| Summary: | 碩士 === 國立成功大學 === 分子醫學研究所 === 106 === Bubbling cell death (BCD) is defined as formation of a bubble from the nucleus per cell and release of this swelling bubble from the cell surface to extracellular space that causes cell death. WW domain-containing oxidoreductase, known as WWOX, is essential for UV/cold shock-induced nuclear damage and bubbling death. Tumor suppressor WWOX with Ser14 phosphorylation is shown to accumulate in the tumor lesions, but functional significance of this regard is largely unknown. We have previously demonstrated that breast cancer 4T1 stem cells undergo shrinkage and then explosion with dead cells in response to UV/cold shock at room temperature, as determined by time-lapse microscopy. Here, we investigated how breast cancer stem cell sphere explosion occurs via the mechanisms of BCD. Ceritinib, an anaplastic lymphoma kinase (ALK) inhibitor for lung cancer, caused toxicity in breast cancer 4T1 cells. When breast cancer 4T1 stem cell spheres were treated with ceritinib, the spheres fully exploded with dead cells via BCD. Ceritinib downregulated the expression of phosphorylated ERK, IκBα and pS14-WWOX, and upregulated pY33-WWOX. UV/cold shock treatment also showed the same result. Treatment of the WWOX7-21 antibody resulted in significantly inhibition of ceritinib-mediated 4T1 sphere explosion. In contrast, synthetic WWOX7-21 peptides enhanced 4T1 sphere death by ceritinib. Notably, when Ser14-phosphorylated WWOX peptide was used to treat the 4T1 spheres, cells significantly acquired resistance to ceritinib. Ceritinib induced ERK and WWOX protein translocation into nucleus. Immunofluorescent microscopy revealed that pS14-WWOX antibody pretreatment enhanced ceritinib-mediated nuclear translocation of endogenous ERK. By Förster resonance energy transfer (FRET) microscopy, we observed that ceritinib reduced the WWOX/ERK/IκBα complex formation. In summary, phosphorylation at Ser14 turns WWOX from tumor suppressor into tumor promoter and regulates the cancer stem cell sphere explosion. WWOX/ERK/IκBα complex also plays a role in ceritinib-dependent cancer stem cell explosion.
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