探討新穎EGFR 抑制劑在非小細胞肺癌的療效及克服抗藥性作用

Abstract

肺癌是現在全世界發病率與致死率最高的癌症之一，其中非小細胞肺癌(簡稱NSCLC)是最常見的 肺癌類型，帶有突變與異常表達的上皮生長因子受體(簡稱EGFR)是目前標靶藥物用於治療NSCLC 的 重要標的與依據，上皮生長因子受體-酪胺酸激酶抑制劑(簡稱EGFR-TKIs)包括Gefitinib、Erlotinib 以 及最近2013 年7 月通過美國FDA 的Afatinib，使用於臨床治療NSCLC，EGFR-TKI 藥物主要藉由抑 制EGFR 的酪胺酸激酶活性，阻斷肺癌細胞生長與造成癌細胞凋亡，病人開始使用這些標靶藥物時有 很好的療效，但可惜大多數的NSCLC 在治療一段時間後藥效就會大幅地降低並產生抗藥性。目前已 知EGFR 在第858 胺基酸位置由白胺酸突變成精胺酸時(L858R)，EGFR-TKI 藥物對此種突變EGFR 具 有結合能力對藥物有敏感性，然而癌細胞的EGFR 在第790 胺基酸位點產生第二個突變時，由蘇胺酸 變成蛋胺酸(T790M)，則會產生抗藥性，這是目前臨床治療NSCLC 病患最大的難題，如能改善此類藥 物的治療效果與克服抗藥性問題，將對肺癌病人將有重大的助益。我們篩選出一種新穎的Gefitinib 衍 生物稱為SP101，SP101 具有抑制EGFR 酪胺酸激酶的活性，雖然發現SP101 抑制EGFR 的激酶活 性沒有比Gefitinib 強，但卻意外發現SP101 在抑制抗藥性的肺癌細胞生長與誘發細胞凋亡的能力比 Gefitinib 好，我們推測SP101 除了具有抑制EGFR 活性之外，也同時經由其他非EGFR 路徑抑制肺 癌細胞生長，而對產生抗藥性的肺癌具有療效，因此將進一步比較SP101 與現行EGFR-TKI 藥物 (Gefitinib、Erotinib 及Afatinib)在治療NSCLC 的效果，以及是否能克服抗藥性的問題。 在此計畫中，我們結合化學合成、蛋白激酶分析、電腦分子模擬、癌症標靶藥物、癌症生物學、 細胞及動物實驗，探討新穎EGFR 抑制劑在NSCLC 的療效及抗藥性的問題，包括四個主要研究目標: (1) 比較SP101 與現行EGFR-TKI 藥物對正常肺細胞、藥物敏感性NSCLC 細胞和抗藥性的NSCLC 細胞間，在抗癌活性是否具有選擇性的差異。(2) 比較SP101 與現行EGFR-TKI 藥物在抑制EGFR 的活性與其下游分子(包括AKT、ERK及STAT)的作用，以及誘發細胞凋亡與抑制癌細胞生長的能力。 (3) 探討p53、Survivin 及Securin 的表現對SP101 與EGFR-TKI 藥物在治療NSCLC 所扮演的角色、 (4) 利用動物模式包括異體移植人類抗藥性EGFR (T790M/L858R)的肺癌裸鼠模式與EGFR (T790M/L858R)基因轉殖肺癌小鼠模式，驗證SP101 在抗藥性NSCLC 的療效。 本研究最終目的期望開創新型有效治療 NSCLC 的藥物與策略，能克服EGFR-TKI 藥物的抗藥性 作用，改善這些藥物治療肺癌病人時所產生的抗藥性問題。

Lung cancer has become one of the leading causes of cancer-related morbidity and mortality in the world. Non-small cell lung carcinoma (NSCLC) is the most common type of all lung cancer cases. Mutations and abnormal expression of epidermal growth factor receptor (EGFR) have been used as an important target and guideline for NSCLC therapy. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), including Gefitinib, Erotinib and recently approved by US FDA on Afatinib (July, 2013) are clinically used for treating NSCLC. The action of EGFR-TKIs is inhibiting EGFR tyrosine kinase activity to limit cell growth and to cause apoptosis in cancer cells. EGFR-TKIs have excellent therapeutic effect in early; unfortunately, the effect of those drugs was largely reduced and acquired drug resistance after a period therapy in NSCLC. A mutation that substitutes an arginine for leucine at codon 858 (L858R) is the most common EGFR activating mutation, which increased the sensitivity of lung tumor to EGFR-TKIs. However, EGFR secondary point mutation that substitutes methionine for threonine at position 790 (T790M) of EGFR, which was identified in NSCLC patients acquired resistance. NSCLC patients showed drug resistance is the most challenge in clinical therapy. Development of novel drugs and strategies to improve efficacy and the conquest of drug resistance problems are highly desired and will be benefit to lung cancer patients. We have screened a novel Gefitinib derivative, which named as SP101. SP101 could inhibit EGFR tyrosine kinase activity. Interestingly, SP101 exerted more effective on the cell growth inhibition and apoptotic induction than Gefitinib in the Gefitinib-resistant lung cancer cells, although it was not better on the inhibition of EGFR kinase activity than Gefitinib. We propose that SP101 may also mediate EGFR-independent pathways to inhibit cancer cell growth, which can overcome the drug resistance of EGFR-TKIs. Accordingly, we will be further compared SP101 and EGFR-TKIs (Gefitinib, Erotinib and Afatinib) on therapeutic efficacy and whether can overcome the drug resistance in NSCLC. In this project, we integrate chemical synthesis, protein kinase analysis, computational molecular modeling, cancer targeting drugs, cancer biology, cellular and animal experiments to investigate the novel EGFR inhibitors on the therapeutic efficacy and the overcoming drug resistance in NSCLC. This plan includes four specific aims: 1) To compare SP101 and clinical EGFR-TKIs on the selectivity and anticancer activities among normal lung cells, drug sensitive NSCLC cells and resistant NSCLC cells. 2) To compare SP101 and clinical EGFR-TKIs on the blockade of EGFR activity and its downstream proteins (including AKT, ERK and STAT) to induce cancer cell apoptosis and growth inhibition. 3) To investigate the role of p53, Survivin and Securin in the EGFR-TKI resistant NSCLC by treatment with SP101 and EGFR-TKIs. 4) Using animal models including xenograft T790M-L858R lung cancer of nude mice and transgenic T790M-L858R lung cancer mice to verify the efficacy of SP101 in the drug resistant NSCLC. The final goal of this project is developing novel effective drugs and strategies to overcome EGFR-TKI drug resistance and improve the efficacy of such drugs in NSCLC therapy.