*1.1.1. Human breast stem cells*

The mammary gland structures are capable of self-renewal due to the presence of mammary stem cells (MSCs) and multipotent adult stem cells. Mammary stem cells lead to the growth of the mammary gland during puberty and are responsible for its further development during pregnancy. Besides their intrinsic self-renewal capacity, normal stem cells call for the adoption of other additional mechanisms to protect them from the microenvironmental pressure that may overdrive the stem cell pool [5]. MSCs are able to reconstitute a completely functional mammary gland upon orthotopic transplantation [6]. MSCs can differentiate into mature epithelial cells of either myoepithelial or luminal lineage through a series of lineagerestricted progenitor intermediate cells. Of major importance is also the long-term survival and expansion of MSCs [7] and the additional accumulation of genetic and/or epigenetic alterations in those cells that may increase the susceptibility of neoplastic transformation [8]. Van Keymeulen et al. demonstrated that the mammary gland contains different types of longlived stem cells [9].

In breast cancer, both stem cells and progenitor cells are a potential candidate for tumorigenesis, and this could underpin the extraordinary phenotypic heterogeneity of malignant breast tumors. Also, different breast tumor subtypes might be linked to distinctive mammary stem cells and progenitor cells within the mammary epithelia as suggested by different studies [10]. Understanding how these cell subpopulations influence the normal epithelial differentiation hierarchy and the development of mammary tumors could have a significant impact on the proper taxonomy of these lesions.

Unlike the mouse counterparts, the detection of human breast stem cells may be difficult due to the lack of reliable surface makers. Also, the profile of MSCs is less predictable compared to mouse models [11].

To identify MSCs, several studies have employed multiple methods, including: 5-bromo-2-deoxy-uridine (BrdU) label-retention studies, nonadherent mammosphere cultures, cellsurface markers, such as Sca1 and CD49f, labeling MSCs with lipophilic fluorescent dye PKH26 during mammosphere growth and Hoechst dye efflux [12, 13]. These methods were very helpful for the further detection and characterization of signal transduction pathways such as the Notch, Wnt, and Hedgehog pathways that may be crucial for the self-renewal and fate determination of MSCs.

**1.1. Cell subtypes in human breast cancer and the alterations in calcium** 

The mammary gland is an exocrine, compound tubuloalveolar gland [2]. Each mammary gland has 15–20 glandular lobes in its structure, each lobe being a separate gland with its excretory channel (galactophore channel) that opens at the level of the nipple via the galactophore pore [3]. The glandular lobe consists of glandular lobules delimited by fibrous connective tissue and fat. Lobules have a radiant arrangement, each opening through a lactiferous duct in the nipple, presenting a dilation called lactiferous sinus before opening. Lobules are composed of parenchyma and stroma, and made of loose connective tissue [4]. Each lobule consists of alveoli, lined by cuboidal epithelial cells, which secrete milk, and lactiferous ducts, lined by columnar epithelium, both epithelia surrounded by an outer layer of myoepithelial cells. The stromal cell population is composed of mesenchymal cells, including adipocytes,

The mammary gland structures are capable of self-renewal due to the presence of mammary stem cells (MSCs) and multipotent adult stem cells. Mammary stem cells lead to the growth of the mammary gland during puberty and are responsible for its further development during pregnancy. Besides their intrinsic self-renewal capacity, normal stem cells call for the adoption of other additional mechanisms to protect them from the microenvironmental pressure that may overdrive the stem cell pool [5]. MSCs are able to reconstitute a completely functional mammary gland upon orthotopic transplantation [6]. MSCs can differentiate into mature epithelial cells of either myoepithelial or luminal lineage through a series of lineagerestricted progenitor intermediate cells. Of major importance is also the long-term survival and expansion of MSCs [7] and the additional accumulation of genetic and/or epigenetic alterations in those cells that may increase the susceptibility of neoplastic transformation [8]. Van Keymeulen et al. demonstrated that the mammary gland contains different types of long-

In breast cancer, both stem cells and progenitor cells are a potential candidate for tumorigenesis, and this could underpin the extraordinary phenotypic heterogeneity of malignant breast tumors. Also, different breast tumor subtypes might be linked to distinctive mammary stem cells and progenitor cells within the mammary epithelia as suggested by different studies [10]. Understanding how these cell subpopulations influence the normal epithelial differentiation hierarchy and the development of mammary tumors could have a significant impact

Unlike the mouse counterparts, the detection of human breast stem cells may be difficult due to the lack of reliable surface makers. Also, the profile of MSCs is less predictable compared

To identify MSCs, several studies have employed multiple methods, including: 5-bromo-2-deoxy-uridine (BrdU) label-retention studies, nonadherent mammosphere cultures, cellsurface markers, such as Sca1 and CD49f, labeling MSCs with lipophilic fluorescent dye PKH26 during mammosphere growth and Hoechst dye efflux [12, 13]. These methods were

**homeostasis**

166 Calcium and Signal Transduction

fibroblasts, and immune cells.

*1.1.1. Human breast stem cells*

lived stem cells [9].

to mouse models [11].

on the proper taxonomy of these lesions.

Identification of breast cancer stem cells is strictly dependent on cell-surface markers. Several markers have been proposed such as hyaluronan receptor (CD44), signal transducer CD24 (CD24), CD133 (Prominin-1), integrins CD29 (β1) and CD49f (α6), aldehyde dehydrogenase-1 (ALDH1), as tumor-initiating cells in breast cancer progression with high metastatic potential and in high-grade tumors resistant to therapeutic treatments [14–17]. However, currently, there is no agreement regarding the phenotypic characterization of breast cancer stem cells. In addition to this discord, the great heterogeneity of breast tumors reflected by a myriad of histological subtypes with variable clinical presentations and diverse molecular signatures also contributes to this major shortcoming. The intrinsic molecular taxonomy describes five major subtypes of breast cancer (luminal-A, luminal-B, basal-like, HER2, and normal-like) which overlap with various clinicopathological classification systems and correlate with clinical behavior being vital for patient's management. In addition, different breast cancer stem cells phenotypes have been described contributing to the proper characterization and nomenclature of breast malignant lesions. Several immunohistochemical markers have been characterized showing that the prevalence of stem cell-like markers varies according to tumor histological subtype [18].

In the light of these facts, some studies have proposed ALDH1 as an independent prognostic marker in breast cancer. Ginestier et al. showed a prevalence of 30% for ALDH1 positivity in a cohort comprising 577 breast tumors from two independent tumor sets. They also showed that ALDH1 expression correlates with a high histological grade, human epidermal growth factor receptor type 2 (HER2) overexpression, and absence of estrogen receptor and progesterone receptor expression [19]. A similar study [20] highlighted the worst prognosis of breast cancer patients with ALDH1 expression. There is no agreement in this matter, as other studies fail to find these correlations [21] even in more aggressive breast tumor subtypes such as inflammatory breast cancer.

With all these conflicting results, the reliability of ALDH1 expression as a clinical prognostic factor is doubtful, thus increasing the need for a standard protocol and more rigorous evaluation criteria, as well as consideration of the dissimilarity between whole-tissue staining versus tissue microarray staining [22].

Alterations in calcium homeostasis frequently occur in some pathological conditions such as malignant proliferation and could have a key role to play in the near future of some targeted therapeutic approaches. Some recent studies have shown that exposure of breast cancer cells to chemotherapy (i.e., carboplatin) induces Ca2+ release and leads to an enrichment of breast cancer stem cells [23]. Lu et al. have documented that chemotherapy induces the expression of glutathione S-transferase omega 1 (GSTO1), a factor which is dependent on hypoxia-inducible factor 1 (HIF-1) and HIF-2. In turn, low level of GSTO1 revokes carboplatin-induced breast cancer stem cell enrichment, decreasing tumor initiation and metastatic potential and delaying tumor recurrence after chemotherapy. The authors also found that GSTO1 interacts with the ryanodine receptor (RYR1) and increases calcium release from the endoplasmic reticulum. In this manner, high levels of cytosolic calcium activate proline-rich tyrosine kinase 2 (PYK2)/ tyrosine-protein kinase (SRC)/signal transducers and activators of transcription factors 3 (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 combining effects may improve clinical outcome in breast cancer patients.

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

Alterations in Calcium Signaling Pathways in Breast Cancer

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

169

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

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

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

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

the plasma membrane by calcium-dependent ATPase isoform 1 [39].

D3 plays a crucial role in the regulation of cell proliferation and differentiation [33].

dependent on Ca2+ signaling [30].

cancer incidence.

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

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