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Annals of Oncology Advance Access published online on October 26, 2007
Annals of Oncology, doi:10.1093/annonc/mdm469
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© 2007 European Society for Medical Oncology

Relationship between LAPTM4B gene polymorphism and susceptibility of colorectal and esophageal cancers

X.-J. Cheng1,{dagger}, W. Xu2,{dagger}, Q.-Y. Zhang1,* and R.-L. Zhou3

1 Department of Clinical Laboratory, Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing
2 The Medical School of Bei Hua University, Jilin
3 Department of Cell Biology, The School of Basic Medical Sciences, Peking University, Beijing, China

* Correspondence to: Dr Q.-Y. Zhang, Department of Clinical Laboratory, Peking University School of Oncology, Beijing Cancer Hospital and Institute, 52 Fucheng Road, Haidian District, Beijing 100036, China. Tel: +86-010-88196336; Fax: +86-10-88122437; E-mail: zhqy_208{at}163.com


   Abstract
Top
 Abstract
introduction
 materials and methods
 results
 discussion
 funding
 Acknowledgements
 References
 
Background: Lysosome-associated protein transmembrane 4 beta (LAPTM4B) is a novel gene of the mammalian LAPTM family and has been shown to be overexpressed in human hepatocellular carcinoma. There are two alleles, LAPTM4B*1 and *2, which share the same sequence except for one segment of 19 bp in the 5' untranslated region of the exon 1. LAPTM4B*1 has one 19 bp segment, while LAPTM4B*2 has two tight tandem segments. The current case–control study was aimed to identify relationship between the gene polymorphism of LAPTM4B and the susceptibility of colorectal and esophageal cancers.

Patients and methods: Blood samples were collected from patients with colon, rectal or esophageal cancers and control subjects. Genotypes of LAPTM4B were determined by PCR to detect differences between cancer cases (n = 701) and healthy controls (n = 350). Association between the LAPTM4B polymorphism and the risk of cancer was calculated by unconditional logistic regression models.

Results: We found that there was a significant difference (P = 0.0016) in allelic frequencies of LAPTM4B*2 between colon cancer cases (33.2%) and controls (24.1%). The risk of colon cancer was elevated significantly in cases with *1/2 genotype [odds ratio (OR) = 1.474; 95% confidence interval (CI) = 1.037–2.095] and *2/2 genotype (OR = 2.531; 95% CI = 1.316–4.868) when compared with the *1/1 genotype. No significant difference was observed for LAPTM4B*2 between the rectal or esophageal cancer cases when compared with the controls. The polymorphism in LAPTM4B was associated with increased risk of colon cancer but not of rectal and esophageal cancers.

Conclusions: These results indicate that the genetic polymorphism of LAPTM4B is a potential risk factor for the development of colon cancer.

colorectal cancer, esophageal cancer, LAPTM4B, polymorphism, susceptibility


   introduction
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 Abstract
 introduction
materials and methods
 results
 discussion
 funding
 Acknowledgements
 References
 
Colorectal and esophageal cancers are common malignant diseases around the world. Both have an extremely poor prognosis owing to insidious symptomatology, late clinical presentation and rapid progression and so lead to poor 5-year disease-free survival [1, 2]. They are related not only to living habits such as dietary but also to the susceptibility of heredity, which is implicated as one of the major predisposing factors to tumorigenesis [3–5].

Lysosome-associated protein transmembrane 4 beta (LAPTM4B), a novel oncogene candidate, was screened through fluorescence differential display and its full length was cloned [6, 7]. It is expressed fairly low in normal adult liver but high in most hepatocellular carcinoma tissues. Furthermore, the expression levels are related closely to differentiation status of the hepatocellular carcinoma [6–9].

LAPTM4B has been mapped to chromosome 8q22.1, spanning at least 50 kb, and is composed of seven exons separated by six introns and the full length of messenger RNA is 2245 bp. It is 46% homologous at amino acid level to a human lysosome-associated transmembrane-4 protein, LAPTM4A [7, 10]. LAPTM4B exists as two allelic genes: LAPTM4B*1 and LAPTM4B*2 (GenBank AC: AY219176 and AY219177). Both have the same sequence except for one segment of 19 bp in the 5' untranslated region of exon 1. LAPTM4B*1 has one 19 bp segment, while LAPTM4B*2 has two tight tandem segments (Figure 1A) [11]. The frequency of *2/2 genotype in the hepatocellular carcinoma, lung cancer and gastric cancer is significantly higher than that in healthy adults [11–13]. Therefore, we designed a case–control study to investigate relationship between gene polymorphism of LAPTM4B and susceptibility of colorectal and esophageal cancers.


Figure 1
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  		Figure 1. Schematic diagram showing the lysosome-associated protein transmembrane 4 beta (LAPTM4B) alleles. (A) In exon 1 (indicated by black boxes), which includes the 5' untranslated region, the LAPTM4B*1 allele has one segment of 19 bp (gray box), while LAPTM4B*2 has two tight tandem segments of 19 bp (depicted as two gray boxes). The DNA sequences in the gray boxes are showed in the lower panels. (B) This polymorphism was shown by PCR using specific primers for LPTM4B. The upper panel depicted the amplified PCR products: Lanes 1–3, *2/2 genotype; lanes 4–6, *1/1 genotype; lanes 7–9, *1/2 genotype. The lower panel showed the amplified product of human GAPDH that served as the positive internal control.

 

   materials and methods
Top
 Abstract
 introduction
 materials and methods
results
 discussion
 funding
 Acknowledgements
 References
 
participants
case patients.
We collected 701 cancer cases (253 colon cancer, 237 rectal cancer and 211 esophageal cancer) who were hospitalized in Beijing cancer hospital from January 2004 to August 2006. Each participant had agreed to the written informed consent for this project. Final diagnosis of all patients was confirmed by pathologic investigations. Tumors were staged according to the Tumor, Node, Metastasis (TNM) classification of the American Joint Committee on Cancer and International Union Against Cancer (AJCC 1988, 2003; UICC 1987, 2003).

control subjects.
In all, 350 healthy adults were quoted from the healthy adult data of Liu et al. [13]. The eligible controls were healthy individuals recruited from employees at the Beijing Cancer Hospital and the Health Science Center in the Peking University. They met the same eligibility criteria except that they never have a diagnosis for cancer. The controls from Beijing Cancer Hospital were checked for cancer history through their past medical records, while the others were directly asked for their cancer history. The nurse interviewers explained the aims of this study to the blood donors, who had all provided informed consent for genotyping.

procedures
Anticoagulant blood (1 ml) was taken from the vein and extracted with alcohol phenol/trichlormethane. DNA was obtained by acetic acid amine and dehydrated alcohol precipitation and dissolved in Tris-EDTA buffer at 4°C. Using the primers 5'-GCCGACTAGGGGACTGGCGGA-3' (sense) coding for the 72–92 bp of LAPTM4B and 5'-CGAGAGCTCCGAGCTTCTGCC-3' (antisense) corresponding to the 255–275 bp [13, 14], PCR was done in a reaction mixture (25 µl) containing 0.5 U of Taq (Tiangen, China) and 1 µl of template DNA with a final concentration of 100 ng/µl. For the positive internal control, the primers 5'-TCCACCACCCTGTTGCTGTA-3'(sense) and 5'-ACCACAGTCCATGCCATCAC-3' (antisense) coding for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used. The PCR condition was 95°C denaturation for 5 min, 35 cycles of 30 s at 94°C, 45 s at 62°C and 1 min at 72°C, followed by autoextension at 72°C for 10 min. The amplified products were analyzed by electrophoresis in 10% polyacrylamide gel or 2% agarose (visualized by 0.5 µg/ml ethidium bromide).

statistical analysis
Statistical analysis was carried out by Statistical Analysis System software (version 8.1; SAS Institute, Cary, NC). {chi}2 test or the Fisher's exact test was used to calculate the genotype frequency and other parametric distributions between cancer cases (colon/rectal/esophageal cancer) and controls and P < 0.05 was used as the significance level. Unconditional logistic regression analysis models were used to analyze relationships between different genotypes and cancer risks adjusted by the age and gender status.


   results
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 Abstract
 introduction
 materials and methods
 results
discussion
 funding
 Acknowledgements
 References
 
Using the specific primers for LAPTM4B, three different genotypes were identified in the PCR products. In homozygous individuals, we found consistently a 204-bp fragment coding for LAPTM4B*1/1 and a 223-bp fragment for LAPTM4B*2/2 (Figure 1B). In LAPTM4B*1/2 heterozygous individuals, both fragments were observed. Amplified products for GAPDH existed as a 226-bp fragment in all positive internal controls.

Analyses of the age and gender in various cancer cases showed a significant difference in the distribution of colorectal and esophageal cancers among different age groups (P = 0.0001) and different genders (P < 0.0001) (Table 1).


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  		Table 1. Distribution of age and gender in cancer cases and controls

 
A different allele distribution was noticed among cancer cases and controls (Table 2). In 350 controls, the LAPTM4B*2 allele frequency was 24.1%, which is different from that in the cancer cases (33.2%, 22.5% and 22.7% for colon, rectal and esophageal cancer, respectively). Subjects with LAPTM4B*2 have a significantly higher risk in colon cancer than controls [odds ratio (OR) = 1.523; 95% confidence interval (CI) = 1.173–1.978] but not in rectal and esophageal cancers (OR = 1.049, 95% CI = 0.792–1.389; OR = 0.840, 95% CI = 0.616–1.146, respectively).


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  		Table 2. Distribution of LAPTM4B alleles in cancer cases and controls

 
Distribution of genotypes of LAPTM4B in controls and cancer cases were summarized in Table 3. The distributions of LAPTM4B genotypes in colon, rectal and esophageal cancers and controls all conformed to the Hardy–Weinberg equilibrium (P = 0.484, 0.975, 0.063 and 0.253, respectively). Statistical comparisons showed that there was a significant difference in the distribution of LAPTM4B*1/2 and *2/2 between colon cancer and controls (P = 0.0305 and 0.0054, respectively), but not between rectal or esophageal cancer when compared with corresponding controls (P = 0.5799 and P = 0.1987, respectively). Subjects with genotypes of LAPTM4B*1/2 and *2/2 have 1.474-fold (95% CI = 1.037–2.095) and 2.531-fold (95% CI = 1.316–4.868) higher risk in colon cancer when compared with those carrying LAPTM4B*1/1, respectively. However, no statistical difference was found for rectal or esophageal cancer.


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  		Table 3. Distribution of various genotypes of LAPTM4B in cancer cases and controls

 
Moreover, we had analyzed the distribution of clinical parameters such as gender, age, pathological category of colorectal and esophageal cancers, differentiation degree, classification of TNM and individual smoking and drinking habits of patients in different genotypes of LAPTM4B (Table 4). The relationship between the genotype distribution of LAPTM4B and these clinical parameters failed to reach statistical significance in our study.


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  		Table 4. Distribution of various genotypes of LAPTM4B in relation to clinicopathological and other variables in different cancer cases

 

   discussion
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 Abstract
 introduction
 materials and methods
 results
 discussion
funding
 Acknowledgements
 References
 
One major finding in this study is that subjects with the LAPTM4B*2 allele show a significantly higher risk of colon cancer, but this susceptibility is not observed for rectal and esophageal cancers when compared with control subjects. These results agreed with the findings of Peng et al. [9], which show through immunohistochemical analysis the expression of LAPTM4B protein in cancer tissues derived from single layer cuboidal and columnar epithelia, such as hepatocellular carcinoma, lung cancer, breast cancer, gastric cancer and colon cancer, and a lack of expression in cancer tissues from stratified epithelia, such as esophageal cancer and rectal cancer. Further support comes from the study by Zhou et al. [14], which shows that LAPTM4B protein is also highly expressed in gall-bladder carcinoma. As the process of carcinogenesis is a long-term and cooperative effect of various factors, among these are multiple micro-predisposing genes [15, 16], our findings indicate that the LAPTM4B*2 allele is one of these genes contributing to a higher risk for colon cancer.

The two ATGs of LAPTM4B gene. Open reading frame (ORF), located at nt157 and nt430, may initiate the translation of two different protein isoforms, LAPTM4B-35 (317 aa) and LAPTM4B-24 (226 aa). Liu et al. [17] have utilized a polyclonal antibody (KLH-LAPTM4B-EC2-pAb) to show that these two protein isoforms have different expression status and roles in hepatocellular carcinoma tissue samples and various cell lines. The expression of LAPTM4B-35 rather than LAPTM4B-24 has been indicated to be closely associated with different cancer grades [6, 11]. Shao and Liu et al. [9, 11, 17] have demonstrated that an equilibrium of these two protein isoform expression is involved in the determination of cell fate, through controls of the survival/proliferation and differentiation of the cell; and a disturbance of this equilibrium may cause malignant transformation.

Previous studies have demonstrated that expression of cyclin D1 and cyclin E is markedly up-regulated when LAPTM4B cDNA is transfected into human HLE cells and mouse NIH 3T3 cells [6, 18], indicating that LAPTM4B may promote cell proliferation through cyclin D1 and cyclin E and accelerate malignant transformation [18]. Moreover, the LAPTM4B transfected cells acquire stronger immigration and invasion abilities, and the expression of cyclin E and cyclin D1 has been shown to relate to the degree of pathologic differentiation in some tumors [19–22], providing further supports to the argument that LAPTM4B is involved in malignant transformation. In LAPTM4B, both N- and C-terminus contain the proline-rich domain, forming the ‘PXXP’ motif. Moreover, the N-terminus is flanked by hydrophobic residues beside the proline-rich domain [9], forming a typical structural feature characterizing the SH3-domain ligands. This domain may interact with the SH3 structural domains in many proteins, mediating cell signal transduction, location and isolation of proteins inside the cell [23–26]. All these results indicate a role of LAPTM4B in regulating cell proliferation, carcinogenesis and tumor invasion.

While the ORF of LAPTM4B*1 is expected to encode a 317 amino acid protein, as mentioned above, we deduce that in the LAPTM4B*2 allele, dislocation of the termination codon should result in production of a 370 amino acid protein (Figure 2) [12, 13, 27]. The two different protein isoforms, which arise basically from a 19-bp difference in the first exon of the LAPTM4B gene that alters the ORF, may influence physiological activities and functions of the cancer cell. While all members of the LAPTM protein family including LAPTM4B are localized to the membrane of late endosomes and lysosomes [28, 29], the C-terminus of LAPTM4B may be responsible for various protein sorting for the final localization in the cell [7, 28]. It may also change the signal transduction that affects the clearing pathways of intracellular carcinogenic substances and so induces different oncogenic susceptibility [10, 30]. Moreover, the 19-bp sequence may act as a cis-acting element to participate in genetic transcription or regulation of nuclear proteins.


Figure 2
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  		Figure 2. Comparison of predicted protein sequence encoded by the allele LAPTM4B*1 and LAPTM4B*2. This figure shows the initial segments of exon 1 in LAPTM4B*1 (A) and LAPTM4B*2 (B). The serial number of the first nucleotide in each row is indicated on the left, and the sequence numbers of the predicted amino acid residues are shown on the right. The predicted termination codons are underlined and indicated by symbol #. They are located at nt40, nt78 and nt103 in LAPTM4B*1, rendering translation possible only at nt157. In LAPTM4B*2, these termination codons are absent and so translation can be initiated at nt17, producing a longer protein product than allele *1.

 
To our knowledge, this is the first study which focuses on the possible role of LAPTM4B*2 in colorectal and esophageal cancers. We concluded that while no statistical significance is observed in susceptibility of rectal and esophageal cancers, LAPTM4B*2 is likely contributing to a higher risk of colon cancer.


   funding
Top
 Abstract
 introduction
 materials and methods
 results
 discussion
 funding
Acknowledgements
 References
 
Key Project Grant of Beijing Natural Science Foundation (No. 7041003); General Project Grant of Beijing Natural Science Foundation (No. 5072016), Beijing, China.


   Acknowledgements
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 Abstract
 introduction
 materials and methods
 results
 discussion
 funding
 Acknowledgements
References
 
The authors gratefully acknowledge F. Guoshuang for his assistance with the statistical analysis and thank all the people and patients who had participated in this study.


   Footnotes
 
{dagger} Both authors contributed equally to the work as first coauthors. Back

Received for publication February 7, 2007. Revision received August 22, 2007. Accepted for publication September 4, 2007.


   References
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 Abstract
 introduction
 materials and methods
 results
 discussion
 funding
 Acknowledgements
 References
 
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