Vitamin D endocrine system in breast cancer*

Vitamin D is a steroid hormone of great importance in the human body. It is produced in the skin from 7-dehydrocholesterol, upon UV radiation. In order to exert its functions, vitamin D has to be hydroxylated (via CYP27A1 and CYP27B1 hydroxylases), which is followed by its interaction with the vitamin D receptor (VDR) or retinoic acid-related orphan receptors α or γ (RORα and RORγ). By binding with the vitamin D response elements (VDRE) located in the promoter regions, the vitamin D ligand-receptor complex may regulate vitamin D-related genes. Recently, vitamin D has acquired a great interest for its plausible association with cancer development. This review discusses the potential role of vitamin D, its analogues, and enzymes involved in its metabolism with breast cancer incidence and outcome. According to the literature, alterations in the vitamin D endocrine system, both at the mRNA and protein level, have an impact on breast cancer incidence and prognosis. Moreover, specific enzymes participating in vitamin D metabolism may serve as therapeutic targets. Notably, treatment with vitamin D analogues also gives promising results in experimental research. However, given the fact that breast cancer is heterogenous disease, further studies are needed to thoroughly elucidate the potential of vitamin D and enzymes involved in its metabolism in breast cancer development, progression and therapy. Therefore, plausible effects of vitamin D in cancer therapy or prevention have been the principal aim of numerous studies.


VITAMIN D METABOLISM
Vitamin D is a precursor of 1,25-dihydroxyvitamin D (calcitriol), a steroid hormone that plays a very im-portant role in the body in maintaining calcium and phosphorus homeostasis. There are two major forms of vitamin D:D 2 (ergocalciferol) and D 3 (cholecalciferol). Vitamin D 2 is mainly produced by plants, and can be delivered to the body with the plant components of meals (for example mushrooms and yeast). In turn, vitamin D 3 (cholecalciferol) is of animal origin. The main source of vitamin D 3 for humans is the synthesis from 7-dehydrocholesterol that occurs in the skin exposed to the sun light, mainly to the UVB light (290-315 nm) (Tripkovic et al., 2012;Christakos et al., 2016). An additional source of this vitamin is a diet rich in fish oils, eggs or fortified foods, such as breakfast cereals and fruit juices. The two forms, D 2 and D 3, differ primarily in their side chain structure, however, they are converted in the body to the same biologically active compound -calcitriol (1,25(OH) 2 D 3 ).
Vitamin D, synthesized under the influence of UVB radiation, is released from epidermal cells into the blood and lymphatic vessels located in the deeper layers of the dermis. This form of vitamin D, similarly to the one taken with food, is bound to the vitamin D binding protein (VDBP) and transported to the liver (Christakos et al., 2016). The liver plays a particularly important role in vitamin D first hydroxylation which is carried out by 25-hydroxylase (CYP27A1). This reaction produces calcidiol (25(OH)D 3 ) which is subsequently transported in the bloodstream as a proteinbound VDBP to the kidneys (Christakos et al., 2016). A transmembrane protein, megalin, present in the proximal tubules of kidneys, acts as a VDBP receptor, allowing the uptake of (25(OH)D 3 ) in tubular epithelial cells by endocytic internalization (Christakos et al., 2016). The second hydroxylation and formation of the active form of vitamin D 3 , or calcitriol (1,25(OH) 2 D 3 ), is catalyzed by 1α-hydroxylase (CYP27B1) in the kidneys (Holick, 2017).
The biological activity of calcitriol is based on its interaction with the vitamin D receptor (VDR) (Jones, 2013;Holick, 2017). After binding calcitriol, the VDR receptor heterodimerizes with the retinoid X receptor (RXR) and translocates to the nucleus. The resulting VDR-RXR heterodimer acts as a transcription factorit can bind to a specific DNA sequence present in the promoter regions, referred to as the vitamin D response element (VDRE), which can regulate expression of the target genes (Jones, 2013;Holick, 2017).
It was indicated that only about 15% of 7-dehydrocholesterol transforms into previtamin D 3 in the UVexposed skin. Each subsequent UV light exposure leads to an equilibrium between previtamin D 3 conversion into its further derivatives: lumisterol3 and tachysterol3, and its transformation back into 7-dehydrocholesterol. Furthermore, if vitamin D 3 produced in the skin is exposed to UVB radiation, it can be converted into several suprasterols and 5,6-trans-vitamin D 3 as a result of absorption of this radiation. In addition, previtamin D 3 may be also transformed into several toxisterols. Therefore, regardless of individual sun exposure, there is no risk of vitamin D hypervitaminosis or toxicity due to photodegradation of excess previtamin D 3 and vitamin D 3 to products without calcemic activity (Wacker & Holick, 2013).
Concentration of the active form of vitamin D (1,25(OH) 2 D 3 ) is tightly regulated by hydroxylation of carbon at position C24, carried out by CYP24A1 (1,25-dihydroxyvitamin D 3 hydroxylase) (Annalora et al., 2010;Wasiewicz et al., 2015). Hydroxylation of calcitriol causes a drastic decrease in its biological activity, and further oxidation by CYP24A1, resulting in urinary excretion of the newly formed metabolite -the calcitroic acid (Prosser & Jones, 2004;Wasiewicz et al., 2015). Alternative pathways of vitamin D metabolism have been also identified. One of them is initiated by the CYP11A1 hydroxylase (cholesterol desmolase), where cholesterol is converted to pregnenolone to initiate steroidogenesis (Slominski et al., 2012b;Slominski et al., 2015). The products of this pathway are many hydroxyl derivatives, including 20-hydroxyvitamin D 3 (20(OH)D 3 ), which are biologically active and may act through VDR and alternative receptors (Slominski et al., 2017a;Slominski et al., 2017c). Therefore, vitamin D may undergo alternative activation pathways in the skin or other organs where CYP11A1 is expressed (Slominski et al., 2017a;Slominski et al., 2017c). The classi-cal and alternative pathways of vitamin D metabolism are presented in Fig. 1.

NOVEL RESEARCH ON VITAMIN D DEFICIENCY IN BREAST CANCER
There are a lot of studies indicating that vitamin D influences inhibition of cell proliferation, invasion, metastasis and angiogenesis, as well as induction of apoptosis and tumor cell differentiation (Chakraborti, 2011). Therefore, various cancers, including breast cancer, have been studied in relation to vitamin D deficiency and cancer risk.
Breast cancer is the most common malignancy among women worldwide. Early stage disease without metastases is curable in ~70-80%, while advanced breast cancer with metastases to distant organs is considered to be terminal since currently available therapies are ineffective for those cases (Harbeck et al., 2019).
Although many studies have been conducted to evaluate the relationship between vitamin D deficiency and breast cancer risk, there is still a controversy in the literature about this association. Some studies have shown that there is no association between breast cancer risk and vitamin D levels (Chlebowski et al., 2008), and others show that breast cancer is associated with low vitamin D levels (Janowsky et al., 1999;Abbas et al., 2008;Yousef et al., 2013;Alco et al., 2014;Clark et al., 2014;Song et al., 2019). Interesting results on the association of vitamin D deficiency and breast cancer come from Pakistan, where low levels of vitamin D are detected especially among Abbreviations: CYP27A1, 25-hydroxylase; CYP11A1, cholesterol desmolase; CYP27B1, 1α-hydroxylase; CYP24A1, 1,25-dihydroxyvitamin D3 hydroxylase; VDR, vitamin D receptor; RORα, retinoic acid-related orphan receptor α; RORγ, retinoic acid-related orphan receptor γ; VDRE, vitamin D response elements; 1,25(OH) 2 D 3 , cacitriol; 25(OH)D, calcidiol. Under exposure to UV radiation, 7-dehydrocholesterol transforms into previtamin D 3. After several subsequent transformations, vitamin D 3 can be converted into 25(OH)D 3 or 20(OH)D 3 forms, in reactions catalyzed by CYP27A1 or CYP11A1, respectively. Further hydroxylation led by CYP11A1 results in formation of the 20,23(OH) 2 D 3 derivative. 25(OH)D 3 form can be transformed into 24,25(OH) 2 D 3 or it can be hydroxylated by CYP27B1 to active form of vitamin D, 1,25(OH) 2 D 3 . It can bind to one of the receptors: VDR, RORα, or RORγ, which is followed by translocation to VDRE in the nucleus, where it can impact vitamin D-related genes. 1,25(OH) 2 D 3 can be also converted by CYP24A1 to calcitroic acid; which can be subsequently excreted. Vitamin D endocrine system in breast cancer the female population, due to body covering with clothing and non-exposure of skin to UVB. In a study conducted at Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan, vitamin D deficiency was found in 95.6% of breast cancer patients and 77% in the control group (Shaukat et al., 2017).
High plasma concentrations of 25(OH)D may have beneficial effects in prevention of breast cancer, especially in older women. However, the risk of developing this cancer may also be affected by the level of local conversion of 25(OH)D to 1,25(OH) 2 D 3 in the breast tissue, as well as circulating 1,25(OH) 2 D 3 in the serum (Bertone-Johnson et al., 2005). It is also believed that vitamin D 3 deficiency is associated with a worse prognosis in patients with breast cancer (Goodwin et al., 2009). The observational study evaluating the association between serum 25(OH)D levels and breast cancer risk, involving a group of 1 760 individuals, found that serum 25(OH)D levels above 130 nM lead to a 50% reduction in the incidence of this cancer (Garland et al., 2007).

ANALYSES OF ENZYMES IMPLICATED IN VITAMIN D METABOLISM IN BREAST CANCER PATIENTS
As was mentioned above, there is an ambiguous relationship between the vitamin D level and breast cancer incidence. The enzymes which take part in vitamin D metabolism, also serve as the crucial components for maintaining vitamin D concentration in the organism. Therefore, it seems plausible that their impaired activity may be related to the breast cancer occurrence.
It has been demonstrated that in the vitamin D metabolism, enzymes belonging to the cytochrome P450 mixed-function oxidases play the major role (Sugimoto & Shiro, 2012). CYP27A1 is a mitochondrial enzyme responsible for vitamin D 25-hydroxylation. It is also involved in bile acid formation (Lorbek et al., 2012), since it participates in cholesterol transformation to 27-hydroxycholesterol (Kimbung et al., 2017). This specific metabolite serves as a selective estrogen receptor modulator (DuSell et al., 2008), and, therefore, all of the studies reviewed so far, did not analyze CYP27A1 in terms of its prospective implication in the vitamin D level in breast cancer patients. Nevertheless, there are several studies which investigated expression level of CYP27A1 in breast cancer patients. Kimbung and others (Kimbung et al., 2020) conducted a study relating to the immunohistochemical expression of CYP27A1 in breast cancer tumors. Nearly one third of breast cancer tumors expressed high CYP27A1 level. Moreover, the majority of them were high graded tumors, with larger size and without estrogen or progesterone receptors (Kimbung et al., 2020). According to the authors, breast cancer patients with high CYP27A1 displayed poorer overall survival and recurrence-free survival (Kimbung et al., 2020). Another study revealed that an increased CYP27A1 level was predominantly detected in HER2 negative (HER2(-)) breast tumors in grade II with high Ki67 and p53 (Le Cornet et al., 2020). This points out that high CYP27A1 expression appears to be more frequently detected in more aggressive breast cancer types. Therefore, an important question is whether the pathways underlying upregulation of CYP27A in breast tumors are related to vitamin D metabolism.
The subsequent vitamin D hydroxylation -from 25-hydroxyvitamin D (25(OH) D 3 ) to 1,25-dihydroxyvitamin D (1,25(OH) 2 D 3 ) -is driven by the CYP27B1 enzyme (Bikle, 2014). Since 1,25(OH) 2 D 3 is an active form of vitamin D, CYP27B1 is an evident determinant of maintaining the vitamin D level. Nevertheless, there is no general agreement about the CYP27B1 expression level in breast cancer. A breast cancer study analyzing mRNA expression of CYP27B1 in 30 patients revealed its downregulation in comparison to normal breast tissue (Zhalehjoo et al., 2017). Moreover, this decrease was more profound in breast tumors in stage 2, in contrast to those in stage 1 (Zhalehjoo et al., 2017). Similar results were obtained in the study by Segersten and others (Segersten et al., 2005), where CYP27B1 mRNA expression was significantly decreased, though the analysis was performed on only 10 breast tumors. Moreover, an in vitro study revealed that CYP27B1 is expressed in nontransformed human mammary epithelial cells, however, after induced oncogenic transformation, its expression is significantly reduced (Kemmis & Welsh, 2008). Therefore, it is somewhat surprising that some papers indicated that expression of CYP27B1 is increased in breast tumors (Townsend et al., 2005;Friedrich et al., 2006), or not-statistically different between breast tumors and normal breast (Lopes et al., 2010). This demonstrated inconsistency may be linked to the molecular subtype of breast cancer and, possibly, to its own specific vitamin D metabolism. In line with this assumption, several in vitro studies indicated different CYP27B1 expression after exposure of vitamin D analogs in molecularly different breast cancer cell lines Richards et al., 2015). Furthermore, it cannot be excluded that changes in CYP27B1 expression may be related to CY-P27B1 splice variants, since such variants were detected in the breast cancer cell lines Fischer et al., 2007).
Degradation of vitamin D (both forms, 25(OH)D and 1,25(OH)D 3 ) is led by CYP24A1 (Bikle, 2014), and thus its expression is frequently analyzed along with CY-P27B1 in order to obtain the complete insight into vitamin D metabolism in an organism. As it was indicated in an in vitro study, CYP24A1 suppression may impact growth and tumorigenic potential of breast cancer cells (Osanai & Lee, 2016). In the study by Cai and others (Cai et al., 2019) enrolling over 1000 patients from the TCGA-BRCA cohort, low CYP24A1 mRNA expression was significantly correlated with poor breast cancer prognosis, overall survival and relapse-free survival. Interestingly, decreased CYP24A1 expression was also associated with the molecular subtype of breast cancer and hormonal receptors' status. Also, an in vitro study with breast cancer cells reveled that breast cultures corresponding to different molecular subtypes displayed different CYP24A1 mRNA expression levels (Alimirah et al., 2010). These findings support our previous assumption that the vitamin D metabolism may differ depending on a specific subtype of breast cancer. On the other hand, another study indicated that there is a relationship between inhibition of CYP24A1 and increased anticancer influence of 1,25(OH) 2 D 3 (Sheng et al., 2016). This inconsistency may be potentially linked with single nucleotide polymorphisms (SNPs) of CYP24A1, which were reported in breast cancer patients (Cao et al., 2020).
There has been an increasing amount of literature on deregulation of CYP27B1 and CYP24A1 in breast cancer, suggesting that the interaction of 1,25(OH) 2 D 3 with its specific receptors may be also disturbed in the course of this malignancy. Since the main vitamin D receptor -VDR -was identified in breast epithelial cells (Zinser & Welsh, 2004), and since there are hundreds of vitamin D-related genes (Nurminen et al., 2019), alterations in the VDR level may be plausibly associated with breast tumorigenesis. In fact, findings from previous papers support this hypothesis. Based on analysis of over 700 invasive breast tumors, Huss and others (Huss et al., 2019) indicated that high VDR expression is strongly related to favorable prognosis: smaller size and lower grade of tumor, and a decreased mortality risk. Moreover, high VDR expression was also more frequently detected in tumors with estrogen and progesterone receptor expression (Lopes et al., 2010;Huss et al., 2019), which are found to have better prognosis. Comparing the VDR level among the different types of breast cancer, the highest VDR expression is observed in benign lesions, and decreases with tumor progression (Lopes et al., 2010) and more aggressive phenotype (Al-Azhri et al., 2017). An in vitro study by Kemmis and Welsh (Kemmis & Welsh, 2008) also indicated that a provoked malignant transformation of normal breast cells has significantly decreased the VDR expression. However, 1,25(OH) 2 D 3 supplementation to normal cells and breast cancer ones evoked VDR downregulation only in one healthy and in one tumorigenic cell line, with no effect in the majority of the rest of breast cultures (Beaudin et al., 2015). Based on these findings, it seems that another mechanism may be implicated in regulation of the tumorigenic potential of breast cancer cells. According to Singh and Adams (Singh & Adams, 2017), several miRNAs may regulate the VDR level in breast cancer. Based on a literature review and in silico analysis, the authors proposed three mRNAs: miR-23, miR-124 and miR-125, since they play crucial roles in breast carcinogenesis. However, further work is required to establish their function in terms of the VDR level in breast cancer development.
Although vitamin D mainly interacts with VDR, there is a growing evidence of its possible transport via retinoic acid-related orphan receptors α and γ (RORα and RORγ). Vitamin D derivatives 20(OH)D 3 , 20(OH)D 2 and 20,23(OH) 2 D 3 can interact with RORα and RORγ in an antagonistic or inverse agonistic manner (Slominski et al., 2014c;Slominski et al., 2017c). A considerable amount of literature has been published on the plausible connection between impaired expression of nuclear receptors and breast cancer development (Riggins et al., 2010;Muscat et al., 2013;Doan et al., 2014). Although RORα was also found to be expressed in normal breast (Zhu et al., 2006), both receptors are mainly investigated in breast tumors. Expression of RORα is reduced in breast cancer (Zhu et al., 2006;Lu et al., 2007), and there are several studies investigating its role in breast carcinogenesis. According to in vitro research, RORα may impact an increase in aromatase expression in breast cancer cells, thus augmenting their proliferation (Odawara et al., 2009). Given that aromatase can convert androgens to estrogens, this enzyme may play central role in breast cancer development, since estrogens are involved in growth of the breast cancer cells (Saha et al., 2019). The molecular mechanism underlying RORα's impact on inhibition of breast cancer cell proliferation is related to its ability to recruit transcription factors. Both, RORα and RORγ, have an ability to bind corepressors or coactivators in regulatory regions of the transcribed genes, and thus they can influence gene expression (Jetten, 2009). Another in vitro study demonstrated that RORα may bind transcription factor E2F1, which is responsible for cell cycle regulation, and hence for cell proliferation (Xiong & Xu, 2014). Moreover, RORα was also indicated as a potential breast tumor suppressor, as it can regulate the tumor suppressor microenvironmental factor: semaphorin 3F (SEMA3F) in breast cancer cells (Xiong et al., 2012). Expression level of RORγ is also reduced in aggressive types of breast cancers, and decreases with histological grade (Muscat et al., 2013;Oh et al., 2016). Moreover, high RORγ is correlated with distance metastasis-free survival and better outcome of breast cancer (Oh et al., 2014). The molecular mechanism associated with RORγ and breast cancer development is plausibly linked with a DNA repair pathway or TGF-β induced epithelial mesenchymal transition (EMT) pathway (Oh et al., 2016), which leads to metastasis (Imamura et al., 2012). The aforementioned findings suggest that RORα and RORγ may be prospective factors in breast cancer therapy.
As was mentioned above, active form of vitamin D can be hydroxylated by CYP11A1, followed by production of approximately 10 vitamin D derivatives, including 20(OH)D 3 or 20,23(OH) 2 D 3 (Slominski et al., 2014a) . However, CYP11A1 is also a crucial enzyme in cholesterol metabolism, thus it can convert cholesterol to pregnenolone which is an initial step in steroid hormones' synthesis (Miller & Bose, 2011). Therefore, CYP11A1 expression in breast cancer is mainly analyzed from that point of view. Nevertheless, several studies reported that genetic polymorphisms of this gene are prospectively related to breast cancer risk (Zheng et al., 2004;Setiawan et al., 2006;Yaspan et al., 2007;Sun et al., 2012). It cannot be excluded that CYP11A1 gene polymorphisms may be also associated with implications of vitamin D metabolism in breast cancer. However, in order to answer entirely whether CYP11A1 significantly implicates vitamin D metabolism in breast cancer, it is necessary to analyze the vitamin D 3 analogues' level.

POSSIBLE EPIGENETIC IMPACT ON CHANGES IN VITAMIN D METABOLISM OBSERVED IN BREAST CANCER PATIENTS
Epigenetic processes are proven to have an impact on transcription regulation (Weinhold, 2006). Moreover, there is a general agreement that disturbances in epigenetic mechanisms are associated with cancer initiation (Baylin & Jones, 2011). The most fundamental and widely described epigenetic processes are associated with DNA methylation and a variety of histone modifications. DNA hypomethylation occurs in many types of cancer, including breast cancer (Feinberg & Vogelstein, 1983), moreover, changes in DNA methylation are associated with molecular subtypes of breast cancer (Holm et al., 2016), suggesting an important role of impaired DNA methylation in breast carcinogenesis. Additionally, it was proven that alterations in DNA methylation of BRCA, p53 or ESR1 are involved in breast cancer progression (Karsli-Ceppioglu et al., 2014). Therefore, it seems plausible that genes implicated in vitamin D metabolism may be also epigenetically changed during breast cancer development. In line with this hypothesis, a comprehensive cohort study has been recently published (O'Brien et al., 2018). The authors examined 198 CpG loci in or near vitamin D-related genes in women with diagnosed breast cancer or with breast cancer diagnosed in their sisters. The study indicated a significant correlation between methylation of RORα and 25(OH)D level with regard to breast cancer incidence. Furthermore, significant relationship was also noticed for CpG methylation of CYP24A1, CYP27A1 and VDR (O'Brien et al., 2018). Similar results were also found in a previous study, which demonstrated that VDR is significantly hypermethylated in breast tumors in comparison to normal mammary glands (Marik et al., 2010). Changes in methylation of vitamin D-related genes were also detected in breast cancer cell lines. Vitamin D endocrine system in breast cancer In these studies, CYP27B1 (Shi et al., 2002) and VDR (Marik et al., 2010) were found to be hypermethylated. Moreover, such changes were reversible upon treatment with 5-aza-2-deoxycytidine (5-aza-dC). Interestingly, supplementation of 1,25(OH) 2 D 3 did not impact the methylation status in breast cancer cells (Marik et al., 2010). However, 1,25(OH) 2 D 3 treatment in MDA-MB-231 cells was related to Cadherin 1 demethylation, and this effect was significantly higher than after treatment with 5-aza-dC (Lopes et al., 2012). These findings highlight the unambiguous relationship between DNA methylation and breast cancer in terms of vitamin D metabolism.
It was conclusively demonstrated that vitamin D exerts its effect by binding in its active form to VDR. Additionally, it was indicated that VDR has an ability to form a dimer with RORα which can subsequently bind to the vitamin D response elements (VDRE) in the DNA (Cheskis & Freedman, 1994;Nishikawa et al., 1994). This complex impacts transcription through interactions with histone acetyltransferases (HAT), followed by chromatin changes (Campbell et al., 2010). An increasing body of evidence reveals that histones' modifications (including methylation and acetylation) are involved in breast cancer metastasis (extensively reviewed in Nandy et al., 2020;Zhuang et al., 2020). According to Saramäki and others (Saramäki et al., 2009) both the histone acetylation and methylation processes are involved in cyclic chromatin looping during regulation of p21 expression after 1,25(OH) 2 D 3 supplementation to breast cancer cells. It should be also mentioned that the histone deacetylase inhibitors, along with 1,25(OH) 2 D 3 , caused significant changes in colony formation and expression of vitamin D-related genes in breast cancer cell lines (Hossain et al., 2020). These data demonstrate that the active form of vitamin D may be considered as a potential epigenetic drug.

VITAMIN D AND ITS ANALOGUES AS POTENTIAL THERAPEUTIC DRUGS IN BREAST CANCER
The use of 1,25(OH) 2 D 3 at therapeutic doses is limited due to calcemic effects. Thus, the studies are focused on identification or synthesis of its derivatives showing anticancer properties and reduced calcemic effects. Already almost 30 years ago Colston and others (Colston et al., 1992) reported that calcipotriol, a vitamin D analogue, has significantly inhibited proliferation of breast cancer cells in vitro, inhibited tumor progression in vivo and had 100-200 folds lower hypercalcemic effects. The same group also showed that other vitamin D analogues, EB1089 and CB1093, resulted in inhibition of breast cancer growth (Colston et al., 1998;Xie et al., 1999). UVB1 and EM1, novel non-hypercalcemic vitamin D analogues, with higher binding affinity to VDR, caused a decrease in viability of cells derived from triple negative breast cancers and organoids in patient-derived xenografts (PDXs) model of breast cancer. The inhibitory effect was stronger than the one observed for calcitriol (Ferronato et al., 2021). BXL0124, a vitamin D analog with hypercalcemic toxicity, decreased proliferation of breast cancer cells in an in vivo model and inhibited the ductal carcinoma in situ progression to invasive ductal carcinoma (Wahler et al., 2014). Recently discovered CY-P11A1-dereived hydroxyderivatives of vitamin D 3 , such as mono-, dihydroxy-and trihydroxy-forms with or without the hydroxyl group at position C1α, show antiproliferative, pro-differentiation, and anti-inflammatory actions (reviewed in Slominski et al., 2017a;Slominski et al., 2017b;Slominski et al., 2017c;Chaiprasongsuk et al., 2019). The anticancer activity of these derivatives is at least as strong as that of 1,25(OH) 2 D 3 or even stronger (Zbytek et al., 2008;Janjetovic et al., 2009;Li et al., 2010;Slominski et al., 2011;Slominski et al., 2012a;Slominski et al., 2013;Slominski et al., 2013;Slominski et al., 2017c;Tuckey et al., 2011;Lu et al., 2012;Lin et al., 2015;Lin et al., 2016a;Lin et al., 2016b;Lin et al., 2018;Chaiprasongsuk et al., 2019), while the calcemic effects are weaker or are not observed Slominski et al., 2013;Slominski et al., 2014a;Slominski et al., 2014b;Wang et al., 2012). The antitumor effects were observed in different cancers, including non-melanoma skin cancer , oral squamous cell cancers (Oak et al., 2020), melanomas (Wasiewicz et al., 2015;Slominski et al., 2018) and others. Antiproliferative activity of a non-calcemic vitamin D derivative, 20(OH)D 3 , also displayed inhibitory effects on proliferation of breast cancer cells .
In summary, these studies support the hypothesis related to the potential use of these vitamin D analogues as antitumor agents to treat breast cancers.

CLINICAL RESEARCH ON BREAST CANCER AND VITAMIN D
Since experimental studies demonstrated a very promising data, some clinical trials have been established. Currently, 84 clinical trials for breast cancers and vitamin D are registered at clinicaltrials.gov: 16 are recruiting, 5 are active but not recruiting, 8 are terminated, 48 are completed, 2 are withdrawn and for 5 the status is unknown; 13 of these trials are observational and are interventional, 18 of them have the results, but only some of them are published. Some studies showed that vitamin D supplementation did not change the mammographic density, considered as an indicator of breast cancer risk (Brisson et al., 2017;Alipour et al., 2018;Crew et al., 2019). These clinical trials showed that the vitamin D level was not related to the relapse-free survival, breast cancer-specific survival and overall survival (Lohmann et al., 2015). As Charehbili and others (Charehbili et al., 2016) had shown, the vitamin D serum level decreased during treatment with chemotherapy, but no effects on pathological complete response were found. On the other hand, clinical trials support the importance of vitamin D supplementation in the reduction of angiogenic growth factors, such as vascular endothelial growth factor A, angiopoietin 2 and hypoxia-inducible factor 1 in breast cancer patients (Shahvegharasl et al., 2020).

CONCLUSIONS
The currently available data suggest that vitamin D and its related genes may be of clinical significance in breast carcinogenesis. Deregulation of hydroxylases implicated in vitamin D metabolism may abrogate the effect of local 1,25(OH) 2 D 3 production in tumors. Moreover, enzymes involved in vitamin D metabolism in the breast tissue may be important targets for both, prevention and treatment of breast cancer, including epigenetic therapy. Therefore, the plausible effects of vitamin D in cancer therapy or prevention have been the principal aim of numerous studies. However, there is still a need for further studies in this field, especially for analysis of vitamin D-related processes in specific molecular subtypes of breast cancer, as it is possible that different bio-