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研究生： 莎凡莉
Shaifali Singh
論文名稱： WDR4促進PTPN23泛素化以抑制EGFR及c-MET 降解而成為肺癌治療靶點
PTPN23 ubiquitination by WDR4 suppresses EGFR and c-MET degradation to define a lung cancer therapeutic target
指導教授： 陳瑞華
Chen, Ruey-Hwa
王雯靜
Wang, Wen-Ching
口試委員: 陳光超
Chen, Guang Chao
王 憶卿
Wang, Yi Ching
賴品光
Lai, Charles P.
學位類別： 博士
Doctor
系所名稱： 生命科學暨醫學院 - 分子與細胞生物研究所
Institute of Molecular and Cellular Biology
論文出版年： 2023
畢業學年度： 112
語文別： 英文
論文頁數： 148
中文關鍵詞： 酪胺酸激酶抑制劑 、受體的降解
外文關鍵詞： Tyrosine Kinase Inhibitor, Receptor degradation
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    • 超過50%的NSCLC的EGFR因為有異常的過度表達，而成為肺部腫瘤發生的主要因素，這也通常與較差的預後有相關性。酪胺酸激酶抑制劑 (TKI) 常被用於抑制EGFR的訊號傳導，但患者在接受治療以後更容易出現EGFR的二次突變，進而產生後天抗藥性，也可能導致包括c-MET在內的不同酪胺酸激酶受體的擴增。於是，TKI療效的降低成為對抗NSCLC腫瘤的一大挑戰。在本篇論文，我們鑑定了WDR4 (Cullin4泛素化家族的E3連接酶的底物接頭蛋白)介導PTPN23 (一種ESCRT相關蛋白) 的泛素化和降解。PTPN23的缺失會降低EGFR和c-MET受體的降解，進而導致EGFR和c-MET調節的下游訊號路徑被長期激活，促進NSCLC的細胞增殖、幹細胞特性和癌症轉移等能力。我們發現WDR4的缺失會促進野生型/突變型EGFR和c-MET受體的降解。此外，WDR4和PTPN23的表現呈現負相關，並與不良預後有關。透過胜肽破壞WDR4和PTPN23的相互作用可以防止WDR4介導的PTPN23泛素化和降解，增強EGFR和c-MET受體的降解，從而降低下游信號通路的激活，抑制NSCLC的癌症進展。不止如此，增加突變型EGFR受體的降解，也有助於克服TKI的抗藥性。因此，我們目前的研究確定了WDR4/PTPN23軸在調節EGFR和c-MET受體降解中的核心作用，將其作為潛在的治療標的，進行進一步研究。同時，我們的研究還確定了一種透過靶向肺癌信號傳導受體的降解機制，來抑制肺部腫瘤生成的新方法。

    More than 50% of NSCLC exhibit aberrant overexpression of EGFR, which acts as a major factor in lung tumorigenesis and is frequently associated with worse prognosis. Tyrosine kinase inhibitors are used to suppress the EGFR signaling but after treatment, patients are more prone to develop secondary mutations in EGFR which causes the development of acquired resistance through amplification of different tyrosine kinase receptors including c-MET. The reduction in TKI effectiveness poses a major challenge to counter tumor progression in NSCLC. Here, we identified WDR4, the substrate adaptor protein of the Cullin4-based E3 ligase, mediates ubiquitination and degradation of PTPN23, an ESCRT-associated protein. PTPN23 depletion results in impairment of EGFR and c-MET receptor degradation that causes prolonged activation of EGFR and c-MET regulated downstream signaling pathways which promotes cell proliferation, stemness, and metastasis in NSCLC. We have identified that WDR4 depletion promotes wild-type/mutant EGFR and c-MET receptor degradation. Furthermore, WDR4 and PTPN23 expressions are negatively correlated with each other and associated with poor prognosis. Moreover, disrupting WDR4 and PTPN23 interaction by a peptide prevents WDR4-mediated PTPN23 ubiquitination and degradation which leads to the enhancement of EGFR and c-MET receptor degradation thus attenuating the activation of downstream signaling pathways thus inhibiting the progression of NSCLC. An increase in the degradation of mutant EGFR help to overcome TKI resistance. Hence, our present study identifies a central role of the WDR4/PTPN23 axis in regulating EGFR and c-MET receptor degradation which can be further explored as a potential therapeutic target. Furthermore, our study also identifies a novel approach to target signaling receptors in lung cancer by targeting their degradation mechanism to inhibit lung tumorigenesis.

    Acknowledgement i Abstract iv List of Abbreviation vi Chapter 1: Introduction 1 1-1 Lung cancer 1 1-1-2 Factors causing lung cancer 1 1-1-3 Current Treatment opportunities and challenges 3 1-1-4 Significance of EGFR (Epidermal Growth Factor Receptor) in lung tumorigenesis 4 1-1-5 Mechanism of EGFR activation 5 1-1-6 Mechanism of EGFR sorting process 6 1-1-7 Current treatment approaches for EGFR 7 1-1-8 Current strategy to overcome resistance 8 1-1-9 Significance of HGFR (Hepatocyte Growth Factor Receptor) in lung tumorigenesis 8 1-1-10 Mechanism of c-MET activation 9 1-1-11 Treatment approaches for c-MET receptor 10 1-1-12 PI3K/Akt pathway 11 1-1-13 ERK (Extracellular signal-regulated kinases) pathway 12 1-2 Ubiquitination 13 1-2-1 Basic description of the ubiquitination process 13 1-2-1 Mechanism of action 15 1-2-3 Types of Ubiquitination 15 1-2-4 Function of different ubiquitin chains 16 1-2-5 E3 ligase 17 1-2-6 Cullin-RING Family 18 1-2-7 WDR4 19 1-3 ESCRT (Endosomal Complex Required for Transport) Complex 21 1-3-1 General description of ESCRT complex 21 1-3-1 Sub-complexes of ESCRTs 22 1-3-1-1 ESCRT-0 22 1-3-1-2 ESCRT-I 22 1-3-1-3 ESCRT- II 23 1-3-1-4 ESCRT-III 23 1-3-1-5 VPS4 complex 24 1-3-2 Function of the ESCRT complex 24 1-3-3 PTPN23 25 1-4 Targeted protein degradation 27 1-4-1 Targeted protein degradation via proteasome 28 1-4-2 Targeted protein degradation via Lysosomes 28 1-4-3 Benefits and Challenges of Targeted Protein Degradation 29 1-5 Key questions 29 Chapter 2: Material and Methods 31 2-1 Cell Culture and transfection 31 2-2 Plasmid 31 2-3 Antibodies and reagents 31 2-4 RNA interference, Lentivirus production, and transfection 31 2-5 Generation of CRISPR knockout (KO) cell line 32 2-6 Western blot 32 2-7 Ubiquitination Assay 33 2-8 Immunoprecipitation Assay 34 2-9 Purification of His/ Flag-PTPN23 and GST-WDR4 34 2-10 In-Vitro binding assay 34 2-11 EGFR and c-MET degradation assay 35 2-12 Quantitative real-time PCR (RT-qPCR) 35 2-13 Proximity ligation assay 36 2-14 EGF internalization assay 36 2-15 Immunofluorescence Assay 36 2-16 Cell viability assay 37 2-17 BrdU Cell Proliferation Assay 37 2-18 Tumor sphere formation assay 37 2-19 Immunohistochemsitry (IHC) 38 2-20 Animal experiments 38 2-21 Generation of cell-penetrating Bro40 peptide 39 2-22 Statistic 40 2-23 Study approval 40 Chapter 3: Results 41 PTPN23 can be a potential substrate for Cul4-based WDR4 E3 ligase 41 WDR4 negatively regulates PTPN23 expression 43 WDR4 can degrade PTPN23 through proteasome 44 WDR4/PTPN23 axis regulates EGFR (Wild-type/ Mutant) degradation 44 WDR4/PTPN23 axis regulates EGFR degradation through lysosomes 46 WDR4/PTPN23 axis attenuates EGFR downstream signaling pathways 47 WDR4/PTPN23 axis regulates c-MET degradation through lysosomes 47 WDR4/PTPN23 axis attenuates c-MET downstream signaling pathways 48 WDR4/PTPN23 axis induce pro-tumorigenic role in NSCLC 49 WDR4/PTPN23 axis predicts poor prognosis of the patients 50 WDR4 interacts with PTPN23 through Bro1 domain 50 Overexpression of the GFP-Bro40 can prevent WDR4-mediated PTPN23 ubiquitination and proteasomal degradation 52 Overexpression of the GFP-Bro40 can promote EGFR/c-MET degradation 52 Overexpression of GFP-Bro40 can attenuate EGFR and c-MET signaling pathways 53 Overexpression of GFP-Bro40 can suppress WDR4/PTPN23 oncogenic effect 54 Overexpression of GFP-Bro40 can suppress lung metastasis in TKI-resistant lung cancer cells 55 Overexpression of GFP-Bro40 can reduce tumor growth in TKI-resistant lung cancer cells 55 Overexpression of purified GFP-Bro40 peptide can reduce tumor growth in TKI-resistant cancer cells 56 Chapter 4: Discussion 58 Chapter 5: References 64 Chapter 6: Figures 74 Chapter 7: Appendix figures 132 Chapter 8: Table 144

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