臺灣博碩士論文加值系統

背景
KIT基因突變為約80%胃腸道基質瘤(gastrointestinal stromal tumors)提供主要驅使致癌訊息(driven oncogenic signal)。目前基立克(Gleevec®; imatinib mesylate, IM)為治療轉移性胃腸道基質瘤的一線藥物，在80%的病人中可達到治療效果。然而，在持續的用藥過程中，胃腸道基質瘤之KIT基因可產生繼發性突變，導致IM抗藥性的發生。而第二線KIT酪氨酸激酶抑制劑舒癌特(Sutent; sunitinib malate, SU)，對具IM抗藥性且繼發性突變位於KIT 外顯子17的胃腸道基質瘤之療效則相當有限。有許多針對KIT酪氨酸激酶抑制劑與熱休克蛋白90抑制劑，用於對IM或SU有抗藥性之病人的研究在進行中，然而目前尚無突破性發展。在本研究中，我們使用市面上可取得之酪氨酸激酶抑制劑與新一類熱休克蛋白90抑制劑AUY922，探討其用於治療對IM或SU有抗藥性之胃腸道基質瘤病人的可行性，以及相關的作用機轉。
方法與結果
首先，我們在COS-1 細胞株中建立了在病人中常見的原發性+/-繼發性之KIT 突變基因的抗藥性篩檢平台，並以市面上可取得的酪氨酸激酶抑制劑，依其臨床上的血中穩定濃度，來比較這些藥物對突變株 KIT蛋白質磷酸化的抑制效果。另外，針對藥物對外顯子11/17突變KIT的結果，我們使用GIST48細胞株的生長抑制效果來做進一步確認，同時利用分子模型來探討抗藥性之機轉。數據顯示，相較於繼發性突變於外顯子17的KIT 蛋白，SU可有效抑制繼發性突變位於外顯子13或14的KIT 蛋白質磷酸化程度，此結果與臨床上的發現一致。另外，相較於其他酪氨酸激酶抑制劑，nilotinib和sorafenib可有效抑制繼發性突變位於外顯子17的KIT 蛋白質磷酸化程度與GIST48細胞的生長。分子模型的研究則發現，同時具有外顯子11大片段缺失與外顯子17點突變的KIT蛋白，結構上會從原本的不活化態趨向完全活化態，並與nilotinib和sorafenib形成較穩定的結合。
另一方面，我們證明AUY922會造成對IM敏感的GIST882細胞與對IM具抗藥性的GIST48細胞中，突變 KIT蛋白質表現總量與磷酸化程度的減少，並引發細胞凋亡，進而抑制細胞生長。利用cyclohexamide處理抑制細胞內蛋白質之合成後，加入AUY922會加速GIST細胞內KIT蛋白質降解。若利用藥物抑制proteasome或是自噬作用等蛋白質降解路徑，均可減少AUY922造成KIT降解的程度。除此之外，抑制自噬作用會造成未經AUY922處理GIST細胞KIT蛋白質表現量的增加，也證實自噬作用參與內生性KIT蛋白質的循環。自噬作用在KIT內生性循環與AUY922所引起降解過程中的角色，也進一步經由KIT蛋白質與MAP1LC3B、acridine orange、SQSTM1所標記的autophagosome共位現象，與干擾autophagsome形成之必要蛋白質BECN1或ATG5生成等實驗中獲得證實。此外，實驗結果也指出，AUY922亦會導致KIT mRNA表現量的減少，然其機轉並非影響KIT mRNA的降解，而是透過降低轉錄作用造成。數據顯示，在AUY922處理後，GIST細胞中多種轉錄因子，如CEBP、TP53、RELA與HIF1A的活性及細胞核中的表現量均會降低。 利用DNA結合力沉澱法與染色質免疫沉澱法，我們進一步證實TP53可結合至KIT promoter上-365到-30的區域，並且在AUY922處理後，造成結合至KIT promoter上的TP53減少。
結論
總結來說，我們發現對IM具有抗藥性且繼發性突變位於外顯子17的胃腸道基質瘤，nilotinib和sorafenib比SU有更佳的抑制效果。另外，AUY922的結果不止強調其用於治療有KIT表現之胃腸道基質瘤的可行性，同時也率先證實在胃腸道基質瘤細胞株GIST882與GIST48中，自噬作用參與內生性與熱休克蛋白90抑制劑所造成的KIT蛋白降解。

Background
Advanced gastrointestinal stromal tumors (GIST), a KIT oncogene-driven tumor, on imatinib mesylate (IM) treatment may develop secondary KIT mutations to confer IM-resistant phenotype. Second-line sunitinib malate (SU) therapy is largely ineffective for IM-resistant GISTs with secondary exon 17 (activation-loop domain) mutations. Nowadays, several KIT tyrosine kinase inhibitors (TKIs) and heat shock protein 90 (HSP90AA1) inhibitors are under investigation for IM and/or SU-resistant GIST patients. However, there is no notable improvement. In this study, we used commercial available TKIs and a new class of HSP90AA1 inhibitor, NVP-AUY922 (AUY922), to evaluate their potencies for treatment on IM and/or SU-resistant GISTs and to clarify the detailed mechanisms.
Methods and Results
First, we established an in vitro cell-based platform consisting of a series of COS-1 cells expressing KIT cDNA constructs encoding common primary±secondary mutations observed in GISTs, to compare the activity of several commercially available TKIs on inhibiting the phosphorylation of mutant KIT proteins at their clinically achievable plasma steady-state concentration (Css). The inhibitory efficacies on KIT exon 11/17 mutants were further validated by growth inhibition assay on GIST48 cells, and underlying molecular-structure mechanisms were investigated by molecular modeling. Our results showed that SU more effectively inhibited mutant KIT with secondary exon 13 or 14 mutations than those with secondary exon 17 mutations, as clinically indicated. On contrary, at individual Css, nilotinib and sorafenib more profoundly inhibited the phosphorylation of KIT with secondary exon 17 mutations and the growth of GIST48 cells than IM, SU, and dasatinib. Molecular modeling analysis showed fragment deletion of exon 11 and point mutation on exon 17 would lead to a shift of KIT conformational equilibrium toward active form, for which nilotinib and sorafenib bound more stably than IM and SU.
In the other hand, we demonstrated that AUY922 is effective in inhibiting the growth of GIST cells expressing mutant KIT protein, the IM-sensitive GIST882 and IM-resistant GIST48 cells. The growth inhibition was accompanied with a sustained reduction of both total and phospho-KIT proteins and the induction of apoptosis in both cell lines. Surprisingly, AUY922-induced KIT reduction could be partially reversed by pharmacological inhibition of either autophagy or proteasome degradation pathway. The blockade of autophagy alone led to the accumulation of the KIT protein, highlighting the role of autophagy in endogenous KIT turnover. The involvement of autophagy in endogenous and AUY922-induced KIT protein turnover was further confirmed by the colocalization of KIT with MAP1LC3B-, acridine orange-, or SQSTM1-labeled autophagosome, and by the accumulation of KIT in GIST cells by silencing either BECN1 or ATG5 to disrupt autophagosome activity. In addition, AUY922 could reduce KIT mRNA at transcription level without affecting its mRNA stability. Further studies showed that AUY922 treatment would reduce the nuclear activities and protein levels of several transcription factors, such as CEBP, TP53, RELA, and HIF1A in GIST cells. Experiments using DNA affinity precipitation and chromatin immune-precipitation assays showed that TP53 could bind on KIT promoter region (from -365 to -30 nucleotides upstream of the transcriptional start site), and its binding activity was significantly reduced after AUY922 treatment.
Conclusions
Taken together, we show that nilotinib and sorafenib are more active in IM-resistant GISTs with secondary exon 17 mutation than SU that deserve further clinical investigation. Moreover, the results of AUY922 not only highlight its potential application for the treatment of KIT-expressing GISTs, but also provide the first evidence for the involvement of autophagy in endogenous and HSP90AA1 inhibitor-induced KIT degradation in GIST882 and GIST48 cells.

中文摘要 1
Abstract 3
Acknowledgement 6
Contents 8
Table of contents 10
Figure of contents 11
Appendixes of contents 12
Abbreviation 13
Introduction 14
Gastrointestinal stromal tumor 14
Immunohistochemical markers in the diagnosis of GIST 16
Current therapies for GIST 19
Resistance mechanisms to IM 20
Second-line treatment: Sutent 22
Developing therapy for IN/SU-resistant GISTs 22
Aims and overview of Thesis 25
Material and methods 27
Cell lines and reagents 27
Construction of the KIT mutants 28
Transient transfection 28
Drug treatment 29
Immunoblotting studies 30
Growth inhibition assay 31
Clonogenic assay 31
Milliplex analysis 31
Molecular modeling and docking 32
RNA interference 33
Fluorescent immunostaining and acridine orange staining 33
Flow cytometry 34
RNA extraction and quantitative analysis of KIT mRNA 34
Protein fractionation 35
Transcription factor array 35
DNA affinity binding assay 36
Chromatin immunoprecipitation (ChIP) assay 37
Results 39
Integration efforts for TKIs potencies to imatinib-resistant KIT mutants : KIT as a model 39
Effects of TKIs on KIT with single mutation expressed in COS-1 cells 39
Effects of TKIs on KIT mutants with secondary ATP-binding domain mutations 40
Effects of TKIs on KIT mutants with secondary activation-loop domain mutations
41
Virtual molecular modeling/docking analysis 41
Screening new compounds targeted to mutant KIT proteins 43
Inhibition of heat-shock protein 90 induced GIST cells death and downregulated KIT transcriptional and posttranslation level 43
AUY922 downregulated phospho- and total KIT expression 43
The cytotoxic effect of AUY922 in GIST48 and GIST882 cells 45
The enhancement of KIT protein degradation by AUY922 46
AUY922-induced autophagy led to KIT reduction 46
Autophagy was also involved in endogenous KIT turnover in GIST cells 47
AUY922 reduced KIT mRNA expression level and downregulated the TP53 transcriptional activity, nuclear partition, and its binding to KIT promoter 48
Discussion 50
Integration efforts for TKIs potencies to imatinib-resistant KIT mutants : KIT as a model 50
Inhibition of heat-shock protein 90 induced GIST cells death and downregulated KIT transcriptional and posttranslation level 52
Conclusions 57
References 58
Tables 69
Figures 72
Appendixes 92

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