Abstract ADP-ribosylation, a post-translational modification in which the ADP-ribose of NAD is transferred to specific acceptors, is used by bacterial toxins to disrupt host cell biosynthetic, regulatory and metabolic pathways. Mammalian cells also use ADP-ribosylation reactions to regulate protein activity. The content of ADP-ribosylated acceptors is tightly regulated by enzymes that transfer ADP-ribose to acceptors i.e., ADP-ribosyltransferases (ARTs), and those that cleave the ADP-ribose acceptor bond, i.e., ADP-ribose-acceptor hydrolases (e.g., ARHs). ADP-ribosyl-acceptor hydrolase 1 (ARH1) is responsible for the hydrolysis of the α-ADP-ribosylarginine(protein) bond. We reported previously that ARH1-deficient mouse embryonic fibroblasts (MEFs) had greater proliferation rates, and formed more colonies in soft agar and tumors in nude mice than their wild-type (WT) counterparts. Consistent with these results with MEFs, ARH1-deficient (ARH1-/-) and heterozygous (ARH1+/-) mice developed tumors more frequently than their WT counterparts (Kato et al, 2011 Cancer Res.). Furthermore, the ARH1 gene from tumors isolated from ARH1+/- MEFs, and from tumors isolated from ARH1+/- mice contained mutations that affected function. Because loss of the functioning allele in ARH1+/- MEFs and mice resulted in tumorigenesis, we infer that ARH1 is a tumor suppressor gene. Female ARH1-/- and ARH1+/- mice developed tumors and metastasis more frequently than male mice (e.g., adenocarcinomas, lymphomas), and developed tumors at a younger age than male mice. Consistent with the gender effect on incidence of tumors in ARH1-/- mice, tumor growth by ARH1-/- MEFs injected subcutaneously in an “estrogen group” (i.e., female, female ovariectomized [FO] supplemented with 17-β-estradiol [E2], male supplemented with E2) of nude mice was significantly faster than that seen in a “non-estrogen group” (i.e., male, FO) of mice. Pulmonary metastasis following intravenous injection of ARH1-/- MEFs in an “estrogen group” of nude mice were larger in size than those in a “non-estrogen group”. Further, estrogen promoted the survival of ARH1-/- MEFs in the circulation and thus enhanced metastatic potential. Our data demonstrate that control of protein-ADP-ribosylation by ARH1 is important for cell proliferation and tumorigenesis. Estrogen enhanced pulmonary metastasis, tumor growth and survival of circulating tumor cells. Funding: Intramural Research Program, NIH, NHLBI. Citation Format: Jiro Kato, Xiangning Bu, Joel Moss. Estrogen promotes tumorigenesis by ADP-ribosyl-acceptor hydrolase 1 (ARH1)-deficient cells and mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2445. doi:10.1158/1538-7445.AM2014-2445