非小細胞肺癌中小分子藥物調控致癌蛋白質降解之機轉探討

Abstract

研究表皮生長因子受體酪胺酸抑制劑的抗藥性是肺癌中最主要的研究方向，因此我們欲找尋新的抗癌藥物以及研發新的聯合治療模式來克服抗藥性。首先在本論文中，我們會探討一個以細胞為基底的高通量藥物篩選模式，尋找能夠抑制非小細胞肺癌生長的小分子藥物，藉由表型篩選，尋找目標蛋白以及結構活性相關分析，我們發現一個以furanonaphthoquinone結構為主的小分子AS4583不只對於酪胺酸抑制劑敏感型與抗藥型細胞具有潛在活性(半抑制濃度為77nM)，甚至是異體移植的老鼠實驗也有良好效果。多層次蛋白質體分析與機轉研究顯示AS4583抑制細胞週期，降低minichromosomal maintenance protein (MCM) 蛋白複合物表現，進而干擾DNA複製。經由結構活性相關分析與電腦模擬AS4583與MCM接合模型，我們找到RJ-LC-07-48更能夠有效抑制抗藥性肺癌細胞H1975 (半抑制濃度為17nM) 與老鼠腫瘤的生長。第二部分中，最近的研究顯示表皮生長因子受體(EGFR) C797S突變賦予癌細胞對於T790M酪胺酸抑制劑產生抗藥性。為了抗衡C797S突變，新一代酪胺酸抑制劑如brigatinib也被改變用途地視為肺癌新藥，但是在長期治療後產生的抗藥性卻是無法避免的。組織蛋白去乙醯酶抑制劑vorinostat擁有抗增生效果，但是對於vorinostat and brigatinib聯合治療在C797S/T790M/激活突變 (三突變)的肺腺癌中卻從未被評估過。我們的數據顯示聯合 vorinostat and brigatinib的治療在動物實驗中加強抑制EGFR-3M (L858R/T790M/C797S)腫瘤的效果，可能途徑是經由降解EGFR與磷酸化EGFR蛋白表現量。如果在細胞中降低熱休克伴隨蛋白70 (HSP70)相關的泛素連接酶STUB1會增加被vorinostat所調控的EGFR-3M蛋白量。 STUB1 與 HSP70 共同調控EGFR 泛素化作用而降解，此現象在突變型的EGFR(L858R, T790M, Del19, L858R/T790M, L858R/T790M/C797S)更為明顯。而在肺腺癌病人中，低表現 STUB1的病人相較於高表現量的病人則有較差的存活期。因此我們的研究顯示給予vorinostat 與brigatinib藉由引發依賴EGFR的癌細胞死亡來顯著地提高EGFR-3M (L858R/T790M/C797S)癌細胞對於EGFR酪胺酸抑制劑的敏感度。綜合上述，我們的結果顯示針對致癌蛋白(MCM與EGFR)降解的小分子藥物可以提供未來治療肺癌病人的新選擇。

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) resistance has been a major threat in lung cancer therapies that necessitates the development of new strategies to overcome this problem. Therefore, we identified new anti-cancer drug and develop novel combination therapy to overcome TKI resistance. First, we report here a cell-based high-throughput screen of a library of 2-milliom molecules for the molecules that inhibit the proliferation of non-small-cell lung cancer (NSCLC). Through the process of phenotypic screening, target deconvolution and structure-activity relationship (SAR) analysis, a compound of furanonaphthoquinone-based small molecule, AS4583, was identified which exhibited potent activity in TKI–sensitive and TKI–resistant NSCLC cells (IC50 77 nM) and in xenograft mice. Multilayered proteomic profiling and mechanistic studies revealed that AS4583 inhibited cell-cycle progression and reduced DNA replication by disrupting the formation of the minichromosomal maintenance protein (MCM) complex. Subsequent SAR study and in silico modeling of AS4583 in complex with MCM gave compound RJ-LC-07-48 which exhibited greater potency in drug-resistant NSCLC cells (H1975) with L858R/T790M EGFR mutations (IC50 17nM) and in mice with H1975 xenograft tumor. Moreover, recent reports demonstrated that the C797S mutation confers drug resistance to T790M-targeting EGFR TKIs in lung adenocarcinomas. To contend with the C797S mutation, a new generation of TKIs, including brigatinib, were repurposed for EGFR, but acquired resistance is unavoidable after prolonged treatment. The histone deacetylase inhibitor, vorinostat, possesses anti-proliferative effects but the combined therapeutic effect of vorinostat and brigatinib in C797S/T790M/activating mutations (triple-mutation)-harboring lung adenocarcinomas has not been assessed yet. Our data shows that the combined treatment of vorinostat and brigatinib produces an enhanced anti-tumor effect on xenografts with EGFR-3M (L858R/T790M/C797S) cells by downregulating the total and phosphorylated EGFR expression. Knockdown of the heat shock protein 70 (HSP70) chaperone-associated ubiquitin ligase, STUB1, increases the EGFR-3M level regulated by vorinostat. STUB1 and HSP70 mediates EGFR ubiquitination and degradation, with preferable modulation towards EGFR mutants (L858R, T790M, Del19, L858R/T790M, L858R/T790M/C797S). Low STUB1 expression had a significantly poorer overall survival than high STUB1 expression in patients with lung adenocarcinoma. Therefore, our findings suggest that treatment with vorinostat coupled with brigatinib significantly improved EGFR-TKI sensitivity in EGFR-3M (L858R/T790M/C797S) cells by inducing EGFR-dependent cell death. Collectively, our findings provided evidence of small molecular target oncoproteins, such as MCM and EGFR degradation that may be novel therapeutic strategies.