新穎的控制因子SPANXA1調節肺癌的表皮間葉細胞轉型過程

Abstract

SPANX (Sperm protein associated with the nucleus, X-linked) 基因家族表現在精子生成的過程。在過去的研究中SPANX被認為是一種CTA (Cancer/Testis Antigen)，高度的表現在睪丸以及很多的癌症，像是膠質母細胞瘤、黑色素細胞瘤、睪丸生殖細胞瘤和惡性血液疾病等。然而，SPANX的功能以及牽涉在腫瘤進程中的機制卻仍然未知。我們先前的研究中顯示，SPANX gene family member A1 (SPANXA1) 在低侵襲性的肺癌細胞株CL1-0呈現高表現量，在高侵襲性細胞株CL1-5則表現較低。從體外細胞實驗中發現，穩定表現 SPANX1的CL1-5細胞株和陰性對照組株相比，其細胞侵襲的能力下降。為了進一步釐清SPANA1下游可能的傳遞訊息，我們使用基因表現微陣列晶片比較SPANXA1穩定表現細胞株與陰性對照組細胞株的基因表現差異，而實驗結果顯示在前十名的路徑中，有六個為表皮間葉細胞轉型過程(EMT)有關的訊息傳遞路徑，也發現細胞型態產生巨大的變化，從本來的間質型態變成表皮型態，呈現典型的反轉表皮間葉細胞轉型過程(MET)，此現象與其侵襲能力下降吻合。在SPANXA1穩定表現的細胞株中，我們使用免疫墨點法確定表皮型標誌蛋白E-cadherin表現量上升而間質型標誌蛋白vimentin表現量則下降。透過定量反轉錄聚合酶連鎖反應去驗證在個別路徑中重要的基因表現。在穩定表現SPNANXA1的細胞株中，調控表皮間葉細胞轉型過程的轉錄因子Slug有下降的趨勢，而在靜默SPANXA1的細胞株中，Slug有上升的趨勢，Slug在過去的研究中顯示其可以作用在E-cadherin的啟動子上進而抑制其轉錄。綜合上述實驗結果都顯示出SPANXA1表現在肺癌時，為表皮間葉轉型過程中一個重要的調控因子，這也顯示出SPANXA1在未來有潛力發展在標靶治療。

SPANX (Sperm protein associated with the nucleus, X-linked) gene family express throughout spermatogenesis in agreement with a potential role in sperm development. It also has been reported as a CTA (Cancer/Testis Antigen), which was highly expressed in testis and several cancers, such as glioblastoma, melanoma, testicular germ tumor and hematologic malignancies. Nevertheless, the function and molecular mechanism of SPANX involved in the cancer progression are greatly unknown. We found that the SPANX gene family member A1 (SPANXA1) was high-expressed in CL1-0, a lower invasive lung cancer cell line, and low-expressed in CL1-5, a higher invasive cell line. In vitro assays showed that the overexpression of SPANXA1 in CL1-5 cells suppresses invasive activity. To identify the underlying mechanism of SPANXA1, we preformed gene expression microarrays and the results indicated that EMT-related pathways are enriched in top ten pathways. Cell morphology is dramatically changed from mesenchymal-like to epithelial-like (MET). In presence of SPANXA1, the epithelial marker, E-cadherin, was up-regulated and the mesenchymal marker, Vimentin, was down-regulated. Quantitative RT-PCR assays were used to validate the differential expression of important genes in the identified pathways. The EMT regulating transcription factor, Slug, which acts a repressor for E-cadherin expression, was decreased in SPANXA1-overexpressed cells and increased in CL1-0 SPANXA1-slienced CL1-0 cells. These data suggest that SPANXA1 is a novel EMT modulator involved in lung cancer progression by mediating Slug expression and implies the therapeutic role of SPANXA1 in the future.