離氨酸缺陷型蛋白激酶-1(WNK1)是一類具有非典型激酶域的離氨酸-絲氨酸蛋白激酶，先前研究發現WNK1在癌細胞大量表現，並已在小鼠和斑馬魚模式中證實WNK1參與胚胎的血管新生。有許多證據支持胚胎血管新生與腫瘤誘導血管新生為相同的分子路徑所調控，我因此想了解WNK1是否參與腫瘤誘導血管新生，並進一步探討WNK1作為癌症治療標地的可能性。
首先我以嗎啉基反義寡核苷酸技術，敲弱WNK1在斑馬魚的同源基因wnk1a和wnk1b，再利用斑馬魚胚胎模式進行腫瘤細胞株的異種移植，觀察腫瘤誘導血管新生作用及腫瘤細胞增生。我發現敲弱 wnk1時，會抑制腫瘤誘導血管新生作用，並且顯著降低了腫瘤細胞的增生。與前人研究中wnk1對胚胎血管新生作用類似，抑制wnk1a比抑制wnk1b更具有減少腫瘤誘導血管新生及降低腫瘤細胞增生的效果。這些初步的結果顯示：WNK1很可能具有作為癌症治療標靶的潛力。
為驗證此一假說，我再次以斑馬魚胚胎異種移植模式，測試WNK抑制劑對於腫瘤誘導血管新生的影響。兩種具有不同機轉的WNK抑制劑WNK463和Closantel，皆有效抑制胚胎血管新生以及降低腫瘤細胞增生。此外我們也利用Tg (ifabp:RPIA; myl7:EGFP)轉基因斑馬魚的腸癌模式，口服餵食WNK抑制劑，以即時聚合酶鏈式反應 (QPCR) 與病理切片分析的結果顯示：餵食一個月的WNK463或 Closantel對大腸直腸癌的生成具有療效。
我的實驗結果顯示：WNK1在腫瘤誘導血管新生的過程中扮演相當重要的角色，藉由抑制WNK1在活體中的表現量或是活性能夠降低腫瘤的增生與形成，這證實了WNK1能夠做為癌症治療的標靶之一。我亦成功建立於血管中大量表現wnk1a以及在血管中條件式剔除wnk1a表現的轉基因斑馬魚，將與RPIA轉基因魚腸癌模型及HBx,Src轉基因魚肝癌模型交配，更進一步研究WNK1在癌症形成中的功能及其作為癌症治療標地的可能性。

The with-no-lysine (K) kinase I (WNK1), is a serine/threonine protein kinase with atypical location of conserved catalytic lysine in kinase domain. Previous study showed that WNK1 is overexpressed in cancer and is involved in embryonic angiogenesis in mouse and zebrafish model. Abundant evidence supports that molecules and pathways are shared during embryonic and tumor-induced angiogenesis, therefore I set out to investigate the role of WNK1 in tumor-induced angiogenesis and whether WNK1 serves as a cancer therapeutic target.
First of all, I used morpholino-antisense oligonucleotide to knockdown wnk1a and wnk1b (two WNK1 orthologue in zebrafish), then performed xenotransplantation of hepatoma cells to zebrafish morphants to examine the effects on tumor-induced angiogenesis and tumor cell proliferation. I found wnk1 knockdown decreased tumor-induced ectopic vessel formation as well as tumor proliferation compared to the control. Similar to our previous study of wnk1 on embryonic angiogenesis, wnk1a knockdown had more significant effects on tumor-induced angiogenesis than wnk1b knockdown. The result demonstrated that knockdown wnk1 reduced tumor-induced angiogenesis and prevented tumor cell proliferation, it shed a light on WNK1 as a cancer therapeutic target.
Next, two different WNK1 inhibitors (WNK463 and Closantel) were examined by using zebrafish xenotransplantation model for their anti-angiogenesis and anti-tumor cell proliferation effects. Both of the WNK1 inhibitors are effective to inhibit embryonic angiogenesis and tumor cell proliferation. In order to validate the anti-cancer effect of WNK1 inhibitors, adult transgenic fish Tg (ifabp:RPIA; myl7:EGFP) with colorectal cancer and Tg (fabp10a:HBx-mCherry, Src; myl7:EGFP) with hepatocellular carcinoma were oral gavaged for one month, and intestine or liver specimens were collected for molecular and pathological examination. I found that the RPIA transgenic fish reduced intestinal tumorigenesis and liver carcinogenesis after treated with WNK463 or Closantel compared to the control.
These results demonstrated that WNK1 plays an important role in tumor-induced angiogenesis, and inhibition of WNK1 reduces tumor cell proliferation and cancer formation in vivo, proving WNK1 could be a cancer therapeutic target. I also had established genetic modified zebrafish with wnk1a overexpression or conditional knockout in endothelial cell, and will be crossed with RPIA transgenic fish with colon cancer or HBx, Src transgenic fish with liver cancer to investigate the role of WNK1 in cancer development and therapeutic target.

Abbreviation list (according to order of alphabet) I
中文摘要 III
Abstract IV
Table of contents V
Chapter 1 Introduction 1
1.1 Lysine deficient protein kinase 1; With-no-lysine (K) kinase 1 (WNK1) 1
1.1.1 WNK family 1
1.1.2 WNK Function 1
1.1.3 WNK1 and Angiogenesis 2
1.1.4 WNK1 inhibitors 3
1.2 Zebrafish model 4
1.2.1 Advantages of zebrafish model 4
1.2.2 Transgenic fish lines used in this study 4
1.2.3 Angiogenesis in zebrafish 5
1.2.4 Xenotransplantation model 6
Chapter 2 Materials and Methods 7
2.1 Zebrafish husbandry 7
2.2 Zebrafish lines 7
2.3 Embryos collection 7
2.4 Microinjection 7
2.5 Cell culture 8
2.6 Establishment of Hep3B_LifeactRFP stable line 8
2.8 Examine the inhibitory effect on tumor-induced angiogenesis via xenotransplantation using morphants 9
2.9 Resource of compounds 10
2.10 Sub-lethal dose test 10
2.11 Inhibition of embryonic angiogenesis 11
2.12 Using xenotransplantation assay to examine the anti-tumor cell proliferation 11
2.13 Confocal microscopy 12
2.14 Oral gavage 12
2.15 Tissue collection and paraffin section 13
2.16 Hematoxylin and eosin stain 14
2.17 Immunohistochemistry stain 15
2.18 Total RNA isolation 16
2.19 Reverse transcription polymerase chain reaction (RT-PCR) 17
2.20 Real time quantitative polymerase chain reaction (QPCR) 18
2.21 Gateway cloning 19
2.22 TALEN-mediated homologous recombination 20
2.23 In vitro transcription 22
2.24 Statistical analysis 23
Chapter 3 Results 24
3.1 Identification of tumor cells with higher angiogenic factor expression for xenotransplantation study. 24
3.2 Establishment of Hep3B_LifeactRFP stable line 24
3.3 Hep3B_LifeAct-RFP stable line are effective to induce angiogenesis in zebrafish embryos 25
3.4 wnk1a/b knockdown reduced tumor-induced angiogenesis and prevent tumor cell proliferation in zebrafish embryos 25
3.5 WNK1 inhibitors-WNK463 and Closantel prevent embryonic angiogenesis 26
3.6 Determine the survival curve and half maximal lethal concentration (LC50) of WNK463 and Closantel for xenotransplantation assay 27
3.7 WNK1 inhibitors-WNK463 and Closantel exhibited stronger anti-proliferation activity than VEGFR inhibitor (PTK787) in xenotransplantation assay 27
3.8 Oral gavage WNK1 inhibitors-WNK463 and Closantel reduced tumorigenesis on adult zebrafish colorectal cancer (CRC) model 28
3.9 Oral gavage WNK1 inhibitors on adult zebrafish reduced tumorigenesis on adult zebrafish hepatocellular carcinoma (HCC) model 29
Chapter 4 Discussion 31
4.1 Possible mechanism of wnk1 involved tumor induced angiogenesis 31
4.2 Comparison of WNK1 inhibitors versus other anti-angiogenesis therapeutics 33
4.3 Future perspective 34
Figures and Tables 36
Figure 1. Identification of tumor cells exhibiting higher angiogenic factors for xenotransplantation study 36
Figure 2. Establishment of Hep3B_LifeactRFP stable line 38
Figure 3. Hep3B_LifeactRFP stable line induced angiogenesis in zebrafish embryos 40
Figure 4. wnk1 knockdown reduced tumor-induced angiogenesis in zebrafish embryos 42
Figure 5. wnk1 knockdown prevented tumor cell proliferation in zebrafish embryos 44
Figure 6. Anti-angiogenesis effect of WNK463 46
Figure 7. Anti-angiogenesis effect of Closantel 48
Figure 8. Sub-lethal test and determination LC50 of WNK463 for xenotransplantation assay 50
Figure 9. Sub-lethal test and determination the LC50 of Closantel for xenotransplantation assay 52
Figure 10. Comparing the anti-proliferation ability of PTK787, WNK463 and Closantel by xenotransplantation assay. 54
Figure 11. Measurement of body length, body width, body weight of RPIA transgenic fish before and after oral gavage 56
Figure 12. QPCR analysis of RPIA transgenic fish treated with WNK1 inhibitors 58
Figure 13. H&E stain analysis of RPIA transgenic fish treated with WNK1 inhibitors 60
Figure 14. PCNA expression revealed by IHC analysis for RPIA transgenic fish treated with WNK1 inhibitors 62
Figure 15. Measurement of body length, body width, body weight of Src, HBx transgenic fish before and after oral gavage 64
Figure 16. QPCR analysis of HBx, Src transgenic fish treated with WNK1 inhibitors 66
Figure 17. H&E stain analysis of HBx, Src transgenic fish treated with WNK1 inhibitors 68
Table 1. The primer information for qPCR analysis in human cell line 70
Table 2. The primer information for qPCR analysis in zebrafish 71
Supplementary Information 72
Supplementary Figure 1. FASC of un-transfected and rLVUbi-LifeAct-TagRFP transfected Hep3B cell lines 72
Supplementary Figure 2. Gene expression pattern of RPIA transgenic fish treated with WNK1 inhibitors 74
Supplementary Figure 3. Gene expression pattern of HBx, Src transgenic fish treated with WNK1 inhibitors 77
Supplementary Figure 4. Schematic diagram of generation endothelial wnk1a conditional knockout fish using the Cre-loxP System 80
Supplementary Data 1 Cloning of Tg (fli1:wnk1a;myl7:EGFP) transgenic fish 82
Supplementary Data 2 Cloning of Tg (fli1:CreERT2;myl7:EGFP) transgenic fish 86
Supplementary Data 3 Cloning of Tg (LoxP-wnk1a-DsRed-LoxP) transgenic fish 88
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