex have been shown to directly interact with β-catenin and mediate Wnt-dependent transcription of target genes such as AXIN (14). Of the genes that reduced aberrant Wnt pathway response in DLD-1 cells by >50% or more (Ctnnb1, Tcf7, and Med15), two specifically reduced DLD-1 growth (Ctnnb1 and Tcf7) in a clonal expansion assay that depends on Wnt pathway activity (9, 15). The identification of Tcf7 as a positive regulator in CRC is surprising given its suggested role as a tumor suppressor (16) but is consistent with the absence of TCF7 mutations in CRC tumors (6, 17).

We identified three candidate negative regulators of the Wnt/β-catenin pathway that enhanced aberrant Wnt/β-catenin pathway activity by more than threefold when targeted with siRNAs in DLD-1 and HCT116 cells (Fwbx10, Axin2, and Tcf7l2) (Table 1). Axin2 is a known suppressor of the Wnt/β-catenin pathway in CRC (18), whereas the F-box protein Fbwx10 likely contributes to ubiquitin-dependent destruction of β-catenin. However, the negative influence of Tcf7l2 on STF activity is surprising given its proposed role in promoting transcriptional activation of growth-promoting genes after loss of APC function in CRC (79). Notably, loss of Tcf7l2 results not only in a dramatic increase in STF reporter activity in CRC cells, but also an increase in cell growth potential. When taken together, these observations suggest that TCF7L2 should be assigned a tumor suppressor role in CRC.

CRC-Associated Mutations in TCF7L2 Abrogate Its Ability to Restrict CRC Cell Growth.

Given the strong association between the Wnt/β-catenin pathway and CRC, we identified potential tumor suppressors by focusing on genes in our candidate pathway suppressor dataset that harbor CRC-associated mutations (6, 17). The most potent pathway suppressor identified in our screen (APC), predictably harbored the largest number of CRC-associated mutations as identified from recent cancer genome sequencing results (Table 1). Surprisingly, the same studies also found a significant number of mutations in TCF7L2, a gene presumed to be necessary for activating growth-promoting target genes subsequent to APC loss (Table 1 and Fig. S6).

We investigated the impact of these CRC-associated mutations in TCF7L2 on its ability to restrict the growth of DLD-1 cells. Of eight CRC mutations in TCF7L2 previously identified (Fig. S6), we introduced four of these into an expression plasmid encoding the predominantly expressed form of Tcf7l2 in CRC cells, the “E” form (19) (Fig. 2A and Fig. S7). Three of these mutations abolish the ability of Tcf7l2 to suppress growth of DLD-1 cells (Fig. 2B). Unlike Mut1 and -2, which are likely to be prematurely terminated molecules, Mut4 harbors a single amino acid change in the DNA-binding domain (HMG box) and is readily expressed as a full-length molecule (Fig. 2C). Nevertheless, Mut4 fails to suppress DLD-1 cell growth, implying that the effects of overexpressing Tcf7l2 on cell growth depends on its ability to induce changes in the transcription of TCF target genes. The Mut3 protein behaves like a WT Tcf7l2 molecule, suggesting the mutation it harbors is irrelevant in cancer or that our reporter assay does not sufficiently measure all aspects of Tcf7l2 function.

Fig. 2.
TCF7L2 mutations identified in CRC abrogate Tcf7l2 pathway repressor function. (A) Predicated changes in Tcf7l2 E variant protein structure resulting from CRC mutations in the TCF7L2 gene (6, 17). NLS, nuclear localization signal; HMG box, high-mobility ...

We tested the influence of the same four CRC-associated mutations on the transcriptional activity of Tcf7l2, using the STF reporter. Expression of either Tcf7 or Tcf7l2, respectively, augmented or repressed STF activity in HeLa and DLD-1 cells, consistent with the roles that we had assigned to them (Fig. 2D). Of the four Tcf7l2 mutant proteins tested by using this assay, the three that failed to suppress DLD-1 cell growth also failed to repress STF activity suggesting that mutations in Tcf7l2 frequently result in loss of transcriptional repressor function (Fig. 2E). Taken together, our data suggest that a cell-growth promoting program is activated subsequent to acquisition of CRC-associated mutations in Tcf7l2.

A Repressor Domain in Tcf7l2.

Two distinct regions of Tcf7l2 family members are known to mediate interactions with the transcriptional corepressors Transducin-like enhancer of split protein [TLE or Groucho-related gene (Grg)], and C-terminal binding proteins (Ctbp) (Fig. 3A). Whereas the TLE binding domains are present in all TCF/LEF proteins, Ctbp interaction sequences are only present in the long C-terminal tail variants of Tcf7l2 (E forms), and Tcf7l1/Tcf3 (20). Indeed, exchanging the Tcf7 N-terminal sequence that includes the TLE domain for the corresponding sequence from Tcf7l2 is insufficient to confer repressor function to the DNA binding sequence of Tcf7 (Fig. 3A). However, fusing C-terminal protein sequence from Tcf7l2, which includes the entire E tail, transforms Tcf7 into a repressor of STF reporter activity (7–7L2). A Tcf7 protein that lacks the C-terminal sequence deleted in the Tcf7-Tcf7l2 fusion molecules exhibits WT levels of transcriptional activity (data not shown), arguing against the inadvertent deletion of transcriptional activation determinants in these fusion molecules. Although deletion of both Ctbp binding sites decreased the repressor capability of 7–7L2 (7–7L2Δ 3 and 7–7L2Δ 4), a remaining 41-aa sequence (amino acids 417–457) provided by Tcf7l2 was sufficient to confer some repressor ability on Tcf7 (Fig. 3 B and C). Interestingly, the sequence we have identified encompasses a DNA binding domain (the “cysteine clamp”), which functions with the HMG domain to confer target gene specificity to TCF family members (21) and is frequently altered in colon cancers with microsatellite-instability (22). Nevertheless, targeting of both Ctbp1 and 2 with RNAi resulted in an increase in cellular responsiveness to Wnt3A consistent with previous observations that the Ctbp binding sequences in Tcf7l2 contribute to its repressor function (Fig. 3D). Thus, we conclude that the cysteine clamp sequence in additional to other protein determinants in Tcf7l2 contribute to its repressor function.

Fig. 3.
A repressor sequence in Tcf7l2. (A) C-terminal sequences in Tcf7l2 confer repressor function on Tcf7. C-terminal sequences containing the Ctbp binding sites (7–7L2) but not sequences containing the TLE binding domain (7L2–7 #1 and 7L2–7 ...

The Functions of Tcf7l2 and Tcf7 in the Wnt Pathway Are Cell-Type Specific.

Our genetically based interrogation of Tcf7l2 and Tcf7 function in a variety of human cell lines suggests that these transcriptional effectors often have contrasting regulatory roles within the Wnt pathway (Fig. 4A). In the case of Tcf7l2, its transcriptional repressor function in human CRC cells appears to differ from that of an activator as described in the mouse small intestinal tissue (4). We investigated whether or not the basis for this difference is species specificity in Tcf7l2 function. We observed contrasting roles for Tcf7l2 in two different mouse cell lines suggesting that the function of Tcf7l2 is cell-type but not necessarily species specific (Fig. 4B). Consistent with the requirement for the E tail in Tcf7l2-mediated repressor function, the predominant form of Tcf7l2 expressed in L-cells is likely the “B” form that lacks this sequence (Fig. 4C). The biochemical and genetic evidence presented here supports a repressor role of Tcf7l2 that is not only dictated by the presence of an E tail sequence but likely the presence of effector(s) that act on this sequence. Our assignment of Tcf7 as positive transcriptional regulator of Wnt/β-catenin pathway response in CRC cells is likewise different from its assigned role as a tumor suppressor (16). In contrast to the mouse small intestinal epithelium, where Tcf7 is expressed as a protein that lacks the β-catenin binding domain (16), the major isoform of Tcf7 expressed in several human cell lines appears to contain this domain, consistent with its positive transcriptional role in these cells (Fig. 4 D and E).

Fig. 4.
The function of TCF family members is cell-type-specific. (A) Tcf7l2 functions as a transcriptional repressor in many cell types. Effects of decreased Tcf7l2 function on normal Wnt pathway response were measured by using the STF reporter in HeLa and HEK293T ...

Tcf7l2 Repressor and Positively Acting TCF/LEF Family Members Control Distinct Transcriptional Programs in CRC.

Our analysis of Tcf7l2 function, using the STF reporter, suggests that the increase in DLD-1 cell growth upon loss of Tcf7l2 may be attributable to the activation of a growth-promoting transcriptional program. We first used genome-wide expression analysis to identify potential Tcf7l2 target genes (Fig. 5A). Next, we determined whether our candidate Tcf7l2 target genes might also be controlled by positively acting TCF family members by comparing expression profiling data from the cell lines lacking Tcf7l2 with those lacking β-catenin, an essential coactivator of TCF-mediated transcription (Fig. 5A). Although we detected some overlap in these datasets, none of the genes sensitive to both RNAi-based manipulations were previously identified TCF targets. Indeed we were able to observe transcriptional changes of known or predicted TCF target genes, such as Myc and Lef1, only in cells treated with Ctnnb1 but not Tcf7l2 siRNAs despite the effectiveness of both sets of siRNA reagents (Fig. 5 A and B; see Table S6 and Table S7). The absence of known TCF target genes that exhibit altered expression levels upon loss of Tcf7l2 likely reflects the reliance of previous studies on dominant-negative TCF proteins to assign target genes to individual TCF proteins (23). Given the similarities of TCF/LEF family members in DNA-binding specificity, those strategies likely would broadly identify TCF target genes rather those specific to each TCF/LEF family member.

Fig. 5.
The cellular program controlled by Tcf7l2 in CRC cells. (A) Positively acting TCF molecules and Tcf7l2 repressor control distinct target genes. Gene expression profiling identified genes regulated by positively acting TCF molecules in Ctnnb1 siRNA-treated ...

Our gene expression analysis also revealed that transcriptional levels of known pathway components are not affected by loss of Tcf7l2 (Fig. 5B, Table S6, and Table S7). Some of these observations were also confirmed by using Western blot analysis (Fig. 5C). Based on these observations, Tcf7l2 likely does not modulate Wnt pathway activity by altering the expression levels of known Wnt pathway components.

We identified among those genes that were up-regulated upon loss of Tcf7l2, two genes that harbor conserved TCF binding enhancer elements in their respective promoters [hepatocyte growth factor (HGF) and the homologue of Drosophila morphogenetic factor headcase (HECA)] (Fig. 5 D and E). However, none of the genes mutually down-regulated upon loss of Ctnnb1 or Tcf7l2 are known or predicted TCF target genes (Fig. 5A). Interestingly, HGF has been shown to promote cell cycle entry in CRC cells (24). Indeed, loss of Hgf or Heca induced by RNAi abrogated the growth promoting effects of Tcf7l2 siRNAs on CRC cells (Fig. 5 F and G). When taken together, our observations suggest that HGF and HECA are a part of a cellular program controlled by Tcf7l2 that regulates CRC cell growth.


Our assignment of TCF7L2 as a tumor suppressor is based on the following: (i) the strong genetic evidence that aberrant Wnt pathway activity is causal to CRC, (ii) loss of Tcf7l2 function enhances, whereas gain of Tcf7l2 function suppresses CRC cell growth, and (iii) CRC mutations identified in primary tumor samples result in the production of Tcf7l2 proteins that are unable to function as transcriptional repressors of cell growth-promoting genes, such as HGF and HECA. The roles for TCF7L2 that we have uncovered in CRCs are different from those described in previous studies. These discrepancies are likely due to the reliance of these prior studies on the use of dominant-negative TCF proteins in lieu of loss-of-function strategies, and possibly the interpretation of phenotypic outcomes of genetically based studies in model organisms. Expression of dominant-negative TCF proteins (proteins lacking β-catenin binding domains) likely suppresses TCF/LEF target gene transcription and growth of CRC cells regardless of their normal cellular functions (9). Also, mice lacking TCF7L2 are able to establish stem cells in the small intestines but cannot maintain them, a defect presumably caused by the loss in expression of Wnt target genes that sustain stem cell self-renewal (4). Based on our observations and recent evidence suggesting that hyperactivation of the Wnt pathway results in loss of stem cells in many tissues (25, 26), the defects observed in TCF7L2 mice could also be caused by stem cell depletion due to de-repression of TCF7L2 target genes. Alternatively, our observations could be explained by differences in the function of TCF molecules in the mouse small intestines and human colon. Indeed, mice lacking TCF7L2 exhibit a loss of homeostatic renewal in the small intestinal epithelium but not in the colon, where most sporadic cancers of the human gastrointestinal tract arise (27).

Using our loss-of-function strategy, we have identified at least two growth-promoting genes that are negatively regulated by Tcf7l2 in CRC cells, HGF and HECA. Increases of either gene product in the serum (28) or feces (29) of CRC patients correlate with poor prognosis. Our findings predict that chemical inhibitors that inhibit the receptor of HGF, the Met receptor kinase, might be particularly useful in tumors with decreased Tcf7l2 function. Interestingly, HGF is known to activate the Wnt pathway by binding to its receptor (the proto-oncogene Met), which in turn induces β-catenin phosphorylation and nuclear localization (24). Thus, long-lasting consequences of Tcf7l2 loss may also include transcriptional activation of Wnt/β-catenin pathway target genes controlled by positively acting TCF molecules.

In light of our findings, the simplest model for normal Wnt/β-catenin response in cells that harbor Tcf7l2 repressor activity is that simultaneous activation of Tcf7 and inactivation of Tcf7l2 proteins is necessary for optimal transcriptional responses to Wnt proteins (Fig. 5H). Although it is unclear at the moment how Wnt proteins might regulate the Tcf7l2 repressor protein, Nemo-like kinase (NLK) may play a role in this regard, because it appears to be a positive mediator of pathway response in at least two cell lines (Table 1) and phosphorylates a consensus sequence found in Tcf7l2 and not Tcf7 (20). In CRC, loss of APC and decreased TCF7L2 function (the mutations analyzed were heterozygous) (Fig. S6) activate two growth-promoting transcriptional programs that result in tumors with more aggressive behavior than that observed in those with only APC mutations (Fig. 5H). Augmentation of Tcf7l2 function or targeting of growth-promoting genes regulated by Tcf7l2 might be therapeutically useful in CRC and possibly breast cancers, which frequently display decreased expression of Tcf7l2 (30).

Although important cell fate determination pathways such as the Wnt/β-catenin pathway are intensely studied, a molecular understanding of their deviant activity in cancer remains incomplete. Further analysis of these pathways, using genome-scale RNAi coupled with data derived from cancer genome sequencing efforts, should allow rapid identification of candidate cancer genes in a broad range of cancers. Ultimately, this integrated approach should improve our ability to realize mechanism-based therapeutic strategies and to personalize treatment regiments based on detailed knowledge of a tumor's molecular underpinnings.

Materials and Methods

Primary screen in HeLa cells.

Approximately 12,000 HeLa cells were plated in 96-well culture plates that contained Effectene (Qiagen) transfection mixes, using all or some the following reagents: (i) control or Wnt3A expression construct; (ii) siRNAs; and (iii) STF, GL, and RL control reporter constructs. Each well received 3 pmol of siRNA. Two days after transfection, culture medium (for GL activity) and lysate (for FL and RL activity) were collected and analyzed for relevant luciferase activities, using either Dual Luciferase kits (Promega) or coelenterazine. The average Wnt induced signal in each screening run (≈800 siRNA pools tested) was at least 20-fold above negative controls. See SI Materials and Methods for criteria used to identify hits.

Secondary Screens.

Secondary screening assays were performed essentially as described above except in a 48-well format, using Effectene transfection reagent and a 3-day posttransfection incubation period either in duplicate or triplicate. For assays involving conditioned medium, cells were transfected with siRNAs 48 h before exchanging normal for medium conditioned from cells producing Wnt3A or control cells. Cut-offs used to identify hits were based on percentage pathway response compared with controls indicated in Fig. 1B and Table S2. For cell growth assays, DLD-1 or HeLa cells were transfected in 96-well format and split two days later to clonal density in 10-cm2 plates (≈12,000 cells). Four days later, cells were trypsin harvested and cellular ATP levels measured by using Cell Titer Glo assay (Promega).

Cell Lines, siRNA Libraries, DNA Constructs, Antibodies, Gene Expression Profiling, and RT-PCR.

See SI Materials and Methods.

Supplementary Material

Supporting Information:


We thank Bert Vogelstein for sharing data before publication; Woodring Wright, Andras Roig, Leanne Jones, Rueyling Lin, and members of the L.L. laboratory for useful discussions; Randall Moon, Jorge Laborda, Raphael Kopan, Gail Johnson, and Marian Waterman for constructs; and Leni Jacob and Baozhi Chen for technical assistance. This work was supported by the National Institutes of Health, the American Cancer Society, the Welch Foundation, and an endowment from Virginia Murchison Linthicum.


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0804709105/DCSupplemental.


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