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研究生： 田博晏
Tien, Po-Yen
論文名稱： 小鼠衍生Alisertib抗藥性人類小細胞肺癌之分子圖譜分析
Molecular profiling of Mouse-derived Alisertib Resistant Small Cell Lung Cancer Cells
指導教授： 陳炯東
Chen, Chiung-Tong
周裕珽
Chou, Yu-Ting
口試委員: 馬念涵
Ma, Nianhan
劉玉麗
Liu, Yu-Li
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 生物科技研究所
Biotechnology
論文出版年： 2025
畢業學年度： 113
語文別： 英文
論文頁數： 105
中文關鍵詞： 小細胞肺癌 、極光激酶A 、上皮細胞間質轉化 、異質性 、抗藥性 、MYC
外文關鍵詞： Alisertib, DBPR728, MYC, drug resistance, Aurora kinase
相關次數： 點閱：49  下載：0 
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    • 小細胞肺癌 (SCLC, small cell lung cancer)是一種侵襲性強且預後不佳的惡性腫瘤，具有極高的增殖和轉移速率。該癌症類型顯示出高度異質性，且在發展出抗藥性後容易復發，導致治療選擇非常有限。因此，迫切需要開發有效的SCLC治療策略。
    小細胞肺癌具有MYC基因高表現以及極光激酶A（AURKA）的高度活化特性。先前研究顯示，AURKA 能與 MYC 家族結合並穩定其功能；因此，抑制AURKA，使其構型改變降低 MYC 的穩定性，這讓AURKA成為SCLC 治療中一個理想目標。Alisertib 和 DBPR728 分別由Puma Biotechnology和國家衛生研究院開發，是AURKA抑制劑，是治療 SCLC 的重要候選藥物。然而，針對這些藥物的抗藥性機制仍有待進一步闡明。
    為了探討 SCLC 對 Alisertib 和 DBPR728 的抗性機制，我們建立了具有藥物抗性的體內小鼠異種移植人類 SCLC 細胞株模型。我們通過 RNA 定序、生物資訊分析和形態學觀察，全面解析了抗性細胞的分子和表型變化。研究結果顯示，具藥物抗性的細胞由神經內分泌型的上皮樣表型轉變為非神經內分泌型的間充質樣細胞，並伴隨 EMT 標誌物（如 Slug、Snail 和 Vimentin）表現的顯著上調。此外，我們發現 Wnt 信號通路的高度活化（包括 β-catenin、LRP6 和 TCF7L2 等關鍵組分的上調）可能是驅動 EMT 的潛在機制，並在藥物抗性發展中起到重要的作用。
    本研究的結果揭示了 SCLC 中 AURKA 抑制劑抗藥性的轉錄組分子機制，並提供了克服抗藥性的潛在治療靶點，鑑定出具有前景的靶點，我們的目標是推進有效治療 SCLC 的發展，解決減少復發和改善患者預後的關鍵重要需求。

    SCLC (small cell lung cancer) is an aggressive malignancy with a poor prognosis, characterized by extremely high rates of proliferation and metastasis. This cancer type demonstrates high heterogeneity and is prone to relapse following the development of drug resistance, leaving limited treatment options. Therefore, effective therapeutic strategies for SCLC are urgently needed. SCLC exhibits high MYC expression and active Aurora kinase A (AURKA) activity. Previous studies have shown that AURKA binds to and stabilizes the MYC family; targeting AURKA destabilizes MYC, making it a suitable choice for SCLC treatment. Alisertib and DBPR728 are AURKA inhibitors developed by Takada (2015) and Puma Biotechnology (2022) and the National Health Research Institutes, respectively, which act as MYC degraders for treating SCLC. However, the mechanisms underlying resistance to these agents of SCLC remain unclear. To investigate the resistance mechanisms of Alisertib and DBPR728 in SCLC, we established in vivo human SCLC models resistant to these drugs. We characterized the molecular and phenotypic changes in resistant cells through RNA sequencing, bioinformatic analyses, and morphological observations. Our findings reveal that drug-resistant sublines undergo a transition from a neuroendocrine, epithelial-like phenotype to a non-neuroendocrine, mesenchymal-like state, accompanied by increased expression of epithelial-to-mesenchymal transition (EMT) markers, such as Slug, Snail, and Vimentin. Furthermore, we identified hyperactivation of the Wnt signaling pathway, including upregulation of key components such as β-catenin, LRP6, and TCF7L2, as a potential driver of EMT, critical for promoting drug resistance. Our study sheds light on the molecular mechanisms of AURKA inhibitor resistance of SCLC at the transcriptome level and provides potential therapeutic targets to overcome drug resistance. By identifying promising targets, we aim to advance the development of effective treatments for SCLC, addressing the critical need to reduce relapse and improve patient outcomes.

    Table of Contents 摘要 4 ABSTRACT 6 Acknowledgments 8 List of Abbreviations 9 Introduction 10 Materials and Methods 13 Cell Culture 13 Establish human SCLC in xenograft animal model 14 Generate drug-resistant in animal model 14 Induction of Stronger Drug Resistance in the Animal Model 15 Xenograft-derived Primary Culture 16 Half Maximal Inhibitory Concentration 18 Real-time PCR 21 Cell Doubling Time Assay 23 RNA sequencing 25 Micro-Western Array Assay 26 Statistical Analysis 30 Bioinformatic Analysis 31 Results 33 Established Drug-Resistant Human SCLC Xenograft Tumors in Mice 33 Isolated Single-Clone Sublines Derived from the Xenograft Tumors 40 Level of Resistance and SCLC Subtype of Single-Clone Sublines Derived from the Xenograft Tumors 41 Drug Resistance Associated Genes Expression in Single-Clone Sublines Derived from Xenograft Tumor 44 Significant Differentially Expressed Genes in Drug-Resistant Single-Clone Sublines Derived from Xenograft Tumors 46 Upregulated Genes across the Single-Clone Sublines Derived from the Xenograft Tumors 50 Putative Biological Pathways Associated with AURKAi Resistance Identified in Resistant Single-Clone Sublines from the Xenograft Tumors 51 Discussion 54 Figures 57 Figure 1A 57 Figure 1B 58 Figure 1C 59 Figure 1D 60 Figure 1 61 Figure 2A 63 Figure 2B 63 Figure 2C 63 Figure 2D 64 Figure 2 65 Figure 3A 67 Figure 3B 67 Figure 3C 68 Figure 3D 68 Figure 3E 68 Figure 3F 69 Figure 3G 69 Figure 3H 69 Figure 4A 74 Figure 4B 74 Figure 4C 76 Figure 4D 77 Figure 4D 78 Figure 4E 79 Figure 4 80 Figure 5A 84 Figure 5B 84 Figure 5C 85 Figure 5D 85 Figure 5E 86 Figure 5 87 References 91 Appendix 97 Appendix Figures 103

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