Published OnlineFirst August 21, 2012; DOI: 10.1158/1541-7786.MCR-12-0189 Molecular Cancer Angiogenesis, Metastasis, and the Cellular Microenvironment Research A Novel Function of YWHAZ/b-Catenin Axis in Promoting Epithelial–Mesenchymal Transition and Lung Cancer Metastasis Ching-Hsien Chen1,4, Show-Mei Chuang1, Meng-Fang Yang1, Jiunn-Wang Liao2, Sung-Liang Yu4, and Jeremy J.W. Chen1,3 Abstract 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 b-catenin–mediated transcription by facilitating the accumulation of b-catenin in cytosol and nucleus. Coimmunoprecipitation assays showed a decrease of ubiquitinated b-catenin in presence of the interaction between YWHAZ and b-catenin. This interaction resulted in disassociating b-catenin from the binding of b-TrCP leading to increase b-catenin stability. Using enforced expression of dominant-negative and -positive b-catenin mutants, we confirmed that S552 phosphorylation of b-catenin increases the b-catenin/YWHAZ complex formation, which is important in pro- moting cell invasiveness and the suppression of ubiquitnated b-catenin. This is the first demonstration showing YWHAZ through its complex with b-catenin in mediating lung cancer malignancy and b-catenin protein stabi- lity. Mol Cancer Res; 10(10); 1319–31. Ó2012 AACR. Introduction by undergoing phenotypic conversion referred to as epithe- – Lung cancer is a malignant tumor with a high incidence lial mesenchymal transition (EMT), which enables them and mortality rate (1). Non–small cell lung cancer (NSCLC) to become motile and to invade adjacent tissues. This process represents 80% of all lung cancers and has an overall 5-year is triggered by various signaling pathways and controlled by a group of transcriptional factors, such as zinc finger survival rate of 10% to 15% (2). Metastasis is the main cause – – of treatment failure and cancer-related deaths. In carcino- proteins and basic helix loop helix factors (4, 5). In lung mas, metastasis is a multiple step process, the first of which cancer, Slug is known to be an important EMT inducer and is invasion (3). Cancer cells acquire their invasive capacity is able to suppress the expression levels of cell adhesion molecules (6–8). b-Catenin binds to the cytoplasmic domain of E-cadherin Authors' Affiliations: Institutes of 1Biomedical Sciences, and 2Veterinary and is essential for the structural organization and function of Pathology and 3Agricultural Biotechnology Center, National Chung Hsing cadherins. b-Catenin also plays a central role as a cotran- University, Taichung; and 4Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, scription factor in both canonical and noncanonical Wnt Taipei, Taiwan, Republic of China signaling (9). In the absence of Wnt signaling, the soluble b Note: Supplementary data for this article are available at Molecular Cancer form of -catenin, which is not associated with cadherins, is Research Online (http://mcr.aacrjournals.org/). phosphorylated by glycogen synthase kinase-3b (10) and is subsequently recognized by the b-transducin repeat-con- S.-L. Yu and J.J.W. Chen codirected the project and contributed equally to b this work. taining protein ( -TrCP), leading to the ubiquitination and degradation of b-catenin by proteasomes (11). Activation of Corresponding Author: Jeremy J.W. Chen, Institute of Biomedical b Sciences, National Chung Hsing University, Taichung 40227, Taiwan, the Wnt pathway inhibits the degradation of -catenin and ROC. Phone: 886-4-22840896, ext. 125; Fax: 886-4-22853469; E-mail: results in its accumulation in the cytosol and subsequent [email protected] nuclear translocation (12). Following nuclear translocation, doi: 10.1158/1541-7786.MCR-12-0189 b-catenin interacts with the transcription factor T-cell fac- Ó2012 American Association for Cancer Research. tor/lymphoid enhancer factor (TCF/LEF) to induce the www.aacrjournals.org 1319 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst August 21, 2012; DOI: 10.1158/1541-7786.MCR-12-0189 Chen et al. transactivation of certain vital genes, including cyclin D1, shYWHAZ-1 and pLKO.1-shYWHAZ-2; TRCN- c-Myc, and Slug (13–15). Although excess b-catenin pro- 0000029404 and TRCN0000029405) were obtained from teins are observed in certain cancers and associated with the National RNAi Core Facility (Academia Sinica, Taipei, dysregulation of ubiquitination, the molecules involved in Taiwan) and prepared in accordance with the standard b-catenin accumulation are largely unknown. protocols. Cells were infected with lentivirus (multiplicity In this study, we identify YWHAZ, also known as 14-3- of infection 5 or 10) in medium containing polybrene 3zeta, as a potential regulator for the function of b-catenin (8 mg/mL). At 24 hours after infection, cells were treated and its turnover. YWHAZ was initially identified as one with 0.75 mg/mL puromycin to select for puromycin-resis- of the metastasis enhancer genes through an integrated tant pooled clones. approach that combined comparative genome hybridization (CGH) with an expression microarray on lung cancer cell Microarray analysis lines with different metastatic potentials (16). YWHAZ Human genomic DNAs from CL1-5 and CL1-0 cells has attracted interest because of its elevated expression asso- were analyzed using array CGH containing 385,000 probes ciated with a variety of cancers (17–24). Recently, it is with a median distance of approximately 6,000 bp (Nim- understood that YWHAZ has critical antiapoptotic func- bleGen) to determine copy number variations in dye-swap tions and is able to suppress anoikis in lung cancer replicate experiments. For expression analysis, cDNA prep- (17, 18, 25). In this communication, we report for the first aration and array hybridization were conducted according to time that YWHAZ amplification is associated with lung the Affymetrix GeneChip expression analysis technical man- cancer malignancy and the importance of YWHAZ/b-cate- ual (see Supplementary Methods). nin axis in preventing b-catenin ubiquitination and degra- dation and subsequently in promoting EMT phenotype Quantitative real-time PCR and invasiveness of cancer cells. 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 in- Materials and Methods structions. The mRNA expression level of YWHAZ and Materials and plasmid constructs CTNNB1 (b-catenin) was also detected by real-time reverse All reagents, antibodies, and plasmid constructs used in transcription polymerase chain reaction (RT-PCR) on this study are described in the Supplementary Information the same system. The primers and probes of YWHAZ in Supplementary Methods. (Hs00237047_m1) and TATA-box binding protein (TBP, Hs00427620_m1) were purchased from Applied Biosystems. Cell culture and transfection The CTNNB1 primers were as follows: forward primer 50-GGCTACTGTTGGATTGATTCGAA-30 and reverse The low invasive and highly invasive human lung adeno- 0 0 carcinoma cell lines, CL1-0 and CL1-5, were established and primer 5 -GCTGGGTATCCTGATGTGCAC-3 .Weused characterized as previously described (16). We tested their the housekeeping gene ACTB as the reference gene in genomic invasiveness for authentication by Matrigel invasion assays real-time PCR assay. The DNA copy number of YWHAZ in our laboratory every month. The cell lines, HEK293, for tumor or normal tissue was presented as relative level to A549, H1299, MCF-7, HeLa, and BEAS2B cells were reference gene. For mRNA analysis, we used TBP as the purchased from the American Type Culture Collection internal control. The relative expression level of YWHAZ (ATCC) that conducts cell line characterizations and pas- and CTNNB1 compared with that of TBP was defined as ÀD ¼½ À saged in our laboratory for fewer than 6 months after CT CTtarget CTTBP . The target/TBP mRNA ratio ÀD receipt. The method of characterization used by ATCC can was calculated as 2 CT Â K,inwhichK is a constant. be found in its website. Cells were cultured in Dulbecco's modified Eagle's medium with 10% FBS and 1% penicillin– Migration and invasion assays streptomycin at 37 C in a humidified atmosphere of 5% In vitro cell migration and invasion assays were conducted CO2. For enforced expression of V5-tagged YWHAZ in the as previously described (26) using transwell chambers (8 mm minimally invasive lung cancer cell line, CL1-0 cells were pore size; Costar). In migration assays, 5 Â 103 cells were transfected with pEF6/V5-HisTOPO-YWHAZ (pEF6/V5- seeded on top of the polycarbonate