BRCA1啟動子甲基化及CSPG4表現與乳癌之相關性探討

BRCA1 promoter methylation and CSPG4 expression in breast cancer

徐仲亨(Nicholas Chung-Heng Hsu)

指導教授 ： 侯明鋒

高雄醫學大學/醫學院/醫學研究所/博士(2013年)

摘要

三陰性乳癌為雌激素受體(ER)、黃體素受體(PR)及第二型表皮細胞接受體(HER2)免疫染色均呈現陰性的乳癌腫瘤，目前約占所有乳癌的百分之十五，是臨床上被認為最棘手難治的乳癌族群。這類腫瘤細胞較具侵襲性，臨床預後差且與轉移相關。新型標靶藥物，包括賀爾蒙和HER2導向治療，在此類腫瘤皆無效用，所以目前治療策略仍以化學治療為主。然而，三陰性乳癌病人於治療後多產生抗藥性、癌症復發和癌細胞迅速擴展。
BRCA1參與DNA的修補機制，在乳癌是極為重要的腫瘤抑制基因，特別是三陰性乳癌中BRCA1突變率高，且BRCA1相關的乳癌也多為三陰性乳癌。相較於西方國家，台灣地區的研究卻發現， BRCA1 突變發生率偏低，並不扮演重要角色。由於DNA甲基化(methylation)是除了基因突變及缺失之外腫瘤抑制基因失去活性的另一種機轉，DNA甲基化異常在多種腫瘤的形成中扮演重要的角色。本研究其一目的為探討BRCA1甲基化在台灣乳癌病人的重要性。
本研究從139位早期乳癌病人的腫瘤標本萃取DNA，然後用甲基特異聚合酶連鎖反應(methylation-specific PCR)進行BRCA1基因啟動子區(promoter) methylation測定，並探討其與乳癌的臨床病理特徵之關係。在139個乳癌病人，共發現78例BRCA1 promoter methylation。卡方檢定顯示，BRCA1 promoter methylation與三陰性乳癌達顯著相關。Kaplan-Meier方法分析發現，BRCA1 promoter methylation與乳癌病人的低整體和無病存活率達顯著相關。經多變項分析方法校正病人的年齡，腫瘤大小，分級，淋巴結轉移也發現，BRCA1 promoter methylation與乳癌病人的整體和無病存活率顯著相關。
有研究推測，化學與放射線治療皆無法殺死的癌症幹細胞的存在是三陰性乳癌病人臨床預後差的部分原因。硫酸軟骨素蛋白多醣4(CSPG4)被報導表現於癌症幹細胞及三陰性乳癌細胞中，實驗結果證明CSPG4抗體抑制三陰性乳癌細胞的生長、粘附和轉移，所以CSPG4蛋白被提出可能成為三陰性乳癌的治療標靶，然而，CSPG4在乳癌的表現頻率為何及其臨床意義尚未知。
本研究的目的之二為探討CSPG4表現與其臨床意義。此研究利用組織微陣列技術及免疫組織化學染色對240名乳癌病人的組織標本進行CSPG4免疫染色分析，並將CSPG4染色結果與臨床、病理特徵、整體和無病存活率做分析。發現與之前的一份報告不一致，本實驗結果指出，CSPG4表現與三陰性乳癌不相關。Kaplan-Meier方法分析發現，CSPG4表現與無病存活率呈顯著相關。用多變項分析方法調整患者疾病分期，腫瘤分級，ER，PR及HER2表現，CSPG4表現仍與較差的整體和無病存活率相關。由於本研究結果顯示CSPG4表現雖與三陰性乳癌並無相關，卻與病人存活率顯著相關，所以未來也許可以針對CSPG4，對較具侵略性的乳癌做治療。
本研究結論為，一、台灣三陰性乳癌與BRCA1 promoter methylation為顯著相關，BRCA1 promoter methylation並與臨床結果差相關。二、不同於先前的研究報告，三陰性乳癌與CSPG4表現並無相關，然而，CSPG4表現可為乳癌預後差的指標。

關鍵字

三陰性乳癌 ； BRCA1 ； 甲基化 ； 硫酸軟骨素蛋白多醣

並列摘要

Triple-negative breast cancer (TNBC) consisted of approximately 15 % of all breast cancer diagnosed and is associated with poor clinical outcome, high rates of recurrence following chemotherapy, and metastasis. Novel targeted agents, including hormonal and HER2-directed therapies, are ineffective in this setting. Cytotoxic chemotherapy remains the backbone of current treatment strategies.
The BRCA1 gene has been shown to be strongly associated with the occurrence of TNBC. Studies conducted in Taiwan had suggested that the mutation of BRCA1 contributes little to the occurrence of breast cancer in the Taiwanese population. The epigenetic transcriptional silencing provides an alternative mechanism for the loss of function of tumor suppressor gene during cancer development. The first objective of the current study was to examine the prevalence and clinical relevance of BRCA1 promoter methylation in Taiwanese women with breast cancer.
Breast tumors from a cohort of 139 early-stage breast cancer patients were obtained during surgery before adjuvant treatment for DNA extraction. Methylation of BRCA1 promoter region was determined by methylation-specific PCR and the results were related to clinical features and outcome of patients using statistical analysis. Methylation of the BRCA1 promoter was detected in 78 of the 139 tumors. Chi-square analysis indicated that BRCA1 promoter methylation correlated significantly with TNBC. The Kaplan-Meier method showed that BRCA1 promoter methylation was significantly associated with poor overall survival (OS) and disease-free survival (DFS). Multivariate analysis which incorporated variables of patients’ age, tumor size, grade, and lymph node metastasis revealed that BRCA1 promoter methylation was associated with OS and DFS.
It has also been speculated that the poor clinical prognosis of TNBC is attributable in part to the presence of substantial chemotherapy- and radiotherapy-resistant cancer stem cells (CSCs) population within tumors. Chondroitin sulfate proteoglycan 4 (CSPG4) was found to be expressed in CSCs and TNBC cells. Anti-CSPG4 antibody inhibited growth, adhesion, and migration of TNBC cells. CSPG4 protein has recently been proposed as a therapeutic target in breast tumors with TNBC phenotype. However, the frequency of the CSPG4 phenotype in TNBC has not yet been determined in a larger set of TNBC patients. Moreover, the biological behavior or significance of CSPG4 in TNBC remained unknown.
This second objective of the study is to examine the expression of CSPG4 and its clinical significance in TNBC. Immunohistochemical analysis of CSPG4 was performed on tissue microarrays constructed from tissue specimens from 240 breast cancer patients. CSPG4 staining was correlated with clinical and pathological characteristics, OS, and DFS. Contradicting to a previous report, our results showed that CSPG4 expression was not related to TNBC. The Kaplan-Meier method showed that high CSPG4 expression was significantly associated with DFS. Patients with high CSPG4 expression had poorer OS and DFS in a multivariate survival analysis after adjustment for stage, tumor grade, ER, PR, and HER2 overexpression. We found no correlation between TNBC and CSPG4 overexpression. However, overexpression of CSPG4 correlates with survival and may represent a therapeutic target for aggressive breast cancers.
In conclusion, this study showed that I). Promoter methylation of BRCA1 gene is associated with TNBC and poorer outcome in Taiwanese patients with breast cancer. II). CSPG4 expression is not related TNBC as reported previously but may be a marker for poor prognosis in breast cancer.

並列關鍵字

Triple-negative breast cancer ； BRCA1 ； methylation ； Chondroitin sulfate proteoglycan

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