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標題: | 阻斷Wnt/β-catenin訊息傳遞路徑以治療肝細胞癌 Targeting the Wnt/β-catenin Signaling Pathway in the Treatment of Hepatocellular Carcinoma |
作者: | Hsiao-Hui Lin 林曉慧 |
指導教授: | 鄭安理(Ann-Lii Cheng) |
共同指導教授: | 徐志宏(Chih-Hung Hsu) |
關鍵字: | Wnt/β-catenin訊息傳遞路徑,肝細胞癌,蕾莎瓦,ICG-001小分子抑制劑,活化突變,CTNNB1基因, Wnt/β-catenin,hepatocellular carcinoma,sorafenib,ICG-001,activating mutation,CTNNB1, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 肝細胞癌(Hepatocellular Carcinoma,簡稱HCC)為十分致命且預後非常差的癌症之一。過去幾十年來,在肝細胞癌的篩檢、早期診斷、以及局部治療上,雖然已有長足的進步;但大多數的肝細胞癌病人仍終將發展至晚期(advanced stage),而必須考慮接受全身性藥物治療。目前晚期肝細胞癌的標準治療,也是唯一經衛生主管機關核可的藥物是sorafenib (商品名為Nexavar【蕾莎瓦】)。Sorafenib是一種多重激酶抑制劑,可抑制Raf激酶及對抗血管內皮生長因子受體。整體而言,sorafenib在肝癌的治療上,腫瘤縮小的機率不高,腫瘤控制時間短暫,其治療效果仍有相當進步的空間。
本論文研究鎖定「Wnt/β-catenin訊息傳遞路徑」做為肝細胞癌嶄新的治療標的。Wnt/β-catenin訊息傳遞路徑在胚胎發育、組織結構的恆定、以及肝細胞癌癌化的過程上,都扮演十分重要的角色。本論文研究嘗試驗證以下兩個假說:一、抑制Wnt/β-catenin訊息傳遞路徑的活性可促進sorafenib對於肝細胞癌的治療療效;二、肝細胞癌β-catenin 基因上特殊的活化性突變,可能導致該癌細胞對特定的Wnt/β-catenin訊息傳遞路徑抑制劑特別敏感。 在本論文的第一部分中,我們證實了無論於細胞培養上或活體內,抑制Wnt/ β-catenin訊息傳遞路徑的活性可促進sorafenib對於肝細胞癌的治療效果。ICG-001是Wnt/β-catenin訊息傳遞路徑的小分子抑制劑,它經由阻斷的β-catenin與轉譯共同激活因子CBP之間的交互作用而達到抑制效果。在多株肝細胞癌的體外培養實驗中,我們發現伴隨著ICG-001使用的濃度提高,肝細胞癌細胞株生長受到sorafenib治療後生長抑制的情況及走向細胞凋亡的細胞比例隨之增加。進一步以RNA干擾的技術抑制β-catenin後,我們亦發現肝細胞癌細胞株Huh7對sorafenib作用的敏感度明顯提升;反之將β-catenin過度表現,則可降低Huh7對sorafenib作用的敏感度。此外,我們發現經短髮夾RNA(shRNA)處理導致β-catenin表現低下之Huh7對sorafenib作用的敏感度提升,再將該細胞的β-catenin過度表現之後,其敏感度則再度降低。就機制上而言,合併使用sorafenib與ICG-001可促使更多肝細胞癌細胞株走向細胞凋亡,並導致Mcl-1表現的更顯著下降。在Huh7肝細胞癌細胞小鼠皮下腫瘤的動物模式,合併使用sorafenib與ICG-001的組別相較於單獨使用sorafenib亦或ICG-001的組別,更能顯著地抑制腫瘤的生長。 在本論文的第二部分中,我們則闡明了CTNNB1基因之第三外顯子具有錯義突變(missense mutation)之肝細胞癌細胞株,對特定的Wnt/β-catenin訊息傳遞路徑抑制劑的抑制作用有較高的敏感度。我們使用SNU398與Huh6(分別於CTNNB1基因的第三外顯子的S37C及G34V有錯義突變)、Huh7及HepG2(在CTNNB1基因第三外顯子處有缺失的片段[interstitial deletion])、及PLC5、Hep3B、HLE及SK-Hep1 (在第三外顯子的位置無基因異常)等肝細胞癌細胞株;並分別處理包括ICG-001、XAV939 (為tankyrase的抑制劑,可藉此達到AXIN-1的穩定,以抑制β-catenin的訊息傳遞活性)、以及LGK974 (為porcupine的抑制劑;porcupine可促使Wnt ligands的成熟與分泌)等Wnt/β-catenin訊息傳遞路徑抑制劑。在XAV939及LGK974的處理下,不同肝細胞癌細胞株生長受到抑制的程度差異並不顯著;然而在ICG-001的處理下,我們發現SNU398的生長抑制作用最為顯著,緊追其後為Huh6。除此之外, ICG-001對於SNU398的細胞聚落形成的抑制力、經ICG-001處理後誘發sub-G1波峰提升的程度,皆高於其他肝細胞癌細胞株。最後,不同的化學治療藥物,包含doxorubicin、cisplatin、及5-fluorouracil對不同肝細胞癌細胞株的抑癌效果,並無發現顯著的差異。 我們的實驗研究結果顯示抑制Wnt/β-catenin訊息傳遞路徑的活性可促進sorafenib對於肝細胞癌的治療療效,而具有β-catenin第三外顯子有錯義突變的肝細胞癌細胞株對ICG-001的抑癌效果最為敏感。本論文研究的發現支持持續鎖定Wnt/β-catenin訊息傳遞路徑研發抑制劑(無論是與sorafenib合併使用,或針對特定分子標記族群做單一治療)以治療肝細胞癌。 Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world. Although a lot of advances in screening, early diagnosis, and loco-regional therapies for HCC have been made over the past decades, most HCC patients would eventually develop advanced diseases that need systemic therapy. Currently, sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is the only approved drug for HCC. However, its efficacy is limited, with low tumor response rate and short tumor control duration. The current thesis work focuses on developing new therapeutic strategies for HCC by targeting the Wnt/β-catenin signaling pathway, which plays an important role in embryonic development and tissue homeostasis, as well as in hepatocarcinogenesis. The thesis work has tested two hypotheses: (1) inhibition of the Wnt/β-catenin signaling pathway could improve the anti-tumor effects of sorafenib in HCC; (2) activating mutations of β-catenin gene may confer sensitivity to specific Wnt/β-catenin pathway inhibitors in HCC. In the first part of the current thesis work, we demonstrated that inhibition of the Wnt/β-catenin signaling pathway could improve the anti-tumor effects of sorafenib against HCC in vitro and in vivo. ICG-001, a small molecule that blocks the interaction of β-catenin with its transcriptional coactivator CBP, dose-dependently enhanced the growth-suppressive and apoptosis- induction effects of sorafenib in multiple HCC cell lines. Downregulation of β-catenin by RNA interference increased sorafenib sensitivity, whereas overexpression of β-catenin reduced sorafenib sensitivity in Huh7 cells. The sorafenib-sensitization effect of short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was attenuated by β-catenin overexpression. Mechanistically, sorafenib combined with ICG-001 or shRNA- mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a significant downregulation of Mcl-1 in HCC cells. In Huh7 cell mouse xenograft model, the combination of ICG-001 and sorafenib showed a more significant growth-retarding effect than single agent treatment of sorafenib or ICG-001. In the second part of the current thesis work, we demonstrated that HCC cells with point mutation at exon 3 of the CTNNB1 gene exhibited an increased sensitivity to antitumor effects of certain Wnt/β-catenin inhibitors in vitro. SNU398 and Huh6 are HCC cells harboring missense somatic mutations in exon 3 of the CTNNB1 gene, resulting in S37C and G34V mutation, respectively; Huh7 and HepG2 are HCC cells with interstitial deletion of exon 3; while other HCC cells including PLC5, Hep3B, HLE, and SK-Hep1 are HCC cells containing no mutations in exon 3. Several classes of Wnt/β-catenin pathway inhibitors were tested including ICG-001, XAV939 (an inhibitor of tankyrases which would stabilize AXIN-1 and thus β-catenin transactivation activity), and LGK974 (an inhibitor of porcupine which could help maturation and secretion of Wnt ligands). The sensitivity to growth suppressive effects of XAV939 and LGK974 did not vary significantly among all tested HCC cells. However, the growth-suppressive effect to ICG-001 was most sensitive in SNU398 cells, followed by Huh6 cells, and less sensitive in other HCC cells. ICG-001 also induced a more significant suppression of colony-formation, and a more significant increase of sub-G1 fraction by flow cytometry in SNU398 than other HCC cells. Finally, the cytotoxic effects of multiple chemotherapy drugs including doxorubicin, cisplatin, and 5-fluorouracil did not significantly differ among tested HCC cells. Our studies have demonstrated that targeting of Wnt/β-catenin signaling pathway improves the anti-tumor effects of sorafenib against HCC and the presence of a missense mutation in exon 3 of β-catenin may confer increased sensitivity to ICG-001 in certain HCC cells. These data support further investigations on developing Wnt/β-catenin inhibitors as potential cancer therapeutics for HCC by either combination with sorafenib or single-agent approach focusing on biomarker-enriched population. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59667 |
DOI: | 10.6342/NTU201700615 |
全文授權: | 有償授權 |
顯示於系所單位: | 腫瘤醫學研究所 |
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