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SYNOPSIS Ubiquitin-like protein, containing PHD and RING finger domains-1 (UHRF1) is required for cell cycle progression and epigenetic regulation. In this study, we show that depleting cancer cells of UHRF1 causes activation of the DNA damage response pathway, cell cycle arrest in G2/M and apoptosis dependent on caspase-8. The DNA damage response in cells depleted of UHRF1 is illustrated by: phosphorylation of histone H2AX on serine 139, phosphorylation of CHK2 on threonine 68, phosphorylation of CDC25 on serine 216 and phosphorylation of CDK1 on tyrosine 15. Moreover, we find that UHRF1 accumulates at sites of DNA damage suggesting that the cell cycle block in UHRF1 depleted cells is due to an important role in damage repair. The consequence of UHRF1 depletion is apoptosis: cells undergo activation of caspases 8 and 3 and depletion of caspase-8 prevents cell death induced by UHRF1 knock-down. Interestingly, the cell cycle block and apoptosis occurs in p53 containing and deficient cells. From these studies we conclude that UHRF1 links epigenetic regulation with DNA replication.

Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and adjuvant chemotherapy have an expected survival of only 2 years due to disease recurrence, frequently in the liver. We investigated the role of liver macrophages in progression of PDAC micrometastases to identify adjuvant treatment strategies that could prolong survival. A murine splenic injection model of hepatic micrometastatic PDAC was used with five patient-derived PDAC tumors. The impact of liver macrophages on tumor growth was assessed by (i) depleting mouse macrophages in nude mice with liposomal clodronate injection, and (ii) injecting tumor cells into nude versus NOD--gamma mice. Immunohistochemistry and flow cytometry were used to measure CD47 ("don't eat me signal") expression on tumor cells and characterize macrophages in the tumor microenvironment. engulfment assays and mouse experiments were performed with CD47-blocking antibodies to assess macrophage engulfment of tumor cells, progression of micrometastases in the liver and mouse survival. clodronate depletion experiments and NOD--gamma mouse experiments demonstrated that liver macrophages suppress the progression of PDAC micrometastases. Five patient-derived PDAC cell lines expressed variable levels of CD47. In engulfment assays, CD47-blocking antibodies increased the efficiency of PDAC cell clearance by macrophages in a manner which correlated with CD47 receptor surface density. Treatment of mice with CD47-blocking antibodies resulted in increased time-to-progression of metastatic tumors and prolonged survival. These findings suggest that following surgical resection of PDAC, adjuvant immunotherapy with anti-CD47 antibody could lead to substantially improved outcomes for patients. .

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