Association of Caveolin-1 Genotypes with Nasopharyngeal Carcinoma Susceptibility
in Taiwan
Yung-An Tsou1, Chia-Wen Tsai1,2, Ming-Hsui Tsai1, Wen-Shin Chang1,2, Fang-Jing Li1,
Yen-Fang Liu1, Chang-Fang Chiu1, Cheng-Chieh Lin1 and Da-Tian Bau1,3
1
Terry Fox Cancer Research Laboratory, China Medical University Hospital, Taichung;
Graduate Institutes of 2Basic Medical Science, and 3Clinical Medical Science, China
Medical University, Taichung, Taiwan
Correspondence to: Da-Tian Bau Ph.D., Terry Fox Cancer Research Laboratory,
Department of Medical Research, China Medical University Hospital, 2 Yuh-Der Road,
Taichung, 404 Taiwan, R.O.C. Tel: +886422053366 Ext 3312, Fax: +886422053366 Ext
1511, e-mail: [email protected]/[email protected]
Key Words: Cav-1, polymorphism, nasopharyngeal carcinoma, carcinogenesis.
Abstract. Background: Caveolin-1 (Cav-1), which has been proposed as a candidate
tumor suppressor, plays a regulatory role in several signaling pathways. High
expression of Cav-1 in nasopharyngeal carcinoma (NPC) may enhance tumor cell
migration and correlate with poor prognosis of the patients, while the genetic
alterations of Cav-1 during nasopharyngeal carcinogenesis are still largely unknown.
The aim of this study was to evaluate the association between NPC susceptibility and
Cav-1 genotypes. Materials and Methods: One hundred and seventy six patients with
NPC and 176 age- and gender-matched healthy controls recruited in Taiwan were
genotyped and analyzed by PCR-restriction fragment length polymorphism. Results:
There were significant differences between the NPC and control groups in the
distributions of the genotypic (P=0.0019) and allelic frequencies (P= 2.5*10-4) in the
Cav-1 T29107A (rs7804372) polymorphism. Conclusion: In this first report of Cav-1
involvement in NPC the A allele of Cav-1 T29107A is found to be protective against
the development of NPC and may be a novel useful genomic marker for early
Nasopharyngeal carcinoma (NPC) occurs sporadically in the West (with an
age-standardized incidence rate (ASR) < 1/100,000), but is a leading form of tumor in
Southern China (ASR = 30-50/100,000) Southeast Asia (ASR = 9-12/100,000) and
Taiwan (ASR = 8.2-8.4/100,000) (1-3). The geographical pattern of NPC incidence
suggests a unique interaction of environmental and genetic factors. Although the
etiology of NPC remains to be elucidated, Epstein-Barr virus (EBV) infection (4, 5),
environmental risk factors (6), certain dietary factors (7) and genetic differences such
as single nucleotide polymorphisms (SNPs) may all contribute to NPC carcinogenesis
(8, 9).
Three caveolin proteins, caveolin-1, -2 and -3, serve as the structural components
of the caveolae and also function as scaffolding proteins, which are capable of
recruiting numerous signaling molecules to the caveolae and regulating their activity.
It has been reported in a caveolin-deficient animal model that caveolins play a role in
human disease processes, including diabetes, cancer, cardiovascular diseases,
atherosclerosis, pulmonary fibrosis and a variety of degenerative muscular
dystrophies (10). Caveolin-1 (Cav-1), a protein of 178 amino acids, initially was
identified as a tumor suppressor gene (11). It has been demonstrated that Cav-1 is
down-regulated in sarcoma, lung carcinoma and ovarian carcinoma (12-14). However,
metastasis of esophageal squamous cell carcinoma and prostate cancer and negatively
correlated with patient survival (15, 16). These findings indicate that the role of Cav-1
may vary considerably, depending on the tissue involved. However little data are
available which consider Cav-1 for genetic predisposition to carcinogenesis (17, 18,
19).
In 2009, it was reported that highly expression of caveolin-1 in NPC, together
with its downstream protein CD147, enhanced tumor cell migration and correlated
with poor prognosis of the NPC patients (20). Up to now, the association of Cav-1
polymorphism with NPC has not been reported. Thus, the objectives of the current
study were to determine the genotypic frequency of six polymorphisms of the Cav-1
gene at C521A (rs1997623), G14713A (rs3807987), G21985A (12672038), T28608A
(rs3757733), T29107A (rs7804372), and G32124A (rs3807992). To the best of our
knowledge, this is the first study carried out to evaluate the contribution of Cav-1
Materials and Methods
Patient population and sample collection. One hundred and seventy six patients
diagnosed with NPC were recruited at the outpatient clinics of general surgery
between 2003-2009 at the China Medical University Hospital, Taichung, Taiwan,
Republic of China. All the patients voluntarily participated, completed a
self-administered questionnaire and provided peripheral blood samples. The
questionnaire included questions on history and frequency of alcohol consumption,
betel quid chewing and smoking habits and “ever” was defined as more than twice a
week for years. Self-reported alcohol consumption, betel quid chewing and smoking
habits were evaluated and classified as categorical variables. One hundred and
seventy six non-NPC or other types of cancer, healthy people as controls were
selected by matching for age and gender after initial random sampling from the
Health Examination Cohort of the hospital. The study was approved by the
Institutional Review Board of the China Medical University Hospital and
written-informed consent was obtained from all the participants.
PCR-restriction fragment length polymorphism genotyping conditions. Genomic
DNA was prepared from peripheral blood leucocytes using a QIAamp Blood Mini Kit
21-27). Briefly, the following primers were used for Cav-1 C521A (rs1997623):
5’-GTGTCCGCTTCTGC TATCTG-3’ and 5’-GCCAAGATGCAGAAGGAG TT-3’;
for Cav-1 G14713A (rs3807987): 5’-CCTTCCAGTAAGCAAGCTGT-3’ and
5’-CCTCTCAATCTTGCCATAGT-3’;
for Cav-1 G21985A (12672038): 5’-GGTGTCAGCAAGGCTATGCT-3’ and
5’-CCAGACACTCAGAATGTGAC-3’;
for Cav-1 T28608A (rs3757733): 5’-GCTCAACCTCATCTGAGGCA-3’ and
5’-GGCCTATTGTTGAGTGGATG-3’;
for Cav-1 T29107A (rs7804372): 5’-GCCTGAATTGCAATCCTGTG-3’ and
5’-ACGGTGTGAACACGGACATT-3’
and for Cav-1 G32124A (rs3807992): 5’-GGTGTCTTGCAGTTGAATG-3’ and
5’-ACGGAGCTACTCAGTGCCAA-3’. The following cycling conditions were
performed: one cycle at 94oC for 5 min; 35 cycles of 94oC for 30 sec, 55oC for 30 sec,
and 72oC for 30 sec; and a final extension at 72oC for 10 min. The PCR products were
studied after digestion with Avr II, Bfa I, Hae III, Tsp509 I, Sau3AI and Nla III,
restriction enzymes for Cav-1 C521A (cut from 485 bp C type into 170+315 bp A
type), Cav-1 G14713A (cut from 268 bp A type into 66+202 bp G type), Cav-1
G21985A (cut from 251+43 bp A type into 153+98+43 bp G type), Cav-1 T28608A
A type into 172+164 bp T type) and Cav-1 G32124A (cut from 213+142+67 bp A
type into 142+118+95+67 bp G type), respectively.
Statistical analyses. Only those matches with all SNP data (case/control =176/176)
were selected into final 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 Cav-1 SNP in the control subjects
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 (where number in
any cell was less than five) was used to compare the distribution of the Cav-1
genotypes between the cases and controls. Data was recognized as significant when
Results
There were no significant differences between the two groups in their age, sex and or
individual behavior factors (Table I). The frequencies of the genotypes for Cav-1
C521A, G14713A, G21985A, T28608A, T29107A and G32124A between the
controls and NPC patients is shown in Table II. The genotype distribution of the
various genetic polymorphisms of Cav-1 T29107A were significantly different
between the NPC and control groups (P=0.0019), while those for Cav-1 C521A,
G14713A, G21985A, T28608A and G32124A were not significant (P>0.05) (Table
II).
The frequencies of the alleles for Cav-1 C521A, G14713A, G21985A, T28608A,
T29107A and G32124A between the controls and NPC patients are shown in Table
III. The T29107A genotype of Cav-1 found to be associated with NPC cancer in
Table II was also found to be associated with higher NPC susceptibility in the allele
frequency analysis (P=2.5*10-4). As for other five SNPs, the distributions of their
allele frequencies were not significantly different in the controls and NPC patients
Discussion
The present study revealed that Cav-1 T29107A (rs7804372) polymorphisms
were associated with the susceptibility to NPC (Table II and III), while the other five
polymorphisms investigated were not. Although these genetic variations do not
directly result in amino acid coding change, it is plausible to suspect modifications
such as alternative spicing, may happen during carcinogenesis via influencing the
expression level or stability of the Cav-1 protein.
The sample sizes of NPC investigations are often not as large as other types of
cancer and as many as possible were enrolled in our hospital during these years. The
similar trends of significant data after age- and behavior-adjustments strengthen the
accuracy and reliability of the present findings, and the frequencies of the Cav-1
polymorphisms variant alleles were similar to those reported in the National Center
Biotechnology Information (NCBI) website in other Asian population studies. For
instance, the minor A allele frequencies of Cav-1T29107A were 34.9% in the present
control group, close to those of 31.1~31.8% for the Tokyo population in the NCBI,
which strongly suggested no selection bias for subject enrolments in terms of
genotypes.
supporting the NPC tumorigenic contribution of Cav-1, of which the polymorphisms
of T29107A were the most significantly associated. Additional functional analyses of
the gene and polymorphisms would be useful for exploring the mechanisms by which
Cav-1 and its regulated proteins affect NPC risk.
In conclusion, Cav-1 T29107A, but not C521A, G14713A, G21985A, T28608A
or G32124A, was associated with higher susceptibility to NPC. The A allele of Cav-1
T29107A might become a novel biomarker for NPC oncology early screening and
prediction.
Acknowledgements
We are grateful to Hui-Ni Cheng, Tzu-Hsin Weng and Tissue Bank personnel in
China Medical University Hospital for their technical assistance. This study was
supported by research grants from China Medical University and Hospital
(DMR-100-046), Terry Fox Cancer Research Foundation, National Science Council
(NSC 98-2320-B-039-010-MY3) and Department of Health, Execution Yuan
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Characteristics Controls (n = 176) Patients (n = 176) Pa n % Mean (SD) n % Mean (SD) Age (y) 49.3 (9.4) 48.2 (11.1) 0.6851 Gender 1.0000 Male 128 72.7% 128 72.7% Female 48 27.3% 48 27.3% Indulgence Cigarette smokers 73 43.8% 77 43.8% 0.7465
Betel quid chewers 54 31.3% 55 31.3% 1.0000
Alcohol drinkers 72 45.5% 80 45.5% 0.5414
a
Genotype Controls Patients Pa C521A rs1997623 1.0000 CC 171 97.2% 171 97.2% AC 5 2.8% 5 2.8% AA 0 0.0% 0 0.0% G14713A rs3807987 0.9441 GG 116 65.9% 113 64.2% AG 45 25.6% 47 26.7% AA 15 8.5% 16 9.1% G21985A rs12672038 0.9090 GG 106 60.2% 102 58.0% AG 57 32.4% 60 34.1% AA 13 7.4% 14 8.0% T28608A rs3757733 0.7621 TT 100 56.8% 106 60.2% AT 59 33.5% 56 31.8% AA 17 9.7% 14 8.0% T29107A rs7804372 0.0019 TT 86 48.9% 109 61.9% AT 57 32.4% 55 31.3% AA 33 18.7% 12 6.8% G32124A rs3807992 0.6834 GG 88 50.0% 82 46.6% AG 68 38.6% 76 43.2% AA 20 11.4% 18 10.2%
Allele Controls Patients Pa C521A rs1997623 1.0000 Allele C 347 98.6% 347 98.6% Allele A 5 1.4% 5 1.4% G14713A rs3807987 0.7154 Allele G 277 78.7% 273 77.6% Allele A 75 21.3% 79 22.4% G21985A rs12672038 0.6603 Allele G 269 76.4% 264 75.0% Allele A 83 23.6% 88 25.0% T28608A rs3757733 0.4343 Allele T 259 73.6% 268 76.1% Allele A 93 26.4% 84 23.9% T29107A rs7804372 2.5*10-4 Allele T 229 65.1% 273 77.6% Allele A 123 34.9% 79 22.4% G32124A rs3807992 0.7450 Allele G 244 69.3% 240 68.2% Allele A 108 30.7% 112 31.8% a
