Promoter methylation inhibits expression of tumor suppressor KIBRA in human clear cell renal cell carcinoma

Abstract

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. 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. We 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 Sss I resulted in complete abrogation of transcriptional activity ( p < 0.001), while partial methylation by Hpa II 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). We 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.