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系統識別號 U0026-2208201420031100
論文名稱(中文) KIT基因外顯子557-558缺失突變在胃腸道基質瘤肝轉移中的調控機制
論文名稱(英文) The mechanism of KIT exon 11 557-558 deletion for liver metastasis in gastrointestinal stromal tumors
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 102
學期 2
出版年 103
研究生(中文) 李姿瑩
研究生(英文) Tzu-Ying Li
學號 s96014019
學位類別 碩士
語文別 英文
論文頁數 65頁
口試委員 指導教授-許凱熙
口試委員-沈延盛
口試委員-陳立宗
口試委員-顏家瑞
中文關鍵字 胃腸道基質瘤  肝轉移  酪氨酸蛋白激酶  轉錄因子  細胞激素接受器 
英文關鍵字 Gastrointestinal stromal tumors  liver metastasis  KIT  ETV1  CXCR4 
學科別分類
中文摘要 胃腸道基質瘤為胃腸道中最常見的基質腫瘤,95%胃腸道基質瘤表現KIT,配體與KIT的結合會活化下游訊息傳遞路徑以調節細胞功能,當KIT突變會導致下游訊息傳遞路徑持續地活化,65%胃腸道基質瘤病患出現KIT exon 11,目前已知KIT exon 11缺失突變與惡性病徵及較差的預後密切相關。肝臟為胃腸道基質瘤患者最常見的轉移器官(50–60%),同時肝轉移會導致治療困難度增加及較差的預後。然而,KIT突變圖譜與肝轉移間的關聯及機制至今仍不清楚,於本研究中期望能夠找出KIT突變與肝轉移之間的關聯性,並探討其調控機制。臨床檢體分析發現多數胃腸道基質瘤伴隨肝轉移的患者帶有KIT exon 11缺失突變,並發現肝轉移患者其缺失突變多集中於KIT exon 11 557及558此兩胺基酸。過去研究發現ETV1轉錄因子調控許多與腫瘤侵犯相關基因,並於胃腸道基質瘤中高度表現並受KIT下游訊息調控,此外,CXCR4化學激素接受器同樣地被發現與腫瘤轉移相關。實驗研究結果發現KIT exon 11 557及558缺失突變增加細胞侵犯能力,並增加ETV1及CXCR4表現,從臨床檢體免疫染色分析同樣發現ETV1及CXCR4於發生肝轉移及KIT exon 11缺失突變檢體中有較高的表現,說明KIT exon 11 557及558缺失突變藉由調控ETV1及CXCR4增加細胞侵犯能力。過去研究發現CXCR4其配體CXCL12於肝臟中大量表現,細胞實驗發現KIT exon 11 557及558缺失突變會受CXCL12濃度影響其化學趨向性,抑制ETV1或CXCR4則明顯降低化學趨向性,動物實驗證實KIT exon 11 557及558缺失突變增加肝轉移的發生,以上實驗結果證實KIT exon 11 557及558缺失突變藉由增強ETV1及CXCR4表現,使其增加對CXCL12的化學趨向性,指出特定器官中高表現的CXCL12可能做為趨化物,誘使CXCR4高度表現的腫瘤細胞發生特定器官的轉移。染色質免疫沉澱實驗說明ETV1於KIT exon 11 557及558缺失突變的胃腸道基質瘤細胞株中,可直接結合於CXCR4啟動子上。總結目前研究結果,KIT exon 11 557及558缺失突變增加ETV1的表現,並藉由ETV1與CXCR4啟動子的結合增加CXCR4表現,使其增加對CXCL12的化學趨向性,進而促進肝轉移可能性,因此,抑制ETV1的活性以及CXCL12/CXCR4訊息調控將可作為抗轉移治療的新標的。
英文摘要 Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the digestive tract. KIT, a receptor tyrosine kinase, is expressed in the most of GISTs (95%). Binding of the stem cell factor (SCF), the ligand of KIT, activate downstream signaling pathways, including MAPK, AKT, STAT1, and STAT3. In GISTs, about 65% of mutations occur in KIT exon 11, and it has been demonstrated that deletions in KIT exon 11 are associated with more malignant features and poor prognosis. Malignant GISTs commonly metastasize into liver (50–60%). However, the correlation between KIT mutations pattern and liver metastasis in GISTs is still unclear. Here, we purposed to examine the correlation between the KIT exon 11 deletion and liver metastasis behavior in GISTs. In our clinical data, the deletion in KIT exon 11 Trp-557 and Lys-558 was the most common mutation among KIT exon 11 mutations in GISTs with liver metastasis. ETV1, a transcription factor, participates in tumor invasion by activating the transcription of several genes. Previous studies have reported that ETV1 is highly expressed and regulated by activated KIT in GIST cells. The chemokine receptor CXCR4 has been reported to play a central role in tumor metastasis. In our study, we showed that KIT exon 11 557-558 deletion enhanced cell invasiveness and increased the expressions of ETV1 and CXCR4. In clinical GIST specimens, ETV1 and CXCR4 were closely associated with liver metastasis and KIT exon 11 deletion. The CXCR4 ligand CXCL12 is strongly expressed in liver, the most common metastatic site for GISTs. In vitro results show that KIT exon 11 557-558 deletion in GIST cells mediated chemotactic and invasive responses toward CXCL12 gradient. Inhibition of ETV1 and CXCR4 abrogated the effects. In vivo results show that KIT exon 11 557-558 deletion enhanced the liver metastatic potential of GIST cells. These findings indicate that CXCL12, highly expressed in liver, played as a chemoattractant that attract CXCR4-expressed tumor cells towards liver. In addition, we also found that ETV1 is able to directly bind to CXCR4 promoter in GIST cells with KIT exon 11 557-558 deletion by chromatin immunoprecipitation assay. In conclusion, KIT exon 11 557-558 deletion upregulated the expression of CXCR4 through the enhanced binding of ETV1 to CXCR4 promoter, and thus promoted liver metastasis via CXCL12/CXCR4axis. Our study provides insight that inhibition of the transcription activity of ETV1 and blockade of the CXCL12/CXCR4 axis may be a novel anti-metastatic therapy in GISTs.
論文目次 中文摘要 I
Abstract III
Acknowledgement V
Contents VII
Abbreviation list X
Introduction 1
1. Gastrointestinal stromal tumor 1
Clinical features 1
Pathogenesis and oncogenic kinase mutations 1
Management of GISTs 3
2. ETS transcription factors 4
Modulation of ETS function 5
The role of PEA3 in cancer 6
3. Chemokine and Chemokine receptor 7
CXCL12/CXCR4 axis in cancer metastasis 8
Therapeutic targeting based on the CXCL12/CXCR4 axis 10
Research motive 12
1. Rationale 12
2. Specific Aims 14
3. Significance 14
Materials and Methods 15
Cell lines 15
Transformation 15
Plasmid isolation 16
Transient transfection 17
Protein extraction 17
Western blotting 18
RNA preparation 18
Reverse transcription PCR 19
Real-time quantitative PCR 19
Invasion assay 20
Migration assay 21
Chemotaxis and chemoinvasion assays 21
Immunohistochemical analysis 22
Chromatin immunoprecipitation (ChIP) assay 23
Animal model 23
Patients 24
Statistical analysis 24
Results 25
GIST patients with KIT exon11 deletion and liver metastasis have poor prognosis 25
KIT exon 11 Trp-557 and Lys-558 deletion correlates with the development of metastases in GISTs 26
KIT exon 11 Trp-557 and Lys-558 deletion promotes cell mobility and EMT in GIST cells 27
KIT exon 11 557-558 deletion enhances liver metastasis of human GIST in vivo 27
KIT exon 11 557-558 deletion increases ETV1 and CXCR4 expression in GIST cells 28
ETV1 and CXCR4 are closely associated with clinical liver metastatic GISTs and KIT exon 11 deletion 28
Depletion of ETV1 and CXCR4 reduce the migration ability in GIST cells 29
Expression of ETV1, CXCR4 and CXCL12 in normal livers, primary GISTs and liver metastatic GISTs 29
KIT exon 11 557-558 deletion accelerates CXCL12-mediated migration and invasion in GIST cells 30
KIT / ETV1 / CXCR4 axis in GISTs 30
KIT exon 11 557-558 deletion regulates ETV1 via ERK signal pathway 31
ETV1 may upregulate CXCR4 expression transcriptionally 31
Discussion and Conclusion 33
References 36
Figures 43
Tables 57
Appendix 60
Appendix I. List of antibodys 60
Appendix II. List of primers 62
Appendix III. List of shRNA TRC number 64
Curriculum vitae 65
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