ROLE OF CANCER STEM CELLS IN BREAST CANCER Sahil Nagpal 1 and Navkiran Kaur* 1 Student, B.Sc (Hons) Medical Biotechnology and *Assistant Professor,

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1 INTRODUCTION ROLE OF CANCER STEM CELLS IN BREAST CANCER Sahil Nagpal 1 and Navkiran Kaur* 1 Student, B.Sc (Hons) Medical Biotechnology and *Assistant Professor, Amity Institute of Biotechnology, Amity University, Noida navkirank@amity.edu Two major models of tumor cell initiation have been proposed, namely, the stochastic model, in which each cell contains a low but similar probability to acquire the accidental genetic mutations resulting in the capacity of proliferation and survival and the stem cell model, in which tumor initiation is driven by cancer stem cells (CSC). The term stem cell is referred to undifferentiated cells that have two basic properties: the capacity to self-renew and the capacity to generate daughter cells that can differentiate in different cell lineages. Normal adult stem cells have relatively long telomeres compared to more differentiated somatic cells, they are usually quiescent or proliferate more slowly than their differentiated progeny, and they have increased longevity; for this reason, they are exposed to more damaging agents than more differentiated cells over time. Thus, they accumulate mutations that are then transmitted to the rapidly proliferating progeny. The stem cell origin of cancer hypothesis considers that stem cells or other differentiated cells that have acquired self-renewal ability tend to accumulate genetic or epigenetic alterations and evade the strict control of their microenvironment and these cancer stem cells (CSCs) could be responsible for the malignant transformation and the progression of the disease. The CSCs are a population of cells that are more tumourigenic than the bulk tumor population and can be defined mainly through the expression of unique properties, such as specific detoxification enzyme systems, molecular surface markers, and embryonic signaling pathways [1]. The main hallmarks of CSCs are their properties of self-renewal, their ability to generate tumors from very few cells, their slow cell division rate, their ability to give rise to phenotypically diverse progeny, and their selective resistance to radio- and chemotherapy [2]. MAMMARY GLAND AND MAMMARY STEM CELLS (MaSCs) The human mammary gland is a compound tubuloalveolar gland. Within each breast is a mammary gland, which is a modified sudoriferous or sweat gland, that produces milk. A mammary gland consists of lobes separated by a variable amount of adipose tissue. Each lobe is made up of smaller lobules which are composed of milk-secreting glands, termed alveoli, embedded in connective tissue.the mammary epithelium is composed of two lineages of epithelial cells: luminal cells (that differentiates into alveolar and ductal cells) and myoepithelial cells. A small number of ductal basally positioned small undifferentiated cells (also called electron-lucent cells when observed by electron microscopy) represent the normal mammary stem cells. These normal mammary stem cells provide the capacity for extensive cellular expansion associated with pregnancy and also generate differentiated cells that support lactation [3]. The origin of mammary gland based in stem cells has been demonstrated in several experiments that show how one single stem cell can generate the entire mammary gland. The primitive breast stem cells are estrogen receptor negative. These cells generate progenitor cells that finally differentiate into luminal and myoepithelial lineages which are defined by specific sets of markers. CHARACTERIZATION OF MAMMARY STEM CELLS An exact identification of a human mammary epithelial stem cell has yet to be solidified, but many groups have identified putative mammary epithelial progenitor cells. Technical challenges have arisen due to the complex nature of the hormonal requirements for MaSC differentiation and also for a suitable environment to support growth. Work with human breast stem cells builds on the foundations of experiments investigating the murine population. Kuperwasser et al., has reported that in the development of a humanized murine fat pad representing the human breast stroma injected with a mixture of irradiated and nonirradiated human mammary epithelial cells allow for the successful engraftment of the stromal cells and for the creation of a humanized environment [4]. More recently, a new model has been described by Eirew et al., where fibroblast and putative mammary stem cells are engrafted in a collagen plug under the murine kidney capsule. The outgrowths observed recapitulate the hierarchal nature of the normal human mammary gland [5]. Through the use of these assays, CD49f hi EpCAM has been established as the fraction containing the human breast stem cell population. To complement these cell surface markers, a functional marker, aldehyde dehydrogenase 1A1 (ALDH+) (Fig. 1) has been established as a functional marker for mammary stem cells among others [6]. Page 37

2 Fig. 1 The Aldefluor assay The Aldefluor assay is a fluorometric assay that detects the enzymatic activity of aldehyde dehydrogenase 1 (ALDH1) (StemCell Technologies, Vancouver, BC, Canada). Cells are incubated with the intrinsically fluorescent ALDH substrate, BODIPYaminoacetaldehyde (BAAA). BAAA is a neutral molecule and enters the cell through passive diffusion, where it is then converted into BAA by ALDH and is unable to leave the cell due to its negative charge. The active removal of BAA by ATP Binding Cassettes is quenched through the use of the assay buffer and through incubation of cells between 2 and 8 C. The resulting fluorescence of the cells is then assessed by flow cytometry, providing single cell analysis of ALDH activity. As a negative control, the activity of ALDH is quenched by the addition of diethylaminobenzaldehyde (DEAB), and the fluorescence of these cells is assessed by flow cytometry. The population observed the DEAB sample is used to create the gate for the ALDH+ cells, whereby cells are only included if they demonstrate higher levels of fluorescence compared to the DEAB sample. Adapted from StemCell Technologies. BREAST CANCER AND CANCER STEM CELLS (CSCS) Breast cancer may originate from either the glands or the ducts of the breast and is termed lobular or ductal carcinoma respectively. When the cancer extends beyond it s immediate surroundings, it is known as invasive cancer. Cancer that has not crossed beyond the involved lobule or tubule is called in-situ carcinoma. Types of breast cancers include ductal carcinoma in situ, lobular carcinoma in situ, invasive ductal carcinoma and invasive lobular carcinoma. Less common types of breast cancers include inflammatory breast cancer, triplenegative breast cancer, paget disease of the nipple, phyllodes tumor and angiosarcoma. The cancer stem cell hypothesis proposes a different model based on a hierarchical organization [7]. According to this hypothesis, a neoplasia originates from the malignant transformation of an adult stem cell through the deregulation of the normally tightly regulated self-renewal program [8]. This model holds that a breast carcinoma may contain genetically and morphologically diverse populations of cells, including primitive stem cells, luminal or basal progenitor cells (also called sometimes transient amplifying cells), and terminally differentiated cells Perhaps, a neoplasia arises from a tumorigenic CSCs rather than from the much larger population of neoplastic progenitor cells. This means that mutations in one stem cell could be transmitted to descendant cells, which can then launch new clonal successions. Conversely, mutation then strikes the genomes of transit-amplifying cells cannot be transmitted further, because these cells only have a limited replicative ability. Therefore, the CSCs represent a minority of the neoplastic cells in tumor masses, while the progenitor and differentiated neoplastic cells represent the majority of the bulk of the tumour. Most cancer cells have only limited proliferative potential, but CSCs have self-renewal capacity that could drive the tumorigenesis process. Page 38

3 In human breast cancer, different reports try to prove how the stem cells are targets for malignant transformation, by the demonstration of mutational changes in genes or in pathways essential for the self-renewal process. BRCA1 gene (Breast Cancer tumor suppressor gene 1), implicated in inherited breast cancer, plays an important role in stem cell self-renewal and in differentiation of a progenitor cell. Because of the BRCA1 gene function in DNA repair and in maintaining chromosome stability, researchers have proposed that the loss of BRCA1 function may produce genetically unstable stem or progenitor cells that serve as prime target for further carcinogenic events [9]. It was proposed that breast carcinogenesis may be initiated by epigenetic changes such as silencing of p16 INK4a. Since p16 INK4a is known to be a downstream target of the polycomb gene BMI1 which regulates stem cell selfrenewal [10]. STEM CELL NICHES Stem cell niches are defined as locations in a tissue which specifically can support the existence of somatic stem cells. These niches contain stromal cells, fibroblasts, and immune cells and these cells maintain the growth and allow repopulation of stem cells in case of depletion of the stem cell compartment. Recently the mesenchymal stem cells (MSC) have been implicated in the breast CSCs niches. This heterogeneous and multipotent subset of mesenchymal stroma cells have fibroblast-like morphology, form colonies that can differentiate into adypocytes, osteocytes, and chondrocytes; are important in the control of niches through its recruitment from bone-marrow to this niches by the signals of IL-6 and IL-8 and its receptors in the stem cells (CXCL7, CXCR1, respectively). The interaction between MSC and CSC has been demonstrated both in vitro and in in vivo mouse models. The cytokine network involving CSC and the microenvironment stimulates self-renewal of breast CSC and accelerates the growth of human breast cancer, and has focused the attention of investigators to target this novel pathway [11]. It is possible that tumour therapy that disrupts the stem cell niche through ablation of the surrounding differentiated cells could lead to the subsequent death of the cancer stem cells. Alternatively, tumour therapy that depletes stem cells, but does not eradicate the stem cell niche, could lead to repopulation of the stem cell niche with additional cancer stem cells. IDENTIFICATION OF CANCER STEM CELLS Advances in cell culture approaches have been important in identifying and studying mammary stem cells. In order to validate the method selected as an appropriate technique to isolate CSC, it is crucial to use assays that can assess the stem cell properties of its self-renewal and its differentiation. Xenograft The xenograft model is based on the orthotopic injection of human cancer cells into the humanized cleared fat pad of immunodeficient mice. It can initiate and maintain the tumor growth upon serial passages. It is presently the most robust model for demonstrating stem cell properties. In addition to self-renewal, CSC also retain the ability to differentiate, albeit abnormally, also generating non-self-renewing cell population that constitutes the bulk of the tumor [12]. In Vitro assays The study of mammary stem cells in vitro has been based upon work identifying neural stem cells through a cell culture assay known as the neurosphere assay, which makes use of serumfree medium supplemented with epidermal growth factor and basic fibroblast growth factor [13, 14]. Application of the neurosphere assay culture conditions has been used to identify undifferentiated human mammary stem cells grown in culture [15] known as mammospheres. Tumorosphere-initiating cells have stem cell properties including the ability to survive and grow in suspension in serum-free conditions. In contrast, more differentiated tumor cells are anchorage-dependent and undergo anoikis in these conditions [16]. The tumorosphere culture has also been used in different studies to screen for drugs capable of targeting the cancer stem cell populations. Side Population Technique This method is useful to identify stem cell population in breast cancer cell lines. This method is based on the overexpression of transmembrane transporters, such as the adenosine triphosphate (ATP)-binding cassettes molecule ABCG2/BCRP1 in stem cells. These molecules actively exclude vital dyes such as Hoechst or Rhodamine 123, a property not found in differentiated cells that retain the dye [17]. Page 39

4 MARKERS USED TO IDENTIFY CSCS Selectable markers are either found on the cell surface or confer functional properties that are characteristics of normal stem cells that have extended to malignant stem cell populations. The current definition of a breast CSC is CD44 + CD24 - and/or ALDH +. THE ROLE OF CSCS IN METASTASIS Metastatic cascade consists of a series of processes that move tumor cells from the primary tumor to distant location. Various factors are involved in intravasation, extravasation, and survival in bloodstream and in the target organ. The induction of epithelial-to-mesenchymal transition in breast cancer cells results in the acquisition of stem-cell properties, including the ability to form mammospheres, resistance to apoptotic signals, facilitates the blood intravasation, and generation of circulating tumor cells. Different studies show that a significant proportion of circulating tumor cells shows stem cell phenotype, such as expression of NOTCH1, ALDH1, and are typically triple negative (estrogen and progesterone receptor-negative and HER2- negative) [20]. The most common site of breast cancer metastasis is the bone, but metastatic lesions are also found in the lymph nodes, liver, lungs, and brain. Interestingly, both HA and osteopontin, common ligands for CD44, are expressed in the bone and other common sites of breast cancer metastasis [21], suggesting a possible adhesive interaction for circulating tumor cell arrest. Experimentally, CD44 has been shown to mediate the attachment of metastatic breast cancer cells to human bone marrow endothelial cells [22]. Additionally, breast cancer cell lines exhibit different levels of C-X-C chemokine receptor type 4 (CXCR4), which appears to correlate with CSC proportions and the tendency to metastasize [19, 23]. BREAST CSCS AND THERAPY RESISTANCE Recent studies have indicated that breast CSCs and other CSCs are more resistant to radiation and chemotherapy as compared to cancer cells. A study in human leukemia revealed that CSCs are often quiescent, and remain in the G0 phase, conferring resistance to many chemotherapy agents as they often target actively replicating cells [24]. Possible mechanisms for this include the expression of cell surface pumps, including ABCG2/BCRP1, capable of expelling chemotherapeutic drugs [25]. Additionally, the presence and activity of ALDH allows CSCs to metabolize cytotoxics such as cyclophosphamide [19]. Other factors potentially prolonging the lifespan of CSCs include the increased expression of anti-apoptotic molecules such as BCL2 and surviving [26, 27]. Furthermore, the radiotherapy resistance of CSCs may be due to the decreased levels of pro-oxidants in the CD44+CD24 population or through Wnt/bcatenin pathway signaling [28]. New therapeutics aimed at eliminating cancer stem cells could also be achieved through a variety of methods: THERAPEUTIC STRATEGIES TO TARGET CSCS Reactive oxygen species (ROS), critical mediators of ionizing radiation-induced cell killing, are shown to be present at lower levels in some subsets of the CSC population in breast cancer. Lower ROS levels in CSCs are associated with an increased expression of free radical scavenging systems predisposing CSCs to develop less DNA damage and preferential sparing after irradiation. Pharmacological depletion of ROS scavengers in CSCs significantly decreases their clonogenicity and results in radiosensitization [29]. New therapeutics aimed at eliminating cancer stem cells could also be achieved through a variety of methods: targeting the self-renewal signaling pathways critical for cancer stem cells, targeting the ABC drug transporters that cancer stem cells use to evade chemotherapy, or inducing the immune system to eliminate the cancer stem cells through various immunotherapeutic interventions REFERENCES 1. Alison, M.; Islam, S. & Wright, N. (2010). Stem cells in cancer: instigators and propagators? Journal of Cell Science, 123, 14, pp Reya, T.; Morrison, S.J.; Clarke, M.F. & Weissman, I.L. (2001). Stem cells, cancer, and cancer stem cells. Nature, 414, pp Stingl J, Eirew P, Ricketson I, et al. (2006) Purification and unique properties of mammary epithelial stem cells. Nature 439: Kuperwasser C, Chavarria T, Wu M, Magrane G, Gray JW, Carey L, Richardson A, Weinberg RA (2004) Reconstruction of functionally normal and malignant human breast tissues in mice. Proc Natl Acad Sci USA 101 (14): doi: /pnas [pii] 5. Eirew P, Stingl J, Raouf A, Turashvili G, Aparicio S, Emerman JT, Eaves CJ (2008) A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability. Nat Med 14 (12): doi:nm.1791 [pii] /nm Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) Aldh1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell stem cell 1 (5): Page 40

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