Cancer stem cells as a potential therapeutic target in breast cancer

Introduction

Breast cancer (BC) is one of the leading causes of death among females globally (1). Although increased rates of early diagnosis of BC led to a significant reduction in mortality in recent years, many patients have recurrent BC (2,3). Thus, there is an urgent need to improve our understanding of the mechanisms of BC progression.

In recent years, experimental data indicate that BC is composed of heterogeneous cell populations with different biological properties (4-6). The tumorigenic process is preserved by a minor subset of cells in the tumor called cancer stem cells (CSCs) (7,8). CSCs are defined by their ability by their ability for self-renewal and multipotency (9-11). These features displayed by CSCs are important for a better understanding of the BC initiation and progression. Accumulating evidence suggests the presence of CSCs in BC, which may contribute to the failure of existing therapies to consistently eradicate malignant tumors (12,13). Therefore, therapeutic targeting of CSCs may provide a novel strategy that is more effective than the current drugs targeting the bulk mature cancer cells in treatment of BC.

This review will discuss the evidence for the existence of CSCs, novel molecular biomarkers and self-renewal pathways related to CSCs, as well as the possibility of targeting CSCs as a potential therapeutic strategy for BC.

The CSCs hypothesis

To fully appreciate the theory of CSCs, it is essential to understand the basic concepts of the biology of normal stem cells. Stem cells from normal tissues are characterized by the following essential properties: self-renew, giving rise to daughter cells that have limited proliferative potential and intended to differentiate, and the number of stem cells in normal tissues must be under strict genetic control in order to prevent uncontrolled expansion (14-16). Understanding of the basic biology of stem cells is crucial for the development of CSCs hypothesis. The emerging and controversial CSCs theory proposes that there is a small fraction of cancer cells which constitute a reservoir of self-sustaining cells with the exclusive ability to self-renew and maintain the tumor (10,17,18). These cells with the properties resembling stem cells are called CSCs (9,18,19). Currently, the widely accepted definition of a CSC is a cell within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor (10).

The CSCs hypothesis has gained wide attention in recent years after the identification of subpopulations of tumor-initiating cells in hematological malignancies and solid cancers such as breast, colon, pancreas, lung, prostate and brain cancers (20-26). Serial xenotransplantation of a putative CSCs-enriched population in immunodeficient mice is the primary assay used in demonstrating CSCs (10). Tumorigenic capacity of stem cells can also be characterized based on the expression of defined cell surface makers and intracellular enzymes such as aldehyde dehydrogenase (ALDH) (10,27).

In BC, CSCs are identified by the presence of cell surface marker protein CD44, with low levels of CD24 (6,28,29). Breast stem cells can be easily identified by their ability to grow in serum-free suspension cultures which called mammospheres, an in vitro alternative test for self-renewal (30,31). Besides, they could also be identified by the ability of retaining bromodeoxyuridine or H3-thymidine (32,33). In BC, the CSCs hypothesis could have profound implications in the prevention, detection and treatment of the disease (34). In addition, the heterogeneity of BC is attributed by some researchers as a function of CSCs (35). It is also suggested that the CSCs hypothesis could be incorporated into the molecular staging of BC (36,37). Growing evidence suggests that CSCs may be responsible for therapy resistance and relapse of BC. For instance, a higher proportion of CD44⁺ CD24^–/low cells of BC are associated with shorter relapse-free and overall survival with increased distant metastases (38-41).

Biomakers of CSCs in BC

Distinct and specific surface biomarker phenotypes can be used to distinguish CSCs from other tumor cells and normal stem cells. Currently, the most common method used to identify CSCs is fluorescence-activated cell sorting based on cell surface markers or intracellular molecules.

CD44

CD44 is a type I transmembrane glycoprotein that binds hyaluronan and a variety of extracellular as well as cell-surface ligands (42,43). The molecule exists in multiple spliced forms and shows enormous variability in glycosylation (42-44). The CD44 protein contains four major domains, including the conserved extracellular hyaluronan-binding and variably spliced regions, the transmembrane sequence, and the intracellular cytoskeletal-signaling domain (44,45). CD44 plays an important role in adhesion, motility, proliferation and cell survival (46). It’s a useful marker for identifying CSCs in breast tumors as well as in various other tumors (6,24,47-49). It’s reported that ESA⁺ CD44⁺ CD24^–/low Lin^– cells were identified as breast CSCs (6). They found that, this population has a greater capacity for tumor formation in immunodeficient mice compared to other cell populations (6).

CD24

CD24 is a heavily and variably glycosylated 35-60 kDa GPI-linked sialoprotein that is expressed on B cells, T cells, and keratinocytes (50). It is also a marker for exosomes released into the urine and amniotic fluid. CD24 binds to P-Selectin on activated platelets and vascular endothelial cells (51,52). The expression of CD24 is a hallmark of a wide range of epithelial cancers and has recently been used as an indicator of metastasis (53-55). The presence or absence of CD24 on the cell surface has been used as a marker for putative CSCs, which seems to be tissue specific (56). The CD44⁺ CD24^–/low population of cancer cells were defined as breast CSCs (6). They were found to have increased adhesion, invasion, and migration characteristics when compared with CD24 expressing cells (54,57). Recent reports showed that breast CSCs have a mesenchymal phenotype (58). Also, transformed BC cells could be able to switch between the mesenchymal and epithelial phenotypes (58).

ALDH activity

ALDH belongs to the ALDH family which is a group of enzymes involved in oxidizing a wide variety of intracellular aldehydes into their corresponding carboxylic acids (59). There are different isoforms of ALDH and ALDH1 is a detoxifying enzyme responsible for the oxidation of aldehydes intracellular. The Aldefluor assay system has been developed to detect the activity of the ALDH1 isoform (60). ALDH1 activity showed to be increased in CSCs and has since been successfully used to isolate CSCs in different cancers (61-65). Normal and cancer human mammary epithelial cells with increased ALDH activity have stem/progenitor properties by utilizing in vitro and in vivo experimental systems (13). In breast carcinomas, high ALDH activity identifies the tumorigenic cell fraction, which is capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor (13,66). Also, it showed that ALDH1 was a predictor of poor clinical outcome of BC patients (64). In conclusion, ALDH1 activity has been widely used as a functional stem cell marker to isolate CSCs in BC.

Side population (SP)

Hoechst 33342 is a DNA dye historically used for flow cytometric analysis of the DNA content of live cells (67). Hoechst is able to penetrate intact cell membranes and it could be also transported out of cells by ATP-binding cassette (ABC) transporters (68). SP cells can be identified using dual wavelength flow cytometry combined with Hoechst 33342 dye efflux. These cells have been detected in various human solid malignant tumors including BC (69-71). It’s reported that SP cells have increased resistance to chemotherapeutic agents and apoptotic stimuli (72,73). Also, SP cells have increased migratory potential and thus may play an important role in the metastatic spread of BC (74). However, recent studies have shown arising problems in using SP cells as a CSCs fraction because of conflicting results due to cross-contamination of the SP and non-SP fractions (75).

Other biomarkers

Additional markers useful in characterizing breast CSCs were recently reported. CD133, identified for breast CSCs isolated from cell lines generated from mouse mammary tumors, is a known marker of CSCs in several solid tumors (76-78). An additional marker, PROCR, identified using gene expression profiling of primary BCs, is also a known marker of hematopoietic, neural, and embryonic stem cells (79,80). Other surface markers such as CXCR4 and ABCG2 may be associated with CSC characteristics. CXCR4 was reported to promote metastasis in BCs (81). Recently, a highly tumorigenic subpopulation expressing PROCR⁺ESA⁺ was identified (82), and which may provide a CSC molecular signature in BC.

Key signaling pathways

The signaling pathways that regulate self-renewal and differentiation of CSCs are not well understood. However, it seems that there are some overlap in the key signaling pathways between CSCs and normal adult stem cells. Here we summarized some of these signaling pathways such as Wnt/β-catenin, Hedgehog (Hh), and Notch signaling that play a vital role in regulating BCSCs.

Wnt/β-catenin signaling pathway

The Wnt signaling pathway is critical for the regulation of embryogenesis, cell fate determination, self-renewal and differentiation of stem cells (83). It causes an accumulation of β-catenin in the cytoplasm and its eventual translocation into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family (84). In the absence of Wnt signal, β-catenin is targeted by coordinated phosphorylation by CK1 and the APC/Axin/GSK-3β complex leading to its ubiquitination and proteasomal degradation. However, in the presence of Wnt ligand, the co-receptor LRP5/6 is brought in complex with Wnt-bound Frizzled. This leads to activation of Dishevelled (Dvl) by sequential phosphorylation, poly-ubiquitination, and polymerization, which displaces GSK-3β from APC/Axin through an unclear mechanism. In addition, it allows β-catenin to accumulate and localize to the nucleus and subsequently induce a cellular response via gene transduction alongside the TCF/LEF transcription factors (84). Wnt/β-catenin signaling is implicated in the maintenance of CSCs of a variety of cancers including BC (83,85). For example, upregulation of β-catenin in stem cell survival pathway was shown to mediate the resistance of mouse mammary stem/progenitor cells to radiation (73). MMTV-Wnt1 transgenic mice could develop premalignant mammary hyperplasia with elevated stem cell numbers, and their subsequent carcinomas contain a CSC population defined by methods similar to those applied to human BCs (86). It’s also reported that Wnt proteins could act directly on mouse mammary stem cells to promote their self-renewal or expansion (87).

Notch signaling

Notch signaling is an evolutionarily conserved pathway in multicellular organisms that regulates cell-fate determination during development and maintains adult tissue homeostasis (88). The Notch transmembrane signaling proteins are expressed in both stem cells and early progenitor cells. It has four different notch receptors, referred to as NOTCH1-4, which play an important role in normal breast development, cell fate, and stem cell self-renewal (89). Aberrant Notch signaling has been implicated in the development and progression of both preinvasive ductal carcinoma in situ and invasive BC (88,90). Notch signaling pathway is believed to be dysregulated in CSCs, ultimately leading to CSCs uncontrolled self-renewal. For example, it was shown that Notch signaling play an important role in the self-renewal function of malignant breast CSCs (88,91). Notch 4 is critical for normal mammary development, which could suppress differentiation of breast epithelial cells in vitro and development of normal mammary glands while promoting the development of mammary tumors in vivo (91). These observations suggest that alterations of Notch 4 signaling might be involved in the transformation of normal mammary stem cell to CSCs.

Hh signaling

Hh signaling pathway is a highly conserved pathway that plays a critical role in embryonic growth and cell fate determination during development (92). Vertebrates consist of three main Hh homologs: Indian hedgehog (Ihh), Desert hedgehog (Dhh) and Sonic hedgehog (Shh). Pathway activation is initiated by binding of one of the three Hh homologs to Patched (Ptch), an Hh receptor necessary for proliferation, differentiation and cell fate (93). Hh signaling is triggered by binding of ligands with transmembrane receptor Ptch and is subsequently mediated by transcriptional effectors belonging to the Gli family, whose functions is tuned by a number of molecular interactions and post-synthetic modifications (93). Hh signaling pathway is another major pathway that is involved in breast stem cell self-renewal (94). It’s reported that the Hh pathway takes part in regulating self-renewal of normal and malignant human mammary stem cells (92). Accumulating evidence also suggests that inhibition of Hh signaling in breast tumors may interfere with the maintenance of a putative CSCs subpopulation (94). Human breast CSCs, as identified by the CD44⁺ CD24^–/low Lin^– phenotype, show increased gene expression of PTCH1, GLI1 and GLI2 compared to remaining tumor cells isolated from primary BCs (95). Additionally, it has been found that inhibition of Hh signaling increases the response of cancer cell lines to classical chemotherapies (96).

Breast CSCs as therapeutic targets

Accumulating studies have demonstrated a small subpopulation of CSCs exist in the cancer cell population. CSCs have powerful self-renewal capacity and tumor-initiating ability, and are resistant to conventional cancer treatment such as chemotherapy and radiation (9). These conventional anticancer therapies are effective at debulking the tumor mass but spare the relatively quiescent CSCs, which are responsible for cancer recurrence. So it is necessary to develop therapeutic strategies acting specifically on CSCs. Therapeutic targeting of CSCs may therefore provide a novel strategy that is more effective than the current drugs targeting the bulk mature cancer cells in treatment of BC. Numerous therapeutic approaches aiming at eradicating CSCs have been developed in recent years such as targeting molecular markers and key signaling pathways, as well as inducing the differentiation of BCSCs.

The first approach is to target molecular markers of CSCs. CD44 is a CSCs surface marker and is upregulated in invasive breast carcinoma (6). It’s reported that targeting CD44 with the specific antibody P245 significantly inhibited the growth of human BC xenografts (97). Treatment with this antibody prevents tumor relapse after chemotherapy-induced remission in a basal-like human BC xenografts (7,97). Moreover, in the treatment of MCF-7 BC, an anti-CD44 antibody-conjugated gold nanorod has been used to target and photo-ablate CD44⁺ cells, which display significant CSC characteristics (98,99).

A second approach is to target key signaling pathways of CSCs. The stem cell signaling pathways play important roles in CSCs renewal and maintenance such as Notch, Wnt/β-catenin and Hh pathways. Small molecules of gamma secretase inhibitors (GSI) or Notch 4 neutralizing antibody have been shown to reduce the population of CSCs (100). GSIs are currently undergoing clinical trials for the treatment of advanced BC. It showed that oral GSI was well tolerated at a weekly dosing, but no clinical benefit was observed in patients with BC (101). Furthermore, inhibition of Wnt signaling by dietary polyphenols curcumin and piperine has been shown to decrease mammosphere formation and percentage of ALDH1-positive cells (102). Some studies also demonstrate that inhibitors of Wnt/β-catenin signaling eradicated breast tumor-initiating cells in vitro and in vivo, which provide a compelling rationale for developing such antagonists for BC therapy (103). Finally, recent studies demonstrate that Hh signaling pathway plays an essential role in maintaining the CD44⁺ CD24^–/low subpopulation, and this pathway might represent a new candidate for BC therapy targeting CSCs (104).

Inducing differentiation of BCSCs is another approach to target CSCs. It will result in the loss of the potential to self-renewal in the CSCs. Enforced expression of let-7 miRNA induced differentiation of CD44⁺ CD24^–/low CSCs and inhibited their ability to form tumors in mice (105). Most recently, Gupta et al. used a high-throughput screening approach to determine the anticancer activity of approximately 16,000 compounds. It was identified that salinomycin could selectively target CD44⁺ CD24^–/low CSCs (106). Treatment of mice with salinomycin induced epithelial differentiation of tumor cells and resulted in inhibition of tumor growth (106). These findings suggest that inducing differentiation of CSCs might be a promising approach for breast cancer therapy.

Conclusions

In summary, recent studies have identified a small population of highly tumorigenic cells with stem cell properties in human BC that are referred to as BCSCs. They are considered to be the source of tumor initiation and maintenance. Also, growing evidence suggests that CSCs may be responsible for therapy resistance and relapse of BC. Current treatments of BC have shown efficacy in removing the bulk of differentiated cancer cells while failing to eliminate the BCSCs, targeting BCSCs might be a promising approach to treat BC metastasis and relapse.

Acknowledgements

Funding: This study was supported by funds from the National Natural Science Foundation of China (Grant No. 81172118), China Postdoctoral Science Foundation (Grant No. 2013M540574) and Youth innovation funds project of the first affiliated hospital of Zhengzhou university (to Z Li).

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

References

1. Porter PL. Global trends in breast cancer incidence and mortality. Salud Publica Mex 2009;51 Suppl 2:s141-6. [PubMed]
2. Gonzalez-Angulo AM, Morales-Vasquez F, Hortobagyi GN. Overview of resistance to systemic therapy in patients with breast cancer. Adv Exp Med Biol 2007;608:1-22. [PubMed]
3. Bouganim N, Tsvetkova E, Clemons M, et al. Evolution of sites of recurrence after early breast cancer over the last 20 years: implications for patient care and future research. Breast Cancer Res Treat 2013;139:603-6. [PubMed]
4. Duru N, Candas D, Jiang G, et al. Breast cancer adaptive resistance: HER2 and cancer stem cell repopulation in a heterogeneous tumor society. J Cancer Res Clin Oncol 2014;140:1-14. [PubMed]
5. Dontu G, Al-Hajj M, Abdallah WM, et al. Stem cells in normal breast development and breast cancer. Cell Prolif 2003;36 Suppl 1:59-72. [PubMed]
6. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100:3983-8. [PubMed]
7. Economopoulou P, Kaklamani VG, Siziopikou K. The role of cancer stem cells in breast cancer initiation and progression: potential cancer stem cell-directed therapies. Oncologist 2012;17:1394-401. [PubMed]
8. Shackleton M, Quintana E, Fearon ER, et al. Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell 2009;138:822-9. [PubMed]
9. Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med 2007;58:267-84. [PubMed]
10. Clarke MF, Dick JE, Dirks PB, et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006;66:9339-44. [PubMed]
11. Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl J Med 2006;355:1253-61. [PubMed]
12. Wicha MS. Cancer stem cells and metastasis: lethal seeds. Clin Cancer Res 2006;12:5606-7. [PubMed]
13. Ginestier C, Hur MH, Charafe-Jauffret E, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007;1:555-67. [PubMed]
14. Pessina A, Gribaldo L. The key role of adult stem cells: therapeutic perspectives. Curr Med Res Opin 2006;22:2287-300. [PubMed]
15. Vats A, Bielby RC, Tolley NS, et al. Stem cells. Lancet 2005;366:592-602. [PubMed]
16. Vats A, Tolley NS, Polak JM, et al. Stem cells: sources and applications. Clin Otolaryngol Allied Sci 2002;27:227-32. [PubMed]
17. O’Connor ML. Cancer stem cells: A contentious hypothesis now moving forward. Cancer Lett 2014;344:180-7. [PubMed]
18. Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105-11. [PubMed]
19. Adams JM, Kelly PN, Dakic A, et al. Role of “cancer stem cells” and cell survival in tumor development and maintenance. Cold Spring Harb Symp Quant Biol 2008;73:451-9. [PubMed]
20. Bao S, Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006;444:756-60. [PubMed]
21. Tan S, Chen JS, Sun LJ, et al. Selective enrichment of hepatocellular cancer stem cells by chemotherapy. J Int Med Res 2009;37:1046-56. [PubMed]
22. Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identification and expansion of human colon-cancer-initiating cells. Nature 2007;445:111-5. [PubMed]
23. Li C, Lee CJ, Simeone DM. Identification of human pancreatic cancer stem cells. Methods Mol Biol 2009;568:161-73. [PubMed]
24. Dalerba P, Dylla SJ, Park IK, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A 2007;104:10158-63. [PubMed]
25. Collins AT, Berry PA, Hyde C, et al. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 2005;65:10946-51. [PubMed]
26. Dontu G, El-Ashry D, Wicha MS. Breast cancer, stem/progenitor cells and the estrogen receptor. Trends Endocrinol Metab 2004;15:193-7. [PubMed]
27. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 2008;8:755-68. [PubMed]
28. Takahashi RU, Takeshita F, Fujiwara T, et al. Cancer stem cells in breast cancer. Cancers (Basel) 2011;3:1311-28. [PubMed]
29. Battula VL, Shi Y, Evans KW, et al. Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis. J Clin Invest 2012;122:2066-78. [PubMed]
30. Dontu G, Abdallah WM, Foley JM, et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003;17:1253-70. [PubMed]
31. Dontu G, Wicha MS. Survival of mammary stem cells in suspension culture: implications for stem cell biology and neoplasia. J Mammary Gland Biol Neoplasia 2005;10:75-86. [PubMed]
32. Kenney NJ, Smith GH, Lawrence E, et al. Identification of Stem Cell Units in the Terminal End Bud and Duct of the Mouse Mammary Gland. J Biomed Biotechnol 2001;1:133-43. [PubMed]
33. Smith GH. Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands. Development 2005;132:681-7. [PubMed]
34. Kakarala M, Wicha MS. Implications of the cancer stem-cell hypothesis for breast cancer prevention and therapy. J Clin Oncol 2008;26:2813-20. [PubMed]
35. Pece S, Tosoni D, Confalonieri S, et al. Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content. Cell 2010;140:62-73. [PubMed]
36. Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A 2003;100:8418-23. [PubMed]
37. Stingl J, Caldas C. Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer 2007;7:791-9. [PubMed]
38. Iqbal J, Chong PY, Tan PH. Breast cancer stem cells: an update. J Clin Pathol 2013;66:485-90. [PubMed]
39. García Bueno JM, Ocaña A, Castro-García P, et al. An update on the biology of cancer stem cells in breast cancer. Clin Transl Oncol 2008;10:786-93. [PubMed]
40. Jaggupilli A, Elkord E. Significance of CD44 and CD24 as cancer stem cell markers: an enduring ambiguity. Clin Dev Immunol 2012;2012:708036.
41. Frank NY, Schatton T, Frank MH. The therapeutic promise of the cancer stem cell concept. J Clin Invest 2010;120:41-50. [PubMed]
42. Tölg C, Hofmann M, Herrlich P, et al. Splicing choice from ten variant exons establishes CD44 variability. Nucleic Acids Res 1993;21:1225-9. [PubMed]
43. Afify A, Purnell P, Nguyen L. Role of CD44s and CD44v6 on human breast cancer cell adhesion, migration, and invasion. Exp Mol Pathol 2009;86:95-100. [PubMed]
44. Haylock DN, Nilsson SK. The role of hyaluronic acid in hemopoietic stem cell biology. Regen Med 2006;1:437-45. [PubMed]
45. Marhaba R, Zöller M. CD44 in cancer progression: adhesion, migration and growth regulation. J Mol Histol 2004;35:211-31. [PubMed]
46. Liu H, Patel MR, Prescher JA, et al. Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models. Proc Natl Acad Sci U S A 2010;107:18115-20. [PubMed]
47. Prince ME, Sivanandan R, Kaczorowski A, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A 2007;104:973-8. [PubMed]
48. Takaishi S, Okumura T, Tu S, et al. Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells 2009;27:1006-20. [PubMed]
49. Hurt EM, Kawasaki BT, Klarmann GJ, et al. CD44+ CD24(-) prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis. Br J Cancer 2008;98:756-65. [PubMed]
50. Pirruccello SJ, LeBien TW. The human B cell-associated antigen CD24 is a single chain sialoglycoprotein. J Immunol 1986;136:3779-84. [PubMed]
51. Aigner S, Sthoeger ZM, Fogel M, et al. CD24, a mucin-type glycoprotein, is a ligand for P-selectin on human tumor cells. Blood 1997;89:3385-95. [PubMed]
52. Fischer GF, Majdic O, Gadd S, et al. Signal transduction in lymphocytic and myeloid cells via CD24, a new member of phosphoinositol-anchored membrane molecules. J Immunol 1990;144:638-41. [PubMed]
53. Sano A, Kato H, Sakurai S, et al. CD24 expression is a novel prognostic factor in esophageal squamous cell carcinoma. Ann Surg Oncol 2009;16:506-14. [PubMed]
54. Baumann P, Cremers N, Kroese F, et al. CD24 expression causes the acquisition of multiple cellular properties associated with tumor growth and metastasis. Cancer Res 2005;65:10783-93. [PubMed]
55. Yang XR, Xu Y, Yu B, et al. CD24 is a novel predictor for poor prognosis of hepatocellular carcinoma after surgery. Clin Cancer Res 2009;15:5518-27. [PubMed]
56. Lim SC. CD24 and human carcinoma: tumor biological aspects. Biomed Pharmacother 2005;59 Suppl 2:S351-4. [PubMed]
57. Croker AK, Goodale D, Chu J, et al. High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability. J Cell Mol Med 2009;13:2236-52. [PubMed]
58. Meyer MJ, Fleming JM, Ali MA, et al. Dynamic regulation of CD24 and the invasive, CD44posCD24neg phenotype in breast cancer cell lines. Breast Cancer Res 2009;11:R82. [PubMed]
59. Vasiliou V, Pappa A, Petersen DR. Role of aldehyde dehydrogenases in endogenous and xenobiotic metabolism. Chem Biol Interact 2000;129:1-19. [PubMed]
60. Storms RW, Trujillo AP, Springer JB, et al. Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity. Proc Natl Acad Sci U S A 1999;96:9118-23. [PubMed]
61. Kim MP, Fleming JB, Wang H, et al. ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma. PLoS One 2011;6:e20636. [PubMed]
62. Deng S, Yang X, Lassus H, et al. Distinct expression levels and patterns of stem cell marker, aldehyde dehydrogenase isoform 1 (ALDH1), in human epithelial cancers. PLoS One 2010;5:e10277. [PubMed]
63. Li T, Su Y, Mei Y, et al. ALDH1A1 is a marker for malignant prostate stem cells and predictor of prostate cancer patients’ outcome. Lab Invest 2010;90:234-44. [PubMed]
64. Balicki D. Moving forward in human mammary stem cell biology and breast cancer prognostication using ALDH1. Cell Stem Cell 2007;1:485-7. [PubMed]
65. Lohberger B, Rinner B, Stuendl N, et al. Aldehyde dehydrogenase 1, a potential marker for cancer stem cells in human sarcoma. PLoS One 2012;7:e43664. [PubMed]
66. Keysar SB, Jimeno A. More than markers: biological significance of cancer stem cell-defining molecules. Mol Cancer Ther 2010;9:2450-7. [PubMed]
67. Zhou S, Morris JJ, Barnes Y, et al. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A 2002;99:12339-44. [PubMed]
68. Montanaro F, Liadaki K, Schienda J, et al. Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters. Exp Cell Res 2004;298:144-54. [PubMed]
69. Petriz J. Flow cytometry of the side population (SP). Curr Protoc Cytom 2007;Chapter 9:Unit9.23.
70. Ho MM, Ng AV, Lam S, et al. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 2007;67:4827-33. [PubMed]
71. Hadnagy A, Gaboury L, Beaulieu R, et al. SP analysis may be used to identify cancer stem cell populations. Exp Cell Res 2006;312:3701-10. [PubMed]
72. Steiniger SC, Coppinger JA, Krüger JA, et al. Quantitative mass spectrometry identifies drug targets in cancer stem cell-containing side population. Stem Cells 2008;26:3037-46. [PubMed]
73. Woodward WA, Chen MS, Behbod F, et al. WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci U S A 2007;104:618-23. [PubMed]
74. Patrawala L, Calhoun T, Schneider-Broussard R, et al. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res 2005;65:6207-19. [PubMed]
75. Wu C, Alman BA. Side population cells in human cancers. Cancer Lett 2008;268:1-9. [PubMed]
76. Yin S, Li J, Hu C, et al. CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. Int J Cancer 2007;120:1444-50. [PubMed]
77. Wright MH, Calcagno AM, Salcido CD, et al. Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics. Breast Cancer Res 2008;10:R10. [PubMed]
78. Vercauteren SM, Sutherland HJ. CD133 (AC133) expression on AML cells and progenitors. Cytotherapy 2001;3:449-59. [PubMed]
79. Ivanova NB, Dimos JT, Schaniel C, et al. A stem cell molecular signature. Science 2002;298:601-4. [PubMed]
80. Hwang-Verslues WW, Kuo WH, Chang PH, et al. Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers. PLoS One 2009;4:e8377. [PubMed]
81. Kang Y, Siegel PM, Shu W, et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 2003;3:537-49. [PubMed]
82. Hwang-Verslues WW, Chang PH, Wei PC, et al. miR-495 is upregulated by E12/E47 in breast cancer stem cells, and promotes oncogenesis and hypoxia resistance via downregulation of E-cadherin and REDD1. Oncogene 2011;30:2463-74. [PubMed]
83. Howe LR, Brown AM. Wnt signaling and breast cancer. Cancer Biol Ther 2004;3:36-41. [PubMed]
84. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006;127:469-80. [PubMed]
85. Khramtsov AI, Khramtsova GF, Tretiakova M, et al. Wnt/beta-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome. Am J Pathol 2010;176:2911-20. [PubMed]
86. Cho RW, Wang X, Diehn M, et al. Isolation and molecular characterization of cancer stem cells in MMTV-Wnt-1 murine breast tumors. Stem Cells 2008;26:364-71. [PubMed]
87. Liu BY, McDermott SP, Khwaja SS, et al. The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells. Proc Natl Acad Sci U S A 2004;101:4158-63. [PubMed]
88. Dontu G, Jackson KW, McNicholas E, et al. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res 2004;6:R605-15. [PubMed]
89. Qi H, Rand MD, Wu X, et al. Processing of the notch ligand delta by the metalloprotease Kuzbanian. Science 1999;283:91-4. [PubMed]
90. Pece S, Serresi M, Santolini E, et al. Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis. J Cell Biol 2004;167:215-21. [PubMed]
91. Harrison H, Farnie G, Howell SJ, et al. Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res 2010;70:709-18. [PubMed]
92. Brechbiel J, Miller-Moslin K, Adjei AA. Crosstalk between hedgehog and other signaling pathways as a basis for combination therapies in cancer. Cancer Treat Rev 2014;40:750-9. [PubMed]
93. Briscoe J, Thérond PP. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 2013;14:416-29. [PubMed]
94. Coni S, Infante P, Gulino A. Control of stem cells and cancer stem cells by Hedgehog signaling: pharmacologic clues from pathway dissection. Biochem Pharmacol 2013;85:623-8. [PubMed]
95. Liu S, Dontu G, Mantle ID, et al. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res 2006;66:6063-71. [PubMed]
96. Chai F, Zhou J, Chen C, et al. The Hedgehog inhibitor cyclopamine antagonizes chemoresistance of breast cancer cells. Onco Targets Ther 2013;6:1643-7. [PubMed]
97. Marangoni E, Lecomte N, Durand L, et al. CD44 targeting reduces tumour growth and prevents post-chemotherapy relapse of human breast cancers xenografts. Br J Cancer 2009;100:918-22. [PubMed]
98. Chen K, Huang YH, Chen JL. Understanding and targeting cancer stem cells: therapeutic implications and challenges. Acta Pharmacol Sin 2013;34:732-40. [PubMed]
99. Alkilany AM, Thompson LB, Boulos SP, et al. Gold nanorods: their potential for photothermal therapeutics and drug delivery, tempered by the complexity of their biological interactions. Adv Drug Deliv Rev 2012;64:190-9. [PubMed]
100. Grudzien P, Lo S, Albain KS, et al. Inhibition of Notch signaling reduces the stem-like population of breast cancer cells and prevents mammosphere formation. Anticancer Res 2010;30:3853-67. [PubMed]
101. Krop I, Demuth T, Guthrie T, et al. Phase I pharmacologic and pharmacodynamic study of the gamma secretase (Notch) inhibitor MK-0752 in adult patients with advanced solid tumors. J Clin Oncol 2012;30:2307-13. [PubMed]
102. Kakarala M, Brenner DE, Korkaya H, et al. Targeting breast stem cells with the cancer preventive compounds curcumin and piperine. Breast Cancer Res Treat 2010;122:777-85. [PubMed]
103. Hallett RM, Kondratyev MK, Giacomelli AO, et al. Small molecule antagonists of the Wnt/β-catenin signaling pathway target breast tumor-initiating cells in a Her2/Neu mouse model of breast cancer. PLoS One 2012;7:e33976. [PubMed]
104. Tanaka H, Nakamura M, Kameda C, et al. The Hedgehog signaling pathway plays an essential role in maintaining the CD44+CD24-/low subpopulation and the side population of breast cancer cells. Anticancer Res 2009;29:2147-57. [PubMed]
105. Yu F, Yao H, Zhu P, et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 2007;131:1109-23. [PubMed]
106. Gupta PB, Onder TT, Jiang G, et al. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 2009;138:645-59. [PubMed]