Abstract
KIBRA has been suggested as a key regulator of the hippo pathway, regulating organ size, cell contact inhibition as well as tissue regeneration and tumorigenesis. Recently, alterations of KIBRA expression caused by promotor methylation have been reported for several types of cancer. Our current study aimed to design an artificial transcription factor capable of re-activating expression of the tumor suppressor KIBRA and the hippo pathway. We engineered a new gene named ‘ZFP226′ encoding for a ~23 kDa fusion protein. ZFP226 belongs to the Cys2-His2 zinc finger type and recognizes a nine base-pair DNA sequence 5′-GGC-GGC-GGC-3′ in the KIBRA core promoter P1a. ZFP226 showed nuclear localization in human immortalized kidney epithelial cells and activated the KIBRA core promoter (p < 0.001) resulting in significantly increased KIBRA mRNA and protein levels (p < 0.001). Furthermore, ZFP226 led to activation of hippo signaling marked by elevated YAP and LATS phosphorylation. In Annexin V flow cytometry assays ZFP226 overexpression showed strong pro-apoptotic capacity on MCF-7 breast cancer cells (p < 0.01 early-, p < 0.001 late-apoptotic cells). We conclude that the artificial transcription factor ZFP226 can be used for target KIBRA and hippo pathway activation. This novel molecule may represent a molecular tool for the development of future applications in cancer treatment.
Highlights
KIBRA (WWC1), a WW and C2 domain-containing protein has been identified as an upstream regulatory component of the hippo pathway, which regulates cell number by modulating proliferation, apoptosis, and differentiation[1,2,3,4]
We observed that inactivated KIBRA expression depends on promoter methylation in ccRCC27
We recently reported that human KIBRA expression depends on a complex alternative CpG-rich promoter system[30] with inactivated KIBRA expression induced by promoter methylation in ccRCC27
Summary
KIBRA (WWC1), a WW and C2 domain-containing protein has been identified as an upstream regulatory component of the hippo pathway ( known as Salvador/Warts/Hippo tumor suppressor network), which regulates cell number by modulating proliferation, apoptosis, and differentiation[1,2,3,4]. The gene expression profiles of 101 ccRCC and adjacent tissue sample pairs of the K2 series suggested KIBRA downregulation in this series using locus-specific probes[26] In this line, we observed that inactivated KIBRA expression depends on promoter methylation in ccRCC27. Novel and more precise medical strategies may involve artificial TFs for locus-specific modulation of gene expression and the reactivation of tumor suppressor function. This approach has been successfully used for the experimental treatment of breast, ovarian and cervical cancer cell lines with an artificial TF re-activating EPB41L3 expression even when expression was silenced by promoter hypermethylation[29]. ZFP226 was capable of reducing the viability of MCF-7 breast cancer cells
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