| Abstract: | 自 2001 年以來，本研究室發表一系列之論文指出 HPV 16/18 感染可能參與台灣肺癌的發生，尤其是不抽煙之女性肺癌。我們在患者肺腫瘤連續切片中發現 HPV16/18 E6 與 p53 蛋白表現呈負相關性。同時我們由患者之胸水培養出具有 HPV16 E6 表現之 TL-1, TL-2 和 TL-3 肺癌細胞株，證實 E6 會與 p53 蛋白結合，使其降解失去轉錄活化下游 p21, MDM2 mRNA 表現之能力和促進腫瘤細胞之增生。但本研究至今並沒有找到參與 HPV 感染之肺腫瘤化的重要基因，而能做為患者之臨床預後因子。DDX3 和 miR-218 已被發現參與病毒相關之肝癌和子宮頸癌的形成。但是 DDX3 和 miR-218 在 HPV 感染之肺腫瘤化之作用分子機制，至今仍然不清楚。本研究假設 HPV E6 可能抑制 DDX3 和增加 miR-218 之表現，而促進腫瘤生長和轉移。因此預期患者可能有較差之臨床預後。為了證實此假設，本研究以過度表現和剔除方式改變 E6 在肺癌細胞之表現，以了解 DDX3 和 miR-218 之表現是否會受到 E6 之調控? 若直接改變 DDX3 和 miR-218 之表現，是否會改變 p21、MDM2、Slug 和 E-cadherin 之表現，而促進細胞生長、細胞群落形成、軟瓊酯細胞群落形成以及細胞侵襲轉移等能力。結果發現 p53 會調控 DDX3 之轉錄。若剔除 DDX3 則會經由抑制 p21 之表現而促進細胞之生長。同時會經由 MDM2/Slug/E-cadherin 路徑促進細胞之侵襲能力。並發現E6 會抑制 DDX3 和促進 cIAP2 之表現，造成肺癌細胞對 cisplatin 的敏感性降低。同時以 144 個肺腫瘤進行組織免疫染色 (immunohistochemistry) 以及 real-time PCR 分析 DDX3 和 miR-218 與其下游基因之表現。以 Kaplan-Meier 法和 Cox 回歸分析 DDX3 和 miR-218 表現高低對患者之 Overall survival (OS) 和 Relapse-free survival (RFS) 之影響。結果發現 DDX3 低表現者有較差之 OS 和 RFS。另外發現 HPV E6 會經由抑制 miR-218 而增加 paxillin (PXN) 的表現，促進肺癌細胞之生長和侵襲。同時發現 PXN 的過度表現會增加與 BCL-2 之結合，而降低肺腫瘤細胞對 cisplatin 的敏感性。若腫瘤有較低之 miR -218 以及有較高表現之 PXN 差之患者則有最差之臨床預後。綜合以上之結果，本研究首次證實 HPV E6 蛋白會經由 p53 去活化，而抑制 DDX3 和 miR-218 之表現以及增加 PXN 之表現，並參與促進腫瘤之生長與轉移。同時發現 DDX3 和 PXN 之表現都與患者之臨床預後有關，因此 DDX3 和 miR- 218 都適合做為臨床治療之分子標靶，期望能有效改善 HPV 感染之肺癌患者的臨床預後和生命品質。
cells were established from patients’ pleural effusions and demonstrated that p53 was degraded by E6 to downregulate p21 and MDM2 mRNA expression and consequently promoted cell proliferation and colony formation. However, the crucial molecules involved in HPV-associated lung tumorigenesis and may predict patients’ outcome remain to be identified. In recent study, the involvement of DDX3 and miR-218 in viral-associated tumorigenesis has been reported, such as hepatitis virus-infected hepatocellular and cervical carcinoma. However, the role of DDX3 and miR-218 in HPV-associated lung tumorigenesis remains unknown. In the present study, we suspected that the modulation of DDX3 and miR-218 by E6 may promote tumor growth and metastasis; subsequently, the reduction of DDX3 and miR-218 may predict poorer survival and relapse in non-small cell lung cancer (NSCLC). Changes of DDX3 and miR-218 expressions in lung cancer cells were conducted by transfection with E6 cDNA and small interenfence or hairpin RNA plasmids. The p21, MDM2, Slug, E-cadherin, and paxillin expression, candidate downstream gene of DDX3 and miR-218, were examined in cell models by transfection with overexpression and knockdown plasmids. Cell growth and invasion were examined by the doubling time, colony formation assay, soft-agar and Boyden chamber assay. In lung tumors from lung cancer patients, expressions of miR-218, DDX3, p21, MDM2, Slug, and E-cadherin were evaluated by real-time PCR and immunohistochemical analysis, respectively. The impact of DDX3 and miR-218 expression on overall survival (OS) and relapse-free survival (RFS) was analyzed by the Kaplan-Meier test and Cox regression analysis. In present study, we showed that DDX3 was the direct target for p53 regulation in lung cancer by luciferase reporter asssy. DDX3 reduction increased cell proliferation, colony formation, soft-agar colony growth, and invasion capability. Cell growth and invasion capability increased by DDX3 reduction may be through decreased p21 expression and MDM2/Slug/E-cadherin pathway, respectively. Additionally, DDX3 reduction may reduce cisplatin sensitivity via upregulation of cIAP-2. In clinical results, we found that patients with low DDX3 tumors had poorer survival and relapse in lung cancer patients. It suggests that low DDX3 expression in tumor not only promotes tumor growth, but also enhances tumor recurrence and/or metastasis. We also provided the evidence to show that paxillin (PXN) overexpression by miR-218 reduction promotes lung cancer cell growth and invasion capability. In addition, the susceptibility to cisplatin was decreased by PXN overxpression, and this was partially through increased the interaction between PXN and BCL-2. Patients with low miR-218 and PXN-positive had the worst OS and RFS among the four combinations. In conclusion, the reduction of DDX3 and miR-218 by E6 may promote tumor malignancy and patients with poor outcome. Therefore, we suggest that DDX3 and miR-218 molecules may be potential therapeutic targets to improve outcome and life quality in HPV-infected lung cancer patients. |
