尋找克服尿路上皮癌化學治療抗藥性的治療策略──探討組織蛋白去乙醯酶抑制劑及去泛素化酶抑制劑治療之機轉

Explore new treatment strategies for overcoming the chemoresistance of urothelial carcinoma ── Using histone deacetylase inhibitor and deubiquitinating enzymes inhibitor as new treatment targets

許富順(Fu-Shun Hsu)

指導教授 ： 黃國皓

共同指導教授 ： 林家齊(Chia-Chi Lin)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200029

摘要

尿路上皮癌（Urothelial carcinoma, UC）在全世界癌症發生率排名前十位，在台灣發生率也有上升的趨勢。接受根除性手術後，仍有 20-50% 的患者會復發和轉移。當患者出現轉移腫瘤時，五年存活率將不到20%。目前的一線化學治療藥物（包括cisplatin和gemcitabine），總體反應率只有約為 50-60%。雖然已開發出用於晚期和轉移性 UC 的二線化療藥物與免疫治療，然而大部分患者的腫瘤仍持續出現抗藥性，最終治療失敗而造成死亡。因此，尋找新的治療方法來克服UC的抗藥性刻不容緩的。
在本研究第一與第二部分中，我們目標在研究組織蛋白去乙醯酶（Histone deacetylase；HDAC）抑制劑: 曲古抑菌素-A （Trichostatin A， TSA） ，在UC的上抗腫瘤效果，並探討TSA能否增加化療藥物對UC的毒殺作用，進一步去釐清其機轉。我們使用了三種膀胱尿路上皮癌細胞株（NTUB1，T24和BFTC-905）以及一種發生於腎臟的上泌尿道尿路上皮癌細胞株（BFTC-909）。實驗證實，TSA 與第一線化療藥物：cisplatin、gemcitabine、doxorubicin和第二線化療藥物 paclitaxel等四種化療藥物合併使用後，皆在UC細胞毒殺作用中產生了顯著的協同作用（Combination index < 1）；TSA並同時抑制了化療藥物所活化的MAPK/ERK抗藥機轉。而此細胞實驗結果，也在腫瘤異體移植的動物實驗中獲得了相同驗證。此外我們也發現，在臨床上對化療反應不佳的病人之UC檢體上，MAPK/ERK磷酸化表現量明顯高於對化療反應良好的UC病人檢體。前兩部分研究的結果證實：TSA與化療藥物合併使用下，經由抑制MAPK/ERK 磷酸化的抗藥機轉，進而增強化療藥物毒殺UC的效用。
在第三部分中，我們探討了去泛素化酶（deubiquitinating enzymes, DUB）抑製劑: PR-619，在cisplatin-resistant UC 中的抗腫瘤作用。首先，在免疫組織化學染色的結果顯示，USP14 和 USP21在化療無效的UC病人檢體中有高度表現，說明了DUB可能與腫瘤抗藥性有正相關，並有潛力成為克服化學抗藥性的新標靶。我們建立了一株對cisplatin具有抗藥性的UC細胞株T24/R。我們發現，PR-619和cisplatin併用時，增強了對T24/R的細胞毒殺作用和凋亡。此外，我們也證明了PR-619可經由抑制c-Myc致癌基因，逆轉UC抗藥性細胞株的抗藥性，進而明顯增強cisplatin對抗藥性UC地毒殺效果。這些結果也在腫瘤異種移植的動物實驗中，獲得相同結果。
總結以上的研究，我們證實了組織蛋白去乙醯酶抑製劑和去泛素化酶抑製劑，除了單獨使用下能有效毒殺UC細胞之外，在兩者個別合併化療藥物使用下，也顯著增強了化療藥物毒殺UC的效果，並抑制了腫瘤細胞抗藥性機轉的產生。這些成果釐清了藥物機轉之外，也提供了克服 UC 抗藥性的新治療策略，具有相當高的臨床應用價值。

關鍵字

尿路上皮癌 ； 化學抗藥性 ； 克莫抗癌 ； 組織蛋白去乙醯酶 ； 去泛素化酶

並列摘要

Urothelial carcinoma (UC) ranks fourth in cancer incidence among men and eleventh in women worldwide. Recurrence and metastasis occurs in 20%–50% of patients after radical surgery. The overall response rate to treatment with first-line regimen, including cisplatin and gemcitabine, remains at 50%–60%. Second-line chemotherapy, including paclitaxel and vinflunine, have been developed for advanced and metastatic UC. Most patients experience chemoresistance, however, which leads to disease relapse. Therefore, the development of innovative UC treatments that address chemoresistance is urgently needed.
In chapter 1 and 2 of this study, we evaluated the antitumor effects of trichostatin A (TSA), an antifungal antibiotic that inhibits histone deacetylases (HDACs), alone or in combination with other chemotherapeutic agents, including cisplatin, gemcitabine, doxorubicin, and paclitaxel, for the treatment of human UC. Cytotoxicity and apoptotic effects were assessed in vitro using fluorescence-activated flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, and the underlying mechanism was measured by western blotting. The data showed that TSA co-treatment with any one of the four chemotherapeutic agents induced synergistic cytotoxicity (combination index < 1) and concomitantly suppressed chemotherapeutic drug-induced activation of the mitogen-activated protein kinase (MAPK) / extracellular signal-regulated kinase (ERK) pathway. These observations were confirmed in a xenograft nude mouse model. The activated MAPK/ERK pathway was also observed in human bladder specimens from chemoresistant UC patients. These findings support that TSA elicited a synergistic cytotoxic response in combination with chemotherapy by targeting the MAPK/ERK pathway.
In chapter 3, we investigated the antitumor effect of the deubiquitylating enzyme inhibitor PR-619 in cisplatin-resistant bladder UC cells. By deubiquitinase (ubiquitin-specific protease 14 [USP14] and USP21) immunohistochemical staining, deubiquitination was related to chemoresistance in patients with metastatic UC. PR-619 was found to enhance the cytotoxic and apoptotic effects of cisplatin in cisplatin-resistant T24/R cells. Mitigated cisplatin chemoresistance was associated with concurrent suppression of c-Myc expression in T24/R cells. Moreover, c-Myc expression was upregulated in human bladder specimens from chemoresistant UC patients. In the xenograft nude mouse model, PR-619 also enhanced the antitumor effects of cisplatin. Our findings suggest that, by disrupting the c-Myc pathway, therapeutic strategies can prevent UC chemoresistance through the combined use of chemotherapeutic agents and deubiquitinating inhibitors (PR-619).
This study describes novel therapeutic strategies to overcome chemoresistance in UC. Our results substantiate the potential clinical applications of histone deacetylase inhibitors and deubiquitinating enzyme inhibitors, and the importance of identifying and developing chemosensitizers that improve the therapeutic efficacy of treatments for chemoresistant UC cells.

並列關鍵字

Urothelial carcinoma ； chemoresistance ； cisplatin ； paclitaxel ； histone deacetylase ； deubiquitinating enzyme ； MAPK pathway

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