Abstract 2167: The tumor suppressor CDC73/parafibromin is required for the maintenance of histone 2B monoubiquitination both in vitro and in vivo

Abstract

Abstract CDC73 (cell division cycle 73), also known as parafibromin, is a ubiquitously expressed tumor suppressor that is mutated in the germline of patients with the familial disorder Hyperparathyroidism Jaw Tumor syndrome, as well as somatically mutated in sporadic parathyroid cancer. Furthermore, CDC73 has been implicated in breast, gastric, renal and colorectal tumorigenesis. CDC73 is a member of the PAF1 (RNA polymerase II-associated factor 1) transcriptional complex (PAF1c), along with PAF1, CTR9, LEO1, and SKI8. Consistent with roles in transcription, CDC73 is localized to the nucleus, and has also been reported in the nucleolus. This tumor suppressor has been shown to function as a negative regulator of cell cycle progression and to promote apoptosis, as well as have a role in the regulation of 3′ processing of histone mRNA. We used CDC73 as bait in a yeast two-hybrid assay and identified the E3 ubiquitin ligase ring finger proteins RNF20 and RNF40 as CDC73 binding partners. RNF20 and RNF40 exist in a heterodimeric complex that functions to monoubiquitinate histone H2B at lysine 120 (H2B-K120). Monoubiquitination of H2B-K120 is associated with transcription in undamaged cells, is induced after DNA damage, and has a role in the maintenance of replication-dependent histone mRNA 3′ - end processing. We have confirmed the interaction of CDC73 with both RNF20 and RNF40 in mammalian cells by co-immunoprecipitation of overexpressed and endogenous proteins from human embryonic kidney (HEK293) cells. Down-regulation of endogenous CDC73 by siRNA in HEK293 cells and the breast cancer cell line MCF7 led to a significant reduction of monoubiquitinated H2B-K120. Furthermore, in a series of eleven parathyroid carcinomas harboring mutations in CDC73 that led to loss of nuclear CDC73, monoubiquitinated H2B-K120 was either absent or significantly reduced relative to benign parathyroid tumors that expressed nuclear CDC73. siRNA down-regulation of other PAF1c members in MCF7 cells also led to reduction in levels of monoubiquitinated H2B-K120. Given that CDC73 is the only PAF1c member in which cancer-associated mutations have been reported, the translational significance of these findings are unclear. Loss of the CTR9 locus has been reported in pancreatic cancer, as well as overexpression of PAF1. PAF1c members may have a greater role in malignancy than previously described. This is the first report to demonstrate pathogenic mutations affecting monoubiquitination of a histone. We propose that loss of H2B-K120 monoubiquitination is a major mechanism whereby CDC73 mutations exert their tumorigenic effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2167. doi:1538-7445.AM2012-2167