I. IL-10 在肺癌患者對 HPV 感染之感受性研究 II. P53 突變、p53 codon72 和 MDM2 SNP309 基因多形性對早期肺癌患者腫瘤復發與臨床預後之影響研究

I. The role of IL-10 on the susceptibility to HPV infection of lung cancer II. The impacts of p53 mutation status, p53 Pro72 and MDM2 SNP 309 polymorphisms on tumor recurrence and clinical outcome of early-stage lung cancer

簡文斌(Wen-Pin Chien)

指導教授 ： 李 輝

中山醫學大學/醫學院/醫學分子毒理學研究所/博士(2009年)

摘要

第一部份中文摘要
過去本研究室發現台灣不抽煙之女性肺癌患者較男性患者，有較高之 HPV 16 與 18 的感染率，但為何女性肺癌有較高之 HPV 感染率，則需進一步之探討。 Interleukin 10 (IL-10) 為維持 TH1 及 TH2 兩大類細胞激素平衡之重要細胞激素。在動物實驗已證實 IL-10 與病毒持續性感染有關。因此假設 (1) 女性肺癌患者有較男性為高之HPV感染率，可能是女性有較高之IL-10 mRNA表現，(2) HPV感染之台灣女性肺癌可能較沒有感染之女性患者，有較高之 IL-10 mRNA表現。本研究以 real-time PCR 分析 121 位肺癌患者腫瘤組織中 IL-10 mRNA 之表現，結果發現在女性、不抽菸之患者，有較高 IL-10 mRNA 表現 (61.7% vs 37.7%, p = 0.008 for gender; 55.8% vs 34.3%, p = 0.032 for smoking status)，且在女性肺癌患者之 IL-10 mRNA 表現與 HPV 感染具有正相關性 (p = 0.022)。為了解為何女性患者較男性患者有較高之 IL-10 轉錄活性？本研究以 luciferase- reporter assay和ChIP之結果顯示， p53 可能是 IL-10 之主要轉錄因子，其次發現雌性素可能會經由 ER 路徑以及組織蛋白之醯基化，而促進 IL-10 之轉錄活性。因此推測女性較男性患者有較高之 IL-10 mRNA 表現，可能與其腫瘤之 p53 突變率較低和體內有較高之雌性素有關。為了解 IL-10 mRNA 表現是否與 HPV 之清除有關？以 real-time PCR 分析 60 位 HPV 有感染肺腫瘤組織之 IL-10 mRNA表現與 HPV DNA 拷貝數，結果發現腫瘤組織中 IL-10 mRNA表現高之患者，有較高之 HPV DNA 拷貝數，兩者間呈正相關性 (p = 0.030)，而 HPV 感染之 TL-1、TL-2 肺癌細胞亦有較高之 IL-10 表現。若以 PHA 處理健康者之 PBMC 培養於 TL-1 之condition medium (CM)，則發現 T-bet 和 IFNγ 被誘發之表現會顯著降低，但以 IL-10 抗體中和，則又恢復表現。本研究發現 IL-10 mRNA 表現高之 TL-1 細胞，被腫瘤細胞毒殺之阻抗性，顯著高於 IL-10 mRNA 低表現之 TL-4 細胞，若同樣以 IL-10 抗體中和後，此抑制現象亦顯著恢復。顯示 IL-10 除了會抑制 T-bet 和 IFNγ 表現，亦會降低免疫細胞清除病毒之能力。在臨床預後分析結果顯示， IL-10 mRNA 高表現又同時有 HPV 16/18 E6 蛋白表現之女性肺癌患者，較 IL-10 低表現又同時沒有HPV16/18 E6 蛋白之患者，有較差之臨床預後。因此 IL-10 表現高可能會抑制清除 HPV 感染以及毒殺腫瘤細胞之免疫能力，而促進 HPV 引起之腫瘤形成。

第二部分中文摘要:
雖然診斷技術日益改進與抗癌新藥不斷開發，非小細胞肺癌患者之存活率至今仍僅有 15% 左右，顯示建立有效之生物標記，預測早期肺癌患者之腫瘤復發或轉移機率以及其臨床預後，應是提升肺癌臨床治療之重要策略。已知 p53 是決定腫瘤發展、藥物反應、以及腫瘤轉移的主要調控者。因此本研究收集306位肺癌患者，以自動 DNA 定序儀分析肺腫瘤組織中之 p53 基因突變，以 RFLP 方法分析 306 肺癌患者非腫瘤組織中之 MDM2 SNP 309 基因型，並以 real-time PCR 分析其中 198 位可獲得腫瘤 mRNA 患者MDM2 及 GADD45 mRNA 表現作為評估 p53 功能之指標。同時以 RFLP 方法分析 266 位患者 p53 codon 72 基因型，這些資料用來了解肺腫瘤組織中之 p53 轉錄活性及功能，是否可用來評估早期肺癌之腫瘤復發率和臨床預後？結果發現 p53 基因正常合併 MDM2 GG 基因型之患者，MDM2 和 GADD45 mRNA 表現量顯著高於 p53 基因正常合併 MDM2 TT 型患者，顯示 p53 基因正常和 MDM2 GG 基因型之患者具有較佳之 p53 轉錄活性或功能，臨床上亦可觀察到早期肺癌患者 p53 基因正常和 MDM2 TT 基因型 (HR=2.9, p = 0.01) 和 p53 突變之患者 (HR=2.5, p = 0.01) 有較差之臨床預後。在追蹤患者腫瘤復發率時發現，早期肺癌患者 p53 基因正常合併 MDM2 GG 型患者復發率，較 TT/TG 型或 p53 突變患者為低，但因早期患者人數過少，未達到統計上差異 (p = 0.087)。在分析 p53 codon 72 基因型發現， Pro allele 之患者較 Arg/Arg 基因型患者有較高之腫瘤復發率 (25% vs 7.5%, p = 0.022)，在 p53 突變合併 Pro allele 較 p53 正常合併 Arg/Arg 之肺癌患者，則同樣有較高腫瘤復發之傾向 (31% vs 10.3%, p = 0.052)，在第一期肺癌患者亦可觀察到同樣之趨勢 (36.4% vs 5.9%, p = 0.052)。在臨床預後上，可觀察到 p53 突變合併 Pro allele 之第一期患者，較 p53 基因正常合併 Arg/Arg 之患者有較差之臨床預後 (HR = 2.66, 95% CI, 1.21-5.85, p = 0.02)。因此藉由簡易偵測 p53 相關之三種生物標記，用來評估早期肺癌患者之腫瘤復發和其臨床預後，將有助於建立較好之臨床治療策略。

關鍵字

肺癌 ； 介白素-10 ； 人類乳突瘤病毒 ； 預後

並列摘要

Part I
Our previous report indicated that HPV16/18 infection prevalence in female lung tumors was significantly higher than in male tumors. However, the different susceptibility to HPV infection between genders remains to be elucidated. IL-10 has been shown to suppress virus clearance to lead virus persistent infection in animal model, and it is conceivable that IL-10 is an important cytokine to regulate the homeostasis between TH1 and TH2 cytokine secretions. We therefore hypothesize that IL-10 mRNA expression level in female lung tumors may be higher than that in male tumors to explain why female has higher susceptibility to HPV infection. Herein, 121 lung tumors were enrolled to examine IL-10 mRNA and HPV DNA copy number by real-time PCR to verify whether IL-10 mRNA level is positively correlated with HPV infection and virus clearance. Our data showed that IL-10 mRNA levels in tumors from female and nonsmokers were higher than those in tumors from male and smokers (61.7% vs 37.7%, p = 0.008 for genders; 55.8% vs 34.3%, p = 0.032 for smoking status), and IL-10 mRNA levels were positively correlated with HPV infection in female tumors (p = 0.022), not in male tumors. Moreover, lung tumors with higher IL-10 mRNA levels had higher HPV DNA copy number than those with lower IL-10 mRNA levels (p = 0.030), suggesting that IL-10 expression may be associated with HPV persistent infection in lung tumors. To verify which molecule(s) could be involved in IL-10 transcriptional regulation, luciferase reporter and ChIP analysis showing that p53 may be an important transcription factor to upregulate IL-10 transcription. Additionally, 17-βestrodiol may synergistically enhance IL-10 transcriptional upregulation by p53 in p53 wild-type lung cancer cells. To elucidate whether lung cancer cells with higher IL-10 expression may be more suppression of TH1 cytokines expressions than those with lower IL-10 expression to modulate the the capability of virus clearance and tumor surveillance, two TH1 biomarkers, T-bet and IFN-γ were examined in PBMC from healthy donor which were cultured in the condition medium of TL-1 cells with higher IL-10 in comparison with those in the condition medium of TL-4 cells with lower IL-10 expression. Our data showed that T-bet and IFN-γ expressions in the condition medium of TL-1 cells were more suppressed than those in the condition medium of TL-4 cells. However, the suppression can be restored by the neutralization of IL-10 antiboy. The cytotoxicity of PBMC to TL-1 cells was significantly lower than that of PBMC to TL-4 cells. We also observed that patients with higher IL-10 mRNA plus HPV16/18 E6-positive expression had poorer prognosis than those with lower IL-10 mRNA plus HPV16/18 E6-negative expression. In summary, high expression of IL-10 may suppress the capability of virus clearance and tumor cell cytotoxicity to promote HPV-associated lung tumorigenesis.

Part II
The 5-year survival rate of non-small cell lung cancer remains around 15%, even though the improvement of the diagnostic techinology and new drug discovery are progress since two-decades. P53 is considered to play a key regulator on tumor progression, tumor recurrence, and chemo-therapeutic response. In the present study, 306 lung tumors and adjacent normal lung tissues were enrolled for p53 mutations and MDM2 309SNP by direct sequencing and PCR-RFLP, respectively. Among these, 266 adjacent normal lung tissues were collected to evaluate p53 codon 72 polymorphisms by PCR-RFLP, and 198 lung tumors were collected to determine MDM2 and GADD45 mRNA expressions by real-time PCR for assessment of p53 transcriptional activity. These data were used to verify whether p53 mutation, p53 codon72 genotypes, and MDM2 SNP309 genotypes and their combinations could be associated with tumor recurrence and prognosis. Our data indicated that patients harbored wild-type p53 combined with MDM2 GG genotype had the highest MDM2 and GADD45 mRNA expression levels among the four combinations, and stage-I patients harbored wild-type p53 combined with MDM2 GG genotype had more favorable prognosis than those with wild-type p53 combined with MDM2 TT genotype and p53 mutations. On the contrast, patients with p53 mutations had worser prognosis than those with wild-type p53. In cohort study, stage-I patients harbored wild-type p53 combined with MDM2 GG genotype had lower tumor recurrence rate compared with those harbored MDM2 TT/TG genotype or p53 mutation, but it did not reach statistically significance because a few case numbers were available (p = 0.087). On the other hand, patients with p53 codon 72 Pro allele had higher tumor recurrence rate than those with Arg/Arg genotype (p = 0.022). In addition, patients harbored p53 mutation combined with Pro allele had a higher trend in tumor recurrence rate compared with those harbored wild-type p53 combined with Arg/Arg genotype (p = 0.052), and a similar trend was also observed in stage-I patients (p = 0.052). In the prognostic significance of p53 codon 72 poymorphism, stage I patients harbored p53 mutation combined with Pro allele had poorer prognosis than those harbored wild-type p53 combined with Arg/Arg genotype (HR=2.66, 95% CI, 1.21-5.85, p = 0.02). Therefore, the information of p53 mutation, p53 codon 72 polymorphisms, and MDM2 SNP309 genotype obtained by simple methods may be feasible to assess the tumor recurrence and the clinical outcome of stage I lung cancer, and helpful to choose a favorable clinical strategy to improve patients’ outcome.

並列關鍵字

Lung cancer ； IL-10 ； HPV ； p53 ； MDM2 ； Survival

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