臺灣博碩士論文加值系統

KRAS 可以活化細胞內不同的訊號傳遞路徑進而促使細胞的存活以及增生。過去 有許多報導發現 KRAS 的突變對於非小細胞肺癌的形成以及進程扮演很重要的角 色。在非小細胞肺癌當中，第 12 號氨基酸位點甘安酸(glycine) 轉變為半胱胺酸 (cysteine)的突變，大約佔了全部突變的 42%，因此許多的研究利用這樣的特性，發 展出針對此種突變的共價鍵抑制劑。AMG510 為目前眾多 KRAS 共價鍵抑制劑中， 最具有潛力的其中一個藥物。然而我發現即使是同樣帶有 G12C 突變的細胞株， AMG510 能夠抑制生長的效果卻有著很大的差異，因此我認為 AMG510 可能有著 除了 KRAS 以外的作用標的來發揮藥效。在此篇研究中，我利用抗體以及質譜儀 的方式找到了 AMG510 的新作用標的蛋白(ICC1B)。我發現 ICC1B 的蛋白質表現 量多寡會影響細胞對於藥物的感受性。進一步研究發現，AMG510 會透過和 ICC1B 的結合進而引發細胞凋亡。最後，我發現在對 AMG510 有抗性的細胞中，ICC1B 蛋白質表現量有顯著的下降，這可能代表著藥物作用標的蛋白質表現量減低會使 細胞對於藥物產生抗藥性。綜合以上結果，顯示 AMG510 在肺癌細胞當中有不只 一個作用標的蛋白。此篇研究對於往後使用 AMG510 來治療癌症提供了更多的資 訊，提升了癌症治療的精準性。

KRAS is a small G protein that can activate several downstream signaling pathways to promote cell survival and proliferation. The mutation of KRAS has been reported to play a role in cancer development and progression including non-small cell lung cancer (NSCLC). In NSCLC, KRAS G12C mutation accounts for approximately 42% of all mutations. The cysteine residue of this mutant has been exploited to develop covalent inhibitors. AMG510 is one of the most promising KRAS covalent inhibitors and is undergoing phase III clinical trials. However, I found that AMG510 had a differential effect on inhibiting cell viability of KRASG12C cells. The results suggest that AMG510 may have other important targets to exert the therapeutic efficacy. To isolate the important targets, we used an anti-AMG510 antibody to pull down AMG510-targeted proteins in KRASG12C cells. The results from LC-MS/MS analysis showed that 13 proteins were exclusively found in AMG510-sensitive LU65 and H358 cells. Among them, a novel protein, ICC1B, caught our attention because of the high scores and the fact that it participates in lung cancer progression. Moreover, the immunoprecipitation results showed that AMG510 could covalently bind to ICC1B. In addition, overexpression or knockdown of ICC1B affected drug sensitivity of the cells. We also found that AMG510 could induce cell apoptosis through targeting ICC1B by using flow cytometry. Finally, we generated the AMG510-resistant cells and found that ICC1B protein expression levels were decreased. The results indicate that down-regulation of AMG510-targeted proteins might contribute to a resistant phenotype. Our results demonstrate that AMG510 has its unique target profiles in lung cancer cells. The study provides more insights to improve AMG510 usages, which will improve precision medicine in cancer treatment.

口試委員會審定書 I
中文摘要 II
Abstract III
Table of contents V
Introduction 1
Non-small cell lung cancer 1
Kristen Rat Sarcoma viral oncogene –KRAS 2
AMG510 (Sotorasib) 3
Actin-related protein 2/3 complex (ARP 2/3 complex) 4
Research motivation 5
Materials and Methods 7
Results 20
Analysis of the cytotoxicity effect of AMG510 on different lung cancer cells 20
Examination of AMG510 effects on the migration and invasion of H358, LU65 and LU99 lung cancer cells 21
Examination of the importance of KRASG12C in AMG510-suppressed lung cancer cell growth and motility 22
Establishment of an anti-AMG510 antibody and characterization of the specificity of the anti-AMG510 antibody 23
Identification of novel AMG510-targeted proteins in LU65, LU99, and H358 cells using immunoprecipitation and LC-MS/MS analysis 24
Identification of ICC1B as a novel target of AMG510 25
Delineation of ICC1B role in lung cancer cell growth and drug sensitivity 27
Examination of AMG510 effects on the cell cycle of ICC1B -silencing LU65 cells 28
Analysis of the correlation between ICC1B expression levels and an AMG510-resistant phenotype 28
Discussion 30
Figures 34
Figure 1. Cytotoxicity effect of AMG510 on different lung cancer cells. 34
Figure 2. Inhibitory effects of AMG510 on the viability and motility of H358, LU65, and LU99 lung cancer cells. 37
Figure 3. Examination of the effects of KRAS silencing on AMG510-suppressed growth, viability and invasion of LU65 and H358 cells. 39
Figure 4. Examination of anti-AMG510 antibody specificity on AMG510-labelled proteins in lung cancer LU99 cells. 41
Figure 5. Identification of novel AMG510-targeted proteins in LU65, LU99, and H358 cells using immunoprecipitation and LC-MS/MS analysis. 43
Figure 6. Analysis of ICC1B to be a novel target of AMG510 in lung cancer cells. 45
Figure 7. The presence or absence of ICC1B protein partially determines the drug efficacy. 47
Figure 8. ICC1B is crucial for AMG510 to induce cell apoptosis in LU65 cells. 49
Figure 9. Down-regulation of ICC1B correlates with the resistant phenotype. 51
Figure 10. Schematic model of the mechanism of actions of AMG510 on KRASG12C lung cancer cells. 53
References 54

Bray, F., et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2018. 68(6): p. 394-424.
Zheng, M., Classification and Pathology of Lung Cancer. Surg Oncol Clin N Am, 2016. 25(3): p. 447-68.
Zappa, C. and S.A. Mousa, Non-small cell lung cancer: current treatment and future advances. Transl Lung Cancer Res, 2016. 5(3): p. 288-300.
Kohno, T., et al., Beyond ALK-RET, ROS1 and other oncogene fusions in lung cancer. Transl Lung Cancer Res, 2015. 4(2): p. 156-64.
Chan, B.A. and B.G. Hughes, Targeted therapy for non-small cell lung cancer: current standards and the promise of the future. Transl Lung Cancer Res, 2015. 4(1): p. 36-54.
Roman, M., et al., KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target. Mol Cancer, 2018. 17(1): p. 33.
Ferrer, I., et al., KRAS-Mutant non-small cell lung cancer: From biology to therapy. Lung Cancer, 2018. 124: p. 53-64.
Nagasaka, M., et al., KRAS G12C Game of Thrones, which direct KRAS inhibitor will claim the iron throne? Cancer Treat Rev, 2020. 84: p. 101974.
Karachaliou, N., et al., KRAS mutations in lung cancer. Clin Lung Cancer, 2013. 14(3): p. 205-14.
Liu, P., Y. Wang, and X. Li, Targeting the untargetable KRAS in cancer therapy. Acta Pharm Sin B, 2019. 9(5): p. 871-879.
Khan, I., J.M. Rhett, and J.P. O'Bryan, Therapeutic targeting of RAS: New hope for drugging the "undruggable". Biochim Biophys Acta Mol Cell Res, 2020. 1867(2): p. 118570.
Cox, A.D., et al., Drugging the undruggable RAS: Mission possible? Nat Rev Drug Discov, 2014. 13(11): p. 828-51.
Ryan, M.B. and R.B. Corcoran, Therapeutic strategies to target RAS-mutant cancers. Nat Rev Clin Oncol, 2018. 15(11): p. 709-720.
Ostrem, J.M., et al., K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature, 2013. 503(7477): p. 548-51.
Janes, M.R., et al., Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor. Cell, 2018. 172(3): p. 578-589 e17.
Hallin, J., et al., The KRAS(G12C) Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer Discov, 2020. 10(1): p. 54-71.
Canon, J., et al., The clinical KRAS (G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature, 2019. 575(7781): p. 217-223.
Hong, D.S., et al., KRAS(G12C) Inhibition with Sotorasib in Advanced Solid Tumors. N Engl J Med, 2020. 383(13): p. 1207-1217.
Rotty, J.D., C. Wu, and J.E. Bear, New insights into the regulation and cellular functions of the ARP2/3 complex. Nat Rev Mol Cell Biol, 2013. 14(1): p. 7-12.
Molinie, N., et al., Cortical branched actin determines cell cycle progression. Cell Res, 2019. 29(6): p. 432-445.
Robinson, R.C., et al., Crystal structure of Arp2/3 complex. Science, 2001. 294(5547): p. 1679-84.
Zimmet, A., et al., Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism. Sci Adv, 2020. 6(23).
Rodnick-Smith, M., et al., Identification of an ATP-controlled allosteric switch that controls actin filament nucleation by Arp2/3 complex. Nat Commun, 2016. 7: p. 12226.
Krause, M. and A. Gautreau, Steering cell migration: lamellipodium dynamics and the regulation of directional persistence. Nat Rev Mol Cell Biol, 2014. 15(9): p. 577-90.
Rohatgi, R., H.Y. Ho, and M.W. Kirschner, Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate. J Cell Biol, 2000. 150(6): p. 1299-310.
Sossey-Alaoui, K., et al., WAVE3-mediated cell migration and lamellipodia formation are regulated downstream of phosphatidylinositol 3-kinase. J Biol Chem, 2005. 280(23): p. 21748-55.
Lorenz, M., et al., Imaging sites of N-wasp activity in lamellipodia and invadopodia of carcinoma cells. Curr Biol, 2004. 14(8): p. 697-703.
Iwaya, K., K. Norio, and K. Mukai, Coexpression of Arp2 and WAVE2 predicts poor outcome in invasive breast carcinoma. Mod Pathol, 2007. 20(3): p. 339-43.
Semba, S., et al., Coexpression of actin-related protein 2 and Wiskott-Aldrich syndrome family verproline-homologous protein 2 in adenocarcinoma of the lung. Clin Cancer Res, 2006. 12(8): p. 2449-54.
Iwaya, K., et al., Correlation between liver metastasis of the colocalization of actin-related protein 2 and 3 complex and WAVE2 in colorectal carcinoma. Cancer Sci, 2007. 98(7): p. 992-9.
Lin, A., et al., Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials. Sci Transl Med, 2019. 11(509).
Lin, A., et al., CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials. Elife, 2017. 6.
Tu, H.F., et al., Afatinib exerts immunomodulatory effects by targeting the pyrimidine biosynthesis enzyme CAD. Cancer Res, 2021.
Mestres, J., et al., Data completeness--the Achilles heel of drug-target networks. Nat Biotechnol, 2008. 26(9): p. 983-4.
Adachi, Y., et al., Epithelial-to-Mesenchymal Transition is a Cause of Both Intrinsic and Acquired Resistance to KRAS G12C Inhibitor in KRAS G12C-Mutant Non-Small Cell Lung Cancer. Clin Cancer Res, 2020. 26(22): p. 5962-5973.
Gyorffy, B., et al., Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PLoS One, 2013. 8(12): p. e82241.
Ahearn, I., M. Zhou, and M.R. Philips, Posttranslational Modifications of RAS Proteins. Cold Spring Harb Perspect Med, 2018. 8(11).
Berndt, N. and S.M. Sebti, Measurement of protein farnesylation and geranylgeranylation in vitro, in cultured cells and in biopsies, and the effects of prenyl transferase inhibitors. Nat Protoc, 2011. 6(11): p. 1775-91.
Yu, C.H., et al., Antibody-assisted target identification reveals afatinib, an EGFR covalent inhibitor, down-regulating ribonucleotide reductase. Oncotarget, 2018. 9(30): p. 21512-21529.
Cascone, T., M.P. Morelli, and F. Ciardiello, Small molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer. Ann Oncol, 2006. 17 Suppl 2: p. ii46-48.
Yuan, M., et al., The emerging treatment landscape of targeted therapy in non-small-cell lung cancer. Signal Transduct Target Ther, 2019. 4: p. 61.
Wang, J.C., Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol, 2002. 3(6): p. 430-40.
McClendon, A.K. and N. Osheroff, DNA topoisomerase II, genotoxicity, and cancer. Mutat Res, 2007. 623(1-2): p. 83-97.
Bejarano, L., M.J.C. Jordao, and J.A. Joyce, Therapeutic Targeting of the Tumor Microenvironment. Cancer Discov, 2021. 11(4): p. 933-959.
Whiteside, T.L., The tumor microenvironment and its role in promoting tumor growth. Oncogene, 2008. 27(45): p. 5904-12.
Tran, D.T., et al., Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo. Nat Commun, 2015. 6: p. 10098.