Neddylation抑制物MLN4924在泌尿上皮細胞癌及子宮頸癌藥物治療上所扮演之角色

Abstract

泌尿上皮細胞癌 (urothelial carcinoma; UC)是泌尿系統常見的惡性腫瘤，在台灣發生率偏高。轉移性的泌尿上皮細胞癌，需接受化學治療，但終會因化學治療抗藥性而導致病患死亡。子宮頸上皮細胞癌(cervical carcinoma, CC)是女性生殖系統常見的惡性腫瘤，全世界發生率仍在女性生殖系統上維持前三名。轉移性的子宮頸上皮癌病患中，許多病人會因抗藥性而死亡，如何抑制這兩種癌症進展及抗藥性是臨床上的重要課題。 泛素-蛋白酶系統（Ubiquitin-proteasome system）負責調控細胞內蛋白質降解及活性，與基因表達、細胞增殖、週期、分化、凋亡、DNA修復皆有相關，若調控失調常會引起腫瘤的發生。Ubiquitin-protein ligase， E3 是一個關鍵的酵素，Ubiquitin ligases 的活化又會與其成分cullin 與neural precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) 結合（Nedd8分子接合作用；Neddylation）有關。利用新的藥物（MLN4924）阻斷neddylation，是治療腫瘤的新機轉，並且已經被報告與治療癌症轉移及降低化學抗藥性（chemoresistance）有關，而且其相關機制之調控推測為克服腫瘤進展及抗藥性的重要標的。吾人之假設為調控neddylation inhibition可抑制尿路上皮細胞癌及子宮頸癌之生長,並嘗試於本研究中探討細胞週期調控蛋白之變化與neddylation inhibition之相關機轉與如何調節cullin及其變化之機制，並期望能進一步在 in vivo 的動物模式中驗證。 在本研究中，我們起初發現MLN4924能有效的抑制三株泌尿上皮腫瘤細胞 NTUB1, T24 和 RT4 cell lines 的生長，且與細胞凋亡及細胞週期調控蛋白變化有關。此外也發現neddylation inhibition與其他細胞壓力反應及內質網壓力也相關。由於泌尿上皮細胞癌是transitional cell origin,我們也將MLN4924運用在兩種不同型態之子宮頸癌細胞株如ME180 (鱗狀上皮細胞癌squamous cell carcinoma) 及HeLa (腺細胞癌adenocarcinoma) cell lines上,發現MLN4924亦能抑制其生長，並證實此藥物在泌尿上皮細胞癌和子宮頸癌細胞之小鼠轉殖動物模式上，具有其活體治療之效果。更進一步，合併neddylation inhibition 與其他化療藥物（如cisplatin）亦有加成之抑癌治療效果在子宮頸癌細胞上。 吾人相信，藉由研究neddylation modulation 治療泌尿上皮細胞癌及不同型態之子宮頸上皮細胞癌，能找到新的治療模式，並能解決臨床上抗藥性及腫瘤進展的問題。

Urothelial carcinoma (UC) is a common cancer of urinary tract and the prevalence rate is relatively high especially in Taiwan. Patients with metastatic UC are usually treated with systemic chemotherapy but still some patients with advanced UC are not responsive to chemotherapy and die of the disease. Cervical carcinoma (CC) of the uterus is a common malignant tumor of female reproductive system. Incidence of CC in the world is still in the top three on the female reproductive system. Patients with metastatic CC usually die of the disease due to tumor recurrence and resistance to the chemotherapy. To overcome the tumor progression and chemoresistance of these two kinds of cancer is still the clinical important issue. Ubiquitin-proteasome system is responsible for intracellular protein degradation and regulation of cell activity, gene expression, cell proliferation, cell cycle regulation, differentiation, apoptosis and DNA repair. Regulation dysfunction of ubiquitin-proteasome system is associated with tumor genesis. Ubiquitin-protein ligase E3 is one major enzyme of ubiquitin ligases and neural-precursor-cell-expressed developmentally down-regulated protein 8 (NEDD8) combining with cullin, the scaffold of some E3, is essential for E3 activation. Access of the new drug (MLN4924) to block the neddylation leads to new treatments for cancer and MLN4924 has been reported to inhibit cancer cell growth and to reduce chemoresistance. The regulation of the associated molecular mechanism is important to overcome tumor progression and chemoresistance for cancer therapy. Our hypothesis is manipulation of neddylation inhibition by MLN4924 can suppress the tumor growth of UC and CC. We try to explore the changes of cell cycle regulatory proteins and neddylation inhibition associated mechanism and the control of cullin to further verify in animal models. In this study, we initially found that MLN4924 can effectively inhibit the growth of transitional cell origin NTUB1, T24 and RT4 cell lines and associated with cell apoptosis and cell cycle regulation. In addition, the neddylation inhibition can induce cell stress reactions and endoplasmic reticulum (ER) stress related molecule expression. We also treated MLN4924 on the two different cell lines as ME180 (squamous cell carcinoma) and HeLa (adenocarcinoma) of the CC and found the similar change of tumor growth suppression. We also investigated MLN4924 by the xenograft animal model of mice on UC and CC cells to confirm MLN4924 treatment effect in vivo. Furthermore, to merge neddylation inhibitor and other chemotherapy drug such as cisplatin, we also demonstrated the combining and synergistic antitumor therapeutic efficacy on CC. In the future, we believe that by studying the neddylation modulation on UC and CC, we will find the new treatment model to overcome the clinical issue of chemoresistance and tumor progression of many kinds of carcinomas and improve the clinical treatment efficacy for cancer.