Abstract
BackgroundKIBRA has been suggested as a key regulator of the Hippo signaling pathway, regulating organ size, cell contact inhibition, tissue regeneration as well as tumorigenesis and cystogenesis. We recently reported that human KIBRA expression depends on a complex alternative CpG-rich promoter system. Our current study aimed at the identification of epigenetic mechanisms associated with alterations in KIBRA expression regulation.ResultsWe identified two separated methylation-sensitive CpG islands located to independent KIBRA promoter regions. In vitro promoter methylation analysis using human neuroblastoma (SH-SY5Y) and immortalized kidney cells (IHKE) revealed that total promoter methylation by CpG methyltransferase SssI resulted in complete abrogation of transcriptional activity (p < 0.001), while partial methylation by HpaII selectively repressed KIBRA core promoter activity in kidney cells (p < 0.001). Cell culture-based experiments demonstrated that 5-azacitidine may be used to restore KIBRA mRNA and protein levels, while overexpression of transcription factor SP1 also induced KIBRA upregulation (all p < 0.001). Furthermore, SP1 transactivation of KIBRA transcription was largely prevented by methylation of KIBRA regulatory elements (p < 0.001). Analysis of human kidney biopsies revealed that KIBRA promoter methylation was associated with human clear cell renal cell carcinoma (ccRCC; n = 8 vs 16 controls, OR = 1.921, [CI 95% = 1.369–2.695]). The subsequent determination of KIBRA mRNA levels by real-time PCR in a larger patient sample confirmed significantly reduced KIBRA expression in ccRCC (n = 32) compared to non-neoplastic human kidney tissue samples (controls, n = 32, p < 0.001).ConclusionWe conclude that epigenetic downregulation of tumor suppressor KIBRA may involve impaired SP1 binding to functional methylation-sensitive KIBRA promoter elements as observed in human kidney clear cell carcinoma. Our findings provide a pathophysiological basis for future studies on altered KIBRA regulation in clinical disease entities such as renal cancer.
Highlights
Kidney and brain (KIBRA) has been suggested as a key regulator of the Hippo signaling pathway, regulating organ size, cell contact inhibition, tissue regeneration as well as tumorigenesis and cystogenesis
To characterize the methylation state of CpG I (63 CG dinucleotides within P1a), we evaluated methylation patterns in 16 non-neoplastic human kidney tissue samples and eight human clear cell renal cell carcinoma (ccRCC) samples in detail by bisulfite sequencing. ccRCC cells are arranged in compact nests, sheets, alveolar, or acinar structures and have clear cytoplasm which distinguishes them microscopically from adjacent benign regions (Additional file 1: Figure S2) [26]
Bisulfite sequencing and dichotomous analysis revealed that KIBRA CpG methylation occurred significantly more often in ccRCC samples compared to control samples
Summary
KIBRA has been suggested as a key regulator of the Hippo signaling pathway, regulating organ size, cell contact inhibition, tissue regeneration as well as tumorigenesis and cystogenesis. 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, Schelleckes et al Clinical Epigenetics (2017) 9:109 pathway negatively regulates the activity of two main downstream mediators: Yes-associated protein (YAP) and its family member the transcriptional co-activator with PDZ-binding motif (WWTR1/TAZ) [7,8,9]. Components of the Hippo pathway have, been suggested to be the target of aberrant gene methylation and epigenetic silencing in humans [13] as already reported for LATS1/2 (large tumor suppressor kinases 1 and 2; human Warts homolog) [17, 18], MST1/2 (serin/ threonine protein kinase 4/3; human Hippo homolog) [19], and KIBRA [20, 21].
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