**3. Vitamin D signalling in the prevention of breast cancer**

#### **3.1. VDR expression in breast**

Several extra-renal epithelial cells of body express VDR, for example, epithelial cells of rat, mouse and human mammary glands. VDR expression is highest in breast tissues during puberty, pregnancy and lactation in women [47]. In mice, the expression is highest in ductal epithelium when compared to terminal end-buds epithelium of mammary gland. In human, VDR-positive cells are found in basal and luminal layer of breast epithelium [39]. Cap cells and stromal compartments of breast are also rich in VDR [48–50]. The presence of VDR in different cells of breast highlights the complexity of vitamin D signalling in breast tissues.

#### **3.2. Mechanism of vitamin D signalling in breast cancer prevention**

Despite these consistent data, the exact mechanism of breast cancer prevention by vitamin D has yet to be discerned. Both 25(OH)D3 and 1,25(OH)2D3 exert its profound effects on normal VDR-positive breast epithelium such as hormone-stimulated growth inhibition, ductal elongation, ductal branching and induction of biomarkers involved in breast differentiation. The expression of VDR and 1-α-hydroxylase in mammary adipocytes also takes part in the prevention of cancer in whole tissue since adipocytes secrete diffusible signals in response to 25(OH)D3, which constrain morphogenesis of the nearby ductal tissues [48].

Furthermore, alteration in cellular energy metabolism, immune responses and other processes of vitamin D signalling in the prevention of breast cancer on non-tumourigenic breast epithelium is described below.

#### *3.2.1. Anti‐proliferation*

Vitamin D causes cell-cycle arrest by direct or indirect involvement of growth factors and does not allow the cell to enter in the S phase from G1 phase [51]. It increases the expression of cyclin-dependent kinases (CDKs) inhibitors, including p21 and p27, and reduces the expression of CDK2, CDK4, cyclin D1, cyclin A1 and cyclin E1, which results in the arrest of cell-cycle progression [52, 53]. It is also involved in the downregulation of c-myc oncoprotein and inhibits the cell proliferation [54]. However, all these consequences describe that vitamin D hampers the cell proliferation by affecting the crucial controllers of cell-cycle progression. Furthermore, vitamin D also enhances the transcription factor CCAT enhancer-binding protein alpha (C/ EBPα), which mediates the anti-proliferative effects of vitamin D observed in *in vitro* study on MCF-7 cells [55]. Tumour suppressor TCF-4 also hinder cell-cycle progression [56]. Beside these, vitamin D also causes the induction of BRCA 1 (breast cancer 1) gene, which is inversely associated with the cell proliferation, promotes tumour suppression and inhibits cell-cycle progression [57].

#### *3.2.2. Growth arrest and pro‐apoptosis*

Vitamin D plays an important role in the induction of apoptosis in mammary tissues, since *in vitro* conditions, such as shrinkage of cell, condensation of chromatin network and fragmentation of DNA, have been observed in MCF-7 cells upon treatment with vitamin D [58]. The mechanism by which vitamin D induced apoptosis has not been fully understood. However, the most probable mechanism is the downregulation of anti-apoptotic protein, called Bcl2 (51). Vitamin D increases the tumour necrotic factor alpha (TNFα) with or without caspase 3 activation. In the caspase 3-independent mechanism, vitamin D-mediated induction of apoptosis in MCF-7 cells is thought to be correlated with mitochondrial disruption, which causes the release of cytochrome C and formation of reactive oxygen species (ROS) resulting in the apoptosis [59]. Other mechanism of caspase-independent apoptosis induced by vitamin D-dependent Ca+ absorption is most likely associated with the increased activation of lysosomal proteases [60]. Finally, vitamin D also acts a pro-oxidant for breast cancer cells, which generally increase the redox potential [61] of carcinogenic cell, may be one of the most important underlying pro-aptototic mechanisms of vitamin D. The pro-oxidant action of vitamin D in MCF-7 cells could result from increased intra-cellular reactive oxygen species production during aerobic metabolism. Vitamin D inhibits the expression of one of the major constituents of the cellular defence system against ROS, like superoxide dismutase (SOD) [62]. This decrease could be one of the mechanisms underlying the pro-oxidant action of vitamin D. Indeed, it was previously reported that overexpression of SOD protects MCF-7 cells from being injured [63, 64] . Decrease in SOD levels would cause a shift in the balance between superoxides and hydrogen peroxide (H2O2). Increased levels of superoxides can, in turn, cause increased oxidative damage attributable to interaction with NO to form the highly toxic peroxynitrite [65] and to increased availability of free iron that supports hydroxyl radical formation through the Fenton reaction [66].

Changes in the redox state could translate into reversible oxidation of cysteines in major proteins that determine cell fate, such as protein kinases, protein tyrosine phosphatases and transcription factors (e.g. Sp1, activator protein-1, nuclear factor-κB and p53) [67–73]. The key components of the apoptotic process, such as mitochondrial permeability transition pores and increase caspases, are also subjected to redox regulation [74]. Oxidation of the cysteine in the active site of GAPDH may be considered a sensitive, easily accessible marker for these processes. It is noteworthy that the increase in the cellular redox potential was shown to abolish the DNA-binding ability of the transcription factors activator protein-1 and nuclear factor-κB [75] can cause apoptosis and prevent breast cancer. Notably, a recent study describes the relationship between p53 and VDR. Mutant P53 (mutp53) converts the Vitamin D proapoptotic activity into anti-apoptotic activity and attain oncogenic activity which demonstrate gain of function (GOF) [76].

#### *3.2.3. Anti‐angiogenesis*

puberty, pregnancy and lactation in women [47]. In mice, the expression is highest in ductal epithelium when compared to terminal end-buds epithelium of mammary gland. In human, VDR-positive cells are found in basal and luminal layer of breast epithelium [39]. Cap cells and stromal compartments of breast are also rich in VDR [48–50]. The presence of VDR in different cells of breast highlights the complexity of vitamin D signalling in breast tissues.

Despite these consistent data, the exact mechanism of breast cancer prevention by vitamin D has yet to be discerned. Both 25(OH)D3 and 1,25(OH)2D3 exert its profound effects on normal VDR-positive breast epithelium such as hormone-stimulated growth inhibition, ductal elongation, ductal branching and induction of biomarkers involved in breast differentiation. The expression of VDR and 1-α-hydroxylase in mammary adipocytes also takes part in the prevention of cancer in whole tissue since adipocytes secrete diffusible signals in response to

Furthermore, alteration in cellular energy metabolism, immune responses and other processes of vitamin D signalling in the prevention of breast cancer on non-tumourigenic breast epithe-

Vitamin D causes cell-cycle arrest by direct or indirect involvement of growth factors and does not allow the cell to enter in the S phase from G1 phase [51]. It increases the expression of cyclin-dependent kinases (CDKs) inhibitors, including p21 and p27, and reduces the expression of CDK2, CDK4, cyclin D1, cyclin A1 and cyclin E1, which results in the arrest of cell-cycle progression [52, 53]. It is also involved in the downregulation of c-myc oncoprotein and inhibits the cell proliferation [54]. However, all these consequences describe that vitamin D hampers the cell proliferation by affecting the crucial controllers of cell-cycle progression. Furthermore, vitamin D also enhances the transcription factor CCAT enhancer-binding protein alpha (C/ EBPα), which mediates the anti-proliferative effects of vitamin D observed in *in vitro* study on MCF-7 cells [55]. Tumour suppressor TCF-4 also hinder cell-cycle progression [56]. Beside these, vitamin D also causes the induction of BRCA 1 (breast cancer 1) gene, which is inversely associated with the cell proliferation, promotes tumour suppression and inhibits cell-cycle

Vitamin D plays an important role in the induction of apoptosis in mammary tissues, since *in vitro* conditions, such as shrinkage of cell, condensation of chromatin network and fragmentation of DNA, have been observed in MCF-7 cells upon treatment with vitamin D [58]. The mechanism by which vitamin D induced apoptosis has not been fully understood. However, the most probable mechanism is the downregulation of anti-apoptotic protein, called Bcl2 (51). Vitamin D increases the tumour necrotic factor alpha (TNFα) with or without caspase 3 activation. In the caspase 3-independent mechanism, vitamin D-mediated induction of

**3.2. Mechanism of vitamin D signalling in breast cancer prevention**

25(OH)D3, which constrain morphogenesis of the nearby ductal tissues [48].

lium is described below.

140 A Critical Evaluation of Vitamin D - Clinical Overview

*3.2.1. Anti‐proliferation*

progression [57].

*3.2.2. Growth arrest and pro‐apoptosis*

Vitamin D inhibits angiogenesis, which is another important feature for tumour growth and progression. It also has the ability to impede angiogenesis at very minute concentration [77] mediated through the downregulation of vascular endothelial growth factor (VEGF), tenascin-C, tumour growth factor α (TGF-α) and epidermal growth factor (EGF) [78, 79].

#### *3.2.4. Anti‐invasion or anti‐metastasis*

Vitamin D inhibits the invasion of tumour in nearby tissues but its deficiency promotes the growth of breast cancer cells in the bones of nude mice and alters the bone micro-environment [80]. This ability of vitamin D is supposed to be caused by the decrease expression of metalloproteinases (MMP-9) and serine proteinases (such as urokinase-type plasminogen activator and tissue-type plasminogen activator) along with the increased expression of their inhibitors [81]. In addition, vitamin D also downregulates P-cadherin [82] and upregulates E-cadherin [83].

#### *3.2.5. Anti‐inflammation*

Vitamin D reduces the expression of cyclooxygenase-2 (COX-2), which plays a crucial role in the synthesis of prostaglandin in many breast cancer cell lines in human. It increases the upregulation of 15-hydroxyprostaglandin dehydrogenase, an enzyme which is involved in catalysing the conversion of active prostaglandins into biologically inactive ketoderivatives [84]. Prostaglandins have been supposed to play a role in the breast cancer development and its progression [85]. Prostaglandins are secreted by the breast cancer cells or surrounding tissues promote tumour progression caused by cell proliferation, resistant to apoptosis, tumour invasion and angiogenesis [85]. An increased expression of COX-2 in breast cancer has been assumed to correlate with high-grade, large tumour size and poor prognosis [86].

#### *3.2.6. Anti‐estrogen*

Vitamin D inhibits estrogen biosynthesis (steroidogenesis) and its biological actions [84]. Vitamin D suppresses the estrogen pathway by inhibiting the expression of gene which encodes aromatase (the enzyme which converts androgens to estrogen) [84]. Vitamin D also reduces the expression of estrogen receptor alpha (ERα-) [87]. The combined actions of vitamin D can decrease the estrogen and the receptor, which mediates their signalling in the prevention of breast cancer.

#### **3.3. Vitamin D deficiency and breast cancer**

The half-life of circulating vitamin D is approximately about 2–3 weeks which is a better indicator of blood vitamin D. Active vitamin D3 (1,25(OH)2D3) is locally synthesized from its precursor (25-(OH)D3) in almost all body cells because of the universal presence of 1αhydroxylases in all cell type including breast [88]. So, the deficiency of 1-α hydroxylase may augment the deficiency of vitamin D and thereby associated with increased breast cancer risk and mortality [89].

Serum vitamin D concentrations and vitamin D supplementations are the independent predictors of breast cancer risk. Serum level of vitamin D of more than 50 ng/ml is associated with the 50% lower risk of breast cancer in women than serum values less than 30 ng/ml [90, 91]. In addition, breast cancer risk reduces in the pre-menopausal women who consume calcium and vitamin D orally [92]. Locally advanced breast cancer patients have more severe vitamin D deficiency than those with early-stage disease [93].

Deficiency of vitamin D is related with secondary hyperparathyroidism, which causes increased bone resorption, release of calcium from bones osteoclasts into the blood and may exacerbate osteoporosis with subsequent harsh effects on bone mineral density (BMD). In breast cancer patients, osteopenia and osteoporosis mostly occur because of early menopause and vitamin D deficiency, which is then augmented by chemotherapy and hormone replacement therapy [94]. Therefore, breast cancer patients are necessary to suffer a baseline metabolic bone evaluation along with circulating vitamin D levels and bone mineral densitometry [94, 95].

Vitamin D deficiency is also associated with the recurrence, tumour size and death from breast cancer. It means that having enough amount of vitamin D may be able to keep a cancer from getting worse. In fact a recent meta-analysis concluded that high circulating level of vitamin D is weakly related with breast cancer incident; however, strong association was found with better breast cancer survival [89]. So, the maintenance of an optimal vitamin D status at the time of diagnosis and during 1-year follow-up period is necessary for improving survival of breast cancer patient.

There are four types of studies which illustrated whether exposure of ultraviolet B (UVB) radiations and low levels of vitamin D decrease the risk of breast cancer.

#### *3.3.1. Geographical studies*

[81]. In addition, vitamin D also downregulates P-cadherin [82] and upregulates E-cadherin

Vitamin D reduces the expression of cyclooxygenase-2 (COX-2), which plays a crucial role in the synthesis of prostaglandin in many breast cancer cell lines in human. It increases the upregulation of 15-hydroxyprostaglandin dehydrogenase, an enzyme which is involved in catalysing the conversion of active prostaglandins into biologically inactive ketoderivatives [84]. Prostaglandins have been supposed to play a role in the breast cancer development and its progression [85]. Prostaglandins are secreted by the breast cancer cells or surrounding tissues promote tumour progression caused by cell proliferation, resistant to apoptosis, tumour invasion and angiogenesis [85]. An increased expression of COX-2 in breast cancer has been assumed to correlate with high-grade, large tumour size and poor prognosis [86].

Vitamin D inhibits estrogen biosynthesis (steroidogenesis) and its biological actions [84]. Vitamin D suppresses the estrogen pathway by inhibiting the expression of gene which encodes aromatase (the enzyme which converts androgens to estrogen) [84]. Vitamin D also reduces the expression of estrogen receptor alpha (ERα-) [87]. The combined actions of vitamin D can decrease the estrogen and the receptor, which mediates their signalling in the prevention

The half-life of circulating vitamin D is approximately about 2–3 weeks which is a better indicator of blood vitamin D. Active vitamin D3 (1,25(OH)2D3) is locally synthesized from its precursor (25-(OH)D3) in almost all body cells because of the universal presence of 1αhydroxylases in all cell type including breast [88]. So, the deficiency of 1-α hydroxylase may augment the deficiency of vitamin D and thereby associated with increased breast cancer risk

Serum vitamin D concentrations and vitamin D supplementations are the independent predictors of breast cancer risk. Serum level of vitamin D of more than 50 ng/ml is associated with the 50% lower risk of breast cancer in women than serum values less than 30 ng/ml [90, 91]. In addition, breast cancer risk reduces in the pre-menopausal women who consume calcium and vitamin D orally [92]. Locally advanced breast cancer patients have more severe

Deficiency of vitamin D is related with secondary hyperparathyroidism, which causes increased bone resorption, release of calcium from bones osteoclasts into the blood and may exacerbate osteoporosis with subsequent harsh effects on bone mineral density (BMD). In breast cancer patients, osteopenia and osteoporosis mostly occur because of early menopause and vitamin D deficiency, which is then augmented by chemotherapy and hormone replacement therapy [94]. Therefore, breast cancer patients are necessary to suffer a baseline metabolic

[83].

*3.2.5. Anti‐inflammation*

142 A Critical Evaluation of Vitamin D - Clinical Overview

*3.2.6. Anti‐estrogen*

of breast cancer.

and mortality [89].

**3.3. Vitamin D deficiency and breast cancer**

vitamin D deficiency than those with early-stage disease [93].

In these studies, the geographical variation in the incidence or mortality of breast cancer is compared statistically with solar UVB radiations. The lower breast cancer incidence rate was found in the regions of high solar UVB radiations such as in Australia, China, France, Nordic countries, Spain and the United States [96].

#### *3.3.2. Observational studies*

Observational studies do comparison of vitamin D levels with the incidence of breast cancer among cases and controls. There are two categories of observational studies:


Only the case-control studies have reported that low levels of vitamin D are associated with breast cancer risk [97]. The reason why nested case-control studies have not reported the same results may be due to


Observational studies have also documented that those females have higher vitamin D levels at the time of diagnosis live longer as compared to those with low vitamin D levels [46, 96]. In addition, the chances of mortality are higher in black women after diagnosis of breast cancer than in white women.

#### *3.3.3. Laboratory studies*

Laboratory studies have focused on the mechanisms of vitamin D in the contribution of reduced risk of breast and other cancer types. According to these studies, vitamin D allows the cells to stay alive if they are the right type and present at the right place, or it helps the cells to commit suicide (apoptosis) if cells are not the right type or not present at the right place. Vitamin D also reduces the formation of blood vessels around tumours and decreases the ability of tumours to invade [98]. According to the randomized controlled trials, vitamin D reduced the risk of cancer, including breast cancer [99, 100].
