細胞冒泡死亡(BCD)被定義為“細胞從細胞核位置形成一顆泡泡，並且泡泡會膨脹到細胞外，進而導致細胞死亡”。含雙色氨酸功能區氧化還原酶(WWOX)在紫外光/冷衝擊造成的核損傷以及冒泡死亡之中是相當重要的。Ser14磷酸化的腫瘤抑制子WWOX被發現會累積在腫瘤組織中，但其功能仍有許多未知性。我們曾經利用室溫的顯微鏡拍攝觀察，發現乳癌幹細胞球體受到紫外光/冷衝擊刺激後會先皺縮再接著爆炸。本篇研究中，我們發現肺癌化療藥物Ceritinib對乳癌細胞具有細胞毒性。進一步使用肺癌化療藥物Ceritinib處理乳癌幹細胞球體，發現其藥物也能夠引發球體爆炸並且細胞行冒泡死亡。我們發現Ceritinib會影響細胞中pERK、IκBα、pS14-WWOX以及pY33-WWOX的蛋白表現量，pERK、IκBα和pS14-WWOX表現下調而pY33-WWOX的表現上升。並且，紫外光/冷衝擊刺激和ceritinib對細胞蛋白表現量的影響有相同的趨勢。從處理WWOX7-21抗體的實驗中，我們發現WWOX7-21抗體會顯著抑制ceritinib介導的球體爆炸，相反地，WWOX7-21胜肽則能夠增強球體爆炸。值得注意的是，pS14-WWOX胜肽處理後，細胞能夠獲得對ceritinib的抗性。免疫螢光顯微鏡揭露了Ceritinib 會誘導ERK和WWOX蛋白轉移進細胞核的表現，並且pS14-WWOX前處理能增強ceritinib介導的內源性ERK蛋白核轉移。我們透過螢光共振能量轉移(FRET)顯微鏡觀察到ceritinib減低WWOX/ERK/IκBα複合體的形成。總而言之，這些結果顯示WWOX參與在癌症幹細胞爆炸之中，而Ser14磷酸化會使腫瘤抑制子WWOX轉換去促進癌症進程。WWOX/ERK/IκBα複合體也在ceritinib介導的癌症幹細胞球體爆炸中扮演重要的角色。

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.

中文摘要 I
Abstract II
致謝 III
Contents IV
Abbreviation VIII
Introduction 1
The goal of this study 1
Cancer stem cells (CSCs) 1
Programmed cell death 2
Bubbling cell death 4
WW domain-containing oxidoreductase (WWOX) 5
Role of WWOX in cancer 7
WWOX in breast cancer 8
HYAL2-WWOX-SMAD4 signaling in cell death 9
WWOX in neurodegeneration 10
Materials and Methods 12
Cell lines and cDNA expression constructs 12
UV irradiation and time-lapse microscopy 12
Preparation of WWOX peptides and antibodies 12
Chemicals and Antibodies 13
MTT assay 13
Cell cycle analysis 13
DNA fragmentation 14
Western Blot and co-immunoprecipitation 14
Nuclear extraction 15
Immunofluorescence staining 15
Time-lapse tri-molecular FRET microscopy 16
Statistical analysis 16
Results 17
ALK inhibitor Ceritinib induced cell death in breast cancer 4T1 cells. 17
Breast cancer stem cell sphere explosion is determined by time-lapse microscopy at room temperature. 17
p53 regulated ceritinib-mediated sphere explosion. Inhibition of phosphatases led to enhanced explosion by ceritinib. 18
Ceritinib affects IκBα/WWOX/ERK expression in 4T1-luc cells. 18
WWOX 7-21 antibody inhibited ceritinib-mediated 4T1 sphere explosion and death. 19
pS14-WWOX7-21 peptide strongly protected breast 4T1 stem cell spheres from explosion and death caused by ceritinib. 20
Suppression of pS14-WWOX on the surface of 4T1 spheres by antibodies increased their susceptibility to attack by activated Z cells. 20
pS14-WWOX antibody pretreatment enhance ceritinib-mediated nuclear translocation of endogenous ERK. 21
Ceritinib-mediated WWOX/ERK/IκBα signal pathway 22
Conclusion 23
Discussion 24
Reference 27
Figures 36
Figure 1. A schematic diagram of WWOX structure. 36
Figure 2. Signaling pathway network of WWOX. 37
Figure 3. The effect of ceritinib on cell death. 38
Figure 4. Breast cancer stem cell sphere explosion is determined by time-lapse microscopy at room temperature. 40
Figure 5. Ceritinib regulates the expression of pro-survival protein and WWOX. 43
Figure 6. The effect of ceritinib is compared with UV/cold-shock treatment in 4T1-luc cells at different temperature. 45
Figure 7. WWOX antibody regulates ceritinib-caused cell death. 47
Figure 8. WWOX 7-21 antibody inhibits ceritinib-mediated 4T1 sphere explosion and death. 48
Figure 9. pS14-WWOX7-21 peptide strongly protects breast 4T1 stem cell spheres from explosion and death caused by ceritinib. 50
Figure 10. Suppression of pS14 -WWOX on the surface of 4T1 spheres by antibodies increases their susceptibility to attack by activated Z cells. 51
Figure 11. UV/cold shock and ceritinib both enhance nuclear translocation of endogenous WWOX. 53
Figure 12. Ceritinib-mediated nuclear translocation of endogenous ERK/WWOX. 55
Figure 13. pS14-WWOX Ab pretreatment enhances ceritinib-mediated nuclear translocation of endogenous ERK. 58
Figure 14. WWOX peptide pretreatment regulates ceritinib-mediated signal pathway WWOX/ERK/IκBα. 60
Figure 15. Ceritinib affects the binding of WWOX with MEK. 61
Figure 16. Time-lapse FRET microscopy for ceritinib-mediated signal pathway WWOX/ERK/IκBα. 62
Figure 17. The potential molecular action of ceritinib-mediated cancer stem cell sphere explosion. 64

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