There is a rich body of evidence supporting vitamin D as a potent immunomodulator [1
]. VDR and vitamin D activating enzyme 1α-hydroxylase is expressed in almost all immune cells [5
]. While enhancing innate immune responses against bacterial infection, vitamin D modulates adaptive immunity by influencing the actions of antigen presenting cells (APCs) and T-helper lymphocytes. Consistent with its roles in mediating tolerant immunity, vitamin D has been linked to a number of autoimmune diseases [6
], and vitamin D supplementation is efficacious in preventing or curing autoimmune diseases in some preclinical studies [1
]. Among the genes upregulated by vitamin D stimulation, it was found that genes involved in autoimmune diseases are particularly enriched [7
]. Moreover, in randomized clinical trials of patients with cardiovascular disease and colorectal adenoma, vitamin D supplementation significantly alters patients’ immune profiles [8
Given the evidence of potential immuno-modulating activities of vitamin D, it is plausible to speculate that there may be interactions between vitamin D and the immune system relevant to the risk of breast cancer. It has been shown in prostate cancer cell lines that the combination of calcitriol with the non-steroidal anti-inflammatory drugs (NSAIDs), including naproxen and ibuprofen, can achieve the same magnitude of cancer cell growth inhibition at 1/2–1/10 the concentrations of the drugs used as a single agent [10
]. Similar synergistic effects have not yet been examined in breast cancer.
A number of epidemiologic studies have examined the role of vitamin D in breast cancer etiology with inconclusive results. One reason for the inconsistent findings might be cancer heterogeneity—the association of a risk factor with cancer may differ by cancer subtypes. For breast cancer, estrogen receptor (ER) is one of a few most important prognostic and predictive biomarkers for breast cancer. It is likely that ER-positive and ER-negative breast cancer follows distinct etiology pathways. We hypothesized that vitamin D is related to breast cancer in an ER-status specific way, which were examined by comparing ER+ vs.
ER− cancer. In our previous study, we found that high blood levels of 25-hydroxyvitamin D (25OHD) were associated with a lower risk of breast cancer, particularly risk of the estrogen receptor (ER)-negative and triple-negative cancer subtype, in premenopausal women [11
]. Similarly to 25OHD, we subsequently found that high levels of plasma cytokines, including interferon α2 (IFNα2), tumor necrosis factor α (TNFα), and interleukin 5 (IL5), as well as several ratios of T-helper type 1 (Th1) to Th2 cytokines, were associated with increased likelihood of ER negative and triple-negative breast cancer compared to ER positive or luminal A tumors in premenopausal women [12
]. Although a few cytokines have been examined in relation to ER- and triple-negative breast cancer, these studies focused mostly on tumor-associated changes in the microenvironment. Our study was the largest examining circulating levels of a variety of cytokines, chemokines and their receptors. Given these previous results and the established literature on the immuno-modulating properties of vitamin D [5
], we speculated that the associations of 25OHD and cytokines with breast cancer characteristics may be inter-correlated and that the combinational associations are stronger than either factor alone.
Among 490 women, newly-diagnosed with invasive breast cancer in our previous studies of 25OHD and cytokines, we examined the inter-dependence of the associations of 25OHD and cytokines and further, their combinational associations with breast cancer ER status.
In this study, we examined the combinational associations of serum levels of 25OHD with plasma levels of three cytokines, IFNα2, TNFα and IL5, as well as ten cytokine ratios, on ER status of breast cancer. There was no correlation between blood levels of 25OHD and any of the cytokines/ratios, and the main effects of 25OHD and cytokines on breast cancer ER status, primarily in premenopausal women, were independent of each other. However, when considered together, premenopausal women with low 25OHD and high TNFα had the highest likelihood of having ER negative breast cancer, which was higher than the addition of main effects of each. Similar synergistic associations were also found for IL5 and several cytokine ratios. On the contrary, there was some evidence for antagonistic associations between 25OHD and IFNα2 on ER status, which was also found for two cytokine ratios where IFNα2 was the numerator. These findings, although preliminary due to the limited sample size and retrospective measurement of the analytes, provide the first evidence for extensive interactions between vitamin D and immune factors, which may influence breast cancer risk and aggressive characteristics.
The strongest synergistic association on breast cancer ER status was found between 25OHD and TNFα. As a pro-inflammatory cytokine from macrophages and tumor microenvironment, TNFα has been implicated in cancer promotion and progression [16
], possibly by mediating epithelial-mesenchymal transition required for tumor invasion and angiogenesis [17
], and has been investigated as a target for cancer therapy [20
]. In breast tumor tissues, high expression of TNFα has been linked with ER negativity and cancer relapse [17
]. While there are numerous studies of either TNFα or vitamin D in breast cancer, studies on their interactions are scarce. In cell culture and animal studies, vitamin D treatment was found to suppress the production of TNFα [22
]. In a small randomized trial among patients with colorectal adenoma, 800 IU/day of vitamin D3 supplementation for over six months only non-significantly decreased TNFα by 13% [9
]; while in two earlier small trials among patients with congestive heart failure or ambulatory adults, 2,000 IU/day of vitamin D3
had no effect on circulating levels of TNFα [8
]. In our study, we did not find a correlation between blood levels of 25OHD and TNFα in breast cancer patients, which was consistent with the results of the two trials. Furthermore, we found that the effects of 25OHD and TNFα on breast cancer ER status were independent from each other. Nevertheless, the group of premenopausal patients with combined low 25OHD and high TNFα levels were at the highest odds of ER negative vs. ER positive breast cancer, much higher than the expected additive effects from each analyte. Given the independence of circulating levels and main effects between 25OHD and TNFα, this observed synergistic association implies separate but inter-connecting pathways underlying the roles of the two analytes on the etiology of breast cancer subtypes. The exact biological mechanisms for the synergy are unclear.
Similarly to the case of TNFα, we also found synergistic associations between 25OHD and IL5 on breast cancer ER status in premenopausal patients. IL5 is produced by Th-2 lymphocytes and plays an important role in B-cell differentiation and eosinophilopoiesis [25
]. IL5 has been linked with anti-tumor activities, specifically, tumor immune surveillance by eosinophils as demonstrated in lung and fibrosarcoma mice models [26
]. In-depth studies of IL5 in breast cancer are lacking. In one small study of 105 breast cancer patients, IL5 expression could not be detected in tumor tissues [21
], while in another study of 35 breast cancer patients and 24 women with benign breast diseases, serum IL5 level was significantly higher in cancer patients [28
]. This finding, as well as the finding from our study that high IL5 level was associated with increased odds of ER negative vs.
ER positive cancer, is inconsistent with the anti-tumor properties of IL5. It is possible that IL5-driven immune surveillance targets more strongly against ER positive cancer cells and thus the higher ratio of ER negative vs.
ER positive odds in patients with high IL5 levels. Alternatively, this observation could be due to a stronger stimulation of IL5 response by ER negative cancer cells. Nonetheless, these observational findings need to be further investigated in prospective studies.
Although vitamin D is known to enhance Th2 immune responses, the relationship between vitamin D and IL5 in human has been ambiguous. In two small trials among ambulatory or obese adults, vitamin D3
supplementation did not increase circulating IL5 concentrations [29
], consistent with our finding of no correlation between 25OHD and IL5 levels in breast cancer patients. The potential biological mechanisms to explain the synergistic associations between 25OHD and IL5 on ER status in our study are unclear, but the observational finding may provide a new clue for future studies of the roles of vitamin D in mediating Th2 immune responses.
In addition to the above synergistic associations, we also found some evidence of antagonistic associations between 25OHD and IFNα2, as well as IFNα2 to Th2 cytokine ratios, on breast cancer ER status in premenopausal patients. IFNα2 belongs to the large family of interferon alpha, which is Th1 cytokine in response primarily to viral infection. Deletion of IFNA
gene has been observed in acute leukemia and glioma cases [31
], and impaired interferon signaling was an immune deficiency common in human cancers, including breast cancer [32
]. Although vitamin D generally suppresses Th1 immune responses, we did not find any correlation between circulating levels of 25OHD and IFNα2. The antagonistic associations on breast cancer ER status could possibly be due to over saturation of the deleterious effects resulting from low vitamin D and high IFNα2 levels.
The synergistic associations between vitamin D and cytokines on breast cancer aggressive characteristics may have important implications to breast cancer prevention and prognosis. Both vitamin D and NSAIDs have been studied separately in relation to reduced breast cancer risk and superior prognosis [33
]. Nevertheless, despite mounting evidence on extensive roles of vitamin D in regulating immune responses, these two potentially “actionable” cancer prevention agents have seldom been considered together. One previous study demonstrated a clear synergistic effect between calcitriol and NSAIDs on inhibiting the growth of prostate cancer cells in vitro
]. Our study based on a cohort of breast cancer patients demonstrates for the first time that such synergistic effects may also exist in breast cancer, and calls for future prospective studies to evaluate the combined use of vitamin D and NSAIDs for cancer prevention and prognosis.
A limitation of our study is that blood samples were drawn at the time of diagnosis, and may not necessarily reflect etiologic events but rather, could be the result of disease onset or progression. Although the average time from diagnosis to the time of blood draw was relatively short (27 days), and the majority of blood samples were collected before any treatment (92%), we still cannot exclude a possibility of reverse causal relationships between blood analytes and breast cancer aggressive characteristics. However, exclusion of the samples collected after the initiation of treatment had little impact on the results. Another limitation of our study is the relatively small number of patients included in the analysis, particularly in subgroup analyses among premenopausal women. As a result, none of the interaction tests reached statistical significance when multiple comparison error was considered. Due to the concern of multiple testing, we refrained from analyzing the interactions between 25OHD and all cytokines and their ratios examined in our previous study, but instead focused on those shown significant main effects. However, it is possible that significant interactions exist even without significant main effects. In our previous analyses of the main effects of 25OHD and cytokines, we examined both ER status and triple-negative subtype vs. luminal A subtype. However, when examining the combinational associations between 25ODH and cytokines, the numbers of triple-negative breast cancer patients were rather limited, especially after stratifying by menopausal status. Although associations for triple negative breast cancer were similar to those reported here for ER status, that women with low 25OHD and high cytokine were most likely to have triple negative breast cancer vs. luminal A subtype (data not shown), the confidence intervals of the ORs were very wide; thus, we reported only the results of ER status.