Association of hypoxia inducible factor-1α polymorphisms with susceptibility to non–small-cell lung cancer

Association of hypoxia inducible factor-1 a polymorphisms with susceptibility to non–small-cell lung cancer WU-HSIEN KUO, CHUEN-MING SHIH*, CHIAO-WEN LIN, WEI-ERH CHENG, SHUO-CHUEH CHEN, WEI CHEN, and YAO-LING LEE* TAIWAN, REPUBLIC OF CHINA

Hypoxia-inducible factor-1a (HIF-1a) is a key regulator of cellular response to hypoxia and has been suggested to play an important role in tumorigenesis and metastasis. The aim of this study was to investigate the role of HIF-1a-1772 C/T (P582S) and -1790 G/A (A588T) polymorphisms in the susceptibility to and severity of non–smallcell lung cancer (NSCLC). Using a case-control study design and polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis, the allele frequencies and genotype distributions of each single nucleotide polymorphism in 285 NSCLC cases and 300 gender-matched controls were compared. The distribution of the genotype frequencies of HIF-1a-1772 C/T and -1790 G/A were significantly different between the NSCLC and the controls. Logistic regression analysis revealed that higher odds ratios (ORs) for lung cancer were observed for individuals with HIF1a-1772 T/T genotype against CC/CT genotypes (an OR of 4.04, 95% confidence interval [CI] 5 2.02–8.08, P 5 0.0001), and HIF-1a-1790 A/A genotype against GG/GA genotypes (an OR of 4.42, 95% CI 2.22–8.78, P , 0.0001). There were no relationship between HIF-1a-1772 C/T or -1790 G/A allele distribution and disease severity of NSCLC (P . 0.05). However, those patients carrying a HIF-1a-1772 T/T genotype or a HIF-1a-1790 A/A had a tendency toward inferior prognosis compared with other patients. (Translational Research 2012;159:42–50) Abbreviations: AD ¼ adenocarcinoma; CI ¼ confidence interval; EDTA ¼ ethylenediaminetetraacetic acid; HIF-1a ¼ hypoxia-inducible factor-1alpha; NSCLC ¼ non–small-cell lung cancer; ODD ¼ oxygen-deprived degradation; OR ¼ odds ratio; PCR ¼ polymerase chain reaction; RFLP ¼ restriction fragment-length polymorphism; SNP ¼ single-nucleotide polymorphism; SQ ¼ squamous carcinoma; VEGF ¼ vascular endothelial growth factor

*

These authors contributed equally to this article.

From the Department of Medicine, Armed-Force Taichung General Hospital, Taiwan, Republic of China; General Education Center, Central Taiwan University of Science and Technology, Taiwan, Republic of China; Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, China Medical University Hospital, Taiwan, Republic of China; Department of Respiratory Therapy, China Medical University, Taiwan, Republic of China; Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taiwan, Republic of China; Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Chia-Yi Christian Hospital, Taiwan, Republic of China; School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taiwan, Republic of China; Clinical Laboratory, Chung Shan Medical University Hospital, Taiwan, Republic of China.

42

Supported in part by Grant CMU99-COL-19, DMR-99-022 from China Medical University and Grant DOH 99-TD-C-111-005 from the Department of Health (The Executive Yuan, Republic of China). Submitted for publication May 5, 2011; revision submitted August 25, 2011; accepted for publication September 13, 2011. Reprint requests: Yao-Ling Lee, School of Laboratory and Biotechnology, Chung Shan Medical University, No.110, Sec. 1, Chien-Kuo N. Road, Taichung City 402, Taiwan, Republic of China; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2012 Mosby, Inc. All rights reserved. doi:10.1016/j.trsl.2011.09.003

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AT A GLANCE COMMENTARY Kuo W, et al. Background

Certain common genetic variants or polymorphisms may have an impact on lung cancer risk. Hypoxia-inducible factor-1a (HIF-1a) is a key regulator of cellular response to hypoxia and has been suggested to play an important role in tumorigenesis and metastasis. The aim of this study was to investigate the relationship between HIF-1a genetic polymorphism and non-small-cell lung cancer. Translational Significance

A case-control study design and polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) method were employed to analyze the allele frequencies and genotype distributions. The genotype frequencies of HIF1a-1772 C/T and -1790 G/A were significantly different between the non-small-cell lung cancer (NSCLC) patients and the controls. HIF-1a polymorphisms may have a significant impact on susceptibility to NSCLC.

Lung cancer is one of the most common malignancies worldwide and the leading cause of cancer deaths in industrial countries.1,2 With a poor prognosis, non–smallcell lung cancer (NSCLC) accounts for approximately 85% of all cases of lung cancer.3,4 Lung cancer development is a multistep process, driven by a series of genetics and environmental alterations that endows cells with a set of hallmark traits required for tumorigenesis.5-7 Oxygen deprivation leading to hypoxia is a common feature of solid tumors. Under these conditions, a signaling pathway involving a key oxygen-response regulator termed the hypoxiainducible factor (HIF) is switched on.8,9 In normoxic cells, HIF-1a protein is degraded by proline hydroxylation. On the contrary, when cells are under hypoxic conditions, HIF-1a will accumulate and heterodimerize with HIF-1b to form the transcription factor (HIF-1). Subsequently, HIF-1 may recognize and bind to the promoters of hypoxia-inducible genes such as vascular endothelial growth factor (VEGF), erythropoietin, and nitric oxide synthase 2 genes. It has been suggested that HIF-1a might play an important role in tumor progression and metastasis through activation of many target genes that are particularly involved in crucial aspects of cancer biology, including angiogenesis, cell survival, evasion of apoptosis, invasion,

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and glucose metabolism.10-14 The overexpression in various solid tumors and correlation with patient prognosis of HIF-1a have been reported.15-23 In addition, enhanced levels of HIF-1a protein have been detected in the cytoplasm and nuclei of 40% to 80% of human carcinoma cases, including NSCLC.24,25 Several investigations revealed the possible association between the HIF-1a polymorphisms and cancer risk.26-33 Although a total of 35 SNPs have been located within the HIF-1a gene,34 2 SNPs were located within exon 12 coding region (P582S and A588T). The C to T transition at nucleotide 1772 leads to an amino acid change of proline to serine at codon 582 (P582S), and the G to A nucleotide substitution at point 1790 gives rise to an alanine/threonine variation at codon 588 (A588T). Previous studies in cells or tissues have reported that both of these 2 variant polymorphisms results in enhanced transcriptional activities, protein stability and protein expression of HIF-1a, compared with that of wild type. These HIF-1a variants exhibit significantly increased numbers and density of microvessels, angiogenic factors in tumors.26,34-36 These data imply that the HIF-1a variant alleles may be associated with tumor susceptibility and clinical significance. Therefore, this study was designed to investigate the impact of the HIF-1a-1772 C/T and 1790 G/A genetic polymorphisms on the susceptibility to and prognosis of NSCLC. METHODS Study population. A total of 285 NSCLC patients, consisting of 193 men and 92 women with a median age of 65.5, who were admitted to China Medical University Hospital, Taichung, Taiwan between June 2006 and January 2010, were included in this study. Of them, 186 patients had adenocarcinomas (ADs) and 99 patients had squamous carcinomas (SQs). The histologic determination, including tumor types and stages, was performed according to the WHO classification (WHO, 1982) and the TNM system (Mountain, 1986), respectively. Meanwhile, 300 matched controls, consisting of 211 men and 89 women with a median age of 65.3, were selected randomly from a pool of healthy volunteers who visited the general health check-up center of China Medical University Hospital during the same period. All controls had no known medical illness or hereditary disorders and were taking no medications. For each subject, a detailed questionnaire, included information on the daily quantity of smoking and smoking history (years), was completed by a trained interviewer. This study was approved by the Research Ethics Committee of China Medical University, and informed consent was

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Table I. Clinical features of the study populations* Variables

NSCLC

Control

P value‡

Subjects Sex (male/female) Age, years Cigarette smoking (Brinkman index†) Tumor type ADs SQs TNM stage I II III IV Tumor size T1 T2 T3 T4 Lymph nodes N0 N1 N2 N3 Metastasis M0 M1

285 193/92 65.5 6 10.4 518.8 6 45.4

300 211/89 65.3 6 10.6 520.4 6 46.2

0.53 0.45 0.28

186 99 22 36 60 167 Fig 1. Polymorphism analysis of the HIF-1a gene by PCR followed by specific restriction enzyme digestion. The polymorphism of HIF1a-1772 C/T (P582S) and -1790 G/A (A588T) were analyzed by PCR followed by BslI restriction enzyme digestion. (A) The SNP of HIF-1a-1772: lane 1, variant homozygous alleles; lanes 2, 4, and 6, heterozygous alleles; lane 3 and 5, wild-type homozygous alleles. (B) The SNP of HIF-1a-1790: lane 1, variant homozygous alleles; lanes 2, 3, and 6, wild-type homozygous alleles; lanes 4 and 5, heterozygous alleles.

54 146 55 30 33 47 169 36 118 167

*Data are presented as number or mean 6 SEM. † Brinkman index 5 daily cigarette numbers multiplied by smoking years. (Brinkman index represents as dose-dependent effects of cigarette smoking.) ‡ P values were calculated using the Mann-Whitney U test.

obtained from each participant prior to the commencement. Basic characteristics, including age and sex, of all participants are summarized in Table I to show no significant differences in the previously mentioned features between patients and controls. Samples extraction.

collection

and

genomic

DNA

Venous blood from each subject was drawn into Vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) containing ethylenediaminetetraacetic acid (EDTA) and stored at 4 C. Genomic DNA was extracted by QIAamp DNA blood mini kits (Qiagen, Valencia, Calif) according to the manufacturer’s instructions. DNA was dissolved in TE buffer [10 mmol/L Tris (pH 7.8), 1 mmol/L EDTA] and then quantitated by a measurement of OD 260. These extracted DNA samples were stored at –20 C and used as templates for polymerase chain reaction (PCR). PCR-restriction fragment length polymorphism (RFLP). Genetic polymorphisms for 1772 C/T (P582S)

and 1790 G/A (A588T) of HIF-1a gene were determined by PCR-RFLP. Primers, 5’-TGTGGCCATTGTAAA AACTCA-3’ (forward) and 5’-CTTGCGGAACTG CCTTCTAA-3’ (reverse) were used for analyses of

1772 C/T of HIF-1a genotype to yield a product of 147 bps. The reverse primer is a mismatch primer (indicated by an underline letter) to create a BslI restriction site. For 1790 G/A polymorphism, PCR was performed with the same forward primer as above and another reverse primer (5’-TTTAATTCATCA GTGGTGGCA-3’ [255 bp]) in a 10-mL volume containing 100 ng DNA template, 1.0 mL 103 PCR buffer (Invitrogen, Carslbad, Calif), 0.25 U of Taq DNA polymerase (Invitrogen), 0.2 mmol/L dNTPs (Promega, Madison, Wisc), and 200 nM of each primer (MDBio Inc, Taipei, Taiwan). The PCR cycling conditions were 5 min at 94 C followed by 35 cycles of 1 min at 94 C, 1 min at 60 C, and 2 min at 72 C, with a final step at 72 C for 20 min to allow a complete extension of all PCR fragments. A 10-mL aliquot of PCR products was subjected to digestion at 55 C for 4 h in a 15-mL reaction containing 5 U of BslI (New England Biolabs, Beverly, Mass) and 1.5 mL 103 buffer. Digested products were separated on a 3% agarose gel and then stained with ethidium bromide. As a result, for1772 C/T polymorphism, the C allele yielded 128- and 19-bp products, whereas the T allele yielded a 147-bp product. For 1790 G/A polymorphism, G allele yielded 143- and 114-bp products, whereas the A allele yielded a 255-bp product (Fig 1). Statistical analysis. Differences in clinical data between the NSCLC patients and the control subjects were examined. All continuous data are expressed as

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Table II. The association between the HIF-1a-1722 C/T polymorphism and the clinicopathologic parameters of the studied subjects Genotypes Characteristics

CC (%)

CT (%)

TT (%)

Total

P value

Odds ratio (95% CI) P value†

Noncancer control Lung cancer Tumor type AD SQ Tumor stage I1II III1IV

216 (72) 153 (54)

73 (24) 94 (33)

11 (4) 38 (13)

300 285

,0.0001*

1.00 4.04 (2.02–8.08) 0.0001

98 (53) 55 (56)

63 (34) 31 (31)

25 (13) 13 (13)

186 99

0.0001* 0.0001*

4.08 (1.96–8.51) 0.0001 3.97 (1.72–9.18) 0.002

35 (60) 118 (52)

16 (28) 78 (34)

7 (12) 31 (14)

58 227

0.51‡

1.15 (0.48–2.77) 0.83

*P value, The frequencies of the genotypes between the cancer and non-cancer control groups were compared with chi-square analysis. † Odds ratios and p value were calculated using logistic regression to measure the association of the variant homozygous genotypes TT with lung cancer risk, with that of the CC/CT genotype being referred to as 1. ‡ P value, The frequencies of the genotypes between lung cancers with different tumor stages were compared with chi-square analysis.

Table III. The association between the HIF-1a-1790 G/A polymorphism and the clinicopathologic parameters of the studied subjects Genotypes Characteristics

GG (%)

GA (%)

AA (%)

Total

P value

Odds ratio (95% CI) P value†

Noncancer control Lung cancer Tumor type AD SQ Tumor stage I1II III1IV

215 (72) 150 (53)

74 (25) 94 (33)

11 (3) 41 (14)

300 285

,0.0001*

1.00 4.42 (2.22–8.78) ,0.0001

97 (52) 53 (54)

63 (34) 31 (31)

26 (14) 15 (15)

186 99

,0.0001* ,0.0001*

4.27 (2.06–8.87) ,0.0001 4.69 (2.08–10.6) ,0.0001

35 (60) 115 (51)

15 (26) 79 (35)

8 (14) 33 (14)

58 227

0.38‡

1.06 (0.46–2.44) 1.00

*P value, The frequencies of the genotypes between the cancer and non-cancer control groups were compared with chi-square analysis. † Odds ratios and p value were calculated using logistic regression to measure the association of the variant homozygous genotypes AA with lung cancer risk, with that of the GG/GA genotype being referred to as 1. ‡ P value, The frequencies of the genotypes between lung cancers with different tumor stages were compared with chi-square analysis.

mean 6 standard deviation and compared using a 2tailed Student’s t test. Categoric variables are reported as a percentage and compared using Chi-square (X2) or the Fisher exact test. A Hardy-Weinberg equilibrium was assessed using a goodness-of-fit X2 test for biallelic markers. The distribution of HIF-1a1772 C/T and 1790 G/A genetic polymorphisms between controls and lung cancer patients was examined by X2 test. Significance was accepted at P , 0.05. Odds ratios (ORs) and 95% confidence intervals (CIs) for lung cancer of each specific genotype were calculated with logistic regression to assess the degree of association observed quantitatively. The Kaplan-Meier method was used to estimate the probability of survival as a function of time and median survival. The log rank test was used to assess the significance of the difference between homozygous variant genotype and other genotypes of HIF-1a-1772 C/T

and 1790 G/A, among pairs of survival probabilities. Significance was accepted at P , 0.05. RESULTS

The association of the HIF-1a-1772 C/T (P582S) and the -1790 G/A (A588T) genetic polymorphisms with certain clinicopathologic parameters of lung cancer patients was analyzed and shown in Table II and III. Overall, genotype distributions of the HIF-1a-1772 C/ T (P582S) and the -1790 G/A (A588T) were significantly different between noncancer controls and lung cancer patients (P , 0.0001). The frequency of HIF-1a-1772 variant homozygote was low as being 0.13 and 0.04 in the cases and control, respectively, compared with that of wild-type allele (0.70 for cases and 0.84 for controls). The frequency of HIF-1a-1790 variant homozygote was also low as being 0.14 and

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observed in patients with homozygous HIF-1a-1790 G/A variant allele (A/A), compared with patients with at least 1 wild-type allele ([G/G] or [G/A]). Unexpectedly, patients carrying a variant homozygous HIF-1a1772 TT genotype or HIF-1a-1790 AA genotype did not had a tendency to NSCLC of higher severity. These differences were not statistically significant between NSCLC patients at early and advanced stages (P . 0.05) (Table II and III). All patients were followed up during the study period. Those patients carrying a variant polymorphic homozygote of HIF-1a-1772 C/ T (P582S) and the -1790 G/A (A588T) had a poorer prognoses compared with other patients (P , 0.0001 and P , 0.0001, by log rank test) (Fig 2). The frequency of HIF-1a-1722 C/T and 1790 G/A polymorphisms in various cancer patients and controls were also studied, as shown in Tables IV and V.28,30,32,35,37-49 Obviously, there is an ethnicityrelated variation in the frequency of both polymorphisms of HIF-1a. However, our results are consistent with result conducted in Malaysia, Ireland, the United States, and Japan,38,28,40,43 indicating that the percentage of cancer patients with a CT or TT genotype of HIF-1a-1722 C/T are significantly higher than controls. Individuals carrying the GG or GA genotype of HIF-1a-1790 G/A are significantly associated with an increased cancer risk, consistent with that of Spain study.42 In other study reports, the genotypes distribution of both polymorphisms (HIF-1a1722 C/T and 1790 G/A) were not different between the cancer patient groups and control groups.

Fig 2. The Kaplan-Meier survival curves with respect to the HIF-1a1772 C/T (A) and 1790 G/A (B) genetic polymorphisms in relation to non–small-cell lung cancer patients. The P value for each analysis is indicated. (Color version of figure is available online.)

DISCUSSION

0.03 in the cases and control, respectively, compared to that of wild-type allele (0.70 for cases and 0.84 for controls). The results of the X2 goodness-of-fit test showed that genotype frequencies of HIF-1a-1772 C/T (P582S) and the -1790 G/A (A588T) were consistent with Hardy-Weinberg equilibrium in the study population. Logistic regression analysis revealed that higher ORs for NSCLC (an OR of 4.04, 95% CI 2.02–8.08, P 5 0.0001), adenocarcinoma (an OR of 4.08, 95% CI 1.96-8.51; P 5 0.0001) and squamous cell carcinoma (an OR of 3.97, 95% CI 1.72–9.18; P 5 0.002), were observed in patients homozygous for HIF-1a-1772 variant allele (T/T), as compared with patients with at least 1 wild-type allele ((C/C) or (C/T)). Also, higher ORs for NSCLC (an OR of 4.42, 95% CI 2.22–8.78, P , 0.0001), adenocarcinoma (an OR of 4.27, 95% CI 2.06–8.87; P , 0.0001) and squamous cell carcinoma (an OR of 4.69, 95% CI 2.08–10.6; P , 0.0001), were

Hypoxia, which is a common phenomenon in human solid tumors, is associated with invasion and metastasis in various tumors. HIFs are key molecules in the hypoxic response and regulate the activation of specific genes to mediate many of the adaptations to hypoxia. The HIF-1a is a transcription factor that plays a crucial role in the cellular response to hypoxia. Overexpression of HIF-1a, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in cancer patients. More detailed studies have indicated that HIF-1a may involved in cell proliferation, invasion, angiogenesis, and metastases.50-53 Hypoxia-HIF-1a could regulate the migration and invasion of lung cancer cells under hypoxic conditions and promote the metastasis of lung cancer.54 The expression level of HIF-1a messenger RNA is associated with disease progression in NSCLC tissues, and is expected to act as a biomarker or therapeutic target.55 Also, HIF-1a and VEGF -C were over-expressed in NSCLC. They may be involved in the carcinogenesis,

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Table IV. The frequency of HIF-1a-1722 C/T polymorphism in various cancers Genotypes n (%) Type of cancer

Lung cancer

Population (n)

Taiwan Japan Turkey

Breast cancer

Korea Malaysia Turkey

Prostate cancer

Ireland USA

OSCC

Taiwan Spain

CRC

Japan Sweden Austria

Cervical carcinoma

Korea

HNSCC

Japan

TCC

Japan

ESCC

China

Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls

CC

CT

TT

Reference

153 (53.7) 216 (72.0) 74 (89.2) 98 (89.1) 110 (78.0) 111 (71.2) 81 (90.0) 93 (91.2) 294 (71.8) 222 (80.7) 79 (77.5) 68 (66.7) 65 (68.4) 175 (93.1) 161 (82.1) 179 (91.3) 163 (93.7) 334 (96.3) 57 (81.4) 113 (76.4) 100 (100.0) 89 (89.0) 167 (84.3) 213 (82.6) 291 (76.4) 1773 (82.2) 177 (88.9) 187 (87.4) 45 (81.8) 98 (89.1) 197 (90.0) 419 (90.9) 84 (88.4) 93 (89.4)

94 (33.0) 73 (24.3) 9 (10.8) 12 (10.9) 31 (22.0) 43 (27.5) 8 (8.9) 9 (8.8) 100 (24.3) 50 (18.2) 21 (20.6) 29 (28.4) 30 (31.6) 13 (6.9) 29 (14.8) 14 (7.2) 10 (5.7) 13 (3.7) 6 (8.6) 27 (18.2) 0 (0) 11 (11.0) 28 (14.1) 43 (16.6) 77 (20.2)* 383 (17.8)* 22 (11.1) 27 (12.6) 10 (18.2) 12 (10.9) 22 (10.0)* 42 (9.1)* 11 (11.6) 11 (10.6)

38 (13.3) 11 (3.7) 0 (0) 0 (0) 0 (0) 2 (1.3) 1 (1.1) 0 (0) 16 (3.9) 3 (1.1) 2 (1.9) 5 (4.9) 0 (0) 0 (0) 6 (3.1) 3 (1.5) 1 (0.6) 0 (0) 7 (10.0) 8 (5.4) 0 (0) 0 (0) 3 (1.6) 2 (0.8)

This article 37 32 30 38 39 28 40 41 42 43 44 45

0 (0) 0 (0) 0 (0) 0 (0)

46 35 47 48

Abbreviations: CRC, colorectal cancer; ESCC, esophageal squamous cell carcinoma; HNSCC, head and neck squamous cell carcinoma; OSCC, oral squamous cell carcinoma; TCC, transitional cell carcinoma of the bladder. *Frequency of genotypes: CT1TT.

as well as in invasion and metastasis of NSCLC. HIF-1a and VEGF-C work synergistically in the process of NSCLC.56 There are two important single-nucleotide polymorphisms (SNPs) of human HIF-1 gene, HIF-1a-1772 C/ T (P582S) and 1790 G/A (A588T), which result in an amino acid substitution of proline to serine and alanine to threonine, respectively. Under normoxic condition, hydroxylation of the Pro402 and Pro564 occurs within the oxygen-dependent degradation (ODD) domain of the HIF-1a, and HIF-1a will be rapidly degraded subsequently. Although P582 and A588 were not the site for hydroxylation, both polymorphsims are located within the ODD/pVHL binding domain in exon 12 of the HIF-1a gene. The polymorphic S582 and T588 variants, with amino acid transition, may enhance the tran-

scriptional activity of HIF-1a gene because of structural changes. Such amino acid changes also increase the stability of HIF-1a protein or its ability to bind the accessory proteins involved the expression of downstream target genes.35,36,57 The studies demonstrated that patients with the variant allele were associated with more advanced cancer development and progression.44,47 Koukourakis et al33 had reported the correlation between the HIF-1a polymorphisms and the patterns of HIF-1a protein expression in 36 patients with NSCLC. In this study, we provided novel information about the effects of genetic polymorphisms of HIF-1 on the susceptibility and clinicopathologic development of NSCLC. The HIF-1a-1772 C/T and -1790 G/A gene variants were evaluated to reveal that these 2 genetic

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Table V. The frequency of HIF-1a-1790 G/A polymorphism in various cancers Genotypes n (%) Type of cancer

Lung cancer

Population (n)

Taiwan Japan

Turkey Breast cancer

Korea Malaysia Turkey

Prostate cancer

USA

OSCC

Taiwan Spain

CRC

Sweden Austria

Cervical carcinoma

Korea Japan

TCC

Japan

Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls Cases Controls

GG

GA

AA

Reference

150 (52.6) 215 (71.7) 72 (86.7) 101 (91.8) 140 (99.3) 154 (98.7) 87 (96.7) 94 (92.1) 332 (80.9) 232 (84.4) 102 (100.0) 98 (96.0) 1,053 (98.8) 1,247 (98.7) 153 (87.9) 333 (96.0) 40 (62.5) 130 (93.5) 189 (95.5) 247 (96.5) 356 (93.4) 2080 (96.5) 187 (94.0) 200 (93.5) 51 (92.7) 101 (91.8) 204 (93.2) 421 (91.3)

94 (33.0) 74 (24.7) 9 (10.9) 9 (8.2) 1 (0.7) 2 (1.3) 3 (3.3) 7 (6.9) 72 (17.6) 41 (14.9) 0 (0) 4 (4.0) 13 (1.2) 17 (1.3) 20 (11.5) 14 (4.0) 21 (32.8) 9 (6.5) 9 (4.5) 9 (3.5) 11 (2.9)* 76 (3.5)* 12 (6.0) 13 (6.0) 4 (7.3) 9 (8.2) 15 (6.8)* 40 (8.7)*

41 (14.4) 11 (3.6) 2 (2.4) 0 (0) 0 (0) 0 (0) 0 (0) 1 (1.0) 6 (1.5) 2 (0.7) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0.6) 3 (4.7) 0 (0) 0 (0) 0 (0)

This article 37 32 30 38 39 49 41 42 44 45

0 (0) 1 (0.5) 0 (0) 0 (0)

46 35 47

Abbreviations: CRC, colorectal cancer; HNSCC, head and neck squamous cell carcinoma; OSCC, oral squamous cell carcinoma; TCC, transitional cell carcinoma of the bladder. *Frequency of genotypes: GA1 AA.

polymorphisms were associated with the susceptibility to NSCLC. Significant difference were found in allele and genotype frequency distribution of HIF-1a-1772 C/T and -1790 G/A between NSCLC patients and controls. Individuals with HIF-1a-1772 T/T homozygotes had a higher risk of 4.04-fold to have NSCLC compared with individuals with C/C homozygotes or C/T heterozygotes. And, individuals with HIF-1a-1790 A/A homozygotes had a higher risk of 4.42-fold to have NSCLC compared with individuals with G/G homozygotes or G/A heterozygotes. However, those carried HIF-1a-1772 T/T homozygotes or -1790 A/A homozygotes did not have a tendency to advanced disease as expected. To our knowledge, only 3 studies investigated the relationship between HIF-1a genetic polymorphism and lung cancer patients, including 1 study that was conducted with patients of both small cell cancer and non– small-cell lung cancer.32,33,37 Their results suggest that the HIF-1a polymorphisms did not relate to lung cancer risk in the Japanese and Turkish population. Because there is an ethnicity-related variation in the frequency

of -1772 C/T (P582S) and -1790 G/A (A588T) polymorphisms of the HIF-1a gene that may be relevant in the context of tumor aggressiveness and progression.58 In this study, a study group was limited to NSCLC and the result demonstrates that the polymorphisms of HIF-1a-1772 C/T and -1790 G/A genes had significant associations with the susceptibility to NSCLC. In addition, those patients carrying a HIF-1a-1772 T/T genotype or a HIF-1a-1790 A/A genotype had a tendency toward poorer prognoses compared with other patients. However, the interpretation of our results was limited by sample size. Another study of larger scale on these genotypes and other polymorphisms is required to analyze haplotypes and their association with both the onset of lung cancer and the development of metastases. SPECULATIONS

In conclusion, by evaluating the polymorphic sites of HIF-1a-1772 C/T and -1790 G/A, a significant association of the polymorphisms with NSCLC was revealed.

Translational Research Volume 159, Number 1

The results of this study support a relation between HIF1a-1772 C/T and -1790 G/A genetic polymorphisms and the susceptibility to and prognosis of NSCLC. REFERENCES

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