口腔癌腫瘤抑制基因的表基因調控: 一個臨床預後預測及潛在治療研究

Epigenetic Silencing of Tumor Suppressor Genes in Oral Cancer: an Outcome-Predicting and Treatment-Implicating Study

林宏益(Hon-Yi Lin)

指導教授 ： 陳永恩

國立中正大學/理學院/生命科學系分子生物研究所/博士(2015年)

摘要

背景介紹及研究目的:
口腔癌的發生率越來越高及重要性越來越增加。在生物學上，口腔癌癌細胞的特徵為基因及表基因的變異。臨床上，我們發現口腔癌的病人經廣泛性手術切除後，經常會有病理切片上不預期性的發現: 癌細胞距離手術邊緣過近的特徵。手術後有這種病理特徵的病人，臨床預後是比較差的。但是在目前的癌症診療指引上，這一類的病人並無特別建議要加強手術後輔助性治療。這一點形成了一個臨床上的困惑點。表示在這一群的病人中，可能混合有預後相對較差及相對較好的病人。因此，本研究的臨床目標為: 嘗試找出並利用表基因生物標記，有效預測病人臨床預後，將潛在預後相對較差的病人找出，以便加強治療。
放射治療是治療口腔癌的重要方式之一。然而，經放射治療後，癌細胞產生輻射抗性，進而引發癌症復發仍然是臨床放射治療的最大難題。放射生物學上，以往認為輻射抗性的主要決定因子應該在癌細胞的基因變異層次。但最近越來越多的證據顯示，放射治療後，表基因的變異及調控不良現象是很顯著的。但是，表基因的變異在輻射抗性上扮演的角色，至今仍然不清楚。因此，本研究的生物學目標為: 嘗試找出在口腔癌經放射治療後，表基因的變異及調控不良現象於輻射抗性產生過程中扮演的角色。
研究材料及方法:
在我們第一部分的實驗中，我們回溯性收集44位惡性頰癌(口腔癌的一種)的病人。每一位病人皆是手術後病理切片發現癌細胞距離刀口邊緣較近(≤5 mm)，每一位病人皆有接受手術後放射治療。我們再次切片病人的病理蠟塊，將其中含癌細胞豐富的區域(>70%)在顯微鏡下切出後，萃取其去氧核醣核酸片段。我們使用甲基化特異性聚合酶鏈鎖反應(Methylation-specific PCR)來偵測四個已知抑癌基因啟動子的甲基化狀態: 包含RASSF1A, DAPK, IRF8, 及SFRP1等。病人經放射治療後的局部區域癌病控制率為主要臨床觀測標的。
在我們第二部分的實驗中，我們先使用分次放射治療從口腔癌癌細胞的原始株中(OML1)，建立具有輻射抗性的子細胞株(OML1-R)。接下來，我們使用甲基化特異性微陣列(methylation-specific microarray-based analysis)來分析兩個細胞株(OML1-R 及OML1)之間的甲基化變異。於微陣列實驗中，發現多個經放射線照射後甲基化標記變多的基因。從中，我們進一步使用MBDcap-PCR 及亞硫酸鹽焦磷酸測序法(bisulphite pyrosequencing)來驗證FHIT 基因啟動子的甲基化狀態。我們也使用染色質免疫共沉澱-聚合酶鏈鎖反應(quantitative ChIP-PCR)來定量FHIT基因啟動子附近的組蛋白甲基化標的狀態(H3K27me3)。之後，在六個細胞株中(OCSL, SAS, SCC4, SCC25, and parental OML1, as well as in OML1-R cells)，我們反向驗證(抑低或拉高)FHIT基因的表現來觀察其對於輻射抗性的影響。我們使用細胞群落形成分析法(colony formation assay)及腫瘤生成分析法(tumorigenic assay)執行體內、外實驗來驗證輻射敏感度。我們使用流式細胞儀(flow cytometry)來分析細胞週期的分布變化(Cell cycle distribution)及細胞凋亡(apoptosis)的程度。另使用表基因調控藥物(如5-Aza, TSA, 及GSK343等)，進行表基因調控介入處置，來觀察表基因變化對於輻射抗性的影響。在臨床驗證方面，我們於2004年至2008年間，回溯性收集40位配對後的口腔癌病人。每一位病人一樣皆有手術後切片發現癌細胞距離刀口邊緣過近(≤5 mm)的病理發現。我們使用亞硫酸鹽焦磷酸測序法(bisulphite pyrosequencing)來偵測啟動子附近甲基化變化的狀態。另使用免疫組織化學染色(Immunohistochemistry stain)來確認蛋白質表現狀態。經放射治療後的局部區域癌病控制率及整體存活率為臨床觀測標的。

研究結果:
在我們第一部分的實驗中，有40位男性及4位女性，中位年齡為53.5歲(範圍,32 – 82歲)。針對癌症局部區域復發，多變項回歸分析發現兩個獨立的預測因子。第一項為手術後病理發現癌細胞非常接近切口邊緣(≤1 mm)，風險比值(HR)為4.96; 95%信賴區間(95% CI)為1.63 – 15.09; P = 0.018。第二項為抑癌基因DAPK啟動子之高度甲基化，風險比值(HR)為2.83; 95%信賴區間(95% CI)為1.05 – 7.63; P = 0.042。最高之癌症局部區域復發率發生在同時具有這兩項因子的病人身上，風險比值(HR)為8.05; 95%信賴區間(95% CI)為2.56 – 25.82; P = 0.002。在這些高復發風險的病人群中，癌症無病存活期也較短，但整體存活期則無統計學上之顯著差異。
在我們第二部分的實驗中，藉由使用甲基化特異性微陣列(methylation-specific microarray-based analysis)來分析兩個細胞株(OML1-R 及OML1)之間的甲基化變異後，我們進一步驗證抑癌基因FHIT。我們發現在OML1-R細胞中 FHIT基因轉錄受抑制的原因有三，分別為啟動子的高度甲基化、抑制性組蛋白標記(H3K27me3)的增加及甲基轉移酶EZH2的過度表現。使用表基因介入處置或去除EZH2能回復抑癌基因FHIT的表現。另一方面，不論在體內或體外實驗，使用外加FHIT表現的人為操作都可以顯著抑制腫瘤的生長，並同時可以使已經具有抗性的癌細胞回復對放射線的敏感性。相關的機轉是藉由回復Chk2的磷酸化及細胞週期檢查站(G2/M arrest)的功能。另外，在臨床上，FHIT啟動子的高度甲基化與病人檢體上的蛋白質表現呈現高度逆相關的關係。並且在40位經配對後的口腔癌病人身上，可以獨立預測癌症局部區域控制率及整體存活率。進一步的老鼠體內治療實驗證實，抑制基因甲基化能有效回復具有輻射抗性腫瘤的放射敏感性。綜合這些實驗結果，我們發現表基因抑制FHIT的表現對於接受放射治療後產生輻射抗性至少具有部分的影響。且藉由偵測FHIT啟動子的甲基化程度可以有效預測臨床預後。合併表基因介入治療及放射治療可建議於臨床試驗中進行後續驗證。
研究結論:
臨床上，我們的研究結果顯示表基因學生物標記，能有效地預測癌症病人的預後，尤其是合併病理預測因子時。在口腔癌病人手術後，有非預期性的癌細胞距離刀口邊緣過近的病理發現時，這種合併預測因子的策略能有效的辨別出預後較差的病人群，進而針對這些高復發風險的病人加強治療，以期增加癌病控制率及存活率。
生物學上，我們的研究結果顯示抑癌基因FHIT受到表基因調控而使其表現遭受抑制這個現象對於口腔癌細胞經放射治療後產生輻射抗性是其中一項重要的調控機制。更重要的是，利用表基因介入治療使FHIT重新表現，可回復口腔癌細胞的輻射敏感度，暗示了其在臨床上的潛在治療應用。尤其是同時使用去甲基化藥物、EZH2抑制劑及放射治療的合併療法更是具有後續臨床試驗的驗證價值。

關鍵字

組蛋白調控 ； 表基因 ； 手術後癌細胞距離刀口邊緣過近 ； 頰癌 ； 頭頸癌 ； 抑癌基因 ； 輻射抗性 ； 去氧核醣核 酸甲基化 ； 口腔癌 ； 放射治療 ； 游離輻射線 ； 輻射敏感性

並列摘要

Background and Aims:
Oral cancer increases its incidence and significance worldwide. Biologically, oral cancers are characterized by both genetic and epigenetic aberrations. Clinically, after radical surgery, unexpected close surgical margins are not uncommon in oral cancer patients, presenting poor clinical outcomes. However, close margin alone does not independently guide post-operative therapies, revealing a clinical debate. As a result, the clinical aim of the present study was to explore epigenetic bio-predictors for stratifying this clinically debating patient population.
Moreover, in managing oral cancers, ionizing radiation (IR) is an essential modality. However, post-irradiation radioresistance is still the main treatment obstacle. The major biological determinant for IR resistance was previously considered at genetic level because DNA is the major target of IR damage. Recently, more accumulative evidence observed significantly epigenetic disturbance after IR. However, the role of such alterations in the development of radioresistance is not fully explored. Hence, the biological aim of the present study was to explore the role of epigenetic disturbance in the process of IR resistance in irradiated oral cancer.
Materials and Methods:
In the Part I experiments, from 2000 to 2008, we retrospectively recruited 44 resected buccal cancer patients with a close surgical margin of ≤5 mm. All patients had post-operative radiotherapy. Genomic DNA was extracted from tumor-enrich areas that contained cancer cells of >70%. Methylation-specific PCR was performed to detect promoter methylation of four tumor suppressor genes, including RASSF1A, DAPK, IRF8, and SFRP1. Post-irradiation locoregional control was defined as the primary end point.
In the Part II experiments, firstly, we used fractionated irradiations to establish a radioresistant cell subline (OML1-R) from parental OML1 oral cancer cells. Next, we used methylation-specific microarray-based analysis to explore differently methylated genes between OML1-R and OML1 oral cancer cells. Of the post-irradiation hypermethylated genes, we used MBDcap-PCR and bisulfite pyrosequencing to further validate FHIT. We also used quantitative ChIP-PCR to examine the histone chromatin status of the promoter region of FHIT. Then, we performed reciprocal experiments to knockdown or overexpress the expression of FHIT in five different oral cancer cell lines, i.e., OCSL, SAS, SCC4, SCC25, and parental OML1, as well as in OML1-R cells. For measuring the level of radiosensitivity, we used in vitro colony formation assay and in vivo tumorigenic assay, respectively. Cell cycle distribution and apoptosis were analyzed by using flow cytometry. Agents that altered epigenetic markers were used for intervention, such as 5-Aza, TSA, and GSK343. For clinical validation, from 2004 to 2008, we retrospectively recruited 40 match-paired oral cancer patients with a close surgical margin of ≤5 mm. Bisulfite pyrosequencing was performed to detect promoter methylation. Immunohistochemistry stain was used to confirm protein expression. Post-irradiation locoregional control and overall survival were defined as study end points.
Results:
In the part I experiments, there were 40 males and 4 females, with a median age of 53.5 years (range, 32 - 82 years). Multivariate analysis identified two independent predictors for locoregional recurrence: very close margin of ≤1 mm (HR: 4.96; 95% CI, 1.63 – 15.09; P = 0.018) and promoter hypermethylation of DAPK (HR: 2.83; 95% CI, 1.05 – 7.63; P = 0.042). The highest risk of locoregional recurrence was observed in patients with both of the two factors (HR, 8.05; 95% CI, 2.56 – 25.82; P = 0.002) when compared with patients with none. Shorter disease-free survival, but not overall survival, was also observed.
In the part II experiments, by using a methylation microarray, we identified promoter hypermethylation of FHIT (fragile histidine triad) in radioresistant OML1-R cells when compared with parental radiosensitive OML1 oral cancer cells. Further analysis confirmed that transcriptional repression of FHIT was due to promoter hypermethylation, H3K27me3 and overexpression of methyltransferase EZH2 in OML1-R cells. Epigenetic interventions or depletion of EZH2 restored FHIT expression. Ectopic expression of FHIT inhibited tumor growth in both in vitro and in vivo models, while also resensitizing radioresistant cancer cells to irradiation, via restoring Chk2 phosphorylation and G2/M arrest. Clinically, promoter hypermethylation of FHIT inversely correlated with its expression and independently predicted both locoregional control and overall survival in 40 match-paired oral cancer patient samples. Further in vivo therapeutic experiments confirmed that inhibition of DNA methylation significantly re-sensitized radioresistant oral cancer cell xenograft tumors. These results show that epigenetic silencing of FHIT contributes partially to radioresistance and predicts clinical outcomes in irradiated oral cancer. The radiosensitizing effect of epigenetic interventions warrants further clinical investigation.
Conclusions:
Clinically, our data demonstrated epigenetic biomarkers is useful in outcome prediction, especially in combination with pathological factors. Remarkably, this combined strategy effectively stratifies oral cancer patients with post-operative unexpected close surgical margins. After effective stratification, more aggressive managements are able to prescribe for high risk patients.
Biologically, our data confirmed an essential role of epigenetic silence of FHIT in the process of post-irradiation radioresistance. Impressively, applying epigenetic interventions to re-express FHIT is able to restore radiosensitivity, implicating a therapeutic role. Further clinical trials are warranted for confirming the treatment efficacy, especially the combination of DNA demethylating agents, EZH2 inhibitors, and radiotherapy.

並列關鍵字

radioresistance ； ionizing radiation ； radiotherapy ； histone modification ； DNA methylation ； close surgical margin ； buccal cancer ； oral cancer ； head and neck cancer ； epigenetics ； radiosensitivity ； tumor suppressor gene ； EZH2 ； DAPK ； RASSF1A ； FHIT

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