Introduction
American women of African ancestry (AA) are more likely to develop breast cancer at a younger age than those with European ancestry (EA) and are more likely to have tumors with aggressive characteristics, including high histological grade, negative estrogen receptor (ER) status, and basal-like - ER
- and/or progesterone receptor (PR)
-, HER2
-, and cytokeratin 5/6
+ and/or HER1
+ -features [
1,
2]. The reasons for these racial disparities are unknown.
It is clear that, among geographically diverse populations, certain genotypic and phenotypic characteristics may be selected for in response to local environmental pressures [
3]. Skin pigmentation, the primary factor that provides protection from ultraviolet (UV) radiation, is correlated with latitude, and dark skin pigmentation is likely to be the original ancestral trait in humans. Migrations to Europe and Asia eventually gave rise to decreased pigmentation and lighter skin [
4‐
6]. As much as 90% of vitamin D is derived from sun exposure, but high skin melanin concentration prevents penetration of UVB light and compromises synthesis efficiency by 10 to 50 times [
7]. Although high pigmentation would reduce absorption of vitamin D, intense sun exposure in sub-Saharan Africa would compensate. However, in high-latitude areas where UVB intensity is low and where more time may be spent indoors (particularly in winter), vitamin D deficiency may result among individuals with higher skin pigmentation. Indeed, in the US, the prevalence of 25-hydroxyvitamin D (25OHD) of less than 15 ng/mL is almost 10 times higher in AA than in EA women [
8], and the prevalence of severe vitamin D deficiency (< 10 ng/mL) among AAs was 29% in 2001 to 2004 [
9]. In contrast, in Guinea-Bissau, the average 25OHD levels in healthy Africans were 34 ng/mL, and the prevalence of severe vitamin D deficiency was as low as 1% [
10].
Endogenous 25OHD may also be affected by variability in metabolic pathways, and synthesis and metabolism catalyzed by two major enzymes, 1α-hydroxylase and 24-hydroxylase, which are encoded by
CYP27B1 and
CYP24A1, respectively. Binding of vitamin D to the vitamin D receptor (VDR) activates or suppresses gene transcription, depending on the type of response elements [
11], and genetic variability in the above genes, known to differ by ancestry [
12], is likely to affect vitamin D signaling.
Laboratory, preclinical, and clinical findings support the hypothesis that low levels of vitamin D are related to breast cancer risk. In the human mammary gland, VDR is expressed in all cell types [
13], and vitamin D treatment inhibits breast cancer cell proliferation, induces cell apoptosis, and prevents carcinogenesis in rodent models [
14,
15]. However, epidemiologic evidence for associations between vitamin D and breast cancer risk is considered 'limited' [
16], and one randomized trial showed little impact of vitamin D supplementation on breast cancer incidence [
17]. These inconclusive findings could be due to tumor heterogeneity, which implies that the effects of vitamin D may only present in specific breast cancer subtypes. In fact,
Vdr knockout mice were more likely than their wild-type littermates to develop
Er/Pr- tumors [
18]. Consistent with results from these preclinical studies, some epidemiologic studies have also indicated that effects of vitamin D may be strongest for breast cancers with poor prognostic characteristics and that lower serum 25OHD levels are found among women with ER
- compared with ER
+ tumors [
19‐
21]. Recently, we reported lower levels of serum 25OHD in women with high- versus low-grade breast tumors and in women with triple-negative versus luminal A breast tumors [
22].
Here, we examined levels of 25OHD in AA and EA women without breast cancer in relation to self-reported race as well as ancestry, which was estimated by using ancestry informative markers (AIMs). 25OHD levels in women with breast cancer could be a result of disease processes, and some samples were obtained after chemotherapy was initiated; thus, we did not compare serum levels of 25OHD between cases and controls. Instead, we evaluated variants in vitamin D activity and major metabolism (VDR, CYP27B1, and CYP24A1) in relation to breast cancer risk, particularly in relation to self-reported race and estrogen receptor status. We also tested whether vitamin D-related genetic variants could explain, in part, the higher prevalence of ER- breast cancer among AA women.
Discussion
In this study, we found that relationships between breast cancer risk and variants in genes associated with vitamin D activity and metabolism,
VDR and
CYP24A1, differed depending upon self-reported race and that associations were most notable for risk of ER
- breast cancer in both AA and EA women. Importantly, we found that rs2209314 and rs2762941 in
CYP24A1 contributed significantly to the higher risk of ER
- breast cancer in AA than EA women. Among controls in the WCHS, serum levels of 25OHD were notably lower in AA women than EA women, the lowest levels were among women with the greatest African ancestry estimated by AIMs, and VDR expression levels, as estimated from published data on cultured lymphoblastoid cells [
32], were also lower in AA women. In a previous study among EA women, we found that low 25OHD levels were associated with increased risk of ER
- breast cancer, both in comparison with controls and with women with ER
+ breast cancer [
22]. Given all of these data, it is possible that low 25OHD levels in AA women, coupled with unique 'at-risk' genetic variants, contribute, in part, to the higher prevalence of ER
- breast cancer among AA women. If these potential associations were to be consistently observed in future studies, our results would support a public health effort for vitamin D supplementation to reduce risk of aggressive breast cancer among AA women.
The finding of an inverse association between African ancestry estimated by AIMs and blood 25OHD levels is consistent with a recent community cohort study of AA men and women [
33]. Our findings of extensive racial differences in allele frequencies and LD patterns for SNPs in
VDR and
CYP24A1 are also consistent with those from an earlier study [
12]. Previous studies on
VDR polymorphisms and breast cancer risk have focused on only a few SNPs. However, we did not find any relationship with Fok1, Bsm1, or Taq1 or a three-SNP haplotype consisting of Bsm1, Apa1, and Taq1 in either AA or EA women. The variant homozygote of Apa1 was associated with increased risk of breast cancer in EA women, but the effect was limited to post-menopausal women. Increased risk of breast cancer was also reported in a previous study for Apa1 [
34]; however, results in the literature are conflicting [
35,
36]. The G allele of Cdx2 was associated with lower risk of breast cancer in AA women in our study and this finding was in contrast to the speculated functional alteration that the variant G allele resulted in lower binding of the Cdx2 protein and thus lower transcriptional activity of VDR [
37,
38]. Similar to a study among women in Germany [
39], our study found no association of Cdx2 with breast cancer risk in EA women.
To date, three studies have examined selected SNPs in
VDR with breast cancer risk in both AA and EA women. Two of them did not find associations for Fok1, Bsm1, Bgl1 (rs739837), or the 3' untranslated region poly(A) microsatellite in either AA or EA women [
40,
41], and a third study found increased risk by Bsm1 variant in EA but not AA women [
42]. However, none of the above studies examined the associations by ER status or used the systematic approach we employed to capture variation across the genes.
We found that four SNPs in VDR and two SNPs in CYP24A1 had differential associations with breast cancer by race (P for interaction was not more than 0.10). The fact that the associations were not consistent in AA and EA populations corroborates the differences in blood levels of vitamin D and frequency and LD pattern of vitamin D-related genetic variants, implying that the race-specific associations might be the result of gene-environment interactions. In further analyses stratified by ER status, one SNP in VDR and seven SNPs in CYP24A1 were specifically associated with ER- but not ER+ cancer risk, and the associations differed between AA and EA women. Controlling for the two SNPs in CYP24A1 in a multivariate model substantially reduced the increased ER- breast cancer risk associated with AA race and made the association no longer significant. This provides the first evidence supporting the contribution of vitamin D-related genetic variants to higher risk of more aggressive breast cancer in AA women.
We found significant associations between breast cancer risk and a number of tag SNPs in
VDR without previously known functionality. However, in our analyses, SNP rs2239186 was associated with increased serum levels of 25OHD in AA women without breast cancer. This SNP and a haplotype containing it were also significantly associated with reduced breast cancer risk in AA women, irrespectively of ER status. This SNP resides in an intronic region and thus is unlikely to be the causal variant. However, it may be a marker for a causal SNP outside of the
VDR gene. This SNP has not been implicated in other breast cancer studies but has been shown to be associated with reduced risk of colorectal cancer in individuals with low vitamin D intake [
43] and was also implicated in type I diabetes [
44]. This SNP may warrant future replication and fine-mapping studies.
The two SNPs, rs2209314 and rs2762941, in
CYP24A1 shown to be associated with racial differences in ER
- breast cancer risk are intronic. Although these SNPs have not been implicated previously in breast cancer, elevated expression of CYP24A1 was found in breast cancer tissues [
45], indicating a potential role in breast cancer etiology. We did not observe associations of these two SNPs in
CYP24A1 with serum 25OHD levels in either AA or EA populations, indicating that these two SNPs themselves or linked causal variants may affect ER
- breast cancer not through altering circulating 25OHD levels but availability of vitamin D in local mammary tissues.
One limitation of our study is the lack of validation for the significant findings. The number of patients with breast cancer and controls was relatively limited, especially after stratification by race and ER status. None of the associations with SNPs, except for rs2239186, remained significant after correction for multiple comparisons. We thus could not exclude the possibility of false-positive findings in our data. However, the fact that rs2239186 was associated with higher serum 25OHD levels in AA women as well as reduced breast cancer risk in this population is biologically coherent and reduces the likelihood of spurious findings for this
VDR SNP. Another limitation is that only three genes in vitamin D-related pathways were included in this study. Although
VDR,
CYP27B1, and
CYP24A1 are the three key genes in this pathway, genes encoding for some other vitamin D metabolizing enzymes, particularly
GC encoding for vitamin D binding protein (which has been related to circulating vitamin D levels), may also be related to breast cancer risk and warrant further studies. AA women are more likely to develop breast cancer at a younger age than EAs; we enrolled all eligible AA women but randomly selected eligible EA women, frequency-matching by 5-year age categories. We also initially limited eligibility to women 65 years or younger because of low participation of older women without breast cancer to case-control studies. Thus, the overall study population is younger than that of some other studies. Although we found no evidence of modification effects by menopausal status for any but two SNPs (Table S5 of Additional file
1), the high proportion of pre-menopausal women in this study needs to be considered in relation to generalizability.
Acknowledgements
This work was supported by grants from the US Army Medical Research and Material Command (DAMD-17-01-1-0334), the National Cancer Institute (R01 CA100598 and P01 CA151135), and the Breast Cancer Research Foundation and a gift from the Philip L Hubbell family. SY was the recipient of a pre-doctoral training award from the Department of Defense Breast Cancer Research Program (W81XWH-08-1-0223). EVB was the recipient of a career transition award from the National Cancer Institute (K22 CA138563). Samples were stored and managed by the Roswell Park Cancer Institute (RPCI) DataBank and BioRepository, and genotyping was performed in the RPCI Genomics Core Facility; both are Cancer Center Support Grant-shared resources supported by P30 CA016056-32. The New Jersey State Cancer Registry (NJSCR) is a participant in the Centers for Disease Control and Prevention's National Program of Cancer Registries and is a National Cancer Institute Surveillance, Epidemiology, and End Results (SEER) Expansion Registry. The NJSCR is supported by the Centers for Disease Control and Prevention under cooperative agreement DP07-703 awarded to the New Jersey Department of Health & Senior Services. The collection of State of New Jersey cancer incidence data is also supported by the National Cancer Institute's SEER Program under contract N01-PC-95001-20. The funding agents played no role in design, in the collection, analysis, and interpretation of data, in the writing of the manuscript, or in the decision to submit the manuscript for publication.
We are grateful to the women who participated in this study and to colleagues, physicians, and clinical staff at participating hospitals in New York who facilitated identification and enrollment of cases into the study: Kandace Amend (i3 Drug Safety), Helena Furberg (Memorial Sloan-Kettering Cancer Center), Thomas Rohan and Joseph Sparano (Albert Einstein College of Medicine), Kitwaw Demissie (University of Medicine and Dentistry of New Jersey), Paul Tartter and Alison Estabrook (St. Luke's Roosevelt Hospital), James Reilly (Kings County Hospital Center), Benjamin Pace, George Raptis, and Christina Weltz (Mount Sinai School of Medicine), Maria Castaldi (Jacob Medical Center), Sheldon Feldman (New York-Presbyterian), and Margaret Kemeny (Queens Hospital Center). We also thank Dr. Eileen M. Dolan for the permission for using the expression data of vitamin D receptor in HapMap lymphoblastoid cell lines.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SY helped to develop the hypothesis, design the study, and perform the data analysis and interpretation. CBA helped to develop the hypothesis, design the study, provide the data and biospecimen from the WCHS, perform the data analysis and interpretation, and provide financial support for the study. GZ helped to provide the data and biospecimen from the WCHS and to perform the data analysis and interpretation. DHB, LJ, MR, GC, WD, KSP, and EVB helped to provide the data and biospecimen from the WCHS. HH provided pathological data of tumor samples. C-CH, LES, LT, CSJ, DLT, SEM and FA helped to perform the data analysis and interpretation. HZ helped to provide financial support for the study. All authors read and approved the final draft of the manuscript.