STK15 polymorphism and breast cancer risk in a population-based study

Kathleen M. Egan7, Polly A. Newcomb1, Christine B. Ambrosone2, Amy Trentham-Dietz3, Linda Titus-Ernstoff4, John M. Hampton5, Makoto T. Kimura6 and Hiroki Nagase6,8

Vanderbilt University Medical Center, Suite 6000, MCE, Nashville, TN 37232-8300, USA, 1 Cancer Prevention Research Group, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, MP 900, Seattle, WA 98104, USA, 2 Department of Epidemiology, Division of Cancer Prevention and Population Science, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA, 3 Comprehensive Cancer Center and Department of Population Health Sciences, University of Wisconsin, WARF Room 701, 610 Walnut Street, Madison, WI 53726, USA, 4 Dartmouth Medical School, Norris Cotton Cancer Center, Lebanon, NH 03756, USA, 5 University of Wisconsin, WARF Room 307, 610 Walnut Street, Madison, WI 53726, USA, 6 Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA

7 To whom correspondence should be addressed. Tel: +1 615 936 1640; Fax: +1 615 936 1269; Email: kathleen.egan{at}vanderbilt.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
STK15 is considered a potential cancer susceptibility gene owing to its functions in normal cell mitosis. Two common coding region polymorphisms in the gene (F31I and V57I) may affect ubiquitin-dependent degradation and thus the half-life of the encoded protein. There are limited data on the relevance of these polymorphisms to population cancer rates. To examine whether functional variation in STK15 may affect breast cancer risk, we genotyped a large series of incident breast cancer cases (n = 941) and age-matched population controls (n = 830) for the F31I and V57I polymorphisms. Individually, neither the F31I polymorphism [odds ratio (OR) 1.54; 95% confidence interval (CI) 0.96–2.47, comparing 31I with 31F homozygotes] nor the V57I polymorphism (OR 0.92; 95% CI 0.50–1.71, comparing 57I with 57V homozygotes) was significantly associated with breast cancer risk. A relatively common genotype, combining the two polymorphisms (31I-57V/31I-57V, 3% of controls) was related to a significant 2-fold increase in the risk of post-menopausal breast cancer (OR 1.96; 95% CI 1.01–3.79). No interaction was detected between STK15 variants and estrogenic risk factors, although the power of these analyses was limited. These results suggest that STK15 may represent a low penetrance type breast cancer susceptibility gene.

Abbreviations: CI, confidence interval; DCIS, ductal carcinoma in situ; OR, odds ratios; SNP, single nucleotide polymorphism


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
STK15 is a serine/threonine kinase the gene for which has been identified as a candidate tumor susceptibility gene using a mouse model of human cancer (1). STK15 protein is a mitotic kinase required in the formation of a bipolar mitotic spindle and it regulates chromosome segregation in mammalian cells (2). STK15 overexpression has been reported in various types of cancer and correlated with a poor prognosis in medulloblastoma (3) and chromosomal instability in breast cancer (4). Its expression is inversely correlated with expression of p53 in bladder tumors and phosphorylation by STK15 induces MDM2-mediated p53 degradation (5).

Two coding region single nucleotide polymorphisms (SNPs) have been identified in STK15. A T/A polymorphism located at nucleotide position 91 encodes a phenylalanine->isoleucine (F31I) substitution at amino acid position 31. A G/A polymorphism at nucleotide 169 encodes valine->isoleucine (V57I) at amino acid position 57. Both polymorphisms are located within two conserved motifs in the N-terminal region of STK15; one is in Aurora Box 1 which contains the KEN box (a region between amino acid positions 5 and 40) and another is in Aurora Box 2, which contains an amino acid sequence with similarity to a destruction box (D-box) (from amino acid 43 to 63). Both motifs could be related to ubiquitin-dependent degradation in early G1 depending on the C-terminal degradation box domain (6,7) and also serve as a localization domain to target the protein to the centrosome in a microtubule-dependent manner (8). STK15 protein localizes to centrosomes only in S phase, and after centrioles have been duplicated the protein is degraded in early G2/M phase (8,9). The F31I and V57I polymorphisms of STK15 occur in an aurora box, which is a domain involved in the Cdh1-enhanced ubiquitination–proteolysis pathway (10). I31 has been associated with the degree of aneuploidy in human colon tumors (11). The E2 ubiquitin conjugating enzyme UBE2N was shown to be a preferential binding partner of the STK15 F31 variant form. This interaction results in co-localization of UBE2N with STK15 at the centrosomes during mitosis and may be associated with ubiquitination of the F31 variant but not the I31 variant. These results are consistent with an important role for the I31 variant of Aurora-A/STK15 in human cancer susceptibility and in loss of UBE2N binding function (1). The F31I amino acid change has been noted in ~8% of Caucasian Americans (1). The functional significance of the V57I amino acid change has not been identified; the allele frequency was ~14% in an unrelated general Japanese population (12). Recently, the Ile/Ile genotype for the F31I variant was reported to be associated with an increased risk of esophageal squamous cell carcinoma compared with the Phe/Phe genotype (13). A recent study also reported a significant association of the I31 allele with ovarian cancer risk (14). No epidemiological study has reported whether the functional F31I polymorphism or the coding polymorphism of V57I segregates with breast cancer risk.

Because the F31I and V57I variants might affect the fidelity of normal mitosis in breast epithelial cells, we examined the association of these polymorphisms with breast cancer risk in a large, population-based case–control study. Results indicate a significant association of breast cancer risk with a relatively common Aurora-A genotype (I31-V57). The analysis suggests that functional variation in STK15 may represent a low penetrance type cancer susceptibility factor in breast cancer.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer cases and controls
To study association of these variants with human cancer, we genotyped DNA collected in a large population-based breast cancer case–control study. Details of the study have been published previously (15). In brief, all English speaking female residents of Wisconsin, New Hampshire and Massachusetts (excluding the four counties that comprise metropolitan Boston) aged 20–74 with a new diagnosis of breast cancer reported to the state's cancer registry were eligible for this study. For comparability with controls, eligibility was limited to women with listed telephone numbers or a drivers' license verified by self-report (if <65 years of age) and/or Medicare beneficiaries (if ≥65 years). Letters were sent to the physician of record for each eligible case to obtain approval to contact the cases. Control women were selected from population lists in each state and frequency matched to the cases on age (in 5 year strata). Subjects completed a telephone interview on known and suspected risk factors in breast cancer. All exposures were assessed prior to a reference date corresponding to the date of diagnosis in cases or, in controls, a date randomly selected from among the diagnosis dates of recently interviewed case women. Women also provided a sample of DNA (by cheek swab or oral rinse). The present analysis is limited to women enrolled in the parent case–control study between 1998 and 1999. A total of 941 women with a recent diagnosis of breast cancer (826 invasive, 115 in situ) and 830 population controls were genotyped for the two STK15 variants (F31I and V57I). All women were Caucasian, most of central European ancestry. The study was reviewed and approved by local investigational review boards at each academic center that enrolled subjects (University of Wisconsin for Wisconsin women, Harvard School of Public Health for Massachusetts women and Dartmouth Medical School for New Hampshire women). All participants provided written informed consent.

STK15 SNP genotyping
For SNP genotyping, pyrosequencing techniques were used following the vendors' instructions. PCR primers used were designed to amplify the STK15 gene specifically. The PCR reaction mix for each of the samples for both the STK15-57 and STK15-31 sequences was performed in a 20 µl mix of 1x Invitrogen buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.5 U Taq polymerase, 0.025 U Pfu Turbo, 0.4 µM A31 specific-F primer, 0.4 µM STK57V/I Bio-R, H2O and genomic DNA. The PCR reaction was initially denatured at 95°C for 2 min, followed by 35 cycles of 94°C for 30 s, 56°C for 45 s and 72°C for 45 s, then an extension of 72°C for 10 min. This optimized condition avoided pseudogene amplification. The primers for pyrosequencing were for A31-F 5'-CAT GAA TGC CAG AAA GTT T-3' and for biotinylated STK15-57V/I Bio-R 5'-CTG CTT CTG ATT CTG AAC CGG CTT G-3'. The sequencing primers for STK15-codon31 were 5'-GTG TTC TCG TGA CTC AGC AA-3' and for STK15-codon57 5'-CTT CAA ATT CTT CCC AGC GC-3'. To determine the genotype combining both polymorphisms, an allele-specific PCR primer STK15-A31-3'-end-specific-A (5'-GTT CTC GTG ACT CAG CAA A-3') was used.

Statistical analysis
Tests of the allele frequencies in controls indicated no significant departures from the Hardy–Weinberg equilibrium for either polymorphism. We examined associations of breast cancer with each of the two polymorphisms. In addition, all subjects were further classified according to STK15 genotype based on the two polymorphisms combined (10 possible). Age- and residence-adjusted and multivariate odds ratios (OR) were obtained using unconditional logistic regression. Multivariate models including terms for age, state of residence and established breast cancer risk factors including parity, age at first giving birth, menopausal status, body mass index (kg/m2), current use of estrogen-containing hormone replacement therapy (as of the reference date) and first degree family history of breast cancer (i.e. breast cancer in a mother, sister or daughter). Because the influence of these polymorphisms on breast cancer risk could be enhanced through the mitogenic influence of estrogen, we tested for multiplicative interaction between STK15 genotype and estrogenic risk factors (total estimated number of lifetime ovulatory cycles, body mass index and use of estrogen hormone replacement therapy); cross-product terms for the risk factor of interest and genotype were incorporated in multivariate models and model log likelihoods were compared for improvements in goodness of fit. We tested for allelic association between the two polymorphisms using the EM algorithm (16); results suggested highly significant (P < 0.0001) linkage between the two loci, in both cases and controls.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cases and controls had median ages of 56.2 and 55.0 years, respectively. Table I shows a comparison of the cases and controls according to known breast cancer risk factors. Cases and controls displayed expected differences in breast cancer risk factors: cases were more likely than controls to report a family history of breast cancer, to be of lower parity and later childbearing and to have a later age at menopause. Cases were more often current users of hormone replacement therapy. Slightly more than half of all the women were post-menopausal.


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Table I. Characteristics of women with breast cancer and controls

 
Table II shows results for the individual polymorphisms and an analysis combining the two STK15 polymorphisms. The prevalence of homozygous carriers in the controls was 4% for the codon 31 polymorphism and 3% for the codon 57 polymorphism. Individually, the polymorphisms were not significantly associated with breast cancer risk. Odds ratios adjusted for age and state of residence were modestly elevated among women homozygous for the codon 31 variant [OR 1.56; 95% confidence interval (CI) 0.98–2.49]. Further adjustment for established breast cancer risk factors had only a modest influence on odds ratios (multivariate OR 1.54; 95% CI 0.96–2.47). Age- and state of residence-adjusted odds ratios were similar in pre-menopausal (OR 1.57; 95% CI 0.68–3.62) and post-menopausal (OR 1.71; 95% CI 0.94–3.12) women. For the less common codon 57 polymorphism the relationship was slightly inverse; the inverse relationship was more pronounced in pre-menopausal women (OR 0.38; 95% CI 0.14–1.07). Combining the two polymorphisms, homozygous I31/V57 carriers (AA + GG genotype, 3.2% of controls) had an overall 60% increase in the odds of breast cancer (age/state-adjusted OR 1.57; 95% CI 0.94–2.62) when compared with the most common genotype in this population (TT + GG, 41% of controls). The association was more pronounced and the OR attained significance in post-menopausal women (OR 1.96; 95% CI 1.01–3.79). None of the other common genotypes were significantly associated with breast cancer risk in this population (see Table II).


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Table II. Relative risk of breast cancer in relation to STK15 polymorphisms

 
We examined possible interactions of the functional F31I polymorphism with estrogenic breast cancer risk factors (body mass, total ovulatory cycles and hormone replacement therapy in post-menopausal women) (Table III). No significant interactions were detected for these risk factors. We also examined potential interactions considering the combined I31/V57 at-risk genotype versus the referent F31/I57 genotype and, similarly, the results indicated no modifying influence of STK15 genotype on association with estrogenic risk factors (data not shown). However, the statistical power of these analyses was limited because of the low prevalence of the at-risk genotypes in the study population.


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Table III. Relative risk for breast cancer according to F31I polymorphism and estrogenic risk factors

 
We also considered the relation of the STK15 polymorphisms to breast cancer subtype (invasive versus in situ) (data not shown). Though based on limited numbers of women with in situ breast cancer, there was evidence that the AA + GG genotype was more strongly associated with this breast cancer subtype: for AA + GG carriers the relative risk of invasive breast cancer (based on 38 exposed cases) was modestly and non-significantly elevated (OR 1.45; 95% CI 0.85–2.48), whereas the risk for in situ disease (based on 6 exposed cases) was increased nearly 3-fold (OR 2.93; 95% CI 1.01–8.53).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To our knowledge, these data provide the first evidence that polymorphic variations in STK15 is a genetic susceptibility factor in breast cancer. Cancer relative risks were significantly increased in homozygous I31 allele carriers but not in heterozygous individuals. Relevant to our findings, the I31 allele has recently been reported to increase the risk of esophageal squamous cell carcinoma (13) and ovarian cancer (14). The I31 allele is virtually devoid of binding activity to UBE2N, whereas the F31 isoform shows binding and co-localization in the centrosome during centrosome duplication. Although it is still not clear whether this interaction leads to activation of STK15 or to inactivation and degradation, overrepresentation of the I31 variant has been shown to induce cell growth and transformation. This observation may explain why breast cancer is more prevalent in homozygous I31 carriers. The SNPs at codons 31 and 57 are in Aurora Box 1 and Aurora Box 2, respectively, in the N-terminal region of STK15, which influence degradation of this kinase. Although the codon 57 polymorphism does not modify STK15 degradation (H.Nagase, unpublished data), the amino acid stretch 56R(V/I)PLQAQKL, which includes amino acid residue 57, is similar to four other consensus RXXL amino acid stretches in the kinase domain and might affect secondary structure and perhaps catalytic activity of the protein. Although there is no evidence that the codon 57 polymorphism induces altered catalytic activity of STK15, the codon 57 SNP could modify the functional effect of codon 31 SNP. A recent publication has also suggested that the aurora kinase A is a key regulatory component of the p53 tumor suppressor gene pathway and that overexpression of aurora kinase A leads to increased degradation of p53, causing down-regulation of checkpoint response pathways and facilitating oncogenic transformation of cells (17).

In this study the risk of breast cancer was enhanced among women with the I31 and V57 genotype, an association that appeared stronger for in situ breast cancer. These results are based on limited numbers and could have arisen by chance. However, potentially relevant to this finding, STK15 has been reported to be a possible mediator of the transition of ductal carcinoma in situ (DCIS) to invasive cancer (18). Hoque et al. reported that overexpression of STK15 protein is found in a high proportion of tissues from normal mammary duct and DCIS; in contrast, they reported expression to be largely absent in invasive ductal tumor at adjacent sites. Based on these observations these authors speculate that elevated expression of STK15 could be an early event in breast carcinogenesis. However, others have reported essentially opposite findings, that STK15 overexpression is nearly universal in invasive ductal carcinomas of the breast, whereas it is largely absent in normal ductal and lobular cells (19). The current results support the view that a misfunctioning centrosome kinase, and resulting genomic instability, may contribute to the development of in situ breast cancers, approximately half of which will progress to invasive disease.

Breast cancer risk is known to be influenced by prolonged estrogen stimulation (20) and the influence of misfunctioning STK15 could potentially be more important in women with higher exposure to the mitogenic influence of estrogen. However, the present data did not confirm an interaction of STK15 polymorphisms with indices of estrogen exposure, including body mass index, total lifetime ovulatory cycles and hormone replacement therapy. It should be stated that our data were limited in detecting such an interaction due to the low prevalence of at-risk genotypes in the study population.

Additional analyses are needed to clarify these relationships and to investigate potential interactions with other genes involved in STK15 activity, as well as other breast cancer risk factors that may work in conjunction with STK15 genotype to affect breast cancer risk. The positive results in this case–control study should stimulate further study of this relatively frequent cancer susceptibility gene.


    Notes
 
8 Correspondence may also be addressed to H.Nagase. Tel: +1 716 845 1546; Fax: +1 716 845 1698; Email: hiroki.nagase{at}roswellpark.org Back


    Acknowledgments
 
The authors wish to thank the interviewers and staff and all of the participants in the Collaborative Breast Cancer Study for their contributions to the study, David S.Chervinsky, Roger L.Eddy Jr, HaiShen Chen and Jun Igarashi for genotyping and laboratory assistance and Adam Morgan for technical assistance in preparing the manuscript. We also acknowledge Dr Wanqing Wen for his assistance in evaluating linkage disequilibrium in the STK15 variants. Finally, we wish to thank Drs Meir Stampfer and Walter Willett for their expert advice and contributions throughout the case–control study. This work was supported by Roswell Park Cancer Institute NCI Cancer Center Support Grant no. CA16056.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 27, 2004; revised June 24, 2004; accepted July 6, 2004.