Squamous cell carcinoma of the head and neck (SCCHN) is the fifth most common cancer worldwide [1], which includes malignancies at the sites of the oral cavity, larynx and pharynx. It was estimated that there were 48,010 new cases and 11,260 deaths of SCCHN in the United States in 2010 [2]. Tobacco smoke and alcohol consumption are the well-recognized risk factors for SCCHN, and more recently human papillomavirus (HPV) is recognized as a major risk factor for the oropharygeal cancer [3]. However, accumulative evidence also shows that genetic factors, including family history and polymorphisms in genes involved in multiple biological pathways, such as carcinogen metabolism, DNA repair, cell cycle regulation, apoptosis and other cellular processes, play important roles in the etiology of SCCHN [4, 5]. Although an increasing number of studies on genetic risk factors for SCCHN have been published, the exact genetic basis of susceptibility to SCCHN is still not well defined.

In the past few years, the wave of genome-wide association studies (GWASs) provided a more robust tool to find novel susceptibility loci or genes for cancer susceptibility by using high-throughput genotyping technology to interrogate a large number of tagging polymorphisms in a high density across the whole genome [6]. To date, more than 50 cancer GWASs have been published, including at least 15 different malignant tumors [7], which has greatly improved our understanding of genetic basis of human cancers. However, only one recent GWAS focused on SCCHN risk and identified five variants at 4q21, 12q24 and ADH gene cluster, significantly associated with risk of upper aerodigestive tract cancers (UATC) including SCCHN [8]. It may be a very small proportion of SNPs associated with SCCHN risk because of the strict criteria for the GWAS significance level (P = 10^-7 or P = 10^-8). Thus, further exploration for the genetic variants that did not reach the GWAS significance level in the development of SCCHN is needed.

In 2010, two large-scale genome-wide association studies [9, 10] simultaneously reported that a new and notable low-penetrance susceptibility locus (rs2274223) located in the phospholipase C epsilon 1 gene (PLCE1) was strongly associated with risk of esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA) in Chinese population. Rs2274223 is a non-synonymous SNP located in exon 26 of the PLCE1 gene, causing the amino acid change from histidine to arginine. Additionally, one study also showed that the positive rates of the PLCE1 protein in ESCC and GCA tissues were significantly higher than that in normal ones, suggesting a biologically plausible role of PLCE1 in carcinogenesis of human cancers [9].

The PLCE1 gene resides on chromosome 10q23 and encodes phospholipase C epsilon 1 (PLCE1), which belongs to the phospholipase family that catalyzes the hydrolysis of polyphosphoinositides to generate secondary messengers, such as inositol-1,4,5 trisphosphate and diacylglycerol. Therefore, PLCE1 is involved in cell growth and differentiation [11]. Studies have reported that PLCE1 functions as an effector of Ras and plays crucial roles in carcinogenesis and progression of several cancers, including cancers of the intestine [12], skin [13], bladder [14], colorectal [15] and head and neck [16]. All these findings further support the biological plausibility that genetic variations, such as single nucleotide polymorphisms (SNPs) in PLCE1 that affect the gene expression or protein functions, may affect the risk of some cancers.

Studies have reported that SCCHN shares some similar risk factors with ESCC and GCA, such as tobacco smoke and alcohol consumption [17–19]. Thus, the exciting results in the GWAS of ESCC and GCA, and the strong biological evidence encouraged us to investigate the association between functional SNPs in PLCE1 and susceptibility to SCCHN. In the present study, we performed genotyping analyses of three potentially functional SNPs in PLCE1 (rs2274223A/G, rs3203713A/G and rs11599672T/G) in non-Hispanic whites and assessed their associations with risk of SCCHN in our ongoing hospital-based case-control study of 1,098 SCCHN cases and 1,090 cancer-free controls matched to cases on age (± 5 years) and sex.

In this case-control study, we investigated the associations between three novel, potentially functional SNPs of PLCE1 (rs2274223, rs3203713 and rs11599672) and risk of SCCHN in a non-Hispanic white population. Although we did not find evidence of a main effect of each PLCE1 SNP on overall SCCHN risk, the joint effect of these variants appeared to contribute to risk of SCCHN in a dose-response manner, especially for cancers arising at non-oropharyngeal sites. Further, the subgroup analysis of SCCHN arising at non-oropharyngeal sites showed that variant genotypes of both rs2274223 and rs11599672 were independently associated with the risk. These findings suggested, for the first time, that potentially functional polymorphisms of PLCE1 may play a role in the development of SCCHN, particularly of those tumors at non-oropharyngeal sites.

PLCE1 is known to be a direct downstream effector of small GTPases (Ras, Rap1 and Rap2), with the presence of two Ras-associating domains at its C terminus and a CDC25 guanine exchange factor (CDC25 gef) domain at its N terminus [11, 23–25]. Since mutations in the RAS gene family are associated with about 30% of all human cancers, several studies have investigated the possible role of PLCE1 in cancer development and progression [13–16, 26]. It has been reported that PLCE1 has an oncogenic role in carcinogenesis of several human cancers, including SCCHN, through distinct mechanisms, such as inflammation, binding to the Ras family small GTPase, and augmentation of angiogenesis [13–16, 26]. Recent evidence has demonstrated that PLCE1 mutations might cause the nephritic syndrome [23] and diffuse mesangial sclerosis (DMS) [27]; however, few studies have investigated the association between genetic variants of PLCE1 and risk of human cancers.

Most recently, for the first time, Wang et al. found that rs2274223, a nonsynonymous SNP of the PLCE1 gene, was associated with an increased risk of ESCC and GCA in a Chinese population by a two-stage GWAS in 9,053 ESCC cases, 2,766 GCA cases and 11,013 controls [8]. Likewise, in another GWAS including 2,115 ESCC cases, 2,240 gastric cancer cases and 3,302 controls in another Chinese population, Abnet et al. also reported that rs2274223 was a notable signal for susceptibility to ESCC and GCA [10]. Despite the present study with 1,098 SCCHN cases and 1,090 controls may have a limited power to detect weak associations between polymorphisms of PLCE1 including rs2274223 and overall risk of SCCHN compared with the published GWA studies [9, 10], our results did show that subjects carrying more risk alleles in PLCE1 (rs2274223G, rs3203713G and rs11599672T) had a higher risk of SCCHN than those with zero to one risk allele, especially for SCCHN arising at non-oropharyngeal sites, suggesting a joint effect of these SNPs on susceptibility to SCCHN. Given only a modest effect of each SNP individually, evaluating their combined effects may help us better understand any role of PLCE1 SNPs in cancer etiology. Furthermore, different carcinogenic mechanisms between esophageal and gastric cardia cancers and SCCHN or genetic difference in different populations may result in the discrepancy for the main effect of rs2274223 between our study on SCCHN and two GWASs of esophageal and gastric cancers.

Studies have shown that the oropharynx is the most common site for HPV-associated SCCHN [28, 29]; in contrast, SCCHN arising at non-oropharyngeal sites have much lower HPV seroprevalence [28, 30] and thus are more likely to be caused by smoking and alcohol drinking, similar to ESCC and GCA. In fact, 36% of our oropharygeal cancer patients were never smokers compared to only 19% of patients with SCCHN arising at non-oropharygeal sites. In our study, we found that rs2274223 and rs11599672 had an independent effect on risk of smoking and drinking related SCCHN arising at non-oropharyngeal sites but not HPV-related oropharyngeal cancer. Even after Bonferroni corrections, the association of rs11599672 with non-oropharyngeal SCCHN and association of rs2274223 with drinking at non-orpharyngeal sites remained significant, suggesting different roles of these polymorphisms in the etiology of two different tumor subsites. Additionally, subgroup analyses restricted to oropharygeal cancer did find a similar pattern of risk (though non-significant) associated with variant genotypes of rs2274223, rs11599672 and combined risk alleles among smokers but not among never smokers (data not shown). The findings from GWASs and our study implied that polymorphisms of PLCE1 are likely to be associated with the development of human cancers related to tobacco and alcohol exposure, which will need the validation from large studies on different cancers.

Further, compared to the most common T _rs11599672A _rs2274223A _rs3203713 haplotype, both T_rs11599672G _rs2274223A _rs3203713 and G _rs11599672A _rs2274223A _rs3203713 haplotypes had a significant association with risk of SCCHN arising at non-oropharyngeal sites, which further reflected the main effect of rs2274223 G and rs11599672 G alleles on risk of SCCHN arising at non-oropharyngeal sites. We also found that the effects of rs2274223 and rs11599672 on risk of SCCHN arising at non-oropharyngeal sites were segregated between the subgroups by smoking or drinking status, indicating the possibility of gene-environment interactions. Indeed, we detected a significant interaction between variant genotypes of rs2274223 and alcohol status with a relatively small sample size, supporting the role of a gene-environment interaction in the development of SCCHN.

It has been identified that rs2274223 is a non-synonymous SNP of PLCE1, and bioinformatics tools show that rs3203713 in PLCE1 is located in the 3'UTR (http://snpinfo.niehs.nih.gov/snpfunc.htm), which may affect the binding of miRNA and target gene PLCE1. Further, rs11599672 is located in the transcription factor binding site (TFBS) of PLCE1 (http://snpinfo.niehs.nih.gov/snpfunc.htm) and may result in the variation of transcription activity and expression of PLCE1. Considering the potentially functional significance of these SNPs, it is biologically plausible that PLCE1 polymorphisms may contribute to the development of SCCHN. However, we cannot exclude the possibility that the findings from a subgroup analysis could be false positive because of a limited sample size. More rigorous studies with larger sample sizes, detailed HPV data and SNP functional relevance are warranted to replicate our findings and identify the underlying mechanism(s) of these SNPs in the etiology of SCCHN.

Some limitations of this study need to be addressed. Firstly, it is a hospital-based case-control study, and inherent selection bias cannot be completely excluded. However, the agreement of observed genotype distributions with Hardy-Weinberg equilibrium and similar allele frequencies of our controls with those reported in CEU populations from the dbSNP database (0.320 vs. 0.314 for rs2274223 G allele, 0.162 vs. 0.120 for rs3203713 G allele, and 0.297 vs. 0.347 for rs11599672, respectively) suggested that selection bias in terms of genotype distribution would not be substantial, if any. Secondly, it is uncertain whether the results from other populations such as Chinese populations in the reported GWASs [9, 10] are generalizable to our study population; yet similar findings from our non-Hispanic white subjects further support a similar biological plausibility of these SNPs. Thirdly, while we explored associations with smoking and drinking exposure, we were not able to stratify our data by HPV exposure. Finally, 3 SNPs were included in this study and we cannot rule out the possibility of false-positive associations because of multiple tests. Actually, only P values for the effect of rs11599672 variant genotypes on tumors arising at non-oropharyngeal sites and the association of rs2274223 variant genotypes with drinking for non-oropharyngeal SCCHN risk remained significant after Bonferroni corrections.

In summary, in this hospital-based case-control study, we found that three PLCE1 SNPs may have a joint effect on the risk of SCCHN, especially those arising at non-oropharyngeal sites, including oral cavity, hypopharynx or larynx, associated with smoking and alcohol exposure. Furthermore, those results from subgroup analysis suggest that variant genotypes of rs2274223 and rs11599672 may independently affect the risk of SCCHN arising at non-oropharyngeal sites. Additional larger studies in different populations are needed to validate our findings.