HNF-1[beta] targets in ovarian clear cell carcinomas

Abstract

Ovarian clear cell carcinoma (OCCC) is a type of epithelial ovarian cancer that is associated with poor prognosis, chemoresistance, lack of available targeted treatment and relative high prevalence in Asia. The transcription factor hepatocyte nuclear factor-1β (HNF-1β) is commonly overexpressed in OCCCs and plays a pivotal role in OCCC carcinogenesis. This study was conducted to investigate the downstream targets of HNF-1β, their molecular functions and their feasibility to be selectively targeted.

Mapping of the genomic regions enriched by HNF-1β in OVMANA, an OCCC cell line with high HNF-1β expression, was achieved by ChIP-sequencing. Many of the 382 HNF-1β directly enriched genes were involved in forming integral components of the plasma membrane and validated that the genes CACNG7, TPO and TRPC6 were also enriched in another HNF-1β-high OCCC cell line, OVISE, by ChIP-qPCR analyses.

The deregulated transcriptional targets of HNF-1β were identified by RNA-sequencing. This revealed a cohort of 124 candidate genes that were largely involved in cellular processes such as proliferation, and protein- kinase binding and activity. Two candidate transcripts were selected for in-depth analysis given their reported cellular functions – FGFR1 and PRR5-ARHGAP8.

ChIP-qPCR analyses suggested that HNF-1β binds directly within promoter region of FGFR1 and found a higher FGFR1 expression in ovarian carcinomas compared to benign and borderline tumours. Moreover, the FGFR-specific inhibitor, AZD4547, selectively inhibited the FGFR-MAPK pathway in OCCC cell lines with high basal levels of FGFR phosphorylation to exert antiproliferative effects, which were reflected by the induction of apoptosis and cell cycle arrest.

PRR5-ARHGAP8 is a read-through transcript generated from the chimeric expression of two individual genes, PRR5 and ARHGAP8. PRR5-ARHGAP8 RNA expression was augmented upon the knockdown of HNF-1β in OCCC cell lines. Lower cDNA copy numbers of PRR5-ARHGAP8 were observed in the tumour samples derived from patients with OCCCs compared to their respective non-tumour samples. The opposite association was observed with samples from patients with serous carcinomas. Ectopic PRR5-ARHGAP8 expression in 293T cells reduced cellular viability and migration with cells accumulating in the G2/M phase of the cell cycle.

Overall, this study successfully mapped the genomic and transcriptomic landscape of OCCCs and identified several putative HNF-1β downstream transcriptional targets that may be implicated in OCCC carcinogenesis. The findings advocate that targeting FGFR phosphorylation using AZD4547 has therapeutic implications given its cytotoxic effect in OCCCs and that PRR5-ARHGAP8 may be a novel biomarker for identifying the induction of OCCC carcinogenesis. This study provides an alternative direction to pursue in the study of ovarian carcinomas.