Ras Stimulation of E2F Activity and a Consequent E2F Regulation of Integrin α₆β₄ Promote the Invasion of Breast Carcinoma Cells

Active Ras proteins play key roles in cell invasion and metastasis (1, 2). In normal cells, extracellular stimuli, such as growth factors, transiently activate Ras proteins through the activation of cell surface receptors. Activated Ras proteins, in turn, regulate multiple downstream effectors that modulate cell proliferation, survival, and differentiation. In contrast to normal cells, many carcinoma cells have aberrantly activated Ras proteins that result from mutations in the ras genes. Ras mutations are found in 90% of pancreatic cancers, 50% of colon cancers, and 50% of thyroid cancers (3). Of interest, sustained activation of Ras is observed in a significant fraction of invasive breast carcinomas but ras mutations in these tumors are relatively infrequent (3). It is thought that constitutive signaling through growth factor receptors, such as the epidermal growth factor (EGF) receptor family, sustains Ras activation in many breast tumors (4).

Breast carcinoma cells express H-Ras, K-Ras, and N-Ras. H-Ras has been linked to their invasion, migration, and proliferation (5, 6). Amplification of the K-Ras gene is associated with mammary tumor progression and N-Ras has been linked to anchorage-independent growth and proliferation (6, 7). The best-characterized downstream targets of Ras that influence carcinoma progression are Raf/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/AKT (1, 8). Ras binds to and stimulates Raf, which activates the mitogen-activated protein/ERK kinase (MEK)/ERK pathway. ERK activity is higher in metastatic cancer cells compared with nonmetastatic cancer cells. ERK activates a host of transcriptional factors, including activating protein-1 (AP1), which increases extracellular matrix-degrading enzymes, such as matrix metalloproteinases (MMP) and urokinase-type plasminogen activator (1, 9). Ras also regulates PI3K kinases that regulate phosphoinositide lipid metabolism and activate AKT, a kinase that affects the migration and survival of carcinoma cells. Among its many functions, AKT can activate the small GTPase Rac and increase transcription factor nuclear factor-κB-dependent MMP secretion (1, 10). In addition to Raf/ERK and PI3K/AKT, Ras proteins also regulate other molecules that are important for carcinoma invasion. Ras increases expression of the hepatocyte growth factor/scatter factor (HGF/SF) receptor, Met, possibly by a mechanism that involves the Ets-1 transcription factor. HGF/SF and Met are often overexpressed in metastases and aberrant Met-HGF/SF signaling increases motility and invasion (1, 11).

A theme that emerges from the existing literature is that Ras can regulate the expression and activity of specific transcription factors that have been implicated in cancer progression. In this direction, we were intrigued by the report that Ras can also regulate the expression of E2F (12). The E2F family of transcription factors plays a pivotal role in cell proliferation by regulating the expression of genes involved in G₁-S transition and DNA synthesis (13). Among the E2F family, E2F1, E2F2, and E2F3 are potent transcriptional activators. E2F4 and E2F5, in contrast, mediate the active repression of E2F-responsive genes by recruiting the pocket proteins. In addition to their roles in G₁-S transition and DNA synthesis, E2Fs regulate a variety of genes that are involved in mitosis, chromosome segregation, mitotic spindle check points, DNA repair, chromatin assembly/condensation, differentiation, and development (14). Little is known, however, about the relationship between E2F and invasion. Recent studies indicate that there is a trend toward increased E2F1 expression in metastatic progression of colorectal carcinoma (15), and E2F3 is frequently amplified and overexpressed in invasively growing bladder cancer (16).

In this study, we examined the hypothesis that one mechanism by which Ras facilitates breast carcinoma invasion is by increasing E2F expression and activity. Moreover, we postulated that the active E2Fs (E2Fs1-3) stimulate invasion. The data we obtained substantiate this hypothesis. In addition, we provide evidence that the active E2Fs can regulate expression of the integrin α₆β₄, which has been implicated in breast carcinoma invasion, and that this E2F-regulation of α₆β₄ is dependent on active Ras.

Cells and reagents. SUM-159 breast carcinoma cells were obtained from Dr. Steve Ethier (Karmanos Cancer Institute, Detroit, MI). Cells were maintained in Ham's F12 supplemented with 5% fetal bovine serum (FBS), 5 μg/mL insulin, 1 μg/mL hydrocortisone, 100 units/mL penicillin, and 100 μg/mL streptomycin. β₄ short hairpin RNA (shRNA)-pSuper.Retro and β₄ Scr-pSuper.Retro vectors were generated, and these were stably expressed in SUM-159 cells as described previously (17). The sorting and subsequent surface labeling analysis of the β₄-deficient cell line was done with 3E1, a mouse anti-β₄ integrin antibody (Chemicon). Decreased β₄ expression in this cell line was confirmed by immunoblotting using a rabbit polyclonal β₄ antibody. MDA-MB-231 breast carcinoma cells were obtained from the Lombardi Breast Cancer Depository at Georgetown University. Cells were maintained in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. A function-blocking integrin α₆ monoclonal antibody (GoH3) was obtained from Immunotech and an actin antibody was purchased from Sigma.

Ras assay. Ras activity was measured using a glutathione S-transferase (GST) fusion protein containing the Ras-binding domain (RBD) of Raf-1 as described (18). The GST-Raf1-RBD construct was kindly provided by Dr. Johannes L. Bos (University Medical Centre Utrecht, Utrecht, the Netherlands). The plasmid was transformed into Escherichia coli strain BL21 and protein production was initiated by adding isopropyl-d-thiogalactopyranoside to the cultures. Bacteria were resuspended in sonication buffer [20% sucrose, 10% glycerol, 50 mmol/L Tris-HCl (pH 8.0), 2 mmol/L DTT, 2 mmol/L MgCl₂, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL leupeptin, and 2 μg/mL aprotinin] and lysed by sonication. The lysate was centrifuged at 12,000 × g for 1 hour in the cold. Bacterially produced GST-Raf1-RBD was precoupled to glutathione-agarose beads and washed in cell lysis buffer [1% NP40, 10% glycerol, 50 mmol/L Tris (pH 7.4), 200 mmol/L NaCl, 2.5 mmol/L MgCl₂, 1 mmol/L PMSF, 2 mmol/L sodium orthovanadate, 1 μg/mL leupeptin, 2 μg/mL aprotinin, 10 μg/mL trypsin inhibitor, and 10 μg/mL NaF]. The cells were extracted in the cell lysis buffer and cleared extracts were split and used for the determination of Ras activity or for the analysis of protein expression. For Ras assay, cleared extracts were incubated with the beads-GST mixture for 45 minutes. The bead mixtures were then washed four times with cell lysis buffer and bound proteins were eluted with SDS-PAGE sample buffer. Samples were separated by SDS-PAGE (12.5% polyacrylamide), blotted, and probed with a Ras antibody.

Invasion assays. The upper chambers of Transwells (Corning Costar) were coated with 0.5 μg Matrigel (Collaborative Research, Bedford, MA) that had been diluted with cold water, and allowed to air dry. Subsequently, the coated Transwell membranes were incubated with DMEM for 1 hour and 5 × 10⁴ cells were plated in the upper chambers. The lower chambers contained NIH-3T3 conditioned medium. The inserts were incubated for 3 hours. The cells that had invaded the lower surface of the membrane were fixed with methanol and stained with 0.2% crystal violet in 2% ethanol. The number of cells that had invaded was quantified using a light microscope equipped with a reticle. For the detection of green fluorescent protein–positive cells, invaded cells were fixed with formaldehyde and quantified using a fluorescence microscope.

Plasmids and transfections. Dominant-active H-ras (V12 H-ras) and dominant-negative H-Ras (N17 H-Ras) were kindly provided by Dr. Kun-Liang Guan (University of Michigan, Ann Arbor, MI). The [E2F]X4 luciferase reporter and DNA-binding mutant E2F construct (E2F1-E132) were kind gifts of Dr. Young-Chae Chang (Catholic University, Daegu, South Korea) and Dr. Joseph R. Nevins (Duke University, Durham, NC), respectively. Cells were transfected with LipofectAMINE 2000 reagent (Invitrogen) according to the protocol of the manufacturer.

Luciferase assay. Cells were cotransfected with a pGL3-[E2F]X4 luciferase reporter plasmid and a control renilla luciferase construct using LipofectAMINE 2000. After 36 hours, luciferase activity was measured according to the instruction of the manufacturer (Promega) using a luminometer.

Analysis of RNA expression. Reverse transcription-PCR (RT-PCR) and Northern blot analysis were done for RNA analysis. Total cellular RNA was purified from cultured cells using the RNeasy mini kit (Qiagen) following the protocol of the manufacturer. For RT-PCR analysis, a one-step RT-PCR kit (Qiagen) was used. For Northern blot analysis, RNA were electrophoresed on 1% agarose gels containing 6% formaldehyde and transferred to Hybond-N membrane (Amersham Biosciences) by capillary transfer. The membrane was fixed using an optimized UV cross-linking procedure. Prehybridization and hybridization were done at 68°C in ExpressHyb hybridization solution (Clontech). cDNA probes were labeled with [³²P]dCTP (3000 Ci/mmol, Perkin-Elmer) using a random primer kit (New England Biolabs). The blot was then washed twice with 20× SSC [300 mmol/L NaCl, 30 mmol/L sodium citrate (pH 7.0)] containing 0.05% SDS at 25°C, and 0.1× SSC containing 0.1% SDS at 55°C in order, and autoradiographed at −70°C.

Analysis of protein expression. Cells were analyzed for their expression of specific proteins by either flow cytometry or immunoblotting. For flow cytometric analysis, cells were washed twice with ice-cold PBS containing 0.2% bovine serum albumin (BSA). Aliquots of cells were incubated for 1 hour at 4°C with antibodies in the PBS/BSA solution. The cells were washed thrice with PBS/BSA and then incubated with secondary antibodies coupled to R-phycoerythrin for 1 hour at 4°C. After washing thrice with PBS/BSA, the cells were resuspended in PBS and analyzed using a FACScan (Becton Dickinson).

For immunoblot analysis, the cells were extracted in a buffer [20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L EGTA, 10% glycerol, 1 mmol/L sodium orthovanadate, 1 mmol/L NaF, 2 mmol/L PMSF, 2 mg/mL aprotinin, 2 mg/mL leupeptin, and 1 mg/mL pepstatin], and samples were resuspended in reducing buffer [5×: 60 mmol/L Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 14.4 mmol/L 2-mercaptoethanol, and 0.1% bromophenol blue]. These were boiled for 5 minutes and electrophoresed by SDS-PAGE. Proteins were then transferred to Hybond-ECL (Amersham Biosciences). The membranes were blocked with a TBST [25 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, and 0.05% Tween 20] containing 5% nonfat dried milk or 5% BSA and probed with primary antibodies for overnight and secondary antibodies coupled to peroxidase for 1 hour. Blots were developed using the enhanced chemiluminescence system (Amersham Biosciences).

Apoptosis assays. Cells were washed once with serum-containing medium, once with PBS, once with Annexin V-FITC buffer [10 mmol/L HEPES-NaOH (pH 7.4), 140 mmol/L NaCl, and 2.5 mmol/L CaCl₂], and then incubated for 15 minutes at room temperature with 5 μg/mL Annexin V-FITC (Biosource International). After washing once with Annexin V buffer, the samples were resuspended in the same buffer. Immediately before analysis, 5 μg/mL propidium iodide (Biosource International) was added to distinguish apoptotic cells from necrotic cells, and the cells were analyzed using a flow cytometer.

Cell adhesion assays. Six-well plates were coated with type I collagen, fibronectin, or laminin-1. Nonspecific binding was blocked by PBS containing 2% BSA for 2 hours at room temperature. Cells were then plated on coated culture plates and incubated for 30 minutes. Plates were washed twice with PBS and adherent cells were fixed with either methanol or formaldehyde.

Cell surface biotinylation and immunoprecipitation. Cells were washed with PBS and HEPES buffer [20 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L KCl, 0.8 mmol/L MgCl2, 1.0 mmol/L CaCl₂ (pH 7.45)]. The cells were then incubated on ice with HEPES buffer containing 0.5 mg/mL EZ-Link Sulfo-NHS-LC-Biotin (Pierce) for 30 minutes. Cells were washed thrice with HEPES buffer and extracted with radioimmune precipitation (RIPA) buffer [50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 10 mmol/L EDTA, 1% NP40, 0.1% SDS, 2 mmol/L PMSF, 5 μg/mL aprotinin, leupeptin, and pepstatin] for 15 minutes. After centrifugation, the supernatants were collected and the total protein concentration was determined. The extracts were preabsorbed with IgG and protein G-Sepharose (Amersham Biosciences.). Immunoprecipitation was done with β₁ (MC13), α₆ (GoH3), and β₄ (439-9B) antibodies. Immunocomplexes were precipitated with protein G-Sepharose, washed four times with RIPA buffer, and eluted with 2× reducing SDS-PAGE sample buffer for α₆ and β₄ integrin or 2× nonreducing sample buffer for β₁ integrin. Samples were separated by SDS-PAGE, blotted, and probed with peroxidase-conjugated streptavidin.

H-Ras increases carcinoma invasion through an E2F-dependent pathway. A significant fraction of human breast carcinomas harbor activated Ras (3) and this characteristic extends to human breast carcinoma cell lines. As shown in Fig. 1A, SUM159 and MDA-MB-231 cells, which possess properties associated with invasive breast carcinoma, exhibit a significantly higher level of constitutive Ras activation than MCF10A cells, a breast cell line considered to be relatively normal. The importance of active H-Ras for invasion was verified by expressing a dominant-negative (N17) H-Ras in SUM-159 cells. Expression of this construct inhibited the ability of these cells to invade Matrigel significantly (Fig. 1B). Conversely, expression of a constitutively active (V12) H-Ras increased invasion (Fig. 1B).

To assess the mechanism by which H-Ras promotes invasion, we focused on the relationship between H-Ras and the E2F transcription factor because a previous report had indicated that H-Ras can stimulate the expression of transcriptionally active E2F (12). Indeed, we verified this report by showing that expression of dominant-active H-Ras in SUM-159 cells increased E2F-1 mRNA, protein, and activity as assessed by RT-PCR, immunoblot analysis, and E2F-dependent reporter gene expression, respectively (Fig. 2A).

Given that H-Ras stimulates both the expression of active E2F and invasion, we assessed a possible role for E2F in H-Ras-mediated invasion. The first approach involved the use of a DNA-binding mutant of E2F that inhibits its transcriptional activity. As shown in Fig. 2B, expression of this mutant E2F impeded the ability of dominant-active H-Ras to stimulate invasion significantly. This E2F mutant was also effective in inhibiting the invasion of MDA-MB-231 cells (Fig. 2C). As an alternative approach to examining the contribution of the E2Fs to invasion, we expressed each of the active E2Fs1-3 and the repressor E2Fs4-5 individually in SUM-159 cells (Fig. 3A). We also assessed the activity of these expressed E2Fs using a reporter construct containing E2F binding sites. Only the active E2Fs1-3 increased E2F activity markedly. Expression of either E2F4 or E2F5, in contrast, did not affect E2F activity significantly (Fig. 3B). The increase in E2F activity that resulted from expression of E2Fs1-3 correlated with a significant increase in Matrigel invasion (Fig. 3C). No such increase in invasion was observed in cells expressing E2Fs4-5. Of note, expression of the E2Fs did not affect apoptosis (Fig. 3D).

E2Fs1-3 facilitate adhesion to laminin and Matrigel invasion by increasing expression of the integrin α₆β₄. Adhesion to matrix proteins is a key component of the invasive process (19). For this reason, we assessed the effect of increasing E2F1 expression on the ability of SUM-159 cells to adhere to type I collagen, fibronectin, and laminin-1. As shown in Fig. 4A, expression of E2F1 significantly increased cell adhesion to laminin-1 compared with either collagen or fibronectin. Similar results were obtained by increasing the expression of either E2F2 or E2F3 (Fig. 4B). Increasing the expression of either E2F3 or E2F4, however, did not influence adhesion to laminin (Fig. 4B).

The finding that expression of the active E2Fs increased adhesion to laminin, as well as invasion through Matrigel, is intriguing in light of the report that E2F null keratinocytes exhibit defects in adhesion to basement membranes and diminished expression of the α₆β₄ integrin, a laminin receptor (20). We examined, therefore, whether the E2Fs could regulate integrin β₄ expression in SUM-159 cells. As shown in Fig. 4C, increasing the expression of E2F1, E2F2, or E2F3 resulted in a significant increase in expression of β₄ mRNA and protein. Moreover, these increases were manifested in increases in β₄ surface expression as assessed by flow cytometry (Fig. 4D). Consistent with our functional data, increasing the expression of E2F4 and E2F5 did not alter β₄ expression (Fig. 4C and D).

The above findings prompted us to examine the possibility that E2Fs may increase carcinoma invasion by a mechanism that involves α₆β₄. For this purpose, we used a retrovirus containing a shRNA to deplete β₄ expression. This approach, which we described previously (17), resulted in the generation of an integrin β₄-RNA interference cell line that exhibited a substantial reduction in total and cell surface integrin β₄ expression (Fig. 5A). When we increased the expression of E2Fs1-3 in integrin β₄-shRNA cells, we did not detect a significant increase in β₄ mRNA expression or an increase in invasion as we did in the parental cells (Fig. 5B). We also examined the ability of an antibody specific for the α₆ integrin to impede the invasion of cells transfected with E2Fs1-3. This antibody caused an approximate 30% decrease in invasion (Fig. 5C). SUM-159 cells have little, if any, α₆β₁, as evidenced by immunoprecipitation and immunoblot analysis of integrin α₆, β₄, and β₁ after biotinylation of cells (Fig. 5D). Specifically, no β₁ integrin was detected in α₆ immunoprecipitates, but α₆ was detected in β₄ immunoprecipitates. These data indicate that α₆β₄ is the predominant α₆ integrin expressed in SUM-159 cells and that it mediates E2F-induced invasion.

H-Ras promotes carcinoma invasion by E2F-dependent regulation of α₆β₄. Given our results that H-Ras can regulate E2F activity and that E2F can regulate β₄ expression, we hypothesized that H-Ras can influence the expression of β₄ in SUM-159 cells. Indeed, dominant-negative H-Ras decreased and dominant-active H-Ras increased integrin β₄ mRNA and protein levels (Fig. 6A). To assess whether H-Ras regulates β₄ mRNA expression through an E2F-dependent pathway, we used a DNA-binding mutant of E2F. As shown in Fig. 6B, dominant-active H-Ras did not increase β₄ mRNA expression in the presence of this mutant. In addition, this E2F mutant decreased the expression of integrin β₄ protein in MDA-MB-231 cells (Fig. 6C). Dominant active H-Ras did not increase invasion of SUM159 cells significantly in the presence of an α₆ function blocking antibody (Fig. 6D), indicating that integrin is important for H-Ras-mediated invasion.

The data reported here highlight H-Ras-mediated regulation of E2Fs as a potential mechanism by which H-Ras promotes tumor invasion. Moreover, they indicate that H-Ras can stimulate the E2F-dependent expression of the α₆β₄ integrin, which is known to promote the invasion of carcinoma cells. Our conclusions are substantiated by the fact that the repressor E2Fs (E2F4 and E2F5) were unable to stimulate either expression of α₆β₄ or invasion. This pivotal role for E2Fs of linking H-Ras to α₆β₄ and invasion differs considerably from most studies on E2Fs that have implicated them in the transcriptional regulation of proteins involved in cell cycle regulation and apoptosis.

A key issue that derives from our data is the mechanism by which H-Ras stimulates E2F activity. Our data indicate that active H-Ras enhances the expression of E2F-1 mRNA and transcriptionally active protein, a finding that substantiates a previous report that Ras can increase expression of transcriptionally active E2F-1 by stabilizing E2F-1 mRNA (12). The mechanism by which Ras stabilizes E2F-1 mRNA seems to involve MEK and Akt, and it is independent of Rb, which is known to bind to and inhibit E2F in its unphosphorylated state. There is also evidence that Ras can induce E2F expression by activating c-myc, which can induce E2F transcription (21). It will be of interest to test whether Ras-induced E2F activation is mediated by c-myc because this oncogene is frequently activated in invasive breast cancer and it is associated with high nuclear grade, lymph node metastasis, and poorer disease outcome (22). In addition, there is evidence that c-myc can regulate the expression of α₆β₄ (see below).

Our finding that increased expression of E2Fs in SUM-159 cells can stimulate their ability to invade meshes with previous work that implicated these transcription factors in the oncogenic properties of breast cancer cells. For example, E2F1 negatively regulates the tumor suppressor gene ARHI in breast cancer cells, and these cells have elevated E2F DNA-binding activity compared with normal breast epithelial cells (23). Moreover, E2F1 expression is significantly higher in invasive ductal carcinomas and ductal carcinoma in situ than in normal breast (24) and it is also associated with poor survival of lymph node–positive patients (25).

Our observation that increased expression of E2F enhances expression of the α₆β₄ integrin suggests a novel E2F target, the β₄ integrin subunit. Of interest, E2F-1(−/−) keratinocytes exhibit markedly reduced integrin β₄ expression, as well as impaired migration and decreased adhesion to extracellular matrix proteins (20). The existing data, therefore, indicate quite strongly that E2F can regulate expression of the β₄ integrin subunit. It does not seem, however, that E2F mediates β₄ transcription directly because the β₄ promoter lacks E2F binding sites (20). Most likely, this regulation is indirect and remains to be elucidated. Nonetheless, other targets of E2F may also promote invasion and metastasis. Stanelle et al. (26) reported that E2F1 increases the expression of mRNA for the membrane-type metalloproteinase 16, which belongs to the group of MT-MMPs that activate MMP-2, a key regulator of invasion and metastasis. E2F1 can also induce expression of the mRNA for vascular endothelial growth factor-B (VEGF-B; ref. 26). VEGF-B is expressed in most breast carcinomas and its expression in human primary breast cancers is associated with lymph node metastasis (27, 28).

The fact that the α₆β₄ integrin seems to be a facilitator of E2F-mediated invasion adds to the numerous reports that have implicated this integrin in the invasion of carcinoma cells (29–32). Interestingly, α₆β₄ expression is often enhanced in carcinoma cells and there is compelling evidence that α₆β₄ promotes the formation of some carcinomas, as well as the migration, invasion, and survival of carcinoma cells (29–33). An emerging consensus is that α₆β₄ synergizes with specific growth factor receptors and other molecules to activate key signaling pathways, especially the PI3K/Akt pathway (33). Despite these important functions of α₆β₄, relatively little is known about the mechanisms that regulate the expression of this integrin. There is evidence that exogenous stimuli such as EGF, retinoic acid, and wounding can increase β₄ expression in specific cell types (34–36). More directly, AP1 and Ets have been shown to be important for the activity of the β₄ promoter (37), which has a high G+C content and does not contain either TATA or CAAT boxes (37). Of particular interest, c-myc has been shown to up-regulate β₄ expression in colon carcinoma cells (38) and, as mentioned above, c-myc has been reported to induce E2F DNA binding activity not only by the induction of cyclin D/Cdk4 and/or cyclin E/Cdk2, but also by the direct activation of the E2F1, E2F2, and E2F3 genes (21). Therefore, based on these data and our findings it seems likely that c-myc-induced β₄ expression could be mediated at least, in part, by the activation of E2Fs1-3. It will also be informative to test whether H-Ras-induced E2F activation, integrin β₄ expression, and invasion are mediated by H-Ras-induced c-myc activation.

In summary, the data reported here indicate a novel mechanism by which H-Ras can promote the invasion of breast carcinoma cells. This mechanism links active H-Ras, transcriptionally active E2F, and the α₆β₄ integrin in a common pathway that culminates in enhanced α₆β₄-dependent invasion. Although numerous studies have implicated α₆β₄ in invasion, ours is one of the few studies that identify upstream regulators of this integrin that are also associated with breast cancer progression. Further studies on this mode of α₆β₄ regulation should prove insightful for deciphering the biology of invasion and for identifying potential therapeutic targets.

Grant support: NIH grant CA 80789 supported this work.

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.

We thank Drs. Steve Ethier, Joseph R. Nevins, Johannes L. Bos, Kun-Liang Guan, and Young-Chae Chang for providing reagents.