SLIT2及其相關基因變異與肺癌形成之機制探討

Abstract

研究背景:自1982年起，癌症即為台灣地區十大死亡原因之首位，其中肺癌的死亡率不論在女性或男性都高居癌症死亡率的首位，儘管目前醫學已相當進步，但對於分子致癌機制仍未完全釐清。目前所知，癌症形成的原因，是由於一連串基因發生變異所造成，和癌症有關的抑癌基因 (tumor suppressor gene) 經研究證實之所以會致癌往往是因其兩個基因座同時發生變異導致失去活性，變異方式多為一基因座產生點突變、小片段鹼基缺失或啟動子過度甲基化 (promoter hypermethylation)，而另一基因座發生抑癌基因及鄰近區域DNA大片段缺失 (loss of heterozygosity)，導致抑癌基因活性降低而致癌。因此，抑癌基因變異的研究有助於了解癌症形成的機制。 SLIT2蛋白質是一個分泌於細胞膜外的醣蛋白，在神經細胞發育過程當中，可以控制軸突的分支、遷移，並能引導其生長方向，藉此調節神經細胞的發育。已有許多文獻報導指出SLIT2基因在肺癌、乳癌與大腸癌皆有嚴重的基因大片段缺失的情形，以說明此基因在腫瘤形成或轉移過程的重要性。然而，目前並無同時針對位在SLIT2訊號傳導路徑的重要三者成員如: SLIT2、ROBO1和SRGAP1，在癌症病人的完整分子致癌機轉與變異情形的探討文獻。因此，本研究針對台灣地區的肺癌病人其SLIT2、ROBO1和SRGAP1三個基因變異情形以及SLIT2蛋白與癌細胞轉移的關係做深入的研究。 材料與方法:為了探討SLIT2、ROBO1和SRGAP1三基因在肺癌病人中變異情形以及與病人病歷資料的相關性，本研究分析了92位台灣地區非小細胞肺癌 (non-small cell lung cancer, NSCLC) 的病人檢體，利用免疫組織染色法 (immunohistochemistry) 觀察病人SLIT2、ROBO1和SRGAP1蛋白的表現，再以反轉錄-聚合酵素鏈反應 (Reverse-transcriptase polymerase chain reaction) 分析組織細胞中SLIT2、ROBO1和SRGAP1三基因mRNA轉錄是否異常，續以聚合酵素鏈反應為基礎的甲基化分析 (methylation-specific PCR) 偵測SLIT2、ROBO1和SRGAP1三基因的啟動子過度甲基化頻率。此外，SLIT2基因在癌症轉移扮演的角色亦利用細胞模式實驗來釐清。 結果: (1) 實驗結果發現所分析的NSCLC病人中，SLIT2和ROBO1蛋白低表達頻率分別達41.3% (38/92) 與20.1% (19/92)，而SLIT2、ROBO1和SRGAP1 mRNA 低表達情形分別為45% (41/92)、11% (10/92) 和34% (31/92)，且SLIT2、ROBO1與SRGAP1啟動子甲基化頻率各為63% (58/92)、37% (34/92) 和40.2% (37/92)。(2) SLIT2、ROBO1和SRGAP1蛋白質/mRNA、mRNA/啟動子甲基化、蛋白質/啟動子甲基化表現彼此間都呈現統計上的顯著相關性 (P<0.05)。(3) 此外，SLIT2 mRNA與蛋白屬於低表達者大多是癌症分期晚期的病人 (mRNA, P=0.05; protein, P=0.003) 與有遠端臟器轉移的病人 (mRNA, P=0.023; protein, P=0.013)。(4) 我們也發現，原本有SLIT2基因變異的肺癌細胞株在經過去甲基化藥物的處理之後，會使肺癌細胞株的SLIT2基因啟動子去甲基化、mRNA與蛋白質表現量上升，以及細胞株爬行能力顯著降低 (P<0.001)；另一方面，原本爬行潛力不高的肺癌細胞株將SLIT2基因knock down後，會使細胞株的SLIT2 mRNA與蛋白質表現量下降、細胞株爬行能力提高、附著能力下降 (P<0.001)。 結論:本研究證實，SLIT2與SRGAP1基因變異情形確實在肺癌形成過程中（尤其是癌症轉移）扮演一個很重要的角色，其表現變異的機制可能主要透過啟動子過度甲基化所致，我們的研究是第一篇在癌組織樣本中，同時探討SLIT2、ROBO1和SRGAP1三者參與癌症形成的研究。Background: The SLIT2 gene encodes a membrane-associated glycoprotein, which mediates the repulsive axons migration during neural development. It is frequently altered in lung, breast, and colorectal tumors. However, the comprehensive clinical correlation and molecular study in genes involved in the SLIT2/ROBO1/SRGAP1 pathway have never been examined. Purpose and study design: To investigate the etiological association of the SLIT2, ROBO1, and SRGAP1 alterations in lung cancer and their clinical significance, we detected the alteration of protein and mRNA expressions of SLIT2, ROBO1, and SRGAP1, as well as promoter hypermethylation of SLIT2, ROBO1, and SRGAP1 in 92 primary lung tumors by immunohistochemistry assay, reverse-transcriptase polymerase chain reaction assay, and methylation-specific PCR assay, respectively. In addition, the role of SLIT2 in cancer metastasis was examined in the lung cell model. Results: (1) The frequencies of low protein expression in lung tumors of SLIT2 and ROBO1 were 41.3% (38/92) and 20.1% (19/92), respectively. Low mRNA expression for SLIT2, ROBO1, and SRGAP1 was found in 45% (41/92), 11% (10/92), and 34% (31/92) of lung tumors, respectively. The promoter hypermethylation of SLIT2, ROBO1 and SRGAP1 was 63% (58/92), 37% (34/92) and 40% (37/92), respectively; (2) High concordances were observed between low protein/mRNA expression and promoter hypermethylation for the SLIT2, ROBO1, and SRGAP1 genes (P< 0.05); and (3) The low mRNA/protein expression of SLIT2 was significantly associated with advance-staged patients (mRNA, P = 0.05; protein, P = 0.003) and distant metastasis (mRNA, P = 0.023; protein, P = 0.013); (4) We found that SLIT2 mRNA/protein re-expression and de-methylation along with a decrease in metastasis potential after demethylation reagent 5’-aza -2’-deoxycytidine treatment in lung cancer cell lines (P<0.001). In addition, SLIT2 mRNA/protein loss of expression along with an increase in metastasis potential and a decrease in adhesion potential after knock down SLIT2 gene in lung cancer cell lines were observed (P<0.001). Conclusion: The frequency of alteration in any one protein among the three genes in the SLIT2 pathway was 63% (58/92). Among the three SLIT2 pathway genes, alterations of SLIT2 and SRGAP1 contribute predominantly to lung tumorigenesis. Our data reveal the importance of SLIT2 alteration especially in cancer metastasis.