PDF file - 354K, Supplementary data, supplementary methods and supplementary references S1. YWHAZ expression promotes tumor growth. S2. YWHAZ induces cell protrusion and branching formation. S3. Analysis of beta-catenin stability in the mock and YWHAZ transfectants of CL1-0 cells. S4. YWHAZ overexpression reduces the interaction of beta-catenin with Myc-tagged ubiquitin. S5. Confirmation of the cytosolic interaction between YWHAZ and beta-catenin. S6. Effects of endogenous beta-catenin Ser552 phosphorylation on its interaction with endogenous YWHAZ. Table S1. Summary of pathological incidence of lungs in mice by intravenous injection with YWHAZ-expressing cells or mock control. Table S2. Comparison of the incidence of tumorigenesis or metastasis between mock and YWHAZ transfectant in SCID mice by orthotopic lung implantation model.
ARTICLE 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 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.
