AIM: To evaluate the association between genetic polymorphisms of the gene encoding AMP-activated protein kinase (PRKAA1) and the risk of gastric cancer.

METHODS: The study subjects consisted of 477 age- and sex-matched case-control pairs. Genotyping was performed for 5 tag single nucleotide polymorphisms (SNPs): rs13361707, rs154268, rs3805486, rs6882903, and rs10074991. Associations between gastric cancer and putative risk factors (including the SNPs) were analyzed with multivariate conditional logistic regression models, after adjusting for potential confounding factors. Multiple testing corrections were implemented following methodology for controlling the false discovery rate. Gene-based association tests were performed by using the versatile gene-based association study (VEGAS) method.

RESULTS: In the dominant model, SNPs rs13361707 [odds ratio (OR) = 1.51, 95%CI: 1.07-2.11)], rs154268 (OR = 1.65, 95%CI: 1.22-2.22), rs6882903 (OR = 1.48, 95%CI: 1.09-2.00), and rs10074991 (OR = 1.53, 95%CI: 1.09-2.16) were significantly associated with an increased risk of gastric cancer. In the recessive model, SNPs rs154268 (OR = 1.66, 95%CI: 1.22-2.26), rs3805486 (OR = 0.63, 95%CI: 0.46-0.85), and rs10074991 (OR = 1.47, 95%CI: 1.15-1.88) were significant risk or protective factors for gastric cancer. In the codominant model, the ORs of each of the 5 SNPs were statistically significant. All SNPs in the model showed a dose-response relationship between the minor allele frequency and the risk of gastric cancer. Most notably, subjects with a homozygous minor allele in SNP rs10074991 showed 2.15 times the risk of gastric cancer as subjects without a minor allele. The PRKAA1 gene showed a significant gene-based association with gastric cancer in the VEGAS test.

CONCLUSION: All 5 tested tag SNPs of the PRKAA1 gene (rs13361707, rs154268, rs3805486, rs6882903, and rs10074991) were significantly associated with gastric cancer.

Gastric cancer is the second most common cause of cancer-related mortality worldwide[1]. Since the 1980s, South Korea, Japan, China, and other Asian countries have had a particularly high incidence of this disease, despite general trends of decreasing incidence and mortality[2,3].

A model of gastric carcinogenesis in humans has been derived based on evidence from various epidemiological and pathological studies. According to this model, gastric cancer arises in a sequence of stages: chronic gastritis, atrophic gastritis, intestinal metaplasia, and dysplasia[4]. Helicobacter pylori, high salt intake, alcohol intake, smoking, diet, and genetic factors have been reported to be involved in gastric carcinogenesis[5-9].

AMP-activated protein kinase (AMPK) is an energy sensing/signaling intracellular protein, and a conserved serine/threonine kinase that regulates energy homeostasis and metabolic stress[10]. AMPK is activated by phosphorylation when the AMP/ATP ratio is high[11]. Activated AMPK switches on ATP-generating (catabolic) pathways and switches off ATP-consuming (anabolic) pathways[12,13]. AMPK activation is known to inhibit the accumulation of lipid in the body, decrease the biosynthesis of fatty acids and cholesterol, and increase the oxidation of fatty acids[12].

Considerable evidence indicates that AMPK activation suppresses cell proliferation in both tumor and non-malignant cells. These results of AMPK activation are mediated through various mechanisms, including G1 phase arrest in the cell cycle[14], and the inhibition of fatty acid and protein synthesis[13,15]. Regulation of the cell cycle by AMPK is mediated through activation of the p53-p21 axis pathway, activation of tumor suppressor LKB1, inhibition of the mammalian target of rapamycin pathway, and other similar mechanisms[16]. Based on this evidence, research on AMPK function has focused on its important role in development, and on its potential use as a therapeutic target for some cancers[16,17]. It is possible that AMPK plays an important role in gastric carcinogenesis and, therefore, polymorphic alleles of the encoding gene could modify individual susceptibility to gastric cancer. Recently, Song et al[18] reported that the rs13361707 single nucleotide polymorphism (SNP) of the protein kinase, AMP-activated alpha 1 catalytic subunit (PRKAA1) gene was associated with an increased risk of gastric cancer in the Korean population. However, because their study only examined the rs13361707 SNP of the PRKAA1 gene, it remains important to elucidate the associations between other SNPs of PRKAA1 and gastric cancer.

Accordingly, the aim of the present study was to evaluate the associations between 5 polymorphic alleles of PRKAA1, the gene that encodes AMPK, and gastric carcinogenesis in Koreans.

We explored the associations between 5 DNA polymorphisms in the PRKAA1 gene, which encodes AMPK, and gastric cancer risk. A total of 954 subjects were included in the analysis, comprising 477 gastric cancer cases and equal number of matched controls. There were no significant differences between the two groups in terms of age, gender, smoking status, or alcohol intake.

Table 1 presents the frequencies of the 5 selected SNPs in the study subjects. The genotype distributions at all 5 SNPs were in Hardy-Weinberg equilibrium, with non-significant χ² values.

Haplotype linkage disequilibrium block and haplotype frequencies for PRKAA1 are presented in Figure 1. Common haplotypes (frequency > 5%) of the block were found in 93.6% of cases, and 95.7% of controls.

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Figure 1 Haplotype linkage disequilibrium blocks and haplotype frequencies for PRKAA1. A: Linkage disequilibrium blocks and correlation coefficients among PRKAA1 polymorphisms. Black squares indicate statistically significant allelic association between the pair of single nucleotide polymorphisms (SNPs), as measured by using the D statistic; darker gray indicate higher values of D, up to a maximum of 1; B: Haplotype frequencies of PRKAA1 polymorphisms in the case and control groups. Freq: Allele frequency.

The frequencies and distribution of genotypes are presented in Tables 2 and 3, which also report the odds ratios for the associations between each polymorphism and gastric cancer. We performed the PRKAA1 gene association analysis with dominant, recessive, and codominant models by using conditional logistic regression. In the dominant model, 4 of 5 SNPs we observed were significantly associated with an increased risk of gastric cancer. SNPs rs13361707 (“C” allele, OR = 1.51, 95%CI: 1.07-2.11, P = 0.018, FDR Q = 0.023), rs154268 (“C” allele, OR = 1.65, 95%CI: 1.22-2.22, P = 0.001, FDR Q = 0.006), rs6882903 (“A” allele, OR = 1.48, 95%CI: 1.09-2.00, P = 0.012, FDR Q = 0.023), and rs10074991 (“G” allele, OR = 1.53, 95%CI: 1.09-2.16, P = 0.014, FDR Q = 0.023) were significantly associated with an increased risk of gastric cancer. In the recessive model, SNP rs154268 (OR = 1.66, 95%CI: 1.22-2.26, P = 0.001, FDR Q = 0.005), rs3805486 (OR = 0.63, 95%CI: 0.46-0.85, P = 0.003, FDR Q = 0.005) and rs10074991 (OR = 1.47, 95%CI: 1.15-1.88, P = 0.002, FDR Q = 0.005) were significant risk factors for gastric cancer (Table 2). In the codominant model, the odds ratios were statistically significant for all 5 SNPs. All SNPs in the model showed dose-response relationships between minor allele frequency and the risk of gastric cancer. Most notably, subjects with a homozygous minor allele in SNP rs10074991 showed 2.15 times of risk for gastric cancer, compared with subjects who did not have a minor allele (Table 3).

To evaluate the association between gastric cancer and all SNPs within the PRKAA1 gene (rather than each SNP individually) we performed a gene-based analysis following the VEGAS method, the results of which indicated that SNPs in PRKAA1 had a statistically significant association with gastric cancer in all 3 models (P = 0.0054, 0.0001, and 0.0004 for the dominant, recessive, and codominant models, respectively).

The haplotype block was also evaluated for an association with the risk of gastric cancer (Table 4), but none of the results were significant in each of the 3 models.

In this study, we hypothesized that genetic polymorphisms in PRKAA1 might contribute to gastric cancer development by affecting the regulation of energy metabolism. Activated AMPK inactivates a number of metabolic enzymes involved in ATP-consuming cellular events (such as fatty acid, cholesterol, and protein synthesis) and also activates ATP-generating processes (such as the uptake and oxidation of glucose and fatty acids)[12,13]. Besides energy metabolism, AMPK also functions as a suppressor of cell proliferation[14]. Consequently, some research on AMPK has focused on its potential role as a therapeutic target for cancer.

A recent genome-wide association study identified a new SNP (rs13361707) in the PRKAA1 gene that is significantly associated with increased susceptibility to gastric cancer[21,22]. Additionally, Song et al[18] reported that the rs13361707 SNP was associated with an increased risk of gastric cancer in the Korean population. In their replication study, rs13361707 TC and CC genotypes were associated with a significantly increased risk of gastric cancer (OR = 1.29 for TC vs TT, OR = 1.68 for CC vs TT). In agreement with these findings, our result also showed that the rs13361707 SNP is associated with gastric cancer (OR = 1.29 for TC vs TT, OR = 2.05 for CC vs TT). Together, these results suggest that rs13361707 SNP might play an important role in the development of gastric cancer. However, since rs13361707 is not the only SNPs found in this gene, it remained important to examine associations between other SNPs of PRKAA1 and gastric cancer development.

In the present study, we evaluated the associations of 5 SNPs of PRKAA1 gene with gastric cancer. Interestingly, we found that all 5 of the tested SNPs of PRKAA1 we tested were associated with a significantly increased risk of gastric cancer. Most notably, subjects with a homozygous minor allele in SNP rs10074991 were at 2.15 times the risk of gastric cancer, compared with subjects who did not have a minor allele. After controlling the FDR, the associations of these SNPs remained statistically significant in the codominant model. In a gene-based association test, the PRKAA1 gene was found to be significantly associated with gastric cancer. These results suggest that genetic polymorphism of PRKAA1 might play an important role in gastric carcinogenesis.

Although the biological mechanism underlying the association between PRKAA1 and gastric cancer has not been clarified, these significant associations could potentially be explained by the ability of activated AMPK phosphorylates p53 to induce G₁/S arrest. Further, the AMPK-p53 connection may represent a cell cycle checkpoint[23]. Therefore, individuals with mutant PRKAA1 alleles, which encode inactive AMPK, may be vulnerable to gastric cancer. Anti-inflammatory action by AMPK could provide another explanation of the association between SNPs of PRKAA1 and gastric cancer. A recent study has reported that activated AMPK can counter-regulate macrophage inflammatory function[24] and activate some anti-inflammatory agents[25]. Loss of anti-inflammatory action by AMPK in the body of individuals with mutant PRKAA1 alleles results in more severe injury of the epithelium[26]. Bone marrow-derived cells are recruited at these sites of epithelial damage, and these cells can be a potential source of malignancy[27]. To our knowledge, there is no study addressed association between PRKAA1 gene and gastric cancer except in Chinese and Korean population. Since SNPs frequency are different according to the population, it is need to further study in different races other than Asian.

The present study has several limitations. First, a relatively small number of patients and controls were enrolled in this study. Second, we could not obtain detailed data on the histological tumor types for the cases of gastric cancer. Finally, because the data of environmental factor for gastric cancer such as H. pylori infection and diet was not available in this study, we could not evaluate the gene-environmental interaction. It is needed further study about it.

In summary, the PRKAA1 gene and 5 of its SNPs (rs13361707, rs154268, rs3805486, rs6882903, and rs10074991) were associated with an increased risk of gastric cancer in Koreans.