臺灣博碩士論文加值系統

放射線療法是頭頸癌治療中相當重要的一環，然而放射線抗性卻是一個使療效大幅降低的主要因素。為了全面性的探討影響放射線抗性的重要基因，我們整併了轉錄體學資料庫、頭頸癌臨床的外顯子陣列晶片資料庫以及單維梯度膠體蛋白質電泳資料庫，進一步針對ITGA6 和 GDF15如何影響放射線敏感性做進一步的探討。我們首先確認THBS1和ITGA6會藉由受體與受器的關係影響放射線抗性，接著發現兩分子結合後會藉由活化FAK和NRF2分別執行其下游的功能，包括影響細胞骨架達到改變細胞移形能力以及轉錄GCLC次單元來減低放射線造成的細胞內氧化壓力。同時我們發現GDF15可以經由SMAD訊息傳遞路徑的磷酸化去降低細胞內反應性氧族進而使CD44癌症幹細胞表面抗原增加以及調控ALDH1族群分布達到癌症幹細胞轉換的目的。而在動物實驗中也可以看到GDF15可以明顯促進裸鼠異體移植腫瘤的放射線抗性。在臨床方面，我們也藉由觀察頭頸癌病患臨床檢體以及TCGA資料庫中的癌症基因體檔案，發現THBS1、ITGA6以及GDF15在癌症病患中有高量表現並降低了整體存活率。總結來說，我們發現THBS1、ITGA6和GDF15會影響頭頸癌的放射線抗性，並且藉由不同的訊息傳遞路徑達到促使細胞惡性化及減少放射線造成的氧化壓力傷害。

Radiotherapy is an integral part for the treatment of head and neck cancer (HNC), while radioresistance is a major cause leads to treatment failure. We have previously identified THBS1, ITGA6 and GDF15 as potential radioresistance associated gene by transcriptomic and proteomic approaches. We found the cross talk between THBS1 and ITGA6 contribute to both radioresistance and invasive ability via FAK-NRF2-GCLC axis and regulating tubulin cytoskeleton protein expression; which GCLC assess ROS clearance and tubulin participate in cancer cell migration. Simultaneously, we discovered GDF15 can contribute to cancer stem cells conversion by elevated CD44 and ALDH1 population via SMAD-associated pathway and further decrease intracellular ROS. In vivo xenograft tumor model also demonstrated that GDF15 possess radioresistance ability of HNC cells. In clinical, we analyzed HNC patient plasma samples and cancer genomics from TCGA database, found higher THBS1, ITGA6 and GDF15 expression in cancer patients and lead to worse overall survival in various types of cancer. Taken together, our results clarify that panel molecules THBS1, ITGA6 and GDF15 contribute to tumor malignancy and radioresistance ability via distinguish signaling pathway, focus on these molecules in future may provide better clinical outcome for patient receive radiotherapy.

指導教授推薦書
口試委員會審定書
致謝 iii
摘要 iv
ABSTRACT v
CONTENT vi
BACKGROUND AND SIGNIFICANCE 1
Head and neck cancer 1
Radiotherapy is an integral part for the treatment of HNC 1
Molecules and phenotypes associated with radioresistance 3
ITGA6, and GDF15 are potential radioresistance genes in HNC 4
SPECIFIC AIMS 6
MATERIALS AND METHODS 7
RESULTS AND DISCUSSION 16
PART I: ITGA6 contributes to radioresistance of head and neck cancer by regulating cellular reactive oxygen species and cytoskeleton via a FAK-associated NRF2 signaling pathway 16
PART II: GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway 31
REFERENCES 45
TABLES AND FIGURES 54


List of Tables
Table 1. Functional classification of 470 genes differentially expressed in the RR sublines compare to the parental HNC cells. 54
Table 2. Functional gene pathways associated with the RR phenotype of HNC. 55
Table 3. Differentially expressed genes (up-regulated) in HNC radio-resistance sublines as analyzed by microarray. 56
Table 4. Differentially expressed genes (down-regulated) in HNC radio-resistance sublines as analyzed by microarray. 58
Table 5. Candidate genes up-regulated in cDNA microarray and clinical tissue array. 61
Table 6. List of genes and primer sequences used in this study. 62
Table 7. List of antibody sources used in this study. 63
Table 8. Clinical characteristics of HNC patients used in GDF15 study. 64


List of Figures
Figure 1. Integration of RR transcriptomic dataset and clinical HNC exon-array dataset.. 67
Figure 2. Profiling and validation of the genes differentially expressed in RR sublines.. 69
Figure 3. Cross talk between THBS1 and ITGA6. 71
Figure 4. THBS1 and ITGA6 protein level expression in RR sublines. 72
Figure 5. ITGA6hi HNC cells show higher radioresistance ability. 73
Figure 6. Knock down THBS1 and ITGA6 can enhance radio-sensitivity in HNC. 74
Figure 7. ITGA6 and THBS1 reduction contribute to higher intracellular ROS under oxidative stress. 75
Figure 8. ITGA6 may effect intracellular ROS via regulating antioxidants precursor enzymes. 76
Figure 9. RhTHBS1 can induce GCLC expression. 77
Figure 10. Knock down GCLC lead to radiosensitive in HNC cells.. 798
Figure 11. THBS1 and ITGA6 can affect NRF2 binding to antioxidant response element individually. 79
Figure 12. Treatment of rhTHBS1 phosphorylated NRF2 in HNC cells. 80
Figure 13. HNC RR sublines show higher phopho-NRF2 expression compare to parental cells. …………………………………………………..………..81
Figure 14. Inhibition of FAK and PKC pathway can reduce the ARE luciferase activity. 82
Figure 15. Inhibition of FAK pathway decreased GCLC expression in HNC cells. 83
Figure 16. FAK binds and activates NRF2. 84
Figure 17. Treatment of NRF2 antibody block the FAK-induced NRF2 translocation and GCLC expression.…………………………………………….85
Figure 18. Knock down ITGA6 decrease THBS1-ITGA6-NRF2 axis induce GCLC expression. 86
Figure 19. Inhibition of FAK by PF573228 decreased radioresistance ability in HNC cells. 87
Figure 20. Treatment of PF573228 increase intracellular ROS level. 88
Figure 21. THBS1-ITGA6 axis may affect tubulin cytoskeleton via FAK signaling. 89
Figure 22. Knock down GCLC contribute to tubulin repression….......90
Figure 23. Oxidative stress induced by H2O2 descending cytoskeleton expression in HNC cells. 91
Figure 24. THBS1 can partially rescue tubulin catastrophe by preventing H2O2 induced oxidative stress. 92
Figure 25. Knock down ITGA6 significantly reduce invasion and migration ability. 93
Figure 26. THBS1 can partially rescue cancer cell migration ability damaged by H2O2-induced oxidative stress. 94
Figure 27. Fig 27. HNC Cancer cells migration reduced after blockade of NRF2 or GCLC expression……………………………………………………….95
Figure 28. THBS1 is elevated in plasma samples from HNC patients. 96
Figure 29. Cancer genomics analysis of overall survival within different cancer by cBioPortal software with both THBS1 and ITGA6 alteration.………………………………………………………………………………….……97
Figure 30. Diagram of the mechanism by which THBS1 and ITGA6 contributes to cancer cell radioresistance via FAK-NRF2 axis pathway. 98
Figure 31. GDF15 may regulate radiosensitivity in HNC cells. 99
Figure 32. GDF15 suppresses cellular level of reactive oxygen species (ROS). 100
Figure 33. Knockdown of GDF15 increase mitochondria membrane potential by mitocapture de binding assay. 101
Figure 34. Introduction of GDF15 reduces K+ ion effluxing out of cells via KCNJ2. 102
Figure 35. Treatment of antioxidant agent led to radioresistance in HNC cells. 103
Figure 36. Treatment of antioxidant agent increase CD44+ cell population. 104
Figure 37. GDF15 contribute to upregulation of ALDH1+ population. 105
Figure 38. Treatment of GDF15 contribute to higher sphere formation ability under oxidative stress. 106
Figure 39. CD44+/CD24- sub-population of HNC cells show higher GDF15 expression than CD44-/CD24- sub-population in mRNA and secrete form protein level. 107
Figure 40. GDF15 contribute to CSCs phenotype by regulating ALDH1+ population. 108
Figure 41. ALDH1 population was affected by GDF15. 109
Figure 42. GDF15+ population exhibit higher sphere forming ability. 110
Figure 43. Silencing of GDF15 reduced formation of spheroid size and number. 111
Figure 44. GDF15 lead to elevation of invasion and migration ability in HNC cells. 113
Figure 45. GDF15 expression was reduced after treatment of TGF-β inhibitor. 114
Figure 46. GDF15 share the signal pathway with TGF-β. 115
Figure 47. GDF15 is associate with SMAD signaling pathway. 116
Figure48. GDF15 regulates cellular functions through similar downstream pathway of TGF-β. 117
Figure 49. Xenograft tumor model design. 118
Figure50. Xenograft tumor model demonstrates IR resistance phenotype induced by GDF15. 119
Figure 51. GDF15 is elevated in plasma samples from HNC patients. 121
Figure 52. Diagram of the mechanism by which GDF15 contributes to radioresistance and cancer stemness through regulating ROS levels via a SMAD-associated pathway. 122

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