PO-120 Proline rich homeodomain protein is required for cholangiocarcinoma tumour growth

Introduction Proline-Rich Homeodomain protein/Haematopoietically Expressed Homeobox (PRH/HHEX) is a transcription factor that regulates cell proliferation, migration, and differentiation in multiple tissues. PRH is essential for embryonic development of the liver and bile ducts and has tumour suppressor activity in hepatocellular carcinoma (HCC). Mis-regulation of PRH is associated with several cancers including HCC, breast cancer, prostate cancer, and leukaemia. Our objective is to determine whether PRH has a role in cholangiocarcinoma (cancer of the bile duct). Material and methods PRH was over-expressed in cholangiocarcinoma (CCA) cell lines and in primary bile duct epithelial cells (BECs) using an adenovirus expressing myc-tagged PRH and knockdown of PRH was achieved by stable expression of PRH shRNA. Cell proliferation was measured by EdU incorporation. RNA sequencing (RNA-seq) was used to determine genes and pathways regulated by PRH in CCA. Quantitative RT-PCR (qPCR) was used to confirm these changes and Western blotting and immunohistochemistry was used to examine the expression and localisation of the corresponding proteins. Mining of TCGA transcriptomics and genomics data was performed using UCSC Xena. Results and discussions Western blotting and immunohistochemical staining reveals that PRH protein is elevated in CCA compared to cholangiocytes and TCGA expression data indicate that PRH mRNA is commonly upregulated in CCA. Over-expression (OE) of PRH increases the proliferation of CCLP1 and CCSW1 CCA cell lines and BECs. Conversely, PRH KD decreases CCLP1 proliferation and alters the morphological phenotype of CCLP1 cells from mesenchymal to epithelial. Xenograft experiments with CCLP1 PRH knockdown (KD) cells compared to control cells shows that depletion of PRH significantly decreases tumour growth. Gene ontology and gene set enrichment analysis of RNA-seq data from CCLP1 PRH KD and OE cells indicates differential expression of genes involved in proliferation, adhesion, and migration including genes associated with Wnt signalling, and epithelial- mesenchymal transition (EMT). Western blotting confirms changes in protein expression in the KD cells in accord with the RNA-seq data. Examination of β-catenin subcellular localisation shows that PRH KD cells have decreased nuclear β-catenin. Conclusion Our data suggest that the PRH protein is intimately involved in the development of CCA.