β-Catenin與Tcf4分子在前列腺癌細胞的探討

β-Catenin/Tcf4 signaling pathway in prostate cancer cells

古勝友(Sheng-Yu Ku)

指導教授 ： 王記慧

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2009年)

https://doi.org/10.6832/KMU.2009.00088

摘要

β-Catenin為一個在Wnt訊息傳遞路徑中最主要的調控因子，控制哺乳類動物細胞型態變化與癌症的形成。在我們的研究中，我們建構了一株能持續表現??-catenin的攝護腺癌細胞株＜PC-3-flag-mut-β-Catenin(T41A、S45A)＞，使得此細胞株在癌化過程中，其Wnt 訊息傳遞路徑持續地處於活化狀態。有趣的是，從我們的細胞模式觀察到，此細胞株＜PC-3-flag-mut-β-Catenin(T41A、S45A)＞與一般的前列腺癌細胞 ＜PC-3＞相較起來具有較強的侵入能力以及比較鬆散的細胞邊界。之後在微陣列晶片分析法(microarray)以及即時聚合酶反應(real-time PCR)中，顯示MMP2、MMP7、Osteoprogerin(OPG) PC-3-flag-mut-β-Catenin(T41A、S45A)細胞中具有較高的表現量。從此結果中，我們推測β-Catenin/Tcf4可能參與攝護腺癌細胞的骨轉移作用。因此我們利用osteotropic模式動物來了解此議題。從我們的結果顯示，過度表現的β-Catenin(T41A、S45A)並不影響皮下(Subcutaneous)癌細胞的生長速率；但β-Catenin(T41A、S45A)卻改變了股骨(femoral)內的癌細胞生長速率：PC-3-flag-mut-β-Catenin(T41A、S45A) 100%能在股骨內生長；PC3-mock 0%在股骨內生長。更重要的是，我們也在裸鼠動物模式觀察到成骨性病變(Osteoblastic lesion)以及蝕骨性病變(Osteolytic lesion)的現象。根據這些結果，我們認為β-Catenin/Tcf4訊息傳遞路徑能促使osteotropic攝護腺癌細胞的生長，並且有可能是造成成骨性病變(Osteoblastic lesion)以及蝕骨性病變(Osteolytic lesion)的原因。
除此之外，我們也深入地探討於β-Catenin與Tcf4此兩分子之間的研究。我們建構了PC3-flag-Tcf4以及β-Catenin結合位缺陷的PC3-flag-△Tcf4兩株細胞株。透過PC3-flag-Tcf4、PC3-flag-△Tcf4、以及PC-3-flag-mut-β-Catenin(T41A、S45A)三株細胞株，我們想了解Tcf4/repressor複合體如何轉換成β-Catenin/Tcf4複合體。從Electrophoretic Mobility Shift Assay分析結果顯示，在缺少??-catenin的存在下，Tcf4會持續與repressor形成複合體；但在??-catenin情況下，β-Catenin會與Tcf4形成較具優勢的複合體。更重要地我們發現在in vitro條件下，β-Catenin是無法競爭掉與Tcf4結合的repressor。因此我們推測Tcf4/repressor複合體轉換成β-Catenin/Tcf4複合體的過程是相當複雜的。除此之外，實驗結果也顯示與本系統檢測到的Tcf4結合的repressor並非先前其他研究團隊所報告的TLE 與 CtBP1。

關鍵字

Wnt訊息傳遞路徑

並列摘要

β-Cateninis a main factor controlling the canonical Wnt signaling pathways involved in morphogenesis and cancer development in mammalian cells(Johnson and Rajamannan, 2006; Takahashi-Yanaga and Sasaguri, 2007). We have established one stable cell line, PC3-f-β-Catenin-(T41A, S45A), that displays constitutively activeβ-Catenin/Tcf4 signaling to pursuit this pathways in development of prostate cancers. Interestingly, our cell model exhibits a less tight cell-cell adhesion with stronger invasiveness in comparison with that of PC3-control cells, which carry one empty vector to serve as the vehicle control. We further performed microarray analysis and real-time PCR in this cell model, demonstrating that MMP-2, MMP-7 and OPG (osteoprotegerin) are up-regulated byβ-Catenin/Tcf4 signaling. These results suggested β-Catenin/Tcf4 might involve in osteoblastic metastasis of prostate cancer. We are using osteotropic xenograft model in nude mice to address this issue. Our results demonstrated that over-expression ofβ-Catenin(T41A、S45A) did not alter growth rate of subcutaneous(SC) tumors, and in contrast, it dramatically changed the tumor of formation in bone environment, which were 100% for PC3-f-β-Catenin-(T41A, S45A) cells and 0% for PC3-mock cells. Significantly, we observed both osteoblastic and osteolytic lesions in this osteotropic xenograft tumor. Together, our results suggested that β-Catenin/Tcf4 signaling promoted osteotropic PCa growth and might determine formation of osteoblastic and osteolytic lesions.
In addition, we also obtained the other two stable lines, PC3-f-Tcf4 and PC3-f-ΔTcf4, that express the full-length and deletion (β-Catenin binding domain) form of Tcf4, respectively. In combination of those two lines with PC3-f-β-Catenin-(T41A, S45A), we asked how Tcf4/repressor complex convert into Tcf4/β-catenin complex. In electronic mobility shift assay (EMSA), we demonstrated that Tcf4/repressor complex always exists in the absence of ??-catenin, and Tcf4/β-catenin complex becomes dominant in the presence of β-Catenin. Significantly, β-catenin can not replace the repressor to interact with Tcf4 once Tcf4/repressor binds to DNA in vitro. These results suggested that the mechanism by which Tcf4/repressor complex convert into Tcf4/β-catenin complex is much more complicated than that of the previous proposal. Moreover, our results also showed that the repressor of Tcf4 in PC3 cells is not the repressor identified by other groups in different cell system

並列關鍵字

β-Catenin ； Tcf4

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