- 學位論文
以基因轉殖小鼠模式研究BCL2過度表現對於肺癌形成及藥物治療的影響
The role of BCL-2 overexpression in lung tumorigenesis and drug treatments in murine models of lung cancer
摘要
肺癌是全世界最普遍與致命的癌症。在非小細胞肺癌(NSCLC)中，表皮生長受體(EGFR)的突變是誘發肺癌的主要原因之一。其中一種超過40%的肺癌病患均有的常見點突變便是EGFR L858R，不過EGFR L858R已經能被初代的標靶藥物給有效地抑制了。不幸地，有60%的EGFR L858R肺癌病患都會產生EGFR T790M-L858R (TL)的二次突變復發。二次突變不但對先前的標靶藥物產生了後天抗藥性，同時也讓肺癌患者比先前更加地惡化。因此研究者們又開發出了新一代的標靶藥物，像是能治療EGFR T790M-L858R的AZD9291 (osimertinib)。另外，有些EGFR突變的肺癌病患也出現了抗細胞凋亡的BCL-2蛋白過度表現的現象。為了瞭解BCL-2的過度表現是否會對腫瘤形成與EGFR的標靶療法產生影響，於是我們的研究便使用了包含BCL-2過度表現的EGFR T790M-L858R (TLB)小鼠模型來探討BCL-2的過度表現在肺腫瘤形成中所扮演的角色，以及是否會對AZD9291的單獨使用或與BCL-2抑制劑的ABT-199結合使用產生標靶藥物抗性。在我們研究中的肺癌轉基因小鼠模型會服用doxycycline來誘導腫瘤形成，以便探討肺癌小鼠的腫瘤發展、存活率和藥物抗性。透過核磁共振造影(MRI)在治療前後來檢驗小鼠的肺部。而從小鼠身上取得的肺部樣本則藉由蘇木精與伊紅染色法(H&E stain)來做組織學的檢驗，也藉由西方墨點法(Western blot)來研究訊號傳遞路徑。最後，我們的數據顯示，BCL-2的過度表現在含有EGFR T790M-L858R突變的非小細胞肺癌小鼠模型中，扮演了促進腫瘤生成、降低存活率和減少AZD9291單獨使用的敏感性。帶有BCL-2過度表現的EGFR TL小鼠有25.2週的存活率，比沒帶有BCL-2過度表現的EGFR TL小鼠的29.9週存活率來得短。在藥物治療方面，BCL-2在EGFR TL的過度表現會對AZD9291的單獨使用減少20%的敏感性，而AZD9291與ABT-199的組合使用則又比AZD9291在帶有BCL-2過度表現的EGFR TL單獨使用上少了20%的敏感性。所以，我們不只表示BCL-2在EGFR TL中的過度表現會減少AZD9291的敏感性，同時也偶然地發現加入ABT-199的組合療法會降低AZD9291的敏感性。期望我們的研究未來將能對肺癌的研究與治療有所貢獻。
並列摘要
Lung cancer is a common and fatal cancer worldwide. In non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) mutant is one of the common oncogenes found in lung cancer. One of the common point mutants in over 40% of patients is EGFR L858R. It can be effectively inhibited with the early generation targeted drugs. Unfortunately, EGFR T790M-L858R (TL) emerges in 60% of EGFR L858R patients. Not only does the secondary mutant acquire resistance to previously targeted drugs easily, but also aggravates in lung cancer patients. Therefore, researchers have developed next generation target drugs, such as AZD9291 (osimertinib) to effectively treat EGFR T790M-L858R mutant. In addition, some lung cancer patients with EGFR mutations also show over-expressions of anti-apoptotic BCL-2 protein. To understand whether BCL-2 overexpression is a crucial factor in tumorigenesis in EGFR-mutated lung cancer, we investigated the role of BCL-2 overexpression in lung tumorigenesis and targeted drug resistance for AZD9291 alone or combined with a BCL-2 inhibitor (ABT-199) in a mouse model harboring EGFR T790M-L858R with BCL-2 overexpression (TLB). The lung cancer transgenic mouse model in our study were administrated with doxycycline to induce tumorigenesis for investigating tumor development, survival rate, and drug resistance. Magnetic resonance imaging (MRI) was done to compare the before and after treatment. Lung samples received from mice were analyzed through hematoxylin and eosin (H&E) stain for histology, and Western blots for the signal pathway. Finally, our data demonstrated that BCL-2 overexpression plays an important role in NSCLC harboring EGFR T790M-L858R mutant in promoting tumorigenesis, decreasing the survival rate and reducing drug sensitivity for AZD9291 alone. The survival rate of EGFR TL mice is 25.2 weeks which is less than the 29.9 weeks in the models without BCL-2 overexpression. In drug treatment, BCL-2 overexpression in EGFR TL reduced 20% sensitivity on AZD9291 alone. Meanwhile, AZD9291 combined with ABT-199 further reduced 20% sensitivity. Therefore, we not only demonstrated BCL-2 overexpression in EGFR TL can reduce sensitivity of AZD9291, but also discovered that AZD9291 combined with ABT-199 would decrease drug efficacy. We expect our study will contribute to researches and therapies for lung cancer in the future.