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

https://doi.org/10.1016/j.taap.2014.08.016Get rights and content

Highlights

  • MT2A gene expression and metal content in laryngeal cancer tissues

  • Association between SNP (rs28366003) and expression of MT2A

  • Significant associations between the SNP and Cd, Zn and Cu levels

  • Negative correlation between MT2A gene expression and Cd, Zn and Cu levels

Abstract

Metallothioneins (MTs) are low molecular weight, cysteine-rich heavy metal-binding proteins which participate in the mechanisms of Zn homeostasis, and protect against toxic metals. MTs contain metal-thiolate cluster groups and suppress metal toxicity by binding to them. The aim of this study was to determine the − 5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene and to investigate its effect on allele-specific gene expression and Cd, Zn and Cu content in squamous cell laryngeal cancer (SCC) and non-cancerous laryngeal mucosa (NCM) as a control. The MT2A promoter region − 5 A/G SNP was determined by restriction fragment length polymorphism using 323 SCC and 116 NCM. MT2A gene analysis was performed by quantitative real-time PCR. The frequency of A allele carriage was 94.2% and 91.8% in SCC and NCM, respectively, while G allele carriage was detected in 5.8% and 8.2% of SCC and NCM samples, respectively. As a result, a significant association was identified between the − 5 A/G SNP in the MT2A gene with mRNA expression in both groups. Metal levels were analyzed by flame atomic absorption spectrometry. The significant differences were identified between A/A and both the A/G and G/G genotypes, with regard to the concentration of the contaminating metal. The Spearman rank correlation results showed that the MT2A expression and Cd, Zn, Cu levels were negatively correlated. Results obtained in this study suggest that − 5 A/G SNP in MT2A gene may have an effect on allele-specific gene expression and accumulation of metal 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.

Section snippets

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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