A Shared Susceptibility Locus in the p53 Gene for both Gastric and Esophageal Cancers in a Northwestern Chinese Population

Abstract

Background:

Upper gastrointestinal tract cancers are the leading causes of cancer-related deaths in Northwest China and they share many similarities in terms of histological type, risk factors, and genetic variants. We hypothesized that shared common single-nucleotide polymorphisms (SNPs) in the p53 pathway exist between patients with gastric and esophageal cancer (EC) patients.

Materials and Methods:

A case-control study to examine genetic variants in the p53 pathway was conducted with subjects from a high-incidence area for upper gastrointestinal cancers of China. Multiple logistic regression analyses were used to estimate the association of genotypes with gastric cancer and EC risks. Median survival was estimated by using the Kaplan-Meier method and compared by using the log-rank test.

Results:

Compared with the rs1042522 Pro allele, the rs1042522 Arg allele was associated with an increased risk of gastric cancer (1.810×) and an increased risk of EC (2.285×). The rs1042522 Arg allele carriers who also smoked or consumed alcohol had a further increased risk for gastric cancer odds ratios (OR_smoking = 2.422, OR_drinking = 5.152) and EC (OR_smoking = 5.310, OR_drinking = 8.359). No association was found between the rs1042522 genotypes and survival (p > 0.05).

Conclusion:

The p53 rs1042522 arg allele together with tobacco smoking and alcohol drinking, was associated with an increased risk, for gastric cancer and EC, but not the survival among northwestern Chinese patients. These associations warrant confirmatory studies.

Introduction

Gastric cancer and esophageal cancer (EC) are two deadly malignancies worldwide. Both gastric cancer and EC have been prevalent in China for decades, with approximately 50% of the world's gastric cancer-related or EC-related morbidity and mortality (Yang et al., 2018). In 2018, gastric cancer was the fourth most frequently diagnosed cancer, with nearly 456,124 new cases in China and the second leading cause of cancer-related deaths with 275,094 deaths. Similarly, EC ranked the sixth among the most common cancers, with 307,359 new cases and the fourth in mortality leading to 283,433 deaths in China in 2018. However, both gastric cancer and EC have geographical variations in China. The Ningxia region, located in Northwest China, has had the highest incidence of upper gastrointestinal cancers, especially gastric cancer, for decades (Zhang et al., 2008; Yang et al., 2016).

Due to the lack of highly effective biomarkers for early diagnosis and prognosis prediction, advanced stages of gastric cancer and EC are commonly present at the time of initial diagnosis. The age-standardized 5-year relative survival rates were only 27.4% and 21% in Chinese gastric cancer and EC patients, respectively (Zeng et al., 2015). Although many research works have been undertaken to elucidate the genetic factors for the etiology of gastric cancer and EC, the established genes or loci could only explain only a small part of cancer risk. In the remaining large portion of gastric cancer and EC patients, there has been limited success in determining the genetic architecture of gastric cancer and EC.

Some recent studies have focused on investigating about whether gastric cancer and EC share common genetic single-nucleotide polymorphism (SNPs). For example, Yao et al. (2018) explored whether known 21 genetic susceptibility loci for EC were also important in the development of gastric cancer. They finally demonstrated that three genetic variants at 10q23.33, 5q11.2, and 12q24.12 may be useful as biomarkers to identify individuals with high risk for both ECC and gastric cancer. Li et al. (2013) and Abnet et al. (2010) reported that rs2274223 in the phospholipase C epsilon 1 gene and rs1434285 in the epidermal growth factor receptor signaling pathway, which could increase the risk of gastric cancer and EC, were shared polymorphisms for both gastric cancer and EC susceptibility.

The polymorphisms in the p53 pathway have been suggested to contribute to the development of upper gastrointestinal cancer (Wu and Zhang, 2015). rs1042522 in the p53 gene, also called p53 codon 72, has been widely studied for its association with the development of cancers (Zhou et al., 2010; Surekha et al., 2011). The 2nd base of p53 codon 72 has a G/C variation, resulting in the replacement of arginine (Arg) with proline (Pro) amino acids (Ara et al., 1990). Some studies have supported that the rs1042522 Arg allele increases cancer risk, including lung cancer, breast cancer, and gastric cancer (Souto-Garcia et al., 2012; Chen et al., 2014; Hebert-Magee et al., 2019). Other investigations showed that the rs1042522 Pro allele enhanced the risk of lung cancer, gastric cancer, and EC (Jiang et al., 2011; Qiao & Hu et al., 2013; Li et al., 2017). Accordingly, how p53 codon 72 works in cancer susceptibility remains inconclusive. In addition, several studies investigated the relationship between the rs1042522 and the prognosis of cancer patients and reported that rs1042522 was an independent prognostic factor for cancers (Toyama et al., 2007; Huang et al., 2008). Hence, rs1042522 may be an independent factor affecting the prognosis of upper gastrointestinal cancer.

Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A), also called p21^Waf1/Cip1, the main downstream regulator of tumor suppressor p53, functions as a unique link from p53 to cell-cycle arrest and DNA repair (El-Deiry et al., 1993; Rati et al., 2004). Thus, mutations or polymorphisms in the CDKN1A gene may result in alteration of CDKN1A expression and/or activity, thereby modulating susceptibility to cancer. Seven tagging SNPs, including rs4135234, rs3829963, rs3829964, rs762624, rs2395655, rs730506, and rs3176320, were identified as risk factors for EC in Henan Province, located in East-Central China. However, their distributions and the associations with cancers in other Chinese populations are still very limited.

Here, we hypothesized that Ningxia gastric cancer and EC patients shared common genetic SNPs among eight SNPs in the p53 pathway. To prove our hypothesis, we genotyped eight SNPs to evaluate their associations with both gastric cancer and EC. Their crossover effects with smoking or drinking status on cancers, as well as their roles in the prognostic survival among cancer patients were also analyzed in a hospital-based northwestern Chinese population.

Materials and Methods

Subjects for the case-control study

A total of 180 gastric cancer cases (all the gastric cancer patients are gastric adenocarcinoma patients) and 113 EC cases (all the EC patients are esophageal squamous carcinoma patients) were enrolled in this study and they were histopathologically confirmed between 2012 and 2013 in the General Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, China. Subjects with previous cancer and previous chemotherapy or radiotherapy were excluded. The 358 cancer-free control subjects were randomly recruited from the health check-up center at the General Hospital during the same period. The inclusion criterion for the controls was free of cancer history. The recruited subjects were confirmed to be free of contraindications regarding endoscopic and cytology examinations.

Demographic data, including the age at diagnosis, gender, alcohol drinking, and cigarette smoking of each subject, were personally questioned through trained interviewers by using a pretested questionnaire. Those who smoked one cigarette per day for more than 1 year were defined as smokers, and those who had three times or more alcoholic drinks a week for more than 6 months were defined as alcohol drinkers. After the interview, 2-mL samples of venous blood were collected from each participant for DNA preparation and genotyping. The study was approved by the Ningxia Medical University Ethical Review Committee, and signed informed consent was obtained before entry into this study according to the regulations.

Cancer-related genes and selected SNPs

Eight SNPs were selected for the analysis, including rs1042522, rs2395655, rs3176320, rs3829963, rs3829964, rs4135234, rs730506, and rs762624, are in p53 or CDKN1A, both of which play a critical role in carcinogenesis in the p53 pathway. For example, rs1042522, one of the most studied SNPs for human malignancy, is located in the p53 gene (at 17p13) (Dahabreh et al., 2013). Seven of the remaining SNPs are located in the promoter region of the CDKN1A gene (at 6p21), six of which have been analyzed for their associations with longevity, EC, or lung cancer except rs3176320 (Gravina et al., 2009; Warfel and El-Deiry, 2013; Hwang et al., 2018).

DNA preparation and genotyping

Genomic DNA was extracted from peripheral blood leukocytes of the participants by using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. SNP genotyping was performed by using an improved multiplex ligase detection reaction method (iMLDR, Genesky Bio-Tech Cod., Ltd., Shanghai, China). The polymerase chain reaction (PCR) primer pairs used to amplify the eight SNPs were the same as earlier dECribed (Dong et al., 2015) and were designed by Oligo5.0 software.

PCR was performed in a 25 μL reaction mixture containing 1.0 μL of DNA, 1.0 μL of each primer, 2.0 μL of dNTP, 2.0 μL of MgCl₂, 0.5 units of Taq DNA polymerase (Promega, Madison, WI), and 10 of reaction buffer. The PCR conditions consisted of an initial melting step of 2 min at 94°C, followed by 35 cycles of 40 s at 94°C, 40 s at 52°C-61°C, and 50 s at 72°C, and a final elongation step of 10 min at 72°C.

The polymorphisms were identified by direct sequencing of the PCR products using an ABI 3700 DNA sequencer (PE Applied Biosystems) with the forward primers. The sequencing chromatograms were base-called with Phred (version 0.990722), assembled with Phrap (version 0.990329), and scanned by PolyPhred (version3.5), and the results were viewed with Consed (version 9.0). Sequence variations were confirmed by two authors independently.

Follow-up

The information about the survival status, death time, and cause of death was collected for all eligible gastric cancer and EC patients from the hospital database, medical records, or by telephone calling or text messages to patients or their family members. All patients were followed up through January of 2019. Patients with one-call failure were contacted at least three times afterward. Those patients who still cannot be contacted after three attempts or were noticed to be with wrong phone numbers were regarded as lost to follow-up. Those who were alive, lost to follow-up at the closing date were considered censored data. The survival data were available for 103 of 180 gastric cancer cases (57.2%) and 85 of 113 EC cases (75.2%). Overall survival was calculated from the operation time to the date of death or the last follow-up.

Immunohistochemical analysis

For further immunohistochemical analysis, 103 gastric cancer cases and 85 EC cases were retrieved from the 180 gastric cancer and 113 EC surgical patients mentioned earlier. All the specimens had been routinely formalin-fixed, paraffin-embedded, and serially sectioned at 4 μm in thickness. p53 protein expression in gastric cancer and EC tissues was examined by using the S-P immunohistochemical method. Briefly, after routine deparaffinization and hydration, slides were treated with 1% hydrogen dioxide and then heated in EDTA (pH 8.0) for antigen retrieval. After blocking in 10% goat serum, tissue sections were then incubated with mouse anti-Human p53 protein (Biogenex, San Raman, CA) at 4°C overnight. After rinsing, sections were subsequently incubated with goat anti-mouse biotin-conjugated IgG for 15 min and then with streptavidin-peroxidase conjugate for 15 min. The signal was developed with diaminobenzidine, and slides were counterstained with 5% hematoxylin. The brown signals located in the nucleus represent positive staining for P53 protein. Sections without monoclonal antibody treatment were used as negative controls. The staining was scored on a scale from 0 to III as follows: 0, <10% cells were stained; I, 10%-25% cells were stained; II, 26%-50% cells were stained; and III, >50% cells were stained. Scores I-III were classified as positive, whereas Score 0 was negative.

Statistical analysis

Descriptive statistical analysis and unconditional logistic regression were performed by using the Statistical Package for the Social Sciences 17.0 (SPSS., Inc., Chicago IL, Microsoft) and Statistical Analysis System software (version 8.01; SAS Institute, Cary, NC). Genotype frequencies in the control subjects for each SNP were tested for departure from Hardy-Weinberg equilibrium (HWE) by using the goodness-of-fit χ²-test. Multiple logistic regression analyses were used to estimate the association between genotypes and gastric cancer or EC risks. Odds ratios (ORs) were adjusted for demographic variables, including age, gender, drinking, and smoking. Crossover analyses were used to investigate the association between rs1042522 and gastric cancer or EC by cigarette smoking or alcohol drinking status.

Cumulative survival rates were calculated from the date of initial diagnosis of gastric cancer or EC to the date of death or last follow-up. Median survival was estimated by using the Kaplan-Meier method and compared by using the log-rank test. Statistical significance was set at p < 0.05, and all statistical tests were two-sided.

Results

Demographic data of the participants

A total of 651 Han people, including 180 gastric cancer cases, 113 EC cases, and 358 cancer-free controls, were recruited in this study. The percentages of males in gastric cancer (71.7%) and EC (75.2%) were higher than those in the control group (57.0%).

Genotype and allele frequencies of the SNPs in the case-control study

The genotype and allele frequencies in the case and control groups are shown in Supplementary Table S1. SNP analyses showed that both p53 rs1042522 in 17p13.1 and CDKN1A rs730506 in 6p2 were significantly associated with EC susceptibility. The rs1042522 Arg allele was associated with an increased risk of EC when compared with the control group (52.7% vs 43.0%, p = 0.011). The distributions of p53 rs1042522 Pro/Pro, Pro/Arg, and Arg/Arg allele estimates for EC patients were 22.1%, 49.6%, and 27.4%, respectively. There were no significant differences in other six SNPs between the gastric cancer or EC and the control group. Genotyping for SNP rs730506 variation revealed genotype frequencies of 3.5% (Pro/Pro), 20.4% (Pro/Arg), and 75.2% (Arg/Arg) in the EC group, and 0.6% (Pro/Pro), 26.3% (Pro/Arg), and 72.9% (Arg/Arg) in the control group.

Associations between SNPs and gastric cancer or EC

A multiple logistic regression model was used to estimate the association between the genotypes and the risk of gastric cancer or EC. The results are listed in Table 1; the results were adjusted by age, gender, cigarette smoking, and alcohol drinking.

Table 1.

Logistic Regression Analysis of Associations Between the Eight Single-Nucleotide Polymorphisms and Gastric Cancer/Esophageal Cancer in a Northwest Chinese Population

Genotypes	Gastric cancer n (%)		Adjusted OR^*	95% CI		p	EC n (%)		Adjusted OR^*	95% CI		p
Genotypes	Case	Control	Adjusted OR^*	Lower	Upper	p	Case	Control	Adjusted OR^*	Lower	Upper	p
p53 rs1042522
Pro/Pro	48 (26.7)	117 (32.7)	1.000 (ref)				25 (22.1)	117 (32.7)	1.000 (ref)
Pro/Arg	89 (49.4)	174 (48.6)	1.591	0.994	2.546	0.053	56 (49.6)	174 (48.6)	1.883	1.052	3.370	0.033
Arg/Arg	43 (23.9)	67 (18.7)	2.064	1.159	3.675	0.014	31 (27.4)	67 (18.7)	2.988	1.486	6.009	0.002
Pro/Arg+Arg/Arg	132 (73.3)	241 (67.3)	1.810	1.151	2.847	0.010	87 (77.0)	241 (67.3)	2.285	1.309	3.989	0.004
CDKN1A rs2395655
GG	41 (22.8)	106 (29.6)	1.000 (ref)				28 (24.8)	106 (29.6)	1.000 (ref)
GA	100 (55.6)	191 (53.4)	1.154	0.720	1.850	0.551	61 (54.0)	191 (53.4)	1.209	0.613	1.922	0.779
AA	39 (21.7)	61 (17.0)	1.597	0.874	2.920	0.128	24 (21.2)	61 (17.0)	1.489	0.755	3.249	0.228
GA/AA	139 (77.3)	252 (70.4)	1.241	0.787	1.958	0.353	85 (75.2)	252 (70.4)	1.277	0.679	2.030	0.566
CDKN1A rs3176320
AA	108 (60.0)	192 (53.6)	1.000 (ref)				62 (54.9)	192 (53.6)	1.000 (ref)
GA	62 (34.4)	153 (42.7)	0.812	0.537	1.228	0.324	44 (38.9)	153 (42.7)	0.891	0.601	1.618	0.957
GG	10 (5.6)	13 (3.6)	0.869	0.313	2.408	0.787	6 (5.3)	13 (3.6)	1.429	0.269	3.113	0.887
GA/GG	72 (40.0)	166 (46.3)	0.822	0.551	1.225	0.335	50 (44.2)	166 (46.3)	0.933	0.611	1.587	0.951
CDKN1A rs3829963
CC	66 (36.7)	127 (35.5)	1.000 (ref)				42 (37.2)	127 (35.5)	1.000 (ref)
CA	80 (44.4)	170 (47.5)	0.971	0.627	1.503	0.894	50 (44.2)	170 (47.5)	0.889	0.602	1.711	0.956
AA	34 (18.9)	60 (16.8)	1.165	0.651	2.087	0.607	21 (18.6)	60 (16.8)	1.058	0.568	2.303	0.708
CA/AA	114 (63.3)	230 (64.3)	1.010	0.670	1.523	0.962	71 (62.8)	230 (64.3)	0.933	0.639	1.713	0.856
CDKN1A rs3829964
CC	79 (43.9)	171 (47.8)	1.000 (ref)				53 (46.9)	171 (47.8)	1.000 (ref)
CT	84 (46.7)	155 (43.3)	1.207	0.800	1.821	0.371	48 (42.5)	155 (43.3)	0.999	0.640	1.730	0.841
TT	17 (9.4)	31 (8.7)	0.948	0.459	1.957	0.884	12 (10.6)	31 (8.7)	1.249	0.437	2.433	0.945
CT/TT	101 (56.1)	186 (52.0)	1.179	0.794	1.751	0.414	60 (53.1)	186 (52.0)	1.041	0.657	1.696	0.822
CDKN1A rs4135234
GG	143 (79.4)	279 (77.9)	1.000 (ref)				87 (77.0)	279 (77.9)	1.000 (ref)
GA	36 (20.0)	74 (20.7)	0.811	0.494	1.333	0.409	24 (21.2)	74 (20.7)	1.040	0.625	2.014	0.701
AA	1 (0.6)	4 (1.1)	0.723	0.062	8.459	0.796	0 (0.0)	4 (1.1)	0.000	0.000	—	0.999
GA/AA	37 (20.6)	78 (21.8)	0.806	0.493	1.316	0.389	24 (21.2)	78 (21.8)	0.987	0.486	1.560	0.643
CDKN1A rs762624
CC	64 (35.6)	141 (39.4)	1.000 (ref)				42 (37.2)	141 (39.4)	1.000 (ref)
CA	85 (47.2)	155 (43.3)	1.116	0.726	1.716	0.617	51 (45.1)	155 (43.3)	1.105	0.657	1.855	0.710
AA	31 (17.2)	62 (17.3)	0.909	0.503	1.643	0.752	20 (17.7)	62 (17.3)	1.083	0.498	2.082	0.961
CA/AA	116 (64.4)	217 (60.6)	1.071	0.715	1.606	0.739	71 (62.8)	217 (60.6)	1.098	0.657	1.744	0.786
CDKN1A rs730506
GG	138 (76.7)	261 (72.9)	1.000 (ref)				85 (75.2)	261 (72.9)	1.000 (ref)
GC	38 (21.1)	94 (26.3)	0.866	0.542	1.384	0.548	23 (20.4)	94 (26.3)	0.751	0.412	1.337	0.322
CC	4 (2.2)	2 (0.6)	2.886	0.441	18.874	0.269	4 (3.5)	2 (0.6)	6.141	0.713	25.542	0.112
GC/CC	42 (23.3)	96 (26.9)	0.919	0.583	1.448	0.716	27 (23.9)	96 (26.9)	0.864	0.496	1.505	0.606

Adjusted for age, gender, cigarette smoking and alcohol drinking. Bold font means the significance at p < 0.05 level.

Arg, arginine; CI, confidence interval; EC, esophageal cancer; OR, odds ratio; Pro, proline.

Only one of the eight SNPs, rs1042522 in 17p13.1, was associated with both gastric cancer and EC risks. The gastric cancer risk of rs1042522 Arg homozygote carriers was 2.064 times that of rs1042522 Pro homozygotes (adjusted OR = 2.064, 95% CI, 1.159-3.675, p = 0.014). The rs1042522 Arg allele was also a risk factor for gastric cancer (adjusted OR = 1.810, 95% CI, 1.151-2.847, p = 0.010). When compared with the rs1042522 Pro homozygote, the rs1042522 Arg allele was associated with higher susceptibility to EC, even after adjustment for multiple conventional EC risk factors (adjusted OR = 2.988, 95% CI, 1.486-6.009, p = 0.002; adjusted OR = 1.883, 95% CI, 1.052-3.370, p = 0.033). The rs1042522 Arg allele was also associated with an increased risk of EC when compared with the rs1042522 Pro allele (adjusted OR = 2.285, 95% CI, 1.309-3.989, p = 0.004). However, no association between the other seven SNPs and gastric cancer or EC risk was observed in this study.

Crossover analysis by cigarette smoking or alcohol drinking status

The results are illustrated in Table 2. The results indicated that there was a significantly increased risk of gastric cancer among participants with the rs1042522 Arg allele who were also cigarette smokers or alcohol drinkers when compared with those non-cigarette smokers or non-alcohol drinkers who carried the rs1042522 Pro homozygote (adjusted OR = 2.422, 95% CI, 1.145-5.123, p < 0.001, and adjusted OR = 5.152, 95% CI, 2.038-13.024, p < 0.001, respectively).

Table 2.

Interactions Between p53 rs1042522 and Environmental Risk Factors in Gastric Cancer/Esophageal Cancer

Environment factors	Genotype	Case (n)	Control (n)	χ²	p	OR (95% CI)
Gastric cancer
Cigarette smoking
(−)	Pro/Pro	23	75	—	—	1.000 (ref)
(−)	Pro/Arg+Arg/Arg	59	185	0.019	0.889	1.095 (0.608-1.972)
(+)	Pro/Pro	25	42	3.697	0.055	1.613 (0.569-4.577)
(+)	Pro/Arg+Arg/Arg	73	56	25.031	0.000	2.422 (1.145-5.123)
Alcohol drinking
(−)	Pro/Pro	29	88	—	—	1.000 (ref)
(−)	Pro/Arg+Arg/Arg	90	224	0.871	0.351	1.232 (0.731-2.074)
(+)	Pro/Pro	19	29	3.961	0.047	1.437 (0.521-3.966)
(+)	Pro/Arg+Arg/Arg	42	17	36.071	0.000	5.152 (2.038-13.024)
EC
Cigarette smoking
(−)	Pro/Pro	12	75	—	—	1.000 (ref)
(−)	Pro/Arg+Arg/Arg	39	185	0.598	0.439	1.316 (0.663-2.746)
(+)	Pro/Pro	12	42	1.676	0.104	1.127 (0.557-2.386)
(+)	Pro/Arg+Arg/Arg	48	56	23.025	0.000	5.310 (2.623-11.300)
Alcohol drinking
(−)	Pro/Pro	20	88	—	—	1.000 (ref)
(−)	Pro/Arg+Arg/Arg	54	224	0.041	0.839	1.061 (0.605-1.906)
(+)	Pro/Pro	5	29	0.259	0.611	0.760 (0.236-2.139)
(+)	Pro/Arg+Arg/Arg	33	17	34.56	0.000	8.395 (3.961-18.380)

Adjusted for age, gender, cigarette smoking and alcohol drinking. Bold font means the significance at p < 0.05 level.

When focusing on rs1042522 Arg allele carriers who were also cigarette smokers or alcohol drinkers, the risk of EC increased (adjusted OR = 5.310, 95% CI, 2.623-11.300, p < 0.001, and adjusted OR = 8.395, 95% CI, 3.961-18.380, p < 0.001, respectively).

Correlation of the rs1042522 genotypes with the P53 expression of gastric cancer or EC patients

To further analyze the effects of the rs1042522 genotype on P53 expression in patient tissues, we examined the expression of P53 protein in 103 cases of gastric cancer and 85 cases of EC (Supplementary Table S2). Representative examples of negative P53 staining and positive I and above are shown in Supplementary Figure S1A-H. The brownish signals represent positive staining and were found mainly in the nucleus of tumor cells and the parabasal layer of normal gastric or esophageal mucosa. There was no statistical correlation between P53 expression and rs1042522, as shown in Supplementary Table S3.

Survival analysis of overall survival

In this retrospective study, there were a total of 103 and 85 survival data available out of the 180 gastric cancer cases (57.2%) and 113 EC cases (75.2%), respectively. The median follow-up time was 43.0 months, and the 5-year overall survival rate in gastric cancer patients was 48.5%. In the EC patients, the median follow-up time was 45.5 months, and the 5-year overall survival rate was 30.6%. The univariate analysis showed that rs1042522 had no effect on gastric cancer and EC patients' prognosis, (Supplementary Table S4 and Fig. 1A, B).

FIG. 1.

Survival curves in gastric cancer and EC patients. (A) The survival curve of gastric cancer patients in rs1042522 (HR = 1.492, p = 0.444); (B) The survival curves of EC patients in rs1042522 (HR = 1.316, p = 0.639). EC, esophageal cancer; HR, hazard ratio.

Discussion

In the current study, we focused on investigating eight SNPs, including rs1042522 in the p53 gene and seven polymorphisms of CDKN1A, among Chinese Ningxia gastric cancer and EC patients and cancer-free controls.

It was shown that rs1042522 was the only common genetic locus shared by both Chinese Ningxia gastric cancer and EC patients among eight SNPs. Compared with the rs1042522 Pro allele, the rs1042522 Arg allele increased the gastric cancer risk by 1.810 times, and the EC risk by 2.285 times as well. A group of studies supported that rs1042522 Arg was a potential cancer genetic risk factor, such as in breast cancer in Turkish patients and cutaneous melanoma in Caucasians (Buyru et al., 2003; Geng et al., 2015). Chen et al. (2014) reported that the rs1042522 Arg allele increased the gastric cancer risk by 1.17-fold compared with the rs1042522 Pro allele in a meta-analysis with 7,444 gastric cancer cases and 9,984 controls among Eastern Asians.

Our previous study also found that the rs1042522 Arg allele contributed to an elevated esophageal squamous cell carcinoma risk by 6.48 times among a northern Chinese population (Yang et al., 2008). These two studies were consistent with our current study. The functional mechanism behind this may result from the changing functions of the two alleles of rs1042522 in different cell types. Pim and Banks (2004) used an inducible switch system for expressing both rs1042522 Arg and rs1042522 Pro to investigate how the 2 forms of rs1042522 brought about a cessation of cell growth. They found that within the primary Saos-2 cells, the rs1042522 Arg allele increased the ability of p53 to locate to mitochondria, which induced apoptosis more efficiently than the rs1042522 Pro allele.

However, Schneider-Stock et al. (2004) reported that in squamous cell carcinomas of the head and neck, the rs1042522 Arg allele was associated with insufficient or absent apoptosis, because it lacked the apoptosis-related protein activities, such as the co-expression of Fas and FasL or high expression of Bcl2 protein. Further, Garima et al. found that compared with the rs1042522 Pro allele, the rs1042522 Arg allele led to reduced expression of the cell proliferation inhibitor p21 and increased angiogenesis-mediating VEGF expression, promoting carcinoma growth through increased cell proliferation and vascularity in tumors arising in patients (Yagnik et al., 2017). Therefore, these changes in rs1042522 may affect the function of the p53 protein, attenuate the loss or alteration of p53 binding capacity to the targets, and induce aberrant cell amplification accompanying cellular transformation, which leads to the occurrence of different cancers (Batinac et al., 2003).

Some studies have also shown that the rs1042522 Arg allele represents a significant risk factor in the development of HPV-associated cancers. Habbous et al. (2012) evaluated the role of the rs1042522 polymorphism and HPV status in the initiation, progression, and development of cervical cancer. They found that there were significantly higher odds of progression from squamous intraepithelial lesions (SIL) to cervical cancer with the rs1042522 Arg allele in HPV-positive white and East Asian individuals. Storey et al. (1998) reported that there was a marked overrepresentation of homozygous rs1042522 Arg when compared with heterozygous or homozygous rs1042522 Pro, making individuals homozygous for rs1042522 Arg 7 times more likely to develop HPV-associated cervical cancer than individuals having one or more rs1042522 Pro alleles. This could be explained by the studies from Storey et al. (1998) and Thomas et al. (1999), who showed that rs1042522 Arg was significantly more susceptible to the degradation induced by human papillomavirus (HPV) E6 protein than rs1042522 Pro. Therefore, the rs1042522 Arg allele represents a significant risk factor in the development of HPV-associated cancers.

However, several other studies reported that the rs1042522 Pro allele was associated with the risk of cancer. For instance, Katkoori et al. (2009) reported that African Americans, but not Caucasians, with the rs1042522 Pro homozygote had significantly higher mortality and 2.15-fold colorectal adenocarcinoma risk than those with the rs1042522 Arg allele. A group of studies also reported that the rs1042522 Pro allele increased the susceptibility of gastric cancer or EC among Asian patients from South China and Mongolia (Peng et al., 2015; Li et al., 2017).

The reasons for the contradictory results may be that the inherent genetic variant was distributed unevenly among different ethnic populations or even different people within the same ethnic group. Shi et al. (2009) observed that the frequency of rs1042522 Arg allele expression was associated with low average winter temperature and that populations living in different latitudinal regions have a strong selection of different alleles of rs1042522, especially because the latitude-dependent distribution of the genotypes was observed in the same ethnic group. In addition, different environmental exposures or inadequate sample sizes may also be attributable to the inconsistent results.

Gastric cancer and EC are multifactorial disorders. Some common risk factors for gastric cancer include tobacco smoking, alcohol consumption, foods preserved by salting, older age, male gender, race, family history, low physical activity, low fiber intake, and radiation (Karimi et al., 2014). Several studies demonstrated that cigarette smoking and alcohol consumption were risk factors for gastric cancer and EC (Agrawal et al., 2012; Lin et al., 2013) and may also lead to death from other smoking-related complications (Tabuchi et al., 2013). In the current study, the crossover analysis by cigarette smoking or alcohol drinking status indicated that rs1042522 Arg allele carriers together with cigarette smoking or alcohol drinking further increased gastric cancer and EC risk.

However, some limitations need to be addressed in this study. First, all of the participants were enrolled from a hospital, and the subjects may not be representative of all upper gastrointestinal patients in this region. Accordingly, selection bias cannot be completely excluded. Second, our sample size was admittedly limited, allowing us to draw only preliminary conclusions. Third, HPV infection was not detected among our patients. Therefore, further investigations with HPV detection, larger sample sizes, and multiple populations are warranted to uncover the genetic basis of gastric cancer and EC.

Conclusion

In conclusion, we comprehensively analyzed the associations between eight SNPs and their association with both gastric cancer and EC patients from a northwestern Chinese population and found that p53 rs1042522 was a common genetic locus shared by gastric cancer and EC patients. In addition, tobacco smoking and alcohol drinking could further enhance the risk of cancer susceptibility in our study.

Footnotes

Authors' Contributions

J.Y., H.Z., and J.Y. collected patient samples and provided technical expertise. J.C. and C.T. interpreted the collected data and generated tables and figures for the data. J.C. wrote the article. Z.C. edited the article. W.Y. supervised the study. All authors have read and approved the article.

Acknowledgments

The authors thank Wiley for the linguistic assistance during the preparation of this article. They also thank all the participants of this study.

Author Disclosure Statement

The authors declare that they have no conflict of interest.

Funding Information

The research was supported by the grants from the National Natural Science Foundation of China (No. 81760525, 81160249), PI foundation by Ningxia Medical University.

Supplementary Material

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