Abstract P5-04-02: DYRK2 regulates breast cancer invasion via Snail/E-cadherin pathway

Abstract

Abstract Introduction: Epithelial-Mesenchymal Transition (EMT) is characterized by the loss of cellular adhesion molecule E-cadherin, and plays a fundamental role during early steps of breast cancer invasion. An important regulator of E-cadherin expression is Snail, a zinc finger transcriptional repressor. Snail is posphorylated by GSK3β and then degraded by βTrCP mediated ubiquitination. Here we found another kinase, DYRK2, regulates the stability of Snail. Knockdown of DYRK2 promoted the EMT and cancer invasion both in vitro and in vivo experiments. Methods: Cell lines: MCF-7 cells were transfected with pSuper vector (pSuper control) and pSuper shRNA DYRK2 (shRNA-DYRK2) with G418 to isolate stable cell lines. Immunoblotting: Expression of pertinent molecular markers was determined by western blotting. RT-PCR: RNA was extracted using TRIsure and 500 ng of total RNA was amplified using Primer Script One Step RT-PCR Kit Ver.2. Invasion assay: Various cancer cell lines were seeded on the top of the upper chamber with serum free medium while the bottom chambers were filled with medium containing 10% FBS. In vivo metastasis assay: Cells in PBS were injected into the left ventricle of 7-week-old female nude mice. Metastases to distant organs were confirmed by IVIS2000. Immunohistochemistry: We acquired paraffin-embedded tissue sections from primary breast tumor cores and from Jikei University of Medicine. Immunohistochemistry for DYRK2 was performed. Results: In MCF-7 cells, knockdown of DYRK2 increased Snail in the protein level but not in mRNA level. Upon DYRK2 knockdown, MG132 treatment had no effect on additional increase in Snail. DYRK2 knockdown decreased Snail ubiquitination. Stable DYRK2 depletion led to Snail accumulation and E-cadherin abrogation. Fibroblast marker, Vimentin, emerged in DYRK2 depleted cells. For further analysis, we carried out invasion assays. The invasion potential in DYRK2-depleted cells was substantially higher than that in control cells. In xenograft model, we used nude mice received intracardial injections of pSuper control or shRNA-DYRK2 cells. 6 weeks after injections, a significant increase in bone and lung metastasis was observed in shRNA-DYRK2 group. The patients with tumors expressed low DYRK2 showed significantly poor prognosis. Conclusion: In breast cancer cells, Snail is phosphorylated by DYRK2 and then acquires the ability to be ubiquitinated. In the absence of DYRK2, Snail is unable to be degraded by ubiquitin-proteasome machinery. Accumulation of Snail promotes EMT and cancer invasion, so low expression of DYRK2 leads to poor prognosis in breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-02.