Breast cancer stem cells: Biology and therapeutic implications
Introduction
Breast cancer accounts for the second largest cause of cancer-related mortality among women worldwide. Over past few decades, there has been a significant decrease in mortality rates of breast cancer patients due to advancements in the diagnosis and development of novel radiation and targeted chemotherapies (Siegel et al., 2017). Distinct chemotherapies have been devised based on subtype, gene expression profile and mutational status such as hormonal therapies for hormone receptor-positive luminal A and luminal B subtypes (Rouzier et al., 2005), human epidermal growth factor receptor 2 (Her2) inhibitors for Her2-enriched breast cancer (Nixon et al., 2018), and poly (ADP-ribose) polymerase (PARP) inhibitors for targeting BRCA1-mutant tumors and triple-negative breast cancer (TNBC) (Fong et al., 2009). Even though patients show an initial response to the chemotherapies, many women still experience drug resistance and tumor relapse and, the recurrent form of breast cancer remains to be dreadful and incurable. Ostensibly, drug resistance emerges due to a minute population of cancer cells known as cancer stem cells (CSCs) which show stem-like properties.
Breast cancer stem cells (BCSCs) exhibit the expression of specific molecular signatures such as CD44+/CD24−, Aldehyde dehydrogenase 1high (ALDH1high), CD133+, Ganglioside 2+ (GD2+), etc. (Li et al., 2017; Liu et al., 2013a). Several pathways are operative in BCSCs to maintain their stemness such as Notch, Hedgehog, Wnt, etc. (Takebe et al., 2015). Accumulated evidence suggests that hypoxia along with stromal cells such as fibroblasts, macrophages, mesenchymal stem cells (MSCs) and tumor-associated endothelial cells play an imperative role in driving these pathways for enrichment and maintenance of BCSCs (Korkaya et al., 2011). Self-renewal and differentiation are hallmarks of CSCs and it is important for maintaining the heterogeneity of tumor. BCSCs are known to undergo differentiation into endothelial cells to support the formation of new blood vessels, a process termed as vasculogenic mimicry (VM) (Delgado-Bellido et al., 2017). Tumor vascularization is essential for supplying nutrients and O2 to support vigorously growing tumor. In addition, BCSC-derived endothelial cells might be more resistant to treatment as compared to normal endothelial cells. BCSCs are resistance to anoikis, a programmed cell death that occurs in anchorage-dependent cells (Kim et al., 2012). Hence, these cells migrate through blood circulation and form secondary tumors at distant sites by a process known as metastasis (Economopoulou et al., 2012). BCSCs also promote angiogenesis by secreting various proangiogenic and angiogenic factors like stromal cell-derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) (Ping and Bian, 2011). By virtue of their tumorigenic potential and drug resistance phenotype, CSCs have emerged as one of the potential therapeutic targets for breast cancer treatment. In this review, we have discussed the biology of CSCs and their implications in angiogenesis, metastasis and drug resistance. In addition, we have reviewed the different approaches of targeting BCSCs for the diminution of drug resistance and tumor relapse to improve the patients’ survival rates.
There are two separable but closely related hypotheses which explain the tumor heterogeneity and origin of BCSCs (Lindeman and Visvader, 2010; Shackleton et al., 2009). According to clonal evolution or stochastic model, all the cells in the tumor have a similar tumorigenic potential and tumor heterogeneity arises as a result of the generation of intra-tumoral clones through the sequential mutations. This model presumes that CSCs can be generated from differentiated mammary cells by virtue of mutations that occur in course of the disease. Exposure to detrimental environmental factors such as radiation and chemotherapies induce genetic alterations in non-malignant somatic cells that prime the de novo generation of CSCs by the de-differentiation process (Lindeman and Visvader, 2010). Several reports also suggest that microenvironmental cues induce the malignant transformation of differentiated cells into BCSCs. Hierarchical or CSC model postulates that only a small proportion of tumor cells reside in the tumor has a tumor-propagating potential. These cells exhibit self-renewal properties and are capable of reiterating tumor hierarchy (Fig. 1; Kreso and Dick, 2014; Sin and Lim, 2017). The concept of BCSCs arising from the progenitors/stem cells seems to be more plausible (Kreso and Dick, 2014; Bao et al., 2015). Exhibition of similar phenotypic features and expression of stem cell markers by BCSCs to their lineage-specific normal stem cells supports this hypothesis. For example, mammary stem cells show a CD44+/CD24− signature which is also a molecular determinant of BCSCs (Liu et al., 2013a). The BCSC population also shares the specific properties including self-renewal with their lineage-specific normal stem cells or partially differentiated mammary progenitor cells (Kreso and Dick, 2014).
Development of CSC-specific biomarkers has facilitated the identification and validation of same in vitro and in vivo breast cancer models, as well as in patients. The molecular markers which routinely used for identification and validation purposes are CD44, CD24, ALDH1 and CD133 (deBeça et al., 2013). CD44 is a cell surface glycoprotein which is known to play a prominent role in cell signaling, adhesion and migration (Aruffo et al., 1990; Senbanjo and Chellaiah, 2017). Several shreds of evidence suggest that it regulates cancer cell proliferation, angiogenesis, invasion, and metastasis (Senbanjo and Chellaiah, 2017). In recent reports, it has been shown that CD44 interact with hyaluronic acid and/or osteopontin (OPN) to exert various functions like cell survival, invasion, angiogenesis and metastasis (Aruffo et al., 1990; Rangaswami et al., 2006). A recent study has reported that OPN/CD44 signaling axis in the perivascular niche promotes CSC phenotype in glioblastoma (Pietras et al., 2014). Several reports show that tumor or stroma-derived OPN also induces CD44 expression thereby controls CSC phenotype in different types of cancer (Butti et al., 2015).
CD24 is an adhesion glycoprotein expressed on the surface of many cell types and it’s a recently discovered ligand for P-selectin (Schäck et al., 2016). The CD24 expression is found in highly differentiated tumor cells (luminol-type) (Kwon et al., 2015). Higher expression of CD44 and lower expression of CD24 marks the CSC population (Li et al., 2017). Combination of CD44 and CD24 expression has been extensively used as a BCSC marker along with epithelial-specific antigen (ESA). As low as 200 ESA+/CD44+/CD24− cells derived from breast tumor were able to form tumors when they administered orthotopically into immunosuppressed mice. However, 100-fold more cells without these markers isolated from the same tumors were not able to form a tumor in the mouse. In addition, it was observed that the tumors generated from ESA+/CD44+/CD24− cells were able to recapitulate heterogeneity of initial tumors (Al-Hajj et al., 2003). Furthermore, implantation of CD44+CD24- cells in immunocompromised mice shows higher bone metastasis (Patanè et al., 2013). A recent report has shown that CD44+/CD24- tumors are associated with poor clinical outcome in ER-ve patients whereas it is associated with longer survival rates in ER + ve patients (Kim et al., 2011). This might be due to higher chemo-resistant property of CD44+/CD24- tumors in ER-ve patients than their counterparts.
ALDH1 is also a marker of BCSCs. It is a detoxifying enzyme that catalyses the oxidation of intracellular aldehydes to carboxylic acids. The activity of ALDH1 in cells is associated with stem cell phenotype and assessed by ALDEFLOUR assay. Hence, ALDH1 is widely used as a marker of BCSCs and its expression is associated with poor clinical outcome in breast cancer. The ALDH1 expression is also linked to drug resistance in breast cancer (Moreb et al., 2012). However, a recent study has reported that CD44+/CD24− CSC population is anatomically different from ALDH1+ve CSCs. Molecular profiling of these populations had shown that the CD44+/CD24− sub-population exhibits mesenchymal phenotype with quiescent state whereas ALDH1+ve sub-population shows epithelial phenotype with higher proliferative potential (Liu et al., 2013a). CD133 also acts as CSC-specific marker in triple-negative breast cancer (TNBC) and BRCA-1 mutant tumors. Even though specific functions of CD133 have not been established, different splice variants of CD133 are known to interact with cholesterol and thus, have a role in Hedgehog signaling pathway (Liu et al., 2013b). The tumor-initiating cells are also defined by CD49f and CD61 in Her2/neu-induced mammary tumors that developed from the luminal progenitor cells in mice (Lo et al., 2012).
Embryonic development and stem cell maintenance are complex and highly regulated processes. Numerous signaling pathways are essential for embryonic development and maintenance of stem cells in adult tissues (Burdon et al., 2002). Several key signaling pathways mainly Notch, Wnt and Hedgehog and their crosstalks dictate the stem cell-specific properties (Katoh, 2007; Takebe et al., 2011). Dysfunction of these stemness signaling pathways moderates self-renewal characteristics thus leads to the detainment of CSC phenotype. Aberrant activation or mutation in stemness-related genes is frequently reported and associated with aggressiveness and cancer relapse (Takebe et al., 2011).
Section snippets
Notch Signalling in the regulation of breast cancer stem cell phenotype
The Notch is an essential transmembrane signaling receptor, required throughout the embryogenesis and involved in the determination of stem cell fate, cell differentiation, apoptosis and cell cycle progression (Bouras et al., 2008). Notch family consists of Notch1–4 receptors. These are known to bind with five different ligands such as jagged proteins (JAG1 and JAG2) and delta-like ligands (DLL1, DLL3, and DLL4). Both Notch receptors and their ligands are transmembrane proteins expressed in
Wnt signaling in the maintenance of breast cancer stem cells
Wnt signaling is an evolutionarily conserved pathway which plays a vital role in various physiological and pathological functions including embryonic development, tissue homeostasis and cancer (Wang and Wynshaw-Boris, 2004). Wnt is a glycoprotein that serves as a ligand for Frizzled (FZD), a seven transmembrane serpentine receptor and low-density receptor-related protein 5/6 (LRP5/6) (MacDonald and He, 2012). Nineteen Wnt ligands have been identified so far. Wnt possesses a cysteine-rich
Hedgehog signaling in the regulation of Breast cancer stem cell phenotype
Hedgehog (HH) signaling plays an important role in various cellular processes during embryonic development and tissue homeostasis and also a key regulator of cell fate and self-renewal (Briscoe and Thérond, 2013). In mammals, three Hedgehog homologs (ligands) has been reported such as Sonic Hedgehog, Indian Hedgehog and Desert Hedgehog (Scales and de Sauvage, 2009; Takebe et al., 2011). These proteins were synthesized as a precursor protein and further activated by various post-translational
Other signaling and their crosstalk in the regulation of breast cancer stem cells
Other signaling pathways and their cross-talk including PI3K/Akt/mTOR and JAK/STAT are involved in CSC enrichment and maintenance. Aberrant regulation of individual signaling could result in breast cancer, but these signaling pathways hardly ever drive in isolation. The interplay between these signaling pathways shows the potential to maintain CSC phenotype with the company of external stimuli. Dysregulation of RTK (receptor tyrosine kinases) signaling is a key contributor to breast cancer
Role of breast cancer stem cells in promoting metastasis
Metastasis is the dissemination of cancer cells from primary tumor to distant parts of the body. The process of metastasis is tightly regulated by multiple signaling pathways. It is a complex process where cancer cells enter the blood circulation by the process of invasion and move through the circulation to establish a new tumor in distant organs. Increasing evidence suggests that CSCs are responsible for metastasis, because of its inherent anoikis-resistant property. Anoikis is a regulated
Therapeutic targeting breast cancer stem cells for the management of breast cancer
CSCs have been implicated in tumor angiogenesis, metastasis, and drug resistance thus therapeutically targeting CSCs might beneficial for the treatment of breast cancer patients. BCSCs can be targeted by affecting different functional and molecular aspects of the BCSCs such as BCSC markers, signaling pathways responsible for self-renewal, tumor-stroma interaction and CSC-driven drug resistance pathways.
Conclusion and future prospective
Several therapeutic interventions have been devised for the management of breast cancer based on its subtypes, molecular signature and mutational status. Nonetheless, intrinsic or acquired resistance to treatment modalities poses major confronts for anticancer therapy and facilitates the tumor recurrence. Decades of research on failure of chemotherapies have identified minute population of drug resistant cells that reside in solid tumors. This population shows stem-like properties and high
Funding
None.
Competing financial interests
The authors declare no competing financial interests.
Acknowledgments
The Fellowship of R.B. is supported by Council of Scientific and Industrial Research (CSIR), Govt. of India. The Post-Doctoral fellowship of V.P.G is supported from Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India. The Fellowship of T.V.S.K is supported by University Grant Commission, Govt. of India. The Fellowship of P.B is supported by Department of Biotechnology, Govt. of India.
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