臺灣博碩士論文加值系統

一般而言，睪丸生殖細胞腫瘤對化學治療的反應良好，但若轉錄因子OCT4表現低下則會造成化療抗性與較差的預後。已知低氧會誘發睪丸生殖細胞腫瘤產生藥物抗性，然而低氧是否造成OCT4的減少與藥物抗性的機轉則尚未清楚。我們於本研究中指出，低氧會降低胚胎癌細胞中OCT4的表現程度與增加對順鉑與博來霉素的藥物抗性。此外，我們驗證了低氧透過SUMO1與SUMO1的胜肽酶SENP1對OCT4進行小泛素化修飾而促使OCT4減少。於低氧條件下，大量表現SUMO1gg（活化態之SUMO1）不僅可以增加小泛素化的OCT4，同時也降低了OCT4蛋白質的穩定性。更進一步，大量表現SENP1亦可降低上述之小泛素化的OCT4表現，進而維持OCT4表現的程度與增加對化學治療的感受性。於低氧條件下，我們發現大量表現的OCT4-K123R點突變蛋白質可以有效的抑制小泛素化的OCT4生成，證實小泛素化修飾發生於OCT4蛋白質上123位置的離胺酸。這些實驗數據顯示，低氧會降低胚胎癌細胞中OCT4的表現，並增加藥物抗性。研究揭示由低氧所降低的藥物感受性是可以被消彌的。我們同時也發現在治療具藥物抗性的睪丸生殖細胞腫瘤上，SENP1是一個具有潛力的治療標的。

Testicular germ cell tumors (TGCTs) generally respond well to chemotherapy, but tumors that express low levels of the transcription factor OCT4 are associated with chemoresistance and poor prognosis. Hypoxia is known to induce drug resistance in TGCTs, however, the mechanistic basis for reduced expression of OCT4 and drug resistance is unclear. Here we demonstrate that hypoxia reduces OCT4 levels and increases the resistance of embryonal carcinoma (EC) cells to cisplatin and bleomycin. Furthermore, we show that the loss of OCT4 expression under hypoxia can be triggered by sumoylation, which was regulated by SUMO1 and the SUMO1 peptidase SENP1. Under hypoxic conditions, overexpression of SUMO1gg (the active form of SUMO1) not only increased the level of sumoylated OCT4 (Su-OCT4), but also decreased the stability of OCT4 protein. Additionally, overexpression of SENP1 reduced the Su-OCT4 level induced by SUMO1gg overexpression, thereby maintaining OCT4 levels and enhancing chemosensitivity. Mechanistic investigations revealed that OCT4 sumoylation occurred at K123, as overexpression of an OCT4-K123R mutant effectively reduced the level of Su-OCT4 under hypoxic conditions. Taken together, our results demonstrated that hypoxia reduces OCT4 expression levels in ECs to increase drug resistance, and that these effects could be countered to ablate the suppressive effects of hypoxia on chemosensitivity. Our findings also highlight SENP1 as a potential therapeutic target for drug-resistant TGCTs.

LIST OF TABLES III
LIST OF FIGURES IV
ABBREVIATION VI
摘要 VII
Abstract VIII
Chapter 1 Introduction 1
1.1 Testicular germ cell tumor 2
1.2 Hypoxia stress and pluripotent cells 3
1.3 Sumoylation 3
1.4 Hypoxia and sumoylation 4
1.5 Sumoylation and cancer 5
1.6 Sumoylation and OCT4 6
1.7 Significance 6
Chapter 2 Materials and Methods 8
2.1 Cell culture and hypoxic treatment 9
2.2 Plasmid construction and site-directed mutagenesis 9
2.3 Short hairpin RNA and lentivirus production 10
2.4 RNA extraction, reverse transcription and quantitative real-time polymerase chain reaction 10
2.5 Immunoprecipitation and Western blot analysis 11
2.6 Immunostaining 13
2.7 Cell viability assay 14
2.8 In vitro sumoylation assay 14
2.9 Protein stability assays 15
2.10 Xenograft tumor models 16
2.11 Statistical analysis 17
Chapter 3 Results 18
3.1 Hypoxia induces drug resistance and reduces OCT4 protein in EC cells 19
3.2 Hypoxia regulates the stability of the OCT4 protein in EC cells through sumoylation 20
3.3 Sumoylation of OCT4 protein occurs at K123 21
3.4 SENP1 suppresses OCT4 sumoylation and increases OCT4 stability under hypoxic conditions 25
3.5 SENP1 increases the drug sensitivity of EC cells in hypoxic conditions 27
3.6 SENP1 enhances the drug sensitivity of EC cells by maintaining the level of the OCT4 protein 29
Chapter 4 Discussion 31
Chapter 5 References 36
Chapter 6 Tables and Figures 45
APPENDIX 81

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