YWHAZ, also known as 14-3-3zeta, has been reportedly elevated in many human tumors, including non–small cell lung carcinoma (NSCLC) but little is known about its specific contribution to lung cancer malignancy. Through a combined array-based comparative genomic hybridization and expression microarray analysis, we identified YWHAZ as a potential metastasis enhancer in lung cancer. Ectopic expression of YWHAZ on low invasive cancer cells showed enhanced cell invasion, migration in vitro, and both the tumorigenic and metastatic potentials in vivo. Gene array analysis has indicated these changes associated with an elevation of pathways relevant to epithelial–mesenchymal transition (EMT), with an increase of cell protrusions and branchings. Conversely, knockdown of YWHAZ levels with siRNA or short hairpin RNA (shRNA) in invasive cancer cells led to a reversal of EMT. We observed that high levels of YWHAZ protein are capable of activating β-catenin–mediated transcription by facilitating the accumulation of β-catenin in cytosol and nucleus. Coimmunoprecipitation assays showed a decrease of ubiquitinated β-catenin in presence of the interaction between YWHAZ and β-catenin. This interaction resulted in disassociating β-catenin from the binding of β-TrCP leading to increase β-catenin stability. Using enforced expression of dominant-negative and -positive β-catenin mutants, we confirmed that S552 phosphorylation of β-catenin increases the β-catenin/YWHAZ complex formation, which is important in promoting cell invasiveness and the suppression of ubiquitnated β-catenin. This is the first demonstration showing YWHAZ through its complex with β-catenin in mediating lung cancer malignancy and β-catenin protein stability. Mol Cancer Res; 10(10); 1319–31. ©2012 AACR.

Lung cancer is a malignant tumor with a high incidence and mortality rate (1). Non–small cell lung cancer (NSCLC) represents 80% of all lung cancers and has an overall 5-year survival rate of 10% to 15% (2). Metastasis is the main cause of treatment failure and cancer-related deaths. In carcinomas, metastasis is a multiple step process, the first of which is invasion (3). Cancer cells acquire their invasive capacity by undergoing phenotypic conversion referred to as epithelial–mesenchymal transition (EMT), which enables them to become motile and to invade adjacent tissues. This process is triggered by various signaling pathways and controlled by a group of transcriptional factors, such as zinc finger proteins and basic helix–loop–helix factors (4, 5). In lung cancer, Slug is known to be an important EMT inducer and is able to suppress the expression levels of cell adhesion molecules (6–8).

β-Catenin binds to the cytoplasmic domain of E-cadherin and is essential for the structural organization and function of cadherins. β-Catenin also plays a central role as a cotranscription factor in both canonical and noncanonical Wnt signaling (9). In the absence of Wnt signaling, the soluble form of β-catenin, which is not associated with cadherins, is phosphorylated by glycogen synthase kinase-3β (10) and is subsequently recognized by the β-transducin repeat-containing protein (β-TrCP), leading to the ubiquitination and degradation of β-catenin by proteasomes (11). Activation of the Wnt pathway inhibits the degradation of β-catenin and results in its accumulation in the cytosol and subsequent nuclear translocation (12). Following nuclear translocation, β-catenin interacts with the transcription factor T-cell factor/lymphoid enhancer factor (TCF/LEF) to induce the transactivation of certain vital genes, including cyclin D1, c-Myc, and Slug (13–15). Although excess β-catenin proteins are observed in certain cancers and associated with dysregulation of ubiquitination, the molecules involved in β-catenin accumulation are largely unknown.

In this study, we identify YWHAZ, also known as 14-3-3zeta, as a potential regulator for the function of β-catenin and its turnover. YWHAZ was initially identified as one of the metastasis enhancer genes through an integrated approach that combined comparative genome hybridization (CGH) with an expression microarray on lung cancer cell lines with different metastatic potentials (16). YWHAZ has attracted interest because of its elevated expression associated with a variety of cancers (17–24). Recently, it is understood that YWHAZ has critical antiapoptotic functions and is able to suppress anoikis in lung cancer (17, 18, 25). In this communication, we report for the first time that YWHAZ amplification is associated with lung cancer malignancy and the importance of YWHAZ/β-catenin axis in preventing β-catenin ubiquitination and degradation and subsequently in promoting EMT phenotype and invasiveness of cancer cells.

Materials and plasmid constructs

All reagents, antibodies, and plasmid constructs used in this study are described in the Supplementary Information in Supplementary Methods.

Cell culture and transfection

The low invasive and highly invasive human lung adenocarcinoma cell lines, CL1-0 and CL1-5, were established and characterized as previously described (16). We tested their invasiveness for authentication by Matrigel invasion assays in our laboratory every month. The cell lines, HEK293, A549, H1299, MCF-7, HeLa, and BEAS2B cells were purchased from the American Type Culture Collection (ATCC) that conducts cell line characterizations and passaged in our laboratory for fewer than 6 months after receipt. The method of characterization used by ATCC can be found in its website. Cells were cultured in Dulbecco's modified Eagle's medium with 10% FBS and 1% penicillin–streptomycin at 37°C in a humidified atmosphere of 5% CO2. For enforced expression of V5-tagged YWHAZ in the minimally invasive lung cancer cell line, CL1-0 cells were transfected with pEF6/V5-HisTOPO-YWHAZ (pEF6/V5-YWHAZ) or pEF6/V5-HisTOPO vector using lipofectamine reagent, according to the manufacturer's protocol. After culturing in medium containing 10 μg/mL of blasticidine for 2 to 3 weeks, individual clones were isolated. Clones that expressed the YWHAZ cDNA coding region were maintained in medium containing 5 μg/mL of blasticidine and used for further investigation.

Lentiviral shRNA–mediated knockdown

Luciferase (pLKO.1-shLuc; TRCN00000072244) and 2 YWHAZ-shRNA–containing lentiviral vectors (pLKO.1 shYWHAZ-1 and pLKO.1-shYWHAZ-2; TRCN0000029404 and TRCN0000029405) were obtained from the National RNAi Core Facility (Academia Sinica, Taipei, Taiwan) and prepared in accordance with the standard protocols. Cells were infected with lentivirus (multiplicity of infection 5 or 10) in medium containing polybrene (8 μg/mL). At 24 hours after infection, cells were treated with 0.75 μg/mL puromycin to select for puromycin-resistant pooled clones.

Microarray analysis

Human genomic DNAs from CL1-5 and CL1-0 cells were analyzed using array CGH containing 385,000 probes with a median distance of approximately 6,000 bp (NimbleGen) to determine copy number variations in dye-swap replicate experiments. For expression analysis, cDNA preparation and array hybridization were conducted according to the Affymetrix GeneChip expression analysis technical manual (see Supplementary Methods).

Quantitative real-time PCR

The DNA copy number of YWHAZ was detected by real-time PCR on the Prism 7900 Sequence Detection System (Applied Biosystems), according to the manufacturer's instructions. The mRNA expression level of YWHAZ and CTNNB1 (β-catenin) was also detected by real-time reverse transcription polymerase chain reaction (RT-PCR) on the same system. The primers and probes of YWHAZ (Hs00237047_m1) and TATA-box binding protein (TBP, Hs00427620_m1) were purchased from Applied Biosystems. The CTNNB1 primers were as follows: forward primer 5′-GGCTACTGTTGGATTGATTCGAA-3′ and reverse primer 5′-GCTGGGTATCCTGATGTGCAC-3′. We used the housekeeping gene ACTB as the reference gene in genomic real-time PCR assay. The DNA copy number of YWHAZ for tumor or normal tissue was presented as relative level to reference gene. For mRNA analysis, we used TBP as the internal control. The relative expression level of YWHAZ and CTNNB1 compared with that of TBP was defined as $- \Delta {\rm {C_T} = - [ {C_{T_{\rm target}}} - {C_{T_{\rm TBP}}}}]$⁠. The target/TBP mRNA ratio was calculated as ${\rm 2^{ - \Delta C_T}} \times K$⁠, in which K is a constant.

Migration and invasion assays

In vitro cell migration and invasion assays were conducted as previously described (26) using transwell chambers (8 μm pore size; Costar). In migration assays, 5 × 103 cells were seeded on top of the polycarbonate filters and incubated for 12 hours. Filters were swabbed with a cotton swab, fixed with methanol, and then stained with Giemza solution (Sigma). For the invasion assays, filters were coated with Matrigel (Becton Dickinson), and 2 × 104 cells were seeded onto the Matrigel and incubated for 20 hours. The cells attached to the lower surface of the filter were counted under a light microscope (× 100 magnification).

In vivo tumorigenesis and metastasis

Six-week-old nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice (supplied by the animal center of the College of Medicine, National Taiwan University, Taipei, Taiwan) were housed 4 mice per cage and fed autoclaved food ad libitum. Detailed information on subcutaneous implantation, intravenous tail injection, and orthotopic implantation of tumors are included in the Supplemental Methods. Mouse experiments were approved by the Institutional Animal Care and Use Committee of National Chung Hsing University (Taichung, Taiwan).

Immunoprecipitation and immunoblotting

The preparation of whole-cell lysates, cytoplasmic and nuclear extracts, and Western blot analysis was described previously (27). Cells were lysed in lysis buffer (50 mmol/L Tris/HCl (pH 7.4), 1% Triton-X 100, 10% glycerol, 150 mmol/L NaCl, 1 mmol/L EDTA, 20 μg/mL leupeptin, 1 mmol/L PMSF, 20 μg/mL aprotinin, and 20 μg/mL pepstatin) and cleaned by preincubation with protein A-Sepharose beads to remove nonspecifically bound proteins. After precipitation with appropriate antibodies and protein A-Sepharose beads, the immunoprecipitated complexes were washed and separated by SDS-PAGE. Immunoblotting was done with appropriate antibodies using the Amersham Biosciences Enhanced Chemiluminescence System for detection.

Immunofluorescent staining

Cells cultured on 12 mm glass cover-slips were fixed for 15 minutes in PBS containing 4% paraformaldehyde and 2% sucrose and then permeabilized in PBS containing 0.3% Triton X-100 for 2 minutes. Cover-slips were reacted with primary antibody against β-catenin and fluorescein isothiocynate (FITC)-labeled antimouse secondary antibody. F-actin was stained with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin, and nuclei were demarcated with 4′,6-diamidino-2-phenylindole (DAPI) staining. The cells were mounted onto slides and visualized using fluorescence microscopy (model Axiovert 100; Carl Zeiss) or a Zeiss LSM510 laser-scanning confocal microscope image system.

Luciferase reporter assay

TOPFLASH or FOPFLASH (Millipore) plasmid was transfected into CL1-0 cells or CL1-5 cells together with the indicated plasmids. Renilla luciferase plasmid (pRL-TK) was cotransfected as an internal control. Cell lysates were collected 48 hours after transfection, and the dual-luciferase assay was conducted according to the manufacturer's protocol.

Statistical analysis

Data are presented as the mean ± SD for at least 3 independent experiments. The quantitative in vitro and in vivo data were analyzed using the Student t test. All analyses were conducted using SPSS software (v10.0; SPSS, Inc.) and SAS v 9.1 software (SAS Institute, Inc.). All statistical tests were 2-sided, and P values <0.05 were considered statistically significant.

YWHAZ expression enhances lung cancer cell invasion and migration in vitro

To identify metastasis-promoting genes caused by genomic amplification, array CGH and an expression microarray were used to study a lung cancer invasion model. Genes with an amplified copy number (log2 > 0.3) and upregulated expression (>5-fold) in the highly invasive cell line, CL1-5, were selected for further analysis and compared with the corresponding genes in a less invasive cell line, CL1-0. Of the genes identified, YWHAZ is known to have oncogenic effects in various types of cancers (28). The gene copy number, transcript expression, and protein expression of YWHAZ in both the CL1-0 and CL1-5 cell lines were consistent with the results obtained from array CGH and microarray approaches (Fig. 1A). We further examined the expression of YWHAZ in various cell lines, and Western blot analysis showed that YWHAZ was widely expressed in different tumor types, such as lung, breast, and cervical cancer cells (Supplementary Fig. S1A); however, its expression was relatively low in the nonmalignant human epithelial cell lines, BEAS2B and HEK293. Ectopic expression of V5-tagged YWHAZ in the low invasive cell line, CL1-0 (Supplementary Fig. S1B), showed higher cell proliferation and enhanced anchorage-dependent and -independent growth as compared with the mock control cells (Supplementary Fig. S1C and S1D). Furthermore, in vitro cell invasiveness (Fig. 1B) and migration (Fig. 1C) were all increased by 2.4- and 2.5-fold in these cells stably transfected with V5-tagged YWHAZ construct. These results suggest oncogenic nature of YWHAZ in mediating lung cancer cell metastatic potential in vitro.

Figure 1.

YWHAZ is associated with invasion and migration of lung cancer cells. A, YWHAZ copy number, mRNA, and protein expression. Top, YWHAZ copy number was detected by real-time genomic PCR (n = 6; *, P < 0.001 vs. mock). Middle, mRNA was measured by qRT-PCR (n = 3; *, P < 0.001 vs. mock). TATA-box binding protein (TBP) served as the internal control. Bottom, YWHAZ expression was measured by Western blot analysis. β-Tubulin served as the internal control. B, invasion capability of V5-tagged YWHAZ-transfectants as analyzed by Matrigel invasion assays (n = 3; *, P < 0.004 vs. mock). Mock: vector only transfectant; YWHAZ #1, YWHAZ #2, and YWHAZ #3: single clone; YWHAZ #mix: mixed clone. C, migration ability of cells with constitutive V5-tagged YWHAZ expression as determined by in vitro transwell migration assays (n = 3; *, P < 0.002 vs. mock).

Figure 1.

YWHAZ is associated with invasion and migration of lung cancer cells. A, YWHAZ copy number, mRNA, and protein expression. Top, YWHAZ copy number was detected by real-time genomic PCR (n = 6; *, P < 0.001 vs. mock). Middle, mRNA was measured by qRT-PCR (n = 3; *, P < 0.001 vs. mock). TATA-box binding protein (TBP) served as the internal control. Bottom, YWHAZ expression was measured by Western blot analysis. β-Tubulin served as the internal control. B, invasion capability of V5-tagged YWHAZ-transfectants as analyzed by Matrigel invasion assays (n = 3; *, P < 0.004 vs. mock). Mock: vector only transfectant; YWHAZ #1, YWHAZ #2, and YWHAZ #3: single clone; YWHAZ #mix: mixed clone. C, migration ability of cells with constitutive V5-tagged YWHAZ expression as determined by in vitro transwell migration assays (n = 3; *, P < 0.002 vs. mock).

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YWHAZ expression promotes lung cancer malignancy in vivo

To determine whether YWHAZ possesses oncogenic activities in vivo, 3 approaches were carried out. Cells from 2 YWHAZ stably expressing clones (YWHAZ #1 and YWHAZ #3) and 1 mock control were: (i) subcutaneously implanted into the dorsal regions of NOD SCID mice, (ii) directly injected to the circulation of mice to eschew the initial steps of local invasion and intravasation, and (iii) orthotopically injected into 1 lobe of mouse lung. As shown in Fig. 2A, hematoxylin and eosin (H&E) staining and immunohistochemistry revealed that the lungs of mice implanted by YWHAZ transfectants contained many more micrometastatic lesions than the mock controls. The average human-vimentin stain burden was 23 to 29 per lung in mice inoculated with the YWHAZ transfectants (Fig. 2B). This result implied that YWHAZ is able to promote cell metastasis in vivo. For circulation injection, mice with YWHAZ transfectants developed more pulmonary metastasis nodules than with the mock control (Fig. 2C). The nodule numbers of YWHAZ #1 and YWHAZ #3 groups were 12 ± 1.72 and 11.3 ± 1.91, respectively, but none in mock controls (Fig. 2D). Macrometastatic lesions in various lung sections are shown in Supplementary Table S1. For the orthotopical lung injection with YWHAZ stably expressing clone cells, we have observed a significant increase in tumorigenesis at the site of injection, and an increase in local metastasis to the adjunct lobe of the lung, and distant metastasis to the liver, compared with the mock control cell injections (Fig. 2E and Supplementary Table S2). These in vivo studies, which are consistent with previous in vitro findings strongly support the oncogenic nature of YWHAZ in promoting cancer metastasis.

Figure 2.

YWHAZ is a metastasis-promoting gene in lung cancer. A, micrometastatic analysis of V5-tagged YWHAZ transfectants. Top, representative H&E–stained sections of lungs from SCID mice (n = 6) with subcutaneous tumors. The black arrow indicates the micrometastasis of CL1-0 cells expressing V5-YWHAZ. Bottom, immunohistochemical stain showing micrometastatic lesions by using antihuman vimentin antibody. B, quantification of the average numbers of human-vimentin stains in the lungs of these mice with subcutaneous tumors (*, P < 0.01 vs. mock). C, macrometastatic analysis of V5-tagged YWHAZ transfectants. Appearance and representative H&E–stained sections of the lungs from SCID mice injected intravenously with V5-tagged YWHAZ-expressing cells. The arrows indicate the metastasis ranges for each group (n = 6); magnification, × 20. D, quantification of the average pulmonary metastasis nodules from these mice with intravenously injected cancer cells (*, P < 0.002 vs. mock). E, gross and H&E staining of sections of lungs and livers from mice after orthotopic lung implantation (n = 6). The black arrow indicates tumor mass.

Figure 2.

YWHAZ is a metastasis-promoting gene in lung cancer. A, micrometastatic analysis of V5-tagged YWHAZ transfectants. Top, representative H&E–stained sections of lungs from SCID mice (n = 6) with subcutaneous tumors. The black arrow indicates the micrometastasis of CL1-0 cells expressing V5-YWHAZ. Bottom, immunohistochemical stain showing micrometastatic lesions by using antihuman vimentin antibody. B, quantification of the average numbers of human-vimentin stains in the lungs of these mice with subcutaneous tumors (*, P < 0.01 vs. mock). C, macrometastatic analysis of V5-tagged YWHAZ transfectants. Appearance and representative H&E–stained sections of the lungs from SCID mice injected intravenously with V5-tagged YWHAZ-expressing cells. The arrows indicate the metastasis ranges for each group (n = 6); magnification, × 20. D, quantification of the average pulmonary metastasis nodules from these mice with intravenously injected cancer cells (*, P < 0.002 vs. mock). E, gross and H&E staining of sections of lungs and livers from mice after orthotopic lung implantation (n = 6). The black arrow indicates tumor mass.

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YWHAZ induces a neuron-like morphologic change and EMT

To identify the downstream genes regulated by YWHAZ, differentially gene expression profilings were carried out in microarray gene chips (GEO series accession no. GSE20318), followed by the analysis with our in-house pathway analysis tool (29) as well as a commercial software, GeneGo (http://www.genego.com/metacore). These pathway analyses had identified 4 EMT-related pathways in the top 10 pathways that were significantly affected by YWHAZ. The differentially expressed genes involved in these pathways were validated using quantitative real-time PCR (qRT-PCR; Table 1). Among them, the Wnt signaling pathway was highly activated in cells stably expressing V5-tagged YWHAZ. Morphologically, there was also a change from the cobble-like appearance of low invasive CL1-0 and/or mock control cells to a neuron-like morphology of cells in these V5-tagged YWHAZ-expressing clones (Fig. 3A, top). Colonies formed by V5-tagged YWHAZ-expressing cells exhibited a well-spread morphology. Cell surface protrusions and branches were evident after phalloidin and DAPI staining (Fig. 3A, bottom). Quantitative data had shown a 70% increase in the ratio of cell branching by YWHAZ expression as compared with mock control cells (Supplementary Fig. S2). Western blot analysis showed that the levels of N-cadherin, vimentin, and Slug increased in all of the YWHAZ transfectants compared with the mock control (Fig. 3B). Conversely, knockdown of endogenous YWHAZ expression in 2 highly YWHAZ-expressing cell lines, CL1-5 and A549, by siRNA increased E-cadherin expression and reduced Slug expression (Fig. 3C). To rule out the effects of off-target, we silenced YWHAZ gene in the highly invasive CL1-5 cells by using 2 independent YWHAZ-specific short hairpin RNAs (shRNA) and found that knockdown of YWHAZ resulted in a reciprocal alteration in EMT markers (Fig. 3D). These ectopic expression and knockdown approaches have showed the oncogenic nature of YWHAZ in mediating EMT phenotype.

Figure 3.

YWHAZ expression is crucial for inducing EMT. A, effect of YWHAZ overexpression on cell morphology and protrusions formation as well as branchings. The morphology of CL1-0 cells expressing either the control vector or V5-YWHAZ was examined by phase contrast microscopy after plating for 24 hours (top). Bottom, immunofluorescent staining of the transfectants. F-actin was stained with phalloidin, indicating the cell morphology; and nucleus was counter-stained with DAPI. B, expression of EMT markers in mock and YWHAZ transfectants, as measured by immunoblotting. β-Tubulin served as a loading control. C, Western blot analysis of epithelial and mesenchymal proteins in CL1-5 and A549 cells after transient transfection with YWHAZ-specific siRNA for 48 hours. D, the effects of YWHAZ silencing on EMT proteins determined by 2 independent YWHAZ–shRNAs (sh-YWHAZ-1 and sh-YWHAZ-2) transfectants in CL1-5 cells.

Figure 3.

YWHAZ expression is crucial for inducing EMT. A, effect of YWHAZ overexpression on cell morphology and protrusions formation as well as branchings. The morphology of CL1-0 cells expressing either the control vector or V5-YWHAZ was examined by phase contrast microscopy after plating for 24 hours (top). Bottom, immunofluorescent staining of the transfectants. F-actin was stained with phalloidin, indicating the cell morphology; and nucleus was counter-stained with DAPI. B, expression of EMT markers in mock and YWHAZ transfectants, as measured by immunoblotting. β-Tubulin served as a loading control. C, Western blot analysis of epithelial and mesenchymal proteins in CL1-5 and A549 cells after transient transfection with YWHAZ-specific siRNA for 48 hours. D, the effects of YWHAZ silencing on EMT proteins determined by 2 independent YWHAZ–shRNAs (sh-YWHAZ-1 and sh-YWHAZ-2) transfectants in CL1-5 cells.

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Table 1.

EMT-related pathways involved in CL1-0 cells following YWHAZ gene introduction

YWHAZ # mix/mock (fold change)
Accession codeGene symbolGene nameFunctionAffymetrixReal-time RT–PCR
Wnt signaling pathway 
NM_003507 FZD7 Frizzled homolog 7 (DrosophilaWnt receptor 2.976 1.507 
NM_006183 NTS Neurotensin Neurotransmitter 5.439 6.274 
NM_003199 TCF4 Transcription factor 4 Transcription factor 2.679 2.606 
NM_053056 CCND1 Cyclin D1 Regulators of CDK kinases 4.698 3.581 
NM_012342 BAMBI BMP and activin membrane-bound inhibitor homolog Signal transduction 2.925 2.337 
NM_001002880 CBY1 Chibby homolog 1 (DrosophilaInhibiting β-catenin–mediated transcriptional activation 0.831 0.731 
TGF-β signaling pathway 
NM_000660 TGFB1 Transforming growth factor, β–1 Inducing transformation 1.368 1.521 
NM_001015886 HMGA2 High mobility group AT-hook 2 Transcriptional regulating factor 1.935 1.301 
NM_002317 LOX Lysyl oxidase Tumor suppressor gene 0.352 0.111 
NM_006079 CITED2 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 Angiogenesis and development 3.58 2.861 
Focal adhesion 
NM_002210 ITGAV Integrin, αV Cell adhesion and facilitate signal transduction 2.668 2.487 
NM_002086 GRB2 Growth factor receptor-bound protein 2 Signal transduction 1.867 1.667 
NM_000090 COL3A1 Collagen, type III, α1 Extracellular matrix 4.193 2.536 
NM_001753 CAV1 Caveolin 1 Negative regulator of the Ras-p42/44 MAP kinase cascade 0.154 0.311 
NM_001233 CAV2 Caveolin 2 Negative regulator of the Ras-p42/44 MAP kinase cascade 0.101 0.111 
NM_001945 HBEGF Heparin-binding EGF-like growth factor Angiogenesis and signal transduction 4.176 3.044 
Regulation of actin cytoskeleton 
NM_002356 MARCKS Myristoylated alanine-rich protein kinase C substrate Cell motility 5.819 2.408 
NM_012250 RRAS2 Related RAS viral (r-ras) oncogene homolog 2 Inducing transformation 2.151 1.54 
NM_004447 EPS8 EGF receptor pathway substrate 8 Signal transduction 1.708 1.427 
NM_004342 CALD1 Caldesmon 1 Regulation of cell contraction 1.995 1.386 
YWHAZ # mix/mock (fold change)
Accession codeGene symbolGene nameFunctionAffymetrixReal-time RT–PCR
Wnt signaling pathway 
NM_003507 FZD7 Frizzled homolog 7 (DrosophilaWnt receptor 2.976 1.507 
NM_006183 NTS Neurotensin Neurotransmitter 5.439 6.274 
NM_003199 TCF4 Transcription factor 4 Transcription factor 2.679 2.606 
NM_053056 CCND1 Cyclin D1 Regulators of CDK kinases 4.698 3.581 
NM_012342 BAMBI BMP and activin membrane-bound inhibitor homolog Signal transduction 2.925 2.337 
NM_001002880 CBY1 Chibby homolog 1 (DrosophilaInhibiting β-catenin–mediated transcriptional activation 0.831 0.731 
TGF-β signaling pathway 
NM_000660 TGFB1 Transforming growth factor, β–1 Inducing transformation 1.368 1.521 
NM_001015886 HMGA2 High mobility group AT-hook 2 Transcriptional regulating factor 1.935 1.301 
NM_002317 LOX Lysyl oxidase Tumor suppressor gene 0.352 0.111 
NM_006079 CITED2 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 Angiogenesis and development 3.58 2.861 
Focal adhesion 
NM_002210 ITGAV Integrin, αV Cell adhesion and facilitate signal transduction 2.668 2.487 
NM_002086 GRB2 Growth factor receptor-bound protein 2 Signal transduction 1.867 1.667 
NM_000090 COL3A1 Collagen, type III, α1 Extracellular matrix 4.193 2.536 
NM_001753 CAV1 Caveolin 1 Negative regulator of the Ras-p42/44 MAP kinase cascade 0.154 0.311 
NM_001233 CAV2 Caveolin 2 Negative regulator of the Ras-p42/44 MAP kinase cascade 0.101 0.111 
NM_001945 HBEGF Heparin-binding EGF-like growth factor Angiogenesis and signal transduction 4.176 3.044 
Regulation of actin cytoskeleton 
NM_002356 MARCKS Myristoylated alanine-rich protein kinase C substrate Cell motility 5.819 2.408 
NM_012250 RRAS2 Related RAS viral (r-ras) oncogene homolog 2 Inducing transformation 2.151 1.54 
NM_004447 EPS8 EGF receptor pathway substrate 8 Signal transduction 1.708 1.427 
NM_004342 CALD1 Caldesmon 1 Regulation of cell contraction 1.995 1.386 

NOTE: NM_003406 YWHAZ, Affymetrix (YWHAZ # mix/mock): 1.753-fold; real-time RT-PCR (YWHAZ # mix/mock): 2.773-fold.

YWHAZ facilitates activation of β-catenin and β-catenin–mediated invasiveness

Wnt/β-catenin signaling pathway is well-established in EMT and cell invasiveness. On the basis of the findings of gene array analysis, we focused on the effects of YWHAZ on regulating β-catenin signaling activity. Immunofluorescent staining has shown an increase of β-catenin presence in the nucleus of YWHAZ-transfectant cells (Fig. 4A). Western blot analysis further showed an increase of β-catenin proteins in both the cytosolic and nuclear fractions of cells that ectopically expressed more with YWHAZ (Fig. 4B). To clarify whether YWHAZ expression modulates nuclear activity of β-catenin in lung cancer cells, CL1-0 cells were cotransfected with the TOPFLASH reporter with or without V5-YWHAZ expression plasmids. Coexpression of CL1-0 cells with V5-YWHAZ promoted a 2.6-fold increase in endogenous β-catenin–dependent transcription (Fig. 4C, left). Moreover, knockdown of YWHAZ expression significantly attenuated TOPFLASH activity in CL1-5 cells (Fig. 4C, right). To further explore whether the metastatic activity of YWHAZ is associated with β-catenin activation, we examined the invasion capability of V5-YWHAZ transfectants with β-catenin silencing by using specific β-catenin siRNAs. For V5-YWHAZ–expressing cells, approximately 50% decrease of invasion ability was found in these cells with low β-catenin expression (Fig. 4D), thus suggesting that YWHAZ enhances cell invasiveness in coordination with β-catenin.

Figure 4.

YWHAZ promotes activation of β-catenin relevant to cell invasiveness. A, the subcellular localization of β-catenin in the V5-tagged YWHAZ-expressing cells, measured by immunofluorescent staining. Green: β-catenin; red: F-actin; blue: nuclear DNA. B, Western blot analysis of β-catenin in the cytosolic and nuclear portions of V5-tagged YWHAZ transfectants. C, YWHAZ expression regulates transcriptional activity of β-catenin/TCF4 in lung cancer cells. CL1-0 (left) or CL1-5 (right) cells in 24-well plates were respectively transfected with 0.5 μg of V5-YWHAZ plasmids or 40 nmol/L YWHAZ-specific siRNAs as indicated. TOPFLASH (TOP) or FOPFLASH (FOP) plasmids (0.3 μg) were used for the reporter assay. pRL-TK was cotransfected as an internal control. The results are presented as the TOP/FOP ratio. *, P < 0.05 compared with control. D, silencing of β-catenin diminishes invasion capability of V5-tagged YWHAZ-transfectants. CL1-0 cells were cotransfected with V5-YWHAZ plasmids and/or β-catenin–specific siRNAs as indicated. Following transfection, cells were subjected to invasion assays at 48 hours (left, n = 5) and then cell lysates were immunoblotted with appropriate antibodies (right).

Figure 4.

YWHAZ promotes activation of β-catenin relevant to cell invasiveness. A, the subcellular localization of β-catenin in the V5-tagged YWHAZ-expressing cells, measured by immunofluorescent staining. Green: β-catenin; red: F-actin; blue: nuclear DNA. B, Western blot analysis of β-catenin in the cytosolic and nuclear portions of V5-tagged YWHAZ transfectants. C, YWHAZ expression regulates transcriptional activity of β-catenin/TCF4 in lung cancer cells. CL1-0 (left) or CL1-5 (right) cells in 24-well plates were respectively transfected with 0.5 μg of V5-YWHAZ plasmids or 40 nmol/L YWHAZ-specific siRNAs as indicated. TOPFLASH (TOP) or FOPFLASH (FOP) plasmids (0.3 μg) were used for the reporter assay. pRL-TK was cotransfected as an internal control. The results are presented as the TOP/FOP ratio. *, P < 0.05 compared with control. D, silencing of β-catenin diminishes invasion capability of V5-tagged YWHAZ-transfectants. CL1-0 cells were cotransfected with V5-YWHAZ plasmids and/or β-catenin–specific siRNAs as indicated. Following transfection, cells were subjected to invasion assays at 48 hours (left, n = 5) and then cell lysates were immunoblotted with appropriate antibodies (right).

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YWHAZ associates with β-catenin to retard degradation of β-catenin via the ubiquitin–proteasome pathway

Because YWHAZ overexpression causes the accumulation of β-catenin in cytosol and nucleus (Fig. 4B), we next ask whether YWHAZ also regulates β-catenin expression. Interestingly, we found that protein levels of β-catenin were elevated in V5-YWHAZ transfectants, but mRNA expression was not proportionally increased in these cells (Fig. 5A), suggesting a potential regulation at the posttranslational level. To explore this possibility, cycloheximide (CHX) was used to characterize the protein turnover in cells. In V5-YWHAZ–transfected cells, the time course turnover of β-catenin levels was much slower than that observed in mock control cells (Supplementary Fig. S3). Next, to investigate whether the accumulation of β-catenin is caused by retardation of 26S proteasome-mediated degradation of ubiquitinated proteins, cells were treated with MG-132. The results showed that MG-132 treatment prevented the degradation of β-catenin in the mock controls to a level almost equal to that of the mixed clone of V5-YWHAZ transfectants (Fig. 5B, left). Immunoprecipitation of β-catenin has shown a reduced level of the MG132-induced ladder forms of β-catenin in cells that were stably expressing V5-YWHAZ (Fig. 5B, right). To address whether the ladder forms of β-catenin came from ubiqutin conjugation, we conducted coimmunoprecipitation followed by Western blot analysis on cell extracts from both mock and V5-YWHAZ transfectants and found a reduction in ubiquitinated β-catenin in presence of V5-YWHAZ–β-catenin interaction in V5-YWHAZ–expressing cells but not in the mock (Fig. 5C). To further confirm that β-catenin degradation is related to ubiquitination, Myc-tagged ubiquitin was transfected into mock and YWHAZ-expressing cells, and immunoprecipitation assays confirmed a marked increase of the ubiquitin conjugates on β-catenin in mock control cells. Constantly, overexpression of YWHAZ attenuated the levels of ubiquitin conjugates on β-catenin (Supplementary Fig. S4). Previous reports (30, 31) and sequences analysis of β-catenin protein (Supplementary Fig. S5A) have indicated an interaction between YWHAZ and β-catenin. However, the function of this interaction has not been addressed. As shown in Fig. 5D and Supplementary Fig. S5B, coimmunoprecipitation and in vitro GST pull-down assays showed a cytosolic association between YWHAZ and β-catenin in lung cancer cells. On the basis of the results, we suggested that the interaction between YWHAZ and β-catenin contributes to a decrease in β-catenin ubiquitination. β-TrCP E3 ligase is responsible for the ubiquitination of β-catenin. Interestingly, the interaction of YWHAZ with β-catenin suppressed β-TrCP binding to β-catenin in V5-YWHAZ–expressing cells, and thus consequently increased β-catenin stability (Fig. 5E). These results showed a competing nature of YWHAZ/β-catenin complex in interfering β-catenin ubiquitination and possibly, the stability of β-catenin in cancer cells.

Figure 5.

YWHAZ binding to β-catenin prevents β-catenin from ubiquitin-mediated degradation. A, an increase of the β-catenin proteins in V5-tagged YWHAZ transfectants. Top, the protein levels of β-catenin and YWHAZ in these transfectants, determined by Western blot analysis. Bottom, the mRNA levels of β-catenin and YWHAZ in the transfectants determined by qRT-PCR. B, YWHAZ overexpression induces the inhibition of β-catenin degradation. Left, the protein level of β-catenin after treatment with 10 μmol/L of proteasome inhibitor (MG-132). Right, the transfectants were treated with 10 μmol/L of MG-132 for 3 hours, followed by immunoprecipitation and immunoblotting. C, the effect of V5-tagged YWHAZ on β-catenin ubiquitination, determined by immunoprecipitation and immunoblotting conducted using the indicated antibodies. D, the cytosolic interaction between YWHAZ and β-catenin in invasive CL1-5 cells. Cytoplasmic lysates were immunoprecipitated using appropriate antibodies and then Western blot analysis was conducted. E, reduced binding activity of β-catenin with β-TrCP in presence of V5-tagged YWHAZ. The immunoprecipitated β-catenin proteins from CL1-0, mock, or V5-YWHAZ–expressing cells were immunoblotted with appropriate antibodies. Fifteen micrograms of cell lysates were used as the input controls.

Figure 5.

YWHAZ binding to β-catenin prevents β-catenin from ubiquitin-mediated degradation. A, an increase of the β-catenin proteins in V5-tagged YWHAZ transfectants. Top, the protein levels of β-catenin and YWHAZ in these transfectants, determined by Western blot analysis. Bottom, the mRNA levels of β-catenin and YWHAZ in the transfectants determined by qRT-PCR. B, YWHAZ overexpression induces the inhibition of β-catenin degradation. Left, the protein level of β-catenin after treatment with 10 μmol/L of proteasome inhibitor (MG-132). Right, the transfectants were treated with 10 μmol/L of MG-132 for 3 hours, followed by immunoprecipitation and immunoblotting. C, the effect of V5-tagged YWHAZ on β-catenin ubiquitination, determined by immunoprecipitation and immunoblotting conducted using the indicated antibodies. D, the cytosolic interaction between YWHAZ and β-catenin in invasive CL1-5 cells. Cytoplasmic lysates were immunoprecipitated using appropriate antibodies and then Western blot analysis was conducted. E, reduced binding activity of β-catenin with β-TrCP in presence of V5-tagged YWHAZ. The immunoprecipitated β-catenin proteins from CL1-0, mock, or V5-YWHAZ–expressing cells were immunoblotted with appropriate antibodies. Fifteen micrograms of cell lysates were used as the input controls.

Close modal

Interaction of YWHAZ and β-catenin enhances cancer invasion

To characterize the interaction and the importance of the YWHAZ/β-catenin complex in cancer cells, especially in the cell invasiveness, V5-tagged wild-type and mutants of β-catenin expression constructs were generated and the mutation changes at serine552 site, such as S552A and S552D, were selected according to the information obtained from the ScanSite software (http://www.motifscan.mit.edu; Supplementary Fig. S5A) and a previous report (32). These constructs were transfected on Myc-tagged YWHAZ-transfectant cells. Immunoprecipitation with anti-Myc antibody on cell extracts of these transfected cells showed a diminished interaction between the S552A mutant of β-catenin and Myc-tagged YWHAZ (Fig. 6A). Reciprocal immunoprecipitation assays with anti-V5 antibody also showed that Myc-tagged YWHAZ binding to mutant protein of V5-tagged S552A β-catenin was decreased by 56.3% ± 8.8% as compared with its binding to wild-type protein of V5-tagged β-catenin (Fig. 6B). Therein, we found that the ubiquitination level on mutant protein of V5-tagged S552A β-catenin was higher than that of wild-type V5–β-catenin and the S552D mutant in these transfected Myc-tagged YWHAZ-expressing transfectants. We also analyzed whether an increase in endogenous S552 phosphorylation of β-catenin promotes its interaction with YWHAZ. Immunoprecipitation of endogenous YWHAZ showed a significant interaction between phospho-β-catenin (Ser552) and YWHAZ in lung cancer cells grown in the medium supplemented with 10% serum. On the contrary, serum starvation downregulated the phosphorylation levels of β-catenin at Ser552 and thereby reduced the binding of phospho-β-catenin (Ser552) with YWHAZ (Supplementary Fig. S6). On the basis of the above observations, we evaluated whether there could be a relationship between S552 phosphorylation levels of β-catenin and β-TrCP binding activity. The results showed that S552 phosphorylation was enhanced by the expression of YWHAZ accompanied with a reduced interaction of β-TrCP with β-catenin (Fig. 6C). Because an increased interaction between YWHAZ and β-catenin was found to reduce β-catenin ubiquitination in these cells, we further examined if this change also affects cell invasiveness. For highly invasive CL1-5 cells, the invasiveness was significantly impaired in S552A β-catenin mutant–transfected cells, as compared with those cells transfected with wild-type β-catenin (Fig. 6D, Top). In Myc-tagged YWHAZ-expressing CL1-0 cells, we found that the invasiveness of the S552A β-catenin mutant–transfected cells was significantly decreased by 48% ± 12% as compared with wild-type β-catenin transfected cells (Fig. 6D, Bottom), suggesting that expression of wild-type or S552D β-catenin promotes cell invasiveness together with YWHAZ. Taken together, these results indicate that the formation of the YWHAZ/β-catenin complex promotes invasion of lung cancer cells.

Figure 6.

Disruption of the interaction between YWHAZ and β-catenin reduces cancer invasion. A, confirmation of the YWHAZ binding site on β-catenin protein. Each pcDNA3.1-CTNNB1 (V5-tagged β-catenin) construct or pcDNA3.1 (mock) was cotransfected with pCMV-Tag3B-YWHAZ (Myc-tagged YWHAZ) construct into HEK293 cells followed by immunoprecipitation and immunoblotting using the indicated antibodies. B, the level of V5-β-catenin ubiquitination in various transfected cells. Wild-type, S552A or S552D β-catenin was coexpressed with Myc-tagged YWHAZ in HEK293 cells. After coexpression of pCMV-Tag3B-YWHAZ and pcDNA3.1-CTNNB1 constructs, the level of ubiquitin binding to V5-tagged β-catenin was examined by immunoprecipitation using an anti-V5 antibody. Twenty micrograms of cell lysates served as the input controls. C, Coimmunoprecipitation analysis of the association between phospho–β-catenin (Ser552) and β-TrCP in V5-YWHAZ–overexpressing cell lines. D, the invasion ability of V5-β-catenin transfectants in lung cancer cells. Empty vector, wild-type, S552A, or S552D β-catenin was expressed in CL1-5 cells (top) or coexpressed with Myc-tagged YWHAZ in CL1-0 cells (bottom). After 24 hours of transfection, the invasion capacity of the transfectants was evaluated with Matrigel invasion assays. E, a hypothetical model for YWHAZ-mediated lung cancer metastasis. YWHAZ associates with the soluble form of β-catenin to protect β-catenin from degradation and subsequently results in an increase in the nuclear accumulation of β-catenin leading to the activation of EMT markers and cancer malignancy.

Figure 6.

Disruption of the interaction between YWHAZ and β-catenin reduces cancer invasion. A, confirmation of the YWHAZ binding site on β-catenin protein. Each pcDNA3.1-CTNNB1 (V5-tagged β-catenin) construct or pcDNA3.1 (mock) was cotransfected with pCMV-Tag3B-YWHAZ (Myc-tagged YWHAZ) construct into HEK293 cells followed by immunoprecipitation and immunoblotting using the indicated antibodies. B, the level of V5-β-catenin ubiquitination in various transfected cells. Wild-type, S552A or S552D β-catenin was coexpressed with Myc-tagged YWHAZ in HEK293 cells. After coexpression of pCMV-Tag3B-YWHAZ and pcDNA3.1-CTNNB1 constructs, the level of ubiquitin binding to V5-tagged β-catenin was examined by immunoprecipitation using an anti-V5 antibody. Twenty micrograms of cell lysates served as the input controls. C, Coimmunoprecipitation analysis of the association between phospho–β-catenin (Ser552) and β-TrCP in V5-YWHAZ–overexpressing cell lines. D, the invasion ability of V5-β-catenin transfectants in lung cancer cells. Empty vector, wild-type, S552A, or S552D β-catenin was expressed in CL1-5 cells (top) or coexpressed with Myc-tagged YWHAZ in CL1-0 cells (bottom). After 24 hours of transfection, the invasion capacity of the transfectants was evaluated with Matrigel invasion assays. E, a hypothetical model for YWHAZ-mediated lung cancer metastasis. YWHAZ associates with the soluble form of β-catenin to protect β-catenin from degradation and subsequently results in an increase in the nuclear accumulation of β-catenin leading to the activation of EMT markers and cancer malignancy.

Close modal

Our results identified a novel pathway of YWHAZ–β-catenin axis in NSCLC cell malignancy. We showed that the metastatic activity of YWHAZ is mediated through the prevention of β-catenin ubiquitination. We showed that YWHAZ associates with phospho-β-catenin at Ser552 to retard β-catenin degradation. Subsequently, β-catenin accumulation in cancer cells contributes to its nuclear translocation and the activation of EMT-related genes (Fig. 6E). Conversely, disruption of the YWHAZ–β-catenin interaction enhances β-catenin ubiquitination and reduces cell invasiveness.

YWHAZ protein expression is well known to be associated with advanced disease grade and poor clinical outcome in Western NSCLC patients (18), but the molecular contribution of YWHAZ in lung cancer metastasis is still unknown. Particularly, it is unclear whether YWHAZ gene amplification is relevant to lung cancer progression. Here, we conducted array CGH and microarray to identify YWHAZ as an invasion-enhancer candidate. Actually, YWHAZ gene amplification was found in highly invasive cell line, CL1-5. We first verified that the amplified DNA copy number of YWHAZ correlates with lung cancer invasiveness. Both in vitro and in vivo studies revealed that YWHAZ promotes cancer invasion/metastasis through stabilizing β-catenin, leading to the induction of EMT (Figs. 1–4). Specifically, our study presents the molecular mechanism by which YWHAZ regulates lung cancer progression. First, YWHAZ associates with the soluble form of β-catenin to protect β-catenin from ubiquitin-mediated degradation (Fig. 5), which suggests a novel mechanism by which β-catenin accumulates in the cytoplasm and is protected from degradation in cancer cells. Second, we present new evidence of β-catenin accumulation via YWHAZ binding to phospho-β-catenin at Ser552 (Fig. 6). Taken together, our results show the importance of YWHAZ/β-catenin axis in the development of NSCLC metastatic potential and EMT phenotype.

During metastasis, cancer cells acquire the ability to become motile and invade adjacent tissue by inducing EMT (33). YWHAZ has been reported to be one of the TGF-β–induced proteins involved in cancer cell transformation (34). So far, the action of YWHAZ on inducing EMT in lung cancer cells is still unclear. Recently, the relationship between YWHAZ and EMT has been verified in breast cancer and head/neck squamous cell carcinoma (20, 23). In breast cancer, it has been verified that YWHAZ cooperates with ErbB2 to induce EMT, but not cell migration, through the TGF-β signaling pathway. In lung cancer, we found that YWHAZ promotes EMT, cell invasion, and migration by activating the YWHAZ/β-catenin axis. Our observation in lung cancer is different from those in breast cancer, which reveals a lung cancer–specific pathway mediated by YWHAZ. To our knowledge, our study is the first to show the role of YWHAZ in inducing EMT to elucidate the metastatic characteristics of YWHAZ in lung cancer.

YWHAZ, a member of the 14-3-3zeta protein family, modulates cellular events by interacting with phosphoserine proteins, including β-catenin (30). Although β-catenin is verified to be a binding partner of YWHAZ, we were surprised to discover that YWHAZ exerts its oncogenic function through its binding with β-catenin in lung cancer cells. β-Catenin is a central effector of Wnt signaling in tumorigenesis and metastasis (35). Indeed, our microarray analysis showed that Wnt signaling is the most likely pathway to be affected by YWHAZ and to cause EMT. A recent article has shown that abnormal β-catenin expression was associated with positive YWHAZ expression in stage-I NSCLC (36). In fact, we confirmed these observations and further elucidated a mechanistic role of YWHAZ in the accumulation of β-catenin. Our coimmunoprecipitation assays revealed that YWHAZ is associated with phospho–β-catenin at S552, consistent with the finding by Fang and colleagues (32). Next, we found that this interaction reduces the binding ability of β-TrCP to β-catenin in cells with enforced expression of YWHAZ. It is reasonable to postulate that higher expression of YWHAZ would compete with β-TrCP in the interaction of β-catenin. Through YWHAZ–β-catenin interaction, we speculated that the binding site of β-TrCP in β-catenin may be masked by YWHAZ, leading to decrease interaction of β-TrCP with β-catenin and increase β-catenin stability. On the other hand, it is possible that YWHAZ binding to β-catenin causes a conformational change of β-catenin and contributes to increased disassociation of β-TrCP and β-catenin. Accordingly, we concluded that YWHAZ may protect the serine-phosphorylated β-catenin from ubiquitination by forming a complex with β-catenin, which may explain why YWHAZ expression promotes the accumulation of β-catenin and activates β-catenin–dependent transcription.

β-Catenin signaling has emerged as a critical pathway in human lung carcinogenesis (37) and malignancies (38–40). In this work, we have shown that YWHAZ promotes activation of β-catenin that is relevant to cell invasiveness in lung cancer. It is consistent with these previous results (31, 32) that YWHAZ directly binds to β-catenin and promotes transcriptional activity of β-catenin. The activation of target genes by β-catenin–TCF/LEF complexes, including cyclin D1 (14), c-Myc (15), and Slug (12, 41), is believed to provide a growth or migration advantage to cells. The results of microarray and real-time RT-PCR showed an increase in cyclin D1 expression, which may explain how YWHAZ promotes cell proliferation and tumorigenesis. Another target gene of β-catenin, Slug, is an important transcription factor that promotes tumor growth, invasion, and drug resistance (8, 42, 43). On the basis of our current data, we also found enhanced expression of Slug in presence of YWHAZ expression. Unlike the findings in breast cancer (23), Slug is an effector in YWHAZ-mediated EMT pathway in lung cancer through activation of β-catenin. Activation of β-catenin is reported to be related to β-catenin phosphorylation. It is well documented that AKT-mediated phosphorylation of β-catenin (Ser552) by EGFR signaling causes β-catenin disengaging from cell contacts and activates transcriptional activity of β-catenin (32). Our studies extended the above observations of β-catenin phosphorylation (Ser552) in lung cancer. Furthermore, we showed a decrease of ubiquitination in phosphorylated β-catenin at Ser552, such as WT or S552D β-catenin upon enforced expression of YWHAZ, implying the importance of YWHAZ expression in enhancing transcriptional activity of phosphorylated β-catenin at Ser552. Fang and colleagues indicated that AKT-mediated phosphorylation of β-catenin at S552 does not alter β-catenin protein stability in 293T cells with low YWHAZ expression. We believe that the conclusion might be from the low expression of YWHAZ in 293T and normal cells.

In summary, we not only provide evidence that YWHAZ participates in lung cancer progression but also illustrate the critical role of the YWHAZ/β-catenin complex in cancer metastasis. Collectively, we show the importance of YWHAZ in lung cancer malignancy and propose that the YWHAZ–β-catenin axis may serve as potential anticancer target.

No potential conflicts of interest were disclosed.

Conception and design: C.-H. Chen, S.-L. Yu, J.J.W. Chen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.-H. Chen, M.-F. Yang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.-H. Chen, M.-F. Yang

Writing, review, and/or revision of the manuscript: C.-H. Chen, S.-M. Chuang, S.-L. Yu, J.J.W. Chen

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.-H. Chen, S.-M. Chuang, M.-F. Yang, J.-W. Liao, J.J.W. Chen

Study supervision: S.-M. Chuang, S.-L. Yu, J.J.W. Chen

The authors thank Drs. G.-C. Tseng (Department of Pathology, China Medical University Hospital, Taiwan) and R. Wu (School of Veterinary Medicine, University of California, Davis, CA) for useful advice and discussion. The shRNA constructs were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research, Academia Sinica (Taipei, Taiwan).

This work was supported by grants from the National Science Council, Taiwan, ROC (NSC 97-2314-B-005-002-MY3), as well as in part by the Ministry of Education, Taiwan, ROC under the ATU plan.

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.

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