the regulatory influence of the transcription factors NFI
and RunX1 in human uveal melanoma.
Manel Benhassinea, Karine Zanioloa and Sylvain L. Guérina,b
aCentre Universitaire d’Ophtalmologie-Recherche (CUO-Recherche), Axe médecine régénératrice, Hôpital du Saint-Sacrement, Centre de Recherche FRQS du CHU de Québec-Université Laval, Québec, Canada; bDépartement d'ophtalmologie, Faculté de Médecine, Université Laval, Québec, Canada
Corresponding author:
Dr. Sylvain L. Guérin, CUO-Recherche, Hôpital du Saint-Sacrement, Centre de recherche FRQS du CHU de Québec, Québec, QC, Canada
Phone: (418) 682-7565 Fax: (418) 682-8000
ABSTRACT
Uveal melanoma (UM) is the most common type of primary intraocular tumor in the adult population. The gene encoding the serotonin receptor 2B (HTR2B) appears to be the most discriminating among the candidates from the class II gene signature as its expression strongly increases in the tumors of UM patients that will progress toward liver metastases. In this study, we investigated the molecular mechanisms that lead to this aberrant expression of HTR2B in metastatic UM cell lines. Transfection analyses revealed that the upstream regulatory regions of the HTR2B promoter is made up of a combination of alternative positive and negative regulatory elements that are functional in HTR2B- but not in HTR23B+ uveal melanoma cell lines. Electrophoretic mobility shift assays (EMSAs) provided evidence that the transcription factor (TF) NFI could interact with the promoter and the upstream negative regulatory element from the HTR2B gene. In addition, the TF RUNX1 was also shown by DMS methylation interference footprinting and EMSA to bind a target site from the distal silencer element. Site-directed mutagenesis analyses indicated that NFI and RUNX1 function respectively as activator and repressor of HTR2B gene transcription. The results of this study will help understand better the molecular mechanisms accounting for the abnormal expression of the HTR2B gene in uveal melanoma.
KEYWORDS
HTR2B, gene promoter, NFI, RUNX1, transcription factor, uveal melanoma
1. INTRODUCTION
Uveal melanoma (UM) is the most common primary ocular malignancy in adults, accounting for 70% of all eye cancers. The overall mean age-adjusted incidence in the United States is 4 to 6 per million individuals [1]. The actuarial 15-year metastatic mortality is approximately 50% despite efficient treatment of the primary ocular tumor [2]. Factors associated with metastatic disease include pathological features [3], cytogenetic abnormalities such as monosomy 3 and 8q gain [4, 5],
BAP1 mutations [6], and the class 2 gene signature [7]. Indeed, a gene expression
signature comprising 12 genes that can discriminate between UM primary tumors at low (class I genes: FXR1, LTA4H, ID2, ROBO1, MTUS1, LMCD1, STAB1 and EIF1B) or high (class II genes: HTR2B, CDH1, RAB31 and ECM1) risk of evolving towards the formation of liver metastases has been described [8]. The 8-year survival probability has been established to be 95% in class 1 patients against only 31% in class 2 patients [8]. The human gene encoding the 5-Hydroxytryptamine receptor 2B (HTR2B), also known as the serotonin receptor 2B, turn out to be the most discriminating among the class II genes in order to identify UM patients at high risk of evolving toward liver metastastic disease [9, 10].
The serotonin receptor HTR2B belongs to a larger family of proteins that comprises seven sub-families (HTR1 to HTR7) [11]. When it binds its ligand serotonin (5-HT), HTR2B activates the G proteins GNAQ, GNA11 and GNA13 and participates to development and both cell proliferation and survival through the activation of a few signal transduction pathways such as PLC, JAK/STAT and RAF/MEK/ERK pathways [6, 11-13]. A recent study conducted in primary cultured hepatocytes also reported the activation of the HTR2B receptor/(Gq)/PLC pathway and the RTK/PI3K/ERK/mTOR signaling pathways in response to the binding of 5-HT to the HTR2B receptor [14].
By preventing differentiation of the neural crest cells, HTR2B also plays a role in embryonic morphogenesis [15]. A few studies ascribed mitogenic and angiogenic properties to serotonin [16-19]. Curiously, the HTR2B receptor has been described as an oncogene in certain types of cancers (hepatocellular and prostate cancers) [19-21] but also as a tumor suppressor in others (such as ovarian cancers) [22]. Moreover, alteration of HTR2B expression in Xenopus embryos was reported to cause alterations in the proliferation rate and survival of retinal precursors in retina that also result in abnormal retinal physiology [23]. Interestingly, excess serotonin signaling that results from the overexpression of the HTR2B receptor was found to cause the formation of irregularly shaped eyes that are inappropriately positioned and oriented in Xenopus embryos [24].
Although the signal transduction cascades activated by the binding of serotonin to HTR2B has been investigated for years, yet no study ever reported the characterization of the regulatory sequences that are critical to ensure proper transcription of the HTR2B gene. In this study, we cloned both the promoter and 5’- flanking sequence from the human HTR2B gene and studied its regulation in human uveal melanoma (UM) cell lines that express this gene to various levels. We identified the presence of both a distal and a proximal silencer that negatively regulate the transcription directed by the HTR2B promoter in the UM cell lines T97, T108 and T143 that do not express this gene, but not in the HTR2B-expressing T142 UM cell line. Members from the NFI family of transcription factors (TFs) were found to activate HTR2B transcription by binding to its promoter region whereas the TF RUNX1 was found to repress HTR2B promoter activity by interacting with the distal silencer element. These results suggest that HTR2B gene expression is likely dictated by subtle alterations in the nuclear ratio of TFs that either repress (RUNX1) or activate (NFI) HTR2B transcription in UM cells.