Abstract
The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is primarily known as a key regulator of calcium and phosphate homeostasis. It exerts its biological functions by binding to the vitamin D receptor (VDR), a transcription factor that regulates gene expression in vitamin D-target tissues such as intestine, kidney and bone. Yet, the VDR is expressed in many additional normal and cancerous tissues, where it moderates the antiproliferative, prodifferentiating and immune-modulating effects of 1,25(OH)2D3. Interestingly, several epidemiological studies show that low levels of 25(OH)D, a biological marker for 1,25(OH)2D3 status, are associated with an increased risk of breast cancer (BC) development. Mendelian randomization studies, however, did not find any relationship between single-nucleotide polymorphisms in genes associated with lower serum 25(OH)D and BC risk. Nevertheless, multiple and in vivo preclinical studies illustrate that 1,25(OH)2D3 or its less calcaemic structural analogues influence diverse cellular processes in BC such as proliferation, differentiation, apoptosis, autophagy and the epithelial–mesenchymal transition. Recent insights also demonstrate that 1,25(OH)2D3 treatment impacts on cell metabolism and on the cancer stem cell population. The presence of VDR in the majority of BCs, together with the various anti-tumoural effects of 1,25(OH)2D3, has supported the evaluation of the effects of vitamin D3 supplementation on BC development. However, most randomized controlled clinical trials do not demonstrate a clear decrease in BC incidence with vitamin D3 supplementation. However, 1,25(OH)2D3 or its analogues seem biologically more active and may have more potential anticancer activity in BC upon combination with existing cancer therapies.
Introduction
Vitamin D3 (cholecalciferol), a natural form of vitamin D, can be obtained from dietary sources (such as fatty fish) but is also generated in the human skin under influence of sunlight (UVB radiation, 290–320 nm) from its precursor, 7-dehydrocholesterol (Leyssens et al. 2013, Christakos et al. 2016, 2019, Jeon & Shin 2018). Since exposure to sunlight is a major trigger for vitamin D3 synthesis in the skin, alterations in sunlight exposure based on season and latitude will influence the ability to synthesise vitamin D3 (Spiro & Buttriss 2014). The biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), is generated by two hydroxylation steps. First, vitamin D3 is hydroxylated at carbon 25 by CYP2R1/CYP27A1 (cytochrome P450 enzymes) in the liver to generate 25-hydroxyvitamin D3 (25(OH)D3), a reliable serum marker for vitamin D status. Next, in the kidney, CYP27B1 hydroxylates 25(OH)D3 at carbon 1 to generate 1,25(OH)2D3. Another important cytochrome P450 enzyme in the kidney is CYP24A1. This enzyme regulates the circulating levels of 1,25(OH)2D3 by hydroxylating carbon 24 not only in 1,25(OH)2D3 but also in 25(OH)D3, thereby decreasing the pool of 25(OH)D3 available for carbon 1 hydroxylation. These 24-hydroxylated products are excreted via urine or faeces (Christakos et al. 2016). 1,25(OH)2D3 exerts its functions after binding to the vitamin D receptor (VDR), which subsequently heterodimerizes with the retinoid X receptor (RXR). Both VDR and RXR are members of the nuclear receptor superfamily. There are three subtypes of RXR (-α, -β and -γ), of which RXRα is the most important isoform for VDR activity (Peehl & Feldman 2004). Binding of VDR to RXR is reported to induce a non-permissive heterodimer complex that cannot be activated by the RXR ligand, 9-cis retinoic acid (RA) (Sanchez-Martinez et al. 2006). Moreover, in vitro studies have shown that 1,25(OH)2D3 enhanced the formation of RXR–VDR heterodimer whereas 9-cis RA reduced their affinity by inducing RXR–RXR homodimerization. Also, the availability of both receptors will influence their responsiveness to the ligands (Lemon & Freedman 1996). However, in vitro studies in MCF7 cells have shown that both 9-cis RA and 1,25(OH)2D3 can induce CYP24A1 gene activation suggesting that the VDR–RXR heterodimer is a conditionally permissive heterodimer rather than a non-permissive heterodimer (Sanchez-Martinez et al. 2006). After binding of VDR to RXR, this complex will migrate to the nucleus and bind to vitamin D response elements (VDREs) in regulatory regions of target genes to regulate gene transcription by recruiting co-activators (p160, DRIP205) and by losing co-repressors (NCoR, SMRT) (Leyssens et al. 2013, Christakos et al. 2016, 2019, Jeon & Shin 2018).
The main function of 1,25(OH)2D3 is to maintain calcium and phosphate homeostasis in the body. Yet, the VDR is not only expressed in intestine, kidney and bone tissue, but also in many other, including cancerous, tissues. Both in vitro and in vivo preclinical studies have illustrated that 1,25(OH)2D3 modulates variable signalling pathways involved in cell proliferation, apoptosis, differentiation, inflammation, invasion and angiogenesis (Welsh 2018) (reviewed in Christakos et al. 2016).
In the 1980s, the group of Garland was the first to describe a possible association between sunlight exposure and breast cancer (BC) risk (Garland et al. 1990, 2006). Thereafter, different epidemiological and preclinical studies have investigated a possible correlation between 25(OH)D serum concentration and the development and progression of BC. However, these studies have shown conflicting results (Hossain et al. 2019).
With 2.3 million diagnoses each year, BC is the most frequently diagnosed cancer in women and the leading cause of cancer-related mortality worldwide (Mattiuzzi & Lippi 2019, Sung et al. 2021). Breast tumours arise from the epithelial cells of the breast located in the milk ducts or lobules (milk-producing glands). Clinically, BC can be subdivided into different subtypes based on the expression of oestrogen receptor (ER), progesterone receptor (PR) and amplification of the human EGF receptor 2 (HER2). These markers allow histological classification of BC tumours into hormone receptor-positive tumours (luminal A and B), HER2 amplified tumours and triple-negative breast cancer (TNBC) tumours (Mueller et al. 2018). The luminal subtype (A–B) is the most common subtype of BC accounting for 60–70% of all BC diagnoses. Luminal A BCs have a better prognosis than luminal B BC as they express lower levels of Ki67-positive cells. TNBC is a subtype of basal-like BC and has the worst prognosis of all BC subtypes. Basal-like BC is characterized by the expression of keratin 5, 14 and 17 and occurs mainly in young premenopausal women under the age of 40, accounting for 15–20% of BC diagnoses (Yin et al. 2020, Johnson et al. 2021).
In this review, we summarize the role of vitamin D in processes such as proliferation, apoptosis, inflammation and autophagy in various in vitro and in vivo BC models. Moreover, we extensively focus on the more recent research that illustrates the effect of 1,25(OH)2D3 on tumour metabolism, metastasis, epithelial–mesenchymal transition (EMT) and cancer stem cells, which represent novel pathways that could be targeted to hamper BC progression. Finally, we discuss potential strategies to overcome the hypercalcaemic side effects, which are associated with the supraphysiological concentrations of 1,25(OH)2D3 required to obtain its anti-neoplastic activity. Indeed, 1,25(OH)2D3-derived synthetic analogues have been developed with the rationale to design analogues with an optimal ratio of anti-neoplastic vs calcaemic effects (Leyssens et al. 2013, 2014, Duffy et al. 2017). These analogues are currently being tested in combination with selective pathway inhibitors in experimental models to block tumour growth and progression. An overview of the described cell lines with their expression of VDR, CYP24A1 and CYP27B1 and the pathways regulated by 1,25(OH)2D3 are presented in Table 1. We used the keywords ‘vitamin D and BC’ to search for papers in the MEDLINE database between 2015 and 2020.
Classification of the different breast cancer cell lines described in this review and the expression of VDR, CYP24A1 and CYP27B1 per cell line. The effects of 1,25(OH)2D3 on different pathways such as cell proliferation, apoptosis, inflammation, cell metabolism, cancer stem cells (CSCs), epithelial–mesenchymal transition (EMT) and metastasis per cell line are described. Used references are included in the last column.
| Classification | Origin | Cell lines | VDR | CYP24A1 | CYP27B1 | Pathways regulated by 1,25(OH)2D3 Inhibition <-> stimulation | References | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Proliferation | Apoptosis | Inflammation | Metabolism | CSCs | EMT and metastasis | |||||||
| ER+, PR+, HER2−(luminal A) | Human | MCF7 | Yes | Yes | Yes | G1-S phase p19, p21, p27 Cyclin D1/3, A1, E1 CDK2/4 pRb C/EBPα miR-125b miR-1204 |
ROS Cytochrome c RAS/MEK/ERK LC3B AMPK/CAMKK2 |
G6PD ROS AMPK TXNIP V-H+-ATPase pump |
Wnt/β-catenin | Mittal et al. 2008 Murray et al. 2017 Sanchez-Martinez et al. 2006 Zinser & Welsh 2004a Verlinden et al. 1998 Jensen et al. 2001 Lopes et al. 2012b Christakos et al. 2016 Dhawan et al. 2009 Klopotowska et al. 2019 Liu et al. 2018 Zheng et al. 2019 Weitsman et al. 2005 Tavera-Mendoza et al. 2017 Hoyer-Hansen et al. 2007 Abu El Maaty et al. 2018 Santos and Hussain 2019 Zheng et al. 2018 |
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| T47D | Yes | Yes | Yes | G6PD | Rodriguez et al. 2016 Zinser & Welsh 2004a Murray et al. 2017 Abu El Maaty et al. 2018 |
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| ZR-75-1 | Yes | Yes | Yes | LC3B | Townsend et al. 2005 Murray et al. 2017 Tavera-Mendoza et al. 2017 |
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| ER+, PR+, HER2+(luminal B) | Human | MCF7-HER18 | Yes | Sub-G1 arrest | Lim et al. 2018 | |||||||
| TNBC | Human | MDA-MB-231 | Yes | Yes | Yes | G6PD AMPK V-H+-ATPase pump |
E-cadherin (CDH1) | Townsend et al. 2005 Murray et al. 2017 Welsh et al. 2017 Abu El Maaty et al. 2018 Santos & Hussain 2019 Lopes et al. 2012a |
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| MDA-MB-453 | Yes | RAS/MEK/ERK | Murray et al. 2017 Zheng et al. 2019 |
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| MDA-MB-468 | Yes | Yes | Yes | Richards et al. 2015 Murray et al. 2017 |
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| HCC1806 | Yes | Yes | Yes | IL-6 | Garcia-Quiroz et al. 2016 Abdel-Mohsen et al. 2019 |
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| SUM159 | Yes | Mammosphere formation CD44, OCT4, Notch1/2/3, JAG1/2 NFkβ |
Thakkar et al. 2016 Shan et al. 2017 |
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| SUM-229PE | Yes | Yes | IL-1β and TNF-α | Garcia-Quiroz et al. 2016 Martinez-Reza et al. 2019 |
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| BT549 | Yes | Murray et al. 2017 | ||||||||||
| MFM-223 | Yes | Thakkar et al. 2016 | ||||||||||
| CAL-148 | Yes | Thakkar et al. 2016 | ||||||||||
| MBCDF-T | Yes | Garcia-Quiroz et al. 2019 | ||||||||||
| TNBC | Murine | 4T1 | Yes | Yes | Yes | Th1 response Th2 response |
OPN (Spp1) TGFβ (Tgfb) SNAIL1 N-cadherin E-cadherin |
Anisiewicz et al. 2018 Williams et al. 2016 Cao et al. 2018 |
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| 168FARN | Yes | Id1 | Williams et al. 2016 | |||||||||
| ERβ+, PR+, HER2+(HER2-enriched) | Murine | E0771 | Yes | Yes | Yes | CD8+ T cell infiltration | Anisiewicz et al. 2020 Karkeni et al. 2019 |
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| Immortalised non-malignant epithelial cells | Human | MCF10A | Yes | Yes | Yes | Beaudin et al. 2015, Welsh et al. 2017 | ||||||
| hTERT-HME1 | Yes | Yes | GLUL GLS1/2 |
Beaudin et al. 2015, Welsh et al. 2017 Beaudin & Welsh 2017 |
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| Transformed malignant epithelial cells | Human | MCF10DCIS | Yes | Yes | CD44 GDF15 |
LNC2 S100A4 KRT6A KRT5 |
Beaudin et al. 2015, So et al. 2011, Wahler et al. 2015 Wilmanski et al. 2016 Shan et al. 2020 |
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| MCF10CA1a | Yes | Yes | Yes | Lipid synthesis | E-cadherin N-cadherin |
Wilmanski et al. 2017a Wilmanski et al. 2016 |
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| H-ras-MCF10A | Yes | Yes | TCA activity LDH SLC1A5 PC |
Zheng et al. 2013 Zhou et al. 2016 Wilmanski et al. 2017b |
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ER, oestrogen receptor; PR, progesterone receptor; HER2, human EGF receptor 2; TNBC, triple-negative breast cancer. Targets inhibited by 1,25(OH)2D3 are indicated in bold and targets stimulated by 1,25(OH)2D3 are underlined.
VDR expression in human breast cancer cells
The VDR is expressed in different cell types of the mammary gland, including the lobular and ductal epithelial cells, where it plays an important role in mammary gland development during puberty, lactation and pregnancy, periods of maximal tissue growth and remodelling (Zinser & Welsh 2004a, Huss et al. 2019). During the pubertal period in mice, VDR expression was highest in differentiated cells in the terminal end buds, while its expression was low in the proliferative zones of the mammary gland (Zinser et al. 2002). In Vdr knock-out (KO) mice, ductal morphogenesis and branching in the mammary glands were accelerated compared to WT mice during the pubertal period. Furthermore, in transgenic MMTV-neu mice, Vdr ablation induced weight loss, atrophy of the mammary fat pad, oestrogen deficiency and reduced survival after 12 months of age (Zinser & Welsh 2004b).
Adipocytes also play an important role in mammary gland development, as Vdr deletion in adipose tissue enhanced the density of the mammary epithelium during hormonally regulated expansion of the mammary gland (Matthews et al. 2016). As adipose tissue is a storage depot for vitamin D metabolites, VDR signalling between adipocytes and epithelial cells plays an important role not only in normal mammary gland development but also in carcinogenesis (Welsh 2017). The VDR is also expressed in cancer-associated fibroblasts (CAFs), in which stimulation with 1,25(OH)2D3 downregulated different genes important during cell proliferation (e.g. Neuregulin NRG1) (Campos et al. 2013).
In human BC tissue, VDR expression has been reported to be inversely correlated with BC aggressiveness. In benign breast lesions, the VDR was significantly more expressed than in breast carcinoma lesions (in situ and invasive) (Lopes et al. 2010). Furthermore, VDR expression was higher in luminal A BC than in TNBC, the most aggressive BC subtype (Welsh 2017, Huss et al. 2019). Different groups demonstrated that VDR expression in BC tissue diminished during tumour progression, rendering them less sensitive to vitamin D3 (Lopes et al. 2010, Welsh 2017). Indeed, BC cell lines with low or no VDR expression were least sensitive to 1,25(OH)2D3 or its analogues (Murray et al. 2017).
Moreover, different groups investigated if VDR expression could be used as a potential biomarker for cancer progression and survival (Al-Azhri et al. 2017, Heublein et al. 2017, Murray et al. 2017, Huss et al. 2019, Xu et al. 2020). Recently, higher total VDR expression in BC lesions (both in nucleus and cytoplasm) was associated with tumour characteristics such as lower grade, smaller size, ER/PR positivity, lower Ki67 expression and with a lower risk of BC mortality (Al-Azhri et al. 2017, Huss et al. 2019). Further analysis distinguishing between BRCA1-mutated BC and sporadic BCs showed significantly higher VDR expression in BRCA1-mutated BCs, which is associated with prolonged overall survival (OS) (Heublein et al. 2017).
A recent meta-analysis containing seven studies reported no correlation between VDR expression and BC OS and disease-free survival (DFS). However, when subgroup analyses based on staining location were performed, high total VDR expression in both nucleus and cytoplasm was correlated with better BC OS. Moreover, when cut-off values for immunoreactive score (IRS) other than IRS > 5 or IRS > 25 were used, high VDR expression was correlated with better BC OS (Xu et al. 2020). IRS is a scoring system used for quantitative evaluation of immunohistochemical stainings and is based on the multiplication of the number of positive cells (0–4) and the staining intensity (0–3), resulting in a score between 0 and 12 (Fedchenko & Reifenrath 2014). Murray et al. (2017) did not confirm the association between VDR expression and BC DFS as a whole, although DFS was positively correlated with VDR expression in luminal A BC patients, whereas no association was observed with basal-like, HER2+ or luminal B BC subtypes. Overall, these findings suggest that VDR expression levels could potentially be used as a biomarker for tumour progression.
Analysis of CYP24A1 and CYP27B1 expression in human BC tissue showed that during de-differentiation and BC progression, vitamin D metabolism and therefore VDR signalling is deregulated (reviewed in Welsh 2017). In most analyses, CYP27B1 expression levels decreased, whereas those of CYP24A1 were increased in more invasive breast carcinomas, suggesting that cancer cells can evade the anti-cancer effects of 1,25(OH)2D3 by decreasing its local production and increasing its local degradation (Lopes et al. 2010, Zhalehjoo et al. 2017). As a result, CYP24A1 is described as an oncogene in BC tissue (Albertson et al. 2000). Next to its expression in breast epithelial tissue, CYP27B1 is also expressed in breast adipose tissue. Breast adipocytes can activate local 25(OH)D3 into 1,25(OH)2D3 and induce paracrine effects on surrounding tissues (Welsh 2017). In CAFs, transcriptional induction of CYP24A1 by 1,25(OH)2D3 is more pronounced than in normal associated fibroblasts, suggesting a faster clearance in the tumour microenvironment (Campos et al. 2013).
However, a more recent study showed that CYP24A1 mRNA levels were lower in breast tumour tissue and inversely correlated with OS (Cai et al. 2019).
Preclinical anti-neoplastic effects of 1,25(OH)2D3 on breast cancer
Effects of 1,25(OH)2D3 on cell proliferation
For many years, 1,25(OH)2D3 has been recognized to hamper the transition of BC cells from G0/G1 to S phase of the cell cycle. 1,25(OH)2D3 mediates these antiproliferative effects through VDR binding, since VDR KO cells are not growth inhibited by 1,25(OH)2D3 (LaPorta & Welsh 2014, Zheng et al. 2017). 1,25(OH)2D3-mediated growth reduction is accompanied by an increased expression of cyclin-dependent kinase inhibitors (CDKIs) such as CDKN2D (p19), CDKN1A (p21) and CDKN1B (p27)and downregulation of cyclins (cyclin D1/3, cyclin A1 and cyclin E1) and CDKs (CDK2/4) (Verlinden et al. 1998, Jensen et al. 2001, Lopes et al. 2012b) (Fig. 1). In addition, upregulation of CDKI expression levels by 1,25(OH)2D3 decreases the activity of CDKs such as CDK4/6 and results in a reduced phosphorylation of retinoblastoma (Rb), a tumour suppressor protein with a crucial role in the regulation of cell cycle progression. Consequently, Rb remains complexed to the E2F transcription factors (TFs) and transcription of E2F-regulated cell cycle genes such as CDK2 decreases (Christakos et al. 2016) (Fig. 1). Furthermore, in ER+ MCF7 cells, the induction of C/EBPα expression and subsequent elevation of VDR transcript levels also contributed to the antiproliferative effects of 1,25(OH)2D3 (Dhawan et al. 2009). In addition, 1,25(OH)2D3 affects expression of miRNAs, short non-coding RNAs that regulate gene expression negatively at the post-transcriptional level. Treatment with 1,25(OH)2D3 or a vitamin D3 analogue (1,24-dihydroxyvitamin D3 (tacalcitol)) decreased the expression of miR-125b. As a consequence, the pro-apoptotic protein BAK1, encoded by the target gene of miR-125b, was increased after treatment with 1,25(OH)2D3 and tacalcitol (Klopotowska et al. 2019). Liu et al. illustrated that increased expression of miR-1204 promoted proliferation, EMT and invasion of BC cells both in vitro and in vivo. Mechanistic studies showed that miR-1204 inhibited VDR expression directly by targeting its 3’ UTR and this VDR suppression contributed to the oncogenic activity of miR-1204. Indeed, silencing of miR-1204 resulted in elevated VDR expression levels and reduced proliferation and invasiveness of BC cells (Liu et al. 2018).
Overview of 1,25(OH)2D3-induced anti-neoplastic effects in pathways involved in the regulation of cell proliferation, apoptosis and inflammation in breast cancer cells. Created with BioRender.com.
Citation: Endocrine-Related Cancer 29, 2; 10.1530/ERC-21-0182
1,25(OH)2D3 has also clear in vivo antiproliferative effects as demonstrated in different (transgenic) mouse models (Welsh 2018). Recently, Rossdeutscher et al. (2015) demonstrated the growth-inhibitory effects of 1,25(OH)2D3 in an MMTV-PyMT model. Mice were subcutaneously implanted with a minipump, delivering continuous doses of 25(OH)D3 or 1,25(OH)2D3. Both treatments significantly decreased cell proliferation (Ki67, ErbB2, cyclin D1) and tumour growth. Interestingly, continuous supplementation with 25(OH)D3 increased local 1,25(OH)2D3 levels in tumour tissues without causing hypercalcaemia, whereas 1,25(OH)2D3 perfusion did induce hypercalcaemia (Rossdeutscher et al. 2015). In contrast, in a xenograft mouse model, derived from highly proliferative tumour tissues, intra-tumoural administration of 1,25(OH)2D3 was unable to decrease BC cell proliferation (BrdU incorporation, Ki67, CDKN1A, CDKN1B) or apoptosis (Bcl-2 expression) (Fonseca-Filho et al. 2017). Next to 1,25(OH)2D3, also its metabolites such as 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) regulate BC cells in vitro and in vivo. 24R,25(OH)2D3 stimulated DNA synthesis in ER+ MCF7 and T47D cells, acting through a caveolae-associated phospholipase D-dependent mechanism via cross-talk with ERs. However, in vivo analysis of an MCF7 xenograft model showed that treatment with 24R,25(OH)2D3 reduced tumour burden and increased animal survival by reducing markers of invasion and metastasis (Snail1, CXCR4/CXCL12) (Verma et al. 2019).
Effects of 1,25(OH)2D3 on apoptosis and autophagy
1,25(OH)2D3 regulates different apoptotic pathways in a BC cell type-dependent manner. In general, 1,25(OH)2D3 decreases the expression of anti-apoptotic factors (Bcl-2, Bcl-XL) and/or increases the pro-apoptotic equivalents (Bax, Bak), which direct cells towards cell death rather than to cell survival (Vanoirbeek et al. 2011). In addition, 1,25(OH)2D3 targets the RAS/MEK/ERK signalling pathway which is an important regulator of cell proliferation and anti-apoptosis (Christakos et al. 2016). Indeed, treatment of MCF7 (ER+) and MDA-MB-453 (ER−) cells with 1,25(OH)2D3 decreased expression of RAS and phosphorylation of MEK and ERK1/2. Furthermore, upregulation of RAS abrogated the antiproliferative effect of 1,25(OH)2D3 (Zheng et al. 2019). In MCF7 cells, pre-treatment with 1,25(OH)2D3 sensitized to reactive oxygen species (ROS)-induced cytotoxicity through a reduction in the inner membrane potential of mitochondria, a subsequent release of cytochrome c and eventually cell death (Weitsman et al. 2005) (Fig. 1).
TP53, encoding for the tumour suppressor gene p53, is often mutated in BC cells. TP53 mutations are most common in TNBC (80%) and HER2-positive cancers (70%), while the prevalence is lower in patients with luminal A type (10%) and luminal B type (30%) BC (Duffy et al. 2018). Interestingly, several studies demonstrated that the VDR gene is a direct target of p53 and its family members (Reichrath et al. 2014). Mutant p53 (mutp53) can interact both functionally and physically with the VDR, thereby converting local vitamin D into an anti-apoptotic agent. This effect was also observed in TNBC cell lines, MDA-MB-231 and MDA-MB-468, that endogenously express mutp53R280K and mutp53R273H, respectively. The exact mechanism responsible for this conversion is not yet known, although in ovarian carcinoma cells, which endogenously express mutp53, vitamin D3 suppressed death receptor-mediated apoptosis. Indeed, additional alterations most likely cooperate with mutp53 to generate the anti-apoptotic response to vitamin D3 (Stambolsky et al. 2010).
Next to apoptosis, treatment of BC cells with 1,25(OH)2D3 affects the autophagy pathway, a well-conserved process aimed at eliminating cellular waste products and dysfunctional organelles (Abu El Maaty & Wolfl 2017). Mammary gland tissue is reported to lose its induced profile of autophagy during BC progression. Mechanistically, in luminal BC cells, VDR constitutively repressed the expression of MAP1LC3B (LC3B), a key gene in the process of autophagy. However, treatment with 1,25(OH)2D3 partially relieved its repression (de-repressed), thereby slightly increasing its expression (Tavera-Mendoza et al. 2017). Another mechanism by which autophagy is induced by 1,25(OH)2D3 in MCF7 cells is via the activation of 5' AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent protein kinase 2 (CAMKK2), both mediating calcium-induced autophagy (Hoyer-Hansen et al. 2007) (Fig. 1).
Effects of 1,25(OH)2D3 on inflammation
1,25(OH)2D3 has well-known anti-inflammatory effects, which are mediated by stimulation of the innate and suppression of the adaptive immune system (Christakos et al. 2016).
Within the adaptive immune system, cytotoxic (CD8+) T lymphocytes are important for the protection against intracellular pathogens including cancer cells. It was previously described that tumour-infiltrating CD8+ lymphocytes (TILs) have anti-tumour activities that are induced by different mechanisms (Martinez-Lostao et al. 2015). Because of the anti-tumour activities of TILs, high tumour infiltration of TILs is associated with better prognosis in TNBC and HER2-enriched BC but not for ER+ BC (Stanton & Disis 2016, Kurozumi et al. 2019, Gao et al. 2020, Oshi et al. 2020). In a recent study, the effect of vitamin D3 supplementation (cholecalciferol, 40 IU/day) on CD8+ T cell infiltration was investigated using a mouse model where murine E0771 (ERβ+, PR+, HER2+) (Le Naour et al. 2020) BC cells were injected in the mammary fat pad (Karkeni et al. 2019). Vitamin D3 supplementation reduced tumour growth and induced the number and activity of CD8+ T cells within the tumour. In contrast, in high-dietary fat conditions, tumour growth was enhanced and CD8+ T cell infiltration was reduced after vitamin D3 supplementation.
The difference in responsiveness to vitamin D3 supplementation between low- and high-dietary fat conditions is probably due to induced expression of Cyp27a1in the adipocytes of obese mice, which led to elevated local levels of 25(OH)D3 but reduced systemic levels (because 25(OH)D3 is diluted in a higher body volume), which could influence CD8+ T cell infiltration. In addition, adipocytes are able to secrete pro-inflammatory cytokines such as IL-6 and CCL5. However, vitamin D3 treatment is able to limit the secretion of inflammatory cytokines from adipose tissue. These data show that the effect of vitamin D3 supplementation on tumour growth and tumour infiltration with cytotoxic T cells is dependent on the fat content of the diet and demonstrated the importance of dietary intake (Karkeni et al. 2019).
Contrary to previous findings, in another BC mouse model where murine 4T1 (TNBC) cells were subcutaneously injected into the flanks, oral treatment with vitamin D3 resulted in accelerated tumour growth and reduced survival. In this model, vitamin D3 suppressed the T helper lymphocytes type 1 (Th1) response, which are TILs with important anti-tumour activities, both systemically and in the tumour microenvironment, which resulted in promotion of tumorigenesis (Cao et al. 2018) (Fig. 1).
Effects of 1,25(OH)2D3 on cell metabolism
Glycolysis
Cancer cell metabolism is characterized by an enhanced uptake of glucose, even in the presence of oxygen (the Warburg effect) (Jang et al. 2013). Aerobic glycolysis enables cancer cells to maintain their energy level and production of nucleotides, amino acids and fatty acids in order to sustain their proliferation capacity (Christakos et al. 2016).
The role of 1,25(OH)2D3 on energy metabolism is an interesting research area as alterations in cellular metabolism might explain its antiproliferative effect (Christakos et al. 2016). Studies in H(arvey)-ras-transformed MCF10A breast epithelial cells illustrated that 1,25(OH)2D3 treatment resulted in an altered glucose consumption. More specifically, 1,25(OH)2D3 treatment reduced glycolysis and lactate production with a reduced tricarboxylic acid (TCA) cycle activity as a consequence. Treatment with 1,25(OH)2D3 reduced the flux of glucose to 3-phosphoglycerate and resulted in decreased intracellular lactate levels by suppressing lactate dehydrogenase (LDH) activity. In addition, the glucose flux to acetyl-coA and oxaloacetate is reduced. Together, these data suggest that 1,25(OH)2D3 has a preventive role in the use of glucose for rapid proliferation during BC progression in a H-ras oncogene-dependent manner (Zheng et al. 2013) (Fig. 2).
Overview of the effects of 1,25(OH)2D3 on cell metabolism in non-transformed mammary epithelial cells (effects shown with blue arrows), transformed mammary epithelial cells (effects shown with orange arrows) and breast cancer cells (effects shown with purple arrows). G6P, glucose 6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; PPP, pentose phosphate pathway; 3PG, 3-phosphoglyceric acid; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; PC, pyruvate carboxylase; FA, fatty acid; αKG, alpha-ketoglutaric acid. Created with BioRender.com.
Citation: Endocrine-Related Cancer 29, 2; 10.1530/ERC-21-0182
The effect of 1,25(OH)2D3 on glucose metabolism appears to be cell type-dependent. Indeed, differences in metabolic response and cellular ATP levels have been demonstrated between luminal BC (MCF7−T47D) and TNBC (MDA-MB-231) cells. However, treatment of both luminal and TNBC cell lines with 1,25(OH)2D3 upregulated the pentose phosphate pathway (PPP) and increased the expression levels of G6PD, a putative oncogene encoding the glucose-6-phosphate dehydrogenase that catalyses the first rate-limiting step of the PPP. Yet, the induction of G6PD by 1,25(OH)2D3did not hamper the anticancer effects of genetic or pharmacological inhibition of G6PD. Treatment of MCF7 cells with 1,25(OH)2D3 resulted furthermore in increased levels of intracellular serine and ROS (Abu El Maaty et al. 2018). In addition, 1,25(OH)2D3 treatment activated the AMPK signalling in MCF7 and MDA-MB-231 cells while levels of thioredoxin-interacting protein (TXNIP), a regulator of redox balance and glucose uptake, were reduced in MCF7 cells (Abu El Maaty et al. 2018) (Fig. 2).
Similar effects of 1,25(OH)2D3 on glycolysis were recently reported in MCF7 and MDA-MB-231 cells (Santos et al. 2018). In a subsequent study, the authors demonstrated that 1,25(OH)2D3 was a potent inhibitor of the V-H+-ATPase proton pump, located at the plasma membrane of metastatic cancer cells, of which the activity is regulated by glucose. Hence, the 1,25(OH)2D3-mediated decrease in glycolytic flux is suggested to contribute to the inhibition of the V-H+-ATPase proton pump. As a result, the extracellular pH increased after treatment with 1,25(OH)2D3, disturbing the optimal pH for cancer cells and ultimately leading to cell death and decreased cancer progression (Santos & Hussain 2019) (Fig. 2).
Glutamine
Treatment of H-ras-transformed MCF10A cells with 1,25(OH)2D3 reduced intracellular glutamine/glutamate and α-ketoglutarate levels, leading to a reduced activity of the TCA cycle. Both mRNA and protein levels of the glutamine transporter, solute linked carrier family 1, member A5 (SLC1A5), were significantly decreased after treatment with 1,25(OH)2D3. Furthermore, reporter studies identified a negative VDRE in the promotor region of the SLC1A5 gene (Zhou et al. 2016). Knockdown (KD) of SLC1A5 increased the number of apoptotic cells in H-ras-transformed MCF10A cells, suggesting that targeting the glutamine pathway with vitamin D decreased BC cell growth. The effect of 1,25(OH)2D3 on glutamine metabolism was also demonstrated in non-transformed mammary epithelial cells (hTERT-HME1). Treatment of hTERT-HME1 cells with 1,25(OH)2D3 decreased the expression of glutamine synthetase (GLUL) and glutaminases (GLS1/2),reducing the amount of glutamine shunt to the TCA cycle (Beaudin & Welsh 2017) (Fig. 2).
TCA cycle
During TCA cycle progression, pyruvate carboxylase (PC) regulates the ATP-dependent carboxylation of pyruvate to oxaloacetate. In BC, PC overexpression is correlated with aggressiveness (Phannasil et al. 2017). 1,25(OH)2D3 decreased PC mRNA and protein expression and thereby induced oxidative stress in H-ras-transformed MCF10A and MCF10A-ErbB2 (HER2) breast epithelial cells (Wilmanski et al. 2017b ). In addition, 1,25(OH)2D3-mediated repression of PC led to reduced synthesis of fatty acids and lipid accumulation in another transformed epithelial cell line, MCF10CA1a (Wilmanski et al. 2017a). Because a functional VDRE is described in the P2 promotor of the PC gene, vitamin D3 is suggested to be an important regulator of epithelial BC cell metabolism (Wilmanski et al. 2017b) (Fig. 2).
Oxidative stress
Ageing Cyp27b1KO mice, which are deficient in circulating 1,25(OH)2D3, had increased oxidative stress and develop BC among other types of cancer. The increased ROS levels in these mice were accompanied by elevated DNA damage and accelerated ageing. Moreover, cellular senescence was increased in the tumour microenvironment. Interestingly, administration of 1,25(OH)2D3 prevented spontaneous tumour development in ageing Cyp27b1KO mice, illustrating the importance of vitamin D deficiency in cancer tumorigenesis (Chen et al. 2018a). Collectively, these data illustrate that the anti-tumour effects of 1,25(OH)2D3 could – at least partially – be explained by modulating metabolic networks of BC cells (Abu El Maaty et al. 2018) (Fig. 2).
Effects of 1,25(OH)2D3 on cancer stem cells
Cancer stem cells (CSCs) were first described in acute myeloid leukaemia as a small subpopulation of cells playing an important role in tumour initiation, progression and recurrence (Bonnet & Dick 1997, Saeg & Anbalagan 2018). Different pathways such as the Notch, Wnt/Frizzled/β-catenin, Hippo and Hedgehog signalling cascades are involved in the formation of CSCs and dysregulation of these pathways is linked to the development of BC. Breast CSCs are characterized by high CD44 and low CD24 expression levels (CD44+/CD24−). CD44, a cell surface adhesion receptor important for recruitment to cell surfaces, is the most commonly used marker for detection of breast CSCs (Senbanjo & Chellaiah 2017, Saeg & Anbalagan 2018). CD24 is a glycosylated cell surface protein that regulates BC metastasis and proliferation because it can function as an alternative ligand of P-selectin, an adhesion receptor on activated endothelial cells, which facilitates the passage of tumour cells in the blood stream during metastasis (Kristiansen et al. 2003, Jaggupilli & Elkord 2012). As CD24 is coexisting with CD44 in different cancers, it gained new interest as CSC marker (Jaggupilli & Elkord 2012). When Al-Hajj et al. (2003) reported that CD44+/CD24−/low cells exhibited more tumorigenic and CSC properties than CD44+/CD24+ cells, CD44+/CD24−/low was widely accepted as breast CSC marker. Next to CD44 and CD24, other markers such as aldehyde dehydrogenase-1 (ALDH-1), EpCAM, CD133 (Prominin-1) and CXCR4 are also used to detect breast CSCs (Song & Farzaneh 2021) (reviewed in So & Suh 2015) (Fig. 3).
Schematic overview of the different effects of VDR signalling on the epithelial–mesenchymal transition process in breast cancer cells. CSCs, cancer stem cells; MMPs, matrix metalloproteinases. Created with BioRender.com.
Citation: Endocrine-Related Cancer 29, 2; 10.1530/ERC-21-0182
Treatment of basal-like MCF710DCIS cells with a Gemini vitamin D3 analogue, BXL0124, reduced mRNA and protein expression of CD44 (So et al. 2011, Wahler et al. 2015). In addition, in TNBC SUM159 cells, 1,25(OH)2D3 and BXL0124 treatment decreased mammosphere formation, a characteristic feature of CSCs, in association with the downregulation of different CSC markers involved in their maintenance (CD44, OCT4; Notch1/2/3; JAG1/2 and NFκB) (Shan et al. 2017). The mammospheres formed after treatment with 1,25(OH)2D3 or BXL0124 had a more organized, symmetrical shape, which was similar to the spheres formed from the non-malignant cell line MCF10A (Wahler et al. 2015). More detailed analysis showed that BXL0124 inhibited the Notch1 signalling pathway in basal-like BC cells by upregulation of HES1, which is an inhibitor of JAG2, ligand for Notch1 (So et al. 2015). Recently, a transcriptomic analysis was performed in MCF10DCIS mammospheres to investigate which pathways were affected by 1,25(OH)2D3 or BXL0124 treatment. Vitamin D3 compounds reduced expression of genes involved in the maintenance of BC stem-like cells (e.g. GDF15), EMT, invasion, metastasis (e.g. LCN2 and S100A4) and chemoresistance (e.g. NGFR, PPP1R1B, and AGR2), while they upregulated genes associated with a basal-like phenotype (e.g. KRT6A and KRT5) and negative regulators of breast tumorigenesis (e.g. EMP1). More detailed pathway analysis identified TP63,a member of the TP53 family of TFs essential for epithelial stem cell development and maintenance, as a major target of vitamin D3 compounds (Shan et al. 2020).
Jeong et al. (2015) investigated the effect of vitamin D3 treatment in an MMTV-wnt1 xenograft mouse model, in which treatment with 1,25(OH)2D3 or vitamin D3 supplementation decreased tumour growth and appearance. From these tumours, CD49fhigh/Epcamlow cells were isolated and spheroid cultures were generated in vitro. Treatment of these spheroids with 1,25(OH)2D3 decreased the capacity of the cells to generate secondary spheroids, suggesting that 1,25(OH)2D3 decreases the self-renewing capacity of CSCs in these tumours. This inhibitory effect of 1,25(OH)2D3 on CSCs is suggested to be regulated by inhibition of the Wnt/β-catenin pathway, an important pathway for the maintenance of CSCs (Jeong et al. 2015). In MCF7 cells, treatment with 1,25(OH)2D3 inhibited Wnt/β-catenin signalling thereby decreasing the population of CSCs (CD133+ cells) and increasing the sensitivity of MCF7 cells to therapy with the ER inhibitor tamoxifen (Zheng et al. 2018). Moreover, 1,25(OH)2D3 affects different pathways of CSC development in other types of cancer (reviewed in So & Suh 2015, Fernandez-Barral et al. 2020).
Effects of 1,25(OH)2D3 on EMT and metastasis
In total, 20–30% of patients with early-stage BC develop metastatic disease. The most common site of metastatic lesions for BC is bone, followed by brain, liver and lung. However, which organ is affected by BC metastasis is highly dependent on the BC subtype (Chen et al. 2018b).
Tumoural VDR expression is reported to protect against BC metastasis in an orthotopic transplantation model of murine 168FARN BC cells, in which Vdr expression was silenced with shRNA. Tumours from Vdr KD cells not only grew significantly faster than tumours from control or Vdr rescue cells but also metastasized to the liver. Gene analysis identified Id1 (DNA-binding protein inhibitor ID-1) as a direct mediator of VDR signalling in murine BC cells, and this relationship was confirmed in humans (Williams et al. 2016).
An important process in the formation of metastatic lesions is EMT. During this process, epithelial cells lose their cell–cell and cell–matrix junctions and undergo a change in gene signature (downregulation of epithelial genes–upregulation of mesenchymal genes) to eventually convert into mesenchymal cells (Larriba et al. 2016) (Fig. 3). The EMT process is activated by different agents and signals, such as the TGF-β, Wnt and Notch signalling, which activate TFs that are important for EMT (EMT-TFs) (Fernandez-Barral et al. 2020). These EMT-TFs include the zinc finger proteins SNAIL1 and SNAIL2, the double zinc finger and homeodomain ZEB1 and ZEB2 as well as TWIST1 and E47, members of the basic-helix-loop family (Larriba et al. 2016). Proteins important for cell adhesion include E-cadherin (hallmark of the epithelial phenotype), the tight junction proteins claudins, occludins and cytokeratins. The mesenchymal phenotype has more fibroblastic-like characteristics with markers such as N-cadherin, vimentin and matrix metalloproteases (MMPs) (Fig. 3).
An association between vitamin D signalling and the EMT process was demonstrated by the finding that the EMT-TF SLUG, member of the SNAIL zinc finger family, repressed the VDR gene in human BC cells, and reduced their sensitivity to the anti-tumour activity of 1,25(OH)2D3. Indeed, introduction of SLUG expression in MDA-MB-468 and MCF7 cells resulted in significant reduction of VDR levels. Moreover, the invasive TNBC BT549 cell line has a high SLUG expression, whereas VDR is not expressed (Mittal et al. 2008).
In an MDA-MB-231 xenograft mouse model, downregulation of miR-1204 decreased distant metastasis not only by reducing cell proliferation after increased VDR expression as described above but also by downregulation of mesenchymal markers (N-cadherin/vimentin), which reduces the EMT process. The critical role of the VDR in the process of EMT was also demonstrated in vivo by injecting MDA-MB-231 cells expressing anti-miR-1204 into nude mice. In these mice, silencing of the VDR increased tumour growth and distal metastasis (Liu et al. 2018).
The induction of epithelial markers such as E-cadherin represents an additional mechanism by which 1,25(OH)2D3 regulates the EMT process and may inhibit cancer progression (Larriba et al. 2016). Also, in TNBC MDA-MB-231 cells, 1,25(OH)2D3 upregulates E-cadherin by CDH1 promoter demethylation (Lopes et al. 2012a) (Fig. 3). Furthermore,1,25(OH)2D3 decreased the bone metastatic potential of MCF10CA1a and MDA-MB-231 cells as illustrated in an in vitro metastasis model. Also, an increased expression of the epithelial marker E-cadherin and decreased expression of N-cadherin suggested a decrease in the process of EMT (Wilmanski et al. 2016).
A recent study described the effect of dietary vitamin D3 deficiency on the CXCL12-CXCR4 axis in MMTV-PyMT mice. CXCL12 is a chemokine involved in cancer progression and increased levels are associated with poor prognosis in BC patients. The receptor of CDXL12, CXCR4, is involved in tumour growth and metastasis. The MMTV-PyMT mouse model spontaneously develops distant metastasis in the lung after 9–10 weeks on a normal diet. However, when mice were fed a vitamin D3-deficient diet, lung metastasis arose already from 8 weeks. Li et al. have demonstrated that vitamin D3 deficiency increased the levels of EMT markers (ZEB1) in the primary tumour tissue and CXCL12 expression in the metastatic long stromal tissue. In addition, vitamin D3 deficiency enhanced CXCL12/CXCR4 colocalization in the lung metastatic tumours, which enhances metastasis formation (Li et al. 2021a).
Furthermore, VDR KD in MDA-MB-231 BC cells promoted cancer cell motility and invasiveness and elevated the bone metastatic potential of MDA-MB-231 cells. VDR KD in MDA-MB-231 cells was accompanied by a reduced expression of epithelial markers such as β-catenin, E-cadherin and F-actin, while mesenchymal markers such as vimentin were increased. These results show that loss of VDR expression in MDA-MB-231 cells induced EMT progression and facilitated the formation of tumour colonies in bone (Horas et al. 2019) (Fig. 3).
In contrast, in young (6–8 weeks old) BALB/c-female mice, treatment with 1,25(OH)2D3 and its low-calcaemic analogues, PRI-2191 and PRI-2205, enhanced the metastatic potential of 4T1 mouse mammary gland cancer cells to the lung without affecting the primary tumour. In tumours from treated mice, osteopontin (OPN, Spp1) secretion was increased (pro-metastatic), while TGFβ (Tgfb) levels were decreased (anti-metastatic). Additionally, treatment with 1,25(OH)2D3 and analogues increased the expression of mesenchymal markers such as SNAIL1 and N-cadherin during tumour progression, whereas E-cadherin expression decreased (Anisiewicz et al. 2018). Further analysis of the immune response in splenocytes and lymph nodes of these mice showed an increased response of T helper lymphocytes type 2 (Th2) with increased activity of regulatory T (Treg) lymphocytes, suggesting that both analogues have an immunosuppressive effect in these mice. Also, the expression of Spp1 and Tgfbin the lung was upregulated by the treatment and was responsible for the immunosuppressive metastatic niche formation (Pawlik et al. 2018). In contrast to these findings, the same research group showed that in old ovariectomized (OVX) mice, 1,25(OH)2D3 and both its analogues reduced the metastatic spread of 4T1 breast carcinoma cells to the lung by decreasing OPN levels. Also, in aged OVX mice, 1,25(OH)2D3 and analogue treatment decreased bone mineralization while this effect was not seen in young mice (Anisiewicz et al. 2019). These data suggest that the activity profile of 1,25(OH)2D3 and analogues is dependent on the age of the mice. However, the researchers do not provide an explanation for the finding that old OVX treated mice responded differently to vitamin D3 treatment than young mice. Important to note is that 4T1 cells are not responsive to 1,25(OH)2D3 treatment in vitro or in vivo, as primary tumour growth was not affected. This suggests that the antimetastatic effects of 1,25(OH)2D3 and analogues were induced by cells in the tumour microenvironment such as fibroblasts or immune cells. Also, previous studies with the same model (4T1) have shown conflicting results regarding the effect of 1,25(OH)2D3 or analogue treatment on primary tumour growth and metastatic formation. Zhang et al. (2014) reported a decrease in the number of lung metastases in 4T1-tumor bearing mice after 1,25(OH)2D3 treatment, while Cao et al. (2018) reported a stimulation of primary tumour growth after treatment with vitamin D3 in an 4T1-subcutaneous mouse model. However, the difference in results could be explained by the different treatment schedules used. Zhang et al. (2014) used an orthotopic model and treated the mice IP once every other day with 1,25(OH)2D3 at a dose of 0.3 µg/kg body weight during 8 weeks while Cao et al. (2018) used a subcutaneous model and treated the mice daily through gavage with vitamin D3 at a dose of 5 µg/kg during 7 days.
Finally, 1,25(OH)2D3 also influences EMT by inhibiting inflammatory cytokines such as IL-6. Previously, Sullivan et al. (2009) have shown that IL-6 induces EMT by activation of STAT3 and downregulation of E-cadherin in ER+ BC cells. In vitro analysis of HCC1806 TNBC cells demonstrated that combined treatment with IL-6 and 1,25(OH)2D3 suppressed the inhibitory effect of 1,25(OH)2D3 on EMT and stemness as E-cadherin was more upregulated after treatment with 1,25(OH)2D3 alone, while IL-6 had no effect on E-cadherin expression (Abdel-Mohsen et al. 2019). Since IL-6 is known to be secreted by adipocytes into the tumour microenvironment in BC, it may impair the anti-cancer effect of 1,25(OH)2D3. However, the exact interplay between IL-6 and 1,25(OH)2D3 was not investigated by the researchers. The downregulation of CYP27B1 by IL-6, as was observed in colon cancer cells, may contribute to these antagonizing effects of IL-6 (Hummel et al. 2014) (Fig. 3).
Combination therapies with 1,25(OH)2D3 or analogues
As 1,25(OH)2D3 and vitamin D3 analogues reduce tumour progression by blocking different pathways, multiple studies investigated possible combinations of approved chemotherapies or other compounds with 1,25(OH)2D3 or vitamin D3 analogues to find a synergistic combination therapy.
As it was previously shown that 1,25(OH)2D3 treatment decreased ERα protein and mRNA levels in MCF7 cells (Swami et al. 2000) and aromatase expression in MCF7 tumour xenografts and surrounding adipose tissue (Krishnan et al. 2010), the combination of 1,25(OH)2D3 with endocrine therapy such as aromatase inhibitors (anastrozole and letrozole) was investigated in an MCF7 xenograft mouse model. Combined treatment with anastrozole was able to significantly reduce tumour volume compared to mono-treatment, and the combination with letrozole significantly decreased tumour volume compared to vehicle treatment (Swami et al. 2011) (Table 2). This suggests that 1,25(OH)2D3 improves the sensitivity to endocrine therapy such as aromatase inhibitors. The combination of 1,25(OH)2D3 with the anti-oestrogen compound tamoxifen showed additive anti-proliferative effects in MCF7 cells (Vink-van Wijngaarden et al. 1994). Also in vivo, combination of tamoxifen with the vitamin D3 analogue 22-oxa-calcitriol (OCT) showed synergistic anti-tumour effects in an MCF7 xenograft model (Abe-Hashimoto et al. 1993) (Table 2). Interestingly, combined targeting of the VDR and the androgen receptor (AR) with agonists proved effective in VDR+ and AR+ TNBC cells by decreasing cell viability, which was even further decreased in combination with chemotherapy (Thakkar et al. 2016) (Table 2). Recently, small molecules affecting cell proliferation and/or cell death pathways were investigated in combination with 1,25(OH)2D3 or vitamin D3 analogues. One such molecule, ruxolitinib, a Janus kinase (JAK) 1 and JAK2 inhibitor, reduced cell proliferation synergistically in combination with 1,25(OH)2D3 in an MCF7-HER18 (ER+, HER2+) BC model by activation of apoptosis and sub-G1 arrest (Lim et al. 2018). Other small molecules such as lapatinib and neratinib, inhibitors of tyrosine kinase activity of the ERBB family (EGFR, HER2 and HER4), inhibited cell growth as well as AKT and ERK phosphorylation (pathways activated by ERBB family members) more effectively after combination with the vitamin D3 analogue, EB1089, in EGFR and/or HER2+ breast cancer cell lines. In addition, apoptosis was increased after these combination treatments in both 2D and 3D cultures (Segovia-Mendoza et al. 2017). In the same subset of BC cell lines, the same group analysed the combination of 1,25(OH)2D3 and vitamin D3 analogues, calcipotriol and EB1089, with another tyrosine kinase inhibitor, gefitinib. Again, the growth inhibitory effect of the combination therapy (1,25(OH)2D3/vitamin D3 analogues and gefitinib) was elicited by downregulation of ERK1/2 MAPK signalling and induction of apoptosis by upregulation of BIM and caspase 3 (Segovia-Mendoza et al. 2015) (Table 2). The same vitamin D3 analogue, EB1089, combined with ionizing radiation reduced tumour growth in an MCF7 breast tumour xenograft model by promoting apoptotic cell death (Sundaram et al. 2003) (Table 2).
Overview of recent studies investigating new combination therapies with 1,25(OH)2D3 or analogues.
| Research group | Vitamin D3 compound | Combined compound | Study model | Observed effect |
|---|---|---|---|---|
| Swami et al. 2011 | 1,25(OH)2D3 | Anastrozole/letrozole | MCF7 xenograft | Decreased ERα and aromatase expression in tumour tissue |
| Vink-van Wijngaarden et al. 1994 | 1,25(OH)2D3 | Tamoxifen | MCF7 cells | Decreased cell proliferation (DNA content) |
| Abe-Hashimoto et al. 1993 | 22-oxa-calcitriol (OCT) | Tamoxifen | MCF7 xenograft | Decreased tumour growth (no mechanism studied) |
| Thakkar et al. 2016 | 1,25(OH)2D3 | Dihydrotestosterone (AR agonist) | MFM-223, CAL-148 | Cell cycle arrest (MFM-223) (BrdU) Apoptosis (CAL-148) (Annexin V-PI) |
| Lim et al. 2018 | 1,25(OH)2D3 | Ruxolitinib | MCF7-HER18 | Decreased cell proliferation (sub-G1 arrest) Increased apoptosis (Annexin V-PI) |
| Segovia-Mendoza et al. 2017 | EB1089 | Lapatinib/neratinib | SUM-229PE | Decreased AKT/ERK phosphorylation Increased apoptosis (activation of caspase 3) Decreased cell proliferation (cell cycle arrest) |
| Segovia-Mendoza et al. 2015 | 1,25(OH)2D3 EB1089 Calcipotriol |
Gefitinib | SUM-229PE | Decreased ERK1/2 MAPK phosphorylation Increased apoptosis (activation of caspase 3) Decreased cell proliferation (G1-S arrest) |
| Sundaram et al. 2003 | EB1089 | Ionizing radiation | MCF7 xenograft | Decreased cell proliferation (Ki67) Increased apoptosis (TUNEL) |
| Tavera-Mendoza et al. 2017 | 1,25(OH)2D3 | Chloroquine | MCF7, ZR-75-1, MDA-MB-231, MCF10A MCF7 xenograft |
Activation of autophagy |
| Garcia-Quiroz et al. 2019 | 1,25(OH)2D3 | Curcumin/resveratrol | TNBC xenograft | Delay tumour onset Reduced microvessel density |
| Attia et al. 2020 | 1,25(OH)2D3 | Paclitaxel and curcumin | MCF7 | Downregulation of MDR1, ALDH-1 Decrease of chemoresistance |
| Friedrich et al. 2018 | 1,25(OH)2D3 | Celecoxib | MCF7, MDA-MB-231 | Decreased cell proliferation (MTT assay) |
| Martinez-Reza et al. 2019 | 1,25(OH)2D3 | TNFα | MCF7, SUM-229PE, HCC1806 | Decreased cell proliferation (XTT assay) |
MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.
Also, chloroquine, an inhibitor of autophagosome acidification, synergistically inhibited cell proliferation in combination with 1,25(OH)2D3 in MCF7 cells not only in vitro but also in vivo. The size of tumour xenografts was substantially smaller after the combination treatment than after the mono-treatment (Tavera-Mendoza et al. 2017) (Table 2).
In addition, combining 1,25(OH)2D3 with curcumin or resveratrol, both angiogenesis blockers, potentiated the action of 1,25(OH)2D3 by facilitating the heterodimerization of VDR with RXR, resulting in a cooperative effect on gene transactivation (Garcia-Quiroz et al. 2019). In an MBCDF-T TNBC xenograft model, combination therapy of 1,25(OH)2D3 with curcumin delayed tumour onset and reduced tumour volume and microvessel density. In addition, tumour endothelial cells were less activated as illustrated by reduced expression and activity of the vitronectin receptor (αvβ3) in the combination groups of 1,25(OH)2D3 with curcumin or resveratrol (Garcia-Quiroz et al. 2019). The combination of curcumin with 1,25(OH)2D3 was also investigated in MCF7 cells in vitro,in combination with paclitaxel. The triple combination reduced gene and protein expression of resistance markers such as multidrug resistance complex 1 (MDR-1) and ALDH-1, suggesting that the addition of curcumin and 1,25(OH)2D3 enhanced the tumour response to paclitaxel treatment by decreasing chemoresistance (Attia et al. 2020) (Table 2).
Another target important in the process of inflammation is cyclooxygenase 2 (COX2). 1,25(OH)2D3 was combined with the COX2 inhibitor, celecoxib, in MCF7 and MDA-MB-231 cells, causing a synergistic growth inhibitory effect in both BC cell lines (Friedrich et al. 2018). Interestingly, 1,25(OH)2D3 regulated the production and secretion of cytokines such as IL-1β and TNF-α in TNBC SUM-229PE cells. Moreover, when combining 1,25(OH)2D3 with TNF-α, the combination was more potent to reduce cell proliferation than either compound alone, probably by potentiating the cytotoxic effect of TNF-α on BC cells (Martinez-Reza et al. 2019) (Table 2).
Epidemiological studies of vitamin D and breast cancer
In the 1980s, the association between sunlight exposure and cancer incidence was suggested based on the observation that geographical areas located far from the equator have a higher incidence of cancer, which could be associated with a decrease in sunlight exposure or vitamin D status (Garland et al. 1990, 2006). This association was further analysed in epidemiological studies in which baseline 25(OH)D levels were measured before start of any treatment (Bilinski & Boyages 2013). Vitamin D deficiency is often seen in BC patients at diagnosis (Peppone et al. 2011, Karthikayan et al. 2018, Machado et al. 2019) and can be correlated to BC subtype, as patients with higher tumour grade, non-luminal and ER− BCs have lower serum 25(OH)D levels than their opposing groups (Peppone et al. 2012, Karthikayan et al. 2018).
Observational studies
Observational studies were set up to investigate a possible causal relationship between different factors such as 25(OH)D levels and vitamin D intake on BC risk, survival and progression. However, these studies often lack sufficient power to prove any association. Therefore, meta-analyses were set up to further investigate this relationship by combining different studies conducted in a specific time frame. Recently, such meta-analysis studies described the association between serum 25(OH)D levels and BC risk and survival (Vaughan-Shaw et al. 2017, Estebanez et al. 2018, Song et al. 2019, Hossain et al. 2019). A protective effect of 25(OH)D levels on BC development was reported in pre-menopausal women (Estebanez et al. 2018), whereas a different meta-analysis described a 6% decrease in BC risk when blood vitamin D levels increased by 5 nmol/L in both pre-and post-menopausal women (Song et al. 2019). However, no association between vitamin D intake and BC risk or development was found in those studies (Estebanez et al. 2018, Song et al. 2019). In addition, another meta-analysis pointed to an association between higher 25(OH)D levels and reduced risk of BC death and disease progression (Vaughan-Shaw et al. 2017). In contrast, Hossain et al. (2019) did not confirm this association, they did however report an association between vitamin D deficiency and BC occurrence. As vitamin D deficiency is often more pronounced in African women, the effect of vitamin D supplementation was specifically investigated in this group of patients. An inverse association between vitamin D supplementation and BC risk was found, with the strongest effect for TNBC in Black individuals. Increased sun exposure was also associated with reduced cancer risk in specific BC subtypes: ER+, ER− and TNBC among Black women (Qin et al. 2020). Although observational studies and their meta-analyses often suggest an association between serum 25(OH)D and BC risk, it remains difficult to prove the causal relationship between vitamin D deficiency and BC risk (Bilinski & Boyages 2013, Feldman et al. 2014).
Mendelian randomization studies
Another way to investigate the effect of vitamin D status on BC risk is by performing Mendelian randomization (MR) studies. These studies analyse the association between single-nucleotide polymorphisms (SNPs) in different genes important in the vitamin D pathway and BC risk. Previously, genetic variants in four genes (GC (vitamin D binding protein), DHCR7 (7-dehydrocholesterol reductase), CYP2R1 and CYP24A1) of the vitamin D signalling pathway were associated with plasma 25(OH)D concentrations. Recently, large-scale genome-wide association studies (GWAS) were performed in 79,366 individuals, which identified 2 additional loci (SEC23A and AMDHD1) associated with serum 25(OH)D levels (Jiang et al. 2018). These 6 loci were then further analysed in 122,977 breast cancer cases, but no association between the genetic variants and BC risk could be observed (Jiang et al. 2019). The latest MR analysis of the same group investigated 138 SNPs in 69 vitamin D-associated loci. Again, there was no evidence for a causal effect of 25(OH)D concentrations on BC risk (Jiang et al. 2021). Despite the increased power of the recent studies performed, until now, no causal association between reduced circulating vitamin D levels and BC risk could be proven (Bouillon et al. 2019).
Randomized controlled trials
To prove a causal role between vitamin D and breast cancer risk, response rate or survival, randomized controlled trials (RCTs) with sufficient power are needed. Recently, the VITamin D and OmegA-3 TriaL (VITAL) was finalized, which is one of the biggest RCTs conducted until now with 25,871 subjects included. This randomized, double-blind, placebo-controlled, 2 × 2 factorial clinical trial investigated the effect of daily vitamin D3 supplementation (2000 IU) alone or combined with marine n-3 (1 g) supplementation, on the prevention of cancer. Of the 25,871 trial participants, 51% were women and the mean age of the participants was 67.1 years. In the cohort, 20% of the participants were Black and 71% were self-declared non-Hispanic White participants. At baseline, the mean 25(OH)D level was 30 ± 10 ng/mL, and after 1 year, the mean 25(OH)D level increased to 41.8 ng/mL in the vitamin D3 group, while there was a minimal change in the placebo group (subgroup analysis of 1644 participants). In the first analysis, where vitamin D3 supplemented patients were compared with placebo controls, the VITAL study did not find any difference in BC incidence between those groups (Manson et al. 2019) (Table 3). Cancer was confirmed based on histological or cytological data.
Overview of randomized controlled trials.
| RCT | Patients/subgroup | Subjects | Study design | 25(OH)D levels (ng/mL) | Vitamin D form and dose | Duration of therapy | Primary outcome | Secondary outcome |
|---|---|---|---|---|---|---|---|---|
| Results: HR (95% CI) | Results: HR (95% CI) | |||||||
| Manson et al. 2019 (VITAL) | Vitamin D3: 12,927 Placebo: 12,944 |
Men (≥50 years) Women (≥55 years) |
Randomized, double-blind, placebo-controlled 2 × 2 factorial clinical trial | Baseline: 30 ± 10 After 1 year: Vitamin D3: 41.8 Placebo: 29.1 |
Vitamin D3: 2000 IU/day ±n-3 fatty acids (1 g) |
5.3 years | - Invasive cancer of any type: 0.96 (0.88–1.06) - Major cardiovascular event 0.97 (0.85–1.12) |
- Site-specific cancer for example, BC 1.02 (0.79–1.31) - Death from cancer 0.83 (0.67–1.02) - Additional cardiovascular events - Metastatic or fatal cancer of any type0.83 (0.69–0.99) |
| Chandler et al. 2020 (VITAL second analysis) | Vitamin D3: 3884 Placebo: 3959 |
Men (≥50 years) Women (≥55 years) With BMI<25 |
Randomized, double-blind, placebo-controlled 2 × 2 factorial clinical trial | Baseline: 30 ± 10 After 1 year: Vitamin D3: 41.8 Placebo: 29.1 |
Vitamin D3: 2000 IU/day | 5.3 years | ND | - Metastatic or fatal cancer incidence with BMI <25 0.62 (0.45–0.86) |
| Lappe et al. 2017 | Vitamin D3+ calcium: 1156 Placebo: 1147 |
Postmenopausal women (≥55 years) | Randomized double-blind, placebo-controlled, population-based clinical trial | Baseline: 32.8 ± 10.5 After 1 year: Vitamin D3: 43.9 Placebo: 31.6 |
Vitamin D3 2000 IU/day + 1500 mg/day calcium | 4 years | - Incidence of all-type cancer0.70 (0.47–1.02) - BC incidence:0.79 (0.43–1.43) |
ND |
| Scragg 2019 (VIDA) | Vitamin D3: 2558 Placebo: 2550 |
Men and women (50–84 years) | Randomized, double-blind, placebo-controlled trial | Baseline: 24.4 ± 9.6 After 1 year: Vitamin D3: 47.7 ± 18 Placebo: 24 ± 11.2 |
Vitamin D3 100,000 IU/month | 3.3 years | - Incidence of cardiovascular disease1.02 (0.87–1.2) | - Acute respiratory infection- Falls and non-vertebral fractures- Cancer incidence 1.01 (0.81–1.25) |
| Crew et al. 2019 | Vitamin D3: 103 Placebo: 105 |
High-risk (5-year risk ≥1.67%, lifetime risk ≥ 20%, lobular carcinoma in situ, prior stage 0–II breast cancer, hereditary breast cancer syndrome, or high MD) pre-menopausal women (18–50 years) | Multicenter randomized double-blind placebo-controlled trial | Baseline: ≤32 | Vitamin D3 20,000 IU/week | 12 months | - Change in mammographic density (MD)P = 0.22 | - Serial blood biomarkers:(25(OH)D3, (1,25(OH)2D3), insulin-like growth factor (IGF)-1, IGF-binding protein-3) - MD change at 24 months P = 0.10 |
| Arnaout et al. 2019 | Vitamin D3: 43 Placebo: 37 |
Newly diagnosed BC patients | Prospective, randomized, phase 2, double-blinded pre-surgical window of opportunity trial | Baseline: 29.2 Post surgery: Vitamin D3: 98.6 Placebo: 24.8 |
Vitamin D3 40,000 IU/day | 2–6 weeks prior to surgery | - Relative change in proliferation (Ki67) and apoptosis (cleaved caspase 3)Ki67: 1.6% vs 16.7%, P = 0.25 CC3: −55.9% vs −45.9%, P = 0.28 |
ND |
HR, hazard ratio; ND, not determined.
However, in a secondary analysis, focusing on the incidence of advanced cancers (metastatic or fatal) and after correcting for BMI, a significant risk reduction was found in the vitamin D3-supplemented group compared to the placebo control group. The strongest risk reduction for advanced cancers was seen in the normal weight group (BMI <25) after supplementation with vitamin D3. Stratification by race did not change the risk for total metastatic or fatal cancer between vitamin D3 or placebo group (Chandler et al. 2020).
A similar RCT conducted by Lappe et al. (2017) aimed at investigating the effect of the same amount of vitamin D3 supplementation (2000 IU/day) but combined with calcium supplementation (1500 mg/day) instead of supplementation with fatty acids. A total of 2303 postmenopausal women were investigated for 4 years, but again, supplementation with vitamin D3 and calcium did not significantly reduce all-type cancer risk over a period of 4 years (Lappe et al. 2017) (Table 3). Recently, in the vitamin D assessment (ViDa) study, a double-blind placebo-controlled trial, the effect of monthly supplementation with vitamin D3 (100,000 IU) on the incidence of acute and chronic diseases was investigated. Also, this study did not support any effect of vitamin D3 supplementation on overall cancer incidence (Scragg 2019) (Table 3). In another RCT, high-dose vitamin D3 supplementation (40,000 IU/day) was given to patients prior to breast cancer surgery to analyse its effect on cell proliferation (Ki67) and apoptosis (cleaved caspase 3). After 2–6 weeks of daily supplementation, there was no effect on BC cell proliferation and apoptosis, despite increased levels of 25(OH)D (Arnaout et al. 2019) (Table 3). Also, the study of Crew et al. (2019) examining vitamin D3 supplementation (20,000 IU/week) in high-risk premenopausal women failed to show a reduced BC risk after 12 months of treatment, as assessed by mammographic density (Table 3). The latter is a well-established predictor for BC risk, as women with higher breast density (75% or more) have four to six times more risk to develop breast cancer (Yaghjyan et al. 2012). In addition, meta-analysis studies performed on eight and seven pooled RCTs could not prove any association between vitamin D3 supplementation and BC risk (Zhou et al. 2020, Li et al. 2021b). During the last 5 years, no RCTs investigated the effect of vitamin D3 supplementation on BC survival or response rate. Currently, one clinical trial is evaluating the effect of neoadjuvant vitamin D3 administration on DFS (5 years) in locally advanced BC (NCT01608451; ClinicalTrials.gov). Another study currently evaluates the effect of adding weekly vitamin D3 supplementation (50,000 IU) to neoadjuvant therapy on pathological complete response (NCT03986268; ClinicalTrials.gov).
So, until now, RCTs do not support any effect for vitamin D3 supplementation on BC risk or incidence. However, as vitamin D3 is a nutrient, a lot of confounders will influence the results of vitamin D3 RCTs. There is still discussion on the optimal threshold for 25(OH)D and the optimal supplementation dose for vitamin D3 (daily/monthly). In addition, the continued self-supplementation and dietary factors during the trial are possible confounding factors (Boucher 2020).
Prevention vs treatment
While in vitro and animal studies show potential anti-cancer effects of 1,25(OH)2D3 in BC, human studies (observational and RCTs) often do not show these effects. However, it is important to acknowledge the differences between the two types of studies. Most preclinical studies investigate the effect of treatment with the active 1,25(OH)2D3 compound either in vitro in BC cell lines or in animal models with existing BC, which enables to study the therapeutic effect of vitamin D3 treatment. In addition, animal studies are performed in a homogenous population under uniform conditions such as cancer type, vitamin D3 status, age of the mice. Moreover, only short time effects are studied in animal experiments due to limited follow-up time. While in human studies, the prevention effects of vitamin D3 supplementation are studied by patient’s follow-up and evaluation of their BC development during a long follow-up period. Furthermore, in animal models, high doses of the active form of vitamin D3, 1,25(OH)2D3, are often used, while this is not possible in human studies due to calcaemic side effects and therefore the dose of active 1,25(OH)2D3, which is targeting the cancer cells and tumour microenvironment is different in animal vs human studies. Importantly, cell culture studies do not encompass the complex cell–cell interactions which may influence the anti-neoplastic effects of 1,25(OH)2D3. For example, adipocytes in breast tissue express CYP27B1, which enables local regulation of 1,25(OH)2D3 synthesis. These autocrine and paracrine effects are important to consider as also CYP24A1 and CYP27B1 are expressed in both normal and cancerous breast tissue.
General conclusions
Numerous preclinical studies illustrated the anti-neoplastic effects of 1,25(OH)2D3 or its less calcaemic structural analogues on cell proliferation, apoptosis, autophagy and inflammation in BC. In addition, 1,25(OH)2D3 influences cellular processes such as CSCs, EMT and cell metabolism, thereby hampering BC progression. Indeed 1,25(OH)2D3-induced downregulation of CD44 and mammosphere formation capacities results in a decreased formation of breast CSCs. Also, upregulation of epithelial markers and downregulation of mesenchymal markers after 1,25(OH)2D3 treatment inhibits the EMT process. Furthermore, 1,25(OH)2D3 affected the PPP pathway and ROS levels in BC cells. These in vitro and in vivo analyses illustrate the importance of VDR expression on the progression of BC and the possible anti-tumour applications for 1,25(OH)2D3 or analogues in BC treatment. However, both observational studies and RCTs in humans do not support a protective role of vitamin D3 on BC risk and development.
Although a possible application for vitamin D3 in the field of BC could be by the use of vitamin D3 analogues in combination with existing cancer therapies such as chemotherapy or small molecules. Nevertheless, more research is required to prove the effectiveness of vitamin D3 analogues in combination therapies to treat different BC subtypes. Although, in vitro and in vivo studies describe promising results for the use of 1,25(OH)2D3 or analogues to decrease BC growth and progression, the translation to humans still needs to be further investigated.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
J Vanhevel receives a PhD fellowship Fundamental Research from Flanders Research Foundation (FWO 11C2921N). H Wildiers is senior clinical investigator of the Fund for Scientific Research (FWO-Vlaanderen). A Verstuyf receives funding from the University of Leuven (C16/18/006) and Flanders Research Foundation (FWO G.0D01.20N, G0D4217N).
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