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[Cancer Research 66, 2279-2286, February 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

SKI-606 Decreases Growth and Motility of Colorectal Cancer Cells by Preventing pp60(c-Src)–Dependent Tyrosine Phosphorylation of ß-Catenin and Its Nuclear Signaling

Addolorata Maria Luce Coluccia1,3, Daniela Benati1, Hafedh Dekhil2, Annamaria De Filippo1, Cathy Lan2 and Carlo Gambacorti-Passerini1,2,3

1 Department of Experimental Oncology, National Cancer Institute, Milan, Italy; 2 McGill University, Montreal, Quebec, Canada; and 3 Department of Clinical Medicine, S. Gerardo Hospital-University of Milano-Bicocca, Monza, Italy

Requests for reprints: Addolorata Maria Luce Coluccia, Department of Clinical Medicine, S. Gerardo Hospital-University of Milano-Bicocca, via Cadore 48, 20052 Monza, Italy. Phone: 39-2-64488059; Fax: 39-2-64488363; E-mail: coluccia{at}istitutotumori.mi.it or malu.coluccia{at}unimib.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibition of deregulated protein tyrosine kinases represents an attractive strategy for controlling cancer growth. However, target specificity is an essential aim of this strategy. In this report, pp60(c-Src) kinase and ß-catenin were found physically associated and constitutively activated on tyrosine residues in human colorectal cancer cells. The use of specific small-interfering RNAs (siRNA) validated pp60(c-Src) as the major kinase responsible for ß-catenin tyrosine phosphorylation in colorectal cancer. Src-dependent activation of ß-catenin was prevented by SKI-606, a novel Src family kinase inhibitor, which also abrogated ß-catenin nuclear function by impairing its binding to the TCF4 transcription factor and its trans-activating ability in colorectal cancer cells. These effects were seemingly specific, as cyclin D1, a crucial ß-catenin/TCF4 target gene, was also down-regulated by SKI-606 in a dose-dependent manner accounting, at least in part, for the reduced growth (IC50, 1.5-2.4 µmol/L) and clonogenic potential of colorectal cancer cells. Protein levels of ß-catenin remained substantially unchanged by SKI-606, which promoted instead a cytosolic/membranous retention of ß-catenin as judged by immunoblotting analysis of cytosolic/nuclear extracts and cell immunofluorescence staining. The SKI-606-mediated relocalization of ß-catenin increased its binding affinity to E-cadherin and adhesion of colorectal cancer cells, with ensuing reduced motility in a wound healing assay. Interestingly, the siRNA-driven knockdown of ß-catenin removed the effect of SKI-606 on cell-to-cell adhesion, which was associated with prolonged stability of E-cadherin protein in a pulse-chase experiment. Thus, our results show that SKI-606 operates a switch between the transcriptional and adhesive function of ß-catenin by inhibiting its pp60(c-Src)–dependent tyrosine phosphorylation; this could constitute a new therapeutic target in colorectal cancer. (Cancer Res 2006; 66(4): 2279-86)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ß-Catenin, a phosphoprotein of 97 kDa, functions both as a structural component of the cell adhesion/actin cytoskeleton network and as a signaling molecule when localized in the nucleus (1, 2). In its signaling role, ß-catenin binds to the members of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family generating a bipartite transcription complex in which ß-catenin provides the trans-activating domain and TCF/LEF provides the DNA-binding region (3–5).

A constitutive ß-catenin-related transcription represents an early event in human colorectal cancer in which deletion of tumor suppressor adenomatous polyposis coli (APC) or "gain-of-function" mutations in the NH2-terminal region of ß-catenin (CTNNB1) gene alters ß-catenin turnover by preventing its serine/threonine phosphorylation by glycogen synthase kinase-3ß, which marks ß-catenin for the ubiquitin/proteosome machinery (6–8). Stabilized ß-catenin accumulates and translocates into the nucleus where it trans-activates the TCF/LEF factors, which normally act as repressors recruiting proteins, such as CtBP (9), Groucho/TLE (10, 11), and histone deacetylase (12). Activation of ß-catenin/TCF4 nuclear complex is crucial for inducing and maintaining a stem cell–like phenotype in intestinal cells by altering the normal balance between cell proliferation, differentiation, and apoptosis during colorectal carcinogenesis (13). In fact, several genes, such as cyclin D1 and c-Myc, contain multiple TCF4-responsive elements in their promoters and are strongly up-regulated in colon cancer (14–16). Silencing of ß-catenin by small-interfering RNAs (siRNA; ref. 17) is required to suppress cyclin D1 expression and thus the growth of colorectal cancer cells in vitro and in vivo (13). Although this represents a reasonable "loss-of-function" approach to reduce oncogenic potential of ß-catenin, there is a growing interest in elucidating other molecular mechanisms and novel class of agents that can potentially inhibit aberrant ß-catenin/TCF4-mediated transcriptional activation (18, 19).

ß-Catenin is also a tyrosine phosphoprotein, and the current prevailing viewpoint is that tyrosine phosphorylation of ß-catenin promotes its dissociation from E-cadherin at cell-to-cell junctions during progression to an invasive and metastatic colorectal cancer disease (20–23). Constitutive tyrosine activation of ß-catenin (24, 25) and several protein tyrosine kinases, such as c-Kit (26), c-erbB2 (27), c-Met (28), and pp60(c-Src) (29–32), is frequently observed in premalignant colorectal lesions. It was also reported that an antisense-mediated down-regulation of pp60(c-Src) expression severely decreased tumorigenicity of HT29 colon adenocarcinoma cells (33), although potential transcriptional targets of pp60(c-Src) still remained elusive.

In this report, we validated pp60(c-Src) as the major kinase responsible for ß-catenin tyrosine activation in intact human colorectal cancer cells (DLD-1 and Ls174T) with different genetic backgrounds. The recent finding that SKI-606 (WAY-173606), a novel substituted 4-anilino-3-quinolinecarbonitrile Src inhibitor (34), impaired colorectal cancer cell growth in vitro and in vivo (35) prompted us to investigate whether inhibition of pp60(c-Src) activation by SKI-606 treatment of colorectal cancer cells is correlated with ß-catenin tyrosine phosphorylation and its signaling activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures and reagents. The human colorectal cancer cell lines Ls174T and DLD-1 were purchased from the American Type Culture Collection (Rockville, MD), maintained in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine (Life Technologies, Paisley, United Kingdom), and incubated at 37°C with 5% CO2 atmosphere.

The dual Src and Abl tyrosine kinase inhibitor SKI-606 (a kind gift from Frank Boschelli, Wyeth Research, Lake George, NY; ref. 34) was dissolved in DMSO (Sigma Chemical Co., St. Louis, MO), which was used as a solvent control in some experiments. After cell cultures were 70% confluent, they were incubated with different concentrations of drugs as reported in the figure legends.

Small-interfering RNA. DLD-1 and Ls174T cells, at 60% to 80% confluence, were washed with Opti-MEM I reduced serum medium (Invitrogen, Groningen, the Netherlands) and transiently transfected with 50 and 150 nmol/L c-Src siRNAs or 150 nmol/L control siRNAs (SMARTpool; Dharmacon, Lafayette, CO) by using Oligofectine (Qiagen, Crawley, United Kingdom) according to the manufacturer's instructions. After 48 hours, the level of Src protein was assessed by Western blotting as reported below. An inducible ß-catenin siRNA was also transfected in DLD-1 cells as described previously by van de Wetering et al. (17). Briefly, DLD-1 cells were stably transfected with the pcDNA6TR plasmid to select clones expressing the Tet repressor and subsequently with the pTER-ß-catenin construct carrying a doxycycline-inducible form of the RNA polymerase III (H1 promoter) to drive expression of specific ß-catenin siRNA. The ß-catenin oligonucleotides (5'-GATCCCGTGGGTGGTATAGAGGCTCTTCAAGAGAGAGCCTCTATACCACCCACTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAGTGGGTGGTATAGAGGCTCTCTCTTGAAGAGCCTCTATACCACCCACGG-3') were annealed and cloned in BglI and HindIII cloning sites of pTER vector. Both pcDNA6TR and pTER vectors were a generous gift from Dr. Hans Clevers (Hubrecht Laboratory, Utrecht, the Netherlands; ref. 17). Expression of ß-catenin siRNA was induced by adding 1 µg/mL doxycycline in culture cell plates.

Luciferase reporter gene assay. Colorectal cancer cells (2 x 105 per well) were transfected by using LipofectAMINE 2000 Transfection kit (Invitrogen) according to the manufacturer's instructions with 5 µg TCF reporter construct TOPflash or FOPflash (Upstate Biotechnology, Lake Placid, NY) and 25 ng pSV-ß-gal construct (Promega, Madison, WI) as an internal control to normalize luciferase activity for transfection efficiency. TOPflash contains three copies of the TCF/LEFG-binding site (AAGATCAAAGGGGGT) upstream of a thymidine kinase (tk) minimal promoter. FOPflash contains a mutated TCF/LEF-binding site (AAGGCCAAAGGGGGT). Twenty-four hours after transfection, cells were treated with SKI-606 for additional 24 hours. Luciferase activity was measured using a luminometer (Turner Designs TD-20/20) and the luciferase assay system (Promega) following the manufacturer's instructions. Results were confirmed by multiple independent assays.

Proliferation assay. Ls174T and DLD-1 cells were seeded in flat-bottomed 96-well plates at 10,000 to 50,000 per well in a volume of 100 µL in supplemented medium. Serial dilutions of SKI-606 were added as indicated in the figure legends and the volume was adjusted to 200 µL. Eight hours before harvesting onto glass fiber filters, 1 µCi [3H]thymidine was added to each well. Incorporation of [3H]thymidine was measured using a filter scintillation counter (1430 MicroBeta, Wallac, Turku, Finland).

Flow cytometric analysis. Cells were harvested, washed with ice-cold PBS, and fixed in 70% cold ethanol. Samples were then treated with DNase-free RNase (Boehringer Mannheim, Indianapolis, IN) and stained with 10 µg/mL propidium iodide (Sigma). DNA content and distribution of individual cells into different phases of the cell cycle was assessed by using FACScalibur flow cytometry and CellQuest software (Becton Dickinson, San Jose, CA). The ranges for G0-G1-, S-, G2-M-, and sub-G1-phase cells were established based on the corresponding DNA content of histograms. At least 10,000 cells per sample were considered in the gate regions used for calculations.

Soft agar clonogenic assay. To test the effect of SKI-606 on anchorage-independent colony formation, 1 x 103 cells (Ls174T and DLD-1) were suspended in complete RPMI 1640 containing 0.5% agar and plated in six-well plates over a basal layer of complete medium containing 1% agar. The plates were cultured at 37°C with 5% CO2 and the clonogenicity was determined in three independent experiments by counting the number of colonies in each well after 21 days of incubation.

Wound healing assay. DLD-1 and Ls174T cells were grown to confluence on six-well tissue culture plates. A "wound" was made by scraping with a P200 pipette tip the middle of the cell monolayer. Floating cells were removed by extensive washing with ice-cold PBS, and fresh complete medium containing DMSO or 1.5 µmol/L SKI-606 was added for 4 days. The rate of motility was quantified by counting the cells migrating into the total wound area of each x10 field by using a Leitz Laborlux D microscope. At least three different fields were randomly chosen across the wound length.

Statistical analysis. Results were statistically compared by simple t test and differences were considered significant if P < 0.05.

Cell fractionation experiments. DLD-1 and Ls174T cells were lysed in a digitonin buffer [1% digitonin, 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgCl2] plus a protease inhibitor cocktail (10 µmol/L benzamidine HCl and 10 µg each of aprotinin, leupeptin, and pepstatin A/mL). The lysates were centrifuged at 13,000 rpm for 10 minutes and supernatants representing cytosolic fractions were saved. The pellets representing nuclear components were lysed in radioimmunoprecipitation assay (RIPA) buffer [150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mmol/L Tris (pH 7.5)]. Equal amounts of proteins were separated on SDS-PAGE gels and analyzed by immunoblotting for ß-catenin.

Indirect ß-catenin immunostaining. DLD-1 cells (1 x 105) were seeded in eight-well chamber slides (Nalge Nunc International, Rochester, NY) and incubated for 2 days at 37°C with 5% CO2 before overnight treatment (16 hours) with 1.5 µmol/L SKI-606. Cells were washed once with PBS and then fixed in 3% paraformaldehyde for 15 minutes at room temperature. Fixed cells were washed with PBS and permeabilized in 100% cold methanol for 1 minute. Cells were washed twice in PBS and covered with blocking buffer (1% bovine serum albumin-1% horse serum in PBS) for 1 hour before staining. The primary ß-catenin antibody (1:300 dilution; BD Transduction Laboratories, Lexington, KY) was visualized with a fluorescent secondary antibody Alexa 488–conjugated goat anti-mouse IgG-FITC (used 1:300; Molecular Probes, Inc., Eugene, OR). After washing, cells were mounted in Vectashield medium (Vector Laboratories, Burlingame, CA) containing 4',6-diamidino-2-phenylindole (DAPI) and observed on a Nikon Eclipse 600 fluorescence microscope with digital camera Nikon DXM 1200.

Immunoprecipitation and Western blotting. After treatment with the indicated drug, cells were harvested, washed once in PBS at 4°C, and resuspended in lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 1% Triton X-100, 5 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L Na3VO4, 1 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (10 µmol/L benzamidine-HCl and 10 µg each of aprotinin, leupeptin, and pepstatin A/mL)] followed by incubation on ice for 30 minutes. Lysates were then clarified by centrifugation at 13,000 x g for 15 minutes at 4°C and transferred into fresh reaction tubes. The protein concentration of cell lysates was determined using the BCA protein assay (Pierce, Rockford, IL) and total proteins (100 µg) were incubated for 1 hour with 1 µg of the indicated antibody at 4°C. Immunocomplexes were precipitated with 30 µg protein A-Sepharose (Pharmacia Biotech, St. Alban, United Kingdom) for 4 hours before separation on SDS-polyacrylamide gels. Immunoblotting was done using Immobilon-P nitrocellulose membrane (Millipore Corp., Bedford, MA). Primary incubations were 1 to 3 hours. Secondary incubations were for 1 hour and antibodies used were horseradish peroxidase–conjugated anti-mouse or anti-rabbit (Amersham, Arlington Heights, IL). Proteins were visualized by chemiluminescence as recommended by the manufacturer (SuperSignal, Pierce). The monoclonal anti-ß-catenin, anti-E-cadherin (clone HECD-1-ICRF), and anti–histone H1B antibodies were purchased from BD Transduction Laboratories. Mouse monoclonal anti-c-Src antibody (clone GD11) was from Upstate (Charlottesville, VA). Mouse monoclonal anti-phosphotyrosine (clone 4G10) and anti-TCF4 (clone 6H5-3) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY), whereas the goat polyclonal anti-ß-actin antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Pulse-chase analysis. DLD-1 cells were cultured to 70% confluence. For each chase time point, 5 x 106 cells were washed once in ice-cold PBS and incubated in RPMI 1640 (without cysteine/methionine; Sigma) for 1 hour at 37°C with 5% CO2. Cells were then pulsed for 30 minutes by using [35S]methionine Promix (10 µCi/106 cells; Amersham Pharmacia). Cells were then chased for the indicated time points in RPMI 1640 in the presence or absence of 1.5 µmol/L SKI-606. Total cell lysates were then prepared and immunoprecipitated with E-cadherin antibody and protein A-Sepharose for 3 hours at 4°C. Immunocomplexes were separated on 8% SDS-PAGE gel. This gel was stained with Coomassie blue (Sigma) as loading control, destained, and incubated with Amplify fluorographic reagent (Amersham Pharmacia), dried, and developed by autoradiography. Densitometric analysis of protein bands was carried out on an Eagle Eye II photodensitometer (Stratagene, La Jolla, CA) and done using the Scion Image analysis software (Scion Corp.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tyrosine phosphorylation of ß-catenin is prevented by Src kinase inhibitor SKI-606 in human colorectal cancer cells. The evidence that ß-catenin can be phosphorylated by recombinant pp60(c-Src) tyrosine kinase by using in vitro kinase assays (20, 23) prompted us to verify whether the novel Src tyrosine kinase inhibitor SKI-606 (34, 35) could prevent ß-catenin tyrosine activation in human colorectal cancer cells.

To this end, tyrosine phosphorylation of ß-catenin was analyzed in Ls174T and DLD-1 cells cultured in the presence of DMSO (-) or 1.5 µmol/L SKI-606 (+) for 2 hours and then immunoprecipitated by using an anti-ß-catenin COOH-terminal antibody (Fig. 1A).


Figure 1
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  		Figure 1. SKI-606 inhibits pp60(c-Src)–mediated ß-catenin tyrosine phosphorylation. A, Ls174T (lanes A and B) and DLD-1 (lanes C and D) cells were treated with DMSO (-) or 1.5 µmol/L SKI-606 (+) for 2 hours and then immunoprecipitated by using an anti-ß-catenin antibody (IP: {alpha} ß-cat). DLD-1 cells (lane E) were also immunoprecipitated with a control IgG antibody (IP: {alpha}IgG). Immunocomplexes were then separated by 8% SDS-PAGE and assessed for tyrosine phosphorylation by using an anti-phosphotyrosine monoclonal antibody (clone 4G10; WB: {alpha} PY). The same blot was stripped and two proteins of 97 and 60 kDa were then identified as ß-catenin (Reblot: {alpha} ß-cat) and pp60(c-Src) tyrosine kinase (Reblot: {alpha} c-Src). HC, heavy chain of IgG antibody (used for immunoprecipitation). B, DLD-1 cells were transiently transfected with 50 or 150 nmol/L specific siRNAs for pp60(c-Src) (lanes D and E), 150 nmol/L control pool of siRNAs (lane C), and Oligofectine used as transfection carrier (lane B) or left untreated (lane A). After 48 hours, total cell lysates were analyzed for the pp60(c-Src) (WB: {alpha} c-Src) and cyclin D1 protein levels (WB: {alpha} cyclin D1) by SDS-PAGE analysis. Levels of ß-actin are shown as loading control (WB: {alpha} ß-actin). C, cell cycle distribution of DLD-1 cells transiently transfected with control siRNA or c-Src siRNA for 48 hours was assessed by propidium iodide staining and flow cytometric analysis as described in Materials and Methods. D, DLD-1 cells transfected with 50 and 150 nmol/L c-Src siRNA or 150 nmol/L control siRNA were immunoprecipitated by using either an anti-ß-catenin antibody (IP: {alpha} ß-cat) or a nonimmunizing IgG antibody (IP: {alpha}IgG) as control. ß-Catenin tyrosine phosphorylation was assessed by anti-phosphotyrosine blotting (p-Tyr ß-cat; top). The amounts of ß-catenin (Reblot: {alpha} ß-cat; middle) and pp60(c-Src) (WB: {alpha} c-Src; bottom) were determined with the indicated monoclonal antibodies.

 
A band of 97 kDa consistent with the molecular weight of ß-catenin (Reblot: {alpha} ß-cat) was detected in Ls174T (lanes A and B) and DLD-1 (lanes C and D) colorectal cancer cell lines by using an anti-phosphotyrosine antibody (WB: {alpha} PY), indicating that ß-catenin is constitutively phosphorylated on tyrosine residues (p-Tyr ß-cat; WB: {alpha} PY; lanes A and C). Treatment with SKI-606 strongly prevented ß-catenin tyrosine phosphorylation (WB: {alpha} PY; lanes B and D). In addition, the PY immunostaining of anti-ß-catenin immunoprecipitates revealed another major band of 60 kDa, which was activated in the DMSO-treated control (WB: {alpha} PY; lanes A and C) but not in SKI-606-positive samples (WB: {alpha} PY; lanes B and D).

Reblotting with a specific anti-c-Src antibody (Reblot: {alpha} c-Src) stained this band, showing that pp60-c-Src kinase and ß-catenin were physically associated. Although SKI-606 treatment did not change this interaction, tyrosine phosphorylation of pp60(c-Src) tyrosine kinase (WB: {alpha} PY; p-Tyr c-Src) was strongly prevented by SKI-606.

To validate this finding, we targeted pp60(c-Src) expression by using specific siRNAs to further probe that the effect of SKI-606 on p-Tyr-ß-catenin was elicited through its inhibitory effect on c-Src kinase activation (35).

As shown in Fig. 1B, when DLD-1 cells were transiently transfected with 50 nmol/L (lane D) or 150 nmol/L (lane E) of a mixture of four selected double-stranded siRNA designed against c-Src gene, they showed a dose-dependent down-regulation of Src expression (70% and 90% of inhibition, respectively) compared with a nonspecific control pool (WB: {alpha} c-Src; lane C). Interestingly, we observed a close relationship between reduced cyclin D1 protein levels (WB: {alpha} cyclin D1) and growth rate of DLD-1 cells on silencing of pp60(c-Src) expression (Fig. 1C). In particular, flow cytometric analysis of DNA amount showed an increase in sub-G1 peak (15.3% versus 1.4%) that was indicative of c-Src siRNA-positive cells undergoing apoptosis compared with control (CTR siRNA) as well as in the percentage of G1-arrested cells (85.2% versus 55.8%), which was further confirmed by decreased uptakes of [3H]thymidine (data not shown). This antiproliferative effect was in keeping with the known function of cyclin D1 as a crucial cell cycle regulator in colon carcinogenesis (13).

DLD-1 siRNA-transfected cells were also immunoprecipitated by using an anti-ß-catenin COOH-terminal antibody (Fig. 1D). The presence of c-Src tyrosine kinase (WB: {alpha} c-Src) was again confirmed in ß-catenin immunoprecipitates from control cells, whereas its protein content was completely (150 nmol/L) or nearly completely (50 nmol/L) down-regulated in cells treated with c-Src-siRNA. In addition, tyrosine phosphorylation of ß-catenin (p-Tyr ß-cat) decreased proportionately to reduction in pp60(c-Src) expression, although an equal amount of ß-catenin was examined in each sample (Reblot: {alpha} ß-cat).

These results therefore defined ß-catenin as a downstream signaling target of pp60-c-Src kinase in intact colorectal cancer cells, implying the Src inhibitor SKI-606 as a potent down-regulator of ß-catenin tyrosine phosphorylation.

SKI-606 disrupts ß-catenin/TCF4 nuclear association and attenuates ß-catenin-related TCF trans-activation in human colorectal cancer cells. To explore the possibility that SKI-606-mediated targeting of ß-catenin tyrosine phosphorylation could interfere with its trans-activating function, the integrity of ß-catenin/TCF4 nuclear complexes was analyzed in SKI-606-treated colorectal cancer cells by doing pull-down experiments.

Ls174T and DLD-1 colorectal cancer cells were treated for 18 hours with vehicle alone (DMSO; -) or 1.5 µmol/L SKI-606 (+) and then immunoprecipitated with an anti-TCF4 antibody (Fig. 2A). Western blot analysis revealed that SKI-606 treatment significantly affected ß-catenin binding to TCF4. In fact, a weak colocalization of ß-catenin and TCF4 was observed in SKI-606-treated samples in both cell lines (WB: {alpha} ß-cat; lanes B and D), whereas a stronger interaction was detected in controls (WB: {alpha} ß-cat; lanes A and C). Comparable amounts of TCF4 were immunoprecipitated from each of the extracts (Reblot: {alpha} TCF4).


Figure 2
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  		Figure 2. Inhibition of ß-catenin tyrosine phosphorylation by SKI-606 correlates with its nuclear transcriptional inactivation in intact colorectal cancer cells. A, Ls174T and DLD-1 cells were treated with DMSO (-) or 1.5 µmol/L SKI-606 (+) for 16 hours. TCF4 and ß-catenin were immunoprecipitated using an anti-TCF4 mouse monoclonal antibody (IP: {alpha} TCF4). ß-Catenin (97 kDa) protein levels were analyzed by immunoblotting (WB: {alpha} ß-cat). The blot was reprobed to assess TCF4 (66 kDa) protein content. HC, heavy chain of IgG antibody (used for immunoprecipitation). B, subconfluent Ls174T and DLD-1 cells were cotransfected with the luciferase reporter constructs TOPflash or FOPflash as described in Materials and Methods. At 24 hours after transfection, cells were treated with the indicated SKI-606 concentrations (0.5-3 µmol/L) or DMSO (-) for additional 24 hours. Luciferase activity was expressed as fold activation compared with carrier control cells. Columns, mean of three representative experiments; bars, SE. C, Ls174T and DLD-1 cells were incubated with the indicated concentrations (µmol/L) of SKI-606 or vehicle alone (-) for 48 hours. Cyclin D1 (38 kDa) expression was assessed by Western blotting analysis of total lysates by using an anti–cyclin D1 (WB: {alpha} cyclin D1) rabbit polyclonal antibody. Total cellular levels of ß-catenin (WB: {alpha} ß-cat) and TCF4 (WB: {alpha} TCF4) were also indicated.

 
These data were further corroborated by measurements of ß-catenin-dependent TCF transcriptional activity (Fig. 2B). Ls174T and DLD-1 cells were transiently transfected with a luciferase gene controlled by a TCF-sensitive tk promoter (TOPflash) or a negative reporter vector (FOPflash) containing the same tk promoter downstream of scrambled TCF-binding sites.

A range of SKI-606 concentrations (0.5-3 µmol/L) or DMSO (-) as control for vehicle alone were then added for 24 hours to transfected cells followed by assay for luciferase activity induction. In DMSO versus SKI-606-treated cells, no significant changes on negative FOPflash reporter activity were observed. In control Ls174T and DLD-1 cells, TOPflash reporter activity averaged 4.2 ± 0.1– and 3.8 ± 0.1–fold activity, respectively, relative to that obtained with negative FOPflash construct, confirming the constitutive activation of ß-catenin/TCF-related transcription in colorectal cancer cells. Conversely, treatment with SKI-606 decreased TOPflash reporter activity in a dose-dependent manner with a 50% reduction occurring ~1.5 µmol/L SKI-606 (IC50, 1.5 ± 0.5 µmol/L) compared with DMSO controls in both cell lines.

A hallmark of ß-catenin/TCF4 oncogenic activation is the overexpression of cyclin D1 oncoprotein, which is a crucial regulator of cell cycle in colorectal cancer cells (13, 14). As reported in Fig. 2C, total cyclin D1 protein levels were also reduced in Ls174T and DLD-1 colorectal cancer cells cultured in the presence of the indicated SKI-606 concentrations for 48 hours. In particular, at 1.5 µmol/L SKI-606, both cell lines were found to have <10% of the initial steady-state level of cyclin D1.

Surprisingly, a simple relationship between SKI-606-reduced cyclin D1 expression and changes in total ß-catenin protein levels was not apparent. In fact, an immunoblotting analysis done with either an anti-ß-catenin (WB: {alpha} ß-cat) or a TCF4 (WB: {alpha} TCF4) antibody revealed that the cellular content of the two proteins remained unchanged on treatment with SKI-606.

The ability of SKI-606 to disrupt ß-catenin/TCF4 nuclear complexes and its oncogenic signaling through cyclin D1 was accompanied by a marked antiproliferative effect of the drug as reflected by decreased uptake of [3H]thymidine of Ls174T and DLD-1 cells cultured in the presence of SKI-606 increasing doses for 2 days (Fig. 3A). The IC50s for SKI-606 were 1.5 ± 0.1 µmol/L (Ls174T) and 2.4 ± 0.1 µmol/L (DLD-1), respectively. In addition, flow cytometric analysis of DNA amount (Fig. 3B) showed a significant increase in the percentage of sub-G1 peak (29.8%) in Ls174T cells treated for 48 hours with 1.5 µmol/L SKI-606 (Fig. 3B, right) compared with control DMSO sample (0.4%; Fig. 3B, left). This effect was indicative of cells undergoing apoptosis with a subdiploid DNA content.


Figure 3
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  		Figure 3. Survival and transforming potential of colorectal cancer cells is reduced by SKI-606. A, proliferation assay of colorectal cancer cell lines DLD-1 and Ls174T cells cultured in the presence of increasing concentrations of SKI-606 (1-20 µmol/L) or vehicle alone for 48 hours. Data were expressed as % of growth inhibition. B, cell cycle distribution of Ls174T cells incubated with DMSO or 1.5 µmol/L SKI-606 for 24 hours was assessed by propidium iodide staining and flow cytometric analysis as described in Materials and Methods. C, number of DLD-1 and Ls174T colonies formed in soft agar containing DMSO or 1.5 µmol/L SKI-606 after 21 days. Columns, mean of three independent experiments; bars, SD. *, P < 0.05.

 
Ls174T and DLD-1 cells exhibited also a lower transforming activity when subjected to soft agar suspension assays in the presence of SKI-606 inhibitor (Fig. 3C). Under these conditions, clonal cells exhibited between 2-fold (Ls174T) and 3-fold (DLD-1) decrease in colony formation compared with DMSO-treated cultures.

Thus, these results indicate that SKI-606 impairs ß-catenin/TCF4 signaling and clonal efficiency of colorectal cancer cells by affecting ß-catenin tyrosine phosphorylation but not its total cellular content.

SKI-606 relocates ß-catenin at the cell-to-cell junction and inhibits colorectal cancer motility. We considered therefore the possibility that SKI-606 could alter cellular distribution of ß-catenin.

To this end, nuclear and cytoplasmic extracts from Ls174T and DLD-1 cells incubated with DMSO alone (-) or 1.5 µmol/L SKI-606 (+) for 8 hours were probed with an anti-ß-catenin antibody (Fig. 4A). ß-Catenin was detected predominantly in the nucleus of DMSO-treated cells (WB: {alpha} ß-cat, lane N compared with lane C), in accordance with a constitutive activation of ß-catenin/TCF4 signaling. On the contrary, ß-catenin was significantly retained in the cytoplasmic fractions of SKI-606-treated cells (WB: {alpha} ß-cat, lane N compared with lane C), showing that cytoplasmic ß-catenin cellular relocation correlates with its phosphotyrosine inhibition.


Figure 4
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  		Figure 4. SKI-606 promotes cytosolic retention of ß-catenin and affects motility of colorectal cancer cells. A, Ls174T and DLD-1 cells were treated with DMSO (-) or 1.5 µmol/L SKI-606 (+) for 8 hours and then fractionated into 1% digitonin (C for cytoplasmic) or RIPA-soluble (N for nuclear) fractions. Equal amounts of each denatured extract (100 µg) were separated by SDS-PAGE and immunoblotted for ß-catenin (WB: {alpha} ß-cat) and histone H1B (WB: {alpha} H1B) as control. B, DLD-1 cells were treated with 1.5 µmol/L SKI-606 or DMSO overnight, stained with a monoclonal antibody for ß-catenin, and visualized with a fluorescent IgG-FITC secondary antibody. DNA was stained with DAPI. Magnification, x40. C, Ls174T and DLD-1 cells were incubated with DMSO (-) or the indicated concentration of SKI-606 for 8 hours and then subjected to immunoprecipitation by using an anti-E-cadherin antibody (IP: {alpha} E-cad). Interaction between ß-catenin and E-cadherin was examined by Western blotting analysis with an anti-ß-catenin (WB: {alpha} ß-cat) and E-cadherin (WB: {alpha} E-cad) antibodies. D, motility of wounded cell monolayers. Columns, mean number of migrating cells after 4 days of exposure to DMSO (-) or 1.5 µmol/L SKI-606 (+) among three independent experiments; bars, SD. *, P < 0.003 for DLD-1; *, P < 0.05 for Ls174T.

 
ß-Catenin subcellular distribution was also determined by indirect immunofluorescence microscopy in DLD-1 cells (Fig. 4B). Treatment with SKI-606 inhibitor was associated with a reduced nuclear ß-catenin staining and a corresponding gain of junctional ß-catenin at cell-to-cell surface contacts. Immunoblotting analysis of E-cadherin immunoprecipitates with an anti-ß-catenin antibody (Fig. 4C) confirmed that treatment with SKI-606 increased the binding of ß-catenin to E-cadherin in a dose-dependent fashion (WB: {alpha} ß-cat, + compared with -).

The hypothesis that the SKI-606-promoted E-cadherin/ß-catenin interaction could affect adhesion and motility of colorectal cancer cell was investigated by using an in vitro monolayer wound healing assay. We observed that exposure to 1.5 µmol/L SKI-606 for 4 days inhibited migration of DLD-1 and Ls174T colorectal cancer cells by ~80% compared with their control counterpart treated with DMSO. The wounded monolayers also exhibited differences of morphology: SKI-606-treated cells were more cohesive than those treated with DMSO alone, reflecting a tightening of cell-to-cell contacts (data not shown).

ß-Catenin is required for SKI-606-induced E-cadherin stability and function. The effect of SKI-606 on colorectal cancer cell motility could be therefore explained by the relocation of tyrosine dephosphorylated ß-catenin to cell-to-cell contacts with a stabilization of the adhesive function of E-cadherin.

As shown in Fig. 5A, total protein content of E-cadherin increased in DLD-1 cells incubated with 1.5 µmol/L SKI-606 for 2 days (WB: {alpha} E-cad, lane B compared with lane A). Interestingly, when ß-catenin was down-regulated by a doxycycline-inducible siRNA (WB: {alpha} ß-cat, lane C compared with lane A), treatment with SKI-606 failed to increase E-cadherin levels (WB: {alpha} E-cad, lane D compared with lane B) as well as the appearance of high packed round-shaped colonies (Fig. 5B).


Figure 5
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  		Figure 5. SKI-606 regulates E-cadherin protein stability through ß-catenin. A, DLD-1 cells carrying a doxycycline (DOX)–inducible ß-catenin siRNA were cultured without (lane A) or with (lane C) 1 µg/mL doxycycline for 72 hours. After 48 hours, 1.5 µmol/L SKI-606 (lanes B and D) was added to cell cultures kept in the absence (lane B) or presence (lane D) of doxycycline for the last 24 hours. E-cadherin (WB: {alpha} E-cad), ß-catenin (WB: {alpha} ß-cat), and ß-actin (WB: {alpha} ß-actin) protein levels were determined from total lysates at 72 hours. B, representative image of cells from one of the six fields captured from each well by using a camera attached to an inverted Nikon (TE300) microscope. C, subconfluent DLD-1 cells were labeled with [35S]methionine and then chased with complete nonradioactive medium for the indicated time points. Cells were then lysed and immunoprecipitated for E-cadherin. Results were analyzed by densitometry and expressed as a percentage of the intensity value at time 0.

 
These findings suggest that SKI-606-induced subcellular relocation of ß-catenin could affect E-cadherin protein turnover in colorectal cancer cells.

To examine this issue, DLD-1 cells were labeled with [35S]methionine and metabolic stability of E-cadherin was analyzed in the absence or presence of SKI-606 by pulse-chase assay. As indicated in Fig. 5C, SKI-606 decreased degradation rate of E-cadherin and densitometric analysis (expressing the intensity of the labeled ß-catenin bands as a percentage of the value at time 0) revealed that the estimated half-life of E-cadherin was ~4 hours in control cells but <8 hours in SKI-606-treated samples.

It can be therefore concluded that SKI-606 activates a cooperative ß-catenin/E-cadherin interaction by enhancing cell-surface relocation of tyrosine dephosphorylated ß-catenin, which binds and contributes to E-cadherin stabilization.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activity of pp60(c-Src) is increased in >80% of colorectal cancer irrespective of the presence or absence of oncogenic mutations (36–39). To our knowledge, the existence of a functional relationship between pp60(c-Src) and ß-catenin has not been studied in human colorectal cancer albeit their separate expression and constitutive tyrosine activation has been documented in the pathogenesis and progression of this disease (26, 31, 33). By using human colorectal cancer cells, we showed that ß-catenin was physically associated to activated pp60(c-Src) kinase and constitutively tyrosine phosphorylated. Silencing of c-Src expression by siRNAs revealed that c-Src, by itself or through related downstream effectors, was required to promote ß-catenin tyrosine phosphorylation in colorectal cancer cells, in accordance with previous biochemical data showing ß-catenin to be a substrate for purified recombinant Src kinases in vitro (20, 23).

The changes on ß-catenin tyrosine activation observed on down-regulation of pp60(c-Src) correlated with impaired growth of DLD-1 colorectal cancer cells and with reduced cyclin D1 protein levels, showing that c-Src could affect the oncogenic TCF4-bounded pool of ß-catenin (13). In this regard, overexpression of aberrantly activated c-Src (v-Src) was reported to enhance ß-catenin/TCF-related gene transcription in SW480 colorectal cancer cells as well as in HEK293T cells under the conditions where a stable mutant form (S37A) of ß-catenin alone resulted ineffective (40). Thus, although pp60(c-Src) seems unable to promote ß-catenin accumulation in cells carrying an intact WNT signaling, modulation of its kinase activity is important to regulate oncogenic ß-catenin/TCF-dependent gene transcription. Other articles also showed that pp60(c-Src) kinase activity contributed to increased tumorigenicity and malignant behavior of human colorectal cancer cells (33, 36). Thus, besides the genetic lesions (e.g., deletion of APC gene and point mutations of ß-catenin itself), which increase the stability of ß-catenin cytosolic pool in colorectal carcinogenesis (6, 7), selective pathways, such as c-Src/ß-catenin signaling, may be important in colorectal cancer growth control. Because colon cancer is defective in the mechanisms that promote serine/threonine phosphorylation of ß-catenin, it is difficult to assess the relevance or dominant effect of ß-catenin tyrosine phosphorylation on its protein turnover, full transcriptional activation, or other post-translational modifications (41); different models, such as the one represented by Bcr/Abl+ leukemic cells (42), will be needed to investigate this aspect. Emerging evidences from in vitro kinase assays done on several deletion mutants of ß-catenin have recently mapped two Src-activated tyrosine residues (Tyr86 and Tyr654), respectively, in the NH2-terminal and COOH-terminal transcriptional domains of ß-catenin (20). Whether these tyrosine residues modulate the degradation or transcriptional activity of ß-catenin will require further investigations. In addition, because the phosphorylation of Tyr654 in the C-tail of ß-catenin was reported to strengthen ß-catenin association to the basal transcription factor TATA-binding protein (43), it will be intriguing to assess how the phosphorylation of one or more tyrosine residues can alter ß-catenin properties in terms of cell-to-cell adhesiveness or binding affinity to the nuclear TCF/LEF transcription factors.

Golas et al. (35) recently studied the antitumoral activity of SKI-606 (WAY-173606) on colorectal cancer cells in vitro and in vivo. We therefore focused on SKI-606 inhibitory mechanisms by testing how its biological effects could correlate with inactivation of c-Src/ß-catenin signaling in human colorectal cancer cells. On incubation with SKI-606, tyrosine phosphorylation of ß-catenin and pp60(c-Src) was significantly inhibited in DLD-1 and Ls174T colorectal cancer cell lines. Although the association between ß-catenin and pp60(c-Src) kinase was not dependent on tyrosine phosphorylation (as it was maintained in SKI-606-treated samples), the activity of pp60(c-Src) kinase was needed to sustain constitutive ß-catenin activation. Tyrosine phosphorylation of ß-catenin decreased concomitantly with its trans-activating ability followed by down-regulation of the pivotal ß-catenin/TCF4 target gene cyclin D1 and proliferation inhibition. It is noteworthy that our findings were remarkably consistent across two human cell lines that recapitulate the early events implicated in colorectal carcinogenesis (i.e., DLD-1 carrying a mutation of the APC gene and Ls174T showing a mutated ß-catenin; ref. 13). Therefore, these conclusions can apply to colorectal cancer cells arising by different genetic backgrounds. Thus far, various studies investigated potential inhibitory agents of the transcriptional activity of ß-catenin in colon cancer, such as curcumin (44), nonsteroidal anti-inflammatory drugs (45), nitric oxide–donating aspirin (46), epigallocatechin-3-gallate (47), flavonone (48), and carnosol (49), but only the latter showed an association between changes in ß-catenin tyrosine phosphorylation and chemoprevention efficacy in a mouse model of colonic tumorigenesis. Thus, based on our results, SKI-606 could define a novel class of small-molecule drugs with a target specificity for ß-catenin tyrosine phosphorylation. In fact, the findings that SKI-606 did not reduce ß-catenin or TCF4 total protein levels indicated that its biological effects were not related to ß-catenin degradation, rather they affected ß-catenin cellular distribution by modulating its phosphotyrosine status. The SKI-606-mediated changes in ß-catenin subcellular localization and colorectal cancer cell adhesion and motility show that the transcriptional and adhesive cellular processes involving ß-catenin can be pharmacologically controlled by agents interfering with ß-catenin tyrosine phosphorylation. An unexpected result was the increased E-cadherin protein stability in colorectal cancer cells treated with SKI-606. Because we showed that ß-catenin knockdown by siRNA removed the stabilizing effect of SKI-606 on E-cadherin levels, the ability of SKI-606 to relocate ß-catenin at cell-to-cell contacts without altering its total cellular content likely represents the cause of the enhanced E-cadherin stability and cell adhesion. We cannot exclude that the inhibitory effect of SKI-606 on additional intracellular pathways might affect colorectal cancer cell-to-cell adhesion and motility. In fact, Golas et al. (35) showed that SKI-606 blocked focal adhesion kinase and AKT survival-promoting kinases in colorectal cancer cells. Interestingly, nine putative TCF/LEF-binding elements have been recently identified in the AKT gene promoter (50), indicating that down-regulation of other ß-catenin target genes might also account for the apoptosis observed on reduced expression of c-Src or its functional inactivation by SKI-606.

In conclusion, this study provides the first evidence in human colorectal cancer cells that molecular targeting of pp60(c-Src)–dependent ß-catenin tyrosine phosphorylation by SKI-606 can effectively interfere with ß-catenin-mediated activation of TCF4 transcription factor diverting its oncogenic nuclear potential toward cell-to-cell adhesion.


   Acknowledgments
 
Grant support: Italian Association for Cancer Research, Min. San. Ricerca Finalizzata (2003), CNR, MIUR-COFIN, and PRIN programs (2003-2004), EU (Prokinase network, 503467), CIHR, CFI, and NCI-C; Fondazione Italiana per la Ricerca sul Cancro fellowship (A.M.L. Coluccia).

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. Frank and Diane Boschelli (Wyeth Research) for kindly providing the SKI-606 (WAY-173606) compound, Dr. Hans Clevers for the pcDNA6TR and pTER vectors, and Dr. Chiara Castelli for critical reading of the article.


   Footnotes
 
Note: A.M.L. Coluccia and D. Benati contributed equally to this work.

Received 6/13/05. Revised 11/23/05. Accepted 12/ 8/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nelson WJ, Nusse R. Convergence of Wnt, ß-catenin, and cadherin pathways. Science 2004;303:1483–7.[Abstract/Free Full Text]
  2. Harris TJ, Peifer M. Decisions, decisions: ß-catenin chooses between adhesion and transcription. Trends Cell Biol 2005;15:234–7.[CrossRef][Medline]
  3. Behrens J, von Kries JP, Kuhl M, et al. Functional interaction of ß-catenin with the transcription factor LEF-1. Nature 1996;382:638–42.[CrossRef][Medline]
  4. Poy F, Lepourcelet M, Shivdasani RA, Eck MJ. Structure of a human Tcf4-ß-catenin complex. Nat Struct Biol 2001;8:1053–7.[CrossRef][Medline]
  5. Klymkowsky MW. ß-Catenin and its regulatory network. Hum Pathol 2005;36:225–7.[CrossRef][Medline]
  6. Oving IM, Clevers HC. Molecular causes of colon cancer. Eur J Clin Invest 2002;32:448–57.[CrossRef][Medline]
  7. Kikuchi A. Tumor formation by genetic mutations in the components of the Wnt signaling pathway. Cancer Sci 2003;94:225–9.[CrossRef][Medline]
  8. Pinto D, Clevers H. Wnt, stem cells and cancer in the intestine. Biol Cell 2005;97:185–96.[CrossRef][Medline]
  9. Waltzer L, Bienz M. Drosophila CBP represses the transcription factor TCF to antagonize Wingless signalling. Nature 1998;395:521–5.[CrossRef][Medline]
  10. Brantjes H, Roose J, van De Wetering M, Clevers H. All Tcf HMG box transcription factors interact with Groucho-related co-repressors. Nucleic Acids Res 2001;29:1410–9.[Abstract/Free Full Text]
  11. Daniels DL, Weis WI. ß-Catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation. Nat Struct Mol Biol 2005;12:364–71.[CrossRef][Medline]
  12. Billin AN, Thirlwell H, Ayer DE. ß-Catenin-histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator. Mol Cell Biol 2000;20:6882–90.[Abstract/Free Full Text]
  13. van de Wetering M, Sancho E, Verweij C, et al. The ß-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 2002;111:241–50.[CrossRef][Medline]
  14. Tetsu O, McCormick F. ß-Catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 1999;398:422–6.[CrossRef][Medline]
  15. He TC, Sparks AB, Rago C, et al. Identification of c-MYC as a target of the APC pathway. Science 1998;281:1509–12.[Abstract/Free Full Text]
  16. Willert J, Epping M, Pollack JR, Brown PO, Nusse R. A transcriptional response to Wnt protein in human embryonic carcinoma cells. BMC Dev Biol 2002;2:814–9.
  17. van de Wetering M, Oving I, Muncan V, et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep 2003;4:609–15.[CrossRef][Medline]
  18. Clapper ML, Coudry J, Chang WC. ß-Catenin-mediated signaling: a molecular target for early chemopreventive intervention. Mutat Res 2004;555:97–105.[Medline]
  19. Lepourcelet M, Chen YN, France DS, et al. Small-molecule antagonists of the oncogenic Tcf/ß-catenin protein complex. Cancer Cell 2004;5:91–102.[CrossRef][Medline]
  20. Piedra J, Martinez D, Castano J, Miravet S, Dunach M, de Herreros AG. Regulation of ß-catenin structure and activity by tyrosine phosphorylation. J Biol Chem 2001;276:20436–43.[Abstract/Free Full Text]
  21. Daniel JM, Reynolds AB. Tyrosine phosphorylation and cadherin/catenin function. BioEssays 1997;19:883–91.[CrossRef][Medline]
  22. Muller T, Choidas A, Reichmann E, Ullrich A. Phosphorylation and free pool of ß-catenin are regulated by tyrosine kinases and tyrosine phosphatases during epithelial cell migration. J Biol Chem 1999;274:10173–83.[Abstract/Free Full Text]
  23. Roura S, Miravet S, Piedra J, Garcia de Herreros A, Dunach M. Regulation of E-cadherin/catenin association by tyrosine phosphorylation. J Biol Chem 1999;274:36734–40.[Abstract/Free Full Text]
  24. Takayama T, Shiozaki H, Doki Y, et al. Aberrant expression and phosphorylation of ß-catenin in human colorectal cancer. Br J Cancer 1998;77:605–13.[Medline]
  25. Wong NA, Pignatelli M. ß-Catenin: a linchpin in colorectal carcinogenesis? Am J Pathol 2002;160:389–401.[Abstract/Free Full Text]
  26. Attoub S, Rivat C, Rodrigues S, et al. The c-kit tyrosine kinase inhibitor STI571 for colorectal cancer therapy. Cancer Res 2002;62:4879–83.[Abstract/Free Full Text]
  27. Nakae S, Shimada E, Urakawa T. Study of c-erbB-2 protein and epidermal growth factor receptor expression and DNA ploidy pattern in colorectal carcinoma. J Surg Oncol 1993;54:246–51.[Medline]
  28. Hiscox SE, Hallett MB, Puntis MC, Nakamura T, Jiang WG. Expression of the HGF/SF receptor, c-met, and its ligand in human colorectal cancers. Cancer Invest 1997;15:513–21.[Medline]
  29. Bolen JB, Veillette A, Schwartz AM, Deseau V, Rosen N. Analysis of pp60c-src in human colon carcinoma and normal human colon mucosal cells. Oncogene Res 1987;1:149–68.[Medline]
  30. Cartwright CA, Meisler AI, Eckhart W. Activation of pp60(c-Src) protein kinase is an early event in colonic carcinogenesis. Proc Natl Acad Sci U S A 1989;87:558–62.
  31. Cartwright CA, Coad CA, Egbert BM. Elevated c-Src tyrosine kinase activity in premalignant epithelia of ulcerative colitis. J Clin Invest 1994;93:509–13.[Medline]
  32. Talamonti MS, Roh MS, Curley SA, Gallick GE. Increase in activity and level of pp60(c-Src) in progressive stages of human colorectal cancer. J Clin Invest 1993;91:53–60.[Medline]
  33. Staley CA, Parikh NU, Gallick GE. Decreased tumorigenicity of a human colon adenocarcinoma cell line by an antisense expression vector specific for c-Src. Cell Growth Differ 1997;8:269–74.[Abstract]
  34. Golas JM, Arndt K, Etienne C, et al. SKI-606, a 4-anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases, is a potent antiproliferative agent against chronic myelogenous leukemia cells in culture and causes regression of K562 xenografts in nude mice. Cancer Res 2003;63:375–81.[Abstract/Free Full Text]
  35. Golas JM, Lucas J, Etienne C, et al. SKI-606, a Src/Abl inhibitor with in vivo activity in HT29 and Colo205 colon tumor xenograft models. Cancer Res 2005;65:5358–64.[Abstract/Free Full Text]
  36. Irby R, Mao W, Coppola D, et al. Overexpression of normal c-Src in poorly metastatic human colon cancer cells enhances primary tumor growth but not metastatic potential. Cell Growth Differ 1997;8:1287–95.[Abstract]
  37. Irby RB, Mao W, Coppola D, et al. Activating SRC mutation in a subset of advanced human colon cancers. Nat Genet 1999;21:187–90.[CrossRef][Medline]
  38. Daigo Y, Furukawa Y, Kawasoe T, et al. Absence of genetic alteration at codon 531 of the human c-src gene in 479 advanced colorectal cancers from Japanese and Caucasian patients. Cancer Res 1999;59:4222–4.[Abstract/Free Full Text]
  39. Laghi L, Bianchi P, Orbetegli O, Gennari L, Roncalli M, Malesci A. Lack of mutation at codon 531 of SRC in advanced colorectal cancers from Italian patients. Br J Cancer 2001;84:196–8.[CrossRef][Medline]
  40. Haraguchi K, Nishida A, Ishidate T, Akiyama T. Activation of ß-catenin-TCF-mediated transcription by non-receptor tyrosine kinase v-Src. Biochem Biophys Res Commun 2004;313:841–4.[CrossRef][Medline]
  41. Levy L, Wei Y, Labalette C, et al. Acetylation of ß-catenin by p300 regulates ß-catenin-Tcf4 interaction. Mol Cell Biol 2004;24:3404–14.[Abstract/Free Full Text]
  42. Jamieson CH, Ailles LE, Dylla SJ, et al. Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N Engl J Med 2004;351:657–67.[Abstract/Free Full Text]
  43. Hecht A, Litterst CM, Huber O, Kemler R. Functional characterization of multiple transactivating elements in ß-catenin, some of which interact with the TATA-binding protein in vitro. J Biol Chem 1999;274:18017–25.[Abstract/Free Full Text]
  44. Jaiswal AS, Marlow BP, Gupta N, Narayan S. ß-Catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells. Oncogene 2002;21:8414–27.[CrossRef][Medline]
  45. Dihlmann S, Siermann A, von Knebel Doeberitz M. The nonsteroidal anti-inflammatory drugs aspirin and indomethacin attenuate ß-catenin/Tcf4 signaling. Oncogene 2001;20:645–53.[CrossRef][Medline]
  46. Nath N, Kashfi K, Chen J, Rigas B. Nitric oxide-donating aspirin inhibits ß-catenin/T cell factor (TCF) signaling in SW480 colon cancer cells by disrupting the nuclear ß-catenin/Tcf association. Proc Natl Acad Sci U S A 2003;1000:12584–9.
  47. Dashwood WM, Orner GA, Dashwood RH. Inhibition of ß-catenin/Tcf activity by white tea, green tea, and epigallocatechin-3-gallate (EGCG): minor contribution of H2O2 at physiologically relevant EGCG concentrations. Biochem Biophys Res Commun 2002;296:584–8.[CrossRef][Medline]
  48. Park CH, Hahm ER, Lee JH, Jung KC, Yang CH. Inhibition of ß-catenin-mediated transactivation by flavonone in AGS gastric cancer cells. Biochem Biophys Res Commun 2005;331:1222–8.[CrossRef][Medline]
  49. Moran AE, Carothers AM, Weyant MJ, Redston M, Bertagnolli MM. Carnosol inhibits ß-catenin tyrosine phosphorylation and prevents adenoma formation in the C57BL/6J/Min/+ (Min/+) mouse. Cancer Res 2005;65:1097–104.[Abstract/Free Full Text]
  50. Dihlmann S, Kloor M, Fallsehr C, von Knebel Doeberitz M. Regulation of AKT1 expression by ß-catenin/Tcf/Lef signaling in colorectal cancer cells. Carcinogenesis 2005;26:1503–12.[Abstract/Free Full Text]



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