魚針草內酯治療頭頸部鱗狀細胞癌的分子機制研究

Investigation of molecular mechanism of Ovatodiolide in the treatment of head and neck squamous cell carcinoma

林群書(Chun-Shu Lin)

指導教授 ： 趙祖怡

臺北醫學大學/醫學院/臨床醫學研究所/博士(2019年)

摘要

動機：頭頸部鱗狀細胞癌是全球常見的癌症，而放射治療是主要治療方式之一，但經常因為腫瘤的放射線抗性，致使療效不佳。其中，癌幹細胞在頭頸部鱗狀細胞癌的放射線抗性、化學藥物抗藥性、局部復發與遠處轉移，皆扮演重要的角色；近年來，組蛋白去甲基酶JARID1B已被證實與癌幹細胞息息相關。
目的：探討JARID1B與頭頸部鱗狀細胞癌的移行與侵襲的相關性，同步分析JARID1B在臨床病人檢體表現量與預後的關連性；並進一步檢驗魚針草內酯抑制頭頸部鱗狀細胞癌JARID1B的分子機制與未來臨床運用的可能性。
研究方法：使用傷口癒合法、基膠質侵入法、磺酰羅丹明B測定法、球狀體形成測定法來分析癌細胞接受處理後(有無放射治療與有無去除JARID1B)的生長狀況；同步評估JARID1B在81名頭頸部鱗狀細胞癌病人的臨床預後相關性。使用魚針草內酯處理癌幹細胞後，通過西方墨點法評估Oct4、Sox-2與Nanog的表現；在癌幹細胞模型中，使用球狀體形成測定法和克隆形成法，來評估魚針草內酯在自我更新能力和克隆形成的影響，並使用小鼠異種移植癌細胞模型來測定魚針草內酯的抗癌效果。
研究結果：使用的頭頸部鱗狀細胞癌包括SAS、FaDu、HSC3、Cal27、TW2.6與SCC4。我們發現將癌細胞的JARID1B去除表現後(shJARID1B)，會明顯抑制癌細胞的移行與侵襲能力、明顯抑制癌幹細胞的相關能力，並增強放射治療的抗腫瘤效果。放射治療合併去除JARID1B後的癌細胞，檢驗信使核醣核酸，會發現NQO1、KEAP1、NRF2、FOXO1、FOXO3、KLF4、OCT4、CD133與Nanog的表現量皆減少，球狀體形成能力也會因缺乏JARID1B而減少，並臨床證實JARID1B過度表現是頭頸部鱗狀細胞癌的獨立預後因子。我們也確定魚針草內酯可顯著的抑制頭頸部鱗狀細胞癌的活性與克隆形成，並且呈現濃度依存效應與時間依存效應；魚針草內酯可明顯抑制乙醛脫氫酶1活性，減少CD44高/乙醛脫氫酶高 的細胞亞群體。另外，魚針草內酯可藉由抑制CD133、Klf4、Oct4A、Nanog與JARID1B達到抑制球狀體形成的效果。我們更進一步的證實，魚針草內酯所媒介的抗癌效果也與STAT3、p-STAT3、pJAK2、pAKT 與 pERK1/2的蛋白質表現量減少有關，並呈現濃度依存效應。魚針草內酯可增強順鉑的抗腫瘤球狀體(包括SAS、FaDu、HSC-3與TW2.6)效果，並呈現藥物間協同作用。在小鼠異種移植癌細胞模型的研究中也發現，魚針草內酯可顯著衰減腫瘤球狀體的初始腫瘤活性。
結論：去除JARID1B表現後的頭頸部鱗狀細胞癌，會抑制腫瘤的移行與侵襲，減少癌幹細胞活性，並促進放射治療敏感性；在放射治療前，去除頭頸部鱗狀細胞癌病人的JARID1B，可作為有潛力的治療策略。我們的結果也顯示魚針草內酯可藉由JAK2/STAT3/JARID1B訊息傳遞路徑，有效的抑制頭頸部鱗狀細胞癌的腫瘤發生與癌幹細胞特性；魚針草內酯可作為未來進一步的臨床運用。

關鍵字

組蛋白去甲基酶JARID1B ； 魚針草內酯 ； 頭頸部鱗狀細胞癌 ； 癌幹細胞 ； 放射治療

並列摘要

Background: Head and neck squamous cell carcinoma (HNSCC) is a major cause of human mortality globally, and radiotherapy is one of the main treatment modalities. However its therapeutic effect is often limited due to radio-resistance. The cancer stem cells (CSCs) play key roles in tumor recurrence, including radio-resistance, chemo-resistance, local recurrence, and distant metastasis after treatment. Recently, histone demethylase JARID1B has been shown to closely relate to CSCs.
Aims: First, we examined the role of JARID1B in migration and invasion of human HNSCC cells lines. We also studied the prognostic role of JARID1B on overall survival of clinical patients. Second, we investigated the anti-JARID1B potential of Ovatodiolide (Ova) in vitro and in vivo. We further examined the molecular mechanism of Ova by JARID1B inhibition on migration and invasion of HNSCC cell lines.
Methods: Wound healing, matrigel invasion, Sulforhodamine B, and spheroid formation assays were used to characterize the signaling pathways of shJARID1B in response to radiation treatment. We evaluated the prognostic relevance of JARID1b expression in a cohort of 81 HNSCC patients. CSCs of HNSCC were treated with Ova, and the expression of pluripotency factors Oct4, Sox-2, and Nanog were evaluated by western blot. Effect of Ova on self-renewal capacity and clonogenicity were assessed with the sphere formation and clonogenic assay in CSCs model derived from HNSCC. The effect of Ova was also investigated in a mouse xenograft model obtained by injecting nude mice with HNSCC-CSCs.
Results: Human HNSCC cell lines, including SAS, FaDu, HSC3, Cal27, TW2.6 and SCC4 cells, were used. shJARID1B cells significantly inhibited migration and invasion ability compared to their vector or wild type counterparts. Silencing shJARID1B significantly inhibited CSCs activity and potentiated the tumor-inhibitory activity of radiation therapy in HNSCC. Radiotherapy coupled with shJARID1B knockdown reduced mRNA levels of NQO1, KEAP1, NRF2, FOXO1, FOXO3, KLF4, OCT4, CD133, and Nanog in malignant HNSCC cells. HNSCC spheroid formation ability was markedly reduced in the shJARID1B cells. JARID1B overexpression is an independent prognostic factor in HNSCC patients. We demonstrated that Ova dose- and time-dependently suppressed HNSCC cell viability and colony formation; Ova markedly inhibited the ALDH1 activities and reduced the CD44high/ALDHhigh cell subpopulation. Additionally, Ova suppressed tumorsphere formation by down-regulating CD133, Klf4, Oct4A, Nanog and JARID1B expression. Furthermore, Ova-mediated anti-cancer effects were associated with the dose-dependent reduction in the expression levels of STAT3, p-STAT3, pJAK2, pAKT and pERK1/2 protein. Moreover, Ova synergistically enhanced the anti-cancer effect of cisplatin against the SAS, FaDu, HSC-3 and TW2.6 tumorspheres. Ova significantly attenuated the tumor-initiating potential of tumorsphere in mouse xenograft model.
Conclusions: Silencing shJARID1B inhibits migration and invasion of human HNSCC cell lines, reduces CSCs activities and potentiates enhancement of radiation sensitivity. JARID1B knockdown prior to radiotherapy is a potential effective therapeutic strategy for the treatment of HNSCC. These results also demonstrate that Ova effectively suppressed head and neck cancer tumorigenesis and stemness properties via JAK2/STAT3/JARID1B signaling. Ova may be considered for future clinical usage.

並列關鍵字

JARID1B ； Ovatodiolide ； head and neck cancer ； cancer stem cell ； radiation therapy

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