Abstract
A single-nucleotide polymorphism (rs2274223: A5780G:His1927Arg) in the phospholipase C epsilon gene (PLCe) was recently identified as a susceptibility locus for esophageal cancer in Chinese subjects. To determine the underlying mechanisms of PLCe and this SNP in esophageal carcinogenesis, we analyzed PLCe genotypes, expression, and their correlation in esophageal cancer cell lines, non-transformed esophageal cells, 58 esophageal squamous cell carcinomas and 10,614 non-cancer subjects from China. We found that the G allele (AG or GG) was associated with increased PLCe mRNA and protein expression in esophageal cancer tissues and in esophageal cancer cell lines. G allele was also associated with higher enzyme activity, which might be associated with increased protein expression. Quantitative analysis of the C2 domain sequences revealed that A:G allelic imbalance was strongly linked to esophageal malignancy. Moreover, the analysis of 10,614 non-cancer subjects demonstrated that the G allele was strongly associated with moderate to severe esophagitis in the subjects from the high-incidence areas of China (OR 6.03, 95% CI 1.59-22.9 in high-incidence area vs. OR 0.74, 95% CI 0.33-1.64 in low-incidence area; P ¼ 0.008). In conclusion, the PLCe gene, particularly the 5780G allele, might play a pivotal role in esophageal carcinogenesis via upregulating PLCe mRNA, protein, and enzyme activity, and augmenting inflammatory process in esophageal epithelium. Thus, 5780G allele may constitute a promising biomarker for esophageal squamous cell carcinoma risk stratification, early detection, and progression prediction. ß 2013 Wiley Periodicals, Inc.
Key words: phospholipase C epsilon; esophageal cancer; esophagitis Additional supporting information may be found in the online version of this article.
Abbreviations: PLCe, phospholipase C epsilon; SCC, squamous cell carcinoma; LPA, 1-Oleoyl-L-a-lysophosphatidic acid; RT-PCR, reverse transcriptional polymerase chain reaction.
INTRODUCTION
Phospholipase C epsilon (PLCe or PLCE 1) is a phosphoinositide-specific enzyme phospholipase C that converts phosphatidylinositol 4,5-biphosphate (PtdIns (4, 5) P2) [1, 2] to two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which regulate protein kinase C activity and calcium mobilization, respectively [3, 4] . These responses are involved in the regulation of cell growth, differentiation, and oncogenesis [5] . To date, six members of the PLC family (PLCb, g, d, e, h, and z) have been identified in mammalian cells. These members share similar catalytic properties and are characterized by distinct regulatory mechanisms [6, 7] , indicating that PLCs exert diverse functions in carcinogenesis. PLCe binds to and is activated by the Ras family GTPases [1, 8, 9] . It participates in murine skin cancer development through augmentation of inflammatory cytokine production and signaling [10] [11] [12] . A more recent report also showed that inhibition of intestinal tumorigenesis in ApcMin/þ mice by loss of PLCe occurs via reducing cytokine-mediated inflammation in local tissue [13] . However, the involvement of PLCe in human carcinogenesis, particularly in esophageal cancer, was not been recognized until a susceptible SNP was recently identified in GWAS studies of more than 20,000 Chinese subjects [14, 15] . This SNP, located within exon 26 of the PLCe gene at chromosome 10q23, leads to a nonsynonymous alteration from CAC (A allele, encoding histidine) to CGC (G allele, encoding arginine). These previous data showed that a G at position 5780 in the C2 domain of PLCe was associated with an increased risk of both squamous cell carcinoma (SCC) and esophagus-cardia gastric adenocarcinoma (ECA). In the current report, we demonstrate that the presence of the G allele at this locus leads to increased PLCe mRNA and protein expression and enzyme activity in esophageal cancer cells in vitro, and that A:G allelic imbalance is strongly associated with esophageal malignancy in primary human tissues. Interestingly, we show that the presence of the G allele is also strongly associated with moderate to severe esophagitis in noncancer subjects from the areas of high-incidence of esophageal cancer in China.
MATERIALS AND METHODS

Human Subjects
Frozen tissues from 58 esophageal SCCs and white blood cell DNA from 10,614 non-cancer subjects were collected from an ongoing hospital-based SCC and EAC case-control study, involving multiple hospitals throughout high-and low-risk areas for esophageal SCC in China since 2007. The genotypes of PLCE in non-SCC patients were based on TaqMan genotyping methods, as described recently [14] . All subjects had undergone esophagogastroduodenoscopy, and non-cancer subjects with esophagitis were determined by endoscopic examination and histopathology. The diagnosis of the degree of esophagitis was made by at least three pathologists. All procedures were conducted according to Declaration of Helsinki principles and had been approved by respective institutional review boards.
DNA Extraction and PCR-Based PCLe Exon 26 Sequencing
Genomic DNA was extracted from the following materials: SCC and adjacent normal esophageal epithelia, esophageal SCC cell lines (TE-1, TE-2, TE-7, TE-8, TE-12, G5, HCE4, HCE7, EC171, EC8712, EC109, EC9706, SHEEC) and non-transformed esophageal epithelial cell lines (HET1A, HEEpic) using a DNA extraction kit (QIAGEN, Valencia, CA). PCR for PLCe exon 26, in which the C2 domain is located, was performed using the following primers: forward (Ex26F): 5 0 -TGTTCTTGGGATTCCTTTGC-3 0 and reverse (Ex26R): 5 0 -TGCTTCTTAATTCAAC-TTCTTTATAGG-3 0 . The PCR product was directly sequenced using an ABI sequencing system (Applied Biosystems, Inc., Foster City, CA). Allelic imbalance was analyzed using Mutation Surveyor software (Softgenetics, College Station, PA).
RNA Extraction, Quantitative Real-Time PCR, Immunoblotting, and Immunohistochemical Staining Total RNA was extracted from human esophageal cancer cell lines and tissues, samples were then subjected to reverse transcription; cDNA was used for quantitative PCR analyses of PLCe expression at the mRNA level. The following primers that cover the C2 domain were used for quantitative PCR analysis: forward 5 0 -TGTGGAACGAGCAGTTTCTG-3 0 and reverse 5 0 -ATCGAAGAGGC TGACATGGT-3 0 . Cell lysate was made from esophageal cancer cell lines, followed by immunoblotting and probing with antiPLCe antibody (provide by Dr. J. Lomasney, Northwestern University, Chicago, IL). Immunoblotting intensities were quantified using Quanty One software (Bio-Rad, Hercules, CA). PLCe immunohistochemical staining was performed as described recently [14] , and immunohistochemical staining was scored by at least three pathologists using the following criteria: 0, no staining or staining area was <10%; 1, positive area was between 10% and 50%; and 2, positive area was more than 50%, similar as we reported [16] .
PLCe Enzyme Activity Assay
PLCe enzymatic activity was determined by measurement of total [ 3 H]inositol phosphates accumulation in esophageal cancer cells containing AG and AA alleles (TE-2, -7, -8, and -12). 1-Oleoyl-L-a-lysophosphatidic acid (LPA) was purchased from SigmaAldrich (St. Louis, MO) and dissolved in water containing 1.0% fatty acid-free bovine serum albumin.
Briefly, cells were seeded in a 24-well plate at a density of 200,000 cells per well. 18 h later, medium was replaced with inositol-and serum-free DMEM containing 1 mCi/well [ 3 H]myo-inositol (American Radiolabeled Chemicals, St. Louis, MO). Phospholipase C activity was quantified 16 h after labeling by incubation in inositol-free DMEM containing 10 mM LiCl, either in the absence of LPA or in the presence of 10 mM LPA. The reaction was stopped after 30 min by aspiration of the medium and addition of an ice-cold buffer containing 0.6 M perchloric acid and 0.2 mM IP 6 . After neutralization with buffer containing 1 M K 2 CO 3 and 40 mM EDTA, the accumulation of [ 3 H] inositol phosphates was quantified by Dowex chromatography. 0.1% Triton X-100/0.1 M NaOH was added to cells in each well to determine total lipids. Each experiment was performed in triplicate, and these triplicate experiments were also repeated independently three times.
RESULTS AND DISCUSSION
To investigate whether the G allele of the PLCe gene SNP at position 5780 alters PLCe expression, we determined PLCe genotypes and mRNA levels in 13 human esophageal squamous cancer cell lines (TE-1, TE-2, TE-7, TE-8, TE-12, G5, HCE4, HCE7,  EC171, EC8712, EC109, EC9706, SHEEC). Six SCC  cell lines (G5, TE-8, TE-12, HCE4, EC171, EC8712) were AA, while the remaining 7 (TE-1, TE-2, TE-7, HCE7, EC109, EC9706, SHEEC) were AG for PLCe at position 5780. Quantitative RT-PCR (qRT-PCR) revealed that PLCe mRNA levels in the seven SCC AG cell lines increased approximately 37-fold relative to the 6 AA SCC cell lines ( Figure 1A ; 271 AE 144 in AG cells vs. 7.3 AE 3.3 in AA cells, P < 0.01). Increased PLCe mRNA levels also correlated with higher PLCe protein levels measured by immunoblotting ( Figure 1B ). The overall (average) intensity of PLCE1 protein in AG cell lines were higher than those in the AA cell lines (P < 0.02). Interestingly, two immortalized human esophageal epithelial cell lines derived from normal cells, HET1A (AG allele) and HEEPic (AA allele) also exhibited a similar association pattern: cells with the AG allele expressed higher levels of PLCe mRNA (23.6 in HET1A vs. 3.6 in HEEPic) and protein (data not shown).
We then examined whether elevated PLCe mRNA and protein levels correlated with PLCe enzyme activity. For these experiments, we chose the two AG cell lines (TE-7 and TE-2) that expressed the highest levels of PLCe and the two AA cell lines (TE-8 and TE-2 and TE-7) than those in the two AA cell lines (TE-8 and TE-12) (76 AE 20 vs. 42 AE 7, P < 0.05). LPA was used as a ligand to stimulate PLCe activity, and PLCe activities were significantly induced (black bars). Bars represent the mean AE SD for three replicates of experiments performed.
TE-12) with the lowest levels of PLCe mRNA and protein. Consistent with findings for protein levels, endogenous PLCe baseline activity was nearly twice as high in the two AG cell lines (TE-2 and TE-7) than those in the two AA cell lines (TE-8 and TE-12) (76 AE 20 vs. 42 AE 7, P < 0.05). The increased enzyme activities might be linked to the increased protein expression. Previous studies have demonstrated that overexpression of activated small Rho family GTPases leads to marked elevation of intracellular inositol phosphate accumulation [17] , and PLCe is a direct effector of activated Rho [18] . Similarly, the expression of G protein a-subunits Ga 12 and Ga 13 resulted in PLCe-dependent accumulation of inositol phosphates [19, 20] . Therefore, agonists of G Protein coupled receptors that couple to Ga 12 and Ga 13, such as LPA (1-Oleoyl-L-a-lysophosphatidic acid), activate PLCe in a Rho-dependent manner [21] . To determine the response of PLCe activity to LPA in cells containing different alleles (AA vs. AG), we treated the two groups of ESCC cell lines with LPA and surprisingly found that AG cell lines exhibited only 2.5-fold average induction of PLCe activity, whereas AA cell lines were induced average fourfold ( Figure 1C ; P < 0.05). This finding may have been due to the failure of adapted AG allele cells to fully activate PLCe after interacting with harmful environmental factors (e.g., bile acids, bacterial infection, carcinogens, or other stressors), which reduces subsequential induction of cytokine and chemokine and development of inflammation in esophageal epithelium as a defensive response to the environmental detrimental stimulation. As a consequence, the lack of fully activated PLCe enzyme could cause epithelial cells to produce more PLCe mRNA and protein as a compensatory response through a feedback mechanism. From analysis of homology modeling of the PLCe C2 domain structure, we observed that changing His1927 to Arg in the C2 domain may affect protein-protein interaction and/or lipid recognition, but is unlikely to have an impact on ion binding by this enzyme (Supplementary Figure 1) . However, further investigations using mutant allele plasmids are needed to confirm this modeling result.
PLCe-mediated cell growth promotion has been reported in various cell types [5, 22, 23] , and the mitogenic effect of PLCe may facilitate cancer progression. We measured PLCe mRNA levels in 26 primary human esophageal SCCs and adjacent normal esophageal epithelial tissues by quantitative RT-PCR. As shown in Figure 2A , PLCe mRNA levels were significantly higher in esophageal SCCs than in adjacent normal tissues (normal, 1.0 vs. SCC, 22.0; P < 0.01). Consistent with these mRNA expression levels, SCC tissues with heterozygous AG expressed 1.5-fold higher protein levels than did homozygous AA tissues (1.5 AE 0.1 vs. 1.0 AE 0.3, P < 0.05; Figure 2B ), assayed by immunohistochemical staining using an anti-PLCe antibody ( Figure 2C ). Our results were different from recent report that showed decreased PLCe mRNA and no changes of protein in esophageal cancer tissues compared to the adjacent normal tissues, assayed by qRT-PCR and immunohistochemical staining, respectively [24] . This discrepancy could be resulted from the patients selection: our patients were from the North Henan area, the most high incidence area of esophageal cancer in the world, the patients used in the recent report were from Shanghai area [24] , the low incidence area of esophageal cancer. The different pattern of PLCe in high-and low-incidence areas was observed in esophagitis ( Figure 3) . We showed the results in detail later. Interestingly, allelic imbalance analysis showed an increased G allele copy number in 44% (15/34) of AG SCC tissues when compared to matching normal esophageal control tissues ( Figure 2D ), using Mutation Surveyor software (Softgenetics, College Station, PA). These findings suggest that G allele is associated with overexpression of PLCe in primary SCC tissues.
Overexpression of PLCe protein and enhancement of PLCe enzyme activity have been reported to activate PKC and induce elevation of intracellular calcium levels [25] , leading to cytokine-or chemokine-mediated inflammation in local tissues [12] . Furthermore, an association between esophagitis and the development of esophageal squamous cell cancer has been recognized and documented previously [26] . To investigate any possible association between A5780G and esophageal inflammation, we correlated SNP genotypes with presence or absence of esophagitis in individuals with cancer (SCC) and without cancer (non-SCC). 52 (89.7%) of the 58 SCC patients exhibited various degrees of esophagitis (mild, moderate, or severe), whereas only 1517 (14.3%) of the 10,614 non-SCC subjects had any esophagitis, determined by endoscopic examination and confirmed by histopathology (P < 0.0001). Importantly, the severity of esophagitis was associated 
E84
with the AG/GG allele in these SCC patients. Eight (62%) of the 13 SCC patients with moderate or severe esophagitis were AG/GG, while only 5 (38%) of the 13 SCC patients with moderate or severe esophagitis had the AA genotype ( Figure 3A) . However, when non-SCC individuals with esophagitis were classified into high-or low-incidence areas for esophageal cancer, a significant association between severe esophagitis and the G allele of the PLCe gene was observed: 77% of the severe esophagitis individuals in high-incidence areas had AG/GG genotypes ( Figure 3B ), versus only 37% of these subjects in low-incidence areas ( Figure 3C ) (OR 6.03 with 95% CI 1.59-22.9 vs. OR 0.74 with 95% CI 0.33-1.64; P ¼ 0.008; Table 1 ). These data support the hypothesis that the interaction of potential environmental factors with PLCe, particularly in the individuals with AG or GG allele, not only exists in high-incidence areas for esophageal cancer development in China, but also correlates with the severity of esophagitis. Currently, this group of individuals is under long-term follow up. Correlations between this risk allele, esophagitis, and cancer development will be clarified in future studies.
In conclusion, PLCe is likely to play a pivotal role in esophageal carcinogenesis: the presence of the 5780G allele may not only predict a high risk of future esophageal cancer development, but may also participate in esophageal cancer growth and progression by upregulating levels of PLCe mRNA, protein, and enzyme activity, ultimately leading to augmentation of the inflammatory process in esophageal epithelium. Thus, the 5780G allele in PLCe may constitute a promising biomarker for esophageal squamous carcinoma risk stratification, early detection, and progression prediction.

{'paper_id': '13775901', 'title': 'A sequence variant in the phospholipase C epsilon C2 domain is associated with esophageal carcinoma and esophagitis', 's2_url': 'https://api.semanticscholar.org/CorpusID:13775901', 'source': 'S2ORC'}