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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 鄭安理,徐志宏 | |
dc.contributor.author | Tzu-Hsuan Lin | en |
dc.contributor.author | 林子軒 | zh_TW |
dc.date.accessioned | 2021-06-17T01:35:57Z | - |
dc.date.available | 2022-09-08 | |
dc.date.copyright | 2017-09-08 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-01 | |
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Journal of Clinical Oncology. 2017;35 (15_suppl): Abs 4097. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67523 | - |
dc.description.abstract | 肝細胞癌是世界上最常見的癌症之一,全世界每年影響超過80萬例。因為只有10〜20%的病患可以使用局部區域性療法治療,所以大多數肝細胞癌患者需要全身性治療。Sorafenib是一個標定Raf激酶以及血管內皮生長因子接受器的多激酶抑制劑,目前已被核准通過治療肝細胞癌的全身性治療用藥,然而,sorafenib在晚期肝細胞癌中的療效不佳,目標腫瘤反應率低,腫瘤進展時間短,表示大多數肝細胞癌患者存在對sorafenib的先天性和後天性抗藥性。
以前探討癌症治療的抗藥性研究通常使用體外癌細胞培養模式,將癌細胞長時間暴露於高劑量的研究藥物,這種忽略腫瘤微環境作用的方法在標定癌細胞和腫瘤血管新生的sorafenib研究受到限制。在本論文的工作中,我們設計了活體內sorafenib抗藥性的兩個實驗模式,以探討活體內sorafenib抗藥性分子機制有助於鑑定肝細胞癌在臨床上有效的sorafenib敏感性策略之假說。 在本論文的第一部分工作中,我們建立了一個來自異種移植人類肝癌細胞Huh7的sorafenib抗藥性亞型細胞株,其被皮下植入免疫功能缺陷老鼠並顯示出對sorafenib的初級抗藥性。從sorafenib抗藥性肝癌異種移植建立初代培養的Huh7子代細胞(定義為Huh7-SR),比較控制組肝癌異種移植建立初代培養的Huh7子代細胞(定義為Huh7-v),Huh7-SR在細胞培養上具有對sorafenib較為增加之抗藥性,50%抑制濃度(IC50)從6±0.8μM增加到9.5±1.0μM。皮下異種移植實驗驗證Huh7-SR細胞在活體內確實保持對sorafenib的抗藥性。利用IPA(生物學路徑分析軟體暨生物資訊資料庫)分析Huh7-SR跟Huh7-v表現差異之基因,顯示許多訊息傳導路徑集中在變形生長因子beta (TGFβ) 接受器。在Huh7-SR細胞中,TGF-β以及pSmad2的表現增加,顯示TGF-β訊息路徑在Huh7-SR被活化。伴隨治療TGF-β接受器激酶抑制劑會促進sorafenib在Huh7-SR細胞中的敏感性;在Huh7-v細胞中外加TGF-β1重組蛋白會增加對於sorafenib的抗藥性。在用sorafenib治療的91例晚期肝癌患者的世代中,我們發現治療前血清TGF-β1高濃度的患者顯示出無惡化存活期(中位數,2.5比4.3個月,P = 0.022)和整體存活期(中位數5.6比11.6個月;P = 0.029),顯著性少於TGF-β1低濃度的患者。此外,在29例sorafenib治療前後惡化的患者其血清樣本中,血清TGF-β1濃度在疾病惡化時顯著性上升(人數 = 29,P = 0.010)。 在本論文的第二部分工作中,我們在用sorafenib治療的人類肝癌細胞小鼠異種移植中應用一組小髮夾的RNA(shRNA)作篩選,並且鑑定參與sorafenib治療效果的基因。這組小髮夾RNAs(shRNAs),總共3920條可以標定1960個基因,裡面包含各種激酶、轉錄因子、磷酸酶以及細胞激素,利用慢病毒感染Huh7肝癌細胞,再將經過shRNAs感染的Huh7細胞,移植到免疫功能缺陷的老鼠皮下,接著餵予控制組溶劑以及sorafenib。從肝癌異種移植腫瘤組織抽取基因組DNAs,利用聚合酶連鎖反應放大shRNA區段,然後將聚合酶連鎖反應的產物做深度定序量化每條shRNA的表現程度。藉由計算在sorafenib治療組異種移植腫瘤組織比較控制組異種移植腫瘤組織每條shRNA表現頻率,我們發現三個候選基因對於sorafenib具有抗藥性,因為它們shRNA的表現在sorafenib治療組異種移植腫瘤組織顯著性降低,其中,只有週期素激酶5(CDK5)做進一步的探討,因為驗證它的表現在sorafenib治療組異種移植腫瘤組織中確實增加。在CDK5表現量相對較高的肝癌細胞PLC5以及Huh7-SR中,利用RNA干擾技術或CDK5的抑制劑roscovitine將CDK5做負調控,會促進sorafenib所誘發抑制生長之敏感性;反之,在Huh7細胞過度表現CDK5會增加對sorafenib的抗藥性。機制的探討上,sorafenib結合shRNA負調控CDK5增強引起細胞凋亡,導致PLC5和Huh7-SR細胞中多種抗細胞凋亡分子如survivin的負調控增加。在Huh7細胞中過度表現CDK5減弱sorafenib所引起的細胞凋亡與survivin的負調控減少。 我們的結果顯示,活化的TGF-β訊息傳導路徑以及CDK5是導致肝癌在活體對於sorafenib產生抗藥性的重要機制,針對TGF-β以及CDK5治療肝細胞癌,需要更深入的研究。 | zh_TW |
dc.description.abstract | Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, affecting more than 800,000 cases per year worldwide. The majority of HCC patients would need systemic therapy because only 10~ 20% of them could be curable by loco-regional therapies. Sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is the approved systemic agent for HCC. However, the efficacy of sorafenib in advanced HCC is modest with a low objective tumor response rate and short time to tumor progression, indicating that the inherent resistance and acquired resistance to sorafenib exist in most HCC patients.
Previous studies investigating the resistance to anticancer therapy have commonly employed in vitro cancer cell culture models by exposing cancer cells to increasing doses of studied drugs for a prolonged period of time. This approach, neglecting the roles of tumor microenvironment, is theoretically defective in studying sorafenib because sorafenib targets both cancer cells and tumor neovascularization. In the current thesis work, we explored two experimental models of in vivo sorafenib resistance to address the hypothesis that delineation of the molecular mechanism underlying in vivo sorafenib resistance would help identify clinically useful sorafenib-sensitizing strategies for HCC. In the first part of this thesis work, we established a sorafenib-resistant subline from one xenograft of Huh7 HCC cells that was subcutaneously implanted on immunocompromised mice and showed primary resistance to sorafenib. Compared with Huh7 cells primarily cultured from one xenograft treated with vehicle of sorafenib (designated as Huh7-v), this sorafenib-resistant Huh7 subline (designated as Huh7-SR) had a moderate increase of resistance to sorafenib in vitro, with 50%-inhibitory concentration (IC50) increased from 6.0±0.8 to 9.5±1.0 μM. Subcutaneous xenograft studies confirmed that Huh7-SR cells retained the phenotype of in vivo resistance to sorafenib. The IPA (ingenuity pathway analysis) of differentially expressed genes, revealed in cDNA microarray, of Huh7-SR versus Huh7-v cells identified several pathway networks centering at transforming growth factor-β (TGF-β) receptor. Huh7-SR had an increased expression of TGF-β and pSmad2, indicating that the TGF-β pathway was activated. The kinase inhibitors of TGF-β receptor improved the sensitivity to sorafenib in Huh7-SR cells; the addition of recombinant TGF-β1 increased resistance to sorafenib in Huh7-v cells. In a cohort of 91 advanced HCC patients treated with sorafenib, we found that patients with high pre-treatment serum TGF-β1 levels exhibited significantly shorter progression-free survival (median, 2.5 vs. 4.3 months; P = 0.022) and overall survival (median 5.6 vs. 11.6 months; P = 0.029) than did patients with low serum TGF-β1 levels. Furthermore, among 29 patients with baseline and post-sorafenib progression serum samples, the serum TGF-β1 levels were significantly increased at disease progression (n = 29, P = 0.010). In the second part of this thesis work, we applied a pooled short hairpin RNA (shRNA) screening in mouse xenografts of human HCC cells treated with sorafenib, and intended to identify genes that are involved in the therapeutic efficacy of sorafenib in vivo. A pool of 3920 shRNAs targeting 1960 genes, including various kinases, transcriptional factors, phosphatases and cytokines, were introduced into Huh7 cells by lentivirus infection. The Huh7 cells with pooled shRNAs were implanted subcutaneously in immunocompromised mice, and were treated with sorafenib or a vehicle. Genomic DNAs were extracted from HCC xenografts, PCR-amplified of the shRNA regions, and deep-sequenced of the PCR products to quantify the abundance of each shRNA. By calculating the abundance frequencies of each shRNA of sorafenib- treated xenografts versus that of vehicle-treated xenografts, we found 3 candidate sorafenib- resistance genes because their shRNA abundance was significantly decreased in sorafenib-treated xenografts. Among them, only the cyclin dependent kinase 5 (CDK5) was further investigated because its expression was confirmed to be increased in sorafenib- treated xenografts. In PLC5 and Huh7-SR cells whose baseline CDK5 expression levels are relatively high, downregulation of CDK5 by RNA interference or roscovitine, a CDK5 inhibitor, enhanced the sensitivity to sorafenib- induced growth-suppression. On the other hand, overexpression of CDK5 increased the resistance to sorafenib in Huh7 cells. Mechanistically, sorafenib combined with shRNA-mediated CDK5 downregulation augmented the induction of apoptosis, and resulted in an increased downregulation of multiple anti-apoptosis molecules including survivin in PLC5 and Huh7-SR cells. Overexpression of CDK5 in Huh7 cells attenuated the induction of apoptosis and down-regulation of survivin induced by sorafenib. Our data indicate that activation of TGF-β signaling pathway and CDK5 are potentially important mechanisms mediating the resistance to sorafenib in HCC in vivo. Targeting TGF-β and CDK5 as sorafenib-enhancing strategies in the treatment of HCC could warrant further studies. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T01:35:57Z (GMT). No. of bitstreams: 1 ntu-106-D99453004-1.pdf: 8931117 bytes, checksum: 81c0ac70aed8771b4a1ab23ab409aef3 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 致謝 i
中文摘要 iii Abstract vi Abbreviations x Contents xii List of Figures xiv List of Tables xvii Chapter I Background Information 1 1.1 Hepatocellular Carcinoma (HCC) 1 1.2 Staging and treatment of HCC 1 1.3 Systemic therapy for advanced HCC 3 1.4 Sorafenib for advanced HCC 5 1.5 Mechanisms of sorafenib resistance in HCC 6 1.6 Hypothesis of the current thesis work 13 Chapter II Activation of TGF-β signaling pathway as a sorafenib-resistance mechanism, identified in a sorafenib-resistant xenograft of human HCC cells 15 2.1 Introduction 15 2.2 Materials and Methods 19 2.3 Results 30 2.4 Discussion 38 Chapter III CDK5 overexpression as a sorafenib-resistance mechanism identified by pooled shRNA screening plus in vivo selection using mouse xenografts of human HCC cells 43 3.1 Introduction 43 3.2 Materials and Methods 46 3.3 Results 58 3.4 Discussion 66 Chapter IV Summary and Discussions 71 Figures 77 Tables 100 References 104 Appendices 111 Supplement Tables 112 | |
dc.language.iso | en | |
dc.title | 利用活體動物模式探討肝細胞癌Sorafenib抗藥性之機轉 | zh_TW |
dc.title | Exploration of Sorafenib Resistance Mechanisms in Hepatocellular Carcinoma Using in vivo Models | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 許駿,葉秀慧,謝森永,趙毅 | |
dc.subject.keyword | 變形生長因子β,週期素激?5,肝細胞癌,蕾莎瓦,抗藥性,小髮夾RNA, | zh_TW |
dc.subject.keyword | TGF-β,CDK5,hepatocellular carcinoma,sorafenib,resistance,shRNA, | en |
dc.relation.page | 141 | |
dc.identifier.doi | 10.6342/NTU201702363 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-01 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 腫瘤醫學研究所 | zh_TW |
顯示於系所單位: | 腫瘤醫學研究所 |
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