尋找第二代EGFR抑制劑的新作用標的與其作用機制之探討

Abstract

先前的研究指出表皮生長因子受體(Epidermal Growth Factor Receptor)基因的異常表現，在許多的癌症發展及惡化過程中扮演著重要的角色，其中包含肺癌、大腸直腸癌等。在臨床治療上，酪胺酸激酶抑制劑（Tyrosine Kinase Inhibitor, TKI），如Iressa (艾瑞莎)及Tarceva (得舒緩)，已被廣泛地運用於治療表皮生長因子突變的癌症患者；然而，許多的臨床數據顯示，大部分患者在使用此類藥物後，最終都會產生抗藥性，使得病情急速惡化，終究無法逃脫癌症復發對生命產生的威脅。引發抗藥性的機制，其中以表皮生長因子產生突變最為常見，此突變會大幅降低藥物與作用標的間的親和性，使得藥物失去作用而無法有效抑制腫瘤的生長與存活。為了克服這樣的問題，發展出第二代酪胺酸激酶抑制劑，Afatinib (妥復克)，此藥物適用於治療因上皮生長因子受體（EGFR）突變之非小細胞肺癌（NSCLC）。相較於前一代抗EGFR藥物，妥復克可以透過與酪胺酸激酶反應產生不可逆的化學共價鍵結，以達到抑制腫瘤生長與延長病人存活的效果。在此研究中，發現妥復克亦可抑制EGFR低表現的乳癌及肺癌細胞株(MCF7, MDA-MB-231, CL 1-0)生長與存活，暗示此藥物在細胞中可作用其他的標的蛋白質，以抑制癌細胞的生存。為了鑑尋這些重要的標的蛋白質，我們利用能專一辨識妥復克的抗體，結合蛋白質免疫共沉澱的技術，發現在MDA-MB-231乳癌細胞中， 有183個特異性蛋白質可與妥復克結合，其中分數最高的是一個可與妥復克結合蛋白(ABP)。利用免疫共沉澱法證實妥復克可與ABP於細胞內產生共價鍵結合。進一步地，從代謝體實驗結果顯示妥復克可以干擾ABP調控參與的嘧啶新生相關的代謝途徑，進而影響中間產物的變化。最後，我們利用Kaplan-Meier plotter與乳癌的組織切片分析，探討ABP與乳癌發展的關聯性。從結果中顯示，ABP的表現量與乳癌的惡化存在著正向的關係。未來，我們將更進一步探討妥復克是否可用於治療因ABP過度表達所引發的癌症或ABP是否可作為未來發展藥物的治療標的物。

Epidermal growth factor receptor (EGFR) has shown to play an important role in cancer development and progression, including lung and colorectal cancer. In clinic, tyrosine kinase inhibitors (TKIs, e.g., Iressa and Tarceva) against EGFR are commonly used for the cancer treatment. However, many cancer patients eventually acquire resistance to the anti-EGFR treatment and the etiology for the drug resistance is frequently due to EGFR mutations that reduce the drug binding affinity. To solve these problems, a second-generation TKI, afatinib (Gilotrif), has been developed and used as a covalent, irreversible inhibitor to treat those patients who fail the first-line treatment. Unexpectedly, I found that afatinib also could inhibit the growth of breast cancer MCF7, MDA-MB-231 and non-small cell lung carcinoma CL 1-0 cells which have low EGFR expression. The results suggest that afatinib may inhibit other important targets in addition to EGFR. To isolate an important target of afatinib, we used an anti-afatinib antibody to pull down afatinib-targeted proteins in MDA-MB-231 cells. The results from LC-MS/MS analysis showed that 183 proteins were identified. Among them, a novel enzyme (Afatinib-binding protein, ABP) received our most attention as one of the potential afatinib targets due to its highest score. Moreover, the co-immunoprecipitation results showed that afatinib could covalently interact with ABP in human cancer cells. In addition, the metabolomics analysis showed that afatinib could interrupt the catalytic reaction of ABP, leading to altering the levels of its intermediates. Finally, we employed Kaplan–Meier analysis and breast tissue microarray to link the expression of ABP with breast cancer progression. The results showed that ABP levels were correlated with poor prognosis and up-regulated in advanced breast tumour tissue. The future works we will further address are to examine whether afatinib can reduce ABP-induced cancer cell proliferation and progression.