Breast Cancer Regulatory Pathways

*Breast Cancer Biology*

[103] Rivera A. Groundwater news. Natural Resources Canada/ESS/Scientific and Technical Publishing Services. 2005

[105] Trinei M et al. A p53-p66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis. Oncogene.

[106] Pinton P, Rizzuto R. p66Shc, oxidative stress and aging: Importing a lifespan determinant into mitochondria.

[107] Drane P et al. Reciprocal downregulation of p53 and SOD2 gene expression-implication in p53 mediated apoptosis. Oncogene. 2001;**20**(4):430-439

mitochondria in NLRP3 inflammasome activation. Nature;**469**(7329):221-225

Cell Cycle. 2008;**7**(3):304-308

[108] Zhou R et al. A role for

[109] Banin S. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science.

[110] Tyler Zarubin QJ, New L, Han J. Identification of eight genes that are potentially involved in tamoxifensensitivity in breast cancer cells. Cell Research. 2005;**15**(6):439-446

[111] Brancho DTN, Jaeschke A, Ventura JJ, Kelkar N, Tanaka Y, Kyuuma M, et al. Mechanism of p38 MAP kinase activation in vivo. Genes & Development. 2003;**17**(16):1969-1978

[112] White CPAE. Does control of mutant p53 by Mdm2 complicate cancer therapy. Genes & Development.

[113] Wheeler TMSK, Lueck JD, Osborne RJ, Lin X, Dirksen RT, Thornton CA. Reversal of RNA

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[104] Sablina AA et al. The antioxidant function of the p53 tumor suppressor. Nature Medicine. 2005;**11**(12):1306-1313 dominance by displacement of protein sequestered on triplet repeat R. Science.

[114] Massarweh S et al. Tamoxifen resistance in breast tumors is driven by growth factor receptor signaling with repression of classic estrogen receptor genomic function. Cancer Research.

overexpression in MCF-7 breast cancer cells produces a tumorigenic, invasive and hormone resistant phenotype. Oncogene. 1999;**18**(44):6063-6070

[116] Cancello RHC, Viguerie N, Taleb S, Poitou C, Rouault C, Coupaye M, et al. Reduction of macrophage infiltration and chemoattractant gene expression changes in white adipose tissue of morbidly obese subjects after surgery-induced weight loss. Diabetes.

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Antoon JW, Fewell C, Zhu Y, Driver JL, et al. Preferential star strand biogenesis of pre-miR-24-2 targets PKC-alpha and suppresses cell survival in MCF-7 breast cancer cells. Molecular Carcinogenesis.

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**26**

**29**

**Chapter 2**

**Abstract**

endocrine disruptors, estrogen

**1. Introduction**

A Potential New Mechanism for

Breast Cancer through Alteration

Signaling in Stem Cells and Their

Endocrine disruptors interfere with endocrine-mediated regulations of cell or organ functions. Estrogens are one of the main hormones altered by endocrine disruptors like bisphenol A (BPA). Stem cells are active from embryogenesis to late stages of adult life. Their unique properties, such as an extended lifespan and low cycling features, render these cell privileged targets of long-term exposure to numerous factors. Therefore, stem cells are likely to be affected following exposure to endocrine disruptors. One of the major signaling pathways involved in stem cell regulation is the bone morphogenetic protein (BMP) pathway. The BMP pathway is known for its involvement in numerous physiological and pathophysiological processes. Exposure of human mammary stem cells to pollutants such as BPA initiates fundamental changes in stem cells, in particular by altering major elements of BMP signaling, such as receptor expression and localization. Lastly, BPA and its substitute bisphenol S (BPS) have similar impacts on BMP signaling despite their different ER-binding properties, supporting the hypothesis that their biological effects cannot be extrapolated only from their interaction with ERα66. We review recent discoveries in this field and discuss their implications for cancer diagnosis, prevention, and treatment, as well as their relevance for studies on endocrine disruptors.

**Keywords:** BMP, bisphenol, stem cells, breast cancer, microenvironment,

Breast cancer is the most common cancer in women and exhibits important phenotypic and genetic diversities associated with different prognoses. Breast cancer subtypes are clinically classified based on histological appearance and expression of hormone receptors such as estrogen (ER) and progesterone (PR) receptors, as well as on the amplification of the HER2 gene coding for a member of the EGF receptor family [1]. Based on these criteria, four major breast cancer subtypes have been defined: luminal A and luminal B (all ER+), HER+ (that can be either ER**−** or ER+),

of Bone Morphogenetic Protein

Microenvironment

*Boris Guyot and Veronique Maguer-Satta*

Bisphenol Molecules to Initiate

#### **Chapter 2**

## A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer through Alteration of Bone Morphogenetic Protein Signaling in Stem Cells and Their Microenvironment

*Boris Guyot and Veronique Maguer-Satta*

### **Abstract**

Endocrine disruptors interfere with endocrine-mediated regulations of cell or organ functions. Estrogens are one of the main hormones altered by endocrine disruptors like bisphenol A (BPA). Stem cells are active from embryogenesis to late stages of adult life. Their unique properties, such as an extended lifespan and low cycling features, render these cell privileged targets of long-term exposure to numerous factors. Therefore, stem cells are likely to be affected following exposure to endocrine disruptors. One of the major signaling pathways involved in stem cell regulation is the bone morphogenetic protein (BMP) pathway. The BMP pathway is known for its involvement in numerous physiological and pathophysiological processes. Exposure of human mammary stem cells to pollutants such as BPA initiates fundamental changes in stem cells, in particular by altering major elements of BMP signaling, such as receptor expression and localization. Lastly, BPA and its substitute bisphenol S (BPS) have similar impacts on BMP signaling despite their different ER-binding properties, supporting the hypothesis that their biological effects cannot be extrapolated only from their interaction with ERα66. We review recent discoveries in this field and discuss their implications for cancer diagnosis, prevention, and treatment, as well as their relevance for studies on endocrine disruptors.

**Keywords:** BMP, bisphenol, stem cells, breast cancer, microenvironment, endocrine disruptors, estrogen

#### **1. Introduction**

Breast cancer is the most common cancer in women and exhibits important phenotypic and genetic diversities associated with different prognoses. Breast cancer subtypes are clinically classified based on histological appearance and expression of hormone receptors such as estrogen (ER) and progesterone (PR) receptors, as well as on the amplification of the HER2 gene coding for a member of the EGF receptor family [1]. Based on these criteria, four major breast cancer subtypes have been defined: luminal A and luminal B (all ER+), HER+ (that can be either ER**−** or ER+), and basal-like (ER**−**) [2, 3]. The most frequent subtype encompasses ER-positive tumors that represent almost 80% of breast cancers. In these tumors, preventing ER activation via hormone therapy is efficient. This can be achieved either by using competitive antagonists of estrogens (e.g., tamoxifen), preventing its binding to and subsequent activation of ER, by using drugs blocking estrogen synthesis (antiaromatase) in postmenopausal women, or by luteinizing hormone-releasing hormone (LHRH) analogs, inhibiting release of female hormones by the ovaries [4].

Breast cancer is a multifactorial disease, and evidences of the involvement of extrinsic factors in the increase of breast cancer risk have been described, such as the environment or lifestyle. Indeed, lack of physical activity, elevated tobacco or alcohol consumption, and the use of contraceptive pills or hormone-replacement therapy (for postmenopausal women) have been shown to increase breast cancer risk [5]. Hormonal status has also been described to play a major role in breast cancer risk. It has been shown that a premature or extensive exposure to endogenous estrogens (due to an early menarche, nulliparity, late age for first full-term pregnancy, or late menopause) increases the risk of breast cancer development.

Several chemical pollutants have been classified as endocrine-disrupting chemicals (EDCs) based on the following definition: "an endocrine disruptor is an exogenous substance or mixture that alters any function(s) of hormone actions and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations" [6–11]. Estrogens are one of the main hormones altered by EDCs. Perturbations in estrogen functions have been identified in a wide spectrum of pathologies, including metabolic, bone, and reproductive disorders, as well as breast, endometrial, or ovarian cancers. Therefore, it is important to consider that the mammary gland is exposed throughout life not only to endogenous hormones but also to EDCs, molecules present in the environment and able to mimic these hormones.

Interest in EDCs is growing rapidly, owing notably to their extensive use in manufactured goods and their release in our environment. Among these environmental pollutants, bisphenol molecules are being increasingly studied in breast cancer due to their estrogen-mimetic properties, enabling them to activate estrogen signaling through their binding to the ER, in particular, bisphenol A (BPA) [12, 13]. Despite rising concerns about its safety [14] and progressive restrictions on its use, several million tons of BPA are still produced worldwide.

#### **2. The major effects of bisphenols on BMP signaling and stem cells**

#### **2.1 BPA and breast cancer**

#### *2.1.1 BPA and estrogen signaling*

BPA is an aromatic organic compound used by the plastic industry as a monomer in the synthesis of polycarbonates and epoxy resins. Polycarbonates are found in consumer plastic-like water bottles, food packaging materials, sport equipment or toys, while epoxy resins are used to coat the inside of food or beverage containers. BPA can also be found in thermal paper. BPA monomers from these compounds can be released into the environment by hydrolysis. At the structural level, BPA is a diphenyl compound with two hydroxyl groups in a "para" position rendering it highly similar to synthetic estrogen (diethylstilbestrol). This thus allows BPA to interact with various physiological receptors similar to estrogen, including ERs.

The classical genomic estrogen signaling pathway is triggered by the binding of estrogen to its α or β receptors that act as transcription factors in the nucleus. In the absence of ligands, these receptors are complexed with inhibitory molecules either

**31**

transformation [29].

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer…*

in the cytoplasm or in the nucleus of the cell. Upon ligand binding, these complexes dissociate resulting in conformational changes that allow DNA binding and recruitment of cofactors to regulate expression of target genes [15]. Both ERα and ERβ are also able to initiate a nongenomic signaling pathway outside of the nucleus depending on their subcellular localization [16]. Moreover, estrogen signaling can also be mediated by other receptors, such as GPR30, EGFR, to list only a few [15, 17].

In line with the current definition of EDCs, BPA was shown to exert its activity by disrupting the estrogen signaling pathway that uses ER as a transcription factor binding to estrogen response element (ERE) sites on DNA [15]. Consequently, estrogen-mimetics (e.g., BPA) were mechanistically thought to primarily act through their binding to ERα66, the main canonical (nuclear) estrogen receptor. This nuclear receptor initiates signaling pathways at the cell membrane and transcriptional responses in the cell nucleus. BPA also upregulates the level of steroid receptor coactivators (SRC-1, SRC-3) and promotes the activity of EREs [18]. However, BPA has also been shown to bind to a number of distinct nuclear and membrane receptors, namely estrogen receptors ERα/β, androgen receptor (AR), G protein-coupled ER (GPER, GPR30), PPAR (especially PPARγ), insulinlike growth factor-1 (IGF1-R) [17, 19, 20]. BPA stimulates the release of EGFR ligands by directly targeting other molecules than ER, like ADAM17 or ADAM10 [21]. Furthermore, the impact of BPA on Ca2+ release or ERK signaling has been highlighted in the pancreas [15]. Altogether, these results indicate that BPA, in addition to its effects on the canonical estrogen pathway, is able to perturb numerous physiological processes through estrogen genomic and nongenomic signaling, as well as nonestrogen-related pathways [19, 22]. Importantly, BPA is at the origin of toxic derivatives (chlorinated bisphenols) and is also processed by cellular and biochemical mechanisms to generate a number of different BPA metabolites. All these BPA derivatives have been reported to have similar or higher toxic effects than BPA [19, 20]. In the context of the mammary gland, it is thus of utmost importance to further elucidate how BPA, its derivatives or metabolites, modulate estrogen- or nonestrogen-related signaling. This should improve our understanding of the tumorigenic potential of BPA, firstly in the luminal breast cancer subtype, and

Evidence gathered from studies in experimental models and human populations has already confirmed that EDCs, including BPA, contribute to increased risk of disease [23, 24]. A positive relationship between exposure to BPA and cancer development is reported in the literature [25]. However, whether BPA is actually harmful for human health remains understudied, similar to our understanding of the molecular mechanisms underlying BPA-dependent effects in cancer development. Given the significant involvement of estrogens in both normal and pathological conditions, EDCs able to interfere with the homeostasis of the estrogen endocrine system are a potential source of several health disorders. In this context, a human population-based study detected a significant increase in serum levels of BPA and established a correlation with breast tissue density measured in mammographies [26]. This finding was attributed to the ability of BPA to increase proliferation of mammary epithelial cells from either normal or breast cancer tissues [27, 28]. Epigenetic data from human tumors or cells exposed to BPA *in vitro* revealed the ability of this EDC to directly induce mammary epithelial cell

Moreover, BPA was correlated with breast cancer patients with high risk profiles and therefore with increased disease relapse [30]. This may be due to the

*DOI: http://dx.doi.org/10.5772/intechopen.90273*

subsequently in other tumor types.

*2.1.2 BPA involvement in breast cancer*

#### *A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer… DOI: http://dx.doi.org/10.5772/intechopen.90273*

in the cytoplasm or in the nucleus of the cell. Upon ligand binding, these complexes dissociate resulting in conformational changes that allow DNA binding and recruitment of cofactors to regulate expression of target genes [15]. Both ERα and ERβ are also able to initiate a nongenomic signaling pathway outside of the nucleus depending on their subcellular localization [16]. Moreover, estrogen signaling can also be mediated by other receptors, such as GPR30, EGFR, to list only a few [15, 17].

In line with the current definition of EDCs, BPA was shown to exert its activity by disrupting the estrogen signaling pathway that uses ER as a transcription factor binding to estrogen response element (ERE) sites on DNA [15]. Consequently, estrogen-mimetics (e.g., BPA) were mechanistically thought to primarily act through their binding to ERα66, the main canonical (nuclear) estrogen receptor. This nuclear receptor initiates signaling pathways at the cell membrane and transcriptional responses in the cell nucleus. BPA also upregulates the level of steroid receptor coactivators (SRC-1, SRC-3) and promotes the activity of EREs [18]. However, BPA has also been shown to bind to a number of distinct nuclear and membrane receptors, namely estrogen receptors ERα/β, androgen receptor (AR), G protein-coupled ER (GPER, GPR30), PPAR (especially PPARγ), insulinlike growth factor-1 (IGF1-R) [17, 19, 20]. BPA stimulates the release of EGFR ligands by directly targeting other molecules than ER, like ADAM17 or ADAM10 [21]. Furthermore, the impact of BPA on Ca2+ release or ERK signaling has been highlighted in the pancreas [15]. Altogether, these results indicate that BPA, in addition to its effects on the canonical estrogen pathway, is able to perturb numerous physiological processes through estrogen genomic and nongenomic signaling, as well as nonestrogen-related pathways [19, 22]. Importantly, BPA is at the origin of toxic derivatives (chlorinated bisphenols) and is also processed by cellular and biochemical mechanisms to generate a number of different BPA metabolites. All these BPA derivatives have been reported to have similar or higher toxic effects than BPA [19, 20]. In the context of the mammary gland, it is thus of utmost importance to further elucidate how BPA, its derivatives or metabolites, modulate estrogen- or nonestrogen-related signaling. This should improve our understanding of the tumorigenic potential of BPA, firstly in the luminal breast cancer subtype, and subsequently in other tumor types.

#### *2.1.2 BPA involvement in breast cancer*

Evidence gathered from studies in experimental models and human populations has already confirmed that EDCs, including BPA, contribute to increased risk of disease [23, 24]. A positive relationship between exposure to BPA and cancer development is reported in the literature [25]. However, whether BPA is actually harmful for human health remains understudied, similar to our understanding of the molecular mechanisms underlying BPA-dependent effects in cancer development.

Given the significant involvement of estrogens in both normal and pathological conditions, EDCs able to interfere with the homeostasis of the estrogen endocrine system are a potential source of several health disorders. In this context, a human population-based study detected a significant increase in serum levels of BPA and established a correlation with breast tissue density measured in mammographies [26]. This finding was attributed to the ability of BPA to increase proliferation of mammary epithelial cells from either normal or breast cancer tissues [27, 28]. Epigenetic data from human tumors or cells exposed to BPA *in vitro* revealed the ability of this EDC to directly induce mammary epithelial cell transformation [29].

Moreover, BPA was correlated with breast cancer patients with high risk profiles and therefore with increased disease relapse [30]. This may be due to the

*Breast Cancer Biology*

and basal-like (ER**−**) [2, 3]. The most frequent subtype encompasses ER-positive tumors that represent almost 80% of breast cancers. In these tumors, preventing ER activation via hormone therapy is efficient. This can be achieved either by using competitive antagonists of estrogens (e.g., tamoxifen), preventing its binding to and subsequent activation of ER, by using drugs blocking estrogen synthesis (antiaromatase) in postmenopausal women, or by luteinizing hormone-releasing hormone (LHRH) analogs, inhibiting release of female hormones by the ovaries [4]. Breast cancer is a multifactorial disease, and evidences of the involvement of extrinsic factors in the increase of breast cancer risk have been described, such as the environment or lifestyle. Indeed, lack of physical activity, elevated tobacco or alcohol consumption, and the use of contraceptive pills or hormone-replacement therapy (for postmenopausal women) have been shown to increase breast cancer risk [5]. Hormonal status has also been described to play a major role in breast cancer risk. It has been shown that a premature or extensive exposure to endogenous estrogens (due to an early menarche, nulliparity, late age for first full-term pregnancy, or late menopause) increases the risk of breast cancer development. Several chemical pollutants have been classified as endocrine-disrupting chemicals (EDCs) based on the following definition: "an endocrine disruptor is an exogenous substance or mixture that alters any function(s) of hormone actions and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations" [6–11]. Estrogens are one of the main hormones altered by EDCs. Perturbations in estrogen functions have been identified in a wide spectrum of pathologies, including metabolic, bone, and reproductive disorders, as well as breast, endometrial, or ovarian cancers. Therefore, it is important to consider that the mammary gland is exposed throughout life not only to endogenous hormones but also to EDCs, molecules present in the environment and able to mimic these hormones. Interest in EDCs is growing rapidly, owing notably to their extensive use in manufactured goods and their release in our environment. Among these environmental pollutants, bisphenol molecules are being increasingly studied in breast cancer due to their estrogen-mimetic properties, enabling them to activate estrogen signaling through their binding to the ER, in particular, bisphenol A (BPA) [12, 13]. Despite rising concerns about its safety [14] and progressive restrictions on its use,

several million tons of BPA are still produced worldwide.

**2.1 BPA and breast cancer**

*2.1.1 BPA and estrogen signaling*

**2. The major effects of bisphenols on BMP signaling and stem cells**

BPA is an aromatic organic compound used by the plastic industry as a monomer in the synthesis of polycarbonates and epoxy resins. Polycarbonates are found in consumer plastic-like water bottles, food packaging materials, sport equipment or toys, while epoxy resins are used to coat the inside of food or beverage containers. BPA can also be found in thermal paper. BPA monomers from these compounds can be released into the environment by hydrolysis. At the structural level, BPA is a diphenyl compound with two hydroxyl groups in a "para" position rendering it highly similar to synthetic estrogen (diethylstilbestrol). This thus allows BPA to interact with various physiological receptors similar to estrogen, including ERs. The classical genomic estrogen signaling pathway is triggered by the binding of estrogen to its α or β receptors that act as transcription factors in the nucleus. In the absence of ligands, these receptors are complexed with inhibitory molecules either

**30**

implication of BPA in breast cancer metastasis. This process has traditionally been associated with late stages of cancer development, though a new hypothesis on its origin has progressively emerged suggesting that it could be an inherent mark of tumor cell [31, 32]. Metastatic dissemination is a dynamic process that involves several steps: local invasion of cells from the primary tumor, intravasation leading to dissemination through the blood or lymph, extravasation to invade new tissues, implantation, and finally new tumor growth. Numerous signaling pathways and programs are activated during this process such as epithelial-to-mesenchymal transition (EMT), anoïkis, migration, and proliferation among others (for review: [33, 34]). It has been shown that ER-negative breast cancers are associated with an increased risk of developing metastases [35]. Indeed, these breast cancers express more mesenchymal markers such as vimentin and N-cadherin or EMT-transcription factors that are required for metastatic initiation. Conversely, ER-positive tumors are associated with a more differentiated luminal phenotype, expressing epithelial markers (E-cadherin, ER, FOXA1 for instance). Accordingly, a downregulation of the luminal-specific transcription factor FOXA1 is induced after BPA treatment in triple-negative tumor cell lines, leading to the induction of EMT and increasing cell motility [36]. In this study, BPA treatment was shown to activate the PI3K/AKT pathway, leading to a downregulation of epithelial genes alongside an upregulation of mesenchymal genes. Another study demonstrated that BPA promotes migration and invasion via GPER, which transduces FAK, Src, and ERK2 signaling pathway activation [37]. Promotion of GPER-induced migration by BPA or BPS occurs via different signaling pathways. Indeed, in contrast to BPA, which acts via the FAK/Scr/ERK2 pathway, it has been shown that BPS induces GPER/Hippo-YAPdependent migration [38]. Effects of BPA, BPS, and BPF on migration and EMT properties of ER-positive tumor cell lines were compared [39]. After treatment, cells lost cell/cell contacts and acquired a fibroblast-like morphology associated with an EMT phenotype. This was further confirmed after analysis of EMTassociated protein expression showing a decrease in E-cadherin and an increase in N-cadherin. Moreover, BPA-, BPS-, and BPF-treated cells displayed a stronger migratory ability. All of these modifications were inhibited after administration of an ER antagonist, demonstrating the ER-dependent effects of these bisphenols [39].

At the mechanistic level, a large number of *in vivo* and *in vitro* studies have highlighted the ability of BPA to disrupt several key signaling pathways that are known to be involved in breast cancer [19, 40]. However, the direct involvement of BPA in breast cancer incidence is difficult to establish and remains controversial [26, 41], owing possibly to the fact that different mechanisms are depicted in either ER-positive or -negative tumors, reflecting the variety of biological effects arising from exposure to BPA [20, 28, 42]. In addition, the combinatorial effects of different pollutants encountered over a life time also complicate these studies. Hence, scientists are faced with a huge challenge in order to formally establish the transforming power of BPA, owing to the different contexts and mechanistic cascades of alterations occurring in the human breast tissue during such long-term exposure.

#### **2.2 BPA target cells**

#### *2.2.1 Stem cells in mammary gland*

Mammary gland development takes place during embryogenesis and is composed of a rudimentary ductal system blocked until puberty. Then, two master reproductive hormones are secreted, namely estrogens and progesterone.

**33**

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer…*

Estrogens control the growth of ducts from their distal extremity called terminal end buds (TEBs) [43–45], while progesterone is involved in lateral branch development [46, 47]. One of the major hormones involved in mammary gland development is estrogen, mostly produced by the ovaries (but also by other tissues). Estrogens, in combination with other hormones, orchestrate the growth of the ductal system and adipose tissue accumulation during puberty and at further

In adults, the mammary gland is formed of ducts and lobules of secreting luminal epithelial cells surrounded by contractile myoepithelial cells. These epithelial cells are embedded in a stroma mainly formed of fibroblasts and adipocytes that secrete several soluble molecules regulating epithelial cell function and differentiation. Epithelial cells of the mammary gland are generated by mammary stem cells (MaSCs) and the stromal compartment by mesenchymal stem cells (MSCs) [48–51]. During adulthood, the mammary gland undergoes functional and structural changes that alternate between phases of proliferation, differentiation, and apoptosis controlled by cyclic hormonal variations due to the estrous/menstrual cycle modulating the stem cell compartment [52]. However, this postpubertal mammary tree remains immature and only achieves full maturation during pregnancy and lactation. These final steps involve alveogenesis and milk production, which take place mostly under the control of progesterone and prolactin [53, 54]. Studies indicate that estrogens do not directly stimulate proliferation of ER-positive luminal cells but act via a paracrine process [55, 56]. Indeed, estrogen acts on luminal ER/ PR-positive cells, leading to the cleavage and liberation of amphiregulin [57, 58], which then affects neighboring ER/PR-negative cells. These ER/PR-negative cells display characteristics of stem cells, in that, their asymmetric division is controlled by growth factors released by stromal cells [59–62]. Conversely, estrogen treatment induces a deficient asymmetric division of a human MaSC cell line (MCF10F) [63]. Ovariectomized mice (or letrozole treated to inhibit endogenous estrogen synthesis and provide a normal stromal and hormonal environment for all other hormones) show a decrease in the ability of MaSCs to repopulate a mammary fat pad and to generate ductal growth and expansion without impacting the size of the MaSCsenriched subpopulation [52]. Collectively, these studies highlight the importance of the estrogen pathway on MaSC regulation through direct and indirect effects and consequently suggest potential sensitivity of these cells to estrogen-mimetics like BPA. Furthermore, stem cells are a unique category of cells active from embryogenesis up to late stages of human adult life, and are thus more prone to be exposed to

*DOI: http://dx.doi.org/10.5772/intechopen.90273*

EDCs, likely altering their normal functions [64–68].

It has been shown that exposure to EDCs occurs throughout life and even during embryogenesis, at the stage of mammary gland establishment. For instance, BPA has been detected in urinary samples but also in maternal and fetal plasma, in colostrum, and in placental tissue at birth. Several studies demonstrated that a prenatal exposure to BPA induces changes in fetal mouse mammary gland, in the epithelial as well as stromal compartments, favoring fat pad maturation and increasing the mammary gland susceptibility to carcinogens [69–71]. This is accompanied by transcriptome modifications, in particular, an increase in the expression of genes belonging to the antiapoptotic family, myoepithelial differentiation, and adipogenesis, and a decrease in those involved in cell adhesion [71]. Exposure to BPA at puberty alters the function of MaSCs, leading to the appearance in the regenerated glands of early neoplastic lesions with molecular alterations similar to those

*2.2.2 BPA, stem cells, and breast cancer*

stages of development [43–45].

#### *A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer… DOI: http://dx.doi.org/10.5772/intechopen.90273*

Estrogens control the growth of ducts from their distal extremity called terminal end buds (TEBs) [43–45], while progesterone is involved in lateral branch development [46, 47]. One of the major hormones involved in mammary gland development is estrogen, mostly produced by the ovaries (but also by other tissues). Estrogens, in combination with other hormones, orchestrate the growth of the ductal system and adipose tissue accumulation during puberty and at further stages of development [43–45].

In adults, the mammary gland is formed of ducts and lobules of secreting luminal epithelial cells surrounded by contractile myoepithelial cells. These epithelial cells are embedded in a stroma mainly formed of fibroblasts and adipocytes that secrete several soluble molecules regulating epithelial cell function and differentiation. Epithelial cells of the mammary gland are generated by mammary stem cells (MaSCs) and the stromal compartment by mesenchymal stem cells (MSCs) [48–51]. During adulthood, the mammary gland undergoes functional and structural changes that alternate between phases of proliferation, differentiation, and apoptosis controlled by cyclic hormonal variations due to the estrous/menstrual cycle modulating the stem cell compartment [52]. However, this postpubertal mammary tree remains immature and only achieves full maturation during pregnancy and lactation. These final steps involve alveogenesis and milk production, which take place mostly under the control of progesterone and prolactin [53, 54]. Studies indicate that estrogens do not directly stimulate proliferation of ER-positive luminal cells but act via a paracrine process [55, 56]. Indeed, estrogen acts on luminal ER/ PR-positive cells, leading to the cleavage and liberation of amphiregulin [57, 58], which then affects neighboring ER/PR-negative cells. These ER/PR-negative cells display characteristics of stem cells, in that, their asymmetric division is controlled by growth factors released by stromal cells [59–62]. Conversely, estrogen treatment induces a deficient asymmetric division of a human MaSC cell line (MCF10F) [63]. Ovariectomized mice (or letrozole treated to inhibit endogenous estrogen synthesis and provide a normal stromal and hormonal environment for all other hormones) show a decrease in the ability of MaSCs to repopulate a mammary fat pad and to generate ductal growth and expansion without impacting the size of the MaSCsenriched subpopulation [52]. Collectively, these studies highlight the importance of the estrogen pathway on MaSC regulation through direct and indirect effects and consequently suggest potential sensitivity of these cells to estrogen-mimetics like BPA. Furthermore, stem cells are a unique category of cells active from embryogenesis up to late stages of human adult life, and are thus more prone to be exposed to EDCs, likely altering their normal functions [64–68].

#### *2.2.2 BPA, stem cells, and breast cancer*

It has been shown that exposure to EDCs occurs throughout life and even during embryogenesis, at the stage of mammary gland establishment. For instance, BPA has been detected in urinary samples but also in maternal and fetal plasma, in colostrum, and in placental tissue at birth. Several studies demonstrated that a prenatal exposure to BPA induces changes in fetal mouse mammary gland, in the epithelial as well as stromal compartments, favoring fat pad maturation and increasing the mammary gland susceptibility to carcinogens [69–71]. This is accompanied by transcriptome modifications, in particular, an increase in the expression of genes belonging to the antiapoptotic family, myoepithelial differentiation, and adipogenesis, and a decrease in those involved in cell adhesion [71]. Exposure to BPA at puberty alters the function of MaSCs, leading to the appearance in the regenerated glands of early neoplastic lesions with molecular alterations similar to those

*Breast Cancer Biology*

implication of BPA in breast cancer metastasis. This process has traditionally been associated with late stages of cancer development, though a new hypothesis on its origin has progressively emerged suggesting that it could be an inherent mark of tumor cell [31, 32]. Metastatic dissemination is a dynamic process that involves several steps: local invasion of cells from the primary tumor, intravasation leading to dissemination through the blood or lymph, extravasation to invade new tissues, implantation, and finally new tumor growth. Numerous signaling pathways and programs are activated during this process such as epithelial-to-mesenchymal transition (EMT), anoïkis, migration, and proliferation among others (for review: [33, 34]). It has been shown that ER-negative breast cancers are associated with an increased risk of developing metastases [35]. Indeed, these breast cancers express more mesenchymal markers such as vimentin and N-cadherin or EMT-transcription factors that are required for metastatic initiation. Conversely, ER-positive tumors are associated with a more differentiated luminal phenotype, expressing epithelial markers (E-cadherin, ER, FOXA1 for instance). Accordingly, a downregulation of the luminal-specific transcription factor FOXA1 is induced after BPA treatment in triple-negative tumor cell lines, leading to the induction of EMT and increasing cell motility [36]. In this study, BPA treatment was shown to activate the PI3K/AKT pathway, leading to a downregulation of epithelial genes alongside an upregulation of mesenchymal genes. Another study demonstrated that BPA promotes migration and invasion via GPER, which transduces FAK, Src, and ERK2 signaling pathway activation [37]. Promotion of GPER-induced migration by BPA or BPS occurs via different signaling pathways. Indeed, in contrast to BPA, which acts via the FAK/Scr/ERK2 pathway, it has been shown that BPS induces GPER/Hippo-YAPdependent migration [38]. Effects of BPA, BPS, and BPF on migration and EMT properties of ER-positive tumor cell lines were compared [39]. After treatment, cells lost cell/cell contacts and acquired a fibroblast-like morphology associated with an EMT phenotype. This was further confirmed after analysis of EMTassociated protein expression showing a decrease in E-cadherin and an increase in N-cadherin. Moreover, BPA-, BPS-, and BPF-treated cells displayed a stronger migratory ability. All of these modifications were inhibited after administration of an ER antagonist, demonstrating the ER-dependent effects of these bisphenols [39]. At the mechanistic level, a large number of *in vivo* and *in vitro* studies have highlighted the ability of BPA to disrupt several key signaling pathways that are known to be involved in breast cancer [19, 40]. However, the direct involvement of BPA in breast cancer incidence is difficult to establish and remains controversial [26, 41], owing possibly to the fact that different mechanisms are depicted in either ER-positive or -negative tumors, reflecting the variety of biological effects arising from exposure to BPA [20, 28, 42]. In addition, the combinatorial effects of different pollutants encountered over a life time also complicate these studies. Hence, scientists are faced with a huge challenge in order to formally establish the transforming power of BPA, owing to the different contexts and mechanistic cascades of alterations occurring in the human breast tissue during such long-term

**32**

exposure.

**2.2 BPA target cells**

*2.2.1 Stem cells in mammary gland*

Mammary gland development takes place during embryogenesis and is composed of a rudimentary ductal system blocked until puberty. Then, two master reproductive hormones are secreted, namely estrogens and progesterone. detected in early neoplastic breast cancer tissues [72]. In a physiological model in which mice were treated at puberty with BPA, estrogen-dependent transcriptional events were perturbed and the number of terminal end buds was altered in a dosedependent fashion [27]. *In vitro* exposure of normal human mammary epithelial cells to BPA was shown to induce their proliferation due to the secretion of autocrine growth factors and allow them to generate bigger mammospheres [73]. Treated cells displayed an increase in DNA hypermethylation of tumor suppressor genes, such as Brca1. These data support that BPA can promote early pretumoral stages corroborating findings in normal human breast epithelial cells (MCF-10F) [29, 64]. Indeed, BPA-treated human MaSC lines, such as MCF-10F, increase their expression of genes involved in DNA repair and decrease proapoptotic gene expression [74]. Chronic exposure of MCF10A cells to BPA at doses similar to those measured in contaminated water lead to major MaSC modifications affecting their stem cell properties and regulation [64]. Importantly, BPA treatment increases stem-like features by inducing the expression of ALDH1 and SOX2 genes, a human MaSCs marker and a master regulator of pluripotency in embryonic stem cells, respectively [75]. BPA also perturbs signals involved in human mammary stem cell (ERα66 negative cells) regulation, like the bone morphogenetic protein (BMP) pathway, which has been identified in their transformation [76], partly by changing BMP membrane receptor availability and priming cells to BMP signaling [64]. These data raised the hypothesis that in ER-positive tumors, under tamoxifen treatment and in a BPA-containing environment, some cells could acquire resistance to treatment by a switch in signaling pathway favoring a stem-like phenotype characterized by a decrease in treatment cytotoxicity and a modification of the stoichiometry of the type of ER (e.g., an increase in ERRγ or ERα isoform expression).

Overall, these observations strongly support that MaSCs are directly sensitive to BPA, which could be involved in their transformation and/or treatment escape [27, 72, 74].

#### **2.3 BMP, stem cells, and cancer**

#### *2.3.1 BMP and mammary epithelial stem cells*

One of the major conserved signaling pathways involved in stem cell regulation from embryogenesis up to adult stages is BMP signaling. There are 21 different soluble BMP molecules that act through serine/threonine kinase BMP receptors (BMPRs). In the context of stem cell regulation, BMP2 and BMP4 are progressively emerging as the most important BMPs. The BMP pathway is involved in numerous physiological and pathological processes [77]. BMPs control MSC regulation, such as lineage specification of adipocytes which are one of the major elements of the mammary gland microenvironment [78–80]. Alterations in BMP signaling have been implicated in metabolic disorders such as obesity in women [81, 82].

During embryogenesis in mice, BMP4 was shown to participate in the early steps of mammary gland development by regulating the dorsoventral axis establishment [83]. The BMP pathway also plays a role in mammary bud formation and outgrowth, as well as in ductal branching morphogenesis initiation. Indeed, BMP4 is expressed in both mesenchymal and epithelial cells of the mammary bud and the use of a BMP4 inhibitor leads to a decrease in bud outgrowth [84]. A link between BMPs and progesterone receptor type A involved in branching morphogenesis during postnatal mammary gland development has also been shown [85]. In addition, BMPs are also involved in the myoepithelial compartmentalization and lumen formation [85]. The knockout of a BMP extracellular antagonist, Twisted, abrogates lumen formation and disorganizes the myoepithelial layer through a decrease in

**35**

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer…*

SMAD1-5-8 phosphorylation and the repression of BMP targets (Msx1, Msx2, and Gata-3) [86]. In human cells, BMP2 regulates luminal epithelial cells by modulating the expression of *GATA-3* and *FOXA1* [76]. Finally, an *in vitro* study using sorted mouse mammary epithelial undifferentiated cells demonstrated the role of BMP signaling in final maturation steps such as lactogenic differentiation [87].

In healthy tissues, epithelial cells, as well as cells within the mammary gland environment (fibroblasts, adipose tissue cells, hematopoietic cells), contribute to the production of soluble BMP2 and BMP4 molecules [76], while distinct subpopulations of normal mammary epithelial cells sorted according to CD10 and EPCAM expression [88] express different elements of the BMP pathway. A role for BMP molecules in MaSC regulation was formally demonstrated by functional assay analyses following exposure of different human cell types to soluble BMP2 or BMP4 [76], and further substantiated by the use of TGF/BMP inhibitors allowing the expansion of immature epithelial basal cells [89]. Interestingly, as in the hematopoietic system [90], BMP2 and BMP4 molecules have distinct functional effects on MaSC regulation despite their strong homology. Indeed, while BMP4 modulates the compartment of MaSC and myoepithelial progenitors, BMP2 allows the commitment and proliferation of luminal progenitors [76]. However, the molecular mechanism by which BMPs interact with estrogen signaling to regulate MaSCs remains to

BMP signaling is also a well-known highly complex pathway that orchestrates the development and homeostasis of adult tissues such as the neural system [91]. The importance of BMP signaling alterations in cancer stem cell features has been revealed in glioblastoma, breast cancer, and leukemia [90, 92–95]. The role of BMPs, especially of BMP2 and BMP4, in breast cancer has been largely documented [96, 97]. Alterations of BMP ligand expression and signaling have been reported and shown to be clinically correlated with breast cancer progression [98, 99] and to play a major role in the development of bone metastases [99–101]. Despite the fact that BMP4 transcripts are expressed at various levels in tumor tissues or breast cancer cell lines [102], high levels of BMP4 are found in 25% of the breast cancer tumors displaying a low proliferation index but high recurrence rate [98]. BMP4 has crucial functions in promoting tumor growth arrest, migration and metastasis by mediating cell cycle arrest in G1 [102], chemokine regulation [103], and inhibition of lumen formation [104] for example. However, the biological effects of BMP4 largely depend on cell context, as they were reported to be either proliferative or antiproliferative in mammary epithelial cells according to cellular density and cooperative factors [105, 106]. The microenvironment of human primary luminal breast tumors produces abnormally high amounts of soluble BMP2 compared to healthy tissue, while higher BMPR1B levels were detected in tumor cells [76, 107]. Chronic exposure to high BMP2 concentrations was demonstrated to initiate MaSC transformation toward a luminal tumor phenotype dependent on a BMPR1B-initiated signaling cascade involved in luminal commitment of normal MaSC. This leads to a FOXA1/FOXC1 transcription factor balance switch in favor of FOXA1, simultaneously with an upregulation of GATA3 [76]. However, while an increase in soluble BMP2 in the tumor microenvironment has been shown in luminal ER-positive tumors where it is correlated with a high BMPR1B tumor expression [76], a strong decrease in BMP2 transcripts was found in ER-negative breast tumors [108]. Also, a downmodulation of the BMPR1B (Alk6) in a basal cell line (MDA-MB-231) increased cell growth *in vitro* [109], suggesting an antiproliferative function for BMPR1B in ER-negative tumors. Interestingly, downregulation of BMPR1A (ALK3)

*DOI: http://dx.doi.org/10.5772/intechopen.90273*

be further deciphered.

*2.3.2 BMP and breast cancer*

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer… DOI: http://dx.doi.org/10.5772/intechopen.90273*

SMAD1-5-8 phosphorylation and the repression of BMP targets (Msx1, Msx2, and Gata-3) [86]. In human cells, BMP2 regulates luminal epithelial cells by modulating the expression of *GATA-3* and *FOXA1* [76]. Finally, an *in vitro* study using sorted mouse mammary epithelial undifferentiated cells demonstrated the role of BMP signaling in final maturation steps such as lactogenic differentiation [87].

In healthy tissues, epithelial cells, as well as cells within the mammary gland environment (fibroblasts, adipose tissue cells, hematopoietic cells), contribute to the production of soluble BMP2 and BMP4 molecules [76], while distinct subpopulations of normal mammary epithelial cells sorted according to CD10 and EPCAM expression [88] express different elements of the BMP pathway. A role for BMP molecules in MaSC regulation was formally demonstrated by functional assay analyses following exposure of different human cell types to soluble BMP2 or BMP4 [76], and further substantiated by the use of TGF/BMP inhibitors allowing the expansion of immature epithelial basal cells [89]. Interestingly, as in the hematopoietic system [90], BMP2 and BMP4 molecules have distinct functional effects on MaSC regulation despite their strong homology. Indeed, while BMP4 modulates the compartment of MaSC and myoepithelial progenitors, BMP2 allows the commitment and proliferation of luminal progenitors [76]. However, the molecular mechanism by which BMPs interact with estrogen signaling to regulate MaSCs remains to be further deciphered.

#### *2.3.2 BMP and breast cancer*

*Breast Cancer Biology*

detected in early neoplastic breast cancer tissues [72]. In a physiological model in which mice were treated at puberty with BPA, estrogen-dependent transcriptional events were perturbed and the number of terminal end buds was altered in a dosedependent fashion [27]. *In vitro* exposure of normal human mammary epithelial cells to BPA was shown to induce their proliferation due to the secretion of autocrine growth factors and allow them to generate bigger mammospheres [73]. Treated cells displayed an increase in DNA hypermethylation of tumor suppressor genes, such as Brca1. These data support that BPA can promote early pretumoral stages corroborating findings in normal human breast epithelial cells (MCF-10F) [29, 64]. Indeed, BPA-treated human MaSC lines, such as MCF-10F, increase their expression of genes involved in DNA repair and decrease proapoptotic gene expression [74]. Chronic exposure of MCF10A cells to BPA at doses similar to those measured in contaminated water lead to major MaSC modifications affecting their stem cell properties and regulation [64]. Importantly, BPA treatment increases stem-like features by inducing the expression of ALDH1 and SOX2 genes, a human MaSCs marker and a master regulator of pluripotency in embryonic stem cells, respectively [75]. BPA also perturbs signals involved in human mammary stem cell (ERα66 negative cells) regulation, like the bone morphogenetic protein (BMP) pathway, which has been identified in their transformation [76], partly by changing BMP membrane receptor availability and priming cells to BMP signaling [64]. These data raised the hypothesis that in ER-positive tumors, under tamoxifen treatment and in a BPA-containing environment, some cells could acquire resistance to treatment by a switch in signaling pathway favoring a stem-like phenotype characterized by a decrease in treatment cytotoxicity and a modification of the stoichiometry of the

type of ER (e.g., an increase in ERRγ or ERα isoform expression).

Overall, these observations strongly support that MaSCs are directly sensitive to BPA, which could be involved in their transformation and/or treatment escape

One of the major conserved signaling pathways involved in stem cell regulation

from embryogenesis up to adult stages is BMP signaling. There are 21 different soluble BMP molecules that act through serine/threonine kinase BMP receptors (BMPRs). In the context of stem cell regulation, BMP2 and BMP4 are progressively emerging as the most important BMPs. The BMP pathway is involved in numerous physiological and pathological processes [77]. BMPs control MSC regulation, such as lineage specification of adipocytes which are one of the major elements of the mammary gland microenvironment [78–80]. Alterations in BMP signaling have been implicated in metabolic disorders such as obesity in women [81, 82].

During embryogenesis in mice, BMP4 was shown to participate in the early steps of mammary gland development by regulating the dorsoventral axis establishment [83]. The BMP pathway also plays a role in mammary bud formation and outgrowth, as well as in ductal branching morphogenesis initiation. Indeed, BMP4 is expressed in both mesenchymal and epithelial cells of the mammary bud and the use of a BMP4 inhibitor leads to a decrease in bud outgrowth [84]. A link between BMPs and progesterone receptor type A involved in branching morphogenesis during postnatal mammary gland development has also been shown [85]. In addition, BMPs are also involved in the myoepithelial compartmentalization and lumen formation [85]. The knockout of a BMP extracellular antagonist, Twisted, abrogates lumen formation and disorganizes the myoepithelial layer through a decrease in

**34**

[27, 72, 74].

**2.3 BMP, stem cells, and cancer**

*2.3.1 BMP and mammary epithelial stem cells*

BMP signaling is also a well-known highly complex pathway that orchestrates the development and homeostasis of adult tissues such as the neural system [91]. The importance of BMP signaling alterations in cancer stem cell features has been revealed in glioblastoma, breast cancer, and leukemia [90, 92–95]. The role of BMPs, especially of BMP2 and BMP4, in breast cancer has been largely documented [96, 97]. Alterations of BMP ligand expression and signaling have been reported and shown to be clinically correlated with breast cancer progression [98, 99] and to play a major role in the development of bone metastases [99–101]. Despite the fact that BMP4 transcripts are expressed at various levels in tumor tissues or breast cancer cell lines [102], high levels of BMP4 are found in 25% of the breast cancer tumors displaying a low proliferation index but high recurrence rate [98]. BMP4 has crucial functions in promoting tumor growth arrest, migration and metastasis by mediating cell cycle arrest in G1 [102], chemokine regulation [103], and inhibition of lumen formation [104] for example. However, the biological effects of BMP4 largely depend on cell context, as they were reported to be either proliferative or antiproliferative in mammary epithelial cells according to cellular density and cooperative factors [105, 106]. The microenvironment of human primary luminal breast tumors produces abnormally high amounts of soluble BMP2 compared to healthy tissue, while higher BMPR1B levels were detected in tumor cells [76, 107]. Chronic exposure to high BMP2 concentrations was demonstrated to initiate MaSC transformation toward a luminal tumor phenotype dependent on a BMPR1B-initiated signaling cascade involved in luminal commitment of normal MaSC. This leads to a FOXA1/FOXC1 transcription factor balance switch in favor of FOXA1, simultaneously with an upregulation of GATA3 [76]. However, while an increase in soluble BMP2 in the tumor microenvironment has been shown in luminal ER-positive tumors where it is correlated with a high BMPR1B tumor expression [76], a strong decrease in BMP2 transcripts was found in ER-negative breast tumors [108]. Also, a downmodulation of the BMPR1B (Alk6) in a basal cell line (MDA-MB-231) increased cell growth *in vitro* [109], suggesting an antiproliferative function for BMPR1B in ER-negative tumors. Interestingly, downregulation of BMPR1A (ALK3)

in MDA-MB231D (a bone metastatic clone of MDA-MB231) basal ER-negative cells inhibited their migration and bone metastatic properties [110]. Therefore, it is very likely that the BMP2/BMPR1B signal is overactivated in the context of ER-positive tumors, while being repressed in ER-negative tumors.

Some of the first steps of carcinogenesis are an increase in proliferation, evasion of apoptosis, and activation of survival signaling pathways. To achieve this, several tumor suppressor genes, like p53 or BRCA1 for instance, need to be inactivated by different mechanisms including epigenetic changes. Modulation of BMP signaling by epigenetic mechanisms [111], such as methylation of BMP-receptor promoters, has been of particular clinical interest to further stratify glioblastoma patients and propose new therapeutic strategies [92]. While different genetic alterations progressively appear following different oncogenic signals, heredity likely accounts for only 10–30% of breast cancers. Based on epidemiological studies, different factors increasing the risk of breast cancer development have been highlighted. They can be intrinsic, like mutations in BRCA1 or 2, Tp53, ATM, or also PTEN, or extrinsic, like environmental factors or lifestyle [112, 113]. In breast cancers with a genetic origin, the most commonly mutated genes are *BRCA1* and *BRCA2,* associated with an increase in cancer risk. BRCA1 and 2 are two major regulators of double-strand breaks (DSB) DNA repair through homologous recombination (HR) and play a crucial role as tumor suppressor genes. In this context, it is interesting to note that a family member and negative regulator of P53, DNp63 has been reported to mediate activation of BMP signaling in order to govern epithelial cell plasticity, EMT, and tumorigenicity during breast cancer initiation and progression [114, 115]. DNp63 has also been identified as a repressor of BRCA1 expression exclusively in ER-positive breast cancer cells [116]. Moreover, a correlation between the BMP pathway and the P53-ATM signaling has been reported [117]. However, the importance of these different signaling crosstalks in the context of breast cancer, exposure to EDCs, and stem cell transformation need to be investigated.

#### **2.4 BMP, estrogen, and bisphenols**

#### *2.4.1 BMP and ER crosstalk*

The BMP signaling pathway is a dynamic and complex pathway, leading to the transduction of various signals depending on the nature of the BMP ligand and of the BMPR complex oligomerization induced (for review: [118, 119]). It has been shown that BMPs may interact with their receptors in two different ways [120, 121]: on the one hand, BMPs induce a BMPR complex formation called BISC (BMPinduced signaling complex), and on the other hand, a preformed BMPR complex is present before BMP fixation, known as PFC (preformed complex). These two different modes of BMP signal initiation lead to two different signaling cascades, namely the canonical SMAD-dependent pathway and the noncanonical SMADindependent pathway [121]. SMAD-phosphorylated proteins then form a complex with SMAD4, leading to its translocation to the nucleus where it acts as a transcription factor on target genes [118, 122]. The SMAD-independent pathway does not simply encompass one signaling pathway but a multitude of downstream cascades, involving p38, Ras/ERK, and PI3K/AKT [123–126]. Interestingly, SMAD1-5-8 phosphorylation is more abundant in undifferentiated murine progenitors and decreases with their differentiation until it is almost fully abrogated in the differentiated cells treated with prolactin [87]. Involvement of the BMPR1A/SMAD1-5-8 pathway in lactogenic differentiation was further confirmed by the lack of expression of a

**37**

pathway have not been thoroughly investigated (**Figure 1**).

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer…*

lactogenic differentiation marker (beta-casein) at the RNA and protein levels in BMPR1A knockdown mammary cell lines [87]. These data demonstrate that the BMP pathway constitutes an important regulator of the mammary gland during embryogenesis but most likely also during adulthood. However, the molecular and functional crosstalk between the BMP and estrogen signaling pathways is poorly understood. A first set of experiments describes the repression of BMP signaling by ER inhibition of BMP production through a direct interaction between SMAD1 and ER [127]. Reciprocally, a BMP2 signal was shown to upregulate the expression of ER receptors, including the induction of specific ER isoforms such as ERα36 [128, 129]. Interestingly, crosstalk between BMP4 and estrogen signaling seems to have opposite effects. Indeed, BMP4 inhibits ERα signaling by promoting receptor degradation through the proteosomal pathway, while estrogens repress BMP4 expression [130]. Similarly, estrogen represses BMP4 expression in cardiomyocytes by preventing BMP4-mediated ERβ expression and JNK activity in this system [131]. In addition, in this context, estrogen inhibition of BMP4 is independent of Smad1/5/8 activity [131]. BMP4, upon activation of its canonical pathway, represses CYP17A1 and induces the transcription of CYP19A1, involved in androgen and estrogen synthesis, respectively [132]. In a rat model of pituitary cells, estrogen stimulates the transcriptional activity of BMP4-specific SMADs through an ER-SMAD1 complex shown to stimulate prolactin production, while having no effect on the TGFβ/SMAD pathway [133]. Similarly, the inhibitory effects of estrogen signaling on the BMP pathway appear to be mediated by a direct physical interaction between ER receptors and the SMAD1 BMP signaling element in a luminal breast cancer cell line model (MCF7). The physical interaction between ERα and SMAD1 requires the DNA binding domain of ERα and this complex formation is dependent on BMP2 and estrogen [127]. Moreover, BMP signaling has also been directly identified in thyroid-lineage specification [134, 135] as well as in thyroid carcinoma [136]. Interestingly, thyroid hormone status interferes with estrogen target gene expression in breast cancer samples in menopausal women [137]. These findings highlight the need to further investigate the importance of the BMP pathway in both thyroid and estrogen signaling in a broader context of exposure to EDCs. More recently, BMP2-mediated luminal transformation of MCF10A was shown to be accompanied by a strong activation of the estrogen signaling pathway despite the absence of ERα66 in those cells [76]. Our understanding of estrogen signaling is hindered by the existence of several isoforms generated by alternative splicing and different promoter usage [138]. Interaction of these isoforms with the BMP signaling elements has not yet being investigated but could be involved in epithelial stem cell response to BMP2. Indeed, the importance of these different ERα isoforms in mammary epithelial SC features and in the context of breast cancer is only just starting to be identified [139, 140]. These isoforms can be expressed in both ERα66-positive and -negative cells and display different subcellular localizations [141, 142]. For example, unlike ERα66, ERα36 is expressed mainly at the plasma membrane and activates estrogen nongenomic signaling by activating the ERK pathway through an interplay with the MKP3 phosphatase [143]. Interestingly, in the context of EDC research, ERα36 displays altered ligand preference and causes distinct effects compared to ERα66. For instance, the tamoxifen drug used as an estrogen antagonist in ERα66 breast cancers behaves as an estrogen agonist for ERα36 [140, 144]. Collectively, these different examples illustrate how BMP signaling through its interaction with estrogen signaling is at the crossroad of a number of fundamental physiological processes. The BMP pathway is therefore directly involved in mammary stem cell regulation and transformation, yet adverse effects of EDCs, like BPA, on the BMP

*DOI: http://dx.doi.org/10.5772/intechopen.90273*

#### *A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer… DOI: http://dx.doi.org/10.5772/intechopen.90273*

lactogenic differentiation marker (beta-casein) at the RNA and protein levels in BMPR1A knockdown mammary cell lines [87]. These data demonstrate that the BMP pathway constitutes an important regulator of the mammary gland during embryogenesis but most likely also during adulthood. However, the molecular and functional crosstalk between the BMP and estrogen signaling pathways is poorly understood. A first set of experiments describes the repression of BMP signaling by ER inhibition of BMP production through a direct interaction between SMAD1 and ER [127]. Reciprocally, a BMP2 signal was shown to upregulate the expression of ER receptors, including the induction of specific ER isoforms such as ERα36 [128, 129]. Interestingly, crosstalk between BMP4 and estrogen signaling seems to have opposite effects. Indeed, BMP4 inhibits ERα signaling by promoting receptor degradation through the proteosomal pathway, while estrogens repress BMP4 expression [130]. Similarly, estrogen represses BMP4 expression in cardiomyocytes by preventing BMP4-mediated ERβ expression and JNK activity in this system [131]. In addition, in this context, estrogen inhibition of BMP4 is independent of Smad1/5/8 activity [131]. BMP4, upon activation of its canonical pathway, represses CYP17A1 and induces the transcription of CYP19A1, involved in androgen and estrogen synthesis, respectively [132]. In a rat model of pituitary cells, estrogen stimulates the transcriptional activity of BMP4-specific SMADs through an ER-SMAD1 complex shown to stimulate prolactin production, while having no effect on the TGFβ/SMAD pathway [133]. Similarly, the inhibitory effects of estrogen signaling on the BMP pathway appear to be mediated by a direct physical interaction between ER receptors and the SMAD1 BMP signaling element in a luminal breast cancer cell line model (MCF7). The physical interaction between ERα and SMAD1 requires the DNA binding domain of ERα and this complex formation is dependent on BMP2 and estrogen [127]. Moreover, BMP signaling has also been directly identified in thyroid-lineage specification [134, 135] as well as in thyroid carcinoma [136]. Interestingly, thyroid hormone status interferes with estrogen target gene expression in breast cancer samples in menopausal women [137]. These findings highlight the need to further investigate the importance of the BMP pathway in both thyroid and estrogen signaling in a broader context of exposure to EDCs.

More recently, BMP2-mediated luminal transformation of MCF10A was shown to be accompanied by a strong activation of the estrogen signaling pathway despite the absence of ERα66 in those cells [76]. Our understanding of estrogen signaling is hindered by the existence of several isoforms generated by alternative splicing and different promoter usage [138]. Interaction of these isoforms with the BMP signaling elements has not yet being investigated but could be involved in epithelial stem cell response to BMP2. Indeed, the importance of these different ERα isoforms in mammary epithelial SC features and in the context of breast cancer is only just starting to be identified [139, 140]. These isoforms can be expressed in both ERα66-positive and -negative cells and display different subcellular localizations [141, 142]. For example, unlike ERα66, ERα36 is expressed mainly at the plasma membrane and activates estrogen nongenomic signaling by activating the ERK pathway through an interplay with the MKP3 phosphatase [143]. Interestingly, in the context of EDC research, ERα36 displays altered ligand preference and causes distinct effects compared to ERα66. For instance, the tamoxifen drug used as an estrogen antagonist in ERα66 breast cancers behaves as an estrogen agonist for ERα36 [140, 144]. Collectively, these different examples illustrate how BMP signaling through its interaction with estrogen signaling is at the crossroad of a number of fundamental physiological processes. The BMP pathway is therefore directly involved in mammary stem cell regulation and transformation, yet adverse effects of EDCs, like BPA, on the BMP pathway have not been thoroughly investigated (**Figure 1**).

*Breast Cancer Biology*

to be investigated.

**2.4 BMP, estrogen, and bisphenols**

*2.4.1 BMP and ER crosstalk*

in MDA-MB231D (a bone metastatic clone of MDA-MB231) basal ER-negative cells inhibited their migration and bone metastatic properties [110]. Therefore, it is very likely that the BMP2/BMPR1B signal is overactivated in the context of ER-positive

Some of the first steps of carcinogenesis are an increase in proliferation, evasion of apoptosis, and activation of survival signaling pathways. To achieve this, several tumor suppressor genes, like p53 or BRCA1 for instance, need to be inactivated by different mechanisms including epigenetic changes. Modulation of BMP signaling by epigenetic mechanisms [111], such as methylation of BMP-receptor promoters, has been of particular clinical interest to further stratify glioblastoma patients and propose new therapeutic strategies [92]. While different genetic alterations progressively appear following different oncogenic signals, heredity likely accounts for only 10–30% of breast cancers. Based on epidemiological studies, different factors increasing the risk of breast cancer development have been highlighted. They can be intrinsic, like mutations in BRCA1 or 2, Tp53, ATM, or also PTEN, or extrinsic, like environmental factors or lifestyle [112, 113]. In breast cancers with a genetic origin, the most commonly mutated genes are *BRCA1* and *BRCA2,* associated with an increase in cancer risk. BRCA1 and 2 are two major regulators of double-strand breaks (DSB) DNA repair through homologous recombination (HR) and play a crucial role as tumor suppressor genes. In this context, it is interesting to note that a family member and negative regulator of P53, DNp63 has been reported to mediate activation of BMP signaling in order to govern epithelial cell plasticity, EMT, and tumorigenicity during breast cancer initiation and progression [114, 115]. DNp63 has also been identified as a repressor of BRCA1 expression exclusively in ER-positive breast cancer cells [116]. Moreover, a correlation between the BMP pathway and the P53-ATM signaling has been reported [117]. However, the importance of these different signaling crosstalks in the context of breast cancer, exposure to EDCs, and stem cell transformation need

The BMP signaling pathway is a dynamic and complex pathway, leading to the transduction of various signals depending on the nature of the BMP ligand and of the BMPR complex oligomerization induced (for review: [118, 119]). It has been shown that BMPs may interact with their receptors in two different ways [120, 121]: on the one hand, BMPs induce a BMPR complex formation called BISC (BMPinduced signaling complex), and on the other hand, a preformed BMPR complex is present before BMP fixation, known as PFC (preformed complex). These two different modes of BMP signal initiation lead to two different signaling cascades, namely the canonical SMAD-dependent pathway and the noncanonical SMADindependent pathway [121]. SMAD-phosphorylated proteins then form a complex with SMAD4, leading to its translocation to the nucleus where it acts as a transcription factor on target genes [118, 122]. The SMAD-independent pathway does not simply encompass one signaling pathway but a multitude of downstream cascades, involving p38, Ras/ERK, and PI3K/AKT [123–126]. Interestingly, SMAD1-5-8 phosphorylation is more abundant in undifferentiated murine progenitors and decreases with their differentiation until it is almost fully abrogated in the differentiated cells treated with prolactin [87]. Involvement of the BMPR1A/SMAD1-5-8 pathway in lactogenic differentiation was further confirmed by the lack of expression of a

tumors, while being repressed in ER-negative tumors.

**36**

**Figure 1.** *Illustration of the main findings that show a crosstalk between BMP and estrogen signaling pathways.*

#### *2.4.2 BMP and bisphenols*

Works from our team and others suggest that bisphenols could act on multiple cell types of the mammary gland, and their effects may converge to provoke major dysregulations of the BMP pathway that could contribute to luminal breast cancer initiation. Indeed, we observed a major impact of BPA on the mammary microenvironment (niche) equilibrium. BPA greatly increases BMP2 production by stromal cells of the human mammary SC microenvironment reaching levels comparable to those measured in luminal breast cancer [76]. Moreover, BPA treatment leads to a decrease in estrogen and BMP15 production in oocytes delaying their maturation [145]. A decrease in BMP2 production through a direct binding of BPA to ERγ was involved in bone loss through a suppression of osteoblast differentiation reverted by inhibition of ERγ [146]. This suggests that the effects and mechanisms of BPA-induced BMP ligand production depend on the estrogen receptor expression profile and are context dependent [147]. However, the molecular mechanism by which BPA induces BMP2 production by stromal cells of the mammary gland BMP2 is not yet known. On the other hand, we have demonstrated that long-term exposure (60 days) to BPA initiates fundamental changes in human mammary stem cells themselves, in particular, by altering major BMP signaling elements such as receptor expression and localization [64]. This results in the "priming" of stem cells to exogenous activating signals of the BMP pathway and sensitizes them to be more sensitive to exogenous soluble BMP ligands. We then demonstrated for the first time that nongenotoxic alterations of both the stem cells and their niche act

**39**

**3. Conclusions**

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer…*

BMP-induced transformation in mammary epithelial stem cells.

Different signaling pathways often engage in complex interactions synergistically mediating an appropriate cellular response. Estrogen signaling is no exception and it is likely involved in a crosstalk with the BMP pathway at multiple levels in the mammary gland. BMPs are secreted proteins active in a very large number of organs and tissues during development, adulthood, and pathogenesis [155]. Previous work suggested a close interaction between ER-mediated estrogen signaling and the BMP pathway in different cell types of the mammary gland. In a model of mammary epithelial stem cells, E2 or known EDCs like BPA or BPS were able to potentiate SMAD activation by BMP2 [64]. This was possibly due to a physical interaction between ERα isoforms and SMAD factors, such as that reported for ERα or ERβ, and could be associated with an increased risk of cell transformation by long-term exposure to BMP2.

synergistically to initiate a transforming process mediated by the BMP signaling perturbation leading to the emergence of ER-positive tumors [76]. Interestingly, these previous studies showed that BPA impacts BMP signaling pathway members in both mammary epithelial and stromal cells that do not express ERα66. At the mechanistic level, the pathways used by BPA to induce these effects in cells remain to be deciphered, focusing notably on their reliance on other ERα isoforms or on

These questions are of great interest for understanding the effects of both BPA and estrogens since it has been reported that some cell lines respond to an estrogen signal despite their very low levels or complete absence of ER [148]. In response to accumulating evidence in favor of adverse health effects following exposure to BPA, likely mediated by its activation of ERα66, alternative bisphenols have been developed such as BPS and BPF that are considered safer due to their very low binding affinity to ERα [149–151]. However, an increasing number of studies show that these alternative bisphenol molecules are not as innocuous as anticipated, including an impact on obesity, steatosis, and reproduction [20]. In a study previously conducted in our team, assessing the impact of bisphenol on BMP2 production by stromal cells of the mammary gland, we were surprised to observe that BPA and BPS displayed very similar effects [76]. Indeed, both BPA (high affinity binding to estrogen receptors) and its substitute BPS (very weak affinity binding to estrogen receptors) induce BMP2 synthesis in the healthy breast stroma, raising concerns as to whether these bisphenols mediate their transforming effects solely through a classical ER-dependent mechanism. Since then, other studies have shown that BPS, as well as BPF, induces similar if not more potent effects than BPA [20, 152, 153]. Moreover, it was reported that BPA treatment increases aromatase expression and its activity in healthy breast fibroblasts, leading to an increase in estrogen biosynthesis and secretion. The same observations were made after treatment with BPS [154]. These results are of particular interest with regards to the important role of the microenvironment in the different steps of carcinogenesis and in the context of MaSC-driven transformation by BMP signaling. Our work thus indicates that the BMP pathway could be altered by several EDCs such as BPA and its proposed alternatives, both at the level of stem cells and their microenvironment. This suggests that early detection of increased BMP2 levels in the mammary microenvironment may constitute a reliable marker of early transformation process and could be a valuable indicator of exposure to EDCs such as bisphenols. In addition, the interplay between BMP and estrogen pathways both at the molecular and functional levels prompt us to further decipher the mechanisms underlying bisphenol- and

*DOI: http://dx.doi.org/10.5772/intechopen.90273*

ER-independent factors.

#### *A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer… DOI: http://dx.doi.org/10.5772/intechopen.90273*

synergistically to initiate a transforming process mediated by the BMP signaling perturbation leading to the emergence of ER-positive tumors [76]. Interestingly, these previous studies showed that BPA impacts BMP signaling pathway members in both mammary epithelial and stromal cells that do not express ERα66. At the mechanistic level, the pathways used by BPA to induce these effects in cells remain to be deciphered, focusing notably on their reliance on other ERα isoforms or on ER-independent factors.

These questions are of great interest for understanding the effects of both BPA and estrogens since it has been reported that some cell lines respond to an estrogen signal despite their very low levels or complete absence of ER [148]. In response to accumulating evidence in favor of adverse health effects following exposure to BPA, likely mediated by its activation of ERα66, alternative bisphenols have been developed such as BPS and BPF that are considered safer due to their very low binding affinity to ERα [149–151]. However, an increasing number of studies show that these alternative bisphenol molecules are not as innocuous as anticipated, including an impact on obesity, steatosis, and reproduction [20]. In a study previously conducted in our team, assessing the impact of bisphenol on BMP2 production by stromal cells of the mammary gland, we were surprised to observe that BPA and BPS displayed very similar effects [76]. Indeed, both BPA (high affinity binding to estrogen receptors) and its substitute BPS (very weak affinity binding to estrogen receptors) induce BMP2 synthesis in the healthy breast stroma, raising concerns as to whether these bisphenols mediate their transforming effects solely through a classical ER-dependent mechanism. Since then, other studies have shown that BPS, as well as BPF, induces similar if not more potent effects than BPA [20, 152, 153]. Moreover, it was reported that BPA treatment increases aromatase expression and its activity in healthy breast fibroblasts, leading to an increase in estrogen biosynthesis and secretion. The same observations were made after treatment with BPS [154]. These results are of particular interest with regards to the important role of the microenvironment in the different steps of carcinogenesis and in the context of MaSC-driven transformation by BMP signaling. Our work thus indicates that the BMP pathway could be altered by several EDCs such as BPA and its proposed alternatives, both at the level of stem cells and their microenvironment. This suggests that early detection of increased BMP2 levels in the mammary microenvironment may constitute a reliable marker of early transformation process and could be a valuable indicator of exposure to EDCs such as bisphenols. In addition, the interplay between BMP and estrogen pathways both at the molecular and functional levels prompt us to further decipher the mechanisms underlying bisphenol- and BMP-induced transformation in mammary epithelial stem cells.

#### **3. Conclusions**

*Breast Cancer Biology*

*2.4.2 BMP and bisphenols*

**Figure 1.**

Works from our team and others suggest that bisphenols could act on multiple cell types of the mammary gland, and their effects may converge to provoke major dysregulations of the BMP pathway that could contribute to luminal breast cancer initiation. Indeed, we observed a major impact of BPA on the mammary microenvironment (niche) equilibrium. BPA greatly increases BMP2 production by stromal cells of the human mammary SC microenvironment reaching levels comparable to those measured in luminal breast cancer [76]. Moreover, BPA treatment leads to a decrease in estrogen and BMP15 production in oocytes delaying their maturation [145]. A decrease in BMP2 production through a direct binding of BPA to ERγ was involved in bone loss through a suppression of osteoblast differentiation reverted by inhibition of ERγ [146]. This suggests that the effects and mechanisms of BPA-induced BMP ligand production depend on the estrogen receptor expression profile and are context dependent [147]. However, the molecular mechanism by which BPA induces BMP2 production by stromal cells of the mammary gland BMP2 is not yet known. On the other hand, we have demonstrated that long-term exposure (60 days) to BPA initiates fundamental changes in human mammary stem cells themselves, in particular, by altering major BMP signaling elements such as receptor expression and localization [64]. This results in the "priming" of stem cells to exogenous activating signals of the BMP pathway and sensitizes them to be more sensitive to exogenous soluble BMP ligands. We then demonstrated for the first time that nongenotoxic alterations of both the stem cells and their niche act

*Illustration of the main findings that show a crosstalk between BMP and estrogen signaling pathways.*

**38**

Different signaling pathways often engage in complex interactions synergistically mediating an appropriate cellular response. Estrogen signaling is no exception and it is likely involved in a crosstalk with the BMP pathway at multiple levels in the mammary gland. BMPs are secreted proteins active in a very large number of organs and tissues during development, adulthood, and pathogenesis [155]. Previous work suggested a close interaction between ER-mediated estrogen signaling and the BMP pathway in different cell types of the mammary gland. In a model of mammary epithelial stem cells, E2 or known EDCs like BPA or BPS were able to potentiate SMAD activation by BMP2 [64]. This was possibly due to a physical interaction between ERα isoforms and SMAD factors, such as that reported for ERα or ERβ, and could be associated with an increased risk of cell transformation by long-term exposure to BMP2.

Deciphering the dysregulations of the BMP signaling pathway has been remarkably useful in identifying its importance in cancer stem cell phenotypes in the neural system [92, 93]. The role of alterations of BMP signaling to sustain cancer stem cell features has been extended by us and others in breast cancer and leukemia [90, 94, 95]. We showed that chronic exposure to high concentrations of BMP2 drives the transformation of mammary stem cells toward the luminal tumor subtype [76] through binding to its BMPR1B receptor. However, downstream mechanisms and crosstalks with estrogen signaling in those mammary stem cells remained to be understood. This is especially important in the context of several studies that demonstrated the involvement of BPA in the proliferation of either ER-positive or -negative cancer cells. In addition, BPA can trigger proliferation via nonclassical estrogen receptors, including the estrogen-related receptor gamma (ERRγ) [156, 157]. We also demonstrated that long-term exposure of human mammary stem cells (ER-negative in terms of ERα-66 expression) to pollutants such as BPA initiates fundamental changes in stem cells by altering major BMP signaling components [64], thus "priming" stem cells to exogenous BMP activation. Complementary to this effect on epithelial stem cells, we revealed an impact of BPA on the tumor microenvironment through the induction of the synthesis of high levels of BMP2 by normal fibroblasts and stromal cells reaching levels similar to those measured in breast tumors [76].

Resistance and relapse can be due to tumor adaptation or evolution. Indeed, therapies elicit a selective pressure on cells, which in turn develop resistance, notably by acquiring mutations. Resistance to tamoxifen of ER-positive tumors can be caused by a loss of ER [158], its mutation, or posttranslational modification [159] among others. It was shown that BPA is involved in chemoresistance [160] and notably in resistance to tamoxifen in ER-positive tumor cell lines [161] by decreasing tamoxifen-induced apoptosis and increasing gene expression of ERRα, which contributes to resistance to tamoxifen [162] and cell proliferation [157]. Another study demonstrated that an ERα variant could be induced by BMP2 [128] and may be involved in resistance to tamoxifen [163]. The addiction of cancer cells toward BMP signaling and the crosstalk with estrogen signaling is currently under consideration as a new therapeutic avenue for ER-positive breast cancer patients [164]. At the clinical level, targeting estrogen signaling has been decisive in improving the outcome of ER-positive breast cancer patients. At the era of immunotherapy, the analysis of the impact of bisphenols on the immune system and on tumor surveillance is crucial. This will need to be pursued to improve our understanding and implementation of antiestrogen therapies in the context of their combination with new immune treatments [165]. Overall, these data indicate that disruption of BMP signaling affects both the stem cells and their niche at different stages of the disease, which could be instrumental in the management of breast cancer.

Several studies demonstrated that BPS promotes breast cancer cell proliferation, notably through an ER-cyclin D1-CDK4/6-pRb-dependent pathway, exclusively in ER-positive breast cancer cells [38, 39, 166]. Moreover, it has also been demonstrated that BPF has the same proliferative action as BPA, BPS, or estrogen treatments on transformed ER-positive cells. Similar to BPS, this proliferative effect relies on cyclin D and E expression through ER-dependent pathways [39]. BPS, as shown for BPA, can also induce epigenetic and transcriptional changes in breast cancer cells, resulting in an increase in the expression of genes implicated in proliferation, cellular attachment as well as adhesion and migration [167]. Lastly, the bioavailability of BPS substitutes might be higher than for BPA. A recent study conducted in pigs, an ideal model for mimicking the human digestive tract, demonstrated the lower plasma clearance of BPS (3.5 lower) compared to BPA and an increased oral systemic exposure exceeding 250-fold [168]. These observations draw our attention and raise concerns about replacing BPA by BPS, as this may result in an increased internal exposure to EDCs.

**41**

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer…*

To conclude, BMP signaling plays a major role in the regulation of SCs and of their microenvironment (niche), in both normal and tumor contexts. Multiple abnormalities of BMP signaling have been observed in cancer, but until recently studies had mostly focused on its role in advanced disease. However, due to the number of studies describing the importance of BMP signaling throughout breast cancer development (from initiation, progression, metastasis up to resistance), we suggest that early detection of BMP signaling alterations, such as increased levels of BMP2 and/or of BMP receptors, may constitute a reliable marker of exposure to BPA. This suggests that further investigations into alterations of the BMP pathway in the context of exposure to bisphenols should improve our understanding of associated side effects.

The authors thank the following funding bodies for their support of the team's work mentioned in this review: the Association pour la Recherche contre le Cancer, La Ligue contre le Cancer, the Déchaine ton cœur association, the Région Rhône-

The author thanks Brigitte Manship for English proofreading and critical reading.

Université Claude Bernard Lyon 1, Cancer Research Center of Lyon-CRCL,

\*Address all correspondence to: veronique.maguer-satta@lyon.unicancer.fr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*DOI: http://dx.doi.org/10.5772/intechopen.90273*

Alpes and the Institut National du Cancer.

**Notes/thanks/other declarations**

Boris Guyot and Veronique Maguer-Satta\*

provided the original work is properly cited.

CNRS UMR5286, Inserm U1052, Lyon Cedex, France

The authors declare no conflict of interest.

**Acknowledgements**

**Conflict of interest**

**Author details**

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer… DOI: http://dx.doi.org/10.5772/intechopen.90273*

To conclude, BMP signaling plays a major role in the regulation of SCs and of their microenvironment (niche), in both normal and tumor contexts. Multiple abnormalities of BMP signaling have been observed in cancer, but until recently studies had mostly focused on its role in advanced disease. However, due to the number of studies describing the importance of BMP signaling throughout breast cancer development (from initiation, progression, metastasis up to resistance), we suggest that early detection of BMP signaling alterations, such as increased levels of BMP2 and/or of BMP receptors, may constitute a reliable marker of exposure to BPA. This suggests that further investigations into alterations of the BMP pathway in the context of exposure to bisphenols should improve our understanding of associated side effects.

#### **Acknowledgements**

*Breast Cancer Biology*

Deciphering the dysregulations of the BMP signaling pathway has been remarkably useful in identifying its importance in cancer stem cell phenotypes in the neural system [92, 93]. The role of alterations of BMP signaling to sustain cancer stem cell features has been extended by us and others in breast cancer and leukemia [90, 94, 95]. We showed that chronic exposure to high concentrations of BMP2 drives the transformation of mammary stem cells toward the luminal tumor subtype [76] through binding to its BMPR1B receptor. However, downstream mechanisms and crosstalks with estrogen signaling in those mammary stem cells remained to be understood. This is especially important in the context of several studies that demonstrated the involvement of BPA in the proliferation of either ER-positive or -negative cancer cells. In addition, BPA can trigger proliferation via nonclassical estrogen receptors, including the estrogen-related receptor gamma (ERRγ) [156, 157]. We also demonstrated that long-term exposure of human mammary stem cells (ER-negative in terms of ERα-66 expression) to pollutants such as BPA initiates fundamental changes in stem cells by altering major BMP signaling components [64], thus "priming" stem cells to exogenous BMP activation. Complementary to this effect on epithelial stem cells, we revealed an impact of BPA on the tumor microenvironment through the induction of the synthesis of high levels of BMP2 by normal fibroblasts and stromal cells reaching

Resistance and relapse can be due to tumor adaptation or evolution. Indeed, therapies elicit a selective pressure on cells, which in turn develop resistance, notably by acquiring mutations. Resistance to tamoxifen of ER-positive tumors can be caused by a loss of ER [158], its mutation, or posttranslational modification [159] among others. It was shown that BPA is involved in chemoresistance [160] and notably in resistance to tamoxifen in ER-positive tumor cell lines [161] by decreasing tamoxifen-induced apoptosis and increasing gene expression of ERRα, which contributes to resistance to tamoxifen [162] and cell proliferation [157]. Another study demonstrated that an ERα variant could be induced by BMP2 [128] and may be involved in resistance to tamoxifen [163]. The addiction of cancer cells toward BMP signaling and the crosstalk with estrogen signaling is currently under consideration as a new therapeutic avenue for ER-positive breast cancer patients [164]. At the clinical level, targeting estrogen signaling has been decisive in improving the outcome of ER-positive breast cancer patients. At the era of immunotherapy, the analysis of the impact of bisphenols on the immune system and on tumor surveillance is crucial. This will need to be pursued to improve our understanding and implementation of antiestrogen therapies in the context of their combination with new immune treatments [165]. Overall, these data indicate that disruption of BMP signaling affects both the stem cells and their niche at different stages of the disease,

levels similar to those measured in breast tumors [76].

which could be instrumental in the management of breast cancer.

Several studies demonstrated that BPS promotes breast cancer cell proliferation, notably through an ER-cyclin D1-CDK4/6-pRb-dependent pathway, exclusively in ER-positive breast cancer cells [38, 39, 166]. Moreover, it has also been demonstrated that BPF has the same proliferative action as BPA, BPS, or estrogen treatments on transformed ER-positive cells. Similar to BPS, this proliferative effect relies on cyclin D and E expression through ER-dependent pathways [39]. BPS, as shown for BPA, can also induce epigenetic and transcriptional changes in breast cancer cells, resulting in an increase in the expression of genes implicated in proliferation, cellular attachment as well as adhesion and migration [167]. Lastly, the bioavailability of BPS substitutes might be higher than for BPA. A recent study conducted in pigs, an ideal model for mimicking the human digestive tract, demonstrated the lower plasma clearance of BPS (3.5 lower) compared to BPA and an increased oral systemic exposure exceeding 250-fold [168]. These observations draw our attention and raise concerns about replacing BPA by BPS, as this may result in an increased internal exposure to EDCs.

**40**

The authors thank the following funding bodies for their support of the team's work mentioned in this review: the Association pour la Recherche contre le Cancer, La Ligue contre le Cancer, the Déchaine ton cœur association, the Région Rhône-Alpes and the Institut National du Cancer.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Notes/thanks/other declarations**

The author thanks Brigitte Manship for English proofreading and critical reading.

#### **Author details**

Boris Guyot and Veronique Maguer-Satta\* Université Claude Bernard Lyon 1, Cancer Research Center of Lyon-CRCL, CNRS UMR5286, Inserm U1052, Lyon Cedex, France

\*Address all correspondence to: veronique.maguer-satta@lyon.unicancer.fr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[16] Hammes SR, Levin ER. Extranuclear steroid receptors: Nature and actions. Endocrine Reviews. 2007;**28**(7):726-741

[17] MacKay H, Abizaid A. A plurality of molecular targets: The receptor ecosystem for bisphenol-A (BPA). Hormones and Behavior. 2018;**101**:59-67

[18] Wang T, Liu B, Guan Y, Gong M, Zhang W, Pan J, et al. Melatonin inhibits the proliferation of breast cancer cells induced by bisphenol A via targeting estrogen receptor-related pathways. Thoracic Cancer. 2018;**9**(3):368-375

[19] Murata M, Kang JH. Bisphenol A (BPA) and cell signaling pathways. Biotechnology Advances. 2018;**36**(1):311-327

[20] Siracusa JS, Yin L, Measel E, Liang S, Yu X. Effects of bisphenol A and its analogs on reproductive health: A mini review. Reproductive Toxicology. 2018;**79**:96-123

[21] Urriola-Munoz P, Li X, Maretzky T, McIlwain DR, Mak TW, Reyes JG, et al. The xenoestrogens biphenol-A and nonylphenol differentially regulate metalloprotease-mediated shedding of EGFR ligands. Journal of Cellular Physiology. 2018;**233**(3):2247-2256

[22] Watson CS, Bulayeva NN, Wozniak AL, Alyea RA. Xenoestrogens are potent activators of nongenomic estrogenic responses. Steroids. 2007;**72**(2):124-134

[23] Holmes D. Breast cancer: Increased risk with concurrent dietary and EDC exposures. Nature Reviews. Endocrinology. 2017;**13**(7):378

[24] Hussain I, Bhan A, Ansari KI, Deb P, Bobzean SA, Perrotti LI, et al. Bisphenol-A induces expression of HOXC6, an estrogen-regulated homeobox-containing gene associated with breast cancer. Biochimica et Biophysica Acta. 2015;**1849**(6):697-708

[25] Hafezi SA, Abdel-Rahman WM. The endocrine disruptor Bisphenol A (BPA) exerts a wide range of effects in carcinogenesis and response to therapy. Current Molecular Pharmacology. 2019;**12**(3):230-238;

[26] Sprague BL, Trentham-Dietz A, Hedman CJ, Wang J, Hemming JD, Hampton JM, et al. Circulating serum xenoestrogens and mammographic breast density. Breast Cancer Research. 2013;**15**(3):R45

[27] Ayyanan A, Laribi O, Schuepbach-Mallepell S, Schrick C, Gutierrez M, Tanos T, et al. Perinatal exposure to bisphenol a increases adult mammary gland progesterone response and cell number. Molecular Endocrinology. 2011;**25**(11):1915-1923

[28] Gao H, Yang BJ, Li N, Feng LM, Shi XY, Zhao WH, et al. Bisphenol A and hormone-associated cancers: Current progress and perspectives. Medicine (Baltimore). 2015;**94**(1):e211

[29] Fernandez SV, Russo J. Estrogen and xenoestrogens in breast cancer. Toxicologic Pathology. 2010;**38**(1):110-122

[30] Dairkee SH, Seok J, Champion S, Sayeed A, Mindrinos M, Xiao W, et al. Bisphenol A induces a profile of tumor aggressiveness in high-risk cells from breast cancer patients. Cancer Research. 2008;**68**(7):2076-2080

[31] Seyfried TN, Huysentruyt LC. On the origin of cancer metastasis. Critical Reviews in Oncogenesis. 2013;**18**(1-2):43-73

**42**

2012

*Breast Cancer Biology*

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[1] Pourteimoor V, Mohammadi-Yeganeh S, Paryan M. Breast cancer classification and prognostication through diverse systems along with recent emerging findings in this respect; the dawn of new perspectives in the clinical applications. Tumour Biology.

Ali A, Lopez de Cerain Salsamendi A, et al. Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: The challenge ahead. Carcinogenesis.

[9] Kabir ER, Rahman MS, Rahman I. A review on endocrine disruptors and their possible impacts on human health. Environmental Toxicology and Pharmacology. 2015;**40**(1):241-258

[10] Gore AC, Chappell VA, Fenton SE, Flaws JA, Nadal A, Prins GS, et al. EDC-2: The endocrine society's second scientific statement on endocrinedisrupting chemicals. Endocrine Reviews. 2015;**36**(6):E1-E150

[11] Zoeller RT, Brown TR, Doan LL, Gore AC, Skakkebaek NE, Soto AM, et al. Endocrine-disrupting chemicals and public health protection: A statement of principles from the Endocrine Society. Endocrinology.

2012;**153**(9):4097-4110

2018;**126**(1):017012

2013;**42**:132-155

[12] Li Y, Perera L, Coons LA,

Burns KA, Tyler Ramsey J, Pelch KE, et al. Differential in vitro biological action, coregulator interactions, and molecular dynamic analysis of Bisphenol A (BPA), BPAF, and BPS ligand-ERalpha complexes. Environmental Health Perspectives.

[13] Routledge EJ, White R, Parker MG,

Sumpter JP. Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta. The Journal of Biological Chemistry. 2000;**275**(46):35986-35993

[14] Rochester JR. Bisphenol A and human health: A review of the literature. Reproductive Toxicology.

2015;**36**(Suppl 1):S254-S296

2016;**37**(11):14479-14499

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Amphiregulin takes center stage. Breast

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[61] Booth BW, Smith GH. ERalpha and PR are expressed in label-retaining mammary epithelial cells that divide asymmetrically and retain their template DNA strands. Breast Cancer

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Anderson LH, Jimenez-Rojo L, Brisken C, Smith GH. Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics. Experimental Cell Research. 2010;**316**(3):422-432

2011;**2**(2):85-90

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Neoplasia. 1999;**4**(1):89-104

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[52] Asselin-Labat ML, Vaillant F, Sheridan JM, Pal B, Wu D, Simpson ER, et al. Control of mammary stem cell function by steroid hormone signalling. Nature.

[53] Brisken C. Hormonal control of alveolar development and its implications for breast carcinogenesis. Journal of Mammary Gland Biology and

2017;**27**(8):556-567

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Neoplasia. 1997;**2**(4):343-354

O'Malley BW, Rosen JM. Use of PRKO mice to study the role of progesterone in mammary gland development. Journal of Mammary Gland Biology and

[55] Mallepell S, Krust A, Chambon P, Brisken C. Paracrine signaling through the epithelial estrogen receptor alpha is required for proliferation and morphogenesis in the mammary gland.

[49] Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, et al. Generation of a functional mammary gland from a single stem cell. Nature.

[50] Inman JL, Robertson C, Mott JD,

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Journal of Mammary Gland Biology and Neoplasia. 1999;**4**(1):89-104

*Breast Cancer Biology*

[32] Weigelt B, Peterse JL, Van't Veer LJ. Breast cancer metastasis: Markers and models. Nature Reviews. signaling of bisphenol A in breast cancer. Environmental Science and Pollution Research International.

[41] Morgan M, Deoraj A, Felty Q, Roy D. Environmental estrogen-like endocrine disrupting chemicals and breast cancer. Molecular and Cellular Endocrinology. 2017;**457**:89-102

[42] Shafei A, Ramzy MM, Hegazy AI, Husseny AK, El-Hadary UG, Taha MM, et al. The molecular mechanisms of action of the endocrine disrupting chemical bisphenol A in the development of cancer. Gene.

[43] Silberstein GB, Van Horn K, Shyamala G, Daniel CW. Essential role of endogenous estrogen in

directly stimulating mammary growth demonstrated by implants containing pure antiestrogens. Endocrinology.

[44] Feng Y, Manka D, Wagner KU, Khan SA. Estrogen receptor-alpha expression in the mammary epithelium is required for ductal and alveolar morphogenesis in mice. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**(37):14718-14723

[45] Daniel CW, Silberstein GB, Strickland P. Direct action of 17 betaestradiol on mouse mammary ducts analyzed by sustained release implants and steroid autoradiography. Cancer Research. 1987;**47**(22):6052-6057

[46] Lydon JP, Sivaraman L, Conneely OM. A reappraisal of progesterone action in the mammary

gland. Journal of Mammary Gland Biology and Neoplasia.

[47] Shyamala G. Progesterone signaling and mammary gland morphogenesis.

2000;**5**(3):325-338

2018;**25**(24):23624-23630

2018;**647**:235-243

1994;**134**(1):84-90

[33] Gupta GP, Massague J. Cancer metastasis: Building a framework. Cell.

[35] Rangel R, Guzman-Rojas L, Kodama T, Kodama M, Newberg JY, Copeland NG, et al. Identification of new tumor suppressor genes in triplenegative breast cancer. Cancer Research.

[34] Jin X, Mu P. Targeting breast cancer metastasis. Breast Cancer (Auckl.).

[36] Zhang XL, Wang HS, Liu N, Ge LC. Bisphenol A stimulates the epithelial mesenchymal transition of estrogen negative breast cancer cells via FOXA1 signals. Archives of Biochemistry and

Cancer. 2005;**5**(8):591-602

2006;**127**(4):679-695

2015;**9**(Suppl 1):23-34

2017;**77**(15): 4089-4101

Biophysics. 2015;**585**:10-16

2016;**29**(3):285-295

2017;**31**(4):358-369

[37] Castillo Sanchez R, Gomez R, Perez Salazar E. Bisphenol A induces migration through a GPER-, FAK-, Src-, and ERK2-dependent pathway in MDA-MB-231 breast cancer cells. Chemical Research in Toxicology.

[38] Deng Q, Jiang G, Wu Y, Li J, Liang W, Chen L, et al. GPER/hippo-YAP signal is involved in Bisphenol S induced migration of triple negative breast cancer (TNBC) cells. Journal of Hazardous Materials. 2018;**355**:1-9

[39] Kim JY, Choi HG, Lee HM, Lee GA, Hwang KA, Choi KC. Effects of bisphenol compounds on the growth and epithelial mesenchymal transition of MCF-7 CV human breast cancer cells. Journal of Biomedical Research.

[40] Shafei A, Matbouly M, Mostafa E, Al Sannat S, Abdelrahman M, Lewis B, et al. Stop eating plastic, molecular

**44**

[48] Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D, et al. Purification and unique properties of mammary epithelial stem cells. Nature. 2006;**439**(7079):993-997

[49] Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, et al. Generation of a functional mammary gland from a single stem cell. Nature. 2006;**439**(7072):84-88

[50] Inman JL, Robertson C, Mott JD, Bissell MJ. Mammary gland development: Cell fate specification, stem cells and the microenvironment. Development. 2015;**142**(6):1028-1042

[51] Lloyd-Lewis B, Harris OB, Watson CJ, Davis FM. Mammary stem cells: Premise, properties, and perspectives. Trends in Cell Biology. 2017;**27**(8):556-567

[52] Asselin-Labat ML, Vaillant F, Sheridan JM, Pal B, Wu D, Simpson ER, et al. Control of mammary stem cell function by steroid hormone signalling. Nature. 2010;**465**(7299):798-802

[53] Brisken C. Hormonal control of alveolar development and its implications for breast carcinogenesis. Journal of Mammary Gland Biology and Neoplasia. 2002;**7**(1):39-48

[54] Humphreys RC, Lydon JP, O'Malley BW, Rosen JM. Use of PRKO mice to study the role of progesterone in mammary gland development. Journal of Mammary Gland Biology and Neoplasia. 1997;**2**(4):343-354

[55] Mallepell S, Krust A, Chambon P, Brisken C. Paracrine signaling through the epithelial estrogen receptor alpha is required for proliferation and morphogenesis in the mammary gland. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(7):2196-2201

[56] Stingl J. Estrogen and progesterone in normal mammary gland development and in cancer. Hormones and Cancer. 2011;**2**(2):85-90

[57] Ciarloni L, Mallepell S, Brisken C. Amphiregulin is an essential mediator of estrogen receptor alpha function in mammary gland development. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**(13):5455-5460

[58] LaMarca HL, Rosen JM. Estrogen regulation of mammary gland development and breast cancer: Amphiregulin takes center stage. Breast Cancer Research. 2007;**9**(4):304

[59] Sternlicht MD, Sunnarborg SW, Kouros-Mehr H, Yu Y, Lee DC, Werb Z. Mammary ductal morphogenesis requires paracrine activation of stromal EGFR via ADAM17-dependent shedding of epithelial amphiregulin. Development. 2005;**132**(17):3923-3933

[60] Villadsen R, Fridriksdottir AJ, Ronnov-Jessen L, Gudjonsson T, Rank F, LaBarge MA, et al. Evidence for a stem cell hierarchy in the adult human breast. Journal of Cell Biology. 2007;**177**(1):87-101

[61] Booth BW, Smith GH. ERalpha and PR are expressed in label-retaining mammary epithelial cells that divide asymmetrically and retain their template DNA strands. Breast Cancer Research. 2006;**8**(4):R49

[62] Booth BW, Boulanger CA, Anderson LH, Jimenez-Rojo L, Brisken C, Smith GH. Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics. Experimental Cell Research. 2010;**316**(3):422-432

[63] Russo J, Snider K, Pereira JS, Russo IH. Estrogen induced breast cancer is the result in the disruption of the asymmetric cell division of the stem cell. Hormone Molecular Biology and Clinical Investigation. 2010;**1**(2):53-65

[64] Clement F, Xu X, Donini CF, Clement A, Omarjee S, Delay E, et al. Long-term exposure to bisphenol A or benzo(a)pyrene alters the fate of human mammary epithelial stem cells in response to BMP2 and BMP4, by preactivating BMP signaling. Cell Death and Differentiation. 2017;**24**(1):155-166

[65] Kopras E, Potluri V, Bermudez ML, Williams K, Belcher S, Kasper S. Actions of endocrine-disrupting chemicals on stem/progenitor cells during development and disease. Endocrine-Related Cancer. 2014;**21**(2):T1-T12

[66] Bateman ME, Strong AL, McLachlan JA, Burow ME, Bunnell BA. The effects of endocrine disruptors on adipogenesis and osteogenesis in mesenchymal stem cells: A review. Frontiers in Endocrinology. 2016;**7**:171

[67] Alonso-Magdalena P, Rivera FJ, Guerrero-Bosagna C. Bisphenol-A and metabolic diseases: Epigenetic, developmental and transgenerational basis. Environmental Epigenetics. 2016;**2**(3):dvw022

[68] Landero-Huerta DA, Vigueras-Villasenor RM, Yokoyama-Rebollar E, Arechaga-Ocampo E, Rojas-Castaneda JC, Jimenez-Trejo F, et al. Epigenetic and risk factors of testicular germ cell tumors: A brief review. Frontiers in Bioscience (Landmark Edition). 2017;**22**:1073-1098

[69] Moral R, Wang R, Russo IH, Lamartiniere CA, Pereira J, Russo J. Effect of prenatal exposure to the endocrine disruptor bisphenol A on mammary gland morphology and gene expression signature. The Journal of Endocrinology. 2008;**196**(1):101-112

[70] Vandenberg LN, Maffini MV, Wadia PR, Sonnenschein C, Rubin BS, Soto AM. Exposure to environmentally relevant doses of the xenoestrogen bisphenol-A alters development of the fetal mouse mammary gland. Endocrinology. 2007;**148**(1):116-127

[71] Wadia PR, Cabaton NJ, Borrero MD, Rubin BS, Sonnenschein C, Shioda T, et al. Low-dose BPA exposure alters the mesenchymal and epithelial transcriptomes of the mouse fetal mammary gland. PLoS One. 2013;**8**(5):e63902

[72] Wang D, Gao H, Bandyopadhyay A, Wu A, Yeh IT, Chen Y, et al. Pubertal bisphenol A exposure alters murine mammary stem cell function leading to early neoplasia in regenerated glands. Cancer Prevention Research (Philadelphia, Pa.). 2014;**7**(4):445-455

[73] Qin XY, Fukuda T, Yang L, Zaha H, Akanuma H, Zeng Q, et al. Effects of bisphenol A exposure on the proliferation and senescence of normal human mammary epithelial cells. Cancer Biology & Therapy. 2012;**13**(5):296-306

[74] Fernandez SV, Huang Y, Snider KE, Zhou Y, Pogash TJ, Russo J. Expression and DNA methylation changes in human breast epithelial cells after bisphenol A exposure. International Journal of Oncology. 2012;**41**(1):369-377

[75] Lillo MA, Nichols C, Seagroves TN, Miranda-Carboni GA, Krum SA. Bisphenol A induces Sox2 in ER(+) breast cancer stem-like cells. Hormones and Cancer. 2017;**8**(2):90-99

[76] Chapellier M, Bachelard-Cascales E, Schmidt X, Clement F, Treilleux I, Delay E, et al. Disequilibrium of BMP2 levels in the breast stem cell niche

**47**

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer…*

the National Academy of Sciences of the United States of America. 2006;**103**(45):16788-16793

2007;**134**(6):1221-1230

[85] Fleming JM, Ginsburg E, Goldhar AS, Plant J, Vonderhaar BK. Progesterone receptor activates Msx2 expression by downregulating TNAP/ Akp2 and activating the Bmp pathway in EpH4 mouse mammary epithelial cells.

PLoS One. 2012;**7**(3):e34058

Biology. 2013;**373**(1):95-106

[86] Forsman CL, Ng BC, Heinze RK, Kuo C, Sergi C, Gopalakrishnan R, et al. BMP-binding protein twisted gastrulation is required in mammary gland epithelium for normal ductal elongation and myoepithelial

compartmentalization. Developmental

[88] Bachelard-Cascales E, Chapellier M,

[87] Perotti C, Karayazi O, Moffat S, Shemanko CS. The bone morphogenetic

protein receptor-1A pathway is required for lactogenic differentiation of mammary epithelial cells in vitro. In Vitro Cellular & Developmental Biology—Animal. 2012;**48**(6):377-384

Delay E, Pochon G, Voeltzel T, Puisieux A, et al. The CD10 enzyme is a key player to identify and regulate human mammary stem cells. Stem Cells.

[89] Mou H, Vinarsky V, Tata PR, Brazauskas K, Choi SH, Crooke AK, et al. Dual SMAD signaling inhibition enables long-term expansion of diverse epithelial basal cells. Cell Stem Cell.

[90] Zylbersztejn F, Flores-Violante M, Voeltzel T, Nicolini FE, Lefort S,

2010;**28**(6):1081-1088

2016;**19**(2):217-231

[84] Hens JR, Dann P, Zhang JP, Harris S, Robinson GW, Wysolmerski J. BMP4 and PTHrP interact to stimulate ductal outgrowth during embryonic mammary development and to inhibit hair follicle induction. Development.

*DOI: http://dx.doi.org/10.5772/intechopen.90273*

launches epithelial transformation by overamplifying BMPR1B cell response. Stem Cell Reports. 2015;**4**(2):239-254

Deng Y, Qiao M, Peabody M, et al. Bone morphogenetic protein (BMP) signaling in development and human diseases. Genes & Diseases. 2014;**1**(1):87-105

[78] Huang RL, Sun Y, Ho CK, Liu K, Tang QQ, Xie Y, et al. IL-6 potentiates BMP-2-induced osteogenesis and adipogenesis via two different BMPR1Amediated pathways. Cell Death &

[79] Zhang X, Guo J, Zhou Y, Wu G. The roles of bone morphogenetic proteins and their signaling in the osteogenesis of adipose-derived stem cells. Tissue Engineering. Part B, Reviews.

[80] Gustafson B, Hammarstedt A, Hedjazifar S, Hoffmann JM, Svensson PA, Grimsby J, et al. BMP4 and BMP antagonists

regulate human white and beige

[81] Ribeiro S, Lopes LR, Paula Costa G, Figueiredo VP, Shrestha D, Batista AP, et al. CXCL-16, IL-17, and bone morphogenetic protein 2 (BMP-2) are associated with overweight and obesity conditions in middle-aged and elderly women. Immunity & Ageing.

adipogenesis. Diabetes. 2015;**64**(5):1670-

[82] Zamani N, Brown CW. Emerging roles for the transforming growth factor-{beta} superfamily in regulating adiposity and energy expenditure. Endocrine Reviews. 2011;**32**(3):387-403

[83] Cho KW, Kim JY, Song SJ, Farrell E, Eblaghie MC, Kim HJ, et al. Molecular interactions between Tbx3 and Bmp4 and a model for dorsoventral positioning of mammary gland development. Proceedings of

Disease. 2018;**9**(2):144

2014;**20**(1):84-92

1681

2017;**14**:6

[77] Wang RN, Green J, Wang Z,

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer… DOI: http://dx.doi.org/10.5772/intechopen.90273*

launches epithelial transformation by overamplifying BMPR1B cell response. Stem Cell Reports. 2015;**4**(2):239-254

*Breast Cancer Biology*

2010;**1**(2):53-65

[63] Russo J, Snider K, Pereira JS, Russo IH. Estrogen induced breast cancer is the result in the disruption of the asymmetric cell division of the stem cell. Hormone Molecular Biology and Clinical Investigation.

expression signature. The Journal of Endocrinology. 2008;**196**(1):101-112

[70] Vandenberg LN, Maffini MV, Wadia PR, Sonnenschein C, Rubin BS, Soto AM. Exposure to environmentally relevant doses of the xenoestrogen bisphenol-A alters development of the fetal mouse mammary gland. Endocrinology. 2007;**148**(1):116-127

[71] Wadia PR, Cabaton NJ, Borrero MD, Rubin BS, Sonnenschein C, Shioda T, et al. Low-dose BPA exposure alters the mesenchymal and epithelial transcriptomes of the mouse fetal mammary gland. PLoS One.

[72] Wang D, Gao H, Bandyopadhyay A, Wu A, Yeh IT, Chen Y, et al. Pubertal bisphenol A exposure alters murine mammary stem cell function leading to early neoplasia in regenerated glands. Cancer Prevention Research (Philadelphia, Pa.). 2014;**7**(4):445-455

[73] Qin XY, Fukuda T, Yang L, Zaha H, Akanuma H, Zeng Q, et al. Effects of bisphenol A exposure on the proliferation and senescence of normal human mammary epithelial cells. Cancer Biology & Therapy.

[74] Fernandez SV, Huang Y, Snider KE, Zhou Y, Pogash TJ, Russo J. Expression and DNA methylation changes in human breast epithelial cells after bisphenol A exposure. International Journal of Oncology. 2012;**41**(1):369-377

[75] Lillo MA, Nichols C, Seagroves TN, Miranda-Carboni GA, Krum SA. Bisphenol A induces Sox2 in ER(+) breast cancer stem-like cells. Hormones

[76] Chapellier M, Bachelard-Cascales E, Schmidt X, Clement F, Treilleux I, Delay E, et al. Disequilibrium of BMP2 levels in the breast stem cell niche

and Cancer. 2017;**8**(2):90-99

2013;**8**(5):e63902

2012;**13**(5):296-306

[64] Clement F, Xu X, Donini CF, Clement A, Omarjee S, Delay E, et al. Long-term exposure to bisphenol A or benzo(a)pyrene alters the fate of human mammary epithelial stem cells in response to BMP2 and BMP4, by preactivating BMP signaling. Cell Death and Differentiation. 2017;**24**(1):155-166

[65] Kopras E, Potluri V, Bermudez ML, Williams K, Belcher S, Kasper S. Actions of endocrine-disrupting chemicals on stem/progenitor cells during development and disease. Endocrine-Related Cancer. 2014;**21**(2):T1-T12

McLachlan JA, Burow ME, Bunnell BA. The effects of endocrine disruptors on adipogenesis and osteogenesis in mesenchymal stem cells: A review. Frontiers in Endocrinology. 2016;**7**:171

[67] Alonso-Magdalena P, Rivera FJ, Guerrero-Bosagna C. Bisphenol-A and metabolic diseases: Epigenetic, developmental and transgenerational basis. Environmental Epigenetics.

[68] Landero-Huerta DA, Vigueras-Villasenor RM, Yokoyama-Rebollar E, Arechaga-Ocampo E, Rojas-Castaneda JC,

Jimenez-Trejo F, et al. Epigenetic and risk factors of testicular germ cell tumors: A brief review. Frontiers in Bioscience (Landmark Edition).

[69] Moral R, Wang R, Russo IH, Lamartiniere CA, Pereira J, Russo J. Effect of prenatal exposure to the endocrine disruptor bisphenol A on mammary gland morphology and gene

2016;**2**(3):dvw022

2017;**22**:1073-1098

[66] Bateman ME, Strong AL,

**46**

[77] Wang RN, Green J, Wang Z, Deng Y, Qiao M, Peabody M, et al. Bone morphogenetic protein (BMP) signaling in development and human diseases. Genes & Diseases. 2014;**1**(1):87-105

[78] Huang RL, Sun Y, Ho CK, Liu K, Tang QQ, Xie Y, et al. IL-6 potentiates BMP-2-induced osteogenesis and adipogenesis via two different BMPR1Amediated pathways. Cell Death & Disease. 2018;**9**(2):144

[79] Zhang X, Guo J, Zhou Y, Wu G. The roles of bone morphogenetic proteins and their signaling in the osteogenesis of adipose-derived stem cells. Tissue Engineering. Part B, Reviews. 2014;**20**(1):84-92

[80] Gustafson B, Hammarstedt A, Hedjazifar S, Hoffmann JM, Svensson PA, Grimsby J, et al. BMP4 and BMP antagonists regulate human white and beige adipogenesis. Diabetes. 2015;**64**(5):1670- 1681

[81] Ribeiro S, Lopes LR, Paula Costa G, Figueiredo VP, Shrestha D, Batista AP, et al. CXCL-16, IL-17, and bone morphogenetic protein 2 (BMP-2) are associated with overweight and obesity conditions in middle-aged and elderly women. Immunity & Ageing. 2017;**14**:6

[82] Zamani N, Brown CW. Emerging roles for the transforming growth factor-{beta} superfamily in regulating adiposity and energy expenditure. Endocrine Reviews. 2011;**32**(3):387-403

[83] Cho KW, Kim JY, Song SJ, Farrell E, Eblaghie MC, Kim HJ, et al. Molecular interactions between Tbx3 and Bmp4 and a model for dorsoventral positioning of mammary gland development. Proceedings of

the National Academy of Sciences of the United States of America. 2006;**103**(45):16788-16793

[84] Hens JR, Dann P, Zhang JP, Harris S, Robinson GW, Wysolmerski J. BMP4 and PTHrP interact to stimulate ductal outgrowth during embryonic mammary development and to inhibit hair follicle induction. Development. 2007;**134**(6):1221-1230

[85] Fleming JM, Ginsburg E, Goldhar AS, Plant J, Vonderhaar BK. Progesterone receptor activates Msx2 expression by downregulating TNAP/ Akp2 and activating the Bmp pathway in EpH4 mouse mammary epithelial cells. PLoS One. 2012;**7**(3):e34058

[86] Forsman CL, Ng BC, Heinze RK, Kuo C, Sergi C, Gopalakrishnan R, et al. BMP-binding protein twisted gastrulation is required in mammary gland epithelium for normal ductal elongation and myoepithelial compartmentalization. Developmental Biology. 2013;**373**(1):95-106

[87] Perotti C, Karayazi O, Moffat S, Shemanko CS. The bone morphogenetic protein receptor-1A pathway is required for lactogenic differentiation of mammary epithelial cells in vitro. In Vitro Cellular & Developmental Biology—Animal. 2012;**48**(6):377-384

[88] Bachelard-Cascales E, Chapellier M, Delay E, Pochon G, Voeltzel T, Puisieux A, et al. The CD10 enzyme is a key player to identify and regulate human mammary stem cells. Stem Cells. 2010;**28**(6):1081-1088

[89] Mou H, Vinarsky V, Tata PR, Brazauskas K, Choi SH, Crooke AK, et al. Dual SMAD signaling inhibition enables long-term expansion of diverse epithelial basal cells. Cell Stem Cell. 2016;**19**(2):217-231

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[94] Jung N, Maguer-Satta V, Guyot B. Early steps of mammary stem cells transformation by exogenous signals, effects of the bisphenols endocrine disruptors. Cancers. 2019;**11**(9): 1351-1368

[95] Chapellier M, Maguer-Satta V. BMP2, a key to uncover luminal breast cancer origin linked to pollutant effects on epithelial stem cells niche. Molecular & Cellular Oncology. 2016;**3**(3):e1026527

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[100] Ye L, Jiang WG. Bone morphogenetic proteins in tumour associated angiogenesis and implication in cancer therapies. Cancer Letters. 2016;**380**(2):586-597

[101] Bellanger A, Donini CF, Vendrell JA, Lavaud J, Machuca-Gayet I, Ruel M, et al. The critical role of the ZNF217 oncogene in promoting breast cancer metastasis to the bone. The Journal of Pathology. 2017;**242**(1):73-89

[102] Ketolainen JM, Alarmo EL, Tuominen VJ, Kallioniemi A. Parallel inhibition of cell growth and induction of cell migration and invasion in breast cancer cells by bone morphogenetic protein 4. Breast Cancer Research and Treatment. 2010;**124**(2):377-386

[103] Owens P, Pickup MW, Novitskiy SV, Chytil A, Gorska AE, Aakre ME, et al. Disruption of bone morphogenetic protein receptor 2 (BMPR2) in mammary tumors promotes metastases through cell autonomous and paracrine mediators. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(8):2814-2819

[104] Montesano R. Bone morphogenetic protein-4 abrogates lumen formation by mammary epithelial cells and promotes invasive growth. Biochemical and Biophysical Research Communications. 2007;**353**(3):817-822

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Journal of Steroid Biochemistry and Molecular Biology. 2007;**105**(1-5):91-97

[112] Lal A, Ramazzotti D, Weng Z,

[113] Lima ZS, Ghadamzadeh M, Arashloo FT, Amjad G, Ebadi MR, Younesi L. Recent advances of therapeutic targets based on the molecular signature in breast cancer: Genetic mutations and implications for current treatment paradigms. Journal of Hematology & Oncology.

[114] Balboni AL, Hutchinson JA, DeCastro AJ, Cherukuri P, Liby K, Sporn MB, et al. DeltaNp63alphamediated activation of bone morphogenetic protein signaling governs stem cell activity and plasticity in normal and malignant mammary epithelial cells. Cancer Research.

characterization of breast tumors with BRCA1 and BRCA2 mutations. BMC Medical Genomics. 2019;**12**(1):84

Liu K, Ford JM, Sidow A. Comprehensive genomic

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2013;**73**(2):1020-1030

2016;**10**(4):575-593

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[116] Amin R, Morita-Fujimura Y, Tawarayama H, Semba K, Chiba N, Fukumoto M, et al. DeltaNp63alpha induces quiescence and downregulates the BRCA1 pathway in estrogen

receptor-positive luminal breast cancer cell line MCF7 but not in other breast cancer cell lines. Molecular Oncology.

[117] Chau JF, Jia D, Wang Z, Liu Z, Hu Y, Zhang X, et al. A crucial role for bone morphogenetic protein-Smad1 signalling in the DNA damage response. Nature Communications. 2012;**3**:836

Balboni A, DiRenzo J. DeltaNP63alpha transcriptionally activates chemokine receptor 4 (CXCR4) expression to regulate breast cancer stem cell activity and chemotaxis. Molecular Cancer Therapeutics. 2015;**14**(1):225-235

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Molecular and Cellular Endocrinology.

Biochemical and Biophysical Research Communications. 2008;**374**(1):164-168

[107] Helms MW, Packeisen J, August C, Schittek B, Boecker W, Brandt BH, et al. First evidence supporting a potential role for the BMP/SMAD pathway in the progression of oestrogen receptorpositive breast cancer. Journal of Pathology. 2005;**206**(3):366-376

[106] Montesano R, Sarkozi R, Schramek H. Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation

of mammary epithelial cells.

[108] Arnold SF, Tims E,

1999;**11**(12):1031-1037

2009;**6**(2):101-108

McGrath BE. Identification of bone morphogenetic proteins and their receptors in human breast cancer cell lines: Importance of BMP2. Cytokine.

[109] Bokobza SM, Ye L, Kynaston HE, Mansel RE, Jiang WG. Reduced expression of BMPR-IB correlates with poor prognosis and increased proliferation of breast cancer cells. Cancer Genomics & Proteomics.

[110] Katsuno Y, Hanyu A, Kanda H, Ishikawa Y, Akiyama F, Iwase T, et al.

Bone morphogenetic protein signaling enhances invasion and bone metastasis of breast cancer cells through Smad pathway. Oncogene.

[111] Zhang M, Wang Q, Yuan W, Yang S, Wang X, Yan JD, et al. Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells. The

2008;**27**(49):6322-6333

2011;**343**(1-2):7-17

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[105] Masuda H, Otsuka F, Matsumoto Y, Takano M, Miyoshi T, Inagaki K, et al. Functional interaction of fibroblast growth factor-8, bone morphogenetic protein and estrogen receptor in breast cancer cell proliferation. Molecular and Cellular Endocrinology. 2011;**343**(1-2):7-17

*Breast Cancer Biology*

Maguer-Satta V. The BMP pathway: A unique tool to decode the origin and progression of leukemia. Experimental et al. Bone morphogenetic protein 4 expression in multiple normal and tumor tissues reveals its importance beyond development. Modern Pathology: An Official Journal of the United States and Canadian Academy of

Pathology, Inc. 2013;**26**(1):10-21

2017;**24**(10):R349-RR66

2016;**380**(2):586-597

[100] Ye L, Jiang WG. Bone

[101] Bellanger A, Donini CF,

[102] Ketolainen JM, Alarmo EL, Tuominen VJ, Kallioniemi A. Parallel inhibition of cell growth and induction of cell migration and invasion in breast cancer cells by bone morphogenetic protein 4. Breast Cancer Research and Treatment. 2010;**124**(2):377-386

[103] Owens P, Pickup MW,

2007;**353**(3):817-822

Novitskiy SV, Chytil A, Gorska AE, Aakre ME, et al. Disruption of bone morphogenetic protein receptor 2 (BMPR2) in mammary tumors promotes metastases through cell autonomous and paracrine mediators. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(8):2814-2819

[104] Montesano R. Bone morphogenetic protein-4 abrogates lumen formation by mammary epithelial cells and promotes invasive growth. Biochemical and Biophysical Research Communications.

morphogenetic proteins in tumour associated angiogenesis and implication in cancer therapies. Cancer Letters.

Vendrell JA, Lavaud J, Machuca-Gayet I, Ruel M, et al. The critical role of the ZNF217 oncogene in promoting breast cancer metastasis to the bone. The Journal of Pathology. 2017;**242**(1):73-89

[99] Zabkiewicz C, Resaul J, Hargest R, Jiang WG, Ye L. Bone morphogenetic proteins, breast cancer, and bone metastases: Striking the right balance. Endocrine-Related Cancer.

[91] Bier E, De Robertis EM. Embryo development. BMP gradients: A paradigm for morphogen-mediated developmental patterning. Science.

[92] Lee J, Son MJ, Woolard K, Donin NM,

Zanetti N, Lamorte G, Binda E, Broggi G, et al. Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature. 2006;**444**(7120):761-765

[94] Jung N, Maguer-Satta V, Guyot B. Early steps of mammary stem cells transformation by exogenous signals, effects of the bisphenols endocrine disruptors. Cancers. 2019;**11**(9):

[95] Chapellier M, Maguer-Satta V. BMP2, a key to uncover luminal breast cancer origin linked to pollutant effects on epithelial stem cells niche. Molecular & Cellular Oncology.

[96] Alarmo EL, Kallioniemi A. Bone morphogenetic proteins in breast cancer: Dual role in tumourigenesis?

[97] Thawani JP, Wang AC, Than KD, Lin CY, La Marca F, Park P. Bone morphogenetic proteins and cancer: Review of the literature. Neurosurgery.

[98] Alarmo EL, Huhtala H, Korhonen T, Pylkkanen L, Holli K, Kuukasjarvi T,

Endocrine-Related Cancer. 2010;**17**(2):R123-RR39

1351-1368

2016;**3**(3):e1026527

2010;**66**(2):233-246

Li A, Cheng CH, et al. Epigeneticmediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells. Cancer Cell. 2008;**13**(1):69-80

[93] Piccirillo SG, Reynolds BA,

Hematology. 2018;**61**:36-44

2015;**348**(6242):aaa5838

**48**

[106] Montesano R, Sarkozi R, Schramek H. Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells. Biochemical and Biophysical Research Communications. 2008;**374**(1):164-168

[107] Helms MW, Packeisen J, August C, Schittek B, Boecker W, Brandt BH, et al. First evidence supporting a potential role for the BMP/SMAD pathway in the progression of oestrogen receptorpositive breast cancer. Journal of Pathology. 2005;**206**(3):366-376

[108] Arnold SF, Tims E, McGrath BE. Identification of bone morphogenetic proteins and their receptors in human breast cancer cell lines: Importance of BMP2. Cytokine. 1999;**11**(12):1031-1037

[109] Bokobza SM, Ye L, Kynaston HE, Mansel RE, Jiang WG. Reduced expression of BMPR-IB correlates with poor prognosis and increased proliferation of breast cancer cells. Cancer Genomics & Proteomics. 2009;**6**(2):101-108

[110] Katsuno Y, Hanyu A, Kanda H, Ishikawa Y, Akiyama F, Iwase T, et al. Bone morphogenetic protein signaling enhances invasion and bone metastasis of breast cancer cells through Smad pathway. Oncogene. 2008;**27**(49):6322-6333

[111] Zhang M, Wang Q, Yuan W, Yang S, Wang X, Yan JD, et al. Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells. The Journal of Steroid Biochemistry and Molecular Biology. 2007;**105**(1-5):91-97

[112] Lal A, Ramazzotti D, Weng Z, Liu K, Ford JM, Sidow A. Comprehensive genomic characterization of breast tumors with BRCA1 and BRCA2 mutations. BMC Medical Genomics. 2019;**12**(1):84

[113] Lima ZS, Ghadamzadeh M, Arashloo FT, Amjad G, Ebadi MR, Younesi L. Recent advances of therapeutic targets based on the molecular signature in breast cancer: Genetic mutations and implications for current treatment paradigms. Journal of Hematology & Oncology. 2019;**12**(1):38

[114] Balboni AL, Hutchinson JA, DeCastro AJ, Cherukuri P, Liby K, Sporn MB, et al. DeltaNp63alphamediated activation of bone morphogenetic protein signaling governs stem cell activity and plasticity in normal and malignant mammary epithelial cells. Cancer Research. 2013;**73**(2):1020-1030

[115] DeCastro AJ, Cherukuri P, Balboni A, DiRenzo J. DeltaNP63alpha transcriptionally activates chemokine receptor 4 (CXCR4) expression to regulate breast cancer stem cell activity and chemotaxis. Molecular Cancer Therapeutics. 2015;**14**(1):225-235

[116] Amin R, Morita-Fujimura Y, Tawarayama H, Semba K, Chiba N, Fukumoto M, et al. DeltaNp63alpha induces quiescence and downregulates the BRCA1 pathway in estrogen receptor-positive luminal breast cancer cell line MCF7 but not in other breast cancer cell lines. Molecular Oncology. 2016;**10**(4):575-593

[117] Chau JF, Jia D, Wang Z, Liu Z, Hu Y, Zhang X, et al. A crucial role for bone morphogenetic protein-Smad1 signalling in the DNA damage response. Nature Communications. 2012;**3**:836

[118] Miyazono K, Maeda S, Imamura T. BMP receptor signaling: Transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine & Growth Factor Reviews. 2005;**16**(3):251-263

[119] Yadin D, Knaus P, Mueller TD. Structural insights into BMP receptors: Specificity, activation and inhibition. Cytokine & Growth Factor Reviews. 2016;**27**:13-34

[120] Nohe A, Hassel S, Ehrlich M, Neubauer F, Sebald W, Henis YI, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. The Journal of Biological Chemistry. 2002;**277**(7):5330-5338

[121] Hassel S, Schmitt S, Hartung A, Roth M, Nohe A, Petersen N, et al. Initiation of Smad-dependent and Smad-independent signaling via distinct BMP-receptor complexes. The Journal of Bone and Joint Surgery. American Volume. 2003;**85-A**(Suppl 3):44-51

[122] Miyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein receptors and signal transduction. Journal of Biochemistry. 2010;**147**(1):35-51

[123] Gamell C, Osses N, Bartrons R, Ruckle T, Camps M, Rosa JL, et al. BMP2 induction of actin cytoskeleton reorganization and cell migration requires PI3-kinase and Cdc42 activity. Journal of Cell Science. 2008;**121**(Pt 23):3960-3970

[124] Guicheux J, Lemonnier J, Ghayor C, Suzuki A, Palmer G, Caverzasio J. Activation of p38 mitogenactivated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation. Journal of Bone and Mineral Research. 2003;**18**(11):2060-2068

[125] Hay E, Lemonnier J, Fromigue O, Marie PJ. Bone morphogenetic protein-2 promotes osteoblast apoptosis through a Smad-independent, protein kinase C-dependent signaling pathway. The Journal of Biological Chemistry. 2001;**276**(31):29028-29036

[126] Vinals F, Lopez-Rovira T, Rosa JL, Ventura F. Inhibition of PI3K/ p70 S6K and p38 MAPK cascades increases osteoblastic differentiation induced by BMP-2. FEBS Letters. 2002;**510**(1-2):99-104

[127] Yamamoto T, Saatcioglu F, Matsuda T. Cross-talk between bone morphogenic proteins and estrogen receptor signaling. Endocrinology. 2002;**143**(7):2635-2642

[128] Wang D, Huang P, Zhu B, Sun L, Huang Q, Wang J. Induction of estrogen receptor alpha-36 expression by bone morphogenetic protein 2 in breast cancer cell lines. Molecular Medicine Reports. 2012;**6**(3):591-596

[129] Matsumoto Y, Otsuka F, Takano M, Mukai T, Yamanaka R, Takeda M, et al. Estrogen and glucocorticoid regulate osteoblast differentiation through the interaction of bone morphogenetic protein-2 and tumor necrosis factor-alpha in C2C12 cells. Molecular and Cellular Endocrinology. 2010;**325**(1-2):118-127

[130] Qian SW, Liu Y, Wang J, Nie JC, Wu MY, Tang Y, et al. BMP4 crosstalks with estrogen/ERalpha signaling to regulate adiposity and glucose metabolism in females. eBioMedicine. 2016;**11**:91-100

[131] Wang YC, Xiao XL, Li N, Yang D, Xing Y, Huo R, et al. Oestrogen inhibits BMP4-induced BMP4 expression in cardiomyocytes: A potential mechanism of oestrogen-mediated protection against cardiac hypertrophy. British Journal of Pharmacology. 2015;**172**(23):5586-5595

[132] Liu Y, Du SY, Ding M, Dou X, Zhang FF, Wu ZY, et al. The

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ER-positive breast cancer stem/ progenitor cells. PLoS One.

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[141] Wang Z, Zhang X, Shen P, Loggie BW, Chang Y, Deuel TF.

Identification, cloning, and expression of human estrogen receptor-alpha36, a novel variant of human estrogen receptor-alpha66. Biochemical and Biophysical Research Communications.

[142] Lee LM, Cao J, Deng H, Chen P, Gatalica Z, Wang ZY. ER-alpha36, a novel variant of ER-alpha, is expressed in ER-positive and -negative human breast carcinomas. Anticancer Research.

[143] Omarjee S, Jacquemetton J, Poulard C, Rochel N, Dejaegere A, Chebaro Y, et al. The molecular mechanisms underlying the ERalpha-36-mediated signaling in breast cancer. Oncogene. 2017;**36**(18):2503-2514

[144] Lin SL, Yan LY, Zhang XT, Yuan J, Li M, Qiao J, et al. ER-alpha36, a variant of ER-alpha, promotes tamoxifen agonist action in endometrial cancer cells via the MAPK/ERK and PI3K/Akt pathways. PLoS One. 2010;**5**(2):e9013

[145] Wang X, Jiang SW, Wang L, Sun Y, Xu F, He H, et al. Interfering effects of bisphenol A on in vitro growth of preantral follicles and maturation of oocyes. Clinica Chimica Acta.

2018;**28**(3):336-358

2005;**336**(4):1023-1027

2008;**28**(1B):479-483

2018;**485**:119-125

[146] Watson CS, Bulayeva NN, Wozniak AL, Finnerty CC. Signaling from the membrane via membrane estrogen receptor-alpha: Estrogens,

2014;**9**(2):e88034

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[133] Giacomini D, Paez-Pereda M, Stalla J, Stalla GK, Arzt E. Molecular interaction of BMP-4, TGF-beta, and estrogens in lactotrophs: Impact on the PRL promoter. Molecular Endocrinology. 2009;**23**(7):1102-1114

[134] Serra M, Alysandratos KD,

regulating lung- versus thyroidlineage specification. Development.

[135] Villacorte M, Delmarcelle AS, Lernoux M, Bouquet M, Lemoine P, Bolsee J, et al. Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly. Development.

[136] Meng X, Zhu P, Li N, Hu J, Wang S, Pang S, et al. Expression of BMP-4 in papillary thyroid carcinoma and its correlation with tumor invasion and progression. Pathology, Research and

2017;**144**(21):3879-3893

2016;**143**(11):1958-1970

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[139] Deng H, Zhang XT, Wang ML,

ER-alpha36-mediated rapid estrogen

2015;**418**(Pt 3):193-206

Zheng HY, Liu LJ, Wang ZY.

signaling positively regulates

[137] Conde SJ, Luvizotto Rde A, de Sibio MT, Nogueira CR. Thyroid hormone status interferes with estrogen target gene expression in breast cancer samples in menopausal women. ISRN Endocrinology. 2014;**2014**:317398

Hawkins F, McCauley KB, Jacob A, Choi J, et al. Pluripotent stem cell differentiation reveals distinct developmental pathways

BMP4-Smad signaling pathway regulates hyperandrogenism

2017;**292**(28):11740-11750

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BMP4-Smad signaling pathway regulates hyperandrogenism development in a female mouse model. The Journal of Biological Chemistry. 2017;**292**(28):11740-11750

*Breast Cancer Biology*

2016;**27**:13-34

[118] Miyazono K, Maeda S, Imamura T. BMP receptor signaling: Transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine & Growth Factor Reviews. 2005;**16**(3):251-263

promotes osteoblast apoptosis through a Smad-independent, protein kinase C-dependent signaling pathway. The Journal of Biological Chemistry.

[126] Vinals F, Lopez-Rovira T, Rosa JL,

2001;**276**(31):29028-29036

Ventura F. Inhibition of PI3K/ p70 S6K and p38 MAPK cascades increases osteoblastic differentiation induced by BMP-2. FEBS Letters.

[127] Yamamoto T, Saatcioglu F, Matsuda T. Cross-talk between bone morphogenic proteins and estrogen receptor signaling. Endocrinology.

[128] Wang D, Huang P, Zhu B, Sun L, Huang Q, Wang J. Induction of estrogen receptor alpha-36 expression by bone morphogenetic protein 2 in breast cancer cell lines. Molecular Medicine

[129] Matsumoto Y, Otsuka F, Takano M, Mukai T, Yamanaka R, Takeda M, et al. Estrogen and glucocorticoid regulate osteoblast differentiation through the interaction of bone morphogenetic protein-2 and tumor necrosis factor-alpha in C2C12 cells. Molecular and Cellular Endocrinology.

[130] Qian SW, Liu Y, Wang J, Nie JC, Wu MY, Tang Y, et al. BMP4 crosstalks with estrogen/ERalpha signaling to regulate adiposity and glucose metabolism in females. eBioMedicine.

[131] Wang YC, Xiao XL, Li N, Yang D, Xing Y, Huo R, et al. Oestrogen inhibits BMP4-induced BMP4 expression in cardiomyocytes: A potential mechanism of oestrogen-mediated protection against cardiac hypertrophy. British Journal of Pharmacology.

2002;**510**(1-2):99-104

2002;**143**(7):2635-2642

Reports. 2012;**6**(3):591-596

2010;**325**(1-2):118-127

2016;**11**:91-100

2015;**172**(23):5586-5595

[132] Liu Y, Du SY, Ding M,

Dou X, Zhang FF, Wu ZY, et al. The

[119] Yadin D, Knaus P, Mueller TD. Structural insights into BMP receptors: Specificity, activation and inhibition. Cytokine & Growth Factor Reviews.

[120] Nohe A, Hassel S, Ehrlich M, Neubauer F, Sebald W, Henis YI, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. The Journal of Biological Chemistry. 2002;**277**(7):5330-5338

[121] Hassel S, Schmitt S, Hartung A, Roth M, Nohe A, Petersen N, et al. Initiation of Smad-dependent and Smad-independent signaling via distinct BMP-receptor complexes. The Journal of Bone and Joint Surgery. American Volume. 2003;**85-A**(Suppl 3):44-51

[122] Miyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein receptors and signal

2010;**147**(1):35-51

23):3960-3970

transduction. Journal of Biochemistry.

[123] Gamell C, Osses N, Bartrons R, Ruckle T, Camps M, Rosa JL, et al. BMP2 induction of actin cytoskeleton reorganization and cell migration requires PI3-kinase and Cdc42 activity. Journal of Cell Science. 2008;**121**(Pt

[124] Guicheux J, Lemonnier J, Ghayor C, Suzuki A, Palmer G,

2003;**18**(11):2060-2068

Caverzasio J. Activation of p38 mitogenactivated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation. Journal of Bone and Mineral Research.

[125] Hay E, Lemonnier J, Fromigue O, Marie PJ. Bone morphogenetic protein-2

**50**

[133] Giacomini D, Paez-Pereda M, Stalla J, Stalla GK, Arzt E. Molecular interaction of BMP-4, TGF-beta, and estrogens in lactotrophs: Impact on the PRL promoter. Molecular Endocrinology. 2009;**23**(7):1102-1114

[134] Serra M, Alysandratos KD, Hawkins F, McCauley KB, Jacob A, Choi J, et al. Pluripotent stem cell differentiation reveals distinct developmental pathways regulating lung- versus thyroidlineage specification. Development. 2017;**144**(21):3879-3893

[135] Villacorte M, Delmarcelle AS, Lernoux M, Bouquet M, Lemoine P, Bolsee J, et al. Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly. Development. 2016;**143**(11):1958-1970

[136] Meng X, Zhu P, Li N, Hu J, Wang S, Pang S, et al. Expression of BMP-4 in papillary thyroid carcinoma and its correlation with tumor invasion and progression. Pathology, Research and Practice. 2017;**213**(4):359-363

[137] Conde SJ, Luvizotto Rde A, de Sibio MT, Nogueira CR. Thyroid hormone status interferes with estrogen target gene expression in breast cancer samples in menopausal women. ISRN Endocrinology. 2014;**2014**:317398

[138] Wang ZY, Yin L. Estrogen receptor alpha-36 (ER-alpha36): A new player in human breast cancer. Molecular and Cellular Endocrinology. 2015;**418**(Pt 3):193-206

[139] Deng H, Zhang XT, Wang ML, Zheng HY, Liu LJ, Wang ZY. ER-alpha36-mediated rapid estrogen signaling positively regulates

ER-positive breast cancer stem/ progenitor cells. PLoS One. 2014;**9**(2):e88034

[140] Wang Q, Jiang J, Ying G, Xie XQ, Zhang X, Xu W, et al. Tamoxifen enhances stemness and promotes metastasis of ERalpha36(+) breast cancer by upregulating ALDH1A1 in cancer cells. Cell Research. 2018;**28**(3):336-358

[141] Wang Z, Zhang X, Shen P, Loggie BW, Chang Y, Deuel TF. Identification, cloning, and expression of human estrogen receptor-alpha36, a novel variant of human estrogen receptor-alpha66. Biochemical and Biophysical Research Communications. 2005;**336**(4):1023-1027

[142] Lee LM, Cao J, Deng H, Chen P, Gatalica Z, Wang ZY. ER-alpha36, a novel variant of ER-alpha, is expressed in ER-positive and -negative human breast carcinomas. Anticancer Research. 2008;**28**(1B):479-483

[143] Omarjee S, Jacquemetton J, Poulard C, Rochel N, Dejaegere A, Chebaro Y, et al. The molecular mechanisms underlying the ERalpha-36-mediated signaling in breast cancer. Oncogene. 2017;**36**(18):2503-2514

[144] Lin SL, Yan LY, Zhang XT, Yuan J, Li M, Qiao J, et al. ER-alpha36, a variant of ER-alpha, promotes tamoxifen agonist action in endometrial cancer cells via the MAPK/ERK and PI3K/Akt pathways. PLoS One. 2010;**5**(2):e9013

[145] Wang X, Jiang SW, Wang L, Sun Y, Xu F, He H, et al. Interfering effects of bisphenol A on in vitro growth of preantral follicles and maturation of oocyes. Clinica Chimica Acta. 2018;**485**:119-125

[146] Watson CS, Bulayeva NN, Wozniak AL, Finnerty CC. Signaling from the membrane via membrane estrogen receptor-alpha: Estrogens,

xenoestrogens, and phytoestrogens. Steroids. 2005;**70**(5-7):364-371

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[150] Huang R, Sakamuru S, Martin MT, Reif DM, Judson RS, Houck KA, et al. Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway. Scientific Reports. 2014;**4**:5664

[151] Peyre L, Rouimi P, de Sousa G, Helies-Toussaint C, Carre B, Barcellini S, et al. Comparative study of bisphenol A and its analogue bisphenol S on human hepatic cells: A focus on their potential involvement in nonalcoholic fatty liver disease. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association. 2014;**70**:9-18

[152] Eladak S, Grisin T, Moison D, Guerquin MJ, N'Tumba-Byn T, Pozzi-Gaudin S, et al. A new chapter in the bisphenol A story: Bisphenol S and bisphenol F are not safe alternatives to this compound. Fertility and Sterility. 2015;**103**(1):11-21

[153] Rochester JR, Bolden AL. Bisphenol S and F: A systematic review and

comparison of the hormonal activity of Bisphenol A substitutes. Environmental Health Perspectives. 2015;**123**(7):643-650

[154] Williams GP, Darbre PD. Lowdose environmental endocrine disruptors, increase aromatase activity, estradiol biosynthesis and cell proliferation in human breast cells. Molecular and Cellular Endocrinology. 2019;**486**:55-64

[155] Katagiri T, Watabe T. Bone morphogenetic proteins. Cold Spring Harbor Perspectives in Biology. 2016;**8**(6)

[156] Pupo M, Pisano A, Lappano R, Santolla MF, De Francesco EM, Abonante S, et al. Bisphenol A induces gene expression changes and proliferative effects through GPER in breast cancer cells and cancer-associated fibroblasts. Environmental Health Perspectives. 2012;**120**(8):1177-1182

[157] Song H, Zhang T, Yang P, Li M, Yang Y, Wang Y, et al. Low doses of bisphenol A stimulate the proliferation of breast cancer cells via ERK1/2/ ERRgamma signals. Toxicology in Vitro. 2015;**30**(1 Pt B):521-528

[158] Kuukasjarvi T, Kononen J, Helin H, Holli K, Isola J. Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy. Journal of Clinical Oncology. 1996;**14**(9):2584-2589

[159] Le Romancer M, Poulard C, Cohen P, Sentis S, Renoir JM, Corbo L. Cracking the estrogen receptor's posttranslational code in breast tumors. Endocrine Reviews. 2011;**32**(5):597-622

[160] Lapensee EW, Tuttle TR, Fox SR, Ben-Jonathan N. Bisphenol A at low nanomolar doses confers chemoresistance in estrogen receptoralpha-positive and -negative breast

**53**

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer…*

[168] Gayrard V, Lacroix MZ, Grandin FC, Collet SH, Mila H, Viguie C, et al. Oral systemic bioavailability of Bisphenol A and Bisphenol S in pigs. Environmental Health Perspectives. 2019;**127**(7):77005

*DOI: http://dx.doi.org/10.5772/intechopen.90273*

cancer cells. Environmental Health Perspectives. 2009;**117**(2):175-180

[161] Huang B, Luo N, Wu X, Xu Z, Wang X, Pan X. The modulatory role of low concentrations of bisphenol A on tamoxifen-induced proliferation and apoptosis in breast cancer cells. Environmental Science and Pollution Research International.

2019;**26**(3):2353-2362

2008;**68**(21):8908-8917

[162] Riggins RB, Lan JP, Zhu Y, Klimach U, Zwart A, Cavalli LR, et al. ERRgamma mediates tamoxifen resistance in novel models of invasive lobular breast cancer. Cancer Research.

[163] Gu W, Dong N, Wang P, Shi C, Yang J, Wang J. Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membraneassociated estrogen receptor ER-alpha36 signaling in vitro. Cell Biology and Toxicology. 2017;**33**(2):183-195

[164] Shee K, Jiang A, Varn FS, Liu S, Traphagen NA, Owens P, et al. Cytokine sensitivity screening highlights BMP4 pathway signaling as a therapeutic opportunity in ER(+) breast cancer. The FASEB Journal. 2019;**33**(2):1644-1657

[165] Welte T, Zhang XH, Rosen JM. Repurposing antiestrogens for tumor immunotherapy. Cancer Discovery.

[166] Lin Z, Zhang X, Zhao F, Ru S. Bisphenol S promotes the cell cycle progression and cell proliferation through ERalpha-cyclin D-CDK4/6-pRb pathway in MCF-7 breast cancer cells. Toxicology and Applied Pharmacology.

[167] Huang W, Zhao C, Zhong H, Zhang S, Xia Y, Cai Z. Bisphenol S induced epigenetic and transcriptional changes in human breast cancer cell line MCF-7. Environmental Pollution.

2017;**7**(1):17-19

2019;**366**:75-82

2019;**246**:697-703

*A Potential New Mechanism for Bisphenol Molecules to Initiate Breast Cancer… DOI: http://dx.doi.org/10.5772/intechopen.90273*

cancer cells. Environmental Health Perspectives. 2009;**117**(2):175-180

*Breast Cancer Biology*

Sciences. 2018;**198**:1-7

2003;**79**(1):95-105

2014;**4**:5664

xenoestrogens, and phytoestrogens. Steroids. 2005;**70**(5-7):364-371

comparison of the hormonal activity of Bisphenol A substitutes. Environmental Health Perspectives.

[154] Williams GP, Darbre PD. Lowdose environmental endocrine disruptors, increase aromatase

[155] Katagiri T, Watabe T. Bone morphogenetic proteins. Cold Spring Harbor Perspectives in Biology.

[156] Pupo M, Pisano A, Lappano R, Santolla MF, De Francesco EM, Abonante S, et al. Bisphenol A induces

proliferative effects through GPER in breast cancer cells and cancer-associated fibroblasts. Environmental Health Perspectives. 2012;**120**(8):1177-1182

[157] Song H, Zhang T, Yang P, Li M, Yang Y, Wang Y, et al. Low doses of bisphenol A stimulate the proliferation of breast cancer cells via ERK1/2/ ERRgamma signals. Toxicology in Vitro.

[158] Kuukasjarvi T, Kononen J, Helin H, Holli K, Isola J. Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy. Journal of Clinical Oncology.

gene expression changes and

2015;**30**(1 Pt B):521-528

1996;**14**(9):2584-2589

2011;**32**(5):597-622

[159] Le Romancer M, Poulard C, Cohen P, Sentis S, Renoir JM, Corbo L. Cracking the estrogen receptor's posttranslational code in breast tumors. Endocrine Reviews.

[160] Lapensee EW, Tuttle TR, Fox SR, Ben-Jonathan N. Bisphenol A at low nanomolar doses confers chemoresistance in estrogen receptoralpha-positive and -negative breast

activity, estradiol biosynthesis and cell proliferation in human breast cells. Molecular and Cellular Endocrinology.

2015;**123**(7):643-650

2019;**486**:55-64

2016;**8**(6)

[147] Thent ZC, Froemming GRA, Muid S. Bisphenol A exposure disturbs the bone metabolism: An evolving interest towards an old culprit. Life

[148] Mei J, Hu H, McEntee M, Plummer H 3rd, Song P, Wang HC. Transformation of non-cancerous human breast epithelial cell line MCF10A by the tobaccospecific carcinogen NNK. Breast Cancer Research and Treatment.

[149] Dreier DA, Connors KA, Brooks BW. Comparative endpoint sensitivity of in vitro estrogen agonist assays. Regulatory Toxicology and Pharmacology. 2015;**72**(2):185-193

[150] Huang R, Sakamuru S, Martin MT, Reif DM, Judson RS, Houck KA, et al. Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha

signaling pathway. Scientific Reports.

[151] Peyre L, Rouimi P, de Sousa G, Helies-Toussaint C, Carre B, Barcellini S, et al. Comparative study of bisphenol A and its analogue bisphenol S on human hepatic cells: A focus on their potential involvement in nonalcoholic fatty liver disease. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association. 2014;**70**:9-18

[152] Eladak S, Grisin T, Moison D, Guerquin MJ, N'Tumba-Byn T,

Pozzi-Gaudin S, et al. A new chapter in the bisphenol A story: Bisphenol S and bisphenol F are not safe alternatives to this compound. Fertility and Sterility.

[153] Rochester JR, Bolden AL. Bisphenol

S and F: A systematic review and

**52**

2015;**103**(1):11-21

[161] Huang B, Luo N, Wu X, Xu Z, Wang X, Pan X. The modulatory role of low concentrations of bisphenol A on tamoxifen-induced proliferation and apoptosis in breast cancer cells. Environmental Science and Pollution Research International. 2019;**26**(3):2353-2362

[162] Riggins RB, Lan JP, Zhu Y, Klimach U, Zwart A, Cavalli LR, et al. ERRgamma mediates tamoxifen resistance in novel models of invasive lobular breast cancer. Cancer Research. 2008;**68**(21):8908-8917

[163] Gu W, Dong N, Wang P, Shi C, Yang J, Wang J. Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membraneassociated estrogen receptor ER-alpha36 signaling in vitro. Cell Biology and Toxicology. 2017;**33**(2):183-195

[164] Shee K, Jiang A, Varn FS, Liu S, Traphagen NA, Owens P, et al. Cytokine sensitivity screening highlights BMP4 pathway signaling as a therapeutic opportunity in ER(+) breast cancer. The FASEB Journal. 2019;**33**(2):1644-1657

[165] Welte T, Zhang XH, Rosen JM. Repurposing antiestrogens for tumor immunotherapy. Cancer Discovery. 2017;**7**(1):17-19

[166] Lin Z, Zhang X, Zhao F, Ru S. Bisphenol S promotes the cell cycle progression and cell proliferation through ERalpha-cyclin D-CDK4/6-pRb pathway in MCF-7 breast cancer cells. Toxicology and Applied Pharmacology. 2019;**366**:75-82

[167] Huang W, Zhao C, Zhong H, Zhang S, Xia Y, Cai Z. Bisphenol S induced epigenetic and transcriptional changes in human breast cancer cell line MCF-7. Environmental Pollution. 2019;**246**:697-703

[168] Gayrard V, Lacroix MZ, Grandin FC, Collet SH, Mila H, Viguie C, et al. Oral systemic bioavailability of Bisphenol A and Bisphenol S in pigs. Environmental Health Perspectives. 2019;**127**(7):77005

**55**

**Chapter 3**

*and Xin Wan*

cancer metastasis.

protein 1 (IQGAP1)

**1. Introduction**

**Abstract**

A Novel SASH1-IQGAP1-E-

*Ding'an Zhou, Xing Zeng, Yadong Li, Zhixiong Wu* 

Breast Cancer Metastasis

Cadherin Signal Cascade Mediates

SAM and SH3 domain-containing protein 1 (SASH1) was previously described as a candidate tumor suppressor gene in breast cancer and colon cancer to mediate tumor metastasis and tumor growth. However, the underlying mechanism that SASH1 implements breast cancer metastasis in most solid cancers remains unexplored. In this study, SASH1 was identified to bind to IQ motif-containing GTPase activating protein 1 (IQGAP1). In breast cancer tissues, there was a correlation between the expressions of SASH1 and IQGAP1 (P < 0.05), and the expressions of SASH1 and IQGAP1 proteins were, respectively, correlated with the expression of E-cadherin (P < 0.001). In addition, the expressions of SASH1 and IQGAP1 proteins were correlated with tumor diameter and tumor grade (all P < 0.05) but without lymph node metastasis (P > 0.05). Therefore, it is suggested that SASH1 may form a new signaling cascade with IQGAP1 and E-cadherin to regulate breast

**Keywords:** breast neoplasms, gene expression regulation, cadherins, SAM and SH3 domain-containing protein 1 (SASH1), IQ motif-containing GTPase activating

Breast neoplasm is the most common cancer in women, which is originated from mammary epithelial tissue. The age of breast cancer is about 40–60 years old or before and after menopause. The morbidity of breast cancer is showed to be an upward trend year by year [1]. There are many factors that trigger breast cancer; however, the genetic factors only account for 10 and 90% of inducing factors of breast cancer remain to be investigated. SASH1 is a novel tumor suppressor gene, which is located in chromosome 6q24.3 [2] and is expressed in most of human tissues and cells except for lymphocytes and dendritic cells [3]. SASH1 was originally identified as a candidate tumor suppressor gene in breast cancer and colon cancer, regulating tumorigenesis of breast and other solid cancers and the adhesive and migratory behavior of cancer cells in tumor formation [4, 5]. Compared with that in normal breast epithelial tissues, SASH1 is downregulated in 74% of epithelial tissues of breast cancer-affected individuals [4, 6]. Some studies indicate that SASH1 downregulation is associated with tumor metastasis [3, 5]. Other studies

#### **Chapter 3**

## A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis

*Ding'an Zhou, Xing Zeng, Yadong Li, Zhixiong Wu and Xin Wan*

#### **Abstract**

SAM and SH3 domain-containing protein 1 (SASH1) was previously described as a candidate tumor suppressor gene in breast cancer and colon cancer to mediate tumor metastasis and tumor growth. However, the underlying mechanism that SASH1 implements breast cancer metastasis in most solid cancers remains unexplored. In this study, SASH1 was identified to bind to IQ motif-containing GTPase activating protein 1 (IQGAP1). In breast cancer tissues, there was a correlation between the expressions of SASH1 and IQGAP1 (P < 0.05), and the expressions of SASH1 and IQGAP1 proteins were, respectively, correlated with the expression of E-cadherin (P < 0.001). In addition, the expressions of SASH1 and IQGAP1 proteins were correlated with tumor diameter and tumor grade (all P < 0.05) but without lymph node metastasis (P > 0.05). Therefore, it is suggested that SASH1 may form a new signaling cascade with IQGAP1 and E-cadherin to regulate breast cancer metastasis.

**Keywords:** breast neoplasms, gene expression regulation, cadherins, SAM and SH3 domain-containing protein 1 (SASH1), IQ motif-containing GTPase activating protein 1 (IQGAP1)

#### **1. Introduction**

Breast neoplasm is the most common cancer in women, which is originated from mammary epithelial tissue. The age of breast cancer is about 40–60 years old or before and after menopause. The morbidity of breast cancer is showed to be an upward trend year by year [1]. There are many factors that trigger breast cancer; however, the genetic factors only account for 10 and 90% of inducing factors of breast cancer remain to be investigated. SASH1 is a novel tumor suppressor gene, which is located in chromosome 6q24.3 [2] and is expressed in most of human tissues and cells except for lymphocytes and dendritic cells [3]. SASH1 was originally identified as a candidate tumor suppressor gene in breast cancer and colon cancer, regulating tumorigenesis of breast and other solid cancers and the adhesive and migratory behavior of cancer cells in tumor formation [4, 5]. Compared with that in normal breast epithelial tissues, SASH1 is downregulated in 74% of epithelial tissues of breast cancer-affected individuals [4, 6]. Some studies indicate that SASH1 downregulation is associated with tumor metastasis [3, 5]. Other studies

indicate downregulated SASH1 promotes metastasis of hepatoma carcinoma cells through Shh signal pathway [7].

IQGAP1 is a scaffolding protein with 189 kDa of molecule weight, which contains multiple protein-interacting domains, such as a calponin homology domain, a polyproline-binding domain, four calmodulin-binding motifs, and a Ras GAP-related domain [8, 9]. The binding players of IQGAP1 proteins are involved in actin, calmodulin, members of the Rho GTPase family (i.e., Rac1 and Cdc42), Rap1, E-cadherin, β-catenin, members of the mitogen-activated protein kinase (MAPK) pathway, and adenomatous polyposis coli [8, 10]. Various basic cellular activities such as cytoskeletal organization, cell-cell adhesion, cell migration, transcription, and signal transduction are mediated by the bindings of IQGAP1 to these proteins [11]. Cell-cell adhesion of epithelial cells is predominantly mediated by E-cadherin and the associated catenin complex [12], which includes α-catenin (102 kDa), β-catenin (92 kDa), and γ-catenin/plakoglobin (83 kDa). β-Catenin combines with E-cadherin, and α-catenin links this E-cadherin/β-catenin complex to the actin cytoskeleton, which is essential for E-cadherin to express its full adhesive function. Remodeling of this adhesive sequence leads to cell detachment or loosening of cell-cell contact, which promotes epithelial cells to move as clusters, and IQGAP1 is involved in the remodeling of the adhesive complexes of epithelial cells [11, 13–15]. Our previous studies suggest that SASH1 is associated with MAP2K2 to cross talk with ERK1/2-CREB cascade to trigger melanin synthesis in the formation of hyperpigmentation plaques of a kind of dyschromatosis [16]. Importantly, our previous studies also indicate that SASH1 not only bind to G alpha S protein (Gαs) but IQGAP1 to form a novel Gαs-SASH1-IQGAP1-Ecadherin cascade and mutated SASH1(s) which mediate E-cadherin expression through the Gαs-SASH1-IQGAP1-E-cadherin cascade to promote directional migration of melanocytes or melanoma cells [17]. So, it is speculated that this mechanism may also exist in breast cancer cells. Taken above, the associations between SASH1 and IQGAP1 in breast cancer cells and the expression of SASH1, IQGAP1, and E-cadherin were analyzed by immunohistochemistry analyses in 80 cases of the affected individuals of breast cancer. Furthermore, the expression relationship among SASH1, IQGAP1, and E-cadherin and the associations between clinical index of breast cancer patients and the expression of SASH1 and IQGAP1, respectively, were assessed to find out novel interference targets for early prevention of breast cancer metastasis.

### **2. Material and methods**

#### **2.1 Plasmid construction of HA-IQGAP1-pcDNA3.0 and pEGFP-C3-SASH1**

The construction of pEGFP-C3-SASH1 recombined vectors was mainly referred to our previous description [17]. IQGAP1 cDNA was obtained from Han Jiahuai Lab, Xiamen University (Xiamen, Fujian, China), and cloned into pcDNA3.0-HA vector. PCR was performed with IQGAP1 cDNA as template using TransTaq® DNA Polymerase High Fidelity (TransGen Biotech, Ltd., Beijing, China) using the following cloning primers of IQGAP1: sense primer, 5'-TAGTCTAGAAT GTCCG CCGCAACGAG-3'(Xba I inserted) and antisense primer, 5′-CCGCTCGAGTTACTTCCCGTAGAACTTTTTG-3′ (Xho I inserted). The amplification conditions were as follows: 95°C 2 min, 95°C 30 s, 58°C 30 s, and 72°C 1 min for 30 cycles and 72°C 5 min and 4°C forever. The recombined vectors were identified by enzyme digestion of endonuclease and CDS of SASH1 and IQGAP1 genes.

**57**

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis*

Human breast cancer cell lines including SK-BR-3 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). After several times of passage, cells were used and cultured in Dulbecco's Modified Eagle's medium (DMEM) (Gibco, Logan, UT), containing 10% BI fetal bovine serum (Bioind, Israel) and 1% penicillin-streptomycin solution at 37°C with 5% CO2. SK-BR-3 cells were subcultured for three times and cultured to logarithmic growth phase for plasmid transfection. The HA-IQGAP1-pcDNA3.0 and pEGFP-C3-SASH1 were transfected into SK-BR-3 cells according to different combinations using PEI prepared by us. The transfected SK-BR-3 cells were divided into three groups, that is, two single-vector transfection groups and one double-vector transfection group. At 48 h after transfection, the transfected SK-BR-3 cells were lysed and collected for

Transfected SK-BR-3 cells were gently washed in PBS three times and then lysed for 25 min using IP-WB lysis buffer (Beyondtime Inc. Ltd., Jiangsu, China) with complete protease inhibitor cocktail per 10-cm dish for 20 min on ice. The cell lysates were transferred to 1.5 ml microcentrifuge tubes. The extracts were centrifuged for 15 min at 12,000 rpm at 4°C. The supernatants were immunoprecipitated using GFP mouse monoclonal antibody (T0005, Affinity Biosciences, Cincinnati, OH, USA) or HA mouse monoclonal antibody (mAb) (Abmart, Shanghai, China) as performed in our previous descriptions [17]. The immunoprecipitates were washed with PBS for three times and subjected to western blotting as previously described [16, 17]. Most of the western blots were mainly performed in our previous reports [17]. The associated HA-IQGAP1 or GFP-SASH1 was detected by western blot along with β-tubulin as loading control. The primary antibodies used in western blot were as follows: anti-GFP, anti-HA, and anti-β-tubulin (10B1) mouse mAb (EarthOx Life

Science, Millbrae, CA, USA or Shanghai Genomics, Shanghai, China).

All breast cancer patients who underwent surgery were followed by treatment in accordance with the National Comprehensive Cancer Network clinical practice guidelines. Fresh primary breast cancer tissues and some of the corresponding adjacent tissues were collected from 80 breast ductal carcinoma patients undergoing resection from May 2015 to June 2016 at the Chongqing Cancer Hospital. Histological diagnosis and tumor-node-metastasis staging of cancer were determined in accordance with the American Joint Committee on Cancer manual criteria for breast cancer. Written informed consent regarding tissue and data used for scientific purposes was obtained from all participating patients. The study was approved by the Research Ethics Committees of the affiliated Hospitals of Guizhou Medical University and Chongqing Cancer Hospital. All of the breast cancer cases were diagnosed by pathological examinations (HE staining and immunohistochemistry analyses). In the clinical cases of breast cancer, 26 cases are with lymph node metastasis, 51 cases are without lymph node metastasis, and 3 cases could not acquire the information of lymph node metastasis. Breast tumor diameters of 16 cases were <1 cm, those of 41 cases were 1.1–2 cm, those of 18 cases were 2.1–3 cm, and those of 5 cases were >3 cm. According to WHO histological classification of breast tumors (2003), 80 cases of breast invasive ductal carcinoma were graded histologically in terms of duct formation, nuclear pleomorphism, and mitosis. Among

*DOI: http://dx.doi.org/10.5772/intechopen.84567*

**2.2 Cell culture and transfection**

immunoprecipitation assays.

**2.4 Clinical cases**

**2.3 Immunoprecipitation and immunoblotting**

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis DOI: http://dx.doi.org/10.5772/intechopen.84567*

#### **2.2 Cell culture and transfection**

*Breast Cancer Biology*

through Shh signal pathway [7].

tion of breast cancer metastasis.

**2. Material and methods**

indicate downregulated SASH1 promotes metastasis of hepatoma carcinoma cells

IQGAP1 is a scaffolding protein with 189 kDa of molecule weight, which contains multiple protein-interacting domains, such as a calponin homology domain, a polyproline-binding domain, four calmodulin-binding motifs, and a Ras GAP-related domain [8, 9]. The binding players of IQGAP1 proteins are involved in actin, calmodulin, members of the Rho GTPase family (i.e., Rac1 and Cdc42), Rap1, E-cadherin, β-catenin, members of the mitogen-activated protein kinase (MAPK) pathway, and adenomatous polyposis coli [8, 10]. Various basic cellular activities such as cytoskeletal organization, cell-cell adhesion, cell migration, transcription, and signal transduction are mediated by the bindings of IQGAP1 to these proteins [11]. Cell-cell adhesion of epithelial cells is predominantly mediated by E-cadherin and the associated catenin complex [12], which includes α-catenin (102 kDa), β-catenin (92 kDa), and γ-catenin/plakoglobin (83 kDa). β-Catenin combines with E-cadherin, and α-catenin links this E-cadherin/β-catenin complex to the actin cytoskeleton, which is essential for E-cadherin to express its full adhesive function. Remodeling of this adhesive sequence leads to cell detachment or loosening of cell-cell contact, which promotes epithelial cells to move as clusters, and IQGAP1 is involved in the remodeling of the adhesive complexes of epithelial cells [11, 13–15]. Our previous studies suggest that SASH1 is associated with MAP2K2 to cross talk with ERK1/2-CREB cascade to trigger melanin synthesis in the formation of hyperpigmentation plaques of a kind of dyschromatosis [16]. Importantly, our previous studies also indicate that SASH1 not only bind to G alpha S protein (Gαs) but IQGAP1 to form a novel Gαs-SASH1-IQGAP1-Ecadherin cascade and mutated SASH1(s) which mediate E-cadherin expression through the Gαs-SASH1-IQGAP1-E-cadherin cascade to promote directional migration of melanocytes or melanoma cells [17]. So, it is speculated that this mechanism may also exist in breast cancer cells. Taken above, the associations between SASH1 and IQGAP1 in breast cancer cells and the expression of SASH1, IQGAP1, and E-cadherin were analyzed by immunohistochemistry analyses in 80 cases of the affected individuals of breast cancer. Furthermore, the expression relationship among SASH1, IQGAP1, and E-cadherin and the associations between clinical index of breast cancer patients and the expression of SASH1 and IQGAP1, respectively, were assessed to find out novel interference targets for early preven-

**2.1 Plasmid construction of HA-IQGAP1-pcDNA3.0 and pEGFP-C3-SASH1**

The construction of pEGFP-C3-SASH1 recombined vectors was mainly referred to our previous description [17]. IQGAP1 cDNA was obtained from Han Jiahuai Lab, Xiamen University (Xiamen, Fujian, China), and cloned into pcDNA3.0-HA vector. PCR was performed with IQGAP1 cDNA as template using TransTaq® DNA Polymerase High Fidelity (TransGen Biotech, Ltd., Beijing, China) using the following cloning primers of IQGAP1: sense primer, 5'-TAGTCTAGAAT GTCCG CCGCAACGAG-3'(Xba I inserted) and antisense primer, 5′-CCGCTCGAGTTACTTCCCGTAGAACTTTTTG-3′ (Xho I inserted). The amplification conditions were as follows: 95°C 2 min, 95°C 30 s, 58°C 30 s, and 72°C 1 min for 30 cycles and 72°C 5 min and 4°C forever. The recombined vectors were identified by enzyme digestion of endonuclease and CDS of SASH1 and

**56**

IQGAP1 genes.

Human breast cancer cell lines including SK-BR-3 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). After several times of passage, cells were used and cultured in Dulbecco's Modified Eagle's medium (DMEM) (Gibco, Logan, UT), containing 10% BI fetal bovine serum (Bioind, Israel) and 1% penicillin-streptomycin solution at 37°C with 5% CO2. SK-BR-3 cells were subcultured for three times and cultured to logarithmic growth phase for plasmid transfection. The HA-IQGAP1-pcDNA3.0 and pEGFP-C3-SASH1 were transfected into SK-BR-3 cells according to different combinations using PEI prepared by us. The transfected SK-BR-3 cells were divided into three groups, that is, two single-vector transfection groups and one double-vector transfection group. At 48 h after transfection, the transfected SK-BR-3 cells were lysed and collected for immunoprecipitation assays.

#### **2.3 Immunoprecipitation and immunoblotting**

Transfected SK-BR-3 cells were gently washed in PBS three times and then lysed for 25 min using IP-WB lysis buffer (Beyondtime Inc. Ltd., Jiangsu, China) with complete protease inhibitor cocktail per 10-cm dish for 20 min on ice. The cell lysates were transferred to 1.5 ml microcentrifuge tubes. The extracts were centrifuged for 15 min at 12,000 rpm at 4°C. The supernatants were immunoprecipitated using GFP mouse monoclonal antibody (T0005, Affinity Biosciences, Cincinnati, OH, USA) or HA mouse monoclonal antibody (mAb) (Abmart, Shanghai, China) as performed in our previous descriptions [17]. The immunoprecipitates were washed with PBS for three times and subjected to western blotting as previously described [16, 17]. Most of the western blots were mainly performed in our previous reports [17]. The associated HA-IQGAP1 or GFP-SASH1 was detected by western blot along with β-tubulin as loading control. The primary antibodies used in western blot were as follows: anti-GFP, anti-HA, and anti-β-tubulin (10B1) mouse mAb (EarthOx Life Science, Millbrae, CA, USA or Shanghai Genomics, Shanghai, China).

#### **2.4 Clinical cases**

All breast cancer patients who underwent surgery were followed by treatment in accordance with the National Comprehensive Cancer Network clinical practice guidelines. Fresh primary breast cancer tissues and some of the corresponding adjacent tissues were collected from 80 breast ductal carcinoma patients undergoing resection from May 2015 to June 2016 at the Chongqing Cancer Hospital. Histological diagnosis and tumor-node-metastasis staging of cancer were determined in accordance with the American Joint Committee on Cancer manual criteria for breast cancer. Written informed consent regarding tissue and data used for scientific purposes was obtained from all participating patients. The study was approved by the Research Ethics Committees of the affiliated Hospitals of Guizhou Medical University and Chongqing Cancer Hospital. All of the breast cancer cases were diagnosed by pathological examinations (HE staining and immunohistochemistry analyses). In the clinical cases of breast cancer, 26 cases are with lymph node metastasis, 51 cases are without lymph node metastasis, and 3 cases could not acquire the information of lymph node metastasis. Breast tumor diameters of 16 cases were <1 cm, those of 41 cases were 1.1–2 cm, those of 18 cases were 2.1–3 cm, and those of 5 cases were >3 cm. According to WHO histological classification of breast tumors (2003), 80 cases of breast invasive ductal carcinoma were graded histologically in terms of duct formation, nuclear pleomorphism, and mitosis. Among

the 80 cases of breast invasive ductal carcinoma, 65 cases were graded into 3 grades: 8 cases belonged to grade I, 47 cases to grade II, and 10 cases to grade III.

#### **2.5 Immunohistochemical analyses of SASH1, IQGAP1, and E-cadherin**

The breast cancer tissues obtained from surgical operation were fixed at 4°C in 10% formaldehyde solution for 24 h. The excess fat and other tissues of breast cancer tissues were removed and embedded with paraffin and made into 5 millimeter (mm) tissue sections. The tissue sections (5 mm) were baked at 56°C and dehydrated and subjected to peroxidase blocking. Tissues of human breast cancer and corresponding adjacent tissues were immunohistochemically stained with SASH1 rabbit polyclonal antibody (pAb) (A302-265A-1, Bethyl Laboratories, Inc., Texas, USA, or Novus Biologicals, USA), IQGAP1 rabbit polyclonal antibody (Bethyl Laboratories, Inc., Texas, USA), and E-Cadherin (24E10) Rabbit mAb (#3195, Cell Signaling Technology). Primary antibodies were added and incubated at 37°C and then for overnight at 4°C. After washing three times for 10 min each with TBS, the sections were incubated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse universal secondary antibodies for 30 min at 37°C. Subsequently, the sections were counterstained with hematoxylin mounted, observed, and photographed under the positive position microscope BX51 at a 100× magnification or a 400× magnification. Finally, the stained slides were observed under a microscope, and images were acquired [17]. The experimental protocols were mainly referred to our previous description [17].

According to the staining intensity of tumor cells, the three proteins, SASH1, IQGAP1, and E-cadherin, were scored and divided into four grades: 0 score (−), 1 score (+), 2 score (++), and 3 score (+++). The three proteins were also scored according to positive cells' percentage of the three proteins and divided into six grades: 0 score (<1%), 1 score (1–20%), 2 score (21–40%), 3 score (41–60%), 4 score (61–80%), 5 score (81–100%). Based on the staining intensity of SASH1, IQGAP1, and E-cadherin, the staining intensity and positive cells' percentage of three proteins were calculated as in our previous description [16]. Total scores of each visual field were determined by the formula: staining intensity scores of positive cells × scores of positive cells' percentage = total scores of each view fields.

#### **2.6 Statistical analyses**

All of experimental results were repeated for three times and statistically analyzed using SPSS 16.0 statistical software. Chi square test was performed to analyze the IHC results of breast cancer tissues and the relationship between expression of SASH1 and IQGAP1 and clinical indicators. Rank-sum test was used to assess the grading relationship between the SASH1 and E-cadherin and IQGAP1 and E-cadherin, respectively. Spearman correlation coefficient method was used to assess the correlation between expressed scores of SASH1 and E-cadherin and IQGAP1 and E-cadherin, respectively. The data are indicated as mean ± standard error of the mean (SEM), and the difference was statistically significant with P < 0.05. Cartograms were plotted with GraphPad Prism 5.

#### **3. Results**

#### **3.1 SASH1 is associated with IQGAP1**

To identify the associations between SASH1 and IQGAP1, HA-IQGAP1-pcDNA3.0 and pEGFP-C3-SASH1 were constructed and were singly or combinedly transfected

**59**

**Figure 1.**

*β-tubulin with loading control.*

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis*

into SK-BR-3 cells and immunoprecipitation; western blot (IP-WB) was performed to identify the associations between exogenous SASH1 and exogenous IQGAP1. HA-IQGAP1 and GFP-SASH1 were singly or in pair transfected into SK-BR-3 cells at 48 h posttransfection, the transfected cells were lysed, and HA-IQGAP1 was immunoprecipitated, and the associated GFP-SASH1 was detected by GFP antibody. The associated HA-IQGAP1 and GFP-SASH1 in the immunoprecipitates and cell lysates (input) were confirmed by western blot. Meanwhile, GFP-SASH1 and HA-IQGAP1 were also either single or in pair transfected into SK-BR-3 cells and after 48 h of transfection, the transfected cells were lysed and were GFP-SASH1 was immunoprecipitated and the associated HA-IQGAP1 was detected by HA antibody. The associated GFP-SASH1 and HA-IQGAP1 in the immunoprecipitates and cell lysates (input) were identified by western blot. Finally, our IP-WB analyses confirmed that exogenous

**3.2 There was a positive correlation between SASH1 and IQGAP1 expression in** 

IHC analyses confirmed that the positive staining of SASH1 and IQGAP1 protein was light brown in breast cancer tissues, the cell nucleus was purple, and the distribution of SASH1 and IQGAP1 was located in the same sites of breast cancer tissues. SASH1 and IQGAP1 show the same or similar expression tendency in breast cancer tissues, i.e., low level of SASH1 expression is followed by low level of IQGAP1 expression and high expression of SASH1 is accompanied by high expression of IQGAP1 (**Figure 2A**). A total of 80 breast cancer tissues were divided into four groups according to the median value of SASH1 and IQGAP1 protein expression scores: SASH1 scores <1.23 were considered as low expression, SASH1 scores ≥1.23 were considered as high expression, IQGAP1 scores <0.78 were maintained as low expression, and SASH1 scores ≥0.78 were maintained as high expression. Statistical analyses indicated that in the 80 cases of breast cancer tissues, cases with low SASH1 expression accounted for 56.3% (45/80) and the cases with low IQGAP1 expression were more than those of high IQGAP1

*SASH1 is associated with IQGAP1. (A) GFP-SASH1 and HA-IQGAP1 were singly or in pair transfected into SK-BR-3 cells, and at 36 h after transfection, the transfected cells were lysed and collected for IP-WB analyses. HA-IQGAP1 was immunoprecipitated, and the associated GFP-SASH1 was detected by western blot using GFP antibody. GFP-SASH1 and HA-IQGAP1 in the cell lysates (input) were detected by western blot along with β-tubulin with loading control. (B) HA-IQGAP1 and GFP-SASH1 were singly or in pair transfected into SK-BR-3 cells, and at 36 h after transfection, the transfected cells were lysed and collected for IP-WB analyses. GFP-SASH1 was immunoprecipitated, and the associated GFP-SASH1 was detected by western blot using GFP antibody. HA-IQGAP1 and GFP-SASH1 in the cell lysates (input) were analyzed by western blot along with* 

*DOI: http://dx.doi.org/10.5772/intechopen.84567*

**breast cancer tissues**

SASH1 was associated with exogenous IQGAP1 (**Figure 1**).

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis DOI: http://dx.doi.org/10.5772/intechopen.84567*

into SK-BR-3 cells and immunoprecipitation; western blot (IP-WB) was performed to identify the associations between exogenous SASH1 and exogenous IQGAP1. HA-IQGAP1 and GFP-SASH1 were singly or in pair transfected into SK-BR-3 cells at 48 h posttransfection, the transfected cells were lysed, and HA-IQGAP1 was immunoprecipitated, and the associated GFP-SASH1 was detected by GFP antibody. The associated HA-IQGAP1 and GFP-SASH1 in the immunoprecipitates and cell lysates (input) were confirmed by western blot. Meanwhile, GFP-SASH1 and HA-IQGAP1 were also either single or in pair transfected into SK-BR-3 cells and after 48 h of transfection, the transfected cells were lysed and were GFP-SASH1 was immunoprecipitated and the associated HA-IQGAP1 was detected by HA antibody. The associated GFP-SASH1 and HA-IQGAP1 in the immunoprecipitates and cell lysates (input) were identified by western blot. Finally, our IP-WB analyses confirmed that exogenous SASH1 was associated with exogenous IQGAP1 (**Figure 1**).

#### **3.2 There was a positive correlation between SASH1 and IQGAP1 expression in breast cancer tissues**

IHC analyses confirmed that the positive staining of SASH1 and IQGAP1 protein was light brown in breast cancer tissues, the cell nucleus was purple, and the distribution of SASH1 and IQGAP1 was located in the same sites of breast cancer tissues. SASH1 and IQGAP1 show the same or similar expression tendency in breast cancer tissues, i.e., low level of SASH1 expression is followed by low level of IQGAP1 expression and high expression of SASH1 is accompanied by high expression of IQGAP1 (**Figure 2A**). A total of 80 breast cancer tissues were divided into four groups according to the median value of SASH1 and IQGAP1 protein expression scores: SASH1 scores <1.23 were considered as low expression, SASH1 scores ≥1.23 were considered as high expression, IQGAP1 scores <0.78 were maintained as low expression, and SASH1 scores ≥0.78 were maintained as high expression. Statistical analyses indicated that in the 80 cases of breast cancer tissues, cases with low SASH1 expression accounted for 56.3% (45/80) and the cases with low IQGAP1 expression were more than those of high IQGAP1

#### **Figure 1.**

*Breast Cancer Biology*

the 80 cases of breast invasive ductal carcinoma, 65 cases were graded into 3 grades:

The breast cancer tissues obtained from surgical operation were fixed at 4°C in 10% formaldehyde solution for 24 h. The excess fat and other tissues of breast cancer tissues were removed and embedded with paraffin and made into 5 millimeter (mm) tissue sections. The tissue sections (5 mm) were baked at 56°C and dehydrated and subjected to peroxidase blocking. Tissues of human breast cancer and corresponding adjacent tissues were immunohistochemically stained with SASH1 rabbit polyclonal antibody (pAb) (A302-265A-1, Bethyl Laboratories, Inc., Texas, USA, or Novus Biologicals, USA), IQGAP1 rabbit polyclonal antibody (Bethyl Laboratories, Inc., Texas, USA), and E-Cadherin (24E10) Rabbit mAb (#3195, Cell Signaling Technology). Primary antibodies were added and incubated at 37°C and then for overnight at 4°C. After washing three times for 10 min each with TBS, the sections were incubated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse universal secondary antibodies for 30 min at 37°C. Subsequently, the sections were counterstained with hematoxylin mounted, observed, and photographed under the positive position microscope BX51 at a 100× magnification or a 400× magnification. Finally, the stained slides were observed under a microscope, and images were acquired [17]. The experimental

According to the staining intensity of tumor cells, the three proteins, SASH1, IQGAP1, and E-cadherin, were scored and divided into four grades: 0 score (−), 1 score (+), 2 score (++), and 3 score (+++). The three proteins were also scored according to positive cells' percentage of the three proteins and divided into six grades: 0 score (<1%), 1 score (1–20%), 2 score (21–40%), 3 score (41–60%), 4 score (61–80%), 5 score (81–100%). Based on the staining intensity of SASH1, IQGAP1, and E-cadherin, the staining intensity and positive cells' percentage of three proteins were calculated as in our previous description [16]. Total scores of each visual field were determined by the formula: staining intensity scores of positive cells × scores of positive cells' percentage = total scores of each view fields.

All of experimental results were repeated for three times and statistically analyzed using SPSS 16.0 statistical software. Chi square test was performed to analyze the IHC results of breast cancer tissues and the relationship between expression of SASH1 and IQGAP1 and clinical indicators. Rank-sum test was used to assess the grading relationship between the SASH1 and E-cadherin and IQGAP1 and E-cadherin, respectively. Spearman correlation coefficient method was used to assess the correlation between expressed scores of SASH1 and E-cadherin and IQGAP1 and E-cadherin, respectively. The data are indicated as mean ± standard error of the mean (SEM), and the difference was statistically significant with

To identify the associations between SASH1 and IQGAP1, HA-IQGAP1-pcDNA3.0 and pEGFP-C3-SASH1 were constructed and were singly or combinedly transfected

8 cases belonged to grade I, 47 cases to grade II, and 10 cases to grade III.

protocols were mainly referred to our previous description [17].

P < 0.05. Cartograms were plotted with GraphPad Prism 5.

**3.1 SASH1 is associated with IQGAP1**

**2.5 Immunohistochemical analyses of SASH1, IQGAP1, and E-cadherin**

**58**

**3. Results**

**2.6 Statistical analyses**

*SASH1 is associated with IQGAP1. (A) GFP-SASH1 and HA-IQGAP1 were singly or in pair transfected into SK-BR-3 cells, and at 36 h after transfection, the transfected cells were lysed and collected for IP-WB analyses. HA-IQGAP1 was immunoprecipitated, and the associated GFP-SASH1 was detected by western blot using GFP antibody. GFP-SASH1 and HA-IQGAP1 in the cell lysates (input) were detected by western blot along with β-tubulin with loading control. (B) HA-IQGAP1 and GFP-SASH1 were singly or in pair transfected into SK-BR-3 cells, and at 36 h after transfection, the transfected cells were lysed and collected for IP-WB analyses. GFP-SASH1 was immunoprecipitated, and the associated GFP-SASH1 was detected by western blot using GFP antibody. HA-IQGAP1 and GFP-SASH1 in the cell lysates (input) were analyzed by western blot along with β-tubulin with loading control.*

expression (>65%, P = 0.015) (**Figure 2B**). And statistical analyses also suggested that in the 80 cases of breast cancer tissues, cases with low IQGAP1 expression accounted for 58.8% (47/80) and the cases with low IQGAP1 expression were more than those of high IQGAP1 expression (>60%, P = 0.011) (**Figure 2B**). Meanwhile, the IHC detection results of SASH1 and IQGAP1 were scored and analyzed by Spearman correlation analyses, and the scores of SASH1 and IQGAP1 were plotted by GraphPad Prism 5 software. In 80 cases of breast cancer tissues, except for 5 cases, SASH1 scores and IQGAP1 scores in most of cases closely

#### **Figure 2.**

*SASH1 expression in 80 cases of breast cancer tissues which is positively correlated with IQGAP1. (A) The expressions of SASH1 and IQGAP1 in 80 cases of breast cancer tissues were detected by immunohistochemical staining method. The cell nucleus was dyed purple and SASH1 and IQGAP1 were dyed pale brown. The left panels were HE staining, and the middle panels and the right panels were IHC staining of SASH1 and IQGAP1. The figures in upper panels were 100× magnification, and one region in the 100× magnification figures was amplified for 400× and framed in black and showed in the bottom panels. (B) The expressions of SASH1 and IQGAP1 were scored, and the score results of SASH1 and IQGAP1 were plotted with GraphPad Prism 5 and analyzed by χ<sup>2</sup> test. The analysis results of SASH1 and IQGAP1 expressions in the left panel indicated that when SASH1 expression was low, the positive percentage of low expressed IQGAP1 was much more than that of high expressed IQGAP1. The expression of SASH1 showed significantly positive correlation with that of IQGAP1 (P = 0.015). And the statistical analyses also suggested that when IQGAP1 expression was low, the positive percentage of low expressed SASH1 was much more than that of high expressed SASH1. The expression of IQGAP1 demonstrated significantly positive correlation with that of SASH1 (P = 0.011). (C) The expressions of SASH1 and IQGAP1 were scored, and the score results of SASH1 and IQGAP1 were plotted with GraphPad Prism 5 and analyzed by Spearman correlation coefficient analyses. Spearman correlation coefficient analyses indicated that except for two score values of SASH1, expression of SASH1 and IQGAP1 showed good similar or same tendency of changes (P = 0.004).*

**61**

*a*

*b*

**Table 2.**

*(n, N = 80).*

*r = 0.461, P < 0.001.*

*r = 0.454, P < 0.001; by rank-sum test.*

*a N = 77. b N = 80. c N = 65.*

**Table 1.**

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis*

**3.3 The expression of SASH1 and IQGAP1 protein in breast cancer was significantly correlated with tumor size and tumor grade**

intersected, which indicated that the SASH1 expression and IQGAP1 expression showed significantly positive correlation (r = 0.308, P = 0.004) (**Figure 2C**).

It has been known that the expression of SASH1 and IQGAP1 is associated with tumor metastasis. So, in this study, we further identify the relationship of expression of SASH1 and IQGAP1 with clinical data of breast cancer-affected individuals. Our analyses (**Table 1**) indicated that in 77 cases of breast cancer with lymph node dissection, the low expression rate of SASH1 protein in lymph node metastasis positive group was slightly higher than that in lymph node metastasis negative group. The low

**Clinical parameters Total SASH1 IQGAP1 P**

Lymph node metastasisa >0.05

Tumor diameter/cmb <0.05

Histological gradec <0.01

Positive 26 17 9 15 11 Negative 51 28 23 30 21

≤1 16 8 8 10 6 1.1–2 41 24 17 23 18 2.1–3 18 9 9 10 8 >3 5 4 1 4 1

I 8 4 4 6 2 II 47 26 21 24 23 III 10 7 3 7 3

*Association of SASH1 and IQGAP1 expressions with the clinical parameters of breast cancer patients (n).*

− 4 6 6 1 17 8 8 1 0 17 + 6 20 2 0 28 12 12 4 0 28 ++ 5 25 3 0 33 12 19 2 0 33 +++ 1 0 1 0 2 1 1 0 0 2 Total 16 51 12 1 80 33 40 7 0 80

*Correlation of SASH1 and IQGAP1 expressions with E-cadherin expression rankin breast cancer tissues* 

**− + ++ ++ Totala − + ++ +++ Totalb**

**E-cadherin SASH1 IQGAP1**

**Low High Low High**

*DOI: http://dx.doi.org/10.5772/intechopen.84567*

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis DOI: http://dx.doi.org/10.5772/intechopen.84567*

intersected, which indicated that the SASH1 expression and IQGAP1 expression showed significantly positive correlation (r = 0.308, P = 0.004) (**Figure 2C**).

#### **3.3 The expression of SASH1 and IQGAP1 protein in breast cancer was significantly correlated with tumor size and tumor grade**

It has been known that the expression of SASH1 and IQGAP1 is associated with tumor metastasis. So, in this study, we further identify the relationship of expression of SASH1 and IQGAP1 with clinical data of breast cancer-affected individuals. Our analyses (**Table 1**) indicated that in 77 cases of breast cancer with lymph node dissection, the low expression rate of SASH1 protein in lymph node metastasis positive group was slightly higher than that in lymph node metastasis negative group. The low


#### **Table 1.**

*Breast Cancer Biology*

expression (>65%, P = 0.015) (**Figure 2B**). And statistical analyses also suggested that in the 80 cases of breast cancer tissues, cases with low IQGAP1 expression accounted for 58.8% (47/80) and the cases with low IQGAP1 expression were more than those of high IQGAP1 expression (>60%, P = 0.011) (**Figure 2B**). Meanwhile, the IHC detection results of SASH1 and IQGAP1 were scored and analyzed by Spearman correlation analyses, and the scores of SASH1 and IQGAP1 were plotted by GraphPad Prism 5 software. In 80 cases of breast cancer tissues, except for 5 cases, SASH1 scores and IQGAP1 scores in most of cases closely

*SASH1 expression in 80 cases of breast cancer tissues which is positively correlated with IQGAP1. (A) The expressions of SASH1 and IQGAP1 in 80 cases of breast cancer tissues were detected by immunohistochemical staining method. The cell nucleus was dyed purple and SASH1 and IQGAP1 were dyed pale brown. The left panels were HE staining, and the middle panels and the right panels were IHC staining of SASH1 and IQGAP1. The figures in upper panels were 100× magnification, and one region in the 100× magnification figures was amplified for 400× and framed in black and showed in the bottom panels. (B) The expressions of SASH1 and IQGAP1 were scored, and the score results of SASH1 and IQGAP1 were plotted with GraphPad* 

*indicated that when SASH1 expression was low, the positive percentage of low expressed IQGAP1 was much more than that of high expressed IQGAP1. The expression of SASH1 showed significantly positive correlation with that of IQGAP1 (P = 0.015). And the statistical analyses also suggested that when IQGAP1 expression was low, the positive percentage of low expressed SASH1 was much more than that of high expressed SASH1. The expression of IQGAP1 demonstrated significantly positive correlation with that of SASH1 (P = 0.011). (C) The expressions of SASH1 and IQGAP1 were scored, and the score results of SASH1 and IQGAP1 were plotted with GraphPad Prism 5 and analyzed by Spearman correlation coefficient analyses. Spearman correlation coefficient analyses indicated that except for two score values of SASH1, expression of SASH1 and* 

*IQGAP1 showed good similar or same tendency of changes (P = 0.004).*

 *test. The analysis results of SASH1 and IQGAP1 expressions in the left panel* 

**60**

**Figure 2.**

*Prism 5 and analyzed by χ<sup>2</sup>*

*Association of SASH1 and IQGAP1 expressions with the clinical parameters of breast cancer patients (n).*


#### **Table 2.**

*Correlation of SASH1 and IQGAP1 expressions with E-cadherin expression rankin breast cancer tissues (n, N = 80).*

expression rate of IQGAP1 protein was slightly lower than that of the negative lymph node metastasis group, but the difference was not statistically significant (65.4% vs. 54.9%, 57.7% vs. 58.8%, all P value > 0.05). In 80 cases of breast cancer, the low expression rate of SASH1 protein was 50.0, 58.5, 50.0, and 80.0%, respectively, in patients with tumor diameter <1.0 cm, 1.1–2.0 cm, 2.1–3.0 cm, and >3.0 cm. The low expression rates of IQGAP1 protein were 62.5, 56.1, 55.6, and 80.0%, respectively. There were significant differences between the two groups (P < 0.05). In 65 cases of breast cancer with histological grading data, the low expression rates of SASH1 protein in histological grading I, II, and III were 50.0, 55.3, and 70.0%, respectively. The low expression rates of IQGAP1 protein were 75.0, 51.1, and 70.0%, respectively. There were significant differences between groups (P < 0.01).

#### **3.4 The expression of SASH1 and IQGAP1 is positively related with E-cadherin, respectively, in breast cancer tissues**

SASH1 and IQGAP1 have been identified to be involved in tumor metastasis. And immunohistochemistry (IHC) analyses were performed to detect the expression of E-cadherin in breast cancer tissues and the relevance of E-cadherin with SASH1 and IQGAP1, respectively. IHC analyses indicated that E-cadherin was mainly located in the cytoplasma membrane of breast cancer tissues. According to the positive intensity of E-cadherin staining, E-cadherin protein expression in breast cancer tissues was graded to four grades, and meanwhile the positive intensity of SASH1 protein staining was also graded to four grades. Statistical analyses suggested that expression of SASH1 protein was significantly positive related to that of E-cadherin (r = 0.461, P < 0.001 (**Table 2** and **Figure 3**)).

#### **Figure 3.**

*SASH1, IQGAP1, and E-cadherin proteins showed consistent changes in the breast cancer tissues. The cell nucleus was dyed purple and SASH1, IQGAP1, and E-cadherin were dyed pale brown, and the magnification is 200×. According to staining intensity of tumor cells and the numbers of positive cells, the immunohistochemical results of SASH1, IQGAP1, and E-cadherin proteins were divided into four grades: negative (−), weakly positive (+), moderately positive (++), and strongly positive (+++). The expression of SASH1 was positively correlated with that of E-cadherin and the expression of IQGAP1 show positive correlation with that of E-cadherin. The cell nucleus was dyed purple, and SASH1, IQGAP1, and, E-cadherin were dyed pale brown, and the magnification is 200×.*

**63**

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis*

SASH1 and E-cadherin staining intensity was moderately positive staining intensity, respectively, which was defined low expression. And further statistical analyses suggested SASH1 and E-cadherin were downregulated in 77 cases (77/80, 96.25%) of breast cancer tissues. All of these indicated that the low expression of SASH1 and the low expression of E-cadherin protein in breast

in 80 cases of breast cancer, the staining intensity of E-cadherin and IQGAP1 was divided into four grades. Statistical analyses demonstrated that expression of IQGAP1 protein was significantly positive related to that of E-cadherin (r = 0.454, P < 0.001 (**Table 2** and **Figure 3**)). Staining intensity of IQGAP1 and E-cadherin was moderately positive staining intensity, respectively, which was defined low expression. And further statistical analyses suggested IQGAP1 and E-cadherin were both downregulated in 78 cases (78/80, 97.5%) of breast cancer tissues. All of these indicated that the low expression of IQGAP1 and the low expression of E-cadherin

According to the staining intensity of IQGAP1 protein and E-cadherin protein

Clinical research indicates that occurrence of breast cancer is associated with many factors including genetic factors, environment, and lifestyle. SASH1, a tumor suppressor gene, is downregulated in most of neoplasms. Decrease or deletion of SASH1 expression is closely related to tumor metastasis [4, 5, 18]. It has been reported that the expression of SASH1 protein in osteosarcoma tissues with lung metastasis is significantly lower than that in osteosarcoma tissues without lung metastasis [19]. Upregulated SASH1 can significantly suppress the migration of cervical carcinoma Hela cells, and, in contrast, knockdown of SASH1 significantly results in reduced adhesion ability of human colon cancer SW480 cells and mouse rectal cancer CMT-93 cells and enhanced migration ability of these tumor cells [3]. Downregulation of SASH1 protein expression in thyroid tumor cells may play an important role in thyroid tumor metastasis [20]. SASH1 mRNA is downregulated in primary liver cancer and thyroid cancer [5]. Compared with corresponding normal tissues, SASH1 protein is downregulated in 37 cases among 50 cases of breast cancer tissues and SASH1 expression loss is associated with breast cancer metastasis [4]. All of these studies suggest that expression loss of SASH1 medicates tumor metastasis. In this study, our IHC analyses identified that in 80 cases of breast cancer tissues, low expression of SASH1 protein in 45 cases (45/80 56.3%) was found, which indicated that SASH1 was downregulated in

IQGAP1 proteins are members of the evolutionarily conserved scaffolding protein family and are more widely expressed than other members of the family [21, 22]. IQGAP1 interacts with specific proteins such as actin, calmodulin, Rho GTPase family members, E-cadherin, and β-catenin. The interactions of IQGAP1 with those specific proteins medicate multiple cell activities such as cell scaffold, intercellular adhesion, metastasis, invasion, transcription, and cell signal transduction. For example, the binding of IQGAP1 to β-catenin to form E-cadherin/β-catenin complex inhibits intercellular adhesion of epithelial cells and promotes β-catenin-mediated transcriptional activation [9]. IQGAP1 protein ,which mediates E-cadherin-mediated-intercellular adhesion, is the key molecule in cell polarization and directed migration [23]. IQGAP1 expression is showed to be of prognostic significance in advanced colorectal carcinoma, and a shorter overall survival of colorectal carcinoma patients can be predicted by diffuse

*DOI: http://dx.doi.org/10.5772/intechopen.84567*

cancer tissue are in good agreement.

**4. Conclusion**

breast cancer.

showed better consistency in breast cancer tissue.

#### *A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis DOI: http://dx.doi.org/10.5772/intechopen.84567*

SASH1 and E-cadherin staining intensity was moderately positive staining intensity, respectively, which was defined low expression. And further statistical analyses suggested SASH1 and E-cadherin were downregulated in 77 cases (77/80, 96.25%) of breast cancer tissues. All of these indicated that the low expression of SASH1 and the low expression of E-cadherin protein in breast cancer tissue are in good agreement.

According to the staining intensity of IQGAP1 protein and E-cadherin protein in 80 cases of breast cancer, the staining intensity of E-cadherin and IQGAP1 was divided into four grades. Statistical analyses demonstrated that expression of IQGAP1 protein was significantly positive related to that of E-cadherin (r = 0.454, P < 0.001 (**Table 2** and **Figure 3**)). Staining intensity of IQGAP1 and E-cadherin was moderately positive staining intensity, respectively, which was defined low expression. And further statistical analyses suggested IQGAP1 and E-cadherin were both downregulated in 78 cases (78/80, 97.5%) of breast cancer tissues. All of these indicated that the low expression of IQGAP1 and the low expression of E-cadherin showed better consistency in breast cancer tissue.

#### **4. Conclusion**

*Breast Cancer Biology*

expression rate of IQGAP1 protein was slightly lower than that of the negative lymph node metastasis group, but the difference was not statistically significant (65.4% vs. 54.9%, 57.7% vs. 58.8%, all P value > 0.05). In 80 cases of breast cancer, the low expression rate of SASH1 protein was 50.0, 58.5, 50.0, and 80.0%, respectively, in patients with tumor diameter <1.0 cm, 1.1–2.0 cm, 2.1–3.0 cm, and >3.0 cm. The low expression rates of IQGAP1 protein were 62.5, 56.1, 55.6, and 80.0%, respectively. There were significant differences between the two groups (P < 0.05). In 65 cases of breast cancer with histological grading data, the low expression rates of SASH1 protein in histological grading I, II, and III were 50.0, 55.3, and 70.0%, respectively. The low expression rates of IQGAP1 protein were 75.0, 51.1, and 70.0%, respectively.

**3.4 The expression of SASH1 and IQGAP1 is positively related with E-cadherin,** 

SASH1 and IQGAP1 have been identified to be involved in tumor metastasis. And immunohistochemistry (IHC) analyses were performed to detect the expression of E-cadherin in breast cancer tissues and the relevance of E-cadherin with SASH1 and IQGAP1, respectively. IHC analyses indicated that E-cadherin was mainly located in the cytoplasma membrane of breast cancer tissues. According to the positive intensity of E-cadherin staining, E-cadherin protein expression in breast cancer tissues was graded to four grades, and meanwhile the positive intensity of SASH1 protein staining was also graded to four grades. Statistical analyses suggested that expression of SASH1 protein was significantly positive related to that of E-cadherin (r = 0.461, P < 0.001 (**Table 2** and **Figure 3**)).

*SASH1, IQGAP1, and E-cadherin proteins showed consistent changes in the breast cancer tissues. The cell nucleus was dyed purple and SASH1, IQGAP1, and E-cadherin were dyed pale brown, and the magnification is 200×. According to staining intensity of tumor cells and the numbers of positive cells, the immunohistochemical results of SASH1, IQGAP1, and E-cadherin proteins were divided into four grades: negative (−), weakly positive (+), moderately positive (++), and strongly positive (+++). The expression of SASH1 was positively correlated with that of E-cadherin and the expression of IQGAP1 show positive correlation with that of E-cadherin. The cell nucleus was dyed purple, and SASH1, IQGAP1, and, E-cadherin* 

*were dyed pale brown, and the magnification is 200×.*

There were significant differences between groups (P < 0.01).

**respectively, in breast cancer tissues**

**62**

**Figure 3.**

Clinical research indicates that occurrence of breast cancer is associated with many factors including genetic factors, environment, and lifestyle. SASH1, a tumor suppressor gene, is downregulated in most of neoplasms. Decrease or deletion of SASH1 expression is closely related to tumor metastasis [4, 5, 18]. It has been reported that the expression of SASH1 protein in osteosarcoma tissues with lung metastasis is significantly lower than that in osteosarcoma tissues without lung metastasis [19]. Upregulated SASH1 can significantly suppress the migration of cervical carcinoma Hela cells, and, in contrast, knockdown of SASH1 significantly results in reduced adhesion ability of human colon cancer SW480 cells and mouse rectal cancer CMT-93 cells and enhanced migration ability of these tumor cells [3]. Downregulation of SASH1 protein expression in thyroid tumor cells may play an important role in thyroid tumor metastasis [20]. SASH1 mRNA is downregulated in primary liver cancer and thyroid cancer [5]. Compared with corresponding normal tissues, SASH1 protein is downregulated in 37 cases among 50 cases of breast cancer tissues and SASH1 expression loss is associated with breast cancer metastasis [4]. All of these studies suggest that expression loss of SASH1 medicates tumor metastasis. In this study, our IHC analyses identified that in 80 cases of breast cancer tissues, low expression of SASH1 protein in 45 cases (45/80 56.3%) was found, which indicated that SASH1 was downregulated in breast cancer.

IQGAP1 proteins are members of the evolutionarily conserved scaffolding protein family and are more widely expressed than other members of the family [21, 22]. IQGAP1 interacts with specific proteins such as actin, calmodulin, Rho GTPase family members, E-cadherin, and β-catenin. The interactions of IQGAP1 with those specific proteins medicate multiple cell activities such as cell scaffold, intercellular adhesion, metastasis, invasion, transcription, and cell signal transduction. For example, the binding of IQGAP1 to β-catenin to form E-cadherin/β-catenin complex inhibits intercellular adhesion of epithelial cells and promotes β-catenin-mediated transcriptional activation [9]. IQGAP1 protein ,which mediates E-cadherin-mediated-intercellular adhesion, is the key molecule in cell polarization and directed migration [23]. IQGAP1 expression is showed to be of prognostic significance in advanced colorectal carcinoma, and a shorter overall survival of colorectal carcinoma patients can be predicted by diffuse

expression pattern of IQGAP1 [11]. In this study, IHC analyses indicated, in 80 cases of breast cancer tissues, IQGAP1 protein level was significantly low in 47 cases accounting for 58.8%, which suggested that IQGAP1 was downregulated in breast cancer.

Multiple endocrine neoplasia type 1 (MEN1) is a dominantly inherited tumor syndrome that results from the mutation of the MEN1 gene that encodes protein menin. MEN1 is revealed to bind to IQGAP1 and increases E-cadherin/β-catenin interaction with IQGAP1 and a novel menin-IQGAP1 pathway that controls cell migration and cell-cell adhesion found in endocrine cells [24]. Activated Rac1 and Cdc42 can bind to IQGAP1, and the bindings of IQGAP1 and Rac1 as well as Cdc42 promote cell mobility and polarization [25, 26]. IQGAP1 is both a downstream effector and an upstream activator of Cdc42, where active Cdc42 antagonizes IQGAP1 dissociation of the cell-cell contacts [27, 28]. Cdc42 inhibits IQGAP1's role in polarized secretion in β-cells or perhaps migration [29]. In this study, IP-WB analyses indicated the protein-protein interactions between SASH1 and IQGAP1. It has been reported that SASH1 expression suppresses cell proliferation and interacts with cytoskeletal proteins, which promotes cell matrix adhesion [3, 4]. Meanwhile, other studies have identified that SASH1 is associated with scaffold proteins and foster tumor migration [3]. Hence, we speculate that the bindings of SASH1 and IQGAP1 co-mediate breast cancer metastasis.

Recurrence or metastasis of breast cancer is the leading cause of breast cancer-related death. It has been identified that epithelial-mesenchymal transition (EMT) plays a pivotal role in tumor metastasis through generation and survival of induced circulating tumor cells [30]. One of the EMT functions is to downregulate and relocate the epithelial cell adhesion protein including the leading actor, E-cadherin [31]. The decreased expression of E-cadherin in breast cancer was associated with high pathological grade, tumor volume enlargement, lymph node metastasis, and distant metastasis and with disease rehabilitation and overall survival time, which indicates that reduced expression or function loss of E-cadherin promotes breast cancer invasion and migration [32]. A dynamic equilibrium of E-cadherin between the E-cadherin-β-catenin-α-catenin complex and the E-cadherin-β-catenin-IQGAP1 complex at sites of cell-cell contact is proposed. The ratio between these two complexes could determine the strength of adhesion [33]. Our previous study found that SASH1 mutations enhanced mutated SASH1 expression, however induced downregulation of E-cadherin in epithelial cells of skin [17]. So it is speculated that there is a connection between SASH1 and E-cadherin. In this study, SASH1 protein level is positively correlated with E-cadherin, and IQGAP1 protein level is also positively correlated with E-cadherin, which also identifies the connection between SASH1 and E-cadherin.

Taken above, we speculate that SASH1 may mediate breast cancer metastasis through a novel SASH1-IQGAP1-E-cadherin signal cascade. When SASH1 and IQGAP1 protein levels in breast cancer tissues and breast cancer cells were low, the protein levels of E-cadherin are also reduced, which causes the reduced cell adhesion ability, the tumor cell ability which is easy to fall off, the enhanced invasion, and the tumor cell metastasis ability to distance. The novel findings about SASH1 will become a novel target to treat breast cancer, which will be conducive to the precision and diversification of breast cancer treatment, effectively improving the prognosis of patients. In this study, we find that low protein levels of SASH1 and IQGAP1 are related to tumor size, and the reduced protein levels of SASH1 and IQGAP1 are associated with tumor grading, which provides a new reference for the rapid diagnosis of tumor grading and tumor size. And our findings on SASH1

**65**

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis*

and IQGAP1 provide a new and more intuitive basis for determining the operation plan and resection range, judging the curative effect of operation and early detection of tumor metastasis and recurrence. However, in this study we find that low expression levels of SASH1 and IQGAP1 are significantly not related to lymph node metastasis, which presumably can be related to a small sample size of breast cancer tissues. The relationship between SASH1 and IQGAP1 with lymph node metastasis

We thank Central Laboratory at Yongchuan Hospital, Chongqing Medical University and Clinical Research Center, and the Affiliated Hospital, Guizhou Medical University for housing experiments. We thank Zhou Xin and Jiang Xiaoli of the Breast Disease Diagnosis and Treatment Center of Chongqing Cancer Hospital for their active help and cooperation in sample collection. This work was supported partly by the High Level Talent Introduction Project of Affiliated Hospital of Guizhou Medical University (grant numbers: 2018I-1 and I-2017-19) and Guizhou Provincial Science and Technology Department Project (grant number: Qian Ke He LH [2017] 7193). This work was supported by the Yongchuan Hospital Project, Chongqing Medical University (YJYJ201347), Chongqing Education Commission Project (KJ1400201), and Guizhou's Introduction Project

Notes: The chapter text was mainly referred to our article entitled as "SASH1- IQGAP1-E-cadherin signal cascade may regulate breast cancer metastasis" (Tumor. 2017;37(6):633–641) which we published in the Chinese Journal Tumor in June 2017. In this chapter, we rewrite the chapter text according to the suggestions of

The figures and tables of this chapter were taken or reedited from the figures and tables of our published article entitled "SASH1-IQGAP1-E-cadherin signal

We thanks the Chinese Journal Tumor allow us reuse the Figures and tables in our article entitled as"SASH1-IQGAP1-E-cadherin signal cascade may regulate breast cancer metastasis" (Tumor, 2017, 37(6): 633~641) which were published in the Chinese Journal Tumor. We are allowed to reuse the Figures, Tables and Text of our article entitled as "SASH1-IQGAP1-E-cadherin signal cascade may regulate breast cancer" under the terms of the Creative Commons Attribution License (CC BY) without having to obtain permission provided that the original source of

*DOI: http://dx.doi.org/10.5772/intechopen.84567*

needs to be further investigated.

**Acknowledgements**

of Million Talents.

reviewers.

**Declarations**

publication.

**Conflict of interest**

No conflict between the authors.

**Notes/thanks/other declarations**

cascade may regulate breast cancer."

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis DOI: http://dx.doi.org/10.5772/intechopen.84567*

and IQGAP1 provide a new and more intuitive basis for determining the operation plan and resection range, judging the curative effect of operation and early detection of tumor metastasis and recurrence. However, in this study we find that low expression levels of SASH1 and IQGAP1 are significantly not related to lymph node metastasis, which presumably can be related to a small sample size of breast cancer tissues. The relationship between SASH1 and IQGAP1 with lymph node metastasis needs to be further investigated.

#### **Acknowledgements**

*Breast Cancer Biology*

breast cancer.

IQGAP1 co-mediate breast cancer metastasis.

expression pattern of IQGAP1 [11]. In this study, IHC analyses indicated, in 80 cases of breast cancer tissues, IQGAP1 protein level was significantly low in 47 cases accounting for 58.8%, which suggested that IQGAP1 was downregulated in

Multiple endocrine neoplasia type 1 (MEN1) is a dominantly inherited tumor syndrome that results from the mutation of the MEN1 gene that encodes protein menin. MEN1 is revealed to bind to IQGAP1 and increases E-cadherin/β-catenin interaction with IQGAP1 and a novel menin-IQGAP1 pathway that controls cell migration and cell-cell adhesion found in endocrine cells [24]. Activated Rac1 and Cdc42 can bind to IQGAP1, and the bindings of IQGAP1 and Rac1 as well as Cdc42 promote cell mobility and polarization [25, 26]. IQGAP1 is both a downstream effector and an upstream activator of Cdc42, where active Cdc42 antagonizes IQGAP1 dissociation of the cell-cell contacts [27, 28]. Cdc42 inhibits IQGAP1's role in polarized secretion in β-cells or perhaps migration [29]. In this study, IP-WB analyses indicated the protein-protein interactions between SASH1 and IQGAP1. It has been reported that SASH1 expression suppresses cell proliferation and interacts with cytoskeletal proteins, which promotes cell matrix adhesion [3, 4]. Meanwhile, other studies have identified that SASH1 is associated with scaffold proteins and foster tumor migration [3]. Hence, we speculate that the bindings of SASH1 and

Recurrence or metastasis of breast cancer is the leading cause of breast cancer-related death. It has been identified that epithelial-mesenchymal transition (EMT) plays a pivotal role in tumor metastasis through generation and survival of induced circulating tumor cells [30]. One of the EMT functions is to downregulate and relocate the epithelial cell adhesion protein including the leading actor, E-cadherin [31]. The decreased expression of E-cadherin in breast cancer was associated with high pathological grade, tumor volume enlargement, lymph node metastasis, and distant metastasis and with disease rehabilitation and overall survival time, which indicates that reduced expression or function loss of E-cadherin promotes breast cancer invasion and migration [32]. A dynamic equilibrium of E-cadherin between the E-cadherin-β-catenin-α-catenin complex and the E-cadherin-β-catenin-IQGAP1 complex at sites of cell-cell contact is proposed. The ratio between these two complexes could determine the strength of adhesion [33]. Our previous study found that SASH1 mutations enhanced mutated SASH1 expression, however induced downregulation of E-cadherin in epithelial cells of skin [17]. So it is speculated that there is a connection between SASH1 and E-cadherin. In this study, SASH1 protein level is positively correlated with E-cadherin, and IQGAP1 protein level is also positively correlated with E-cadherin, which also identifies the connection between SASH1

Taken above, we speculate that SASH1 may mediate breast cancer metastasis through a novel SASH1-IQGAP1-E-cadherin signal cascade. When SASH1 and IQGAP1 protein levels in breast cancer tissues and breast cancer cells were low, the protein levels of E-cadherin are also reduced, which causes the reduced cell adhesion ability, the tumor cell ability which is easy to fall off, the enhanced invasion, and the tumor cell metastasis ability to distance. The novel findings about SASH1 will become a novel target to treat breast cancer, which will be conducive to the precision and diversification of breast cancer treatment, effectively improving the prognosis of patients. In this study, we find that low protein levels of SASH1 and IQGAP1 are related to tumor size, and the reduced protein levels of SASH1 and IQGAP1 are associated with tumor grading, which provides a new reference for the rapid diagnosis of tumor grading and tumor size. And our findings on SASH1

**64**

and E-cadherin.

We thank Central Laboratory at Yongchuan Hospital, Chongqing Medical University and Clinical Research Center, and the Affiliated Hospital, Guizhou Medical University for housing experiments. We thank Zhou Xin and Jiang Xiaoli of the Breast Disease Diagnosis and Treatment Center of Chongqing Cancer Hospital for their active help and cooperation in sample collection. This work was supported partly by the High Level Talent Introduction Project of Affiliated Hospital of Guizhou Medical University (grant numbers: 2018I-1 and I-2017-19) and Guizhou Provincial Science and Technology Department Project (grant number: Qian Ke He LH [2017] 7193). This work was supported by the Yongchuan Hospital Project, Chongqing Medical University (YJYJ201347), Chongqing Education Commission Project (KJ1400201), and Guizhou's Introduction Project of Million Talents.

#### **Conflict of interest**

No conflict between the authors.

#### **Notes/thanks/other declarations**

Notes: The chapter text was mainly referred to our article entitled as "SASH1- IQGAP1-E-cadherin signal cascade may regulate breast cancer metastasis" (Tumor. 2017;37(6):633–641) which we published in the Chinese Journal Tumor in June 2017. In this chapter, we rewrite the chapter text according to the suggestions of reviewers.

The figures and tables of this chapter were taken or reedited from the figures and tables of our published article entitled "SASH1-IQGAP1-E-cadherin signal cascade may regulate breast cancer."

#### **Declarations**

We thanks the Chinese Journal Tumor allow us reuse the Figures and tables in our article entitled as"SASH1-IQGAP1-E-cadherin signal cascade may regulate breast cancer metastasis" (Tumor, 2017, 37(6): 633~641) which were published in the Chinese Journal Tumor. We are allowed to reuse the Figures, Tables and Text of our article entitled as "SASH1-IQGAP1-E-cadherin signal cascade may regulate breast cancer" under the terms of the Creative Commons Attribution License (CC BY) without having to obtain permission provided that the original source of publication.

#### **Acronyms and abbreviations**


#### **Author details**

Ding'an Zhou\*, Xing Zeng, Yadong Li, Zhixiong Wu and Xin Wan Clinical Research Center, The Affiliated Hospital, Guizhou Medical University, Guiyang, Guizhou, The People's Republic of China

\*Address all correspondence to: 460318918@qq.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**67**

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis*

tumorigenesis of human breast

Chemistry. 2008;**283**:1692-1704

[12] Beavon IR. The E-cadherincatenin complex in tumour metastasis: Structure, function and regulation. European Journal of Cancer.

[13] Kuroda S et al. Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherinmediated cell-cell adhesion. Science.

[14] Fukata M et al. Involvement of IQGAP1, an effector of Rac1 and Cdc42 GTPases, in cell-cell dissociation during cell scattering. Molecular and Cellular

[15] Shimao Y, Nabeshima K, Inoue T, Koono M. Complex formation of IQGAP1 with ecadherin/catenin during cohort migration of carcinoma cells. Its possible association with localized release from cell-cell adhesion. Virchows

Biology. 2001;**21**(6):2165-2183

Archiv. 2002;**441**:124-132

2017;**21**(10):2465-2480

[16] Zhou D et al. p53 regulates ERK1/2/CREB cascade via a novel SASH1/MAP2K2 crosstalk to induce hyperpigmentation. Journal of Cellular and Molecular Medicine.

2000;**36**:1607-1620

1998;**281**(5378):832-835

[11] Hiroyuki Hayashi KN, Aoki M, Hamasaki M, Enatsu S, Yamauchi Y, Yamashita Y, et al. Overexpression of IQGAP1 in advanced colorectal cancer correlates with poor prognosis—Critical role in tumor invasion. International Journal of Cancer. 2010;**126**:2563-2574

epithelial cells. The Journal of Biological Chemistry. 2008;**283**(2):1008-1017

[10] Owen D, Campbell L, Littlefield K, Evetts KA, Li Z, Sacks DB, et al. The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: Interfaces differ between the complexes. The Journal of Biological

*DOI: http://dx.doi.org/10.5772/intechopen.84567*

CXCR4 and breast cancer prognosis: A systematic review and meta-analysis.

[1] Zhang Z et al. Expression of

[2] Sheyu L, Hui L, Junyu Z, et al. Promoter methylation assay of SASH 1 gene in breast cancer. Journal of BUON.

[3] Martini M, Gnann A, Scheikl D, Holzmann B, Janssen KP. The candidate tumor suppressor SASH1 interacts with the actin cytoskeleton and stimulates cell-matrix adhesion. The International Journal of Biochemistry & Cell Biology.

[4] Zeller C, Hinzmann B, Seitz S, Prokoph H, Burkhard-Goettges E, Fischer J, et al. SASH1: A

candidate tumor suppressor gene on chromosome 6q24.3 is downregulated

[5] Rimkus C, Martini M, Friederichs J, Rosenberg R, Doll D, Siewert JR, et al. Prognostic significance of downregulated expression of the candidate tumour suppressor gene SASH1 in colon cancer. British Journal of Cancer. 2006;**95**(10):1419-1423

[6] Lin S et al. Effects of SASH1 on melanoma cell proliferation and apoptosis in vitro. Molecular Medicine

[7] He P et al. Overexpression of SASH1 inhibits the proliferation, invasion, and EMT in hepatocarcinoma cells. Oncology Research. 2016;**24**(1):25-32

[8] Brown MD, Sacks DB. IQGAP1 in cellular signaling: Bridging the GAP. Trends in Cell Biology.

[9] Jadeski L et al. IQGAP1 stimulates

2006;**16**(5):242-249

proliferation and enhances

Reports. 2012;**6**(6):1243-1248

in breast cancer. Oncogene. 2003;**22**(19):2972-2983

BMC Cancer. 2014;**14**(1):49

2013;**18**(4):891-898

2011;**43**(11):1630-1640

**References**

*A Novel SASH1-IQGAP1-E-Cadherin Signal Cascade Mediates Breast Cancer Metastasis DOI: http://dx.doi.org/10.5772/intechopen.84567*

#### **References**

*Breast Cancer Biology*

**Acronyms and abbreviations**

DMEM Dulbecco's Modified Eagle's medium EMT epithelial-mesenchymal transition

IQGAP1 IQ motif-containing GTPase activating protein 1

HE staining hematoxylin and eosin staining

IP-WB immunoprecipitation-western blot

SASH1 SAM and SH3 domain-containing 1

SEMs standard error of the means

IHC immunohistochemical

PEI polyethylenimine pAb polyclonal antibody

Gαs guanine nucleotide-binding protein subunit-alpha isoforms short

**66**

**Author details**

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Clinical Research Center, The Affiliated Hospital, Guizhou Medical University,

Ding'an Zhou\*, Xing Zeng, Yadong Li, Zhixiong Wu and Xin Wan

Guiyang, Guizhou, The People's Republic of China

\*Address all correspondence to: 460318918@qq.com

[1] Zhang Z et al. Expression of CXCR4 and breast cancer prognosis: A systematic review and meta-analysis. BMC Cancer. 2014;**14**(1):49

[2] Sheyu L, Hui L, Junyu Z, et al. Promoter methylation assay of SASH 1 gene in breast cancer. Journal of BUON. 2013;**18**(4):891-898

[3] Martini M, Gnann A, Scheikl D, Holzmann B, Janssen KP. The candidate tumor suppressor SASH1 interacts with the actin cytoskeleton and stimulates cell-matrix adhesion. The International Journal of Biochemistry & Cell Biology. 2011;**43**(11):1630-1640

[4] Zeller C, Hinzmann B, Seitz S, Prokoph H, Burkhard-Goettges E, Fischer J, et al. SASH1: A candidate tumor suppressor gene on chromosome 6q24.3 is downregulated in breast cancer. Oncogene. 2003;**22**(19):2972-2983

[5] Rimkus C, Martini M, Friederichs J, Rosenberg R, Doll D, Siewert JR, et al. Prognostic significance of downregulated expression of the candidate tumour suppressor gene SASH1 in colon cancer. British Journal of Cancer. 2006;**95**(10):1419-1423

[6] Lin S et al. Effects of SASH1 on melanoma cell proliferation and apoptosis in vitro. Molecular Medicine Reports. 2012;**6**(6):1243-1248

[7] He P et al. Overexpression of SASH1 inhibits the proliferation, invasion, and EMT in hepatocarcinoma cells. Oncology Research. 2016;**24**(1):25-32

[8] Brown MD, Sacks DB. IQGAP1 in cellular signaling: Bridging the GAP. Trends in Cell Biology. 2006;**16**(5):242-249

[9] Jadeski L et al. IQGAP1 stimulates proliferation and enhances

tumorigenesis of human breast epithelial cells. The Journal of Biological Chemistry. 2008;**283**(2):1008-1017

[10] Owen D, Campbell L, Littlefield K, Evetts KA, Li Z, Sacks DB, et al. The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: Interfaces differ between the complexes. The Journal of Biological Chemistry. 2008;**283**:1692-1704

[11] Hiroyuki Hayashi KN, Aoki M, Hamasaki M, Enatsu S, Yamauchi Y, Yamashita Y, et al. Overexpression of IQGAP1 in advanced colorectal cancer correlates with poor prognosis—Critical role in tumor invasion. International Journal of Cancer. 2010;**126**:2563-2574

[12] Beavon IR. The E-cadherincatenin complex in tumour metastasis: Structure, function and regulation. European Journal of Cancer. 2000;**36**:1607-1620

[13] Kuroda S et al. Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherinmediated cell-cell adhesion. Science. 1998;**281**(5378):832-835

[14] Fukata M et al. Involvement of IQGAP1, an effector of Rac1 and Cdc42 GTPases, in cell-cell dissociation during cell scattering. Molecular and Cellular Biology. 2001;**21**(6):2165-2183

[15] Shimao Y, Nabeshima K, Inoue T, Koono M. Complex formation of IQGAP1 with ecadherin/catenin during cohort migration of carcinoma cells. Its possible association with localized release from cell-cell adhesion. Virchows Archiv. 2002;**441**:124-132

[16] Zhou D et al. p53 regulates ERK1/2/CREB cascade via a novel SASH1/MAP2K2 crosstalk to induce hyperpigmentation. Journal of Cellular and Molecular Medicine. 2017;**21**(10):2465-2480

[17] Zhou D et al. SASH1 regulates melanocyte transepithelial migration through a novel Galphas-SASH1- IQGAP1-E-Cadherin dependent pathway. Cellular Signalling. 2013;**25**(6):1526-1538

[18] Tsatmali M, Ancans J, Yukitake J, Thody AJ. Skin POMC peptides: Their actions at the human MC-1 receptor and roles in the tanning response. Pigment Cell Research, 2000;**13**(Suppl 8):125-129

[19] Meng Q et al. SASH1 regulates proliferation, apoptosis, and invasion of osteosarcoma cell. Molecular and Cellular Biochemistry. 2013;**373**(1-2):201-210

[20] Sun D, Zhou R, Liu H, et al. SASH1 inhibits proliferation and invasion of thyroid cancer cells through PI3K/Akt signaling pathway. International Journal of Clinical and Experimental Pathology. 2015;**8**(10):12276-12283

[21] Briggs MW, Sacks DB. IQGAP1 proteins are integral components of cytoskeletal regulation. EMBO Reports. 2003;**4**(6):571-574

[22] Sanchez-Laorden B, Viros A, Marais R. Mind the IQGAP. Cancer Cell. 2013;**23**(6):715-717

[23] Noritake J et al. IQGAP1: A key regulator of adhesion and migration. Journal of Cell Science. 2005;**118** (Pt 10):2085-2092

[24] Jizhou Yan YY, Zhang H, King C, Kan H-M, Cai Y, Yuan CX, et al. Menin interacts with IQGAP1 to enhance intercellular adhesion of β cells. Oncogene. 2009;**28**(7):973-982

[25] Mataraza JM et al. IQGAP1 promotes cell motility and invasion. The Journal of Biological Chemistry. 2003;**278**(42):41237-41245

[26] Watanabe T et al. Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. Developmental Cell. 2004;**7**(6):871-883

[27] Fukata M et al. Cdc42 and Rac1 regulate the interaction of IQGAP1 with beta-catenin. The Journal of Biological Chemistry. 1999;**274**(37):26044-26050

[28] Lambert M, Choquet D, Mege RM. Dynamics of ligand-induced, Rac1 dependent anchoring of cadherins to the actin cytoskeleton. The Journal of Cell Biology. 2002;**157**(3):469-479

[29] Rittmeyer EN et al. A dual role for IQGAP1 in regulating exocytosis. Journal of Cell Science. 2008;**121** (Pt 3):391-403

[30] Tsai JH, Yang J. Epithelialmesenchymal plasticity in carcinoma metastasis. Genes & Development. 2013;**27**(20):2192-2206

[31] Thiery JP. Epithelialmesenchymal transitions in tumour progression. Nature Reviews. Cancer. 2002;**2**(6):442-454

[32] Memni H et al. E-cadherin genetic variants predict survival outcome in breast cancer patients. Journal of Translational Medicine. 2016;**14**(1):320

[33] Takeuchi H et al. c-MET expression level in primary colon cancer: a predictor of tumor invasion and lymph node metastases. Clinical Cancer Research. 2003;**9**(4):1480-1488

**69**

**Chapter 4**

**Abstract**

treatment target

**1. Introduction**

**1.1 Metastatic breast cancer etiology**

Evidence of BKCa

Channelopathy-Driven Breast

*KCNMA1* encodes the a-subunit of the large conductance, voltage and Ca2+ activated and Voltage-dependent potassium channel (BKCa) and was shown by others and us to be a potential drug target gene in several cancers, including breast cancer. In addition, we studied the role of alternative pre-mRNA splicing events of *KCNMA1* in migration, invasion, proliferation and dispersal of breast cancer cells. It is conceivable that by targeting gene variants we can attenuate processes such as distant metastasis and angiogenesis. Here we reviewed literature on the alternative splicing events specific to breast cancer metastasis to brain, its microenvironment, the biological activity of most alternatively spliced isoforms. We conclude that based on our and others' work *KCNMA1* and other such gene variants contribute to breast cancer dispersion, invasion, growth, and progression in the tumor microenvironment. Thus *KCNMA1*/BKCa channels and their variants are opportunistic

Breast cancer is the most common type of cancer affecting women. Despite great advances in primary breast cancer treatment a significant number of women develop metastases in different organs of the body, especially brain [1], possibly as a result of the emergence of targeted and aggressive systemic cancer therapy. The actual incidence of brain metastases is not precisely known; however, studies suggest that 6–16% of patients with metastatic breast cancer develop brain metastases during their lifetime. Furthermore, autopsy studies have reported brain metastases in 18–30% of patients dying of breast cancer [2]. The majority of women who develop brain metastases have undergone aggressive treatment for stage IV disease [3–5]. Although brain metastasis is the leading cause of breast cancer death, its pathogenesis is poorly understood and the predictors of breast metastasis to brain are yet to be characterized. Albeit recent studies found genes that mediate breast cancer metastasis to brain [6, 7]. Targeting metastatic breast cancer cells in brain is

Cancer Metastasis to Brain

diagnostic, prognostic and treatment targets in breast cancer.

**Keywords:** *KCNMA1* pre-mRNA splicing, BKCa channelopathy, breast cancer-dispersion, invasion, growth, angiogenesis, progression,

*Divya Khaitan and Nagendra Ningaraj*

#### **Chapter 4**

*Breast Cancer Biology*

2013;**25**(6):1526-1538

2013;**373**(1-2):201-210

2015;**8**(10):12276-12283

2003;**4**(6):571-574

2013;**23**(6):715-717

(Pt 10):2085-2092

[17] Zhou D et al. SASH1 regulates melanocyte transepithelial migration through a novel Galphas-SASH1- IQGAP1-E-Cadherin dependent pathway. Cellular Signalling.

actin filaments during cell polarization and migration. Developmental Cell.

[27] Fukata M et al. Cdc42 and Rac1 regulate the interaction of IQGAP1 with beta-catenin. The Journal of Biological Chemistry. 1999;**274**(37):26044-26050

[28] Lambert M, Choquet D, Mege RM. Dynamics of ligand-induced, Rac1 dependent anchoring of cadherins to the actin cytoskeleton. The Journal of Cell

Biology. 2002;**157**(3):469-479

[30] Tsai JH, Yang J. Epithelialmesenchymal plasticity in carcinoma metastasis. Genes & Development.

mesenchymal transitions in tumour progression. Nature Reviews. Cancer.

[32] Memni H et al. E-cadherin genetic variants predict survival outcome in breast cancer patients. Journal of Translational Medicine. 2016;**14**(1):320

[33] Takeuchi H et al. c-MET expression

predictor of tumor invasion and lymph node metastases. Clinical Cancer Research. 2003;**9**(4):1480-1488

level in primary colon cancer: a

2013;**27**(20):2192-2206

[31] Thiery JP. Epithelial-

2002;**2**(6):442-454

(Pt 3):391-403

[29] Rittmeyer EN et al. A dual role for IQGAP1 in regulating exocytosis. Journal of Cell Science. 2008;**121**

2004;**7**(6):871-883

[18] Tsatmali M, Ancans J, Yukitake J, Thody AJ. Skin POMC peptides: Their actions at the human MC-1 receptor and roles in the tanning response. Pigment Cell Research, 2000;**13**(Suppl 8):125-129

[19] Meng Q et al. SASH1 regulates proliferation, apoptosis, and invasion of osteosarcoma cell. Molecular and Cellular Biochemistry.

[20] Sun D, Zhou R, Liu H, et al. SASH1 inhibits proliferation and invasion of thyroid cancer cells through PI3K/Akt signaling pathway. International Journal of Clinical and Experimental Pathology.

[21] Briggs MW, Sacks DB. IQGAP1 proteins are integral components of cytoskeletal regulation. EMBO Reports.

[22] Sanchez-Laorden B, Viros A, Marais R. Mind the IQGAP. Cancer Cell.

[23] Noritake J et al. IQGAP1: A key regulator of adhesion and migration. Journal of Cell Science. 2005;**118**

[24] Jizhou Yan YY, Zhang H, King C, Kan H-M, Cai Y, Yuan CX, et al. Menin interacts with IQGAP1 to enhance intercellular adhesion of β cells. Oncogene. 2009;**28**(7):973-982

[25] Mataraza JM et al. IQGAP1 promotes cell motility and invasion. The Journal of Biological Chemistry.

[26] Watanabe T et al. Interaction with IQGAP1 links APC to Rac1, Cdc42, and

2003;**278**(42):41237-41245

**68**

## Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain

*Divya Khaitan and Nagendra Ningaraj*

#### **Abstract**

*KCNMA1* encodes the a-subunit of the large conductance, voltage and Ca2+ activated and Voltage-dependent potassium channel (BKCa) and was shown by others and us to be a potential drug target gene in several cancers, including breast cancer. In addition, we studied the role of alternative pre-mRNA splicing events of *KCNMA1* in migration, invasion, proliferation and dispersal of breast cancer cells. It is conceivable that by targeting gene variants we can attenuate processes such as distant metastasis and angiogenesis. Here we reviewed literature on the alternative splicing events specific to breast cancer metastasis to brain, its microenvironment, the biological activity of most alternatively spliced isoforms. We conclude that based on our and others' work *KCNMA1* and other such gene variants contribute to breast cancer dispersion, invasion, growth, and progression in the tumor microenvironment. Thus *KCNMA1*/BKCa channels and their variants are opportunistic diagnostic, prognostic and treatment targets in breast cancer.

**Keywords:** *KCNMA1* pre-mRNA splicing, BKCa channelopathy, breast cancer-dispersion, invasion, growth, angiogenesis, progression, treatment target

#### **1. Introduction**

#### **1.1 Metastatic breast cancer etiology**

Breast cancer is the most common type of cancer affecting women. Despite great advances in primary breast cancer treatment a significant number of women develop metastases in different organs of the body, especially brain [1], possibly as a result of the emergence of targeted and aggressive systemic cancer therapy. The actual incidence of brain metastases is not precisely known; however, studies suggest that 6–16% of patients with metastatic breast cancer develop brain metastases during their lifetime. Furthermore, autopsy studies have reported brain metastases in 18–30% of patients dying of breast cancer [2]. The majority of women who develop brain metastases have undergone aggressive treatment for stage IV disease [3–5]. Although brain metastasis is the leading cause of breast cancer death, its pathogenesis is poorly understood and the predictors of breast metastasis to brain are yet to be characterized. Albeit recent studies found genes that mediate breast cancer metastasis to brain [6, 7]. Targeting metastatic breast cancer cells in brain is

extremely difficult as brain provides a "safe haven" for cancer cells. Gene expression profiling has been used to predict metastatic gene-expression signature that is present in a subset of primary breast tumors [8]. However, a reliable profile has not yet been identified that specifically predicts brain metastases. Therefore, it is extremely important to study the genetic changes in breast cancer cells that metastasize to brain and develop specific targeted therapeutic molecular agents.

#### **2. Channelopathy promote breast cancer metastasis**

Cancer research is not only focusing on understanding the possible role of transmembrane-BKCa channels in cancer development and progression but also on development of BKCa channel modulator drugs to attenuate cancer growth. Several researchers, including us have shown that brain tumor cells express BKCa and ATPsensitive potassium (KATP) channels that are highly responsive to minute changes in intracellular Ca2+ and ATP levels. This allows the brain tumor cells to develop pseudopodia for migration through constricted spaces in the brain parenchyma, as depicted in **Figure 1**. Several articles have described the efficacy of BKCa channelinhibiting drugs or molecules in reducing tumors in preclinical mouse tumor models. A recent study has shown the role of intracellular BKCa channels (mitoBKCa) in cancer cell biology [9, 10].

#### **2.1 Ion channels in breast cancer metastasis**

Even now the metastatic breast cancers are incurable. Extensive research has shown that breast cancer metastasis to other organs, including brain is a complicated process. It is widely believed that breast cancer cells escape the primary site

#### **Figure 1.**

*Anticipated role of BKCa and KATP channels in breast cancer cells that seek brain and colonize in brain parenchyma. The potassium ion channels expressed in breast cancer cells are extremely sensitive to minute surge of extracellular and intracellular Ca2+ and cause K+ efflux through BKCa channels. Similarly, slight imbalance in ADP-ATP levels in the cell causes K<sup>+</sup> efflux through KATP channels. Then the ion imbalance triggers the Ca2+ entry, which promotes cancer cell migration though pseudopodia.*

**71**

**Figure 2.**

*base pair deletion in exon 22 (E22), respectively.*

[Ca2+

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain*

and migrate by lymphatic route to lymph nodes and vascular route to colonize in other organs including brain [11, 12]. Gene-expression profiling studies of breast cancer cells indicate that specific molecular pathways are associated with dissemination of primary tumor cells through a vascular route and not by lymphatic dissemination [12]. There is much interest in studying how and when the cancer cells initiate the metastatic cascade so that a therapeutic intervention can be developed to stop or delay the metastasis. Some cancer researchers [13] believe that targeted treatment of breast cancer with ER/PR modulators (Aromatase inhibitors) and targeted biologics such as Herceptin (Her-2 neu inhibitor) [14] and bevacizumab [15] (anti-vascular). Others argue that the metastasis of cancer cells is triggered by a dysregulated cellular Ca2+ homeostasis and altered Ca2+ signaling caused by imbalanced fluxes through ion channels and transporters [10, 16]. The BKCa channels are more sensitive to Ca2+ ions in cancer cells. In this regard, we studied whether the increased sensitivity of potentially new BKCa channel variant protein encoded by splice variants (**Figure 2**) *KCNMA1ΔE2* and *KCNMA1vE22* to intra and extra cellular Ca2+ in breast cancer [17]. In fact, a recent evidence indicates that KCa-Ca2+ channel complexes were found in cancer cells and contribute to cancerassociated functions such as cell proliferation, cell migration and the capacity to develop metastases [10]. The BKCa channels are unique since its activity is triggered by depolarization and enhanced by an increase in μM range of intracellular calcium

i]. In this regard, we recently showed that BKCa channel variant encoded by

progression to high grade glioblastoma multiforme (GBM) [18]. We also discovered a new splice variant *KCNMA1vE22* in breast cancer cells that contributes to breast cancer metastasis to brain (to be published). Epigenetics play an important role in cancer initiation, growth and progression. Understanding the precise mechanism helps us in developing diagnosis, prognosis and treatment strategies for affected cancer patients. For example, overexpression of Ezh2 plays a role in many cancers,

*BKCa channel is a 7-transmembrane tetramer of four monomeric pore-forming alpha-subunits encoded by KCNMA1. The cytoplasmic C-terminal domain has RCK1 and RCK2 (with calcium bowl) segments. We identified KCNMA1ΔE2 and KCNMA1E22 in human brain-specific metastatic breast cancer cells. Using relevant siRNA designs, we showed that these splice variants are formed by the deletion of exon 2 (E2) and 108* 

i] and causes glioma

a new splice variant *KCNMA1vE22* is highly sensitive to [Ca2+

*DOI: http://dx.doi.org/10.5772/intechopen.84957*

#### *Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain DOI: http://dx.doi.org/10.5772/intechopen.84957*

*Breast Cancer Biology*

in cancer cell biology [9, 10].

**2.1 Ion channels in breast cancer metastasis**

extremely difficult as brain provides a "safe haven" for cancer cells. Gene expression profiling has been used to predict metastatic gene-expression signature that is present in a subset of primary breast tumors [8]. However, a reliable profile has not yet been identified that specifically predicts brain metastases. Therefore, it is extremely important to study the genetic changes in breast cancer cells that metastasize to

Cancer research is not only focusing on understanding the possible role of transmembrane-BKCa channels in cancer development and progression but also on development of BKCa channel modulator drugs to attenuate cancer growth. Several researchers, including us have shown that brain tumor cells express BKCa and ATPsensitive potassium (KATP) channels that are highly responsive to minute changes in intracellular Ca2+ and ATP levels. This allows the brain tumor cells to develop pseudopodia for migration through constricted spaces in the brain parenchyma, as depicted in **Figure 1**. Several articles have described the efficacy of BKCa channelinhibiting drugs or molecules in reducing tumors in preclinical mouse tumor

models. A recent study has shown the role of intracellular BKCa channels (mitoBKCa)

Even now the metastatic breast cancers are incurable. Extensive research has shown that breast cancer metastasis to other organs, including brain is a complicated process. It is widely believed that breast cancer cells escape the primary site

*Anticipated role of BKCa and KATP channels in breast cancer cells that seek brain and colonize in brain parenchyma. The potassium ion channels expressed in breast cancer cells are extremely sensitive to minute surge* 

 *efflux through BKCa channels. Similarly, slight imbalance* 

 *efflux through KATP channels. Then the ion imbalance triggers the Ca2+*

brain and develop specific targeted therapeutic molecular agents.

**2. Channelopathy promote breast cancer metastasis**

**70**

**Figure 1.**

*of extracellular and intracellular Ca2+ and cause K+*

*entry, which promotes cancer cell migration though pseudopodia.*

*in ADP-ATP levels in the cell causes K<sup>+</sup>*

and migrate by lymphatic route to lymph nodes and vascular route to colonize in other organs including brain [11, 12]. Gene-expression profiling studies of breast cancer cells indicate that specific molecular pathways are associated with dissemination of primary tumor cells through a vascular route and not by lymphatic dissemination [12]. There is much interest in studying how and when the cancer cells initiate the metastatic cascade so that a therapeutic intervention can be developed to stop or delay the metastasis. Some cancer researchers [13] believe that targeted treatment of breast cancer with ER/PR modulators (Aromatase inhibitors) and targeted biologics such as Herceptin (Her-2 neu inhibitor) [14] and bevacizumab [15] (anti-vascular). Others argue that the metastasis of cancer cells is triggered by a dysregulated cellular Ca2+ homeostasis and altered Ca2+ signaling caused by imbalanced fluxes through ion channels and transporters [10, 16]. The BKCa channels are more sensitive to Ca2+ ions in cancer cells. In this regard, we studied whether the increased sensitivity of potentially new BKCa channel variant protein encoded by splice variants (**Figure 2**) *KCNMA1ΔE2* and *KCNMA1vE22* to intra and extra cellular Ca2+ in breast cancer [17]. In fact, a recent evidence indicates that KCa-Ca2+ channel complexes were found in cancer cells and contribute to cancerassociated functions such as cell proliferation, cell migration and the capacity to develop metastases [10]. The BKCa channels are unique since its activity is triggered by depolarization and enhanced by an increase in μM range of intracellular calcium [Ca2+ i]. In this regard, we recently showed that BKCa channel variant encoded by a new splice variant *KCNMA1vE22* is highly sensitive to [Ca2+ i] and causes glioma progression to high grade glioblastoma multiforme (GBM) [18]. We also discovered a new splice variant *KCNMA1vE22* in breast cancer cells that contributes to breast cancer metastasis to brain (to be published). Epigenetics play an important role in cancer initiation, growth and progression. Understanding the precise mechanism helps us in developing diagnosis, prognosis and treatment strategies for affected cancer patients. For example, overexpression of Ezh2 plays a role in many cancers,

#### **Figure 2.**

*BKCa channel is a 7-transmembrane tetramer of four monomeric pore-forming alpha-subunits encoded by KCNMA1. The cytoplasmic C-terminal domain has RCK1 and RCK2 (with calcium bowl) segments. We identified KCNMA1ΔE2 and KCNMA1E22 in human brain-specific metastatic breast cancer cells. Using relevant siRNA designs, we showed that these splice variants are formed by the deletion of exon 2 (E2) and 108 base pair deletion in exon 22 (E22), respectively.*

including breast cancer and brain tumors. H3K27M serves as an oncohistone and, if mutated it contributes to tumor development as Ezh2 is no longer able to methylate the histone and gene expression is aberrantly upregulated.

Furthermore, a recent computational analysis of human genomic sequence identified mutations that cause pathogenic splicing abnormalities in breast cancer susceptibility genes, BRCA1, BRCA2 and other genes [19]. Several investigations have reported that voltage gated ion channels are expressed in several cancers and contribute significantly to cell signaling, cell cycle progression and cell volume regulation, cancer cell proliferation, as well as metastasis. Hence, there is a great deal of interest in possible therapeutic potential of voltage gated ion channels as pharmacological targets [20, 21].

#### **2.2 Metastatic breast cancer in brain microenvironment**

Cancer cells have the innate ability to "exploit" the "chaotic" environmental challenges surrounding them and grow uninterrupted by manipulating transportome that regulate proliferation, apoptosis, metabolism, growth factor signaling, migration and invasion. Ion channels and transporters are some of the key modulators of cancer progression in hostile tumor microenvironment that includes hypoxia. It has been suggested that modulation of ion channels by the hypoxic environment may contribute to the aggressive phenotype observed in GBM cells residing in a hypoxic environment [22]. In hostile microenvironment such as hypoxia, BKCa channels are modulated to aid cancer cell invasion and neovascularization. Affymetrix Array analyses of brain tumor cell lines where KCNMA1 was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation, angiogenesis, cell cycle, and invasion [18].

#### **2.3** *KCNMA1***/BKCa channel splice variants in breast cancer**

During the past decade, a number of genes associated with breast cancer have been cloned and identified. Gene expression levels alone cannot fully explain gene function as alternative splicing produce multiple mRNAs and protein isoforms. New molecular insights indicate that the metastatic capacity of breast tumors is an inherent feature, and not necessarily a late, acquired phenotype [23, 24]. Breast cancer cells show alternative mRNA splicing and have prognostic and therapeutic value [21]. Although there are many reports of alternative splicing events specific to breast cancer [25, 26], the biological activity of majority of alternatively spliced isoforms, and specifically their contribution to metastatic breast cancer biology, remains to be investigated. As many researchers are focusing on "Understanding and Preventing Brain Tumor Dispersal", we recently reported on a novel *KCNMA1* mRNA splice variant with a deletion of 108 base pairs (KCNMA1v) mostly overexpressed in high-grade gliomas [18]. In order to understand the role of alternative pre-mRNA splicing events of *KCNMA1/* BKCa channels, we employed specific inhibitors. We showed that the modulation of *KCNMA1/*BKCa channels in brain specific metastatic breast cancer cells (MDA-MB361) resulted in attenuation of migration, invasion [11, 17]. Further, we identified a hitherto unknown *KCNMA1* variant *KCNMA1vE22* (to be published) with a deletion of 108 base pairs of nucleotides and deletion of the entire exon-2 (*KCNMA1ΔE2*) expressed only in metastatic breast tumor cells seeking brain (**Figure 2**). However, biological function of *KCNMA1ΔE2* under different tumor microenvironment is yet to be elucidated. The *KCNMA1* splicing effects and the potential role of *KCNMA1ΔE2* as a critical posttranscriptional regulator of BKCa channel isoform resulting in diversified channel functions merit further investigation.

**73**

in **Table 1**.

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain*

lines with three different phenotypes (to be published).

**3. BKCa channels and neovascularization**

Identifying the most optimal and novel biomarker(s) for breast cancer metastasis to brain is ideal [27, 28] yet challenging because of the multi-factorial nature of the disease. The roles of roles of different ion channels in the development of cancer have been reported [29]. The identification of a potential new biomarker has relied heavily on an increase or decrease in gene expression, but these changes may not always result in altered protein expression. Growing evidence indicates that alternative or aberrant pre-mRNA splicing resulting in protein isoforms with diverse functions occurs during the development, progression, and metastasis of breast cancer [29]. Earlier, we have reported that the BKCa channels play a role in human breast tumor progression, cell proliferation, invasion, and micro-metastases [11, 17]. Nevertheless, the precise role of *KCNMA1* and its splice variants in modification of BKCa channel functions in promoting breast cancer metastasis to brain is still unclear. Therefore, to understand the role of BKCa channels in breast cancer metastasis to brain the we showed that the relative messenger RNA levels in MDA-MB-361 cells derived from human metastatic breast tumor in brain were higher than metastatic breast cancer cells (MDA-MB-231) that prefer other organs and primary breast cancer (MCF-7) cells. In addition, using GeneChip Exon array (Affymetrix) we probed the presence of alternative splicing of *KCNMA1* in MDA-MB-361, MDA-MB-231 and MCF-7. The array data showed that *KCNMA1* splicing pattern is different among cell

**Invasion Trans-**

Untransfected 1 ± 0.09 1 ± 0.10 1 ± 0.17 1 ± 0.11 1 ± 0.21 Vector-transfected 0.98 ± 0.04 1.01 ± 0.12 0.95 ± 0.13 1.2 ± 0.15 1 ± 0.27

*Data shown are in SEM of n = 3 in triplicates. Biological function assays [11] and functional activity of BKCa channels were measured by membrane potential assay using FlexStation and in vivo mouse brain tumor models as* 

*Effect of KCNMA1vE22 expression on biological functions of MDA-MB-231BR cell line.*

**endothelial migration**

1.4 ± 0.12 1.9 ± 0.15 1.5 ± 0.19 1.6 ± 0.21 3.8 ± 0.42

**Functional activity**

**Tumor volume at the 5th week**

The PCR results validated the findings of Exon array study. Two distinct splice

variants expressed in breast cell line (MDA-MB-361) metastatic to brain were identified (i) deletion of exon 2 (*KCNMA1ΔE2*) between S0-S1 protein subunit **(Figure 2**) corresponding to the cytoplasmic potential domain of BKCa channel α-subunit and (ii) deletion of 108 bp in exon 22 (*KCNMA1vE22*) between the S9 and S10 protein subunit (C-terminus). However, the biological function of these alternative splice variants in breast cancer remains to be investigated. To our knowledge we believe that our laboratory is the first to report the presence of these variants in metastatic breast cancers. We established that *KCNMA1vE22* plays a key role in several biological functions of MDA-MB-231BR cell line as represented

Altered ion channels could play a pivotal role in physiological angiogenesis in including cancer [30, 31]. BKCa channel inhibitor modulated the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway [32].

*DOI: http://dx.doi.org/10.5772/intechopen.84957*

**(at 72 h)**

**Cell line Proliferation** 

*KCNMA1vE22* transfected

*described by us earlier.*

**Table 1.**

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain DOI: http://dx.doi.org/10.5772/intechopen.84957*


*Data shown are in SEM of n = 3 in triplicates. Biological function assays [11] and functional activity of BKCa channels were measured by membrane potential assay using FlexStation and in vivo mouse brain tumor models as described by us earlier.*

#### **Table 1.**

*Breast Cancer Biology*

pharmacological targets [20, 21].

angiogenesis, cell cycle, and invasion [18].

including breast cancer and brain tumors. H3K27M serves as an oncohistone and, if mutated it contributes to tumor development as Ezh2 is no longer able to methylate

Furthermore, a recent computational analysis of human genomic sequence identified mutations that cause pathogenic splicing abnormalities in breast cancer susceptibility genes, BRCA1, BRCA2 and other genes [19]. Several investigations have reported that voltage gated ion channels are expressed in several cancers and contribute significantly to cell signaling, cell cycle progression and cell volume regulation, cancer cell proliferation, as well as metastasis. Hence, there is a great deal of interest in possible therapeutic potential of voltage gated ion channels as

Cancer cells have the innate ability to "exploit" the "chaotic" environmental challenges surrounding them and grow uninterrupted by manipulating transportome that regulate proliferation, apoptosis, metabolism, growth factor signaling, migration and invasion. Ion channels and transporters are some of the key modulators of cancer progression in hostile tumor microenvironment that includes hypoxia. It has been suggested that modulation of ion channels by the hypoxic environment may contribute to the aggressive phenotype observed in GBM cells residing in a hypoxic environment [22]. In hostile microenvironment such as hypoxia, BKCa channels are modulated to aid cancer cell invasion and neovascularization. Affymetrix Array analyses of brain tumor cell lines where KCNMA1 was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation,

During the past decade, a number of genes associated with breast cancer have been cloned and identified. Gene expression levels alone cannot fully explain gene function as alternative splicing produce multiple mRNAs and protein isoforms. New molecular insights indicate that the metastatic capacity of breast tumors is an inherent feature, and not necessarily a late, acquired phenotype [23, 24]. Breast cancer cells show alternative mRNA splicing and have prognostic and therapeutic value [21]. Although there are many reports of alternative splicing events specific to breast cancer [25, 26], the biological activity of majority of alternatively spliced isoforms, and specifically their contribution to metastatic breast cancer biology, remains to be investigated. As many researchers are focusing on "Understanding and Preventing Brain Tumor Dispersal", we recently reported on a novel *KCNMA1* mRNA splice variant with a deletion of 108 base pairs (KCNMA1v) mostly overexpressed in high-grade gliomas [18]. In order to understand the role of alternative pre-mRNA splicing events of *KCNMA1/* BKCa channels, we employed specific inhibitors. We showed that the modulation of *KCNMA1/*BKCa channels in brain specific metastatic breast cancer cells (MDA-MB361) resulted in attenuation of migration, invasion [11, 17]. Further, we identified a hitherto unknown *KCNMA1* variant *KCNMA1vE22* (to be published) with a deletion of 108 base pairs of nucleotides and deletion of the entire exon-2 (*KCNMA1ΔE2*) expressed only in metastatic breast tumor cells seeking brain (**Figure 2**). However, biological function of *KCNMA1ΔE2* under different tumor microenvironment is yet to be elucidated. The *KCNMA1* splicing effects and the potential role of *KCNMA1ΔE2* as a critical posttranscriptional regulator of BKCa channel isoform resulting in diversified channel functions

the histone and gene expression is aberrantly upregulated.

**2.2 Metastatic breast cancer in brain microenvironment**

**2.3** *KCNMA1***/BKCa channel splice variants in breast cancer**

**72**

merit further investigation.

*Effect of KCNMA1vE22 expression on biological functions of MDA-MB-231BR cell line.*

Identifying the most optimal and novel biomarker(s) for breast cancer metastasis to brain is ideal [27, 28] yet challenging because of the multi-factorial nature of the disease. The roles of roles of different ion channels in the development of cancer have been reported [29]. The identification of a potential new biomarker has relied heavily on an increase or decrease in gene expression, but these changes may not always result in altered protein expression. Growing evidence indicates that alternative or aberrant pre-mRNA splicing resulting in protein isoforms with diverse functions occurs during the development, progression, and metastasis of breast cancer [29]. Earlier, we have reported that the BKCa channels play a role in human breast tumor progression, cell proliferation, invasion, and micro-metastases [11, 17]. Nevertheless, the precise role of *KCNMA1* and its splice variants in modification of BKCa channel functions in promoting breast cancer metastasis to brain is still unclear. Therefore, to understand the role of BKCa channels in breast cancer metastasis to brain the we showed that the relative messenger RNA levels in MDA-MB-361 cells derived from human metastatic breast tumor in brain were higher than metastatic breast cancer cells (MDA-MB-231) that prefer other organs and primary breast cancer (MCF-7) cells. In addition, using GeneChip Exon array (Affymetrix) we probed the presence of alternative splicing of *KCNMA1* in MDA-MB-361, MDA-MB-231 and MCF-7. The array data showed that *KCNMA1* splicing pattern is different among cell lines with three different phenotypes (to be published).

The PCR results validated the findings of Exon array study. Two distinct splice variants expressed in breast cell line (MDA-MB-361) metastatic to brain were identified (i) deletion of exon 2 (*KCNMA1ΔE2*) between S0-S1 protein subunit **(Figure 2**) corresponding to the cytoplasmic potential domain of BKCa channel α-subunit and (ii) deletion of 108 bp in exon 22 (*KCNMA1vE22*) between the S9 and S10 protein subunit (C-terminus). However, the biological function of these alternative splice variants in breast cancer remains to be investigated. To our knowledge we believe that our laboratory is the first to report the presence of these variants in metastatic breast cancers. We established that *KCNMA1vE22* plays a key role in several biological functions of MDA-MB-231BR cell line as represented in **Table 1**.

#### **3. BKCa channels and neovascularization**

Altered ion channels could play a pivotal role in physiological angiogenesis in including cancer [30, 31]. BKCa channel inhibitor modulated the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway [32].

Our work shows an association between the BKCa channel isoform expression and VEGF secretion by breast tumor cells, which might be exacerbated under hypoxia that has implications for vascular permeability and anticancer drug delivery (to be published). Understanding the underlying mechanism and splicing patterns of *KCNMA1* and expression of the splice variant *KCNMA1ΔE2* under normoxia and hypoxia alone and in coculture with brain endothelial cells will shed light on the role of *KCNMA1* alternative splicing in metastatic breast tumor biology. Perhaps the discovery and validation of brain specific metastasis-associated *KCNMA1* alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. In fact, splice variations in a number of genes have already been shown to correlate with malignancy and their occurrence could precede clinical cancer diagnosis [33]. To date, however brain-specific alternate *KCNMA1* splice variants in breast cancer have not been reported. The variant *KCNMA1ΔE2* that we have discovered potentially may fill the gap to serve as a biomarker of breast cancer metastasis to brain. Undoubtedly, the research on the putative association between *KCNMA1* splice variants and breast cancer metastases to brain will prove to be an extremely productive exercise for the identification of a new generation of biomarkers. *KCNAM1*/BKCa channels are hypothesized to be involved in VEGF secretion and neovascularization in brain tumors. We tested this hypothesis by activation and suppression of *KCNMA1* in brain tumor cells and constructed a potential VEGF signaling pathway adapted from KEGG VEGF signaling pathway (**Figure 3**).

We rationalize that *KCNMA1ΔE2* is expressed specifically in metastatic breast tumors in brain, and this requires validation to confirm its role as a potential transformation biomarker of breast cancer metastasis to brain. In metastatic breast tumor cells seeking brain there is an upregulation and constitutive activation of *KCNMA1*, which correlates with increased malignancy [11]. In this context,

#### **Figure 3.**

*Adapted from KEGG-VEGF signaling pathway: we activated and suppressed KCNMA1 in brain tumor cells and constructed a probable VEGF signaling pathway affected by modified KCNAMA1 expression. The genes in rectangular boxes—red represents genes overexpressed by KCNMA1 overexpression and black represents genes downregulated by KCNMA1 inhibition in U-87 (glioma cells)*.

**75**

stimulating*.*

**Figure 4.**

*(brain Mets) cell lines.*

**4. Discussion**

**4.1 Splicing in health and disease**

aspects of tumor biology.

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain*

we showed (**Figure 4**) that the *KCNMA1* is overexpressed in breast cancer cells metastatic to brain (MDA-MB-361) and exhibit differences in expression levels in other non-metastatic (MCF-7) and metastatic to other organs (MDA-MB-231). MDA-MB-231 BR was established from the triple negative MDA-MB-231 cells, which are highly metastatic but have no organ specificity. The MDA-MB 231-BR cell line was derived from MDA-MB-231 cells following sequential rounds of implantation, resection from the brain, and re-injection into mice. Eventually a subline with selectivity for the brain was isolated [34], and exhibit higher KCNMA1 level than parental MDA-MB-231 cells, however the expression was far lower than the

*BKCa channels in breast cancer biology-expression of KCNMA1 by qPCR (A) and alternate splice variants (B) using Affymetrix Genechip Exon Array in MCF-7 (non Mets), MDA-MB-231 (Mets) and MDA-MB-361* 

In addition, alternate splicing of *KCNMA1* [17] including *KCNMA1ΔE2* may provide a mechanism to generate a physiologically diverse complement of functionally and structurally diverse BKCa channel isoform that might affect cell proliferation, cell cycle, migration and micrometastases in brain. Future studies will validate the role of *KCNMA1ΔE2* in brain-specific metastatic process. Inhibiting *KCNMA1ΔE2 in in vitro* and *in vivo* models with shRNA or the variant BKCa channel using specific inhibitor like Iberiotoxin to attenuate breast tumor metastasis to brain using human metastatic breast tumor xenograft mouse models will be very

Many human diseases are implicated to errors in mRNA splicing. These aberrant splicing also provides an opportunity to develop targeted treatment to correct the faulty gene in some genetic disorders, or target aberrant protein encoded by these gene variants in human cancers. Breast cancer-specific biomarkers might generate specific epitopes that offer targets for developing diagnostic, prognostic and immunotherapy [35]. Articles on alternative pre-mRNA splicing regulation in cancer [36] and misregulation of mRNA splicing in cancer [29] highlights the important roles in promoting aberrant splicing, which in turn contributes to all

naturally-selected MDA-MB-361 cells (**Figure 4**).

*DOI: http://dx.doi.org/10.5772/intechopen.84957*

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain DOI: http://dx.doi.org/10.5772/intechopen.84957*

**Figure 4.**

*Breast Cancer Biology*

ing pathway (**Figure 3**).

Our work shows an association between the BKCa channel isoform expression and VEGF secretion by breast tumor cells, which might be exacerbated under hypoxia that has implications for vascular permeability and anticancer drug delivery (to be published). Understanding the underlying mechanism and splicing patterns of *KCNMA1* and expression of the splice variant *KCNMA1ΔE2* under normoxia and hypoxia alone and in coculture with brain endothelial cells will shed light on the role of *KCNMA1* alternative splicing in metastatic breast tumor biology. Perhaps the discovery and validation of brain specific metastasis-associated *KCNMA1* alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. In fact, splice variations in a number of genes have already been shown to correlate with malignancy and their occurrence could precede clinical cancer diagnosis [33]. To date, however brain-specific alternate *KCNMA1* splice variants in breast cancer have not been reported. The variant *KCNMA1ΔE2* that we have discovered potentially may fill the gap to serve as a biomarker of breast cancer metastasis to brain. Undoubtedly, the research on the putative association between *KCNMA1* splice variants and breast cancer metastases to brain will prove to be an extremely productive exercise for the identification of a new generation of biomarkers. *KCNAM1*/BKCa channels are hypothesized to be involved in VEGF secretion and neovascularization in brain tumors. We tested this hypothesis by activation and suppression of *KCNMA1* in brain tumor cells and constructed a potential VEGF signaling pathway adapted from KEGG VEGF signal-

We rationalize that *KCNMA1ΔE2* is expressed specifically in metastatic breast

*Adapted from KEGG-VEGF signaling pathway: we activated and suppressed KCNMA1 in brain tumor cells and constructed a probable VEGF signaling pathway affected by modified KCNAMA1 expression. The genes in rectangular boxes—red represents genes overexpressed by KCNMA1 overexpression and black represents genes* 

*downregulated by KCNMA1 inhibition in U-87 (glioma cells)*.

tumors in brain, and this requires validation to confirm its role as a potential transformation biomarker of breast cancer metastasis to brain. In metastatic breast tumor cells seeking brain there is an upregulation and constitutive activation of *KCNMA1*, which correlates with increased malignancy [11]. In this context,

**74**

**Figure 3.**

*BKCa channels in breast cancer biology-expression of KCNMA1 by qPCR (A) and alternate splice variants (B) using Affymetrix Genechip Exon Array in MCF-7 (non Mets), MDA-MB-231 (Mets) and MDA-MB-361 (brain Mets) cell lines.*

we showed (**Figure 4**) that the *KCNMA1* is overexpressed in breast cancer cells metastatic to brain (MDA-MB-361) and exhibit differences in expression levels in other non-metastatic (MCF-7) and metastatic to other organs (MDA-MB-231). MDA-MB-231 BR was established from the triple negative MDA-MB-231 cells, which are highly metastatic but have no organ specificity. The MDA-MB 231-BR cell line was derived from MDA-MB-231 cells following sequential rounds of implantation, resection from the brain, and re-injection into mice. Eventually a subline with selectivity for the brain was isolated [34], and exhibit higher KCNMA1 level than parental MDA-MB-231 cells, however the expression was far lower than the naturally-selected MDA-MB-361 cells (**Figure 4**).

In addition, alternate splicing of *KCNMA1* [17] including *KCNMA1ΔE2* may provide a mechanism to generate a physiologically diverse complement of functionally and structurally diverse BKCa channel isoform that might affect cell proliferation, cell cycle, migration and micrometastases in brain. Future studies will validate the role of *KCNMA1ΔE2* in brain-specific metastatic process. Inhibiting *KCNMA1ΔE2 in in vitro* and *in vivo* models with shRNA or the variant BKCa channel using specific inhibitor like Iberiotoxin to attenuate breast tumor metastasis to brain using human metastatic breast tumor xenograft mouse models will be very stimulating*.*

#### **4. Discussion**

#### **4.1 Splicing in health and disease**

Many human diseases are implicated to errors in mRNA splicing. These aberrant splicing also provides an opportunity to develop targeted treatment to correct the faulty gene in some genetic disorders, or target aberrant protein encoded by these gene variants in human cancers. Breast cancer-specific biomarkers might generate specific epitopes that offer targets for developing diagnostic, prognostic and immunotherapy [35]. Articles on alternative pre-mRNA splicing regulation in cancer [36] and misregulation of mRNA splicing in cancer [29] highlights the important roles in promoting aberrant splicing, which in turn contributes to all aspects of tumor biology.

#### **4.2 BKCa channels as target to attenuate breast cancer metastasis**

*The* BKCa channels are known to function as oncogenes in certain cancers. These channels besides being sensitive to [Ca2+ i] are highly dependent on amounts of outward K<sup>+</sup> currents, which modulate the transmembrane potential of a cell. The BKCa channels are overexpressed in many types of cancers via gene amplification, alternative splicing or increased protein half-life. A recent study showed that by inhibiting BKCa channels with Iberiotoxin in breast cancer cells, tumorigenicity was reduced by downregulation of β-catenin and (phospho)Akt and HER-2/neu protein levels [37]. Evidence presented above clearly show that over expression of wild type BKCa channels or the presence of BKCa channel variant support breast cancer metastasis to brain. Understanding the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast cancers that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective diagnosis, prognosis and treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy. Activation of BKCa channels was shown to be a novel molecular pathway involved in zoledronic acidinduced apoptosis of MDA-MB-231 cells *in vitro* [32]. Du et al. [8] showed that BKCa promotes growth and metastasis of prostate cancer through facilitating the coupling between αvβ3 integrin and FAK. BKCa channels are shown to support cancer cell migration, invasion and tumorigenesis [11, 17, 18]. Hence it is extremely interesting to explore BKCa channels as putative targets for anti-breast cancer therapies.

#### **4.3 Alternate splicing of BKCa channels in diagnosis and prognosis**

Several articles have highlighted the use of alternative splicing as a promising source for new diagnostic, prognostic, predictive, and therapeutic tools [38–40]. The diversity of RNA species detected through RNA-seq holds the potential of extracellular RNAs as non-invasive diagnostic indicators of disease [41–44]. We recently reported that targeting the KCNMA1 variants may be a clinically beneficial strategy to prevent or at least slow down glioma transformation to GBM [18]. In both human and mouse lymphoma models, researchers have shown that MYC directly induced the transcription of genes encoding core splicing machinery components. They also showed that PRMT5 is involved in MYC-driven tumorigenesis in mice with lymphoma and discovered that tumor development was delayed [44]. Now due to high-throughput New Generation Sequencing (NGS) technologies the splicing diagnostic methodologies have improved. Hence NGS can be utilized in clinical diagnostics of splice variants in diagnosis, prognosis and treatment of breast cancers.

#### **4.4 Perspective of** *KCNMA1* **splice variants**

We believe that future therapies for metastatic breast cancer depend on further investigation into the mechanisms and cellular events caused by oncogene splicing such as *KCNMA1*. Such studies should lead to the development of future therapies for this deadly type of cancer. *KCNMA1* splice variants that are identified in breast tumor patients with brain metastasis will pave for accurate diagnosis and prognostication. Furthermore, they provide potential targets for anticancer drug development. Clinical outcome of *KCNMA1vE22* expression in breast metastasis is expected to reveal the variants' clinical importance. Quantifying the levels of *KCNMA1vE22* could be useful to identify biological process that increases the malignancy and affect prognosis of patients with breast cancer metastasis in the brain.

**77**

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain*

Perhaps the discovery and validation of brain specific metastasis-associated *KCNMA1* alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. In fact, splice variations in a number of genes have already been shown to correlate with malignancy and their occurrence could precede clinical cancer diagnosis. To date, however brain-specific alternate *KCNMA1* splice variants in breast cancer have not been reported. The variant *KCNMA1ΔE2* and *KCNMA1E22* that we have recently discovered potentially may fill the gap to serve as a biomarker of breast cancer metastasis to brain. Undoubtedly, the research on the putative association between *KCNMA1* splice variants and breast cancer metastases to brain will prove to be an extremely productive research to identify new generation of biomarkers for early detection and therapeutic intervention in breast cancer patients with high risk for

The authors thank the Scintilla Group, Bangalore, India; Anderson Cancer Institute and Mercer University Medical Center, Savannah, GA, USA; Vanderbilt-Ingram Cancer Center, Nashville, TN, USA; Cedars-Sinai Medical Center, Los Angeles, CA, USA; American Cancer Society, USA; Georgia Cancer Coalition, Atlanta, GA, USA; and NIH for providing opportunity and research grant support. We also thank Dr. Nagendra of MVIT, Bangalore, for assisting us with the STRING software for the analysis and Michigan State University Research Center, Grand Rapids, MI, USA, for generating KKEG pathway using Affymetrix analysis data.

*DOI: http://dx.doi.org/10.5772/intechopen.84957*

**5. Conclusions**

brain metastases.

**Author details**

Divya Khaitan1

and Nagendra Ningaraj1,2\*

\*Address all correspondence to: sainagendra50@gmail.com

Education and Research, Bangalore, India

provided the original work is properly cited.

1 Department of Molecular Oncology, Scintilla Academy for Applied Sciences'

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Molecular Diagnostics Labs, Scintilla Bio-MARC, Bangalore, India

**Acknowledgements**

### **5. Conclusions**

*Breast Cancer Biology*

outward K<sup>+</sup>

channels besides being sensitive to [Ca2+

**4.2 BKCa channels as target to attenuate breast cancer metastasis**

*The* BKCa channels are known to function as oncogenes in certain cancers. These

BKCa channels are overexpressed in many types of cancers via gene amplification, alternative splicing or increased protein half-life. A recent study showed that by inhibiting BKCa channels with Iberiotoxin in breast cancer cells, tumorigenicity was reduced by downregulation of β-catenin and (phospho)Akt and HER-2/neu protein levels [37]. Evidence presented above clearly show that over expression of wild type BKCa channels or the presence of BKCa channel variant support breast cancer metastasis to brain. Understanding the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast cancers that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective diagnosis, prognosis and treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy. Activation of BKCa channels was shown to be a novel molecular pathway involved in zoledronic acidinduced apoptosis of MDA-MB-231 cells *in vitro* [32]. Du et al. [8] showed that BKCa promotes growth and metastasis of prostate cancer through facilitating the coupling between αvβ3 integrin and FAK. BKCa channels are shown to support cancer cell migration, invasion and tumorigenesis [11, 17, 18]. Hence it is extremely interesting

to explore BKCa channels as putative targets for anti-breast cancer therapies.

Several articles have highlighted the use of alternative splicing as a promising source for new diagnostic, prognostic, predictive, and therapeutic tools [38–40]. The diversity of RNA species detected through RNA-seq holds the potential of extracellular RNAs as non-invasive diagnostic indicators of disease [41–44]. We recently reported that targeting the KCNMA1 variants may be a clinically beneficial strategy to prevent or at least slow down glioma transformation to GBM [18]. In both human and mouse lymphoma models, researchers have shown that MYC directly induced the transcription of genes encoding core splicing machinery components. They also showed that PRMT5 is involved in MYC-driven tumorigenesis in mice with lymphoma and discovered that tumor development was delayed [44]. Now due to high-throughput New Generation Sequencing (NGS) technologies the splicing diagnostic methodologies have improved. Hence NGS can be utilized in clinical diagnostics of splice variants in diagnosis, prognosis and treatment of

We believe that future therapies for metastatic breast cancer depend on further investigation into the mechanisms and cellular events caused by oncogene splicing such as *KCNMA1*. Such studies should lead to the development of future therapies for this deadly type of cancer. *KCNMA1* splice variants that are identified in breast tumor patients with brain metastasis will pave for accurate diagnosis and prognostication. Furthermore, they provide potential targets for anticancer drug development. Clinical outcome of *KCNMA1vE22* expression in breast metastasis is expected to reveal the variants' clinical importance. Quantifying the levels of *KCNMA1vE22* could be useful to identify biological process that increases the malignancy and

affect prognosis of patients with breast cancer metastasis in the brain.

**4.3 Alternate splicing of BKCa channels in diagnosis and prognosis**

currents, which modulate the transmembrane potential of a cell. The

i] are highly dependent on amounts of

**76**

breast cancers.

**4.4 Perspective of** *KCNMA1* **splice variants**

Perhaps the discovery and validation of brain specific metastasis-associated *KCNMA1* alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. In fact, splice variations in a number of genes have already been shown to correlate with malignancy and their occurrence could precede clinical cancer diagnosis. To date, however brain-specific alternate *KCNMA1* splice variants in breast cancer have not been reported. The variant *KCNMA1ΔE2* and *KCNMA1E22* that we have recently discovered potentially may fill the gap to serve as a biomarker of breast cancer metastasis to brain. Undoubtedly, the research on the putative association between *KCNMA1* splice variants and breast cancer metastases to brain will prove to be an extremely productive research to identify new generation of biomarkers for early detection and therapeutic intervention in breast cancer patients with high risk for brain metastases.

### **Acknowledgements**

The authors thank the Scintilla Group, Bangalore, India; Anderson Cancer Institute and Mercer University Medical Center, Savannah, GA, USA; Vanderbilt-Ingram Cancer Center, Nashville, TN, USA; Cedars-Sinai Medical Center, Los Angeles, CA, USA; American Cancer Society, USA; Georgia Cancer Coalition, Atlanta, GA, USA; and NIH for providing opportunity and research grant support. We also thank Dr. Nagendra of MVIT, Bangalore, for assisting us with the STRING software for the analysis and Michigan State University Research Center, Grand Rapids, MI, USA, for generating KKEG pathway using Affymetrix analysis data.

### **Author details**

Divya Khaitan1 and Nagendra Ningaraj1,2\*

1 Department of Molecular Oncology, Scintilla Academy for Applied Sciences' Education and Research, Bangalore, India

2 Molecular Diagnostics Labs, Scintilla Bio-MARC, Bangalore, India

\*Address all correspondence to: sainagendra50@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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10.1186/1471-2407-9-258

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**81**

Section 3

Different Treatment

Strategies for Breast

Cancer

Section 3
