**1. Introduction**

#### **1.1. Breast cancer**

Breast carcinoma is one of the most frequently diagnosed cancers among women worldwide with a high frequency reported in the West [1, 2]. This highest incidence of breast cancer in American whites and in most European countries reveal the long-standing high prevalence of reproductive factors associated with increased risk of breast cancer, including early menarche, late child bearing age, few pregnancies, hormone replacement therapy and increased mammography [3, 4]. In Israel, the increased incidence of breast cancer may reflect the disproportionately high prevalence of BRCA1 and BRCA2 mutations [5, 6].

Western lifestyle is another most important factor for Britain's high number of breast cancer cases fuelled by the women overeating, too much drinking and too little exercise doing in routine life. In addition, breastfeeding is also an important factor, which reduces the chance of developing breast cancer. Eastern women do not drink alcohol than women in the United Kingdom, and obesity ratio is much lower in Asian women than in western women, whereas breastfeeding rates are much higher in Asians (http://www.dailymail.co.uk/news/article-1301445/Western-lifestyle-blame-soaring-breast-cancer-rates.html). Affected women with breast cancer are usually young and often present with advanced disease [7]. According to a World Health Organization (WHO) estimate, around 25.2% people are diagnosed with breast cancer annually. The exact reason why a woman develops breast cancer is still unrevealed; though certain risk factors enhance a person's probability of getting breast cancer.

The factors that play a significant role in the aetiology of breast cancer include genetic [8, 9], hormonal [10, 11], environmental [12], lifestyle [13] and reproductive factors [14]. In addition, ovarian hormones (endogenous estrogen) are the key risk factors for the development of breast cancer and their progression among post-menopausal women [15, 16]. However, it is unclear that to what degree the effects of other risk factors may be mediated by their links with circulating free estradiol. Intake of vegetables and fruits are related with a substantial decrease of breast cancer risk [17, 18]. Vegetables are rich in antioxidants and certain phytochemicals may contribute to the reduced risk of breast cancer [19–21]. Plant-based diets are also high in fibres, which can decrease serum estrogen and could, in this way, contribute to reduced risk of breast cancer [22, 23]. In addition, increased consumption of fruit and vegetables are associated with lower rates of obesity, which is a crucial risk factor for post-menopausal breast cancer [24]. High energy intake, physical sluggishness, high body mass index (BMI) and weight put on are coupled to an increased breast [25] cancer risk. Low levels of HDL-C in breast cancer patients than in control subjects have also been documented [26]. But still, data from prospective studies are very limited (Moorman, 1998). Furthermore, consanguineous marriages are common in certain racial groups, which will increases the risk of breast cancer [27].

Among these contributing factors, vitamin D and its receptor gene polymorphisms may play a pivotal role in the development of mammary gland tumourigenesis [28].

#### **1.2. Vitamin D and vitamin D receptor (VDR)**

Vitamin D and VDR are the two most important participants playing a key role in vitamin D endocrine system in the prevention of breast cancer. Vitamin D is a sunshine vitamin, which is involved in a variety of actions and also reduces the risk of many cancers [29, 30].

VDR is a member of nuclear receptor (NR) superfamily and transcription regulating factor also called NR1I1 or nuclear receptor subfamily 1, group I and member 1. VDR is a high‐affnity, low‐capacity receptor having a molecular weight of about 48–55 kD. VDR is expressed in majority of human tissues. But some cells have decrease or no VDR expression including RBCs, mature cardiac and skeletal muscles and cerebellar Purkinje cells [31]. Its actions are preceded by the formation of heterodimer with retinoid X receptor (RXR), which causes the conforma‐ tional changes in VDR and allow the binding of vitamin D3 at ligand binding domain (LBD). In addition, the heterodimer complex then binds with a specific sequence present in the DNA called vitamin D response element (VDRE). Genomic pathway involves the expression of genes in a tissue‐specific manner [28].

#### *1.2.1. VDR domains*

**1. Introduction**

136 A Critical Evaluation of Vitamin D - Clinical Overview

**1.1. Breast cancer**

Breast carcinoma is one of the most frequently diagnosed cancers among women worldwide with a high frequency reported in the West [1, 2]. This highest incidence of breast cancer in American whites and in most European countries reveal the long-standing high prevalence of reproductive factors associated with increased risk of breast cancer, including early menarche, late child bearing age, few pregnancies, hormone replacement therapy and increased mammography [3, 4]. In Israel, the increased incidence of breast cancer may reflect the dispropor-

Western lifestyle is another most important factor for Britain's high number of breast cancer cases fuelled by the women overeating, too much drinking and too little exercise doing in routine life. In addition, breastfeeding is also an important factor, which reduces the chance of developing breast cancer. Eastern women do not drink alcohol than women in the United Kingdom, and obesity ratio is much lower in Asian women than in western women, whereas breastfeeding rates are much higher in Asians (http://www.dailymail.co.uk/news/article-1301445/Western-lifestyle-blame-soaring-breast-cancer-rates.html). Affected women with breast cancer are usually young and often present with advanced disease [7]. According to a World Health Organization (WHO) estimate, around 25.2% people are diagnosed with breast cancer annually. The exact reason why a woman develops breast cancer is still unrevealed;

though certain risk factors enhance a person's probability of getting breast cancer.

common in certain racial groups, which will increases the risk of breast cancer [27].

a pivotal role in the development of mammary gland tumourigenesis [28].

Among these contributing factors, vitamin D and its receptor gene polymorphisms may play

The factors that play a significant role in the aetiology of breast cancer include genetic [8, 9], hormonal [10, 11], environmental [12], lifestyle [13] and reproductive factors [14]. In addition, ovarian hormones (endogenous estrogen) are the key risk factors for the development of breast cancer and their progression among post-menopausal women [15, 16]. However, it is unclear that to what degree the effects of other risk factors may be mediated by their links with circulating free estradiol. Intake of vegetables and fruits are related with a substantial decrease of breast cancer risk [17, 18]. Vegetables are rich in antioxidants and certain phytochemicals may contribute to the reduced risk of breast cancer [19–21]. Plant-based diets are also high in fibres, which can decrease serum estrogen and could, in this way, contribute to reduced risk of breast cancer [22, 23]. In addition, increased consumption of fruit and vegetables are associated with lower rates of obesity, which is a crucial risk factor for post-menopausal breast cancer [24]. High energy intake, physical sluggishness, high body mass index (BMI) and weight put on are coupled to an increased breast [25] cancer risk. Low levels of HDL-C in breast cancer patients than in control subjects have also been documented [26]. But still, data from prospective studies are very limited (Moorman, 1998). Furthermore, consanguineous marriages are

tionately high prevalence of BRCA1 and BRCA2 mutations [5, 6].

VDR contain five functional domains (**Figure 1**) including:


Both N‐ter and C‐ter has activation function (called AF‐2) in translation [33].

#### *1.2.2. Vitamin D/VDR actions*


**Figure 1.** The crystal structure of VDR showing its functional domains [34]. (A) Schematic representation of VDR domains. (B) LBD of the VDR which contains 12 alpha helices. (C) The binding mode of Vitamin D in the hormone-binding pocket. (D) The DBD of the Vitamin D. The two zinc atoms are represented in blue in colour, whereas beta sheets are represented in yellow colour.

## **2. Bio‐activation and metabolism of Vitamin D in normal breast**

It is already known that vitamin D affects the breast cancer cell growth but limited information is available about its delivery, uptake and metabolism in mammary cells. Vitamin D is either derived from the gastrointestinal (GIT) absorption or synthesized within the skin under the exposure of UVB radiations, which then undergoes the 25-hydroxylation in liver in presence of 25-hydroxylase resulting in the production of 25(OH)D3. 25(OH)D3 is the precursor molecule for the synthesis of active Vitamin D3 (1,25(OH)2D3). It is a major circulating form of vitamin D, which is stored in adipose tissues. It is also an accurate biomarker of vitamin D, which determines the overall status of vitamin D in the body. However, the precursor does not readily binds to the VDR and must be converted into its active form, 1,25(OH)2D3, which has high binding affinity to VDR. The conversion of precursor vitamin D into its active metabolite occurs in the presence of 1-α-hydroxylases. Immunohistochemistry and *in situ* hybridization studies indicated strong expression of 1α-hydroxylase protein and mRNA in the distal convoluted tubule, the cortical and medullary part of the collecting ducts and the papillary epithelia. Lower expression was observed along the thick ascending limb of the loop of Henle and Bowman's capsule. Weaker and more variable expression of 1α-hydroxylase protein and mRNA was seen in proximal convoluted tubules, and no expression was observed in glomeruli or vascular structures [36]. Whereas lesser expression of 1*α*–hydroxylase was also observed in non–renal cells including keratinocytes, macrophages, prostatic epithelium, colonocytes [37, 38] and breast epithelium [39] to lesser extent. Kidneys and non-renal 1-α-hydroxylases are encoded by the same gene mapped on the chromosome 12 [40]. However, the presence of this enzyme on non-renal tissues indicated that the non-renal tissues have the ability of vitamin D bio-activation, responsible to convert 25(OH)D3 into 1,25(OH)2D3. 1,25(OH)2D3 is virtually not detected in human serum under anephric conditions, which means that kidneys are the major source of 1,25(OH)2D3 in circulation. These observations emphasize that 1,25(OH)2D3 produced by the non-renal tissues is not released in the bloodstream. However, they act locally upon binding to VDR on the same tissues from where it is synthesized. Such local actions of vitamin D are likely included in proliferation, differentiation and apoptosis, which are discussed below in later sections.

#### **2.1. Bio‐activation pathways in breast cells**

The above information supports the hypothesis that two distinct pathways may be involved in the bio-synthesis and bio-activation of vitamin D in breast such as 1,25(OH)2D3 and 25(OH)D3 (vitamin D precursor) pathways [41, 42].

#### *2.1.1. Endocrine pathway*

**Figure 1.** The crystal structure of VDR showing its functional domains [34]. (A) Schematic representation of VDR domains. (B) LBD of the VDR which contains 12 alpha helices. (C) The binding mode of Vitamin D in the hormone-binding pocket. (D) The DBD of the Vitamin D. The two zinc atoms are represented in blue in colour, whereas beta sheets

It is already known that vitamin D affects the breast cancer cell growth but limited information is available about its delivery, uptake and metabolism in mammary cells. Vitamin D is either derived from the gastrointestinal (GIT) absorption or synthesized within the skin under the exposure of UVB radiations, which then undergoes the 25-hydroxylation in liver in presence of 25-hydroxylase resulting in the production of 25(OH)D3. 25(OH)D3 is the precursor molecule for the synthesis of active Vitamin D3 (1,25(OH)2D3). It is a major circulating form of vitamin D, which is stored in adipose tissues. It is also an accurate biomarker of vitamin D, which determines the overall status of vitamin D in the body. However, the precursor does not readily binds to the VDR and must be converted into its active form, 1,25(OH)2D3, which has high binding affinity to VDR. The conversion of precursor vitamin D into its active metabolite occurs in the presence of 1-α-hydroxylases. Immunohistochemistry and *in situ* hybridization studies indicated strong expression of 1α-hydroxylase protein and mRNA in the distal convoluted tubule, the cortical and medullary part of the collecting ducts and the papillary epithelia. Lower expression was observed along the thick ascending limb of the loop of Henle and Bowman's capsule. Weaker and more variable expression of 1α-hydroxylase protein and mRNA was seen in proximal convoluted tubules, and no expression was observed in glomeruli or vascular structures [36]. Whereas lesser expression of 1*α*–hydroxylase was also observed in non–renal cells including keratinocytes, macrophages, prostatic epithelium, colonocytes [37, 38] and breast epithelium [39] to lesser extent. Kidneys and non-renal 1-α-hydroxylases are

**2. Bio‐activation and metabolism of Vitamin D in normal breast**

are represented in yellow colour.

138 A Critical Evaluation of Vitamin D - Clinical Overview

The endocrine pathway is involved with the circulation of 1,25(OH)2D3, which reaches the mammary tissues and produces anti-neoplastic effects through genomic pathway.

#### *2.1.2. Autocrine/paracrine pathway*

The other pathway is the autocrine/paracrine pathway involved with the 25(OH)D3, which reaches the mammary gland and converts into 1,25(OH)2D3 [43] in the presence of 1-αhydroxylase to prevent breast cancer [41]. Most of the extra-renal tissues of the body have its own 1-α-hydroxylase enzyme needed for the production of active metabolite of vitamin D [37]. The circulating level of 25(OH)D3 seems to be the key regulator of tissue-specific synthesis of active vitamin D [37, 44]. The locally produced active vitamin D binds with VDRs of mammary epithelium in order to regulate the expression of more than 200 genes, which are involved in controlling cell proliferation, inhibit cell growth, stimulate cell differentiation, induce apoptosis and inhibit angiogenesis [45] and contribute in the prevention of breast tumourogenesis [46]. Moreover, mammary epithelial cells also contain 24-hydroxylase enzyme (CYP24), which converts active vitamin D into less active metabolites including 24,25-dihydrohydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 [43]. For this reason, we can say that breast tissues contain all the elements of vitamin D signalling axis, which involve in the local synthesis as well as metabolism of vitamin D and its signal transduction through VDRs.
