*1.1.2. Human breast epithelial cells*

The lactiferous ducts spread into the breast through a series of branches decreasing in caliber from the nipple to the terminal ductal-lobular units (TDLU) that are surrounded in specialized, hormonally responsive stroma. Extralobular ducts are lined by columnar epithelium that is supported by myoepithelial cells and a basement membrane composed of elastic fibers. The epithelium of the luminal duct can give rise to ductules, and fully formed lobules can originate directly from these anatomic structures in the nipple or at deeper levels of the mammary ductal system [26]. The lobules that consist of groups of alveolar glands encompassed by specialized vascularized stroma are connected by intralobular ductules that combine to form a single TDLU that drains into the extralobular ductal system.

The normal microscopic appearance of the lobules is not constant because the structure and histological appearance of the lobule in the mature breast are subject to individual changes associated with the menstrual cycle, pregnancy, lactation, exogenous hormone administration, therapy, aging, and menopause. The inactive lobular glands are lined by a single layer of cuboidal epithelial cells supported by underlying, loosely connected myoepithelial cells. The intralobular stroma contains more capillaries and is less densely collagenized than the interlobular surrounding stroma. Immunoreactivity for hormone receptors (ER and PR) is also variably expressed in lobules in a checkerboard staining pattern: the intensity and frequency of staining varies considerably from patient to patient and in many individual slides from one area to another. ER PR are sporadically positive in normal luminal epithelial cells, but some areas are, however, completely negative for those receptors. ER, PR, and AR are almost always negative in myoepithelial cells.

The 3D cultures of human breast epithelial cells have been designed in order to mimic the normal and pathological tissue architecture [27].

The regulation of signaling pathways and homeostasis of free intracellular Ca2+ can entail many cellular and physiological consequences, which may lead to changes in Ca2+ levels during lactation [28]. The Ca2+ influx has a decisive part in determining the concentration of Ca2+ in breast epithelial cells. Breast glandular proliferation, differentiation, and lactation are regulated by several local and systemic hormones, of which estrogen is one of the most important hormones. The regulators of estrogen and its receptor are modulators of proliferation and differentiation of breast epithelial cells [29]. The effect of estrogen on epithelial breast cells is done mainly through genomicpathways, but nongenomic mechanisms are particularly dependent on Ca2+ signaling [30].

(STAT3) signaling pathways, leading to an increased expression of pluripotency factors and breast cancer stem cell enrichment. Concurrent HIF inhibition blocks chemotherapy-induced GSTO1 expression and breast cancer enrichment [23]. The authors have concluded that these

Not just chemotherapeutic agents are responsible for the release of free intracellular calcium. Petrou et al. investigated the effect of several ion channel modulators such as amiodarone, dofetilide, furosemide, minoxidil, loxapine, and nicorandil in prostate and breast cancer cell lines, PC3 and MCF7, respectively and found that in all investigated cases, calcium levels were increased by modulator concentrations comparable to those used clinically [24]. However, the

Calcium-and integrin-binding protein (CIB1) depletion impairs cell survival and tumor growth in triple-negative breast cancer by inducing genetic programs that reduce proliferation and survival and mediate differentiation and cancer stem cell function and epithelial to mesenchymal transition [25]. The authors also observed an almost complete cell death in MDA-468 cells after extended CIB1 depletion, suggesting that CIB1-depleted cells do not become stem cells, but

The lactiferous ducts spread into the breast through a series of branches decreasing in caliber from the nipple to the terminal ductal-lobular units (TDLU) that are surrounded in specialized, hormonally responsive stroma. Extralobular ducts are lined by columnar epithelium that is supported by myoepithelial cells and a basement membrane composed of elastic fibers. The epithelium of the luminal duct can give rise to ductules, and fully formed lobules can originate directly from these anatomic structures in the nipple or at deeper levels of the mammary ductal system [26]. The lobules that consist of groups of alveolar glands encompassed by specialized vascularized stroma are connected by intralobular ductules that combine to

The normal microscopic appearance of the lobules is not constant because the structure and histological appearance of the lobule in the mature breast are subject to individual changes associated with the menstrual cycle, pregnancy, lactation, exogenous hormone administration, therapy, aging, and menopause. The inactive lobular glands are lined by a single layer of cuboidal epithelial cells supported by underlying, loosely connected myoepithelial cells. The intralobular stroma contains more capillaries and is less densely collagenized than the interlobular surrounding stroma. Immunoreactivity for hormone receptors (ER and PR) is also variably expressed in lobules in a checkerboard staining pattern: the intensity and frequency of staining varies considerably from patient to patient and in many individual slides from one area to another. ER PR are sporadically positive in normal luminal epithelial cells, but some areas are, however, completely negative for those receptors. ER, PR, and AR are almost

The 3D cultures of human breast epithelial cells have been designed in order to mimic the

combining effects may improve clinical outcome in breast cancer patients.

way these modulators act on breast cancer stem cells remains unknown.

form a single TDLU that drains into the extralobular ductal system.

rather gain some stem-like features as they are dying.

*1.1.2. Human breast epithelial cells*

168 Calcium and Signal Transduction

always negative in myoepithelial cells.

normal and pathological tissue architecture [27].

Some studies on the MCF-7 breast cell line concluded that breast epithelium proliferation is influenced by Ca2+ through activation of mitogen-activated protein kinase (MAPK) by 17β-estradiol [31]. It is known that several Ca2+-related proteins can cause changes in cellular functions, leading to many breast lesions, including cancer and hypercalcemia-related malignancy, which have a poorer prognosis and have often a more aggressive nature been associated with metastasis [32]. The way calcium is involved in the differentiation of breast epithelial cells is closely dependent on vitamin D3. By modulating Ca2+ metabolism, vitamin D3 plays a crucial role in the regulation of cell proliferation and differentiation [33].

Various epidemiological studies [34, 35] suggest that vitamin D3 deficiency might increase cancer incidence, but no spontaneous tumors have been reported in mice models lacking 1,25(OH)2D3 or deficient in its receptor until recently [36]. The authors observed, for the first time, diverse types of spontaneous tumors in vitamin-D3-deficient mice for more than 1 year of age. The authors concluded that the tumors developed due to increased oxidative stress, cellular senescence, and senescence-associated secretory phenotype molecules, such as hepatocyte growth factor, mediated via its receptor c-Met. As such, vitamin D3 prevents tumorigenesis by inhibiting oxidative stress and inducing tumor cellular senescence in mice, and the study provides direct evidence supporting the role of vitamin D deficiency in increasing cancer incidence.

Calcium levels play an important role in mitochondria-induced apoptosis and epithelial breast cell necrosis [37], and reduction of Ca2+ content in the endoplasmic reticulum lumen is associated with resistance to apoptosis [38]. The release of calcium from the endoplasmic reticulum can be triggered by different molecules, even natural ones like resveratrol, a product commonly found in grapes. Resveratrol triggers the release of calcium from the endoplasmic reticulum, which in turn activates the calpain protease that ultimately leads to degradation of the plasma membrane by calcium-dependent ATPase isoform 1 [39].

Human breast epithelial cells with stem-like phenotype have been also demonstrated to be sensitive to the pathophysiological changes in calcium metabolism. To date, Wang et al. showed that how antioxidant medium is superior in terms of prolonged growth for normal breast epithelial cells that expressed stem cell phenotypes. The characteristics of these mammary stem cells include the deficiency in gap junctional intercellular communication, expression of Oct-4, and the ability to differentiate into basal epithelial cells and to form organoid showing mammary ductal and terminal end bud-like structures [40]. Their study concluded that using this new method of growing breast cancer epithelial cell with stem cell phenotype in a medium with low calcium levels and antioxidants will be of real use in the future studies of mammary development, breast carcinogenesis, chemoprevention, and cancer therapy.

The function of myoepithelial cells is strongly dependent on regulation of intracellular calcium. These cells contract in response to oxytocin secretion during lactation to generate the contractile force required for milk ejection. It is difficult to understand whether the alteration of calcium metabolism of myoepithelial cells plays a role, if any, in carcinogenesis. Even if it has been demonstrated that store-operated Ca2+ entry was mediated by a functional Orai3 in

findings on myoepithelial cell remains largely unknown, because it is known that most breast carcinomas originate in the epithelial cells and the spectrum of myoepithelial proliferative

However, disruption in calcium metabolism may alter the functionality of myoepithelial cells. On this issue, some studies have shown the important role of Orai1 store-operated calcium channels in lactation [43]. Davis et al., using genetically modified mouse models, observed that the store-operated Ca2+ channel Orai1 delivers over 50% of the calcium ions present in the secreted milk. They also demonstrated the role of Orai1 as a principal regulator of oxytocinmediated alveolar unit contractility, milk ejection, general myoepithelial function, and survival. S100 protein is expressed in myoepithelial cells. The S100 gene family is a Ca2+-binding protein with low molecular weight [44]. The members of the S100 family have a myriad of cell functions such as cell proliferation, apoptosis, differentiation, cancer invasion, and metastasis. S100A2 is involved in breast tumorigenesis being downregulated in some cases, which led to the invasion of breast cancer cells [45]. The S100A4 expression is associated with tumor progression and metastatic potential [46]. In a similar manner, S100A7 is not only overexpressed in high-grade ductal breast carcinoma but also in in situ high-grade lesion (DCIS), and some studies suggested that the concomitant expression of S100A7, S100A8, and S100A9 in a class

Apart from the epigenetic and genetic changes that occur within epithelial cells leading to breast proliferative lesions, it has shown that tumor initiation and progression also depend on the intricate intercellular dialog between tumor epithelial cells and the surrounding stromal cells [48]. Among the different cell types comprised in the breast stroma, the most abundant are those of adipose origin, mainly mature adipocytes, preadipocytes, and adipose-derived stromal/stem cells (ASCs). Besides the structural role that breast tissue has, it also has an important bioactive function [49, 50]. In pathologic condition, it is of great importance the interaction that is established between tumor cells and stromal adipocytes within the invasive front characteristic of breast cancers [51, 52]. Similar to breast myoepithelial cells, breast adi-

The role of calcium metabolism in such events is poorly understood, but some studies have found that some proteins such as calpains [54] and calpastatin [55] may be altered in adipose-

Vitamin D3 triggers apoptosis in breast cancer cells and adipocytes by inducing an apoptotic signal by increasing the concentration of intracellular Ca2+. This signal acts as an apoptotic

derived stromal cells being responsible for their enhanced invasion potential.

) breast cancer cells [42], the tumorigenesis impact of these

Alterations in Calcium Signaling Pathways in Breast Cancer

http://dx.doi.org/10.5772/intechopen.80811

171

estrogen receptor-expressing (ER<sup>+</sup>

of breast cancers was associated with poor prognosis [47].

pocytes also express s100 protein—**Figure 1** [53].

breast lesions in scarce.

*1.1.4. Human breast adipocytes*
