Lysosome-associated protein transmembrane 4 beta (LAPTM4B), a novel oncoprotein, is overexpressed in several carcinomas. Previous studies revealed that LAPTM4B polymorphisms contribute to the risk of certain types of cancers. The purpose of this study was to investigate the association between different LAPTM4B alleles and the risk of cervical carcinoma.
A case-control analysis was performed in 317 patients with cervical cancer and 416 control subjects. Genomic DNA was extracted from peripheral blood lymphocytes in all participants. LAPTM4B genotypes were determined using polymerase chain reaction. Odds ratios and 95% confidence intervals (CIs) were computed using an unconditional logistic regression model.
There was a significant difference (P<.001) in LAPTM4B*2 allele frequency between cervical cancer cases (35.8%) and controls (26.3%). There was also a significant difference in the overall genotypic distribution between patients and controls (P < .001). Using the LAPTM4B*1/1 genotype as a reference, we found that LAPTM4B allelic variation was associated with a significantly increased risk of cervical cancer, with adjusted odds ratios of 1.60 (95% CI, 1.15-2.22) and 2.12 (95% CI, 1.20-3.76) for the *1/2 and *2/2 genotype, respectively. Stratification analysis indicated that the association was more pronounced in younger subjects, smokers, premenopausal women, and women with more parities. Moreover, multiplicative joint effects were found between the *1/2 or *2/2 genotype and smoking.
Cervical cancer is the second most common cancer in women worldwide, with an increased mortality rate in developing countries.1 Each year, more than 470,000 new cases of cervical cancer are diagnosed, with 80% of the diagnoses occurring in the developing world.2, 3 Some evidence suggests that the oncogenic type of human papillomavirus is an important factor in the development of the precursors of cervical cancer.4-7 However, only a small portion of women infected with human papillomavirus develop cervical cancer, indicating there are other factors that contribute to the progression of cervical cancer.8, 9 Therefore, efforts to identify more molecular markers for detection and diagnosis of cervical cancer are of great clinical importance. Several notable advances have been made in the cervical carcinoma field in recent years.10-12 Many studies have shown that genetic alterations play important roles in the development and progression of cervical carcinoma.13, 14 Among these molecular markers, the relationships between gene polymorphisms and susceptibility to cervical cancer have been studied extensively.15-17
Lysosome-associated protein transmembrane 4 beta (LAPTM4B) was initially identified as a hepatocellular carcinoma-associated gene. LAPTM4B was recently shown to be up-regulated in various human cancers, including breast cancer, lung cancer, gastric cancer, colon cancer, esophageal cancer, rectal cancer, and adrenocorticotropin-secreting and nonfunctioning pituitary adenoma. In addition, LAPTM4B has important roles in multiple biological processes during cancer development and progression, including malignant transformation, apoptosis, invasion, and metastasis through its involvement in several oncogenic signal transduction pathways.
The LAPTM4B gene has been mapped to chromosome 8q22.1 and contains 7 exons and 6 introns.18 There are 2 alleles of the gene, LAPTM4B*1 and LAPTM4B*2 (GenBank accession nos. AY219176 and AY219177, respectively), and they have the same sequence except for a 19-bp segment in the first exon. The LAPTM4B*1 allele has a single 19-bp segment, whereas the LAPTM4B*2 allele has 2 tandem repeats of this segment in close proximity (Figure 1).19 Previous studies have revealed that LAPTM4B allelic variation is associated with increased risk of lung, gastric, and colon cancer, but it is not associated with rectal or esophageal squamous cell carcinoma.20-22
Our previous findings have revealed that LAPTM4B expression is associated with progression and a poor prognosis of cervical carcinoma.23 However, to date, the relationship between LAPTM4B polymorphisms and the risk of cervical cancer has not been investigated. The purpose of this study was to evaluate LAPTM4B polymorphisms and their association with cervical cancer risk. We also examined the association between LAPTM4B allelic variations and prognostic factors in cervical carcinoma.
MATERIALS AND METHODS
Cases consisted of 317 patients who had received a histological diagnosis of cervical cancer and who had first visited the Affiliated Tumor Hospital of Harbin Medical University between January 2007 and March 2010. The final diagnosis for all patients was confirmed via pathologic examination. Tumor stage was assessed according to the International Federation of Gynecology and Obstetrics (FIGO) staging system. The controls were selected from healthy women who visited the same hospital for regular gynecological examinations during the period when women with cervical cancer were enrolled. Control selection criteria included no abnormal findings during the gynecological examination, no history of cancer, age matching with the patients, and residence in Heilongjiang Province.
The Institutional Ethics Committee approved the study before it began, and informed consent was obtained from each patient before sample collection. After informed consent was obtained, participants were asked to complete a standard questionnaire about their age, smoking status, menopausal status, and parity.
DNA Isolation and Polymerase Chain Reaction
Genomic DNA was isolated using standard methods (Axygen, Union City, CA) from the peripheral blood of 317 patients with cervical cancer and 416 healthy female controls. For each sample, the LAPTM4B genotype was determined via polymerase chain reaction (PCR) using the following primers: 5′-GCCGACTAGGGGACTGGCGGA-3′ (forward) and 5′-CGAGAGCTCC GAGCTTCTGCC-3′ (reverse). The PCR reaction mixture (50 μL) contained 0.5 U of Taq DNA polymerase (TaKaRa, Shiga, Japan) and 5 μL of template DNA at a final concentration of 100 ng/μL. Human β-actin was used as a positive internal control using the following oligonucleotide primers: 5′-AACTGGGACGACATGGA GAA-3′ (forward) and 5′-GGTAGTCAGTCAGGTCCC-3′ (reverse). PCR conditions were as follows: 96°C denaturation for 5 minutes, 35 cycles of 30 seconds at 94°C, 30 seconds at 65°C, and 1 minute at 72°C, followed by extension at 72°C for 5 minutes. The amplified products were analyzed via electrophoresis in 2% agarose (visualized with 0.5 μg/mL ethidium bromide). For the PCR-based genotyping assay, 2 research assistants who were blinded to individual case-control status independently read the gel pictures, and the genotyping was repeated if there was a disagreement in the result. We randomly selected 10% of the samples for replication, and the results were 100% concordant. In addition, to confirm the genotyping results, 30 randomly selected, PCR-amplified DNA samples for LAPTM4B gene were examined via DNA sequencing, and these results were also 100% concordant.
The chi-square test or Fisher exact test was used to calculate the genotypic frequency and other parametric distributions between cancer cases and controls. P < .05 was considered significant. Genotypic frequencies were tested for Hardy-Weinberg equilibrium using the chi-square test.24 Associations between genotypes and risk of cervical cancer were estimated using odds ratios (ORs) and 95% confidence intervals (CIs), which were computed by using unconditional logistic regression models. All ORs were adjusted for age, smoking status, menopausal status and parity, as appropriate. We tested the null hypothesis of a multiplicative gene-smoking interaction. A greater than multiplicative joint effect was suggested when OR11 > OR10 × OR01.25 Stratified analysis was used to evaluate the potential interaction effects between LAPTM4B genotypes and selected variables. Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL).
Three different genotypes were identified using PCR primers specific for LAPTM4B.LAPTM4B*1/1 homozygotes were identified as a 204-bp band, and LAPTM4B*2/2 homozygotes were identified by the presence of a 223-bp band. The 204-bp and 223-bp bands were present in LAPTM4B*1/2 heterozygotes. The human β-actin PCR product was 318 bp and appeared in all internal controls. Representative LAPTM4B genotypes from 7 individuals in this study are shown in Figure 1.
Baseline characteristics of the 317 cervical cancer patients and 416 controls are summarized in Table 1. Of the 317 cervical cancer cases, 238 (75.1%) were squamous cell carcinoma and 79 (24.9%) were adenocarcinoma. One hundred twenty-seven patients had cervical carcinoma classified as FIGO stage 1, 114 had FIGO stage 2, 46 had FIGO stage 3, and 30 had FIGO stage 4. Based on the histological grade analysis, 193 cases were G1, 79 were G2, and 45 were G3. No significant differences in age or menopausal status were observed between cases and controls. However, smokers or subjects with parity >2 were significantly more common in cases (P < .001 and P = .001, respectively). These variables were further adjusted for with multivariate logistic regression models.
Table 1. Distributions of Select Characteristics Among Cervical Cancer Cases and Cancer-Free Controls
The LAPTM4B*2 allelic frequency was significantly higher in cases than in controls (35.8% vs 26.3%, respectively; P < .001). LAPTM4B*2 subjects had a significantly higher risk of cervical cancer compared with LAPTM4B*1 subjects (OR, 1.49; 95% CI, 1.17-1.89).
LAPTM4B genotype distributions in cervical cancer patients and controls were all in Hardy-Weinberg equilibrium (P = .37 and .84, respectively). Distributions of LAPTM4B genotypes were significantly different between cases (40.1% for 1/1, 48.3% for 1/2, and 11.7% for 2/2) and controls (54.1% for 1/1, 39.2% for 1/2 and 6.7% for 2/2) (P < .001). In adjusted multivariate logistic regression analyses, subjects with the LAPTM4B*1/2 and *2/2 genotypes had, respectively, 1.60-fold (95% CI, 1.15-2.22) and 2.12-fold (95% CI, 1.20-3.76) increased risk of cervical cancer than those carrying LAPTM4B*1/1. In addition, individuals with at least one LAPTM4B*2 allele (LAPTM4B*1/2 + LAPTM4B*2/2 genotype) had a 1.67-fold (95% CI, 1.22-2.29) increased risk for developing cervical cancer compared with LAPTM4B*1/1 individuals (Table 2).
Table 2. Allele and Genotype Frequencies of LAPTM4B Gene Polymorphism Among Patients and Controls and Their Associations with the Risk of Cervical Cancer
Data were calculated using logistic regression and were adjusted for age, smoking status, menopausal status, and parity.
Two-sided chi-square test.
Stratification analysis showed that the effect of LAPTM4B*1/2 variant genotypes were more evident in younger subjects (≤50 years; adjusted OR, 2.04; 95% CI, 1.39-3.01), smokers (adjusted OR, 3.48; 95% CI, 1.67-7.23), premenopausal women (adjusted OR, 2.25; 95% CI, 1.43-3.56) and women with more parities (adjusted OR, 1.63; 95% CI, 1.03-2.58 for parity >2). Similar results were also apparent for the *2/2 genotype (Table 3).
Table 3. Association Between the LAPTM4B Genotype and the Risk for Cervical Carcinoma Stratified by Age, Smoking Status, Menopausal Status, and Parity
ORs were adjusted for age, smoking status, menopausal status, and parity using a logistic regression model where appropriate.
*1/1 was used as a reference genotype.
Furthermore, a significant gene-smoking interaction was observed between LAPTM4B genotype and smoking. LAPTM4B1/2 individuals or smokers alone had an OR of 1.35 (95% CI, 0.93-1.97) or 2.68 (95% CI, 1.57-4.57), respectively; however, the OR increased to 7.38 (95% CI, 4.17-13.04) in LAPTM4B1/2 individuals who smoked, which was greater than the product of the OR for the LAPTM4B1/2 genotype and the OR for smoke alone (7.38 > 1.35 × 2.68). Similar results were also apparent for the LAPTM4B2/2 genotype. Together, these results suggest that there is a joint effect between LAPTM4B polymorphisms and smoking status (Table 4).
Table 4. Risk of Cervical Cancer Associated with LAPTM4B Genotypes by Smoking Status
We further analyzed the association between different LAPTM4B genotypes and various clinico-pathological features of cervical cancer. However, no significant association was observed between LAPTM4B genotypes and FIGO stage, histological grade, or histological type (data not shown).
In this hospital-based case-control study, we investigated the association between LAPTM4B gene polymorphisms and risk of cervical cancer in Chinese women. We found that the LAPTM4B*2 allele was associated with significantly increased risk of cervical cancer.
Previous studies have demonstrated a possible association between LAPTM4B gene polymorphisms and susceptibility to several cancers, including gastric cancer, lung cancer, and colon cancer. Previous studies have shown that the frequencies of *1/2 and *2/2 genotypes in gastric, lung, and colon cancer patients are significantly higher than in corresponding controls. The allelic frequencies of the *2 allele were 33.9%, 40.1%, and 33.2% in individuals with gastric, lung, and colon cancer, respectively, which were significantly higher than those of corresponding controls. These results demonstrate that the *2 allele is associated with an increased risk of these cancers.20-22 Our study showed similar results, with a significantly elevated risk of developing cervical cancer in women with the *1/2 genotype and the *2/2 genotype compared with the *1/1 genotype. However, studies in esophageal and rectal cancers have failed to show such a result, indicating that allele *2 was specifically associated with an increased susceptibility to certain tumors.20 In addition, our study indicated that the LAPTM4B genetic polymorphism had a significant joint effect with smoking on intensifying the risk of the cancer. Cigarette smoking is an important risk factor for cervical cancer, but the mechanism underlying this association is unknown.26-29 Our results suggest that LAPTM4B may play a role in the development of smoking-related cervical cancer. To the best of our knowledge, this is the first study investigating the association between LAPTM4B polymorphisms and the risk of developing cervical cancer.
The present study demonstrates that an association between cervical cancer risk and LAPTM4B polymorphisms are biologically plausible for the following reasons. The LAPTM4B*1 and LAPTM4B*2 allele sequences are homologous, with the exception of a 19-bp sequence in the first exon. The *1 allele contains 1 copy of the 19-bp sequence in its first exon, whereas the 19-bp sequence is duplicated in tandem as a 38-bp sequence in the *2 allele. Thus, differential susceptibility to cervical cancer could be due to the 19-bp repeat sequence. This also suggests a mechanism for the relationship between LAPTM4B polymorphisms and the susceptibility to several other cancers, such as colon and gastric cancers. Other studies have shown that the 19-bp sequence may act as a cis-acting element, participating in the transcription or regulation of nuclear proteins. The 19-bp duplicated sequence in the first exon of LAPTM4B*2 leads to a different open reading frame (ORF) for LAPTM4B*1 and LAPTM4B*2. The LAPTM4B*1 ORF is predicted to encode a 317-amino acid protein, whereas the LAPTM4B*2 ORF is predicted to encode a 370-amino acid protein, with 53 extra amino acids at the N-terminus (Figure 2). The LAPTM4B N-terminal sequence functions in signal transduction by providing ligand specificity. The extra amino acids (53 aa) at the LAPTM4B*2 N-terminus could alter protein activation, perhaps changing the biological activities and functions of cancer cells, thus inducing oncogenic susceptibility.18, 20-22
LAPTM4B was originally identified as a hepatocellular carcinoma (HCC)-associated gene that belongs to the mammalian LAPTM family.19 Studies have shown that LAPTM4B is up-regulated in many human cancers and may have a dual-functional role in the disease progression of malignant cells and in multidrug resistance.30-36 Li et al37 found that siRNA-mediated knockdown of LAPTM4B in tumor cells sensitizes cells to anthracyclines, while overexpression of LAPTM4B induces anthracycline resistance. Yang et al38 demonstrated that overexpression of LAPTM4B may promote proliferation, migration, and invasion in human HCC lines and enhance the growth and metastasis of HCC xenografts in nude mice. These studies suggest that LAPTM4B may be a suitable target for the development of novel therapeutic agents.
The limitation of predicting the development of cancer based on epidemiological data emphasizes the importance of identifying biomarkers for cancer risk and encourages further investigation. There are also some limitations to the current study. First, because the frequency of the LAPTM4B*2/2 genotype was lower in subjects, only relatively small numbers were available for subgroup analyses, suggesting that a much larger study would be needed to effectively test our conclusion. Second, our study was limited to several known clinico-pathological factors that were examined for association with LAPTM4B genetic variants. More studies are needed to evaluate these genetic variants with other risk factors for cervical cancer, such as human papillomavirus infection. Finally, replication of our results in populations of different ethnicities is required.
However, the strong points in our study are that both the cases and controls were recruited from the same area and matched on age, ethnicity, and residence, and the LAPTM4B genotype distributions were in Hardy-Weinberg equilibrium. Moreover, the cases were pathologically confirmed, and followed by a strict quality control from genotype detection.
In conclusion, this case-control study demonstrates that LAPTM4B polymorphisms are associated with increased risk for cervical cancer among Chinese women. Further investigation of these findings is warranted in analyses involving combinations with other alleles.