探討PDCD5在腫瘤及免疫細胞的角色

Abstract

Programmed Cell Death 5 (PDCD5)又名TFAR19 (TF-1 cell apoptosis-related gene 19)，最早被發現在TF-1細胞凋亡時會大量產生，並進入細胞核內執行作用。但是PDCD5如何被活化及其相關訊息分子，目前仍不清楚。此外，在許多的癌症細胞中，PDCD5的表達都比較低，這被認為和癌症細胞克服細胞凋亡增加生長與存活有關。本研究之目的在探討PDCD5是如何被活化和傳導訊息來執行細胞凋亡。我們採用GAL4/UAS系統建立的轉基因果蠅做交配來找尋可能與PDCD5相關的分子，首先建立PDCD5轉基因果蠅，利用dsRNA選擇性在眼睛抑制PDCD5表現，並藉其眼睛潰爛的表現型和不同分子之轉基因果蠅進行交配，再由觀察眼睛表現型的變化來推測PDCD5可能相關的訊息傳導路徑。然後再將篩選結果在人類細胞做進一步驗證和分析，從而找出調節PDCD5與被PDCD5控制的相關分子。由於現有文獻報告指出PDCD5與癌症形成關連，因此我們首先篩選和癌症相關的訊息傳遞路徑。我們發現mTOR訊息傳遞路徑中，TSC高度表達或抑制S6K活性皆可減輕PDCD5轉基因果蠅眼睛之潰爛，顯示mTOR和PDCD5的訊息傳遞頗有關連。另一方面，我們也發現c-Abl作用增強會加重PDCD5被抑制掉所造成的果蠅眼睛潰爛，說明c-Abl也參與PDCD5的訊息傳遞。Hedgehog與Wnt訊息不僅在細胞發育與生長方面扮演重要角色，也和癌症的形成有關。我們發現Hedgehog訊息傳遞路徑下游的轉錄因子Ci高度表達時可以減緩PDCD5轉基因果蠅眼睛之表現型。而Wnt訊息傳遞路徑中的轉錄因子LEF/TCF高度表達時也有類似的現象。綜合上述，利用GAL4/UAS轉基因果蠅系統，我們篩選到mTOR、c-Abl、Hedgehog和Wnt的訊息傳遞路徑和PDCD5的訊息傳導可能有直接或間接的關係。接下來，我們決定先用BJAB淋巴瘤和AGS胃癌這兩種癌症細胞株來深化機轉的研究，希望透過更瞭解PDCD5的作用和訊息傳遞，藉以研發可以調控PDCD5表達和訊息傳導的藥物，提供癌症治療之新標靶和新策略。

Programmed cell death 5 (PDCD5), also known as TFAR19 (TF-1 cell apoptosis-related gene 19), is a novel apoptosis-related gene, which was first identified as a gene upregulated in TF-1 cells undergoing apoptosis. During apoptosis, PDCD5 rapidly translocates from cytoplasm where it normally resides to the nucleus for its action and this process occurs much earlier than externalization of membrane phosphatidylserine and nuclear DNA fragmentation, suggesting that PDCD5 might be crucial in the initiation of apoptosis. In addition, a decrease of PDCD5 expression, presumably to suppress apoptosis, has been reported in various human tumors, such as breast cancer, hepatocellular carcinoma, and gastric tumor. Nevertheless, the mechanisms of PDCD5 activation and signaling remain largely unclear and this motivates us to conduct an in-depth investigation. We took advantage of the powerful genetics of Drosophila by phenotypic analysis to unravel the associated molecules and pathways with PDCD5. The UAS/GAL4 ectopic gene expression system was utilized to generate a Drosophila line carrying an eye-specific dsRNA knockdown of PDCD5 gene as a convenient readout for screening. By crossing this fly line with other genetically modified flies of a specific molecule of interest, we can determine its potential relationship with PDCD5 through assessment of changes of the eye phenotype. We started to investigate key pathways in tumorigenesis for PDCD5 is often down-regulated in cancer. We found that both gain-of-function of TSC (tuberous sclerosis) and dominant negative mutation of S6K (ribosomal S6 kinase) genes of mTOR pathway rescued the rough-eye phenotype of PDCD5 gene knockdown. In contrast, over-expression of c-Abl enhanced the rough-eye phenotype of PDCD5 knockdown. Hedgehog and Wnt pathways are known to play an important role in not only growth and development but tumorigenesis. We found that both gain-of-function of Ci (Cubitus interruptus), a transcription factor of Hedgehog pathway, and the dominant negative mutation of LEF/TCF (lymphoid enhancer-binding factor 1/T cell-specific transcription factor) gene of Wnt pathway rescued the rough-eye phenotype of PDCD5 knockdown. The results from this initial screening showed that PDCD5 signaling may crosstalk and interact with intermediaries of mTOR, c-Abl, Hedgehog and Wnt signaling pathways, respectively. To validate these findings from Drosophila crosses in mammals, we decided to use BJAB (human Burkitt's lymphoma) and AGS (human gastric adenocarcinoma) cell lines for further investigation. In summary, our data from screening genetically modified flies have provided insights into the anti-apoptotic mechanisms of cancer cells through modulation of PDCD5 and pointed out a PDCD5-based novel strategy for drug discovery to treat cancers.