Osteoprotegerin and breast cancer risk by hormone receptor subtype: a nested case-control study in the EPIC cohort

Osteoprotegerin (OPG) is a homodimeric glycoprotein and a member of the tumor necrosis factor (TNF) receptor superfamily [1]. OPG was first characterized as a negative regulator of bone turnover. Bone homeostasis is maintained by the interplay between the receptor activator of nuclear factor kappa-B (RANK), its soluble activation ligand (RANKL), and OPG. OPG binds RANKL as a decoy receptor, inhibiting the activation of RANK by RANKL and preventing the differentiation of bone marrow precursor (monocyte/macrophage) cells to osteoclasts — cells that are central in the process of bone resorption [2].

Besides bone, OPG is expressed in other tissues, including the stomach, intestines, skin, liver, heart, lung, kidney, and breast. This expression across diverse tissue types indicates that its biologic functions may extend beyond bone metabolism. In relation to breast cancer, OPG was initially investigated as a potential inhibitor of metastasis-related osteolysis [3, 4]. The RANKL inhibitor denosumab acts to reduce skeletal-related events in patients with bone metastases. More recent data show that OPG is also produced in breast tumor cells, and that it can promote tumor growth and metastasis [5, 6]. In vitro studies suggest that OPG, again acting as a decoy receptor, also binds to TNF-related apoptosis-inducing ligand (TRAIL), thereby preventing cancer cell death via apoptosis [7, 8]. Furthermore, OPG can induce cell proliferation and angiogenesis through a variety of cell surface receptors [5, 6].

Besides local expression in many tissue types, OPG is present in blood. Circulating OPG has been examined in the context of chronic disease risk, including impaired glucose tolerance and type 2 diabetes [9], vascular calcification, atherosclerosis and cardiovascular disease [10, 11], cancer [12], and overall mortality [10]. However, to date, only one prospective study has examined the relationship between circulating OPG and breast cancer risk [12]. This study observed reduced breast cancer risk among women with comparatively high OPG concentrations, but given a small number of incident cases (n = 76), risk by tumor subtypes (e.g., by estrogen (ER) and progesterone (PR) receptor status) was not investigated. We provide the first prospective data on OPG and breast cancer risk by hormone receptor status, in a large nested case-control study in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort.

The EPIC cohort was designed to identify risk factors for cancer [13]. Briefly, between 1992 and 2000 about 520,000 apparently healthy men and women (n = 367,993 women), from 10 European countries (Denmark, France, Germany, Greece, Italy, the Netherlands, Norway, Spain, Sweden, and the United Kingdom), generally between ages 35 and 75 years, enrolled in the study. Detailed dietary, anthropometric, lifestyle, and medical history data were collected via standardized methods. Blood samples were collected according to standardized protocols (n = 235,607 women). Samples for this study were stored centrally under liquid nitrogen (–196 °C) at the International Agency for Research on Cancer (IARC), except those from Denmark, which were stored locally at –150 °C.

Incident cancers were identified through linkages with regional cancer registries except in France, Germany, Greece, and Naples (Italy), where follow-up is conducted by review of health insurance records, contacts with cancer and pathology registries, and/or direct contact with cohort members. Vital status is ascertained through linkages with regional and national mortality registries and active follow-up (Germany and Greece). Details on the identification and verification of incident breast cancer cases and collection of hormone receptor status data were reported previously [14].

A total of 462 (23%) case-control pairs were premenopausal at blood donation, whereas 1546 pairs were postmenopausal (Table 1). Among postmenopausal women, a total of 755 pairs (49%) were using exogenous hormones at blood collection. Median age at baseline was 57 (10^th–90^th percentiles: 45–64) years, and among cases the median age at diagnosis was 61 (10^th–90^th percentiles: 50–70) years. Median time between blood collection and diagnosis was 4.7 (10^th–90^th percentiles: 1.2–8.1) years. Of the 2008 total cases, 81% were ER+ (n = 1622; ER–, n = 386).

Serum OPG concentrations were positively correlated with age (r = 0.29, p < 0.01). Among premenopausal women, OPG did not vary by menstrual cycle phase (p = 0.29). OPG concentrations did not vary by menopausal status at blood collection (p = 0.31). However, among women postmenopausal at blood collection, OPG levels differed between current users and non-users of HT (at time of blood collection; p = 0.01), although the absolute differences were small (adjusted geometric mean concentrations, ng/mL, HT non-users: 0.202 [95% CI: 0.197–0.207] vs. HT users: 0.210 [0.204–0.216]). Among HT users, OPG levels did not further differ by type of HT (i.e., unopposed vs. opposed estrogens; p = 0.64).

OPG concentrations were not correlated with age at menopause, number of FTPs, BMI (kg/m²), or alcohol consumption (g/day), nor did concentrations differ between women with and without completed FTP (data not shown). Higher OPG concentrations were observed in women reporting ever OC use, as compared to never users (ng/mL, OC use, never: 0.192 [0.188–0.198]; ever OC users: 0.198 [0.193–0.203]; p = 0.02). Among women with additional hormone measurements available (n: 297–2008) OPG was not correlated (r < |0.14|) with endogenous sex steroid hormones, IGF-I, or prolactin, among either pre- or postmenopausal women, or within strata of postmenopausal HT users and non-users (Additional file 1: Table S2).

We observed significant heterogeneity in the association between OPG and risk of ER+ vs. ER– tumors (p _het = 0.02) (Table 2). Higher concentrations of OPG were associated with increased risk of ER– breast cancer (top vs. bottom tertile RR =1.93 [95% CI 1.24–3.02]; p _trend = 0.03). In contrast, we observed a suggestive inverse association between OPG and ER+ disease (top vs. bottom tertile RR = 0.84 [95% CI 0.68–1.04]; p _trend = 0.18). The association between OPG and ER– disease did not differ by menopausal status (p _het = 0.97). However, we observed suggestive heterogeneity by menopausal status for ER+ disease (p _het = 0.05; premenopausal, RR_log2 = 0.53 [0.31–0.88]); postmenopausal, RR_log2 = 0.97 [0.75–1.25]; Table 3). However, there was evidence of potential non-linearity of the association between OPG and ER+ breast cancer among women premenopausal at blood collection (p = 0.03; Additional file 1: Figure S1), and this association should be interpreted cautiously. We observed no heterogeneity by HT use (among postmenopausal women; p _het ≥ 0.43) or by age at breast cancer diagnosis (age <50 vs. 50+; p _het ≥ 0.30; Table 4). Results were similar in analyses considering both ER and PR status.

We observed no evidence for effect modification by other reproductive and lifestyle factors (e.g., ever full-term pregnancy, OC use, age at menopause (postmenopausal women) or by levels of endogenous hormones (e.g., estradiol, progesterone, prolactin; data not shown), with the exception of suggestive heterogeneity by prolactin concentrations for ER– disease (prolactin < median, RR = 0.78 [0.23–2.69]; ≥ median, RR = 2.98 [1.23–7.23]; p _het = 0.05) and heterogeneity by testosterone concentrations for ER+ disease (testosterone < median, RR = 0.35 [0.15–0.79]; ≥ median, RR = 1.05 [0.52–2.13]; p _het = 0.04). Finally, results were similar in sensitivity analyses restricted to women diagnosed >2 years after blood collection. OPG demonstrated high within-person reproducibility, in terms of the relative ranking of study participants, over 1 year (r = 0.85) and 14 years (r = 0.75).

Higher serum osteoprotegerin (OPG) concentrations were associated with an increased risk of ER– breast cancer, and a modest, suggestive reduction in risk of ER+ tumors, in this large investigation. With a total of 2008 breast cancer cases with documented ER status, this is the first large-scale prospective study to address circulating OPG and subsequent breast cancer risk by hormone receptor subtype.

Vik et al. reported an inverse association between OPG concentrations and breast cancer risk in a case-cohort study of more than 6000 women and men (median 13.5-year follow-up) [12]. This Norwegian investigation included only 76 breast cancer cases and thus could not address risk by hormone receptor status. Most likely, however, the majority of breast cancer cases were ER+, as this is the predominant subtype among European women. The suggestive inverse association between OPG and ER+ breast cancer risk observed in the current study lies in the same direction as in this previous investigation, although the association observed in our study is weaker. In two analyses restricted to BRCA1/2 carriers, higher serum OPG concentrations were associated with lower risk mutation site [22] and lower breast cancer risk (n = 18 cases) [23]. BRCA1/2 mutation status was not available in our study population, and family history of breast cancer is missing for the majority of participants (64%). Therefore, we were unable to evaluate OPG and breast cancer risk restricted to a higher risk population in our study. These three investigations used ELISA assays, while we used an electrochemiluminescence assay. Vik [12] and Oden [23] observed starkly different mean/median OPG concentrations in their two study populations (Vik, reported mean concentration: 3.40 ng/mL; Oden, reported median concentration, 95 ng/mL). Widschwendter et al. [22] did not directly report mean or median OPG concentration. To date, there is no cross-assay standardization protocol for OPG. Thus, between-study comparisons must rely on the within-study relative ranking of participants.

Prior investigations show high levels of OPG protein or mRNA in a variety of breast cancer cell lines [3, 6, 24‐27], with lower levels in primary human mammary epithelial cells [6]. Studies of primary breast tumors also showed variable degrees of OPG protein [3, 6, 24, 28‐30] or mRNA expression [30‐34]. In larger patient series, immunohistochemical analyses [24, 29, 35] indicated OPG protein in 40-45% of invasive primary tumors, an inverse association between OPG expression and tumor grade and stage, and a positive association with ER status [24, 29, 30]. Studies based on mRNA analysis, however, showed a positive [36], a negative [33, 34], or no association [32] between the gene expressions for OPG and ER status. A limited number of studies have examined non-neoplastic mammary tissue. On balance, these investigations observed some level of OPG expression, however, at lower levels than that observed in breast tumor tissue [6, 29, 31, 37]. In a large, multicenter analysis of more than 4500 patients, higher OPG mRNA expression was associated with reduced breast cancer-specific mortality among patients with ER+ tumors (n = 1941), but not among patients with ER– disease (n = 649) [33]. These observations support a role for OPG in breast tumor development and prognosis, with potential differences between ER- and ER+ disease.

We observed increased risk of ER– (and ER–/PR–) breast cancer among women with higher serum OPG concentrations. OPG binding to TRAIL represents one key mechanism through which OPG could promote breast tumor development, particularly of ER– tumors [8, 38]. TRAIL is highly homologous to tumor necrosis factors (TNFs), and induces apoptosis through specific TRAIL receptors in a diverse range of cell lines, and preferentially in cancer cells [39]. Analyses in breast cancer cell lines have shown that triple-negative cells (i.e., ER–/PR–/HER2–) are especially sensitive to TRAIL-induced apoptosis, whereas sensitivity to TRAIL varies across different HER2+ cell lines, and is systematically absent among ER+ cells [7]. Our study population included only 58 triple-negative cases, and we observed no association in this subgroup. We observed a stronger association between OPG and ER– breast cancer risk among women with relatively high circulating prolactin concentrations. Prolactin inhibits TRAIL-induced apoptosis in hormone (androgen)-insensitive PC3 prostate cancer cells [40]. To our knowledge there are no data on prolactin and TRAIL in breast cancer; however, prolactin inhibition has been shown to increase apoptosis in breast cancer cell lines [41], and prolactin interferes with the apoptotic effect of cisplatin chemotherapy [42].

We observed a suggestive inverse association between OPG and ER+ breast cancer. OPG may reduce breast tumor development through binding to RANKL. In addition to regulating bone turnover, RANK and RANKL mediate progesterone- and prolactin-induced changes in the mammary gland, including the expansion of mammary epithelial stem cells [43], proliferation of the mammary epithelium [44, 45], and the development of a lactating mammary gland during pregnancy [46]. Furthermore, experimental animal studies have demonstrated that increased expression of RANKL in PR+ luminal epithelial cells, following autocrine and paracrine stimulation of RANK signaling in both receptor positive and negative epithelial cells, is a mechanism mediating progestin-driven mammary carcinogenesis [43, 44, 47, 48]. Circulating OPG may modulate these effects, with one investigation showing increased mammary epithelial proliferation after intravenous administration of soluble RANKL (sRANKL), and inhibition of proliferation by the administration of OPG, in an animal model [44]; in vitro investigations of administered RANKL and OPG in human breast tumor samples show similar results [49]. It is conceivable that OPG inhibits the development of ER+/PR+ breast tumors by interfering with the growth-promoting effects of RANKL, which depend on PR-mediated local synthesis.

Breast cancer risk increases with higher lifetime number of menstrual cycles, an indicator for cumulative exposure to luteal phase progesterone levels, and is also increased among postmenopausal women using combined estrogen-plus-progestin HT [50‐52]. Interestingly, RANKL expression in both normal and malignant breast tissue is also increased during the luteal phase of the menstrual cycle [53] and in combined HT users [54]. We observed no indication that the association of serum OPG concentrations with breast cancer risk varied between tumors diagnosed before or after age 50 (the median age at menopause in EPIC), by age at menopause, or between users and non-users of HT.

While several tissue types produce OPG, bone marrow cells and vascular endothelial cells may be the predominant sources of circulating OPG [55‐57]. Estrogens, vitamin D3, and growth hormone promote OPG synthesis in vitro (e.g., by osteoblasts) [55, 58, 59]. Limited cross-sectional data in women support no, or only modest, associations between circulating hormones (e.g., estradiol [60], progesterone [22]) and OPG in healthy women. As in other recent studies [22, 61], we observed no systematic variation of serum OPG concentration with phase of menstrual cycle or with menopausal status (after adjustment for age at blood collection) [62], and a gradual rise of OPG levels with increasing age [58, 62, 63]. Prior studies provide mixed results on whether postmenopausal HT use is associated with OPG [64‐66]. We observed somewhat higher OPG concentrations among postmenopausal women using HT, with no differences by HT type (i.e., opposed vs. unopposed estrogens). In subgroup analyses, where additional measurements of endogenous hormones were available, we observed no correlations between OPG and DHEAS, androstenedione, testosterone, estradiol, IGF-I, or prolactin.

In summary, recent research has revealed intriguing parallels in the roles for OPG and the RANK/RANKL axis in bone metabolism and in mammary gland physiology. Our large-scale investigation is the first to document potentially diverse associations of OPG with risks of ER– or ER+ breast cancer. There is increasing interest in denosumab — a humanized antibody that blocks RANKL signaling, which is used routinely to treat osteoporosis — as a drug for preventing breast cancer recurrence among women with early stage breast cancer and primary prevention in high risk women [67]. Although the role of OPG in breast cancer is complex, due to its possible interactions with both TRAIL and RANKL, our data may provide indirect insight into the possible benefits or risks potentially associated with treatments targeting the RANK/RANKL system.