三陰性乳癌分子生物特徵研究與探討

Abstract

中文摘要 導論 三陰性乳癌是指乳癌細胞內部的雌激素受體、黃體激素受體和細胞膜上的人類表皮因子受體皆為陰性的乳癌。與其他類型的乳癌相比，三陰性乳癌本質上較具有侵略性，而且其腫瘤細胞增長的機制也與其他類型的乳癌不同。目前醫療上尚未有一個標靶藥物可對三陰性乳癌進行有效的治療，因此三陰性乳癌的治療模式相對貧乏，且其病患術後遠處轉移復發的比例也較高。近年來有許多的研究資料顯示異常的信使核糖核酸(messenger RNA)和微型核糖核酸(microRNA)基因表現對人類腫瘤細胞的增生和惡化有著重大的影響，然而目前醫學研究上對於三陰性乳癌的基因表現及其內部調控機制仍然不甚明瞭。國外有專家學者提出使用數個關鍵基因的基因表現做為乳癌病患預後的一個指標，但其研究結果通常只適用在雌激素受體和黃體激素受體陽性的乳癌病患族群，無法準確的預估三陰性乳癌病患術後腫瘤轉移的可能性。因此本研究著重於探討利用三陰性乳癌的信使核糖核酸的基因表現，做為三陰性乳癌病患其術後癌細胞遠處轉移預測的一個方式，以及近年來腫瘤醫學的熱門研究重心微型核糖核酸在三陰性乳癌細胞內的表現特徵。 研究方法 本研究中所使用的五十一個三陰性乳癌檢體和一百零六個管腔型乳癌檢體及臨床病理資料皆由台大醫院臨床醫師提供。研究團隊使用寡核苷酸微陣列晶片技術來量測乳癌檢體內的信使核糖核酸基因表現，再將各個檢體內的基因表現量做正規化，接著合併分析三陰性乳癌病患的腫瘤遠處轉移臨床資料和其腫瘤內的信使核糖核酸基因表現以取得其統計上的關聯性。此外研究團隊也使用高通量定序技術對二十四個三陰性乳癌檢體和十四個其周遭正常乳房組織檢體中的微型核糖核酸表現進行量測，之後用百分位正規化的方式為每一個檢體中的微型核糖核酸表現做前置處理，再使用統計檢定分析來找出在三陰性乳癌中有顯著差異表現的微型核糖核酸。研究團隊進一步針對這些微型核糖核酸進行定量即時聚合酶鏈鎖反應的實驗，確認所找到的微型核糖核酸在三陰性乳癌和正常乳房組織中的確有顯著的差異表現。作者應用微型核糖核酸調控基因預測的miRanda演算法，找出可能被這些微型核糖核酸所調控的基因，並用螢光素酶檢測法做進一步的驗證。本論文並深入的探討前人文獻中所提出關於這些顯著表現的微型核糖核酸在其他人類癌症腫瘤內所調控的基因與病理機制。 研究結果與討論 作者使用階層式集群法來分析三陰性乳癌和管腔型乳癌的基因表現，發現大部分(94%)的三陰性乳癌皆帶有顯著高量表現的類基底型角質素基因，管腔型乳癌則無此基因表現特徵。作者接著在遠處腫瘤轉移的三陰性乳癌病患族群中找出顯著表現的信使核糖核酸基因，並使用留一交互檢驗法和接受器操作特性曲線來找出一個由四十五個基因所組成之優化預測模組，其預測準確度在本研究病患族群可達到98%。此外這個四十五個基因的預測模組在另一個獨立的三陰性乳癌病患族群也可以有效的預測其病患術後腫瘤復發的狀況，然而若使用其他已發表的乳癌預後預測基因模組則無法在此病患族群做準確的預測。作者進一步的研究這四十五個基因，發現到有許多的基因都與調控人體免疫反應和發炎機制的轉化生長因子β訊號傳遞密切的相關。在微型核糖核酸的研究方面，團隊使用高通量定序技術找到一組由二十五個顯著差異表現的微型核糖核酸所組成的辨識模組，可藉由此模組的微型核糖核酸表現量精確地分辨三陰性乳癌組織和周遭正常乳房組織。作者並發現許多在三陰性乳癌中顯著表現的微型核糖核酸皆群聚於人類染色體中的七個不同的基因位點，且進一步用定量即時聚合酶鏈鎖反應實驗來驗證這些微型核糖核酸群聚組的表現量。作者並藉由螢光素酶檢測法發現在三陰性乳癌內顯著表現的微型核糖核酸miR-130b-5p會直接附著於CCNG2抑癌基因的3′ 非轉譯區並抑制CCNG2在三陰性乳癌的基因表現，進而增快三陰性乳癌癌細胞的細胞週期。 結論 作者在本篇論文中清楚的的描述信使核糖核酸和微型核糖核酸在三陰性乳癌內的的表現特徵，並提出了一個藉由四十五個預後基因的基因表現來預測三陰性乳癌病患其腫瘤遠處轉移的預測模組。此預測模組在另一個獨立的三陰性乳癌病患族群中也同樣的能夠準確的預估其病患腫瘤復發的可能性。這個由四十五個基因所組成的的預測模型可幫助臨床醫師對三陰性乳癌病患腫瘤遠處轉移的可能性進行初步評估，並以此作為臨床藥物治療的一個輔助參考。此外研究團隊使用高通量定序技術找出在三陰性乳癌和其周遭正常乳房組織有顯著差異表現的微型核糖核酸，並深入探討其在人類癌症腫瘤內的調控機制，此研究成果讓我們對於三陰性乳癌內的微型核糖核酸表現特徵與其對於癌細胞的影響有更清楚的認識。

Abstract Introduction Triple-negative breast cancer, immunohistochemically defined by lacks of expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, is a type of breast cancer with aggressive tumor behavior and distinct disease etiology. Due to the lack of an effective targeted medicine, treatment options for triple-negative breast cancer are few and its recurrence rates are high. Recent studies have shown that deregulated gene expression is heavily involved in the progression of human cancer. However, the gene expression characteristics in triple-negative breast cancer are still poorly understood. Moreover, although various multi-gene prognostic markers have been proposed for the prediction of breast cancer outcome in published literatures, most of them were proven clinically useful only for estrogen receptor-positive breast cancers. Reliable identification of triple-negative patients with a favorable prognosis using gene expression data is not yet possible. Methods In this study, clinicopathological information and gene expression microarray data from 51 triple-negative and 106 luminal breast cancers were collected at National Taiwan University Hospital. Gene expression data of triple-negative breast cancer tissues were collected using Agilent oligonucleotide microarrays. Gene expression data along with distant metastasis follow-up information of the triple-negative breast cancer patients were analyzed in this work. In addition, microRNA (miRNA) expression data of 24 triple-negative breast cancers and 14 adjacent normal tissues were analyzed using deep sequencing technology. Expression levels of miRNA reads from each sample were normalized with the quantile-quantile scaling method. Quantitative reverse transcription PCR was performed for validation of deregulated miRNAs in triple-negative breast cancer. Potential target candidates of miRNAs were predicted using the miRanda target prediction algorithm and were further verified using luciferase reporter assays. Previously validated miRNA target genes of the deregulated miRNAs were investigated and their molecular pathways associated with cancer progression were discussed. Results and discussion Hierarchical clustering analysis of the gene expression data revealed that the majority (94%) of triple-negative breast cancers were tightly clustered together carrying strong basal-like characteristics. A 45-gene prognostic signature giving 98% predictive accuracy in distant recurrence of our triple-negative patients was determined using the receiver operating characteristic analysis and leave-one-out cross validation. External validation of the prognostic signature in an independent microarray dataset of 59 early-stage triple-negative patients also obtained statistical significance (hazard ratio 2.29, 95% confidence interval (CI) 1.04-5.06, Cox p = 0.04), outperforming five other published breast cancer prognostic signatures. The 45-gene signature identified in this study revealed that TGF-β signaling of immune/inflammatory regulation may play an important role in distant metastatic invasion of triple-negative breast cancer. Deep sequencing analyses of miRNA expression revealed that a novel 25-miRNA signature was able to effectively distinguish triple-negative breast cancers from surrounding normal tissues. We documented the evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNA genes in triple-negative breast cancer. Two of these miRNA clusters (miR-143-145 at 5q32 and miR-497-195 at 17p13.1) were markedly down-regulated in triple-negative breast cancer, while the other five miRNA clusters (miR-17-92 at 13q31.3, miR-183-182 at 7q32.2, miR-200-429 at 1p36.33, miR-301b-130b at 22q11.21, and miR-532-502 at Xp11.23) were up-regulated in triple-negative breast cancer. Noticeably, miR-130b-5p from the miR-301b-130b cluster was shown to directly target the cyclin G2 (CCNG2) tumor-suppressor gene in luciferase reporter assays. Overexpression of miR-130b-5p was shown to significantly repress CCNG2 expression and enhance cell cycle progression in triple-negative breast cancer cells. Conclusions Our work delivers a clear picture of the global messenger RNA and miRNA regulatory characteristics in triple-negative breast cancer. A novel 45-gene prognostic signature was found to be statistically predictive in distant metastasis of triple-negative breast cancer. The 45-gene signature, if further validated, may be a clinically useful tool in risk assessment of distant metastasis for early-stage triple-negative patients. Moreover, miRNA expression of triple-negative breast cancer was measured using deep sequencing technology. A panel of 25 differentially expressed miRNAs identified from 24 triple-negative breast cancers and 14 adjacent normal tissues was found to effectively distinguish triple-negative breast cancers from surrounding normal tissues. Real-time PCR validations of the deregulated miRNAs further supported our findings from the sequencing analyses. The miR-130b-5p-CCNG2 axis identified in this study may play a role in the malignant progression of triple-negative breast cancer.