The − 5 A/G single-nucleotide polymorphism in the core promoter region of MT2A and its effect on allele-specific gene expression and Cd, Zn and Cu levels in laryngeal cancer
Introduction
Metallothioneins (MTs) are intracellular low molecular weight, cysteine-rich heavy metal-binding proteins which have unique structural characteristics. Due to their rich thiol groups, they bind to trace metal ions, in particular cadmium, zinc, copper, mercury, nickel, platinum and silver. They also regulate intra- and extracellular metal distribution and donation to various enzymes and transcription factors, and protect against heavy metal toxicity (Mehus et al., 2014, Raudenska et al., 2014). Of the eleven human functional MT isoforms, MT1 and MT2 are extensively expressed. Transcription of MT genes is rapidly up-regulated in response to Cd and Zn, oxidative stress, hormones, selected chemical agents and inflammatory mediators (Mehus et al., 2014, Raudenska et al., 2014, Vasák and Meloni, 2011).
A crucial role in the transcriptional activation of the MT1 and MT2 genes is played by Cys2-His2 six zinc-finger-metal-responsive transcription factor (MTF-1) and short cis-acting DNA metal response elements (MREs), which bind to the proximal MT promoter chromatin in response to metals and oxidative stress (Kling et. al., 2013). Downregulation of constitutive and heavy metal-induced MT expression is controlled by a nuclear factor I (NF-I) protein, which acts by direct interaction with three half-sites within the MT promoter region. Furthermore, overexpression of MTF-1 overcomes NF-I-mediated repression of the MT promoter activity (Kling et al., 2013). A schematic diagram of MT2A gene and the transcription factors binding to the promoter are shown in Fig. 1.
The expression and regulation of MTs can be also affected by genetic variation in the core promoter region and diverse frequency of the essential alleles, which may lead to differences among individuals in terms of heavy metal uptake and metabolism (Kayaaltı et al., 2011, Kayaalti et al., 2010). Most studies emphasize the differential expression of MT isoforms and role of single-nucleotide polymorphisms in inhibiting the binding of nuclear proteins to the core promoter region of the MT gene, and by doing so, determine the role played by MT expression in judging the risk and prognostic value of cancers of various origin (Forma et al., 2012, Kita et al., 2006, Krześlak et al., 2013, Krześlak et al., 2014, Mehus et al., 2014, Pedersen et al., 2009). A literature survey also reveals some evidence for an association of MT1/MT2 expression with the presence of SNPs in MT genes near the TATA box, the carriage of different genotypes with occurrence risk, clinicopathological parameters and resistance to chemotherapy in head and neck cancers, but the final conclusions are ambiguous (Gumulec et al., 2014a, Pastuszewski et al., 2007, Pedersen et al., 2009, Theocharis et al., 2011).
Many metallic agents, heavy metals and their compounds are classified as carcinogenic to humans (Beyersmann and Hartwig, 2008, Navarro Silvera and Rohan, 2007, Qiao et al., 2014, Tokar et al., 2011).
Mechanisms of biological activity and the carcinogenicities of metal compounds are diverse and still the subject of discussion in the literature. Metallic agents and heavy metals can affect cellular activities and signaling pathways during tumor initiation and development; the predominant mechanisms presented in the literature are induction of aberrant gene expression by their ability to inhibit the repair of endogenous and exogenous DNA damage and increase genomic instability, deregulation of cell proliferation, inhibition of apoptosis, and induction of oxidative stress (Beyersmann and Hartwig, 2008, Qiao et al., 2014, Joseph, 2009).
Many reviews also focus largely on the association between metal concentration and risk of cancers, aggressiveness of the tumor and disease outcome. Unfortunately, most of these studies have reported not only contradictory results, inverse or positive interconnections but also no associations between trace element levels and either clinicopathological tumor features or cancer risk (Basu et al., 2013, Navarro Silvera and Rohan, 2007, Park et al., 2012). Moreover, it is hard to find literature data which relates to these parameters in head and neck cancers (Akinmoladun et al., 2013, Kosova et al., 2012). Therefore, it is understood that more studies are needed to elucidate the biological functions of both MTs and trace metals in the carcinogenic process and their possible clinical significance.
The aim of this study was to determine the − 5 A/G (rs28366003) single-nucleotide polymorphism in the core promoter region of the MT2A gene and to investigate its effect on allele-specific gene expression as well as Cd, Zn and Cu content in squamous cell laryngeal cancer tissue.
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Study subjects
In this study, 323 tissue samples from squamous cell laryngeal carcinoma (SCC) cases were investigated from genetically unrelated individuals: 294 male and 29 female, mean age 61.74 ± 9.05 yrs. Individuals were recruited between January 2004 and December 2011 and were under treatment at the Department of Otolaryngology and Laryngological Oncology, Medical University of Łódź, Poland. All patients had a confirmed diagnosis of SCC based on histopathological evaluation and had undergone partial or
Allelic-specificity of MT2A expression
Genomic DNAs from 323 samples from squamous cell laryngeal carcinoma (SCC) and 116 samples from non-cancerous laryngeal mucosa (NCM) were isolated, and the − 5 A/G (rs28366003) single-nucleotide polymorphism in the core promoter region of MT2A was evaluated. The demographic and clinicopathological characteristics of the study subjects are shown in Table 1.
The genotype frequencies of the –5 A/G (rs28366003) SNP in the MT2A gene were found as homozygote typical (A/A), heterozygote (A/G) and
Discussion
The literature offers a considerable body of evidence of the toxic and carcinogenic potential of metallic agents, heavy metals and their complexes and the confirmed associations with various types of cancers (Beyersmann and Hartwig, 2008, Krześlak et al., 2013, Navarro Silvera and Rohan, 2007, Qiao et al., 2014, Tokar et al., 2011). Since − 5 A/G SNP is situated in the core promoter region of the MT2A gene between the TATA box in the center of the consensus sequence TGCACTC and the site of
Conclusions
In conclusion, our results suggest that − 5 A/G (rs28366003) MT2A gene polymorphism can affect the expression of MT2A, and the content of Cd and Cu, in squamous cell laryngeal cancer tissues. Moreover, our findings indicate the importance of the SNP as a significant factor in determining susceptibility to metal toxicity and protective mechanism disturbances, which can contribute to the development and progression of cancer of the larynx. However, additional data on MTs and their isoforms in head
Conflict of interest statement
The authors declare to have no conflict of interests.
Acknowledgments
This work was supported, in part, by the statutory fund of the Department of Cytobiochemistry, University of Łódź, Poland (506/811) and by a grant from the National Science Council, Poland (N403 043 32/2326).
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2018, Journal of Trace Elements in Medicine and BiologyCitation Excerpt :It was shown that cigarette smoking increased plasma MT in the body by about 20%, as a part of the MT’s role in antioxidative defence and pregnancy itself increase MT levels by about 65% [59]. Lower expression of MT2A mRNA in several types of cancerous and related non-cancerous tissues has been found in G allele carriers [16,49,50]. Hattori et al. [17] found no differences in MT2 serum concentrations of G allele carriers in persons of both genders aged 35–69 years, which is in agreement with the present results.
Zinc gluconate toxicity in wild-type vs. MT1/2-deficient mice
2017, NeuroToxicologyCitation Excerpt :In this study, the G variant of the −5 A/G locus was associated with squamous cell laryngeal cancer (odds ratio (OR) 2.9, 95% confidence interval (CI): 1.53-5.21). The minor allele (G) carriers also had a higher stage of laryngeal cancer, including increased aggressiveness and more diffuse tumor growth (Starska et al., 2014). MT1 polymorphisms were investigated for their association with oral squamous cell carcinoma (OSSC).
The effect of metallothionein 2A core promoter region single-nucleotide polymorphism on accumulation of toxic metals in sinonasal inverted papilloma tissues
2015, Toxicology and Applied PharmacologyCitation Excerpt :However, in contrast to the results given above for heterozygotes of the major allele and atypical homozygotes, a considerably high accumulation of both Cd and Cu in IP tissue was reported. These changes in toxic metal levels in neoplastic tissue remain in line with our recent studies on laryngeal cancer, which note that − 5 A/G SNP in the MT2A gene may be related to Cd, Zn and Cu content (Starska et al., 2014a). It should be emphasized that discrepancies exist in the literature with regard to the interpretation of the results, the comparison of MT expression, and the relationship between MT2A SNP and toxic metal tissue content in tumors of various origin, such as carcinomas and benign Schneiderian papillomas.