Contribution of the STAT Family of Transcription Factors to the Expression of the Serotonin 2B (HTR2B) Receptor in Human Uveal Melanoma.

Manel Benhassine,Sylvain L Guérin,Gaëtan Le-Bel

doi:10.3390/ijms23031564

Manel Benhassine, Sylvain L Guérin + Show 1 more

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https://doi.org/10.3390/ijms23031564

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Abstract

Uveal melanoma (UM) remains the most common intraocular malignancy among diseases affecting the adult eye. The primary tumor disseminates to the liver in half of patients and leads to a 6 to 12-month survival rate, making UM a particularly aggressive type of cancer. Genomic analyses have led to the development of gene-expression profiles that can efficiently predict metastatic progression. Among these genes, that encoding the serotonin receptor 2B (HTR2B) represents the most discriminant from this molecular signature, its aberrant expression being the hallmark of UM metastatic progression. Recent evidence suggests that expression of HTR2B might be regulated through the Janus kinase/Signal Transducer and Activator of Transcription proteins (JAK/STAT) intracellular signalization pathway. However, little is actually known about the molecular mechanisms involved in the abnormally elevated expression of the HTR2B gene in metastatic UM and whether activated STAT proteins participates to this mechanism. In this study, we determined the pattern of STAT family members expressed in both primary tumors and UM cell-lines, and evaluated their contribution to HTR2B gene expression. Examination of the HTR2B promoter sequence revealed the presence of a STAT putative target site (5′-TTC (N)3 GAA3′) located 280 bp upstream of the mRNA start site that is completely identical to the high affinity binding site recognized by these TFs. Gene profiling on microarrays provided evidence that metastatic UM cell lines with high levels of HTR2B also express high levels of STAT proteins whereas low levels of these TFs are observed in non-metastatic UM cells with low levels of HTR2B, suggesting that STAT proteins contribute to HTR2B gene expression in UM cells. All UM cell lines tested were found to express their own pattern of STAT proteins in Western blot analyses. Furthermore, T142 and T143 UM cells responded to interleukins IL-4 and IL-6 by increasing the phosphorylation status of STAT1. Most of all, expression of HTR2B also considerably increased in response to both IL-4 and IL-6 therefore providing evidence that HTR2B gene expression is modulated by STAT proteins in UM cells. The binding of STAT proteins to the −280 HTR2B/STAT site was also demonstrated by electrophoretic mobility shift assay (EMSA) analyses and site-directed mutation of that STAT site also abolished both IL-4 and IL-6 responsiveness in in vitro transfection analyses. The results of this study therefore demonstrate that members from the STAT family of TFs positively contribute to the expression of HTR2B in uveal melanoma.

Highlights

Uveal melanoma (UM) is the most common intraocular malignancy in adults, with an incidence of four to six affected individuals per million in the United States [1]
In addition to its function as a neurotransmitter, serotonin plays a role in its development, and most of its biological actions are transmitted within the cell through the activation of a few signal transduction pathways including the phospholipase C (PLC), the Receptor Tyrosin Kinase (RTK)/Phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)/Extracellular signalRegulated Kinase (ERK)/mammalian target of rapamycin, the RAF/Mitogen activated protein Kinase Kinase (MEK)/ERK and the Janus kinase/Signal Transducer and Activator of Transcription proteins (JAK/STAT) pathways [10,11,12,13,14,15]
In order to verify whether transcription of the Hydroxytryptamine receptor 2B (HTR2B) gene might be under the regulatory influence of STAT family members, we first searched for the presence of putative STAT DNA regulatory element (DRE) in both the promoter and 5 -flanking sequence of the HTR2B gene

Summary

Introduction

Uveal melanoma (UM) is the most common intraocular malignancy in adults, with an incidence of four to six affected individuals per million in the United States [1]. Microarray analyses identified 12 genes, designated as the UM gene expression signature, that can distinguish between UM primary tumors that are at low or high risk of progressing towards the liver metastatic disease. The human gene encoding the 5-Hydroxytryptamine receptor 2B (HTR2B), known as the serotonin receptor 2B, turns out to be the most discriminating among the class II genes for the identification of UM patients at high risk of evolving toward formation of liver metastases [4,5,6]. The serotoninmediated activation of the JAK/STAT pathway has been shown to rely essentially on the 5-HT1A or 5-HT2A receptors [14,16,17,18], recent evidence suggests that the HTR2B receptor might participate as well in the activation of this signal transduction pathway in uveal melanoma [19]

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