The Nonsynonymous Polymorphisms Val276Met and Gly393Ser of E2F1 Gene are Strongly Associated with Lung,and Head and Neck Cancers

Introduction

Lung cancer (LC) is the most common cancer worldwide. In India, LC and head and neck cancer (HNC) constitute the highest pool of cancer patients (Jemal et al., 2011). This may be due to high consumption of tobacco and alcohol, lack of awareness about this deadly disease, and socioeconomic status of the population (Rao et al., 2017). Despite these well-known reasons, interindividual variations like single nucleotide polymorphisms (SNPs) in the cell cycle regulator genes are frequently associated with cancer susceptibility (Rao et al., 2017).

The E2F transcription factors are present downstream to the retinoblastoma (RB) protein in RB/E2F pathway and determine cell fate and cancer development (Polager and Ginsberg, 2009). Both RB and E2F are crucial in proliferation/antiproliferative processes of the cell (Polager and Ginsberg, 2008) and are involved in chromatin assembly/condensation, chromosome segregation, DNA damage response, mitotic spindle checkpoint, apoptosis induction, and development (Ishida et al., 2001; Müller et al., 2001; Ren et al., 2002). Many human cancers have altered expression and activity of various E2F family members (Whibley et al., 2009). E2F1, the best-characterized member, regulates the key genes involved in the DNA replication and G1/S transition to promote the cell cycle (Kowalik et al., 1995; Dyson, 1998; Helin, 1998; DeGregori, 2002). E2F1 is an important player in carcinogenesis as it acts as both an oncogene and a tumor suppressor gene (Polager et al., 2008). Deregulated expression of E2F1induces proliferation and p53-dependent apoptosis in transgenic mice (Pierce et al., 1998a, 1998b). Mice lacking E2F1 have increased susceptibility to develop tumors (Polager and Ginsberg, 2008, 2009). Moreover, E2F1transgenic mice with keratin five promoter develop spontaneous neoplasm in a variety of K5-expressing tissues (Pierce et al., 1999). Contrary to this, E2F1 activation leads to apoptosis by both p53-dependent (Kowalik et al., 1995; Hsieh et al., 2002) and p53-independent pathways (Nahle et al., 2002). In addition, etoposide-treated cells lead to checkpoint kinase-2-dependent phosphorylation and activation of E2F1, thus promoting apoptosis (Polager and Ginsberg, 2009). It is well known that hereditary variants of the coding, promoter, and miRNA binding sites can alter gene functions (Gupta et al., 2005; Lu et al., 2012; Randhawa et al., 2015; Agarwal et al., 2016; Vij et al., 2016, 2017). The roles of nonsynonymous SNPs (nsSNPs) of E2F1gene in the susceptibility of LC and HNC have not been described so far. We hypothesized that nsSNPs in E2F1 might alter their function and transform it to an oncogene, which may predispose individuals to risk of LC and HNC. Therefore, we analyzed the association of its variants rs3213173 (C/T) and rs3213176 (G/A) with LC and HNC risk in patients from North Indian population.

Materials and Methods

Genotyping

DNA was isolated from peripheral blood following inorganic method (Miller et al., 1988). We used polymerase chain reaction-restriction fragment length polymorphism for genotyping these variants and optimized conditions are given in Supplementary Data. The representative images of genotyping are given in Figure 1A, B. The details of genotyping are presented in Supplementary Data.

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FIG. 1.

Representative agarose gel images of rs3213173 (C/T) and rs3213176 (G/A) genotyping. (A) For rs3213173 (C/T), PCR-amplified products were digested with MspI. The digestion products of 168, 117, and 51 bp fragments indicated CT heterozygous allele (L4). The presence of 117 and 51 bp indicates the homozygous allele CC (L5, L6, L7, and L8) and presence of single band 168 bp indicates the homozygous mutant allele TT (L2 and L3) M = 100 bp DNA ladder. (B) For rs3213176 (G/A), PCR-amplified products were digested with MspI. The digested products of 262, 191, and 71 bp fragments indicate GA heterozygous allele (L2, L3, L4, L5, and L6). The presence of 191 and 71 bp indicates the homozygous allele GG (L8) and presence of single band 262 bp indicates the homozygous mutant allele AA (L7) M = 100 bp ladder. PCR, polymerase chain reaction.

Results

Discussion

The marked box domain and its adjacent region of E2F1 are responsible for their unique ability to strongly induce apoptosis (Hallstrom and Nevins, 2003). The analyzed nsSNPs of this gene are located in these regions, which interact with Jab1 (apoptosis inducer cofactor factor) (Hallstrom and Nevins, 2006). The selected nsSNPs in this domain could alter its structure and interactions with Jab1, leading to reduced apoptosis, and may play a role in cancer susceptibility. Various studies have reported the role of SNPs in structural and functional changes in the protein and susceptibility of various diseases (Gupta et al., 2005; Randhawa et al., 2015; Agarwal et al., 2016; Vij et al., 2016, 2017). SNPs in the regulatory region of E2F1 and E2F2 were associated with the early onset of squamous cell carcinoma of the HNC in American population (Lu et al., 2012). Moreover, a recent study on Indian population has also shown rs3213172 (C/T), rs3213173 (C/T), and rs3213176 (G/A) as a risk factor for cervical cancer (Singh et al., 2018).

In this study, we observed a significant association of Val276Met and Gly393Ser variants of E2F1 protein with the cancer risk. The allele and genotype frequencies of these two SNPs exhibited significant differences between the case and control groups. This is the first report investigating the role of E2F1 coding region variants in LC and HNC risk. We found a strong association of rs3213176 (G/A) polymorphism with the risk of HNC in the homozygous model (11.39-fold), the heterozygous model (3.469-fold), as well as in the dominant model (4.745-fold). This SNP also increases the risk of LC in the homozygous model (2.750-fold), heterozygous model (2.111-fold), and dominant model (2.214-fold) (Table 1). Furthermore, the risk of HNC (in homozygous, heterozygous, and dominant models) is increased among smokers with rs3213176 (G/A) polymorphism (Table 2).

A study on E2F1 gene polymorphism [rs35301225 (C/A)] in Chinese population revealed its involvement in the occurrence of colorectal cancer by the upregulation of E2F1 gene. Also, this SNP was associated with tumor size, tumor differentiation, and metastasis in colorectal cancer (CRC) patients (Jiang et al., 2017). In our population, we could not find any association with the clinicopathological parameters (TNM clinical staging, tumor size, and lymph node metastasis) (Supplementary Tables S3 and S4).

Conclusion

In this study, we observed a statistically significant association between the rs3213173 (C/T) polymorphism with LC risk and rs3213176 (G/A) polymorphism with LC and HNC risk. These polymorphisms of E2F1 gene might serve as a valuable prognostic biomarker for genetic susceptibility to LC and HNC and hence need to be explored further in replication studies from other populations with larger patient sample size.