Esophageal cancer (EC) comprises of heterogeneous groups of tumors that differ in pathogenesis and etiological and pathological features. EC ranks among the ten most frequent cancers worldwide with regionally dependent incidence rates and histological subtypes [1
]. Statistics indicate that EC mortality rates are very similar to incidence rates due to the relatively late stage of diagnosis, the poor efficacy of treatment [2
], and the poor prognosis of EC result in a five year survival rate of 5-20% [3
]. The most recurrent histological subtype is esophageal squamous cell carcinoma (ESCC), followed by adenocarcinoma (ADC) [4
]. ESCC has a worse prognosis than ADC due to the primary ESCC tumor being in contact with the tracheobronchial tree in 75% of cases, while ADC is found below the tracheal bifurcation in 94% of cases [5
The striking 3-4:1 male predominance of ESCC was previously ascribed to the different patterns of smoking and drinking between males and females. However, more recently Bodelon et al. reported that current users of estrogen and progestin therapy show reduced risk of ESCC [6
]. Previous research supports this finding as several groups have reported estrogen induced gene regulation in esophageal squamous cell carcinoma (ESCC) and Barrett’s esophageal adenocarcinoma (BEAC) [7
]. Moreover, Wang et al. specifically demonstrated that serum level of estradiol of ESCC patients from the high risk areas were significantly lower compared to healthy controls from both high and low risk areas and suggested the use of estrogen analogues as promising targets for the prevention and treatment of ESCC [13
]. Additionally, published scientific data shows that estrogen induces an inhibitory effect on esophageal carcinoma by activating the estrogen receptor (ER) [7
]. The activated ER functions as a transcription factor that binds to a specific TFBS known as the estrogen response element (ERE) [14
]. There are two ER subtypes, ERα and ERβ, that are encoded on human chromosomes 6q25.1 [16
] and chromosome 14q22-24 [17
], respectively. Both ERα and ERβ bind to the same EREs, but ERα does so with an approximately twofold higher affinity [18
]. Additionally, ERβ is known to bind to ERα suppressing ERα function [19
]. The inverse biological effect associated with the two ER subtypes has been confirmed to exist in ESCC [7
]. This collation of research findings suggests that the estrogen based therapies which have improved survival rates of cancer types such as: prostate cancer [21
], lung cancer [22
], brain and spinal cord tumors [23
], and breast cancer [24
], may also improve the outcome of ESCC.
Our current study aims at identifying estrogen responsive genes by using ESCC as a model. Potentially, such genes could be affected by estrogen. We propose a methodology that provides insight into the underlying regulation of estrogen responsive ESCC genes. We mapped EREs to the promoters of 418 ESCC genes using the Dragon ERE Finder version 6.0 (http://apps.sanbi.ac.za/ere/index.php
]. The 418 ESCC genes were divided into two groups: 1) genes whose promoters contain predicted EREs, and 2) genes lacking predicted EREs. These two gene groups were further divided into those known to be experimentally confirmed as estrogen responsive and those that are not. To accomplish this the 418 ESCC genes were cross checked against two databases housing estrogen responsive genes, namely KBERG [26
] and ERtargetDB [27
] databases. At the time of analysis the KBERG database contained 1516 experimentally confirmed estrogen-responsive genes. The ERTargetDB database contained: (a) 40 genes with 48 experimentally verified ERE direct binding sites and 11 experimentally verified ERE tethering sites; (b) 42 genes identified via ChIP-on-chip assay for estrogen binding and (c) 355 genes from gene expression microarrays, all of which were included in this study. However, this study excludes the 2659 computationally predicted estrogen responsive genes included the ERTargetDB, database. Thus this study defines estrogen responsive genes as genes that can be modulated by an external estrogen source.
We classified the 418 ESCC genes into the following four categories (Table ):
ESCC genes categorized based on ERE predictions and experimental evidence of estrogen responsiveness
C1/ESCC genes with predicted EREs in their promoters and known as estrogen responsive,
C2/ESCC genes with predicted EREs in their promoters but not known as estrogen responsive,
C3/ESCC genes having no predicted EREs in their promoters, but known as estrogen responsive,
C4/ESCC genes having no predicted EREs in their promoters and not known as estrogen responsive.
We used these categories to develop a methodology for the identification of co-localized TFBSs (cTFBSs) that characterize the promoters of the known estrogen responsive gene sets (class (C1 and C3)) as opposed to the background set (class C4). These significant cTFBSs were mapped to the promoter sequences of the candidate estrogen responsive ESCC genes in class C2. The genes in class C2 whose promoters contained such cTFBSs were singled out as novel putative estrogen responsive genes in ESCC (class C2A).
To the best of our knowledge our study provided the first computational large-scale analysis of the transcription potential of estrogen responsive ESCC genes and suggests important regulatory potential of these genes. Although we used ESCC as a model, the developed system biology based methodology has a potential to identify hormone responsive genes using other hormone-affected diseases, and provides a framework for identifying hormone responsive genes based on complex diseases.