Wnt訊息傳遞途徑 (Wnt signaling pathway) 參與許多正常的發育和人類疾病形成的過程。最近有許多研究發現，Wnt訊息傳遞途徑在癌細胞轉型獲得幹細胞特性的過程中扮演關鍵性的角色。Secreted Frizzled-related protein (sFRP) 和Dickkopf (Dkk) 家族都是 Wnt訊息傳遞途徑的拮抗劑，然而sFRP4和Dkk1在Wnt訊息傳遞誘發轉型為癌症幹細胞過程所扮演的角色仍然不清楚。
在我們的研究中發現，細胞外的sFRP4並不會和Wnt3a結合。但另一個拮抗劑Dkk1可以藉由直接和Wnt3a結合達到抑制Wnt訊息傳遞，進而降低LRP6-GSK3β訊息傳遞的活化和細胞核內β-catenin的累積。此外，sFRP4甚至可以和細胞質和細胞核內之β-catenin結合，並導致Wnt訊息傳遞所調控之轉錄活性的喪失。藉由降低球體形成 (sphere-forming) 的能力與ABC轉運蛋白家族 (ABC transporter family) 的表現，我們發現sFRP4與β-catenin結合可以降低癌症的發生 (cancer-initiating)。綜合以上結果，我們的研究不只釐清了sFRP4在調控Wnt訊息傳遞誘發轉型為癌症幹細胞過程所扮演的角色，同時也撼動了原本sFRP蛋白家族被認為需與細胞外Wnt配體 (ligand) 結合才能達成拮抗作用的法則。

The Wnt signaling is involved in many normal developmental processes and human diseases. Recent evidence suggests that the Wnt signaling pathway is critical for reprogramming of stem cell properties in cancer cells. The secreted Frizzled-related protein (sFRP) and Dickkopf (Dkk) family are both antagonists of the Wnt signaling pathway. However, the mechanism of sFRP4 and Dkk1 in the Wnt signaling-dependent reprogramming of stem cell properties is still equivocal.
In this study, we found that extracellular sFRP4 did not associate with Wnt3a. But another Wnt antagonist, Dkk1, repressed the Wnt signaling by directly binding to Wnt3a and consequently abolishing the LRP6-GSK3β signaling activation and β-catenin accumulation in the nucleus. Moreover, sFRP4 could even associate with β-catenin in the cytosol and nucleus to loss β-catenin/TCF transcriptional activities. To characterize the biological effects of sFRP4 on reprogramming of stem cells properties, we found that the association of sFRP4 and β-catenin could diminish cancer-initiating capabilities, characterized by capable of decreasing sphere-forming abilities, expression levels of the ABC transporter family. Taken together, these findings not only establish a central role of sFRP4 in regulating the Wnt signaling-elicited reprogramming of stem cell properties in cancer cells but also shake the dogma of the sFRP family being extracellular antagonists of the Wnt signaling via directly binding to Wnt ligands.

1. Introduction
2. Materials and methods
2.1. Cell culture and stably transfected cells
2.2. Conditional medium (CM)
2.3. Western blotting (Immunoblot analysis)
2.4. Nucleus/Cytosol fractionation
2.5. Immunostaining (Immunofluorescence)
2.6. TOP luciferase reporter assay
2.7. Co-immunoprecipitation (Co-IP)
2.8. The sphere-forming assay
2.9. Total RNA extraction
2.10. RT-PCR (Reverse transcription-polymerase chain reaction) and PCR
3. Results
3.1. sFRP4 does not associates with Wnt3a
3.2. sFRP4 does not suppress the phosphorylation of LRP6 at Ser1490
3.3. Wnt3a stimulation promotes nuclear β-catenin accumulation in Mock, sFRP4, sFRP4N and sFRP4C cells
3.4. Identification the β-catenin/TCF transcriptional activities of the Wnt signaling pathway in sFRP4 and Dkk1 cells
3.5. sFRP4 associates with β-catenin after Wnt3a stimulation
3.6. sFRP4 associates with β-catenin through its N-terminal domain
3.7. sFRP4 associates with both phosphorylated and de-phosphorylated β-catenin
3.8. Paracrine mature sFRP4 could enter into the cells and associate with β-catenin after Wnt3a treatment
3.9. sFRP4 and Dkk1 diminish the cancer-initiating capabilities
4. Discussion
5. References
6. Figures

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