| Summary: | The ubiquitin-like with PHD and ring finger domains 1 (UHRF1) is a DNA binding protein, involved in epigenetic regulation. UHRF1 expression varies in different cancers, and its role in pancreatic ductal adenocarcinoma (PDAC) is unknown. PDAC is a devastating disease with an overall 5-year survival rate of less than 5 %. Here we report that UHRF1 is frequently overexpressed in PDAC and negatively regulates Keap1, an important component of the Nrf2-mediated cellular stress response pathway. UHRF1 was expressed in 114 of 132 (86%) pancreatic tumours, was associated with larger tumour size (p= 0.02) and had higher expression in tumours compared to matched preneoplastic (PanIN) lesions (n=9). UHRF1 expression varied at different phases of the cell cycle with a peak level at G2/M. Moreover, siRNA-mediated depletion of UHRF1 was associated with a G2/M phase block and induced apoptosis. UHRF1 protein expression levels varied in different PDAC cell lines and reflected the DNA methylation levels in examined sequences. UHRF1 knockdown reduced global DNA methylation in LINE-1 and Alu-V repetitive elements and tumour suppressor-specific (p16Ink4a and RASSF1) promoter methylation. Combining UHRF1 knockdown with 5-aza-deoxycytidine treatment resulted in restoration of P16 levels compared to controls siRNA samples. KEAP1 expression loss was previously reported in ~70 % of PDAC cases. Here we established that the Keap1 promoter is hypermethylated in pancreatic cancer cells. UHRF1 knockdown was accompanied by a reduction in Keap1 promoter methylation, restoration of KEAP1 protein, loss of NRF2 protein and corresponding loss of NRF2 downstream gene expression. We showed strong evidence that Keap1 expression is regulated by its promoter methylation, as KEAP1 expression was restored following 5-aza-deoxycytidine treatment. In PDAC tumour specimens (n=124), an inverse relationship between UHRF1 and KEAP1 expression was observed (p=0.002). In summary, we have shown a role for UHRF1 in pancreatic cancer growth and promoter methylation. Moreover, we have discovered an important function for UHRF1 in controlling KEAP1 expression and consequently regulating the KEAP1/NRF2 stress response pathway.
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