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    National Tsing Hua University Institutional Repository > 生命科學院  > 分子與細胞生物研究所 > 博碩士論文  >  異常細胞核內表現的SFRPs在Wnt/β-catenin誘發增加癌症幹細胞特性所扮演的角色

    Please use this identifier to cite or link to this item: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/86829

    Title: 異常細胞核內表現的SFRPs在Wnt/β-catenin誘發增加癌症幹細胞特性所扮演的角色
    Authors: 王姿雯
    Wang, Zih-Wun
    Description: GH02102080522
    Date: 2015
    Keywords: 癌症幹細胞
    cancer stem cells
    Abstract:   近期有許多研究顯示 Wnt 訊息傳遞途徑 (Wnt signaling pathway) 會參與癌症幹細胞 (cancer stem cells) 維持其特性及形成之過程。Secreted frizzled-related protein (SFRP) 家族由五個分泌性醣蛋白成員組成 (SFRP1、SFRP2、SFRP3、SFRP4、SFRP5),並在 Wnt 訊息傳遞途徑中扮演負向調控者的角色。因 SFRPs 其 N 端序列具有與 Frizzled 相似的半胱胺酸富含區 (cysteine rich domain, CRD),使 SFRPs 可以和 Wnt 結合,進而阻礙 Wnt 和 Frizzled 之交互作用,抑制 Wnt 訊息傳遞途徑之活化,然而目前仍不清楚 SFRPs 是否只會扮演細胞外調控者的角色,或者可以透過其他機制調節 Wnt 訊息傳遞途徑。

      本論文的研究發現,在過度表現 SFRPs 的 HM20 細胞中,SFRPs 不但可以進入細胞核內,並且可以進一步與 β-catenin 結合;透過免疫共沉澱法發現: SFRPs 與 β-catenin 結合的區域和 T-cell factor (TCF)/Lymphoid enhancer-binding factor (LEF) 與 β-catenin 的結合位置非常相近,因而進一步偵測 TCF4 和 β-catenin 在細胞核內的結合程度。結果說明:雖然 β-catenin 可以累積在過度表現 SFRPs 的 HM20 細胞核內,但是 SFRP1、SFRP2、SFRP4 和 SFRP5 卻明顯抑制 TCF4 和 β-catenin 的結合作用,反之 SFRP3 則會促進 TCF4 和 β-catenin 的結合。隨後,為了測試 SFRPs 是否除了做為細胞外調控者之外,也可以利用細胞核內與 β-catenin 之結合而調控 Wnt 訊息傳遞的轉錄活性及生物性功能,利用 glycogen synthase kinase 3 beta (GSK3β) 專一性抑制劑誘導 Wnt 訊息傳遞活化後,我們發現 SFRP1、SFRP2、SFRP4 和 SFRP5 仍然可以顯著的抑制 β-catenin/TCF 轉錄的活性和癌症幹細胞形成球體 (sphere-forming) 的能力,而 SFRP3 則會增強由 β-catenin/TCF 所調控的轉錄活性及促進癌症幹細胞球體的形成。綜合以上的研究結果,我們提供了不同於傳統的觀點:SFRPs 於 Wnt 訊息傳遞路徑中不只扮演細胞外拮抗劑的角色,SFRPs 也可以在細胞內藉由和 β-catenin 結合,進而影響 TCF4 和 β-catenin 的交互作用,藉此調控 Wnt 訊息傳遞路徑,並更進一步地影響腫瘤形成的能力。
      Recent researches suggest the Wnt signaling pathway is involved in maintaining the characteristics and population of cancer stem cells. Secreted frizzled-related protein (SFRP) family is composed of five secreted glycoproteins (SFRP1 to SFRP5) which play modulators in the Wnt signaling pathway. SFRPs contain the Frizzled-like cysteine-rich domain (CRD) at the N-terminus which interacts with Wnt ligands, resulting in the prevention of Wnt ligand-Frizzled receptor interaction and the Wnt signaling pathway activation. However, little is known about whether SFRPs only act as extracellular modulators or could be through another mechanism to regulate the Wnt signaling pathway.

      In our study, we found that SFRPs could translocate into the nucleus and associate with β-catenin in SFRPs-expressing cells. The interaction domain mapping indicated that the binding region of β-catenin for SFRPs was similar to that for T-cell factor (TCF)/Lymphoid enhancer-binding factor (LEF). Thus, we further investigated the interaction of TCF4 and β-catenin in the nucleus by co-immunoprecipitation. Interestingly, although β-catenin accumulated in the nucleus of SFRPs-expressing cells, SFRP1, SFRP2, SFRP4 and SFRP5 could significantly suppress TCF4 binding β-catenin compared with Mock cells, whereas SFRP3 could promote the recruitment of β-catenin to TCF4. After that, a glycogen synthase kinase 3 beta (GSK3β) specific inhibitor was used to activate the intracellular Wnt signaling cascade and test whether SFRPs could regulate the transcriptional activity of β-catenin/TCF and consequent biological functions beyond acting as extracellular modulators. We found that SFRP1, SFRP2, SFRP4 and SFRP5 significantly inhibited the transcriptional activity of β-catenin/TCF and sphere formation ability after GSK3β specific inhibitor treatment, whereas SFRP3 obviously enhanced the β-catenin/TCF-mediated transcriptional activity and sphere formation ability. Taken together, in addition to the role of extracellular modulators, SFRPs can also regulate the Wnt signaling via associating with β-catenin to modulate the interaction of TCF4 and β-catenin in the nucleus, which impacts on Wnt/β-catenin-elicited the tumor-initiating ability. To all of above, we provide a novel mechanism of SFRPs regulating the Wnt signaling at the intracellular level, which challenges the rule of SFRPs acting as extracellular modulators in the Wnt signaling pathway.
    URI: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/86829
    Source: http://thesis.nthu.edu.tw/cgi-bin/gs/hugsweb.cgi?o=dnthucdr&i=sGH02102080522.id
    Appears in Collections:[分子與細胞生物研究所] 博碩士論文

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