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An investigation of relationships between hypoxia-inducible factor-1α gene polymorphisms and ovarian, cervical and endometrial cancers
Cancer Detection and Prevention 31 (2007) 102–109 www.elsevier.com/locate/cdp
Short communication
An investigation of relationships between hypoxia-inducible factor-1a gene polymorphisms and ovarian, cervical and endometrial cancers Ece Konac PhDa, H. Ilke Onen MSca, Jale Metindir MDb, Ebru Alp MSca, Aydan Asyali Biri MDc, Abdullah Ekmekci PhDa,* b
a Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey Department of Obstetrics and Gynecology, Ankara Oncology Education and Research Hospital, Demetevler, Ankara, Turkey c Department of Obstetrics and Gynecology, Faculty of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey
Accepted 2 January 2007
Abstract Background: DNA sequence variations in HIF-1a gene might yield changes both in the production outcomes and in the activities of the gene. Overexpression of the HIF-1a subunit, resulting from intratumoral hypoxia and genetic alterations, has been demonstrated in common human cancers and is correlated with tumor angiogenesis and patient mortality. In this study, we aimed to determine how the three single nucleotide polymorphisms (SNPs, C1772T and G1790A exon 12, C111A exon 2) in the HIF-1a gene coding regions affect the ovarian, cervical and endometrial cancer patients in the Turkish population. A study on this relationship has not been conducted to date. Method: 102 gynecologic cancer patients and 107 healthy controls were studied. Genotypes of the three polymorphisms were analyzed by PCR-RFLP. Results: There was no significant difference between ovarian cancer patients and controls in terms of the distribution of C1772T genotypes and alleles (P > 0.05). However, there was a highly significant increase in the frequency of both CT 1772 and TT 1772 genotypes in patients with cervical and endometrial cancers compared with healthy controls. In fact, 1772T allele-carriers (CT + TT genotypes) showed an association with the risk of cervical and endometrial cancers compared to the wild type (OR = 3.84, 95% CI: 1.65–8.93; OR = 7.41, 95% CI: 2.33–23.59, respectively). C1772T polymorphism was not associated with family history concerning gynecologic and/or other cancer types, stages (I–IV) and grades of tumor, smoking habits and existence of other diseases that generate a hypoxic microenvironment even after multivariable logistic regression analysis. As for HIF-1a G1790A genotypes, the frequencies of G alleles were 98% in ovarian patients and 100% in the control group. We found no significant difference in the genotype distribution and allele frequencies between the ovarian patients and healthy control subjects. There were no GA and AA genotypes among the cervical and endometrial cancer patients. As for HIF-1a C111A polymorphism, we did not find CA and AA variants of the gene in controls or in any of the three types of patients. Conclusion: Our results suggest that the C1772T polymorphism of the HIF-1a may be associated with cervical and endometrial cancers. # 2007 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. Keywords: HIF-1a; Gynecologic cancers; Polymorphism; Risk factors; Turkish population; Family history; Genomic DNA; Hardy-Weinberg equilibrium
1. Introduction Tumor vascularization supplies nutrition and oxygen to proliferating cells, cellular adaptation to hypoxia, and strongly correlates with the risk of invasion and metastasis [1]. An important mediator of such cellular adaptation is * Corresponding author. Tel.: +90 312 202 69 94; fax: +90 312 212 46 47. E-mail address: [email protected] (A. Ekmekci).
hypoxia-inducible factor-1 (HIF-1), a critical transcription factor [2]. HIF-1 consists of a constitutively expressed HIF1ß subunit and one of three subunits (HIF-1a–HIF-3a). HIF1a forms a heterodimeric complex with HIF-1ß on hypoxic responsive elements and activates transcription of a wide variety of genes which are particularly relevant to cancer [3–6]. Tumors derived from cells lacking HIF-1a or HIF-1ß show significantly reduced vascularization and growth rates compared to parental cells [7,8]. In addition, enhanced
0361-090X/$30.00 # 2007 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cdp.2007.01.001
E. Konac et al. / Cancer Detection and Prevention 31 (2007) 102–109
expression levels of HIF-1a have been reported in human malignancies including colon, breast, stomach, pancreas, prostate, kidney, and esophagus [9–11]. The determination of SNPs is a new means to study the etiology of polygenic disorders with complex inheritance patterns, such as cancer [12]. All regions of the HIF-1a were examined and a total of 35 SNPs in the gene were found [13]. Polymorphisms of the gene have not been associated with some diseases [14], however, some of them were associated with production of HIF protein and reported to be involved in susceptibility to several disorders [14–18]. Angiogenesis of gynecologic cancer is regulated by vascular endothelial growth factor (VEGF), a target gene of HIF-1a [4]. In studies of ovarian cancer, VEGF expression and microvessel density (MVD) have been correlated with poor survival [19]. Birner et al. found that the combination of HIF-1a protein overexpression with nonfunctional p53 indicates a dismal prognosis of ovarian cancer [20]. It has been reported that HIF-1a expression was an independent prognostic marker in early stage cervical carcinoma [21]. Another study suggests that the increase of proliferating cell nuclear antigen (PCNA) index and MVD may enhance development of endometrial carcinoma [22]. Indeed, the role of HIF-1a in the development of ovarian, cervical, and endometrial cancers and their transition to metastases remain to be elucidated. On the basis of these facts, HIF-1a polymorphisms might contribute to the development and progression of ovarian, cervical, and endometrial cancers. Nevertheless, there have been no published studies on any population so far, as regards to their relationship. Therefore, this study was designed to examine the role of three SNPs, which were located in coding regions of the gene- one in exon 2 (S28Y), and two in exon 12 (P582S, A588T) in the development and progress of ovarian, cervical and endometrial cancers in the Turkish population.
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2. Materials and methods We studied a total of 102 gynecologic cancer patients who were selected from patients admitted to the Departments of Obstetrics and Gynecology, Faculty of Medicine, Gazi University and the Ankara Oncology Education and Research Hospital. A total of 107 subjects were enrolled as healthy controls (mean ages of menarche: 13 0.25 and mean age: 48 0.2 years, range 25–65). The study was approved by the Committee of Ethics of the Gazi University. All cases and controls were of Caucasian origin and became subjects of this study after submitting their written consents. Age of menarche and first diagnosis, history of gynecologic and/or other cancer types in the family, stages (I–IV) and grades of tumor (based on the International Federation of Gynecology and Obstetrics (FIGO) stage method), information on smoking habits and existence of other diseases such as stroke and cardiovascular diseases that generate a hypoxic microenvironment were obtained from the patient files. Gynecologic or other cancers, smoking habits and existence of other diseases were not encountered in the family history of the control individuals. Clinicopathological characteristics of the patients are summarized in Table 1. Genomic DNA was isolated from peripheral blood by using a DNA extraction kit (Heliosis1, Metis Biotechnology, Turkey) according to the manufacturer’s instructions. Amplifications of the C1772T and G1790A regions of the HIF-1a gene were carried out by placing in a Mastercycler gradient (Eppendorf, Germany) thermal cycler, a total volume of 50 mL PCR mixture containing 50 ng genomic DNA, 2.5 mM MgCl2, 100 mM dNTP, 50 pmol/ml of each primer and 1.0 U/mL Taq DNA polymerase. For the C1772T, the following pair of primers produced a PCR product of 346 bp: forward 50 -AAG GTG TGG CCA TTG TAA AAA CTC-30 , reverse 50 -GCA CTA GTA GTT TCT TTA TGT ATG-30 (Genbank accession no. AH006957; nucleotides 425–770). We set the PCR cycling conditions for the gene as explained by Ollerenshaw [16]. The
Table 1 Clinicopathological characteristics of the patients Cancer
Number of cases Mean ages of menarche (years) Mean ages of first diagnosis (years) Range (years) History of gynecologic and/or other cancer types in the family Stages of tumora I II III IV Histological grade (G1/G2 and G3) Smoking habits Existence of other diseases a
Ovarian (%)
Cervical (%)
Endometrial (%)
49 (48) 13 0.43 48 0.71 16–74 9 (18.37)
32 (31) 13 0.18 49 0.46 25–68 7 (21.8)
21 (21) 13 0.14 50 0.52 39–81 9 (42.8)
2 (4.08) 5 (10.20) 41 (83.67) 1 (2.05) 2/47 8 (16.33) 11 (22.45)
5 (15.6) 16 (50.0) 10 (31.3) 1 (3.1) 5/27 4 (12.5) 5 (15.6)
10 (47.6) 5 (23.8) 5 (23.8) 1 (4.8) 10/11 1 (4.76) 5 (23.8)
Stages of tumor (staging was performed according to the current classification of the International Federation of Gynecology and Obstetrics (FIGO)).
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Fig. 1. Electropherograms of DNA sequence (forward) data for the S28Y (C111A) polymorphism.
Fig. 2. Electropherograms of DNA sequence (reverse) data for the S28Y (C111A) polymorphism.
E. Konac et al. / Cancer Detection and Prevention 31 (2007) 102–109
experiment was conducted twice for each gene region (C1772T, G1790A and C111A) by two different researchers. For the C1772T region, the experiment was carried out by digesting the PCR products with restriction endonuclease, HphI (Fermantas, USA) and loading them onto 3% agarose gel (containing 0.5% ethidium bromide). For the G1790A, the polymorphism was analyzed by digesting the same PCR products with restriction endonuclease, AciI (Fermantas, USA) and loading them onto 3% agarose gel. The products were separated by electrophoresis and visualized by Gel Logic 100 gel image system (Kodak, USA). The 1772C allele was cut into two fragments of 228 and 118 bp while the 1772T allele remained uncut (346 bp). The G allele produces a single cut site to give two bands, 201 and 145 bp. The substitution from a G to an A abolishes an AciI cut site (346 bp). For the C111A, the following pair of primers were designed to produce a PCR product of 187 bp: forward 50 GGA TAA GTT CTG AAC GTC GA -30 , reverse 50 -ATC CAG AAG TTT CCT CAC AC -30 (Gene bank accession no. AH006957; nucleotides 177–363). Reaction products were sequenced by the Ohio State University (see http:// pmgf.biosci.ohio-state.edu) to confirm our primer designs (Figs. 1 and 2). Amplifications of the C111A regions of the HIF-1a gene were carried out by placing in a Mastercycler gradient (Eppendorf, Germany) thermal cycler, a total volume of 50 mL PCR mixture containing 50 ng genomic DNA, 2.5 mM MgCl2, 100 mM dNTP, 3% dimethylsulfoxide (DMSO), 50 pmol/mL of each primer and 1.0 U/mL Taq DNA polymerase. The conditions for the PCR reaction were denaturation at 95 8C for 5 min, followed by 30 cycles of denaturation at 95 8C for 30 s, annealing at 60 8C for 30 s and extension at 72 8C for 30 s, followed by a final extension at 72 8C for 5 min. The polymorphism was analyzed by digesting the PCR products with restriction endonuclease, BglII (Fermantas, USA) and loading them onto 3% agarose gel. The products were separated by electrophoresis and visualized by Gel Logic 100 gel image system (Kodak, USA). The 111C allele was cut into two fragments of 143 and 44 bp while the 111A allele remained uncut (187 bp) (Fig. 3). Tests for the Hardy-Weinberg equilibrium were performed by the chi-square (x2) test. The differences in genotype distribution and allele frequency among the groups were examined for statistical significance using the Pearson’s two-way chi-square (x2)-test. When the assumption of the x2 was violated, Fisher’s exact test was performed. The relationships between genotypes and/or alleles and gynecologic cancer risks were determined by obtaining the Odds ratios (ORs) through a logistic regression method [OR, 95% confidence interval (CI)]. Adjusted ORs for the clinicopathological covariant factors were determined by using a multivariable logistic regression method. Two-tailed P-values smaller than 0.05 were considered statistically significant. All analysis was conducted by using SPSS V.11.5.
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Fig. 3. PCR-based restriction analysis of HIF-1a gene C111A polymorphism shown on 3% agarose electrophoresis. Lane 1–7: 111C allele (wildtype) yielded two bands (44 basepair (bp) and 143 bp). The left lane (marker) contains a 100 bp ladder and lane 8 contains a 187 bp PCR product (uncut) of the gene.
3. Results In control and patient groups, there were no significant deviations from the Hardy–Weinberg equilibrium (P > 0.05). Out of the 107 healthy control cases, 68 cases were type CC, 37 cases were type CT and 2 cases were type TT. No significant differences in genotype (P = 0.759) and allele (P = 0.637) frequencies for C1772T polymorphism were observed between ovarian cancer patients and controls. However, we found a significant difference in the genotype distribution and allele frequencies between the cervical and endometrial cancer patients and control subjects (P < 0.001). ORs were calculated in comparison with the most common homozygote genotype observed in controls. A statistically significant difference was found for genotypic frequencies of the C1772T polymorphism between cervical and endometrial cancer patients with CT, TT, CT + TT genotypes and those with CC genotype (P < 0.05). Carriers of a 1772T allele (CT + TT genotypes) were more frequent among cervical (68.8%) and endometrial (81%) cancer patients than among controls (36.5%) (Table 2). The occurrence of all three types of gynecologic cancer were not associated with the other epidemiological covariant factors (P > 0.05) (Table 3). As for G1790A genotypes, out of the 49 ovarian patients, 47 patients had the GG genotype, two patients had the GA genotype, none of the patients had AA genotype (Table 4). We found no significant difference in the genotype distribution and allele frequencies between the ovarian patients and controls (P = 0.497 and 0.238, respectively). There were no GA and AA genotypes among the cervical and endometrial cancer patients (Table 4). All 107 control cases were type GG. It, therefore, became meaningless to run a multivariable logistic regression analysis to estimate adjusted ORs with the epidemiological covariant factors. As for C111A polymorphism, the genotype distributions in ovarian, cervical and endometrial cancer patients are
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Table 2 HIF-1a (C1772T) genotype and allele frequencies in ovarian, cervical and endometrial cancer patients and control subjects Cancer types
n (%) Patients
n (%) Controls
Ovarian Genotypes CC CT TT CT + TT Allele Frequency C T Cervical Genotypes CC CT TT CT + TT Allele Frequency C T Endometrial Genotypes CC CT TT CT + TT Allele Frequency C T
n = 49
n = 107
34 (69.4) 14 (28.6) 1 (2.0) 15 (30.6)
68 (63.5) 37 (34.6) 2 (1.9) 39 (36.5)
82 (83.7) 16 (16.3) n = 32
173 (80.1) 41 (19.9) n = 107
10 (31.2) 14 (43.8) 8 (25) 22 (68.8)
68 (63.5) 37 (34.6) 2 (1.9) 39 (36.5)
34 (53.1) 30 (46.9) n = 21
173 (80.1) 41 (19.9) n = 107
4 (19) 12 (57.2) 5 (23.8) 17 (81)
68 (63.5) 37 (34.6) 2 (1.9) 39 (36.5)
20 (47.6) 22 (52.4)
173 (80.1) 41 (19.9)
P-value a
ORb (95% CI)
P-value b
0.759 1.00 0.76 1.00 0.77
(ref) (0.36–1.59) (0.09–11.42) (0.37–1.59)
0.460 1.000 0.477
0.637 1.00 (ref) 0.82 (0.44–1.55)
0.548
<0.001 1.00 (ref) 2.57 (1.04–6.36) 27.20 (5.04–146.78) 3.84 (1.65–8.93)
0.037 <0.001 0.001
1.00 (ref) 3.72 (2.05–6.77)
<0.001
1.00 (ref) 5.51 (1.66–18.31) 42.50 (6.20–291.36) 7.41 (2.33–23.59)
0.003 <0.001 <0.001
1.00 (ref) 4.64 (2.32–9.30)
<0.001
<0.001
<0.001
<0.001
OR, Odds ratio; CI, confidence interval. P values <0.05 and 0.001 are shown in bold. Values in parentheses are percentages. a x2 analysis. b Calculations were performed CC vs. CT, TT, CT + TT.
Table 3 Odds ratios (ORs) of HIF-1a (C1772T) polymorphism in ovarian, cervical and endometrial cancer patients with adjustments Cancer types Ovarian Genotypes CC CT TT CT + TT Cervical Genotypes CC CT TT CT + TT Endometrial Genotypes CC CT TT CT + TT
a,z
OR
(95% CI)
1.00 1.05 1.75 0.96
a,z
b,z
P-value
OR
(95% CI)
(ref) (0.23–4.83) (0.14–21.88) (0.21–4.38)
1.000 0.556 1.000
1.00 1.03 0.40 1.12
1.00 3.60 3.00 3.38
(ref) (0.34–38.48) (0.22–40.93) (0.35–32.64)
0.358 0.559 0.387
1.00 1.00 0.25 0.70
(ref) (0.10–9.61) (0.01–4.73) (0.08–6.22)
1.000 0.524 1.000
b,z
c,z
ORd,z (95% CI)
P-valued,z
(ref) (0.03–2.67) (0.14–21.88) (0.03–2.47)
0.407 0.556 0.406
1.00 0.89 1.50 0.81
(ref) (0.20–3.98) (0.12–18.57) (0.18–3.62)
1.000 1.000 1.000
1.00 0.13 0.33 0.11
(ref) (0.01–1.40) (0.03–4.04) (0.01–1.25)
0.133 0.588 0.079
1.00 0.67 0.57 0.63
(ref) (0.08–5.75) (0.04–7.74) (0.09–4.53)
1.000 1.000 0.637
1.00 0.15 0.33 0.11
(ref) (0.01–2.18) (0.02–5.03) (0.01–1.55)
0.202 0.559 0.133
1.00 2.14 0.33 1.25
(ref) (0.17–27.10) (0.02–5.03) (0.10–15.11)
1.000 0.559 1.000
OR
(ref) (0.18–6.09) (0.03–5.15) (0.19–6.55)
1.000 0.457 1.000
1.00 0.30 1.75 0.28
1.00 0.27 0.09 0.20
(ref) (0.05–1.48) (0.01–1.10) (0.04–0.98)
0.211 0.666 0.056
1.00 1.50 0.75 1.25
(ref) (0.12–19.44) (0.03–17.51) (0.10–15.11)
1.000 1.000 1.000
OR, odds ratio; CI, confidence interval. z Calculations were performed CC vs. CT, TT, CT + TT. a OR adjusted for history of gynecologic and/or other cancer types in the family. b OR adjusted for stages and grades of tumor. c OR adjusted for smoking habits. d OR adjusted for existence of other diseases.
a,b,c,d
P-value
P-value
(95% CI)
c,z
E. Konac et al. / Cancer Detection and Prevention 31 (2007) 102–109
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Table 4 HIF-1a (G1790A) genotype and allele frequencies in ovarian, cervical and endometrial cancer patients and control subjects Cancer types
n (%) Patients
n (%) Controls
Ovarian Genotypes GG GA AA Allele frequency G A Cervical Genotypes GG GA AA Allele frequency G A Endometrial Genotypes GG GA AA Allele frequency G A
n = 49
n = 107
47 (96) 2 (4) 0 (0)
107 (100) 0 (0) 0 (0)
96 (98) 2 (2) n = 32
214 (100) 0 (0) n = 107
32 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
64 (100) 0 (0) n = 21
214 (100) 0 (0) n = 107
21 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
42 (100) 0 (0)
214 (100) 0 (0)
P-valuea
ORb (95% CI)
P-valueb
1.00 (ref) 1.02 (0.99–1.05) Not calculated
0.240 –
1.00 (ref) 0.49 (0.44–0.54)
0.240
1.00 (ref) Not calculated Not calculated
– –
1.00 (ref) Not calculated
–
1.00 (ref) Not calculated Not calculated
– –
1.00 (ref) Not calculated
–
0.497
0.238
1.000
1.000
1.000
1.000
OR, odds ratio; CI, confidence interval. Values in parentheses are percentages. a x2 analysis. b Calculations were performed GG vs. GA.
Table 5 HIF-1a (C111A) genotype and allele frequencies in ovarian, cervical and endometrial cancer patients and control subjects Cancer types
n (%) Patients
n (%) Controls
Ovarian Genotypes CC CA AA Allele frequency C A Cervical Genotypes CC CA AA Allele frequency C A Endometrial Genotypes CC CA AA Allele frequency C A
n = 49
n = 107
49 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
98 (100) 0 (0) n = 32
214 (100) 0 (0) n = 107
32 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
64 (100) 0 (0) n = 21
214 (100) 0 (0) n = 107
21 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
42 (100) 0 (0)
214 (100) 0 (0)
Values in parentheses are percentages.
presented in Table 5. We did not find CA and AA variants of the gene in controls or in any of the three types of patients. Therefore, no comparison was made between the CC genotype patients and CA and AA variants and the adjusted ORs with epidemiological covariant factors were not estimated.
4. Discussion HIF-1a in the regulation of tumor angiogenesis is the target of novel anti-cancer drugs [23]. Therefore, we investigated the polymorphic effects of the changes in the C1772T, G1790A and C111A of the HIF-1a gene on the ovarian, cervical and endometrial cancer patients. C1772T and G1790A polymorphisms have been reported in relation to patients with head and neck squamous cell carcinoma (HNSCC) [15] and renal cell carcinoma (RCC) [16,24]. A highly significant increase in the frequency of both the CC 1772 and GA 1790 genotypes was found in patients with RCC compared with healthy controls [16]. Tanimoto et al. [15], found CC and CT genotypes of C1772T polymorphism, but none of the subjects had the homozygous genotype TT. Furthermore, they showed the elevated transactivation capacity of variant forms of HIF-1a C1772T and G1790A polymorphisms [15]. On the other hand, it was reported that inactivation of pVHL for E3 ubiquitin ligase activity, but not genetic polymorphism of
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HIF-1a, may be one of the mechanisms underlying HIF-1a activations in colorectal carcinomas [25]. In another study, the T allele of the C1772T polymorphism was not detected in patients with colorectal carcinoma and controls [26]. In contrast, our study pointed to a statistically significant increase in the percentages of carriers of T allele (CT, TT and CT + TT genotypes) in cervical and endometrial cancer patients, which indicates that carrying T allele is a risk factor. C allele, on the other hand, is not a risk factor in the development of cervical and endometrial cancers. However, due to the small sample size we cannot suggest a tumor suppressor function for the C allele. The molecular basis for T allele’s high frequency is unclear, but the corresponding protein has been reported to have increased transcriptional activity and stability under normoxic conditions [27]. There was a significant relationship between HIF-1a and VEGF expression in tumors located in the lower lip and in larynx [28], in contrast, not in those located in the oral cavity [28], in HNSCC and in ovarian carcinoma [29–31]. We have earlier demonstrated that VEGF -634 G > C polymorphism is not associated with ovarian, cervical, and endometrial cancers [32]. The present study found no significant difference between ovarian cancer patients and controls in terms of the distribution of C1772T genotypes and alleles (P > 0.05). Since the association of the polymorphism with the three types of gynecologic cancers has not been investigated in any known studies, we were unable to compare our results with those of similar studies covering other populations. In general, the distribution of C1772T genotypes in control groups was approximately similar to those reported in previous studies [15,24–26,33] with regard to most common genotypes in population. We found no evidence of a relationship between clinicopathological characteristics and C1772T polymorphism in ovarian, cervical, and endometrial cancer patients. On the other hand, evaluation of angiogenesis intensity in endometrial cancer also seems to be an independent prognostic factor and statistically correlates with FIGO stage of disease, histological type and grade of tumor, depth of myometrial invasion and metastasis [34]. As for the 1790 gene region of HIF-1a, we found that there were no GA and AA genotypes among the cervical and endometrial cancer patients and only two GA genotypes among the ovarian cancer patients. As a result, the difference in the genotype distribution between ovarian patients and control subjects was not significant (P > 0.05). Allelic and genotypic frequencies of G1790A polymorphism were in close agreement with those previously published in Tanimoto et al. [15]. C111A polymorphism found in bHLH 17–30 aa (protein no: Q16665) has not been shown to be hydroxylated. However, bHLH and PAS domains are required for heterodimerization with HIF-1ß and binding to DNA [35]. Therefore, we hypothesized that if the HIF-1a gene plays any part in the disease process, then there should be differences between all three types of gynecologic cancer
patients and control subjects. However, in our study, A allele was not detected in patients and controls, that is, all samples were found to be CC genotype. It is not clear why we did not detect the 111A allele. Nevertheless, the distribution of C111A polymorphism was consistent with the report of Yamada et al. [13]. In conclusion, this is the first study to report that C1772T polymorphism of HIF-1a gene is associated with the occurrence of cervical and endometrial cancers while G1790A and C111A gene polymorphisms are not associated with any of the three types of gynecologic cancer. Although a limited number of patients were enrolled in the study, which forced us to cope with small sample sizes, our results are expected to contribute to the efforts to better understand the phenotypic effects of SNPs, which are based on direct genetic effects, gene–gene and gene-environment interactions, and to have important implications for understanding the pathogenesis of these tumors and for screening, if confirmed in follow-up studies.
Conflict of Interest None.
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E. Konac et al. / Cancer Detection and Prevention 31 (2007) 102–109 [13] Yamada N, Horikawa Y, Oda N, Iizuka K, Shihara N, Kishi S, et al. Genetic variation in the hypoxia-inducible factor-1 alpha gene is associated with type 2 diabetes in Japanese. J Clin Endocrinol Metab 2005; 90(10):5841–7. [14] Percy MJ, McMullin MF, Lappin TR. Sequence analysis of the 30 hypoxia-responsive element of the human erythropoietin gene in patients with erythrocytosis. Biochem Mol Med 1997; 62(1):132–4. [15] Tanimoto K, Yoshiga K, Eguchi H, Kaneyasu M, Ukon K, Kumazaki T, et al. Hypoxia-inducible factor-1 alpha polymorphisms associated with enhanced transactivation capacity, implying clinical significance. Carcinogenesis 2003; 24(11):1779–83. [16] Ollerenshaw M, Page T, Hammonds J, Demaine A. Polymorphisms in the hypoxia inducible factor-1 alpha gene (HIF1A) are associated with the renal cell carcinoma phenotype. Cancer Genet Cytogenet 2004; 153(2):122–6. [17] Chau CH, Permenter MG, Steinberg SM, Retter AS, Dahut WL, Price DK, et al. Polymorphism in the hypoxia-inducible factor 1alpha gene may confer susceptibility to androgen-independent prostate cancer. Cancer Biol Ther 2005; 4(11):1222–5. [18] Hebert C, Norris K, Parashar P, Ord RA, Nikitakis NG, Sauk JJ. Hypoxia-inducible factor-1 alpha polymorphisms and TSC1/2 mutations are complementary in head and neck cancers. Mol Cancer 2006; 16(5):3. [19] Raspollini MR, Amunni G, Villanucci A, Baroni G, Boddi V, Taddei GL. Prognostic significance of microvessel density and vascular endothelial growth factor expression in advanced ovarian serous carcinoma. Int J Gynecol Cancer 2004; 14(5):815–23. [20] Birner P, Schindl M, Obermair A, Breitenecker G, Oberhuber G. Expression of hypoxia-inducible factor 1 alpha in epithelial ovarian tumors: its impact on prognosis and on response to chemotherapy. Clin Cancer Res 2001; 7(6):1661–8. [21] Birner P, Schindl M, Obermair A, Plank C, Breitenecker G, Oberhuber G. Overexpression of hypoxia-inducible factor 1 alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res 2000; 60(17):4693–6. [22] Xiao L, Tang LD. Significance of proliferating cell nuclear antigen (PCNA) index and microvessel density (MVD) in endometrial carcinoma. Ai Zheng 2005; 24(1):19–22. [23] Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350(23): 2335–42.
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[24] Clifford SC, Astuti D, Hooper L, Maxwell PH, Ratcliffe PJ, Maher ER. The pVHL-associated SCF ubiquitin ligase complex: molecular genetic analysis of elongin B and C, Rbx1 and HIF-1alpha in renal cell carcinoma. Oncogene 2001; 20(36):5067–74. [25] Kuwai T, Kitadai Y, Tanaka S, Hiyama T, Tanimoto K, Chayama K. Mutation of the von Hippel-Lindau (VHL) gene in human colorectal carcinoma: association with cytoplasmic accumulation of hypoxiainducible factor (HIF)-1a. Cancer Sci 2004; 95:149–53. [26] Kuwai T, Kitadai Y, Tanaka S, Kuroda T, Ochiumi T, Matsumura S, et al. Single nucleotide polymorphism in the hypoxia-inducible factor-1alpha gene in colorectal carcinoma. Oncol Rep 2004; 12(5):1033–7. [27] Fu XS, Choi E, Bubley GJ, Balk SP. Identification of hypoxiainducible factor-1alpha (HIF-1alpha) polymorphism as a mutation in prostate cancer that prevents normoxia-induced degradation. Prostate 2005; 63(3):215–21. [28] Kyzas PA, Stefanou D, Batistatou A, Agnantis NJ. Hypoxia-induced tumor angiogenic pathway in head and neck cancer: an in vivo study. Cancer Lett 2005; 225(2):297–304. [29] Nakayama K, Kanzaki A, Hata K, Katabuchi H, Okamura H, Miyazaki K, et al. Hypoxia-inducible factor 1 alpha (HIF-1 alpha) gene expression in human ovarian carcinoma. Cancer Lett 2002; 176(2):215–23. [30] Abulafia O, Ruiz JE, Holcomb K, Dimaio TM, Lee YC, Sherer DM. Angiogenesis in early-invasive and low-malignant-potential epithelial ovarian carcinoma. Obstet Gynecol 2000; 95(4):548–52. [31] Hartenbach EM, Olson TA, Goswitz JJ, Mohanraj D, Twiggs LB, Carson LF, et al. Vascular endothelial growth factor (VEGF) expression and survival in human epithelial ovarian carcinomas. Cancer Lett 1997; 121(2):169–75. [32] Konac E, Onen HI, Metindir J, Almayanlar E, Biri Asyali A, Ekmekci A. The relationship between vascular endothelial growth factor -634 G > C polymorphism and ovarian, cervical and endometrial cancers. Korean J Genetic 2006; 28(2):99–107. [33] Ling TS, Shi RH, Zhang GX, Zhu H, Yu LZ, Ding XF. Common single nucleotide polymorphism of hypoxia-inducible factor-1alpha and its impact on the clinicopathological features of esophageal squamous cell carcinoma. Chin J Dig Dis 2005; 6(4):155–8. [34] Mazurek A, Kuc P. Angiogenesis-prognostic factor in patients with endometrial cancer. Ginekol Pol 2005; 76(10):838–45. [35] Jiang BH, Rue E, Wang GL, Roe R, Semenza GL, Dimerization. DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem 1996; 271(30):17771–8.
Short communication
An investigation of relationships between hypoxia-inducible factor-1a gene polymorphisms and ovarian, cervical and endometrial cancers Ece Konac PhDa, H. Ilke Onen MSca, Jale Metindir MDb, Ebru Alp MSca, Aydan Asyali Biri MDc, Abdullah Ekmekci PhDa,* b
a Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey Department of Obstetrics and Gynecology, Ankara Oncology Education and Research Hospital, Demetevler, Ankara, Turkey c Department of Obstetrics and Gynecology, Faculty of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey
Accepted 2 January 2007
Abstract Background: DNA sequence variations in HIF-1a gene might yield changes both in the production outcomes and in the activities of the gene. Overexpression of the HIF-1a subunit, resulting from intratumoral hypoxia and genetic alterations, has been demonstrated in common human cancers and is correlated with tumor angiogenesis and patient mortality. In this study, we aimed to determine how the three single nucleotide polymorphisms (SNPs, C1772T and G1790A exon 12, C111A exon 2) in the HIF-1a gene coding regions affect the ovarian, cervical and endometrial cancer patients in the Turkish population. A study on this relationship has not been conducted to date. Method: 102 gynecologic cancer patients and 107 healthy controls were studied. Genotypes of the three polymorphisms were analyzed by PCR-RFLP. Results: There was no significant difference between ovarian cancer patients and controls in terms of the distribution of C1772T genotypes and alleles (P > 0.05). However, there was a highly significant increase in the frequency of both CT 1772 and TT 1772 genotypes in patients with cervical and endometrial cancers compared with healthy controls. In fact, 1772T allele-carriers (CT + TT genotypes) showed an association with the risk of cervical and endometrial cancers compared to the wild type (OR = 3.84, 95% CI: 1.65–8.93; OR = 7.41, 95% CI: 2.33–23.59, respectively). C1772T polymorphism was not associated with family history concerning gynecologic and/or other cancer types, stages (I–IV) and grades of tumor, smoking habits and existence of other diseases that generate a hypoxic microenvironment even after multivariable logistic regression analysis. As for HIF-1a G1790A genotypes, the frequencies of G alleles were 98% in ovarian patients and 100% in the control group. We found no significant difference in the genotype distribution and allele frequencies between the ovarian patients and healthy control subjects. There were no GA and AA genotypes among the cervical and endometrial cancer patients. As for HIF-1a C111A polymorphism, we did not find CA and AA variants of the gene in controls or in any of the three types of patients. Conclusion: Our results suggest that the C1772T polymorphism of the HIF-1a may be associated with cervical and endometrial cancers. # 2007 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. Keywords: HIF-1a; Gynecologic cancers; Polymorphism; Risk factors; Turkish population; Family history; Genomic DNA; Hardy-Weinberg equilibrium
1. Introduction Tumor vascularization supplies nutrition and oxygen to proliferating cells, cellular adaptation to hypoxia, and strongly correlates with the risk of invasion and metastasis [1]. An important mediator of such cellular adaptation is * Corresponding author. Tel.: +90 312 202 69 94; fax: +90 312 212 46 47. E-mail address: [email protected] (A. Ekmekci).
hypoxia-inducible factor-1 (HIF-1), a critical transcription factor [2]. HIF-1 consists of a constitutively expressed HIF1ß subunit and one of three subunits (HIF-1a–HIF-3a). HIF1a forms a heterodimeric complex with HIF-1ß on hypoxic responsive elements and activates transcription of a wide variety of genes which are particularly relevant to cancer [3–6]. Tumors derived from cells lacking HIF-1a or HIF-1ß show significantly reduced vascularization and growth rates compared to parental cells [7,8]. In addition, enhanced
0361-090X/$30.00 # 2007 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cdp.2007.01.001
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expression levels of HIF-1a have been reported in human malignancies including colon, breast, stomach, pancreas, prostate, kidney, and esophagus [9–11]. The determination of SNPs is a new means to study the etiology of polygenic disorders with complex inheritance patterns, such as cancer [12]. All regions of the HIF-1a were examined and a total of 35 SNPs in the gene were found [13]. Polymorphisms of the gene have not been associated with some diseases [14], however, some of them were associated with production of HIF protein and reported to be involved in susceptibility to several disorders [14–18]. Angiogenesis of gynecologic cancer is regulated by vascular endothelial growth factor (VEGF), a target gene of HIF-1a [4]. In studies of ovarian cancer, VEGF expression and microvessel density (MVD) have been correlated with poor survival [19]. Birner et al. found that the combination of HIF-1a protein overexpression with nonfunctional p53 indicates a dismal prognosis of ovarian cancer [20]. It has been reported that HIF-1a expression was an independent prognostic marker in early stage cervical carcinoma [21]. Another study suggests that the increase of proliferating cell nuclear antigen (PCNA) index and MVD may enhance development of endometrial carcinoma [22]. Indeed, the role of HIF-1a in the development of ovarian, cervical, and endometrial cancers and their transition to metastases remain to be elucidated. On the basis of these facts, HIF-1a polymorphisms might contribute to the development and progression of ovarian, cervical, and endometrial cancers. Nevertheless, there have been no published studies on any population so far, as regards to their relationship. Therefore, this study was designed to examine the role of three SNPs, which were located in coding regions of the gene- one in exon 2 (S28Y), and two in exon 12 (P582S, A588T) in the development and progress of ovarian, cervical and endometrial cancers in the Turkish population.
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2. Materials and methods We studied a total of 102 gynecologic cancer patients who were selected from patients admitted to the Departments of Obstetrics and Gynecology, Faculty of Medicine, Gazi University and the Ankara Oncology Education and Research Hospital. A total of 107 subjects were enrolled as healthy controls (mean ages of menarche: 13 0.25 and mean age: 48 0.2 years, range 25–65). The study was approved by the Committee of Ethics of the Gazi University. All cases and controls were of Caucasian origin and became subjects of this study after submitting their written consents. Age of menarche and first diagnosis, history of gynecologic and/or other cancer types in the family, stages (I–IV) and grades of tumor (based on the International Federation of Gynecology and Obstetrics (FIGO) stage method), information on smoking habits and existence of other diseases such as stroke and cardiovascular diseases that generate a hypoxic microenvironment were obtained from the patient files. Gynecologic or other cancers, smoking habits and existence of other diseases were not encountered in the family history of the control individuals. Clinicopathological characteristics of the patients are summarized in Table 1. Genomic DNA was isolated from peripheral blood by using a DNA extraction kit (Heliosis1, Metis Biotechnology, Turkey) according to the manufacturer’s instructions. Amplifications of the C1772T and G1790A regions of the HIF-1a gene were carried out by placing in a Mastercycler gradient (Eppendorf, Germany) thermal cycler, a total volume of 50 mL PCR mixture containing 50 ng genomic DNA, 2.5 mM MgCl2, 100 mM dNTP, 50 pmol/ml of each primer and 1.0 U/mL Taq DNA polymerase. For the C1772T, the following pair of primers produced a PCR product of 346 bp: forward 50 -AAG GTG TGG CCA TTG TAA AAA CTC-30 , reverse 50 -GCA CTA GTA GTT TCT TTA TGT ATG-30 (Genbank accession no. AH006957; nucleotides 425–770). We set the PCR cycling conditions for the gene as explained by Ollerenshaw [16]. The
Table 1 Clinicopathological characteristics of the patients Cancer
Number of cases Mean ages of menarche (years) Mean ages of first diagnosis (years) Range (years) History of gynecologic and/or other cancer types in the family Stages of tumora I II III IV Histological grade (G1/G2 and G3) Smoking habits Existence of other diseases a
Ovarian (%)
Cervical (%)
Endometrial (%)
49 (48) 13 0.43 48 0.71 16–74 9 (18.37)
32 (31) 13 0.18 49 0.46 25–68 7 (21.8)
21 (21) 13 0.14 50 0.52 39–81 9 (42.8)
2 (4.08) 5 (10.20) 41 (83.67) 1 (2.05) 2/47 8 (16.33) 11 (22.45)
5 (15.6) 16 (50.0) 10 (31.3) 1 (3.1) 5/27 4 (12.5) 5 (15.6)
10 (47.6) 5 (23.8) 5 (23.8) 1 (4.8) 10/11 1 (4.76) 5 (23.8)
Stages of tumor (staging was performed according to the current classification of the International Federation of Gynecology and Obstetrics (FIGO)).
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Fig. 1. Electropherograms of DNA sequence (forward) data for the S28Y (C111A) polymorphism.
Fig. 2. Electropherograms of DNA sequence (reverse) data for the S28Y (C111A) polymorphism.
E. Konac et al. / Cancer Detection and Prevention 31 (2007) 102–109
experiment was conducted twice for each gene region (C1772T, G1790A and C111A) by two different researchers. For the C1772T region, the experiment was carried out by digesting the PCR products with restriction endonuclease, HphI (Fermantas, USA) and loading them onto 3% agarose gel (containing 0.5% ethidium bromide). For the G1790A, the polymorphism was analyzed by digesting the same PCR products with restriction endonuclease, AciI (Fermantas, USA) and loading them onto 3% agarose gel. The products were separated by electrophoresis and visualized by Gel Logic 100 gel image system (Kodak, USA). The 1772C allele was cut into two fragments of 228 and 118 bp while the 1772T allele remained uncut (346 bp). The G allele produces a single cut site to give two bands, 201 and 145 bp. The substitution from a G to an A abolishes an AciI cut site (346 bp). For the C111A, the following pair of primers were designed to produce a PCR product of 187 bp: forward 50 GGA TAA GTT CTG AAC GTC GA -30 , reverse 50 -ATC CAG AAG TTT CCT CAC AC -30 (Gene bank accession no. AH006957; nucleotides 177–363). Reaction products were sequenced by the Ohio State University (see http:// pmgf.biosci.ohio-state.edu) to confirm our primer designs (Figs. 1 and 2). Amplifications of the C111A regions of the HIF-1a gene were carried out by placing in a Mastercycler gradient (Eppendorf, Germany) thermal cycler, a total volume of 50 mL PCR mixture containing 50 ng genomic DNA, 2.5 mM MgCl2, 100 mM dNTP, 3% dimethylsulfoxide (DMSO), 50 pmol/mL of each primer and 1.0 U/mL Taq DNA polymerase. The conditions for the PCR reaction were denaturation at 95 8C for 5 min, followed by 30 cycles of denaturation at 95 8C for 30 s, annealing at 60 8C for 30 s and extension at 72 8C for 30 s, followed by a final extension at 72 8C for 5 min. The polymorphism was analyzed by digesting the PCR products with restriction endonuclease, BglII (Fermantas, USA) and loading them onto 3% agarose gel. The products were separated by electrophoresis and visualized by Gel Logic 100 gel image system (Kodak, USA). The 111C allele was cut into two fragments of 143 and 44 bp while the 111A allele remained uncut (187 bp) (Fig. 3). Tests for the Hardy-Weinberg equilibrium were performed by the chi-square (x2) test. The differences in genotype distribution and allele frequency among the groups were examined for statistical significance using the Pearson’s two-way chi-square (x2)-test. When the assumption of the x2 was violated, Fisher’s exact test was performed. The relationships between genotypes and/or alleles and gynecologic cancer risks were determined by obtaining the Odds ratios (ORs) through a logistic regression method [OR, 95% confidence interval (CI)]. Adjusted ORs for the clinicopathological covariant factors were determined by using a multivariable logistic regression method. Two-tailed P-values smaller than 0.05 were considered statistically significant. All analysis was conducted by using SPSS V.11.5.
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Fig. 3. PCR-based restriction analysis of HIF-1a gene C111A polymorphism shown on 3% agarose electrophoresis. Lane 1–7: 111C allele (wildtype) yielded two bands (44 basepair (bp) and 143 bp). The left lane (marker) contains a 100 bp ladder and lane 8 contains a 187 bp PCR product (uncut) of the gene.
3. Results In control and patient groups, there were no significant deviations from the Hardy–Weinberg equilibrium (P > 0.05). Out of the 107 healthy control cases, 68 cases were type CC, 37 cases were type CT and 2 cases were type TT. No significant differences in genotype (P = 0.759) and allele (P = 0.637) frequencies for C1772T polymorphism were observed between ovarian cancer patients and controls. However, we found a significant difference in the genotype distribution and allele frequencies between the cervical and endometrial cancer patients and control subjects (P < 0.001). ORs were calculated in comparison with the most common homozygote genotype observed in controls. A statistically significant difference was found for genotypic frequencies of the C1772T polymorphism between cervical and endometrial cancer patients with CT, TT, CT + TT genotypes and those with CC genotype (P < 0.05). Carriers of a 1772T allele (CT + TT genotypes) were more frequent among cervical (68.8%) and endometrial (81%) cancer patients than among controls (36.5%) (Table 2). The occurrence of all three types of gynecologic cancer were not associated with the other epidemiological covariant factors (P > 0.05) (Table 3). As for G1790A genotypes, out of the 49 ovarian patients, 47 patients had the GG genotype, two patients had the GA genotype, none of the patients had AA genotype (Table 4). We found no significant difference in the genotype distribution and allele frequencies between the ovarian patients and controls (P = 0.497 and 0.238, respectively). There were no GA and AA genotypes among the cervical and endometrial cancer patients (Table 4). All 107 control cases were type GG. It, therefore, became meaningless to run a multivariable logistic regression analysis to estimate adjusted ORs with the epidemiological covariant factors. As for C111A polymorphism, the genotype distributions in ovarian, cervical and endometrial cancer patients are
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Table 2 HIF-1a (C1772T) genotype and allele frequencies in ovarian, cervical and endometrial cancer patients and control subjects Cancer types
n (%) Patients
n (%) Controls
Ovarian Genotypes CC CT TT CT + TT Allele Frequency C T Cervical Genotypes CC CT TT CT + TT Allele Frequency C T Endometrial Genotypes CC CT TT CT + TT Allele Frequency C T
n = 49
n = 107
34 (69.4) 14 (28.6) 1 (2.0) 15 (30.6)
68 (63.5) 37 (34.6) 2 (1.9) 39 (36.5)
82 (83.7) 16 (16.3) n = 32
173 (80.1) 41 (19.9) n = 107
10 (31.2) 14 (43.8) 8 (25) 22 (68.8)
68 (63.5) 37 (34.6) 2 (1.9) 39 (36.5)
34 (53.1) 30 (46.9) n = 21
173 (80.1) 41 (19.9) n = 107
4 (19) 12 (57.2) 5 (23.8) 17 (81)
68 (63.5) 37 (34.6) 2 (1.9) 39 (36.5)
20 (47.6) 22 (52.4)
173 (80.1) 41 (19.9)
P-value a
ORb (95% CI)
P-value b
0.759 1.00 0.76 1.00 0.77
(ref) (0.36–1.59) (0.09–11.42) (0.37–1.59)
0.460 1.000 0.477
0.637 1.00 (ref) 0.82 (0.44–1.55)
0.548
<0.001 1.00 (ref) 2.57 (1.04–6.36) 27.20 (5.04–146.78) 3.84 (1.65–8.93)
0.037 <0.001 0.001
1.00 (ref) 3.72 (2.05–6.77)
<0.001
1.00 (ref) 5.51 (1.66–18.31) 42.50 (6.20–291.36) 7.41 (2.33–23.59)
0.003 <0.001 <0.001
1.00 (ref) 4.64 (2.32–9.30)
<0.001
<0.001
<0.001
<0.001
OR, Odds ratio; CI, confidence interval. P values <0.05 and 0.001 are shown in bold. Values in parentheses are percentages. a x2 analysis. b Calculations were performed CC vs. CT, TT, CT + TT.
Table 3 Odds ratios (ORs) of HIF-1a (C1772T) polymorphism in ovarian, cervical and endometrial cancer patients with adjustments Cancer types Ovarian Genotypes CC CT TT CT + TT Cervical Genotypes CC CT TT CT + TT Endometrial Genotypes CC CT TT CT + TT
a,z
OR
(95% CI)
1.00 1.05 1.75 0.96
a,z
b,z
P-value
OR
(95% CI)
(ref) (0.23–4.83) (0.14–21.88) (0.21–4.38)
1.000 0.556 1.000
1.00 1.03 0.40 1.12
1.00 3.60 3.00 3.38
(ref) (0.34–38.48) (0.22–40.93) (0.35–32.64)
0.358 0.559 0.387
1.00 1.00 0.25 0.70
(ref) (0.10–9.61) (0.01–4.73) (0.08–6.22)
1.000 0.524 1.000
b,z
c,z
ORd,z (95% CI)
P-valued,z
(ref) (0.03–2.67) (0.14–21.88) (0.03–2.47)
0.407 0.556 0.406
1.00 0.89 1.50 0.81
(ref) (0.20–3.98) (0.12–18.57) (0.18–3.62)
1.000 1.000 1.000
1.00 0.13 0.33 0.11
(ref) (0.01–1.40) (0.03–4.04) (0.01–1.25)
0.133 0.588 0.079
1.00 0.67 0.57 0.63
(ref) (0.08–5.75) (0.04–7.74) (0.09–4.53)
1.000 1.000 0.637
1.00 0.15 0.33 0.11
(ref) (0.01–2.18) (0.02–5.03) (0.01–1.55)
0.202 0.559 0.133
1.00 2.14 0.33 1.25
(ref) (0.17–27.10) (0.02–5.03) (0.10–15.11)
1.000 0.559 1.000
OR
(ref) (0.18–6.09) (0.03–5.15) (0.19–6.55)
1.000 0.457 1.000
1.00 0.30 1.75 0.28
1.00 0.27 0.09 0.20
(ref) (0.05–1.48) (0.01–1.10) (0.04–0.98)
0.211 0.666 0.056
1.00 1.50 0.75 1.25
(ref) (0.12–19.44) (0.03–17.51) (0.10–15.11)
1.000 1.000 1.000
OR, odds ratio; CI, confidence interval. z Calculations were performed CC vs. CT, TT, CT + TT. a OR adjusted for history of gynecologic and/or other cancer types in the family. b OR adjusted for stages and grades of tumor. c OR adjusted for smoking habits. d OR adjusted for existence of other diseases.
a,b,c,d
P-value
P-value
(95% CI)
c,z
E. Konac et al. / Cancer Detection and Prevention 31 (2007) 102–109
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Table 4 HIF-1a (G1790A) genotype and allele frequencies in ovarian, cervical and endometrial cancer patients and control subjects Cancer types
n (%) Patients
n (%) Controls
Ovarian Genotypes GG GA AA Allele frequency G A Cervical Genotypes GG GA AA Allele frequency G A Endometrial Genotypes GG GA AA Allele frequency G A
n = 49
n = 107
47 (96) 2 (4) 0 (0)
107 (100) 0 (0) 0 (0)
96 (98) 2 (2) n = 32
214 (100) 0 (0) n = 107
32 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
64 (100) 0 (0) n = 21
214 (100) 0 (0) n = 107
21 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
42 (100) 0 (0)
214 (100) 0 (0)
P-valuea
ORb (95% CI)
P-valueb
1.00 (ref) 1.02 (0.99–1.05) Not calculated
0.240 –
1.00 (ref) 0.49 (0.44–0.54)
0.240
1.00 (ref) Not calculated Not calculated
– –
1.00 (ref) Not calculated
–
1.00 (ref) Not calculated Not calculated
– –
1.00 (ref) Not calculated
–
0.497
0.238
1.000
1.000
1.000
1.000
OR, odds ratio; CI, confidence interval. Values in parentheses are percentages. a x2 analysis. b Calculations were performed GG vs. GA.
Table 5 HIF-1a (C111A) genotype and allele frequencies in ovarian, cervical and endometrial cancer patients and control subjects Cancer types
n (%) Patients
n (%) Controls
Ovarian Genotypes CC CA AA Allele frequency C A Cervical Genotypes CC CA AA Allele frequency C A Endometrial Genotypes CC CA AA Allele frequency C A
n = 49
n = 107
49 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
98 (100) 0 (0) n = 32
214 (100) 0 (0) n = 107
32 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
64 (100) 0 (0) n = 21
214 (100) 0 (0) n = 107
21 (100) 0 (0) 0 (0)
107 (100) 0 (0) 0 (0)
42 (100) 0 (0)
214 (100) 0 (0)
Values in parentheses are percentages.
presented in Table 5. We did not find CA and AA variants of the gene in controls or in any of the three types of patients. Therefore, no comparison was made between the CC genotype patients and CA and AA variants and the adjusted ORs with epidemiological covariant factors were not estimated.
4. Discussion HIF-1a in the regulation of tumor angiogenesis is the target of novel anti-cancer drugs [23]. Therefore, we investigated the polymorphic effects of the changes in the C1772T, G1790A and C111A of the HIF-1a gene on the ovarian, cervical and endometrial cancer patients. C1772T and G1790A polymorphisms have been reported in relation to patients with head and neck squamous cell carcinoma (HNSCC) [15] and renal cell carcinoma (RCC) [16,24]. A highly significant increase in the frequency of both the CC 1772 and GA 1790 genotypes was found in patients with RCC compared with healthy controls [16]. Tanimoto et al. [15], found CC and CT genotypes of C1772T polymorphism, but none of the subjects had the homozygous genotype TT. Furthermore, they showed the elevated transactivation capacity of variant forms of HIF-1a C1772T and G1790A polymorphisms [15]. On the other hand, it was reported that inactivation of pVHL for E3 ubiquitin ligase activity, but not genetic polymorphism of
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HIF-1a, may be one of the mechanisms underlying HIF-1a activations in colorectal carcinomas [25]. In another study, the T allele of the C1772T polymorphism was not detected in patients with colorectal carcinoma and controls [26]. In contrast, our study pointed to a statistically significant increase in the percentages of carriers of T allele (CT, TT and CT + TT genotypes) in cervical and endometrial cancer patients, which indicates that carrying T allele is a risk factor. C allele, on the other hand, is not a risk factor in the development of cervical and endometrial cancers. However, due to the small sample size we cannot suggest a tumor suppressor function for the C allele. The molecular basis for T allele’s high frequency is unclear, but the corresponding protein has been reported to have increased transcriptional activity and stability under normoxic conditions [27]. There was a significant relationship between HIF-1a and VEGF expression in tumors located in the lower lip and in larynx [28], in contrast, not in those located in the oral cavity [28], in HNSCC and in ovarian carcinoma [29–31]. We have earlier demonstrated that VEGF -634 G > C polymorphism is not associated with ovarian, cervical, and endometrial cancers [32]. The present study found no significant difference between ovarian cancer patients and controls in terms of the distribution of C1772T genotypes and alleles (P > 0.05). Since the association of the polymorphism with the three types of gynecologic cancers has not been investigated in any known studies, we were unable to compare our results with those of similar studies covering other populations. In general, the distribution of C1772T genotypes in control groups was approximately similar to those reported in previous studies [15,24–26,33] with regard to most common genotypes in population. We found no evidence of a relationship between clinicopathological characteristics and C1772T polymorphism in ovarian, cervical, and endometrial cancer patients. On the other hand, evaluation of angiogenesis intensity in endometrial cancer also seems to be an independent prognostic factor and statistically correlates with FIGO stage of disease, histological type and grade of tumor, depth of myometrial invasion and metastasis [34]. As for the 1790 gene region of HIF-1a, we found that there were no GA and AA genotypes among the cervical and endometrial cancer patients and only two GA genotypes among the ovarian cancer patients. As a result, the difference in the genotype distribution between ovarian patients and control subjects was not significant (P > 0.05). Allelic and genotypic frequencies of G1790A polymorphism were in close agreement with those previously published in Tanimoto et al. [15]. C111A polymorphism found in bHLH 17–30 aa (protein no: Q16665) has not been shown to be hydroxylated. However, bHLH and PAS domains are required for heterodimerization with HIF-1ß and binding to DNA [35]. Therefore, we hypothesized that if the HIF-1a gene plays any part in the disease process, then there should be differences between all three types of gynecologic cancer
patients and control subjects. However, in our study, A allele was not detected in patients and controls, that is, all samples were found to be CC genotype. It is not clear why we did not detect the 111A allele. Nevertheless, the distribution of C111A polymorphism was consistent with the report of Yamada et al. [13]. In conclusion, this is the first study to report that C1772T polymorphism of HIF-1a gene is associated with the occurrence of cervical and endometrial cancers while G1790A and C111A gene polymorphisms are not associated with any of the three types of gynecologic cancer. Although a limited number of patients were enrolled in the study, which forced us to cope with small sample sizes, our results are expected to contribute to the efforts to better understand the phenotypic effects of SNPs, which are based on direct genetic effects, gene–gene and gene-environment interactions, and to have important implications for understanding the pathogenesis of these tumors and for screening, if confirmed in follow-up studies.
Conflict of Interest None.
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