機轉為基礎的肝癌放射治療增敏研究

Abstract

緒論 隨著科技日新月異，放射線治療(放療)逐漸成為肝癌的治療選項之一，藉由抑制放療引發之訊息傳導和次致死傷害修復可能有助於提升療效。Sonic hedgehog (SHH)已被證實在肝癌的腫瘤生成過程扮演重要的調節角色。放療引發之SHH可以藉由自分泌模式，促進DNA修復，保護肝癌細胞。此研究之第一部分目標為探究SHH於放療抵抗性，及SHH抑制劑作為放射增敏劑之可行性。 放療引發之DNA損傷修復可由組織蛋白去乙醯酵素(HDAC)所調控。其中，放射線引發之DNA受損可造成HDAC4與泛素載體蛋白9(Ubc9)和Rad51結合入核進行修補。第二部分，我們研究HDAC4抑制劑增強肝癌之放療效用。 第一型拓樸異構酶(topoisomerase I，TOP1)與放療後之DNA雙股螺旋體之修補有關。在此修補機制中，RNF144A調控DNA-PKcs的泛素化(ubiquitination)。第三部分，我們欲釐清TOP1抑制劑作為放射增敏時，RNF144A於DNA-PKcs之ubiquitination對於不同肝癌之DNA修補調控及相關機制。 方法 細胞實驗中，重組SHH蛋白或SHH抑制劑合併放療，使用人類肝癌細胞株Huh7和PLC5，及小鼠肝癌細胞株BNL。短夾型構造的RNA（short hairpin RNA；shRNA）、HDAC抑制劑、TOP1抑制劑(Lipotecan及irinotecan)和蕾莎瓦(sorafenib)，使用Huh7及PLC5進行合併放療之細胞實驗。以群落形成測定分析癌細胞存活率，以Annexin V染色觀察細胞凋亡。以西方墨點法觀察蛋白質變化。免疫螢光染色、共軛焦顯微鏡和核質分離技術偵測目標蛋白的位置。以免疫沉澱法研究蛋白質間交互作用。使用肝癌的同種原位移植小鼠模式，檢測SHH抑制劑合併放療合併放射線治療可能造成之療效。肝癌的異位異種移植小鼠模式，檢測HDAC抑制劑和shRNA剔除HDAC4合併放療的效果。以異種原位移植免疫缺陷小鼠模式，檢測Lipotecan或蕾莎瓦合併放療於活體之治療效果。 結果 第一部分：細胞實驗中，我們發現使用重組SHH蛋白可以顯著抵抗放射線對於細胞之毒殺效果。合併放射線及SHH抑制劑則明顯地比單一治療更能抑制肝癌細胞生長，其效果主要來自於細胞凋亡的增加。單獨使用放射線會造成肝癌細胞之Gli-1蛋白增加，且隨放射劑量增加，其增加主要在細胞核內。於活體實驗中，亦可觀察到相同之現象。合併放射線及SHH抑制劑(cyclopamine)可減少Gli-1並增加DNA雙股斷裂. 於同種原位移植小鼠模式合併放射線及cyclopamine與單獨放射線治療相比，可多抑制67%腫瘤大小(p < 0.05)。 第二部分：HDAC4抑制劑或shRNA剔除HDAC4合併放療都可以增強放射線造成之癌細胞死亡，並減少蛋白激酶B及同源性重排(Homologous Recombination)之DNA雙股螺旋體斷裂之修補，增加癌細胞凋亡。於活體實驗中，HDAC4抑制劑或shRNA剔除HDAC4合併放療都能夠延緩腫瘤生長。放射線引發之Rad51入核為HDAC4相關之模式，且Rad51與Ubc9直接結合，HDAC4造成Ubc9乙醯侲，入核進行DNA修補。抑制HDAC4，則造成HDAC4/Ubc9/Rad51複合體之分解，進而阻止其入核修補DNA雙股螺旋體斷裂。 第三部分：於肝癌細胞株(Huh7及PLC5)及，我們發現Lipotecan比蕾莎瓦有更佳的放射線增敏效果。異種原位移植小鼠模式中，合併放射線及Lipotecan抑制腫瘤生長效果比單獨放射線多7倍(p < 0.05)，比合併放射線及蕾莎瓦多9倍 (p < 0.01)。Lipotecan造成更多的放射線引起的DNA-PKcs和DNA傷害。TOP1抑制劑作為放射增敏劑，對於抑制放射線引起的DNA-PKcs增加效果，在Huh7細胞優於PLC5細胞。於Huh7細胞，其RNF144A (DNA-PKcs與其E3連接酶)於合併放射線和TOP1抑制劑後有增加的情形，而DNA-PKcs則減少。這些現象，在抑制泛素/蛋白酪系統(ubiquitin/proteasome system)後，則可逆轉。然而，於PLC5細胞中，合併於合併放射線和TOP1抑制劑後，則造成RNF144A入核及DNA-PKcs堆積，以致於較強的放射線抗性。 結論 抑制SHH訊息，可以經由增加凋亡和DNA雙股斷裂，造成肝癌細胞及同種原位移植活體腫瘤之放射增敏作用。抑制HDAC4訊息傳遞則可以增加肝癌之放射線致死效果。其作用主要藉由則抑制HDAC4，進而導致HDAC4/Ubc9/Rad51複合體之分解，進而阻止其入核修補DNA斷裂。TOP1抑制劑之放射增敏效果則是經由RNF144A所調控之DNA-PKcs泛素化。此研究經由SHH、HDAC4和TOP1訊息傳遞路徑，勾畫出可行之放射增敏分子生物學機制。

Introduction With the advances in technologies, radiotherapy (RT) has become one of the treatment choices for hepatocellular carcinoma (HCC). One strategy to enhance the tumoricidal effect of RT is to inhibit radiation-activated signals and overcome sublethal damage repair. Radiosensitizer (RS) is the agent that augments the effect of RT. The most common investigational RSs are chemotherapeutic agents, especially DNA base analogs. Among these agents, CDDP is the typical RS in the treatment of a variety of cancer types. Sonic hedgehog (SHH) is a regulator in tumorigenesis of HCC. RT-induced SHH signaling protects HCC cells against RT in an autocrine manner by facilitating DNA damage repair. The first part of this study aimed to determine the role of SHH in radio-resistance and propose the SHH inhibitor as a RS. The histone deacetylases (HDACs) could mediate the process of DNA damage repair following RT. HDAC4 interacts with ubiquitin-conjugating enzyme 9 (Ubc9) and Rad51 proteins upon RT-induced DNA damage. In the second part, we postulated HDAC4 inhibition to enhance the RT effect on HCC cells. DNA topoisomerase I (TOP1) involves the repair of DNA double-strand break (DSB) after RT. RNF144A mediates ubiquitination of catalytic subunit of DNA protein kinase (DNA-PKcs), a critical factor in DSB repair. In the third part, we aimed to demonstrate the radiosensitization with TOPI inhibition and investigate the differentiating mechanism by DNA-PKcs/RNF144A between HCC cells. Materials and Methods The in vitro effects of combining SHH ligand (recombinant human SHH) or inhibitor (cyclopamine) with RT were evaluated in the human HCC cell lines, Huh7 and PLC5, and murine cell line BNL. The in vitro effects of short hairpin RNA (shRNA) and HDAC inhibitor (HDACi), panobinostat to knock down expression of HDAC4, and TOP1 inhibitors (Lipotecan and irinotecan) or sorafenib with RT were evaluated in Huh7 and PLC5. We evaluated cell survival by a clonogenic assay, and apoptosis by Annexin V staining. Western blotting was used to detect protein expression. We used immunofluorescence staining, confocal microscopy, and cell fractionation to identify the location of target proteins. We evaluated the physical interaction between proteins by immunoprecipitation. We tested the in vivo response to RT and cyclopamine in BALB/c mice with an orthotopic allogeneic tumor. Ectopic xenografts were pretreated with HDACi or shRNA to knockdown expression of HDAC4 and then irradiated. Orthotopic HCC xenografts with severe combined immunodeficient mice were treated with Lipotecan or sorafenib and RT for the in vivo response. Results First part: SHH ligand protected HCC cells from RT on clonogenic cell survival. The combination of RT and cyclopamine produced a more potent inhibitory effect on cell proliferation than either modality alone. RT upregulated the expression of Gli-1 (a transcription factor induced by SHH) dose-dependently, predominantly in the nucleus. When combined with cyclopamine, RT inhibited Gli-1 and increased DNA DSB. Treatment with cyclopamine and RT reduced the mean tumor size of orthotopic tumors by 67% when compared with RT alone (p < 0.05). Second part: HDAC4 knockdown and HDACi both increased RT-induced cell death and lowered homologous recombination repair of DNA DSBs and Akt activation. The combination of HDAC4 knockdown or an HDACi with RT led to increased cancer cell apoptosis. HDAC4 knockdown or an HDACi with RT delayed tumor growth in a xenograft model, significantly. RT triggered the nuclear translocation of Rad51 in an HDAC4-dependent manner. The binding of Ubc9 directly to HDAC4 results in Ubc9 acetylation. Furthermore, these effects were followed by HDAC4/Ubc9/Rad51 complex dissociation through the block of nuclear translocation. Third part: Lipotecan/RT had a superior synergistic impact on sorafenib/RT on HCC cells. Combined RT and Lipotecan reduced the tumor growth by 7-fold than RT alone (p < 0.05) and by 9-fold than sorafenib plus RT (p < 0.01). Lipotecan induced more RT-induced DNA damage and DNA-PKcs signalings than sorafenib. The radiosensitization with TOP1 inhibitors and the inhibition of RT-induced DNA-PKcs were more effective in Huh7 than PLC5 cells. In Huh7 cells, RNF144A, an E3 ubiquitin ligase for DNA-PKcs, increased, and DNA-PKcs decreased after the combination of RT and TOP1 inhibitors. The effect was reversed with the inhibition of the ubiquitin/proteasome system. In comparison, RNF144A decreased through nuclear translocation after the combinational treatment, resulting in accumulated DNA-PKcs and radio-resistance of PLC5 cells. Conclusion The inhibition of SHH signaling significantly sensitizes HCC cells and orthotopic xenografts to radiation by increasing apoptosis and the number of DSBs. HDAC4 signaling blockade enhances radiation-induced lethality in HCC. The effects are accompanied by the dissociation of HDAC4/Rad51/Ubc9 complex to inhibit DNA repair. TOP1 inhibitor radiosensitizes HCC through RNF144A mediated DNA-PKcs ubiquitination. This study delineates the molecular mechanisms of radiosensitization through the inhibition of SHH, HDAC4, and TOP1 signaling pathways.