Association of lysosomal protein transmembrane 4 beta gene polymorphism with pancreatic carcinoma susceptibility in the Chinese population

Abstract

Pancreatic carcinoma is an aggressive malignancy with particularly low 5-year survival rate. In order to improve the dismal survival rate, identification of new potential susceptibility risk factors for the prevention and early detection of pancreatic carcinoma is of utmost importance. Lysosomal protein transmembrane 4 beta has two alleles designated as LAPTM4B*1 and LAPTM4B*2. The aim of this study was to investigate the association between lysosomal protein transmembrane 4 beta gene polymorphism and the risk of pancreatic carcinoma in China. A population-based case-control analysis was performed in 233 patients with pancreatic carcinoma and 842 control subjects. The genotypes of lysosomal protein transmembrane 4 beta were determined by utilizing polymerase chain reaction based on specific primers. The χ² test was used to analyze the differences of categorical variables and Hardy–Weinberg equilibrium. Odds ratio and 95% confidence intervals were computed using an unconditional logistic regression model. A significant difference in the frequency of LAPTM4B*2 was observed between the patients and the controls (33.05% vs 27.55%, p = 0.03). LAPTM4B*2 had a 1.33-fold (95% confidence interval: 1.04–1.71) higher risk for developing pancreatic carcinoma when compared with LAPTM4B*1 carriers. We found that the frequency of LAPTM4B*1/2 + *2/2 in pancreatic carcinoma group was higher than that in the control group (57.94% vs 48.34%, p = 0.01). However, no significant association was observed between lysosomal protein transmembrane 4 beta genotypes and gender, age, family history of cancer, smoking/alcohol status, histopathological differentiation, lymph node metastasis, clinical stage, or serum cancer antigen 19-9 level. These findings indicate that the LAPTM4B*2 allele is associated with the high risk of pancreatic carcinoma and carrying LAPTM4B*2 may be a susceptible factor to Chinese pancreatic carcinoma patients.

Introduction

Pancreatic carcinoma (PC), one of the most aggressive malignancies, is the fourth leading cause of cancer deaths in the United States, being responsible for 7% of all cancer-related deaths in both men and women. The National Cancer Center estimated that there would be 90,100 new cases of PC and 79,400 deaths due to PC in China in 2015.¹ The American Cancer Society estimates that there will be 53,070 new cases of PC and 41,780 deaths due to PC in the United States in 2016.² By the year 2030, it is projected that PC will be the second leading cause of cancer death after lung cancer among the major cancers.³ The initial symptoms of PC are often quite nonspecific and subtle in onset. When someone has symptoms such as abdominal pain, jaundice, anorexia, vomiting, weight loss, fatigue, it means he is in the late stage with poor prognosis, and the 5-year survival rate is around 7%. Therefore, identification of new potential susceptibility risk factors for the prevention and early detection of PC is of utmost importance.

Lysosomal protein transmembrane 4 beta (LAPTM4B) is a novel cancer-related gene which was successfully cloned in human hepatocellular carcinoma (HCC).⁴ It is mapped to chromosome 8q22.1 and consists of seven exons. Similar to other LAPTM family members, LAPTM4B protein has a lysosomal localization motif at the C terminus and co-localizes with markers of late endosomes and lysosomes.^5,6 Previous studies have shown that LAPTM4B was overexpressed in many malignant tissues and had a significant association with unfavorable biological behaviors and the prognosis of cancers, such as HCC,^7–9 PC,¹⁰ extrahepatic cholangiocarcinoma,¹¹ endometrial carcinoma,¹² epithelial ovarian cancer,¹³ breast cancer,¹⁴ gallbladder carcinoma,^15,16 colorectal cancer,¹⁷ small cell lung cancer¹⁸ and gastric cancer.¹⁹ It also has been reported that LAPTM4B gene can promote the cell proliferation by involving in the regulation of cell cycle control and causing tumorigenesis of NIH3T3 cell, and overexpression of LAPTM4B-35 by transfection of LAPTM4B complementary DNA (cDNA) promotes cell proliferation, migration, and invasion in HCC xenografts in nude mice and induces multidrug resistance, indicating that it plays important roles in tumorigenesis.^20,21 Moreover, in vitro, HeLa cells with downregulated LAPTM4B also exhibited decreased migration and invasion activity as well as significantly reduced cyclin-dependent kinase 12 (CDK12), hypoxia-inducible factor 1-alpha (HIF-1α), matrix metalloproteinase 2 (MMP-2), MMP-9, and vascular endothelial growth factor (VEGF) expression.²²

LAPTM4B has two alleles designated as LAPTM4B*1 and LAPTM4B*2 (GenBank No. AY219176 and AY219177).²³ Allele *1 differs from allele *2 in that it contains only one copy of a 19-bp sequence in the first exon, whereas this sequence is duplicated and tandemly arranged in allele *2 (Figure 1). Previous studies showed that the LAPTM4B polymorphism was associated with the susceptibility of HCC,^24,25 lung cancer,²⁶ gastric cancer,²⁷ colorectal cancers,²⁸ lymphoma,²⁹ cervical cancer,³⁰ gallbladder carcinoma,³¹ ovarian carcinoma,³² breast cancer,^33,34 and malignant melanoma.³⁵ Subsequently, a meta-analysis proved that LAPTM4B polymorphism was associated with cancer risk and allele *2 was a risk factor.³⁶

Figure 1.

Schematic diagram of LAPTM4B alleles in exon1. Allele *1 contains one copy of the 19-bp sequence (the box with oblique line), and allele *2 contains two copies of this 19-bp sequences which are tandemly arranged. The lower line represents the corresponding DNA sequences of the boxes with oblique line.

[Figure omitted. See PDF]

Zhang et al.¹⁰ reported that LAPTM4B protein was found to be aberrantly overexpressed in a large proportion of patients with PC and was closely related to disease progression and poor prognosis. LAPTM4B may represent a new molecular target for the clinical evaluation and treatment of PC. Wang and Zhang³⁷ found that the frequencies of LAPTM4B*2 in the case and control groups were 31.09% and 36.21%, respectively, through analysis of the genotypes of 58 PC patients and 156 normal controls, but no significant statistical difference (p = 0.35) was found. Since the incidence of PC is relatively low, the research only collected 58 cases in the limited time; the participants in the survey were not strong enough to represent the entire population. We did not know whether this difference was due to sampling error from the relative small cases. Therefore, the initial findings should be independently verified in a large-scale case-control study. Toward this goal, we intend to expand the study population to clarify the relationship between LAPTM4B gene polymorphism and PC susceptibility.

Materials and methods

Cases

Whole blood samples of patients with PC were derived from patients at Peking University Cancer Hospital (Beijing, China) between September 2009 and December 2013. All 233 patients (149 males, 84 females, and mean age 60.33 ± 11.32 years) had been confirmed to have PC. Disease staging was performed in accordance with the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) stage classification (7th edition).³⁸ Clinicopathological features including gender, age, family history of cancer, smoking/alcohol status, histopathological differentiation, lymph node metastasis, clinical stage, and serum cancer antigen 19-9 (CA 19-9) level were summarized for all patients.

Controls

We enrolled 842 healthy individuals (468 males, 374 females, mean age 57.13 ± 15.34 years) undergoing annual physical examination as controls. None of them had previously been diagnosed with a malignancy.

DNA extraction

Human peripheral blood samples were collected in ethylenediaminetetraacetic acid (EDTA)-K₂ anticoagulation tubes. Genomic DNA was immediately isolated from 800 µL of each blood sample using the RelaxGene blood DNA system (Tiangen, China) according to the protocol provided by the manufacturer and then stored at −20°C for subsequent polymerase chain reaction (PCR) assay.

PCR analysis

The heterozygous LAPTM4B genotype was determined by PCR analysis using the specific primers—F: (forward): 5′-GCCGACTAGGGGACTGGCGGA-3′ and R (reverse): 5′-CGAGAGCTCCGAGCTTCTGCC-3′. The 20 µL PCR reaction included 1 µL genomic DNA, 1 µL reverse primer, 1 µL forward primer, 10 µL 2× EASY Taq mix (TransGen, China), 5 µL double-distilled water (ddH₂O), and 2 µL primers of human β-actin that served as the internal control. The reaction mixture was incubated in a 0.2 mL tube at 95°C for denaturation for 5 min; followed by 37 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s; then it underwent extension at 72°C for 5 min. PCR products were analyzed by electrophoresis in a 2.8% agarose gel and visualized with ethidium bromide. The LAPTM4B*1/1 and LAPTM4B*2/2 genotypes were identified by a 204-bp band and a 223-bp band, respectively. We used human β-actin as the positive inner control, and a 340-bp fragment was generated using the following primers—F: 5′-TCACCAACTGGGACGACAT-3′ and R: 5′-AGGTAGTCAGTCAGGTCCCG-3′.

Statistical analysis

SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA) was used for data analysis. The chi-square test was also used to analyze differences of categorical variables. Genotypic frequencies were tested for Hardy–Weinberg equilibrium. Unconditional logistic regression model was used to evaluate the odds ratio (OR) and the corresponding 95% confidence intervals (CIs) to analyze the association between the LAPTM4B polymorphism and the risk of PC adjusted by gender and age. LAPTM4B*1/1 genotype was held as the reference category in calculating OR. Two-sided p values less than 0.05 were considered to be statistically significant.

Results

LAPTM4B genotypes

Using the specific primers for LAPTM4B, three different genotypic LAPTM4B polymorphisms designated LAPTM4B*1/1, LAPTM4B*2/2, and LAPTM4B*1/2 were identified by PCR assay. As shown in Figure 2, genotype *1/1 was represented by a 204-bp band, *2/2 was represented by a 223-bp band, and genotype *1/2 showed both of these bands. All bands were identified with PCR using specific primers and separated by electrophoresis on a 2.8% agarose gel.

Figure 2.

Genotype of LAPTM4B. Samples were analyzed by separating in a 2.8% agarose gel electrophoresis. The lower band depicted the amplified PCR products—lane M: DNA marker (100, 200, 300, 400, 500, 600, and 700 bp); lanes 1, 4, and 5: *1/1 genotype; lanes 2 and 7: *2/2 genotype; and lanes 3 and 6: *1/2 genotype. The upper band in each lane (340 bp) was the product of human β-actin that served as the positive internal control.

[Figure omitted. See PDF]

LAPTM4B polymorphism and PC susceptibility

We tested 233 PC subjects and 842 normal control subjects for the LAPTM4B gene. LAPTM4B genotype distributions in the PC group and the controls were all in Hardy–Weinberg equilibrium (p > 0.05 separately), indicating that the selected samples could represent the whole population. However, the distributions of gender, age, smoking/alcohol status, family history of cancer, and serum CA 19-9 level between the case and control groups showed a significant difference (p < 0.05; Table 1). These variables were further adjusted for with multivariate logistic regression models.

Table 1.

Distribution of select characteristics in case and control groups.

	Controls (n = 842)	Cases (n = 233)	pa value
Gender (n (%))			0.022
Male	468 (55.58%)	149 (63.95%)
Female	374 (44.42%)	84 (36.05%)
Age (years) (n (%))			0.019
<60	463 (54.99%)	108 (46.35%)
≥60	379 (45.01%)	125 (53.65%)
Smoking status			0.005
Nonsmokers	709 (84.20%)	178 (76.39%)
Smokers	133 (15.80%)	55 (23.61%)
Alcohol status			0.018
Nondrinkers	716 (85.04%)	183 (78.54%)
Drinkers	126 (14.96%)	50 (21.46%)
Family history of cancer			0.001
No	767 (91.09%)	195 (83.69%)
Yes	75 (8.91%)	38 (16.31%)
Serum CA 19-9 level (ng/mL)			0.000
≤37	680 (80.76%)	37 (15.88%)
>37	72 (8.55%)	142 (60.94%)
Unknown	90 (10.69%)	54 (23.18%)

CA 19-9: cancer antigen 19-9.

a

Chi-square test.

As shown in Table 2, we found that the frequencies of the *2 allele were 33.05% in the PC group and 27.55% in the control group, representing a significant difference between the two groups (p = 0.03). It suggests that LAPTM4B*2 is associated with PC risk.

Table 2.

Distribution of genotypes and alleles of LAPTM4B in case and control groups.

	Controls (n (%))	Cases (n (%))	OR (95% CI)a	pa value
Genotype
*1/1	435 (51.66%)	98 (42.06%)
*1/2	350 (41.57%)	116 (49.79%)	1.52 (1.07–2.14)	0.02
*2/2	57 (6.77%)	19 (8.15%)	1.58 (0.84–2.97)	0.16
*1/2 + *2/2	407 (48.34%)	135 (57.94%)	1.52 (1.09–2.13)	0.01
Total	842	233
Allele
*1	1220 (72.45%)	312 (66.95%)
*2	464 (27.55%)	154 (33.05%)	1.33 (1.04–1.71)	0.03
Total	1684	466

OR: odds ratio; CI: confidence interval; CA 19-9: cancer antigen 19-9.

a

Data were calculated by unconditional logistic regression adjusted by gender, age, smoking/alcohol status, family history of cancer, and serum CA 19-9 level.

The distribution of LAPTM4B genotypes was 42.06% for LAPTM4B*1/1, 49.79% for LAPTM4B*1/2, and 8.15% for LAPTM4B*2/2 in patients, and 51.66% for LAPTM4B*1/1, 41.57% for LAPTM4B*1/2, and 6.77% for LAPTM4B*2/2 in controls. The frequencies of LAPTM4B*1/2 and *1/2 + *2/2 in PC group were higher than the control group, and this data represented that there was a significant difference between the two groups (p = 0.02 and p = 0.01), indicating that subjects carrying LAPTM4B*1/2 + *2/2 had a 1.52-fold (95% CI: 1.09–2.13) increased risk of PC when compared with those who carry LAPTM4B*1/1. Though different frequency of LAPTM4B*2/2 was noticed among PC and the control group, no significant statistical difference was found (p = 0.16).

LAPTM4B genotypes and clinicopathological features

The association between LAPTM4B genotypes and various clinicopathological features in the PC group was summarized in Table 3. No significant association was observed between LAPTM4B genotypes and gender, age, family history of cancer, smoking/alcohol status, histopathological differentiation, lymph node metastasis, clinical stage, or serum CA 19-9 level (p > 0.05) in the PC group.

Table 3.

Relationship between LAPTM4B genotype and clinicopathological features of PC.

Characteristics	Patients	LAPTM4B genotype			pa value
		*1/1	*1/2	*2/2
Number of patients	233	98	116	19
Gender					0.128
Male	149	70	68	11
Female	84	28	48	8
Age (years)					0.979
<60	108	46	53	9
≥60	125	52	63	10
Family history of cancer					0.928
No	195	83	96	16
Yes	38	15	20	3
Smoking status					0.761
Nonsmokers	178	73	91	14
Smokers	55	25	25	5
Alcohol status					0.912
Nondrinkers	183	76	91	16
Drinkers	50	22	25	3
Histopathological differentiation					0.192
Well differentiated	9	5	4	0
Moderately differentiated	97	38	49	10
Poorly differentiated	81	36	36	9
Unknown	46	19	27	0
Lymph node metastasis					0.509
Negative	59	21	35	3
Positive	156	69	72	15
Unknown	18	8	9	1
Clinical stage					0.677
I–II	78	30	42	6
III–IV	155	68	74	13
Serum CA 19-9 level (ng/mL)					0.806
≤37	37	15	18	4
>37	142	58	74	10
Unknown	54	25	24	5

PC: pancreatic carcinoma; CA 19-9: cancer antigen 19-9.

a

Chi-square test.

Discussion

Although PC is an important cause of cancer death, genetic factors involved with the etiology of the disease have not been extensively studied. In this study, we have expanded the number of the study population to 1075 to investigate the association of LAPTM4B polymorphism with PC susceptibility. Through the analysis of the genotypes by PCR, we found that the allelic frequencies of the *2 allele were 33.05% in the PC group and 27.55% in the control group, representing a significant difference between the two groups and suggesting that the LAPTM4B*2 allele is associated with significantly increased risk of PC. Similarly, statistical comparisons showed carriage of LAPTM4B*1/*2 + *2/2 was associated with increased risk of PC. In accordance with the previous studies, no significant association between LAPTM4B genotypes and clinicopathological features was observed either. Therefore, we believed that LAPTM4B was an independent risk factor in PC development.

The finding is consistent with our previous work showing that the initial findings³⁷ were not strong enough to represent the entire populations due to insufficient sample size. In addition, our results are in line with the previous results, which showed that the increased risk for individuals carrying LAPTM4B*2 was 1.65-fold for primary liver cancer,²⁵ 1.72-fold for lung cancer,²⁶ 1.61-fold for gastric cancer,²⁷ 1.49-fold for cervical cancer,³⁰ 1.30-fold for breast cancer,³³ and 1.31-fold for malignant melanoma,³⁵ in comparison with LAPTM4B*1 in the Chinese population, but there was no association of LAPTM4B gene polymorphism with the risk of squamous cell carcinomas such as esophageal carcinoma, rectum carcinoma, and nasopharyngeal carcinoma.^28,39 Peng et al.⁹ showed that the expression of LAPTM4B protein in cancer tissues originated from single layer cuboidal and columnar epithelia, such as HCC, gastric cancer, and colon cancer, and the lack of expression in cancer tissues originated from stratified epithelia, such as esophageal cancer and rectal cancer. Cheng et al.⁴⁰ revealed an independent prognostic role of LAPTM4B gene polymorphism in colon cancer patients who received surgical resection but not in rectal and esophageal cancers. But, so far, the underlying molecular mechanisms are not clear; hence, we will continue to study it in the following trials. Moreover, Zhang et al.¹⁰ reported that LAPTM4B was found to be present at high levels in a large proportion of patients with PC. So we believe that our results are convincing and representative.

The relationship between LAPTM4B gene polymorphism and susceptibility of PC could be explained by the following aspects. First, the overexpression of LAPTM4B in PC tissue samples was considered as being caused by gene amplification and transcriptional upregulation. Though there are no typical CCAAT and TATA cassettes in LAPTM4B gene promoter region, various binding sites of transcription factors exist in the upstream region of LAPTM4B promoter, such as cyclic adenosine monophosphate response element binding protein 1 (CREBP1)/c-Jun, c-Ets-1, E2F, LYF-1, and c/v-Myb, some of which are proto-oncogene itself. These transcription factors may specifically regulate LAPTM4B expression. The abnormal expression and activation of these transcription factors in PC cells possibly lead to an unbalanced expression of LAPTM4B proteins. Second, Cheng et al.⁴¹ illustrated that the degree of methylation at multiple CpG sites within the LAPTM4B core promoter region significantly affects its messenger RNA (mRNA) expression level. However, more studies should be carried out to prove this presumption. The last but the most important, the allele LAPTM4B*1 differs from allele LAPTM4B*2 in the 19-bp sequence in the 5′-untranslated region (5′-UTR) of exon 1. Thus, for different alleles of the tumor, different susceptibility can be attributed to the 19-bp sequence. On one hand, this 19-bp sequence in exon 1 can alter the open reading frame (ORF). Shao et al.⁵ showed that LAPTM4B*1 was predicted to encode a 35-kDa protein. In allele *2, the extra 19-bp sequence changed ORF of LAPTM4B gene and made LAPTM4B*2 to encode one more protein isoform, a 40-kDa protein with an extra 53 amino acids at the N terminus (Figure 3). It is likely that this 19-bp sequence plays an important role in genetic transcriptional regulation or that the extra 53 amino acids produced by LAPTM4B*2 may influence physiological activity and function in tumor cells.^4,23 However, the function of the 40-kDa protein encoded by allele *2 and its correlation with disease have not been elaborated so far. On the other hand, the 19-bp sequence may act as a cis-acting element to participate in genetic transcriptional regulation. This area urgently needs further research to prove.

Figure 3.

Comparison of the predicted proteins encoded by LAPTM4B*1 and *2. The sequences shown are the initial segments of first exon in (a) LAPTM4B*1 and (b) LAPTM4B*2. The sequence numbers of the first nucleotide (left) and the final amino acid (right) in each row are demonstrated. The nucleotide sequences are numbered with the putative transcription start site as +1. Termination codons are underlined and marked by symbols # and the 19-bp sequences in both the alleles are underlined. Allele *1 can only start translation at nt 157, because of the termination codons at nt 40 and nt 103. However, allele *2 starts translation at nt 17 which produces an extra 53 amino acids that are shown with gray shading at N terminus of LAPTM4B.

[Figure omitted. See PDF]

As far as we know, this study is the first one to research the possible relationship between LAPTM4B polymorphism and PC risk by PCR assay in such a large population. In the study, higher distribution of LAPTM4B*2/2 genotype and LAPTM4B*2 allele in the PC patients suggests that LAPTM4B*2 is associated with an increased risk of PC in the Chinese population and may be a risk factor of PC. We believe that searching for more genetic factors to PC high-risk areas has great value in PC prevention, and then, exploring the interaction between genes and environmental factors will make a breakthrough in the molecular etiology of PC. Future functional studies on LAPTM4B gene should provide more information on the polymorphism modified the risk for PC at the molecular level.

The authors thank all the people who participated in this study.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. A waiver of informed consent was requested, and the ethical approval was obtained from the Institutional Ethics Committee of Peking University (Beijing, China).

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (No. 81572910).