Xu Jiaqi, Sadahira Takuya, Kinoshita Rie, Li Shun-Ai, Huang Peng, Wada Koichiro, Araki Motoo, Ochiai Kazuhiko, Noguchi Hirofumi, Sakaguchi Masakiyo, Nasu Yasutomo, Watanabe Masami
Department of Urology, Okayama University, Okayama 700-8558, Japan.
Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama 700-8558, Japan.
Oncol Lett. 2017 Nov;14(5):5638-5642. doi: 10.3892/ol.2017.6833. Epub 2017 Aug 28.
The third member of the Dickkopf family (DKK-3), also known as reduced expression in immortalized cells (REIC), is a tumor suppressor present in a variety of tumor cells. Regarding the regulation of the Wnt/β-catenin signaling pathway, exogenous DKK-1 and DKK-2 are reported to inhibit Wnt signaling by binding the associated effectors. However, whether exogenous DKK-3 inhibits Wnt signaling remains unclear. A recombinant protein of human full-length DKK-3 was used to investigate the exogenous effects of the protein in KPK1 human renal cell carcinoma cells. It was demonstrated that the expression of phosphorylated (p-)β-catenin (inactive form as the transcriptional factor) was increased in KPK1 cells treated with the exogenous DKK-3 protein. The levels of non-p-β-catenin (activated form of β-catenin) were consistently decreased. It was revealed that the expression of transcription factor (TCF) 1 and c-Myc, the downstream transcription factors of the Wnt/β-catenin signaling pathway, was inhibited following treatment with DKK-3. A cancer cell viability assay confirmed the anti-proliferative effects of exogenous DKK-3 protein, which was consistent with a suppressed Wnt/β-catenin signaling cascade. In addition, as low-density lipoprotein receptor-related protein 6 (LRP6) is a receptor of DKK-1 and DKK-2 and their interaction on the cell surface inhibits Wnt/β-catenin signaling, it was examined whether the exogenous DKK-3 protein affects LRP6-mediated Wnt/β-catenin signaling. The LRP6 gene was silenced and the effects of DKK-3 on the time course of the upregulation of p-β-catenin expression were subsequently analyzed. Notably, LRP6 depletion elevated the base level of p-β-catenin; however, there was no significant effect on its upregulation course or expression pattern. These findings indicate that exogenous DKK-3 upregulates p-β-catenin and inhibits Wnt/β-catenin signaling in an LRP6-independent manner. Therefore, exogenous DKK-3 protein may inhibit the proliferation of KPK1 cells via inactivating Wnt/β-catenin signaling.
Dickkopf家族的第三个成员（DKK-3），也被称为永生化细胞中表达降低（REIC），是一种存在于多种肿瘤细胞中的肿瘤抑制因子。关于Wnt/β-连环蛋白信号通路的调节，据报道，外源性DKK-1和DKK-2通过结合相关效应器来抑制Wnt信号。然而，外源性DKK-3是否抑制Wnt信号仍不清楚。使用人全长DKK-3重组蛋白研究该蛋白对KPK1人肾癌细胞的外源性作用。结果表明，在用外源性DKK-3蛋白处理的KPK1细胞中，磷酸化（p-）β-连环蛋白（作为转录因子的无活性形式）的表达增加。非p-β-连环蛋白（β-连环蛋白的活化形式）水平持续下降。结果显示，在用DKK-3处理后，Wnt/β-连环蛋白信号通路的下游转录因子转录因子（TCF）1和c-Myc的表达受到抑制。癌细胞活力测定证实了外源性DKK-3蛋白的抗增殖作用，这与Wnt/β-连环蛋白信号级联的抑制一致。此外，由于低密度脂蛋白受体相关蛋白6（LRP6）是DKK-1和DKK-2的受体，它们在细胞表面的相互作用抑制Wnt/β-连环蛋白信号，因此研究了外源性DKK-3蛋白是否影响LRP6介导的Wnt/β-连环蛋白信号。沉默LRP6基因，随后分析DKK-3对p-β-连环蛋白表达上调时间进程的影响。值得注意的是，LRP6缺失提高了p-β-连环蛋白的基础水平；然而，对其上调过程或表达模式没有显著影响。这些发现表明，外源性DKK-3以不依赖LRP6的方式上调p-β-连环蛋白并抑制Wnt/β-连环蛋白信号。因此，外源性DKK-3蛋白可能通过使Wnt/β-连环蛋白信号失活来抑制KPK1细胞的增殖。
