非小細胞肺癌之新藥開發與相關藥物作用標的之研究– 以GRC0321及Monocarboxylate transporter 4為例

Identify Novel Drug and Related Therapeutic Target in Non-Small Cell Lung Cancer - in terms of GRC0321 and Monocarboxylate transporter 4

郭廷群(Ting-Chun Kuo)

指導教授 ： 楊泮池 ； 陳璿宇

國立臺灣大學/醫學院/轉譯醫學博士學位學程/博士(2017年)

https://doi.org/10.6342/NTU201702037

摘要

肺癌至今仍然是全世界致死率最高的癌症之一。儘管藥物或是手術方面於近年皆有長足的進步，但在有效降低肺癌致死率的部分仍不盡理想。歸納造成抗癌藥物無法展現有效的抗癌效果及提供安全的藥物劑量之眾多原因可發現，大多與癌症細胞快速產生抗藥性及抗癌藥物對人體正常細胞產生嚴重的副作用有關。但近年來隨著精準醫學此一觀念在抗癌藥物研究領域之蓬勃發展，越來越多的肺癌診斷標記蛋白及藥物標的在肺癌細胞中被發現，也再次為此領域注入了新的希望與發展方向。
於此博士論文中，我們運用了隨機藥物篩選及機制導向藥物設計兩種策略成功的找到了兩個可做為肺癌藥物標的之蛋白，katanin蛋白及Monocarboxylate transporter 4 (MCT4)蛋白。同時我們亦針對其分別發展出能有效抑制肺癌細胞生長之抗癌藥物，GRC0321及MCT4中和抗體。此兩藥物不論是在細胞內或是動物體內之實驗模型中都展現了良好的抗癌效果及優異的安全性。在詳細的藥物抗癌機轉研究中，我們發現小分子藥物GRC0321是透過影響katanin蛋白來破壞癌症細胞微管的完整性。而後，此一現象將會進一步地造成癌症細胞G2/M細胞週期停滯，並導致癌症細胞走向由caspase-9 蛋白所主導的細胞凋亡途徑。關於MCT4中和抗體造成糖解作用依賴型肺癌細胞生長停滯的機轉部分，我們則發現此一抗體能有效的抑制癌症細胞將堆積於細胞內之多餘乳酸運送到細胞外之機制。此一抑制機制可以進一步的造成癌症細胞細胞內之pH值降低，並同時產生過量之活性氧化物質。而最後則一樣會造成癌症細胞走向由caspase-9 蛋白所主導的細胞凋亡途徑。此外，我們亦發現MCT4中和抗體在MCT4大量表性之肺癌細胞中能展現較好之抗癌能力。詳細研究後發現，因為此類肺癌細胞較依賴糖解作用來得到能量，且會透過Sp1蛋白參與之轉錄機制來調控MCT4蛋白之大量表現，且此現象即使在一般正常氧分壓環境仍可觀察到，因此其對MCT4中和抗體的敏感性較高。
根據目前之實驗結果數據，此兩種藥物具有極大的潛能進行後之藥物發展。但較差的水溶解性及不適當的免疫球蛋白種型可能會是造成日後發展瓶頸的兩大原因。因此，如何針對此兩大問題進行適當的解決及改善勢必是影響此兩種藥物後續能否成功發展成為臨床抗癌藥物的關鍵。慶幸的是，目前在這些藥物開發的領域都有許多新穎的技術及方法被研發出來。因此我們深信，藉由不同領域人員及技術的合作幫忙，GRC0321及MCT4中和抗體必定能在將來發展成為臨床抗癌藥物，提供廣大的肺癌病人族群更多的治療選擇與幫助。

關鍵字

肺癌 ； 細胞微管 ； katanin蛋白 ； 糖解作用 ； MCT4蛋白

並列摘要

Lung cancer is the leading cause of the cancer-related death in the world. Though many improvements in medical and surgical therapies has been done, the advancement in lung cancer mortality remains unsatisfactory. Drug resistance and serious side effects are two important reasons to limit the efficacy and safety of the therapeutic agents targeting lung cancer cells. However, with the introduction of precision medicine, more and more promising diagnostic markers and therapeutic targets have been identified in lung cancer cells recently.
In this dissertation, we adopted two strategies, inclusive of randomizing drug screening and mechanism-based drug design, and successfully identified katanin and Monocarboxylate transporter 4 (MCT4) with corresponding therapeutic agents, GRC0321 and MCT4 neutralizing antibody, for anti-lung cancer treatment. We demonstrated the excellent anti-cancer effects and broad therapeutic windows of these two drugs in the in vitro and in vivo models. Besides, the detail mechanisms of the drugs and the expression regulations of the drug targets in lung cancer cells were also investigated. GRC0321, a small compound, destructed the microtubule structure of lung cancer cells by targeting katanin protein and caused a caspase-9 mediated cancer cell apoptosis through prolonging G2/M cell cycle arrest. On the other hand, the MCT4 neutralizing antibody inhibited the proliferation of glycolysis-dependent subtype of lung cancer cells by blocking the export of excess lactate in the cancer cells. This blockage could reduce the intracellular pH level and increase reactive oxygen species (ROS) production and further activate caspase-9 mediated cancer cell apoptosis. The MCT4 neutralizing therapy revealed excellent effects especially on the MCT4-overexpressed lung cancer cells. To explore the detail mechanisms, we found that this MCT4-overexpressed lung cancer cells had higher dependency on the glycolysis and would express more MCT4 on their cell membranes through a Sp1-mediated transcriptional regulation even in a normoxia condition.
Though all the wonderful experimental results of these two drugs make them deserve further development, poor solubility and improper immunoglobulin isotypes are the two major issues encountered and need to be solved in the near future. Fortunately, more and more progresses have been made in these fields which makes us strongly believe that the two drugs could be developed successfully and become clinical therapies to help the patients with lung cancer.

並列關鍵字

lung cancer ； microtubule ； katanin ； glycolysis ； MCT4

參考文獻

References

1. Ferlay, J.; Soerjomataram, I.; Dikshit, R.; et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 2015, 136(5), E359-386.

2. Chiang, C. J.; Lo, W. C.; Yang, Y. W.; et al. Incidence and survival of adult cancer patients in Taiwan, 2002-2012. J. Formos. Med. Assoc. 2016, 115(12), 1076-1088.

3. 103 年各類癌症健保前 10 大醫療支出統計. Retrieved June 28, 2017, from http://www.nhi.gov.tw/News_Content.aspx?n=FC05EB85BD57C709 sms=587F1A3D9A03E2AD s=B164A72522D27089.

4. Non-Small Cell Lung Cancer - American Cancer Society. Retrieved June 28, 2017, from https://www.cancer.org/cancer/non-small-cell-lung-cancer.html.