Critical Role of the Ubiquitin Ligase Activity of UHRF1, a Nuclear RING Finger Protein, in Tumor Cell Growth

Abstract

Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.

Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1

“…This suggests that pathological overexpression of UHRF1, by repressing permanently the expression of specific tumor suppressor genes, could induce disorders in the G1/S progression and consequently promote tumor development [10]. In agreement with our hypothesis, it has been shown that the activation of different cell cycle checkpoints during DNA damage-induced apoptosis leads to a down-regulation of UHRF1 [9]; such deregulation has been described to be dependent on the p53/p21 WAF1/CIP1 pathway [14]. It should be noted that reduction of UHRF1 expression solely can suppress proliferation and induce apoptosis of cancer cells whose p53 is inactivated [15].…”

“…The consequences of this hypermethylation and its maintenance are not clearly understood but they likely target the expression of specific genes involved in the DNA damage response. We hypothesized that UHRF1 (Ubiquitin-like, containing PHD and RING Finger domains, 1) could be a major effector of the p73 deregulation, since this nuclear protein is known to be over-expressed in numerous cancer cell lines and tissues [8][9][10][11]. 4 Several studies have shown that UHRF1 participates in the control of cell proliferation and cell cycle transition from G1 to S, by regulating the expression of several genes, including RB1 and p16 INK4A [12,13].…”

“…There are several lines of evidence that UHRF1 deregulation may impair the control of G1/S transition during cell cycle checkpoint activation [8,9,14]. Since such activation is known to occur during the DNA damage-related apoptosis of TQ [23][24][25][26], we analyzed the expression levels of UHRF1 and its partners DNMT1 and HDAC1 in TQ-treated Jurkat cells.…”

UHRF1‐mediated tumor suppressor gene inactivation in nonsmall cell lung cancer

“…This is consistent with the reported association between UHRF1, proliferation, 30 and tumor cell growth. 44 E2F1 levels did not change upon UHRF1 knockdown, indicating a lack of a feedback loop. Both our ex vivo and in vitro data suggest that UHRF1 can influence the cell cycle control mainly through the epigenetic silencing of relevant tumor suppressors.…”

“…UHRF1 mRNA was overexpressed in NSCLC tumor tissues in comparison to their normal adjacent tissue, confirming in a large sample set a preliminary observation. 44 UHRF1 overexpression was more profound in squamous carcinomas than adenocarcinomas, further underlying the differences in the molecular evolution of these tumor types. Further research is required to provide evidence on whether this difference could be used in the clinical management of these 2 histological types.…”