Abstract
Background: A growing body of evidence indicates that caveolin-1 (CAV1) may influence the development of human cancer. However, the exact role of CAV1 in childhood leukemia is still controversial. We investigated six novel polymorphic variants of CAV1, namely C521A (rs1997623), G14713A (rs3807987), G21985A (rs12672038), T28608A (rs3757733), T29107A (rs7804372), and G32124A (rs3807992), and analyzed the association of each specific genotype with susceptibility to childhood leukemia. Materials and Methods: In total, 266 patients with childhood leukemia and 266 age-matched healthy controls, recruited from two major medical centers in Taiwan, were genotyped investigating the association of these polymorphisms with childhood leukemia. Results: We found that there were significant differences between childhood leukemia and control groups in the distributions of their genotypes (p=4.1×10-8 and 0.0167) and allelic frequencies (p=4.9×10-10 and 3.7×10-3) in the CAV1 G14713A and T29107A polymorphisms, respectively. As for the haplotype analysis, those who had GG/AT or GG/AA at CAV1 G14713A/T29107A had a reduced risk of childhood leukemia compared to those with GG/TT, while those with any other combinations were at increased risk. Conclusion: The A allele of CAV1 G14713A is risky, while the A allele of CAV1 T29107A is protective for the development of childhood leukemia and these may be novel useful genomic markers for the early detection of childhood leukemia.
Childhood leukemia is the most common type of childhood cancer over the world, accounting for approximately one-third of childhood malignancies and poses a severe issue for every society. The etiology and genomic contributing factors of leukemia are still largely unknown. In recent years, investigators have become interested in caveolae and in defining how these lipid domains participate in the pathogenesis of human cancer and what their possible utility may be for its detection and treatment (1). Caveolae are vesicular invaginations of the plasma membrane, and are believed to play a critical role in transcytosis, communication between cell surface membrane receptors and intracellular signaling protein cascades, such as apoptosis and tumorigenesis (2, 3).
A growing body of evidence indicates that caveolin-1 (CAV1) influences the development of human cancer. However, the exact functional role of CAV1 in leukemogenesis is still controversial. In 2010, CAV1 was found to be overexpressed in adult T-cell leukemia cells (4). In certain cell types, antisense inhibition of CAV1 expression is sufficient to induce oncogenic transformation (5). Targeted down-regulation of CAV1 activates mitogen-activated protein kinases (MAPK) and stimulates anchorage-independent growth of NIH-3T3 cells (5). Loss of CAV1 is required to accelerate tumorigenesis and metastasis; PyMT/CAV1(-/-) mice exhibited accelerated onset of mammary tumors and lung metastasis (6). CAV1 is highly expressed in the normal ovary but markedly down-regulated in human grade 3 serous ovarian carcinoma (7). In addition to ovarian carcinoma, CAV1 is also down-regulated in human tumors derived from the breast and colon, while controversy exits that it is up-regulated in tumor samples from the kidney, prostate, and stomach (7). CAV1 expression is lost in human mammary carcinoma cell lines (5). Expression of CAV1 in a highly metastatic carcinoma-derived cell line suppressed lung metastasis in vivo and reduced cancer invasion in vitro (6). Decreased invasion of CAV1-expressing cells was accompanied by reduction in matrix metalloproteinase-9 (MMP9) and MMP2 secretion and gelatinolytic activity, and reduced extracellular signal-regulated kinases (ERK)1/2 signaling in response to growth factors (6). In addition to lung cancer cell lines, CAV1 re-expression in human breast cancer and in colon carcinoma cell lines also inhibited tumor cell growth (8).
The primer sequences, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) conditions for Caveolin-1 gene polymorphisms.
To the extent of our knowledge, there is no study of the role of CAV1 genotypes in childhood leukemia. The emerging evidence pointing to the role of CAV1 in carcinogenesis led us to study whether different alleles of this gene are associated with leukemogenesis. Thus, the aims of the current study were to determine the genotypic frequency of six single nucleotide polymorphisms (SNPs) of the CAV1 gene at C521A (rs1997623), G14713A (rs3807987), G21985A (rs12672038), T28608A (rs3757733), T29107A (rs7804372), and G32124A (rs3807992), and their association with susceptibility to childhood leukemia. To the best of our knowledge, this is the first study carried out aiming to evaluate the contribution of CAV1 polymorphisms in childhood leukemia.
Materials and Methods
Study population and sample collection. Two hundred and sixty-six patients diagnosed with childhood leukemia (i.e. the population under 18 years old) were recruited from the Pediatric Departments at the China Medical University Hospital and National Taiwan University Hospital, Taiwan, Republic of China in 2005-2009. A non-cancer healthy person was matched by gender and age after initial random sampling from the Health Examination Cohort of the two hospitals for each patient. Patients and controls completed a self-administered questionnaire and provided their peripheral blood samples.
Genotyping assays. Genomic DNA was prepared from peripheral blood leucocytes using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and further processed according to our previous articles (9-19). The polymerase chain reaction (PCR) cycling conditions were: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. Pairs of PCR primer sequences and restriction enzyme for each DNA product are all listed in Table I.
Statistical analyses. Only cases with both the complete clinical data and successful genotypic data (case/control=266/266) were selected for analyzing. To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of CAV1 SNPs in the controls from those expected under the Hardy-Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson’s chi-square test or Fisher’s exact test (when the expected number in any cell was less than five) was used to compare the distribution of the CAV1 genotypes between cases and controls. Data were recognized as significant when the statistical p-value was less than 0.05.
Results
The frequencies of the genotypes for CAV1 C521A (rs1997623), G14713A (rs3807987), G21985A (rs12672038), T28608A (rs3757733), T29107A (rs7804372), and G32124A (rs3807992) for controls and childhood leukemia patients are shown in Table II. Genotypic distributions of the polymorphisms of CAV1 G14713A and T29107A were significantly different between childhood leukemia and control groups (p=4.1×10-8 and 0.0167, respectively), while those for CAV1 C521A, G21985A, T28608A and G32124A were not significant (p>0.05) (Table II). To sum up, the polymorphisms of CAV1 G14713A and T29107A are associated with childhood leukemia risk and may be useful biomarkers for childhood leukemia early detection. The people carrying A allele of CAV1 G14713A and T allele of CAV1 T29107A in their genome seemed to be of higher risk of childhood leukemia in the Taiwanese population. The representative PCR-based restriction analyses for the CAV1 G14713A and T29107A polymorphisms are shown in Figure 1.
Distribution of Caveolin-1 genotypes among childhood leukemia and control groups.
The frequencies of the alleles for the CAV1 C521A, G14713A, G21985A, T28608A, T29107A, and G32124A between controls and patients with childhood leukemia are shown in Table III. The two SNPs of CAV1 found to be associated with childhood leukemia risk are shown in Table II, G14713A and T29107A, are also found to be associated with differential childhood leukemia susceptibility in the analysis of their allelic frequency analysis (p=4.9×10-10 and 3.7×10-3, respectively). As for the other four SNPs, the distributions of their allelic frequencies are not significantly different in controls and patients (Table III). The conclusion deduced from Table II and III is that CAV1 G14713A and T29107A seem to be markers for childhood leukemia.
Considering potential interactions between these significant SNPs of the CAV1 gene and susceptibility to childhood leukemia, the risk of childhood leukemia related to haplotype of CAV1 G14713A and T29107A were further analyzed (Table IV). Both odds ratios (ORs) adjusted for individual age and gender and chi-square p-value were calculated to evaluate the contribution for each haplotype to childhood leukemia. Compared with the major GG/TT haplotype of CAV1 G14713A/T29107A combination, the GG/AT and GG/AA group has a lightly lower risk of childhood leukemia (OR=0.68, 95% confidence interval, CI=0.43-1.12, p=0.1117). Other combinations of AG/TT (OR=2.61, 95% CI=1.60-4.27, p=0.0004) AG/AT or AG/AA (OR=2.92, 95% CI=1.79-4.91, p=0.0886), AA/TT (OR=2.26, 95% CI=1.22-4.17, p=0.0006), and AA/AT or AA/AA (OR=2.34, 95% CI=1.06-5.27, p=0.0459), all have increased childhood leukemia risk compared to the GG/TT haplotype (Table IV).
Distribution of alleles for Caveolin-1 gene among the childhood leukemia and control groups.
Discussion
To our knowledge, there has been no investigation regarding the polymorphisms of CAV1, whose products play an important role in membrane signaling, in childhood leukemia. According to our results, the G14713A and T29107A polymorphisms of the CAV1 gene were found to be associated with childhood leukemia (Tables II and III). In our hypothesis, the variant polymorphisms of intronic G14713A and T29107A on CAV1 may alter the normal expression and/or the protein function of CAV1 by alternative splicing and regulation of mRNA stability. It has been reported that hematological cells express CAV1 in certain states of cell activation (20) and in adult T-cell leukemia, CAV1 was found to be overexpressed (4).
A wealth of evidence suggests a role for CAV1 as a suppressor of tumor growth and metastasis in ovarian, breast and colon human carcinomas (5-8). However, the function of CAV1 might be entirely different in different tissues. CAV1 could exert opposing functions, resulting in tumor progression promotion rather than inhibition. CAV1 expression is increased in tumor samples from the kidney, prostate, stomach with respect to the normal tissues, and re-expression is found in some advanced adenocarcinomas (7). Elevated expression of CAV1 is associated with progression in the prostate, colon, breast, lung carcinoma (7), and adult T-cell leukemia (4). Remarkably, induced re-expression of CAV1 in less invasive caveolin-negative lung cancer cell lines enhanced their invasive capability (21). We postulated that induced CAV1 expression may alter the interactions between cell and extracellular matrix by lipid raft internalization of adhesion molecules, such as catenin and cadherin, and subsequently facilitate tumor metastasis (22). CAV1 protects prostate cancer cells from c-myelocytomatosis oncogene (c-MYC)-induced apoptosis (23). Thus, engagement of CAV1 as a tumor metastasis promoter or tumor metastasis suppressor is strongly determined by the specific cellular context, and, at the molecular level, by the signaling molecules interacting with CAV1 and by the signaling pathways affected and regulated by CAV1. We hypothesize that altered CAV1 expression may somehow lead to failure of homeostatic maintenance, resulting in an increased frequency of childhood leukemogenesis.
Polymerase chain reaction (PCR)-based restriction analysis of the G14713A (A) and T29107A (B) polymorphisms of the Caveolin-1 gene, shown on 3% agarose electrophoresis. M: 100 bp DNA size marker; A: AA: indivisible homozygote, AG: heterozygote, and GG: divisible homozygote. B: AA: Indivisible homozygote, AT: heterozygote, and TT: divisible homozygote.
. Frequencies of combined Caveolin-1 G14713A/T29107A haplotypes among the childhood leukemia and control groups.
In this study, we screened six SNPs of the CAV1 gene and investigated their genotypic associations with childhood leukemia susceptibility. We found that two novel SNPs, G14713A and T29107A of the CAV1 gene, were associated with childhood leukemia. However, the effects of these SNPs of the CAV1 gene need further studies, such as immunohistochemistry or western blotting, in leukemogenesis of different stages (in bone marrow, metastatic sites and peripheral blood), to reveal the role of each section or even each nucleotide in the subtle regulation of the transcriptional, translational, and post-translational expression of the CAV1 gene in different body compartments. Therefore, these two SNPs, together with other SNPs, may be found to play a role in childhood leukemia progression in their joint effects. In addition, an enlarged population size and genomic-environmental combinatorial studies could provide with more comprehensive and realistic data regarding the progression of childhood leukemia.
In conclusion, to our knowledge this is the first report to provide evidence for CAV1 G14713A and T29107A polymorphisms, but not C521A, G21985A, T28608A, or G32124A, being associated with higher susceptibility to childhood leukemia. In addition, the G allele of CAV1 G14713A and A allele of CAV1 T29107A might become potential biomarkers for early detection, prediction and integrative cancer therapy of childhood leukemia.
Acknowledgments
This study was supported by research grants from the China Medical University and Hospital (CMU-101-122) and the Terry Fox Cancer Research Foundation. The assistance from Tsai-Ping Ho in clinical data collection, and from Liang-Yi Lin, Yi-Ting Chang, Hong-Xue Ji in genotyping were highly appreciated by the Authors.
Footnotes
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↵* These Authors contributed equally to the study.
- Received February 26, 2013.
- Revision received March 16, 2013.
- Accepted March 18, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved
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