MCF7 and Hela cells were cultured with DMEM medium supplemented with 10% fetal bovine serum in a humidified CO₂ incubator at 37°C. To generate Twist-expression stable transfectants, Hela and MCF7 cells were transfected with pcDNA3-Twist1, and stable clones were selected with 1000 μg/ml of G418 (CALBIOCHEM) for 4 weeks.

TOPflash or FOPflash plasmid (Upstate, Lake Placid, NY) was transiently transfected into cells with Fugene 6 (Roche, Indianapolis, IN). For measuring the transcription of CD44, pGL3-CD44P was also expressed in cells. To normalize transfection efficiency, cells were also co-transfected with 0.1 μg of the pRL-CMV (Renilla luciferase). Forty-eight hours after transfection, luciferase activity was measured using the Dual-Luciferase Assay kit (Promega, Madison, WI). Three independent experiments were performed, and the calculated means and standard deviations are presented.

To knock down the expression of β-catenin, cells were seeded on 6-well plates and transfected with pGL3-CD44P, along with validated human β-catenin siRNA (Dharmacon) at a final concentration of 100 nM using X-tremeGENE siRNA transfection reagent (Roche) following manufacturer's instructions. After 36 h of transfection, cells were treated with or without PI3K/Akt inhibitors wortmannin (100 nM) for overnight. Luciferase activity was measured as described above. All experiments were performed at least three times in triplicate.

Commercial antibodies used in this study were presented in Table 1.

To prepare the whole-cell extract, cells were washed with PBS once and harvested by scraping them in 1 ml lyses buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 0.2 mM EDTA, 0.2% NP-40, 10% Glycerin, 1M β-Me, 1 μg/ml Aprotin, 0.5 μg/ml Leupetin, 0.1 mM Na₃VO₄, 0.5 mM 4NPP, 0.5 mM NaF, and protease inhibitors). Cellular lysates were centrifuged at 13,200 × g for 5 min at 4°C. Protein content was determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). The extracted proteins were separated in a 10-12% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Amersham Bioscience). The membranes were first blocked with 5% (w/v) nonfat dry milk in PBST and then probed with the indicated primary antibodies with gentle shaking at 4°C overnight. After washing the membranes four times, the membranes were incubated with the appropriate peroxidase-conjugated secondary antibodies for 1 hour. The signals were detected using an enhanced chemiluminescence kit (Amersham Biosciences).

Cells were grown on glass chamber slides fixed with 4% paraformaldehyde in PBS for 30 min. Then cells were permeabilized in 0.1% Triton X-100 for 30 min and blocked with 0.5% bovine serum albumin in PBS for 30 min at room temperature. After washing with PBS, the cells were incubated with specific primary antibodies for 1 hour at room temperature. After being washed with PBST, the cells were incubated with appropriate fluorescein isothiocyanate-conjugated secondary antibodies and then stained with 4', 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics). The images were visualized with an Olympus microscope.

Flow Cytometry Analysis was performed as described previously 23 . Cells were harvested by trypsinization and washed twice with PBS. The cells then were fixed and stained with monoclonal antibodies against CD44, CD24 or an isotype IgG, labeled with Alexa 488-conjugated secondary antibody, and subjected to flow cytometric analysis using a flow cytometer (BD-LSR model, Becton-Dickinson, San Jose, CA).

Single-cell suspensions were suspended at a density of 4,000 cells per milliliter in Dulbecco's modified Eagle's medium/F-12 (Twist/MCF7, MCF7) or Dulbecco's modified Eagle's medium (Twist/HeLa; HeLa) and seeded into six-well plates (2.0 mL per plate) coated with 1.2% poly-Hema. Suspension cultures were continued for 1-2 weeks (7-day for Hela and Twist/Hela cells; 12-day for MCF7 and Twist/MCF7 cells) until the formation of tumorspheres. Colonies were counted at 10 different views under microscope. Experiments were repeated three times with duplication in each experiment.

To examine the role of Twist in EMT induction and the generation of stem-cell like properties, we generated Twist-stable expression clones in cervical cancer Hela and breast cancer MCF7 cells. Expression of Twist induced EMT in these cells as morphological changes from a cobble-stone-like shape to a spindle-like appearance were noted; these cells became elongated in shape and disassociated from their neighboring cells (Figure 1a). Immunofluoresent staining showed the upregulation of mesenchymal markers N-cadherin and vimentin and the downregulation of epithelial markers ZO-1 (Figure 1a). Interestingly, β-catenin was accumulated and translocated into both the cytoplasm and the nucleus. Similar results were further confirmed by Western blotting using specific antibodies against E-cadherin, ZO-1, N-cadherin and vimentin (Figure 1b). Consistent with these molecular changes, cell motility was significantly enhanced in cells expressing Twist than that of parental cells (Figure 2a). These results indicate that expression of Twist can induce EMT in Hela and MCF7 cells, which is accompanied with the downregulation of epithelial markers and upregulation of mesenchymal molecules, and thus, results in the enhancement of cell motility.

The tumorsphere assay, based on the unique property of stem/progenitor cells to survive and grow in serum-free suspension, was successfully used to establish long-term cultures enriched in stem/progenitor cells from invasive tumor samples. To examine whether the expression of Twist induced stem cell-like properties in Hela and MCF7 cells, we performed a tumorsphere formation assay. Surprisingly, the expression of Twist induced about a 24- and 18-fold enhancement in tumorsphere-formation in Hela and MCF7 cells, respectively, compared with that of parental cells (Figure 2b). To further confirm these findings, we also measured the level of aldehyde dehydrogenase 1 (ALDH1), a detoxifying enzyme responsible for the oxidation of retinol to retinoic acid and which has a role in the early differentiation of stem cells. High ALDH1 activity is associated with several types of murine and human hematopoietic and neural stem/progenitor cells. As shown in Figure 2c, the expression of Twist significantly induced the level of ALDH1 in Hela and MCF7 cells.

The CD44^high/CD24^low phenotype has been used to isolate stem cells from the human normal mammary epithelium 2 . It has been shown that as few as 200 of these cells generated tumors in NOD/SCID mice whereas 20,000 cells that did not display this phenotype failed to do so. These cells were able to self-renew, differentiate, and display CSC features. To examine whether expression of Twist induces the expansion of this population of cells, we measured the expression of CD44 by Western blotting, immune-fluorescence staining and FACS analyses. As shown in Figures 3a, b and 3c, expression of Twist dramatically elevated the level of CD44 in Hela and MCF7 cells. Consistent with these observations, when CD44 promoter luciferase plasmid was expressed in these cells, the luciferase activity was significantly elevated in Twist-overexpressing cells than that of parental cells (Figure 3d). Together, these results indicate that the expression of Twist is critical in EMT induction, which confers cells with stem-cell like properties by inducing the expression of CD44 and enhancing tumorsphere formation and ALDH1 activity.

β-catenin plays an important role in a variety of human tumors. Downregulation of E-cadherin expression often results in an increase of β-catenin, which binds to TCF/LEF to participate in transcription regulation. To test whether the β-catenin pathway was activated in cells expressing Twist, we isolated β-catenin from the membrane, the cytoplasm and the nucleus of parental and Twist-overexpressing cells. Although the membrane-bound β-catenin was significantly decreased, the total level of β-catenin, the cytoplasmic and the nuclear β-catenin were greatly increased in cells expressing Twist (Figure 4a). β-catenin is a labile protein, and it subjected to GSK-3β-mediated phosphorylation and proteasome degradation. Interestingly, we found that the phosphorylation of β-catenin was significantly reduced in cells expressing Twist, suggesting that the increase of the cytoplasmic and the nuclear β-catenin from Twist-overexpressing cells resulted from the release of membrane-fraction β-catenin as well as from the inhibition of phosphorylation and degradation of β-catenin in these cells. To further confirm the activation of the β-catenin pathway, we measured the TOP/FOP luciferase activities. Both Twist-overexpressing cell lines have higher luciferase activities than that of the corresponding parental cells (Figure 4b). Taken together, these data showed that EMT induces an accumulation and nuclear translocation of β-catenin and thus activates the Wnt/β-catenin signaling pathway.

We also treated Hela cells with Wnt3a, a ligand known to activate the Wnt/β-catenin pathway. As expected, Wnt3a induced β-catenin stabilization in Hela cells (Figure 5a) and a corresponding upregulation of TOP/FOP luciferase activity (Figure 5b). Although Twist-overexpressing Hela cells contained higher levels of β-catenin, and treatment with Wnt3a did not further elevate the level of β-catenin (Figure 5a), Wnt3a can further enhance the TOP/FOP luciferase by more than 10-fold (Figure 5c); this suggests that EMT can synergize the activation of β-catenin induced by Wnt ligands.

CD44 expression was part of a genetic program controlled by the β-catenin/Tcf-4 signaling pathway 26 . Over-expression of the CD44 family is an early event in the colorectal adenoma-carcinoma process, which suggests β-catenin/Tcf-4 signaling is crucial in initiating tumorigenesis 27 . Masaki et al supported this result with the immunostaining of β-catenin and CD44, suggesting that the up-regulation of CD44 through nuclear β-catenin contributed to the formation of the tumor 28 . Thus, we measured the CD44 luciferase in Twist-overexpressing cells stimulated with Wnt3a. We found that CD44 luciferase levels were further elevated by Wnt3a (Figure 5c), indicating that the activation of the β-catenin pathway plays a critical role in the expansion of CD44⁺ cells with stem-cell like properties.

Twist has been shown to activate the Akt signaling pathway by inducing the expression of Akt 29 . To examine whether the expression of Twist activates the Akt signaling, we measured the phosphorylation of Akt in cells expressing Twist and their corresponding parental cells. We found that Akt was activated in Hela and MCF7 cells expressing Twist (Figure 6a). Serine/threonine protein kinase GSK-3β, a downstream target of PI3K/Akt, was also found to be inactivated by phosphorylation at serine 9, whereas the total GSK-3β level remained changed. As GSK-3β can phosphorylate β-catenin and result in its proteasome degradation, this result was consistent with our finding that β-catenin was stabilized because of the significantly reduced level of phosphorylation (Figure 4a). The activation of Akt and suppression of GSK-3β in Twist-expressing cells were quite interesting, as we showed previously that GSK-3β is the major kinase regulating the protein stability and the cellular localization of Snail 30 . To further extend this finding, we examined the expression of Snail in these cells. We found that the level of Snail was significantly higher in Twist-overexpressing cells than that of parental cells (Figure 6b). Together, our results indicate that expression of Twist can induce the activation of Akt and the suppression of GSK-3β, which results in the stabilization of β-catenin and Snail in Hela and MCF7 cells.

We showed that EMT induced the downregulation of E-cadherin and the detachment of β-catenin from membrane localization. We further showed that EMT activated Akt and suppressed the function of GSK-3β, which is required for the stabilization and nuclear translocation of β-catenin, and thus results in the transcription of CD44. To investigate whether the β-catenin and Akt pathways were critical for the induction of CD44, we knocked down the expression of β-catenin or inhibited the Akt pathway by wortmannin in cells. We found that either the knockdown of β-catenin expression or the inhibition of Akt pathway suppressed the expression of CD44 (Figure 7a). Inhibition of both pathways can further synergistically suppress the expression of CD44, suggesting that the activation of these two pathways is critical for the maintenance of CD44 expression.