Dkk3, downregulated in cervical cancer, functions as a negative regulator of β-catenin

The Wnt/beta-catenin signaling pathway is activated during the malignant transformation of keratinocytes that originate from the human uterine cervix. Dkk1, 2 and 4 have been shown to modulate the Wnt-induced stabilization of the beta-catenin signaling pathway. However, the function of Dkk3 in this pathway is unknown. Comparison of the Dkk3 gene expression profiles in cervical cancer and normal cervical tissue by cDNA microarray and subsequent real-time PCR revealed that the Dkk3 gene is frequently down-regulated in the cancer. Methylation studies showed that the promoter of Dkk3 was methylated in cervical cancer cell lines and 22 (31.4%) of 70 cervical cancer tissue specimens. rhis promoter methylation was associated with reduced expression or Dkk3 mRNA in the paired normal and tumor tissue samples. Further, the reintroduction or Dkk3 into HeLa cervical cancer cells resulted in reduced colony formation and retarded cell growth. The forced expression of Dkk3 markedly attenuated P-catenin-responsive luciferase activity in a dose-dependent manner and decreased the beta-catenin levels. By utilizing a yeast two-hybrid screen, beta TrCP, a negative regulator of beta-catenin was identified as a novel Dkk3-interacting partner. Coexpression with beta TrCP synergistically enhanced the inhibitory function of Dkk3 on beta-catenin. The stable expression of Dkk3 blocks the unclear translocation of 0-catenin, resulting in downregulation of its downstream targets (VEGF and cylcin D), whereas knockdown of Dkk3 abrogates this blocking. We conclude from our finding that Dkk3 is a negative regulator or beta-catenin and its downregulation contribute to an activation of the beta-catenin signaling pathway. (C) 2008 Wiley-Liss, Inc.