ZFP226 is a novel artificial transcription factor for selective activation of tumor suppressor KIBRA

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

Cryptogenic stroke and small fiber neuropathy of unknown etiology in patients with alpha-galactosidase A -10T genotype

Background: Fabry disease (FD) is a multisystemic disorder with typical neurological manifestations such as stroke and small fiber neuropathy (SFN), caused by mutations of the alpha-galactosidase A (GLA) gene. We analyzed 15 patients carrying the GLA haplotype -10C>T [rs2071225], IVS2-81_-77delCAGCC [rs5903184], IVS4-16A>G [rs2071397], and IVS6-22C>T [rs2071228] for potential neurological manifestations. Methods and results: Patients were retrospectively analyzed for stroke, transient ischemic attack (TIA), white matter lesions (WML) and SFN with neuropathic pain. Functional impact of the haplotype was determined by molecular genetic methods including real-time PCR, exon trapping, promoter deletion constructs and electrophoretic mobility shift assays. Symptomatic -10T allele carriers suffered from stroke, TIA, WML, and SFN with neuropathic pain. Patients’ mean GLA mRNA expression level was reduced to ~70% (p < 0.0001) and a dose-dependent effect of the -10T allele on GLA mRNA expression was observed in hemi/homozygous compared to heterozygous patients (p < 0.0001). Molecular analyzes revealed that the -10T allele resulted in a reduced promoter activity and an altered transcription factor binding, while a functional relevance of the co-segregated intronic variants was excluded by exon trapping. Conclusions: Based on this complementary approach of clinical observation and functional testing, we conclude that the GLA -10T allele could be causal for the observed neurological manifestations. Future studies are needed to clarify whether affected patients benefit from GLA enzyme replacement therapy for end-organ damage prevention.<br