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Cancer Epidemiology Biomarkers & Prevention 17, 972, April 1, 2008. doi: 10.1158/1055-9965.EPI-07-2787
© 2008 American Association for Cancer Research
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Short Communication

Polymorphisms in Angiogenesis-Related Genes and Prostate Cancer

Eric J. Jacobs1, Ann W. Hsing2, Elizabeth B. Bain1, Victoria L. Stevens1, Yiting Wang1, Jinbo Chen3, Stephen J. Chanock2, S. Lilly Zheng4, Jianfeng Xu4, Michael J. Thun1, Eugenia E. Calle1 and Carmen Rodriguez1

1 Department of Epidemiology and Surveillance Research, American Cancer Society, Atlanta, Georgia; 2 Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Department of Health and Human Services, Bethesda, Maryland; 3 Department of Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, Pennsylvania; and 4 Center for Human Genomics, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Requests for reprints: Eric J. Jacobs, Epidemiology and Surveillance Research, American Cancer Society, National Home Office, 250 Williams Street, Atlanta, GA 30303-1002. Phone: 404-329-7916; Fax: 404-327-6450. E-mail: Eric.Jacobs{at}cancer.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Angiogenesis is required for development and progression of prostate cancer. Potentially functional single nucleotide polymorphisms (SNP) in genes important in prostate angiogenesis (VEGF, HIF1A, and NOS3) have previously been associated with risk or severity of prostate cancer.

Methods: Prostate cancer cases (n = 1,425) and controls (n = 1,453) were selected from the Cancer Prevention Study II Nutrition Cohort. We examined associations between 58 SNPs in nine angiogenesis-related candidate genes (EGF, LTA, HIF1A, HIF1AN, MMP2, MMP9, NOS2A, NOS3, VEGF) and risk of overall and advanced prostate cancer. Unconditional logistic regression was used to estimate odds ratios, adjusted for matching factors.

Results: Our results did not replicate previously observed associations with SNPs in VEGF, HIF1A, or NOS3, nor did we observe associations with SNPs in EGF, LTA, HIF1AN, MMP9, or NOS2A. In the MMP2 gene, three intronic SNPs, all in linkage disequilibrium, were associated with overall and advanced prostate cancer (for overall prostate cancer, Ptrend = 0.01 for rs1477017, Ptrend = 0.01 for rs17301608, Ptrend = 0.02 for rs11639960). However, two of these SNPs (rs17301608 and rs11639960) were examined and were not associated with prostate cancer in a recent genome-wide association study using prostate cancer cases and controls from the Prostate, Lung, Colorectal, and Ovary study cohort. Furthermore, when we pooled our results for these two SNPs with those from the Prostate, Lung, Colorectal, and Ovary cohort; neither SNP was associated with prostate cancer.

Conclusion: None of the SNPs examined seem likely to be importantly associated with risk of overall or advanced prostate cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(4):972–7)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Angiogenesis, the growth of new blood vessels, is required for the growth of microscopic cancers into larger, clinically relevant tumors (1). The importance of angiogenesis specifically in prostate carcinogenesis is supported by a large body of research, including studies demonstrating altered expression of angiogenic factors in prostate cancer, inhibition of tumor growth in animal models after treatment with angiogenesis inhibitors, and correlations between tumor blood vessel density and both tumor characteristics and clinical outcome (2, 3). Proangiogenic factors important in prostate angiogenesis have been reviewed (2, 3). We selected nine candidate genes, described individually below, which are important in prostate angiogenesis. We then used cases and controls from a large cohort of U.S. men to examine associations between 58 polymorphisms in these genes and risk of advanced and overall prostate cancer.

Vascular endothelial growth factor (VEGF) plays a central role in prostate angiogenesis (2). The G allele of a VEGF promoter region single nucleotide polymorphism (SNP; rs1570360, also known as –1154 G/A), which increases VEGF transcription (4), has been associated with significantly increased risk of prostate cancer in two small case control studies (5, 6).

Hypoxia inducible factor 1 (HIF1A) is a transcription factor that is overexpressed even in early stage prostate cancer and increases transcription of VEGF (7). The minor allele of a SNP encoding an amino acid substitution (rs11549465, also known as P582S) has been associated with increased risk in a relatively small case control study of metastatic prostate cancer (8) and has also been detected as a somatic mutation in prostate cancers (9). However, no statistically significant association between this SNP and prostate cancer risk was found in a recent larger study (10). Hypoxia inducible factor l {alpha} subunit inhibitor (HIF1AN) is a potentially important inhibitor of HIF1A activity (11).

Epidermal growth factor (EGF) can increase VEGF expression in prostate cancer cell lines (12). The minor allele of a SNP in the 5' untranslated region (UTR) of the EGF gene (rs4444903, also known as 61 A/G) has been associated with increased EGF expression as well as risk and/or severity of melanoma and gastric cancer in some studies (reviewed in ref. 13).

Lymphotoxin {alpha} (LTA) increases synthesis of VEGF in prostate cancer cell lines (14). An LTA SNP (rs909253) may increase transcription (15).

Endothelial nitric oxide synthase (NOS3) and inducible nitric oxide synthase (NOS2A) both catalyze the synthesis of nitric oxide, which can be proangiogenic (16), and NOS2A is overexpressed in prostate cancer (17). A small case control study of a SNP in NOS3 (rs1799983, also known as D298E) found no overall association with prostate cancer risk (18) but reported that the minor allele was associated with reduced risk of advanced disease among prostate cancer cases (19).

Matrix metalloproteinase 2 (MMP2) and MMP9 degrade basement membranes and extracellular matrix, processes that are necessary for both angiogenesis and tumor invasion (20). MMP2 is overexpressed in prostate cancer, with higher expression levels predicting poorer survival (21). The minor allele of a MMP2 promoter region SNP (rs243865, also known as –1306 C/T) reduces transcription (22).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Population
Men in this analysis were selected from among the 86,404 male participants in the Cancer Prevention Study II Nutrition Cohort, a prospective study of cancer incidence among U.S. men and women established in 1992 (23). Approximately 99% of male participants were between the ages of 50 and 79 y at the time of enrollment, and ~97% were White (23). Follow-up questionnaires were sent to cohort members in 1997 and every 2 y thereafter to ascertain newly diagnosed cancers. Incident cancers reported on questionnaires were verified through medical records, linkage with state cancer registries, or death certificates. From June 1998 through June 2001, participants in the Nutrition Cohort were invited to provide a blood sample. After obtaining informed consent, blood samples were collected from 17,411 men. The recruitment, characteristics, and follow-up of the Nutrition Cohort are described in greater detail elsewhere (23).

From men who provided a blood sample, we identified 1,476 who had been diagnosed with prostate cancer between 1992 and 2003, and had not been diagnosed with any other cancer (other than nonmelanoma skin cancer). For each case, we randomly selected one control from among those who had provided a blood sample and had no history of cancer on the diagnosis date of the case. Each control was individually matched to their case on birth date (±6 mo), date of blood collection (±6 mo), and race/ethnicity (White, African-American, Hispanic, Asian, and other/unknown). Twenty-nine cases were later excluded because their initial self-report of prostate cancer could not be verified. An additional 22 cases and 23 controls were later excluded because they were found not to have an adequate sample available or because of handling or laboratory errors. A total of 1,425 cases and 1,453 controls remained for analysis.

Of the cases included in this analysis, 62% were diagnosed before they provided a blood sample. However, important survival bias was considered unlikely because only a small proportion (2.9%) of prostate cancer cases diagnosed during the time period included in this analysis died of prostate cancer before collection of blood samples from cohort members was completed. We categorized 559 cases as advanced prostate cancer, defined as stage II cancers with a Gleason score of ≥7, all stage III or stage IV cancers, and prostate cancers that were listed as the underlying cause of death on a death certificate.

SNP Selection and Genotyping
We selected SNPs based on results from previous epidemiologic studies and potential functional importance. In addition, for several genes (EGF, HIF1A, HIF1AN, MMP2, and MMP9), we also selected all SNPs identified as haplotype-tagging SNPs, using the method developed by Gabriel et al. (24), from HapMap data available in January 2006.

Genotyping was done in two phases. The first phase included genotyping of 10 SNPs at the National Cancer Institute's Core Genotyping Facility, using a Taq Man assay. These included all SNPs for the LTA, VEGF, and NOS2A genes, and one of the EGF SNPs (rs4444903). Only cases diagnosed between enrollment in 1992 and June 2001 (n = 1,173) and matched controls (n = 1,187) were included in the first phase, as follow-up through 2003 was not yet available at the time of genotyping.

The second phase included genotyping of an additional 48 SNPs at the Center for Human Genomics (Wake Forest University), using the MassARRAY system (SEQUENOM). All prostate cancer cases (n = 1,425) diagnosed through August 2003 and matched controls (n = 1,453) were included in the second phase, including all the cases and controls that were in the first phase. All 48 SNPs were initially genotyped on a subset of 553 predominantly advanced prostate cancer cases (529 advanced cases that were verified at the time of genotyping as well as an additional 24 not advanced cases) and matched controls (n = 553). To minimize genotyping costs, 15 of these SNPs were then selected for additional genotyping (based on previous literature and the initial genotyping results), using all remaining cases and matched controls.

For quality control, 3.5% of the samples genotyped during both phases of the study were blinded replicates. Concordance for these replicates was 100% for all SNPs.

Statistical Analysis
Odds ratios (OR) and 95% confidence intervals (95% CI) for the association between each SNP and overall and advanced prostate cancer incidence were determined using unconditional logistic regression. Our primary measure of association was the per-allele OR, determined by entering a continuous variable for the number of minor alleles (0, 1, or 2), into the logistic regression model. All models were adjusted for the study matching factors of birth year (single year categories), date of blood draw (single year categories), and race/ethnicity (White or other/unknown, African-American, Hispanic, and Asian). In analyses limited to matched pairs, conditional logistic regression accounting for the original matched pair design yielded similar results as unconditional logistic regression.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Table 1 shows per allele ORs for all 58 SNPs. One HIF1A SNP, rs11549465, also known as P582S, was associated with both advanced (P = 0.047) and overall prostate cancer (P = 0.010). Three SNPs in the MMP2 gene were also associated with both advanced and overall prostate cancer [rs1477017 (located in intron 2), Ptrend = 0.011 for overall cancer; rs17301608 (intron 3), Ptrend = 0.014 for overall prostate cancer; rs11639960 (intron 11), Ptrend = 0.020 for overall prostate cancer]. These three MMP2 SNPs were in linkage disequilibrium and moderately highly correlated. Among controls, the squared correlation coefficient (r2) was 0.92 for the intron 2 and intron 3 SNPs, 0.70 for the intron 2 and intron 11 SNPs, and 0.65 for the intron 3 and 11 SNPs. The linkage disequilibrium coefficient (D') was 0.99 for the intron 2 and intron 3 SNPs, 0.88 for the intron 2 and intron 11 SNPs, and 0.87 for the intron 3 and 11 SNPs.


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  		Table 1. ORs for prostate cancer associated with each additional minor allele, CPS-II Nutrition Cohort 1992 to 2003

 
Analyses including only cases that were stage III, stage IV, or fatal (n = 148) yielded similar results. However, statistical precision was limited.

Table 2 shows more detailed genotype results for the four statistically significant SNPs described above, as well as the VEGF SNP previously reported to be associated with prostate cancer (rs1570360) and the NOS3 SNP previously reported to be associated with severity among prostate cancer cases (rs1799983). Because the per allele analyses shown in Table 1 could miss associations limited to the homozygous variant genotype, we also examined ORs for the homozygous variant genotype (compared with the homozygous wild-type) for the 52 SNPs not shown in Table 2. The homozygous variant of one of these SNPs, HIF1AN rs2295778 (P41A), was associated with a statistically significant reduction in risk of advanced prostate cancer (OR for GG versus CC, 0.74; 95% CI, 0.56-0.99).


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  		Table 2. ORs for prostate cancer by genotype for selected polymorphisms, CPS-II Nutrition Cohort 1992 to 2003

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Few of the SNPs examined were significantly associated with prostate cancer, and all ORs were relatively small. Three linked intronic SNPs in the MMP2 gene, of no known functional significance, were associated with prostate cancer risk in our study. To our knowledge, the association between these SNPs and prostate cancer has not previously been published in a research journal. However, two of these SNPs (rs17301608 and rs11639960) were examined as part of the Cancer Genetic Markers of Susceptibility genome-wide association study, using >1,100 prostate cancer cases and a similar number of controls from the Prostate, Lung, Colorectal, and Ovary (PLCO) cohort (25). Neither of these SNPs was associated with prostate cancer risk in the PLCO cohort. Pooling our data for rs17301608 with those from PLCO using a random effects meta-analysis (26) yielded a per allele OR of 1.07 (95% CI, 0.78-1.45) for advanced prostate cancer and 1.03 (95% CI, 0.83-1.27) for overall prostate cancer. Pooled results for rs11639960 were similarly null. The third SNP (rs1477017), although not directly examined in the PLCO cohort, is highly correlated with rs17301608. Therefore, the totality of the evidence does not support a strong association between any of these SNPs and prostate cancer risk.

The HIF1A rs11549465 SNP (P582S) was associated with a statistically significant reduction in risk of both overall and advanced prostate cancer. However, this association is in the opposite direction from that observed in a previous study that included 196 metastatic prostate cancer cases (8). No association between rs11549465 and prostate cancer was observed in a recent published larger study (10) or in the PLCO cohort (25). The totality of the evidence suggests that rs11549465 is not strongly associated with prostate cancer, although an association with metastatic prostate cancer cannot be ruled out.

In addition to the MMP2 and HIF1A SNPs discussed above, nine additional SNPs included in this analysis (footnoted in Table 1) were also examined in the PLCO cohort (25). None of these SNPs were associated with prostate cancer in either our analysis or in the PLCO cohort.

A limitation of this study is that we cannot rule out associations with more advanced prostate cancer (e.g., stage III or above) because we had relatively few cases this advanced. In addition, we could not examine associations separately by race or ethnicity because nearly all men in our study were White. Strengths of this study are its relatively large size and the inclusion of a considerable number of SNPs for which an association with prostate cancer is biologically plausible and/or has been previously reported.

In conclusion, our results do not support previously reported associations between prostate cancer and SNPs in VEGF, HIF1A, and NOS3. Taking into account recent results from other studies, none of the SNPs we examined seem likely to be strongly associated with risk of prostate cancer.


   Acknowledgments
 
We thank Kimberly Walker-Thurmond and Cari Lichtman (American Cancer Society) for their contributions to making this study possible.


   Footnotes
 
Grant support: Intramural Research Program of the NIH, National Cancer Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/10/07; revised 1/15/08; accepted 1/21/08.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online