Ethmoid carcinomas are uncommon tumors recognized as an occupational disease amongst woodworkers. Current treatment with surgery and radiotherapy is unsatisfactory given the 50% survival at 5 years and the serious side effects. To better understand the molecular events involved in this tumor and to identify potentially novel markers we pioneered a gene expression profiling study of 9 sinonasal adenocarcinomas.
This study, using dedicated-microarrays containing 6864 genes previously known to be involved in cancer, allowed us to select 5 genes (LGALS4, ACS5, CLU, SRI and CCT5) with significant differential expression between tumors and normal tissue. We confirmed by RT-qPCR the overexpression of LGALS4, ACS5, SRI, CCT5 and the down-regulation of CLU. By IHC on an independent set of patients, we focused our interest on the genes with the highest differential expression i.e. LGALS4, ACS5 and CLU, and confirmed the results at the protein level for LGALS4 and CLU.
gene codes for the Galectin 4 protein [38
]. Galectins constitute a family of proteins containing carbohydrate recognition domains (CRD) with high affinity for β galactosides. Their complete physiological functions are not known but they have been reported to be involved in inflammation, apoptosis, cell adhesion and cell growth. LGALS4 in particular has been detected in normal epithelial cells of the oral esophagus, and in the intestinal mucosa [39
]. In tumors, LGALS4
expression increases in liver, gastric, breast cancer and mucinous epithelial ovarian cancer whereas it is down-regulated in colon adenocarcinoma [41
]. The presence of two binding sites for c-Rel, a subunit of NFκ-B, and the experimental data obtained with transgenic mice for c-Rel, suggest that LGALS4 could be a downstream component of the NFκ-B pathway, known to be involved in the regulation of tumorogenesis [44
]. In cancer cell lines LGALS4 is expressed in highly differentiated cell lines which form polarized monolayers while undifferentiated cell lines do not express LGALS4 but Galectin1 [38
]. In our series of ethmoid adenocarcinoma, the LGALS4
is the gene with the highest differential expression and our IHC data are in accordance with the literature, given that we found that LGALS4 is overexpressed in all ethmoid tumors except the high-grade non ITAC tumors which are poorly differentiated. LGALS4 expression seems to be correlated to both histological type and the differentiation status of the adenocarcinoma. This trend was confirmed by the P5 case where LGALS4 was overexpressed only in the "colonic-type" component and not in the poorly differentiated "solid-type" component of the tumor. For patient 6 (P6) we observed a strong overexpression of LGALS4 by IHC, which contrasts with the relative expression obtained by RT-qPCR (fold change 0.45). We therefore hypothesize that, in this "mucinous-type" ITAC containing numerous mucin lakes, the RNA extracted from the tissue was not representative of the tumor.
The highly conserved gene CLU (
apolipoproteinJ, sulfated glycoprotein 2), codes for Clusterin, a sulfated glycoprotein with chaperone activity found in numerous tissues and body fluids. CLU has been reported as being involved in many biological functions such as DNA repair, cell cycle regulation and apoptosis [37
]. CLU is described as being overexpressed in several types of cancers including colon, breast and lung cancer [37
], yet a down-regulation has been found in esophageal squamous cell carcinoma, in some pancreatic, prostate or colon cancers and in HPV-negative squamous cell carcinoma of the head and neck [37
], suggesting a pro-survival or a pro-apoptotic function. The recent description of several isoforms, including the nuclear form (n-CLU) and the cytoplasmic or secreted form (s-CLU), might help to resolve these apparent contradictions and to define the cellular functions of Clusterin as well as its potential use as a biomarker [48
In our series of ethmoid tumors, CLU was highly down-regulated at the RNA level. Although the level of Clusterin detected by IHC in normal tissue was rather low, we confirmed the down-regulation of the protein except in one case (P11). This patient was also the one whose tumor sample showed the least down-regulation of CLU by RT-qPCR. This case is of interest because the patient was exposed to wood and, in contrast with most of the cases reported in the literature, he presented a non-ITAC tumor. The absence of Clusterin in ethmoid tumors suggests a pro-apoptotic function in normal ethmoidal tissue, possibly in response to DNA damage caused by wood dust, or other occupational exposures. It is useful to note that CLU is localized on chromosome 8p21-p12 [51
]. In fact, by comparative genomic hybridization, Ariza et al
. found losses on 8p21 in about 50% of patients with sinonasal adenocarcinomas [20
]. This feature was confirmed by the study of Korinth et al
. who reported a loss of 8p in 61% of cases [21
] in a series of 42 patients. We do not know the cytogenetics of our tumors but it would be worthwhile ascertaining whether the down-regulation of CLU in the tumors studied here is due to deletion on chromosome 8p or if other mechanisms such as epigenetic regulation occur on the CLU
, Acyl coenzyme A synthetase 5 (FACL5
, E.C. 126.96.36.199.), is one isoform of the ACSs, key proteins in lipid metabolism via the activation of fatty acids in acylCoA thioesters. These esters are the metabolites for oxidation, elongation and desaturation of fatty acids as well as for the synthesis of complex lipids. ACS5 is essential for lipid metabolism but it might also play a role in intermediate metabolism and regulation of gene expression [52
]. This gene has been well characterized in the small intestine mucosa by Gassler et al
]. ACS5 is expressed in the enterocytes from the villus tip but not in the crypts and it could be involved in the differentiation and maintenance of crypt-villus axis, by inducing TRAIL apoptosis in apical villi of the mucosa. Within the context of tumorogenesis, few reports have been published on ACS5. In adenoma and adenocarcinoma of the small intestine ACS5 expression is decreased [54
] while it is up-regulated in gliomas [55
], in well-differentiated endometrioid adenocarcinomas [56
] and in certain colorectal adenocarcinomas [57
]. The RT-qPCR data in our panel of tumors revealed an increase in the expression of ACS5 (p = 0.001), eventhough it has not been confirmed by IHC. Whereas some tumors expressed strong ACS5, others had completely lost the expression of this molecule. Moreover, we could not find any correlation between ACS5 expression and histological type, differentiation or collateral exposures.
The other selected genes were not evaluated by immunohistochemistry as their variation in expression was much lower and our primary goal was to find new markers for a better characterization of these tumors with a clear etiology. Nevertheless, we confirmed the transcriptional profiling obtained with the microarray by RT-qPCR.
and CCT5 (
chaperonin-containing complexe peptide 1) are less known genes. Both code for multi-drug resistance proteins and might be involved in the cell detoxification [58
]. These genes were slightly overexpressed in our panel of tumors. This trend could be related to the chemical or particle exposures of the patients. In fact, SRI
has also been identified by Differential Display analysis as being overexpressed in oral cancer mediated by tobacco-chewing [60