- 學位論文
DNA 雙股斷裂修復蛋白質 NBS1 與 AKT 訊息傳遞路徑在肝癌之互動
The interplay of DSB repair protein, NBS1, with AKT signaling in hepatocellular carcinoma
摘要
維持基因組的完整性對於細胞的存活以及其正常功能的執行是不可或缺的。同時，基因組的不穩定也已被證實與腫瘤的發生與進展有關，其中也包含肝癌。DNA 雙股斷裂修復蛋白複合體包含 Mre11，Rad50 與 NBS1，在修復 DNA 雙股斷裂 (DNA double-strand breaks, DSBs) 以維持基因組穩定性中扮演著重要角色。在此蛋白複合體中，NBS1 的主要功能為偵測 DNA 雙股斷裂並啟動 DNA 修補的進行。有爭議的是，在頭頸癌的細胞株中 NBS1 可藉由活化 AKT 訊息傳遞促使細胞轉型。正因為肝癌是一種對化學療法和放射線治療具有普遍抗性的腫瘤，而此類抗性的部份起因已被認為與異常的 DNA 修復能力有關。我們因而想探討 NBS1 表現量的改變是否在肝癌中扮演著某些角色。 我們的研究發現在肝癌組織切片中 NBS1 的細胞質染色強度與 CD34 和疾病復發呈正相關 (P<0.001 與 0.009)；與肝癌病人的腫瘤分期則為負相關 (P=0.018)。然而，Kaplan-Meier 存活率曲線顯示當 NBS1 表現於細胞核而非細胞質時患者會有較差的存活率 (P=0.052)。此外，NBS1 在 60 對肝癌組織中有 65.0% 的癌組織蛋白表現量較鄰近非癌肝臟組織高。由於 NBS1 與 DNA 修復及維持基因完整性有密切的關聯性，我們因此進一步分析癌組織與非癌肝臟組織中 NBS1 與微衛星不穩定 (Microsatellite instability, MSI) 現象之間的相關性。微衛星不穩定分析結果顯示在 69 名病患中約有 30.4 % 的人呈現微衛星不穩定現象，而在此群微衛星不穩定的病患中當 NBS1 低表現時病患有較差的存活率 (P=0.041)。藉由血管生成試驗證實，NBS1 大量表現時會促進人類臍靜脈內皮細胞血管生成的能力。而過度表現 NBS1 時並不能有效增加肝癌細胞株 HepG2 與 Hep3B 增生和侵襲的能力。此外，NBS1 能夠增加 AKT 的磷酸化和下游蛋白質 mTOR 的表現量。同時，NBS1 也能增加 eNOS 的蛋白質和 mRNA 的表現量與一氧化氮 (nitric oxide, NO) 的產量，但對於 VEGF 與 HIF-1 卻無顯著的影響。利用 Wortmannin (PI3K 特異性抑制劑) 與 Rapamycin (mTOR 特異性抑制劑) 可阻斷 NBS1 所改變 eNOS 蛋白質與一氧化氮的表現量。我們的研究發現，大量表現 NBS1 可活化 AKT 訊息傳遞，並增加下游蛋白質 mTOR 與 eNOS 的表現及一氧化氮的產量，並促進血管生成的能力，可能藉此影響肝癌病程進展。 因此，我們相信 NBS1 在肝癌的預後或治療中可能扮演著重要的角色，然而更深入的作用機制仍須進一步去研究探討，希望後續的研究能夠為肝癌的治療與預後上提供一個新的方向。
並列摘要
The maintenance of genomic integrity is essential for cell survival and normal function, while genomic instability has been demonstrated in the development and progression of multistep cancers including hepato- cellular carcinoma (HCC). DNA double strand breaks (DSBs) repair protein complex including Mre11, Rad50 and NBS1, plays an important role in repairing DSBs to maintain genomic integrity. In the protein complex, NBS1 serves as the key player in sensing DSBs. However, controversially, NBS1 can also activate AKT signaling and thereby contribute to transformation activity in head and neck cancer cells. Since HCC has been one of the most chemotherapy- or radiotherapy-resistant cancers and the production of resistance to these therapies is closely associated with alterations of DNA repair activity, we thus aim to explore if the alteration of NBS1 might be involved in HCC development. Our results showed that cytoplasmic NBS1 staining in the lesions was positively correlated with CD34 expression and recurrence (P<0.001 and 0.009), but was inversely correlated with tumor staging (P=0.018) in HCC patients. Rather, the Kaplan-Meier survival curve showed that nuclear but not cytoplasmic NBS1 expression was associated with a poor overall survival rate (P=0.052). In addition, NBS1 protein was increased in 65.0% of 60 HCC tissues as compared with the paired non-cancer liver tissues. Since NBS1 and activated form has been closely associated with DNA repair activity and genomic integrity, we therefore analyzed its correlation with microsatellite instability (MSI) in paired cancerous and non-cancerous tissues. Microsatellite instability analysis showed that 30.4% of 69 patients had microsatellite instability. High microsatellite instability with low expression of NBS1 contributed to poor survival (P=0.041). From tube formation assay demonstrated that ectopic NBS1 overexpression contributed to angiogenesis in human umbilical vein endothelial cells (HUVEC). Ectopic NBS1 overexpression did not significantly increase cell proliferation and invasion in HepG2 and Hep3B cells. However, NBS1 increased phsphorylated AKT levels and the downstream protein, mTOR. Intriguingly, NBS1 also increased the levels of eNOS protein, mRNA expression and nitric oxide (NO) production but unaffected those of VEGF and HIF-1. Both Wortmannin (PI3K-specific inhibitor) and Rapamycin (mTOR-specific inhibitor) abolished the expression of eNOS protein and the production of NO. Our results showed that increased NBS1 might activate AKT signaling, leading to activation of mTOR and increased eNOS expression and NO production and promoted angiogenesis, and thereby contributed to HCC development. In conclusion, we believe that NBS1 may play some critical roles for prognosis and treatment in HCC. Further investigations are required. Hopefully, the study will provide a better understanding of the carcinogenesis and progression of HCC.