Abstract 4947: HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis down-regulated by both deletion and epigenetic mechanisms
Authors: Bouzelfen Abdelilah , Alcantara Marion , Kora Hafid , Bertrand Philippe , Mareschal Sylvain , Bohers Elodie , Maingonnat Catherine , Ruminy Philippe , Adriouch Sahil , Riou Gaetan , Figeac Martin , Fest Thierry , Bastard Christian , Tilly Hervé , Latouche Jean-Baptiste , Jardin Fabrice 
Published: August 2015

Abstract
HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in human tumors such as neuroblastomas and natural killer (NK) lymphomas. HACE1 has been shown to ubiquitylate Rac1, a protein involved in cell proliferation and G2/M cell cycle progression. The function of HACE1 and the factors involved in its transcriptional regulation are largely unknown in the context of B-cell lymphomas. We show here, by RT-qPCR, that HACE1 gene is constitutively expressed in Normal lymph nodes and in normal B-cells isolated from peripheral blood, contrasting with a strong downregulation of its expression in more than 70% (77/111) of diffuse large B-cell lymphoma (DLBCL) cases and in four tested B-Lymphoma cell lines. HACE1 gene copy number was assessed by quantitative multiplex PCR of short fluorescent fragments (QMPSF) and array for comparative genomic hybridization (aCGH) in 91 DLBCL cases. A HACE1 heterozygous deletion was observed in 38.1% and an homozygous deletion in 2.4% of cases. These deletions were associated with a significant gene expression decrease. The molecular epigenetic mechanisms underlying HACE1 downregulation were also investigated. Using pyrosequencing assays, as compared to normal B-cells, we observed an hypermethylation of HACE1 promoter CpG177 island in 60% (68/111) of DLBCL cases and in all tested B-Lymphoma cell lines. However, no significant correlation between promoter methylation status and gene expression level was demonstrated. Furthermore, RT-qPCR assays revealed that the demethylating agent 5′azacytidine (5′AZA) did not induce a HACE1 gene expression increase in the different cell lines. By contrast, the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and LBH589 strongly reactivated the expression of HACE1 in Ramos, Raji and RL cells in which the CpG 177 island was fully methylated. We next performed ChIP experiments to determine whether HACE1 locus chromatin was in an active or inactive conformation in Ramos cell line, the most sensitive cell line to TSA effect. We found that the chromatin of HACE1 gene promoter region was predominantly in the inactive conformation (methylated H3 histones). TSA treatment was able to reverse this pattern, switching the conformation of HACE1 promoter chromatin to an active one predominantly associated with acetylated H3 histones. The putative role of HACE1 in B-cell lymphomagenesis was further investigated using lentiviral transduction (shHACE1). We demonstrated in Ramos and Raji cells that a down-regulation of HACE1 expression was associated with a significant decrease of apoptosis level and cell cycle arrest in G2/M phase.
To conclude, our experiments indicate that HACE1 can act as a haploinsufficient tumor suppressor gene in most B-cell lymphomas and be downregulated by deacetylation and methylation of its promoter region chromatin constituting a potential target for HDAC inhibitors.
Citation Format: Abdelilah Bouzelfen, Marion Alcantara, Hafid Kora, Philippe Bertrand, Sylvain Mareschal, Elodie Bohers, Catherine Maingonnat, Philippe Ruminy, Sahil Adriouch, Gaetan Riou, Martin Figeac, Thierry Fest, Christian Bastard, Hervé Tilly, Jean-Baptiste Latouche, Fabrice Jardin. HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis down-regulated by both deletion and epigenetic mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4947. doi:10.1158/1538-7445.AM2015-4947