Breast Cancer Stem Cells: A Novel Therapeutic Target

Abstract

Breast cancer stem cells (BCSCs), characterized by the CD44⁺/CD24^−/low marker, are attributed with features that are demonstrated by the disease itself, such as growth of tumor, recurrence, metastases, and multiple drug resistance. This review concerns the emergence and expediency of BCSCs in treating relapse and advanced cases of breast cancer. One of the ideal ways of detecting and eliminating BCSCs would be to tweak certain molecular receptors in the desired pathway, which would require extensive and comprehensive knowledge about these cell signaling pathways. Although hedgehog (Hh), Notch, and Wnt signaling are of prime concern, governing tumorigenesis and cancer stem cell (CSC) renewal, designing chemotherapeutic or molecular targeted therapies is still a tricky arena to venture into, as these pathways play a vital role in normal mammary gland development. Thus selective inhibition of pathway receptors needs to be investigated in the future.

Introduction

Breast cancer, the most common form of cancer among women, also has the second highest morbidity rate worldwide (10.9% of all cancers). With an estimated 1.38 million new cancer cases diagnosed in 2008, it is also the most common cancer in both developed and developing regions. About 69,000 new cases have been estimated in each of these regions (population ratio 1:4).

Incidence rates vary from 19.3 per 100,000 women in Eastern Africa to 89.7 per 100,000 women in Western Europe and are high (> 80 per 100,000) in developed regions of the world (except Japan) and low (<40 per 100,000) in most developing regions.

The range of mortality rates is much less (approximately 6-19 per 100,000) because of the more favorable survival of breast cancer in (high-incidence) developed regions. As a result, breast cancer ranks as the fifth cause of death from cancer overall (458,000 deaths), but it is still the most frequent cause of cancer death in women in both developing (269,000 deaths, 12.7% of total) and developed regions, where the estimated 189,000 deaths is almost equal to the estimated number of deaths from lung cancer (188,000 deaths).¹

Statistical analysis suggests that <10% of patients with newly diagnosed breast cancer exhibit advanced or metastatic disease, whereas approximately 30% to 50% of patients who are diagnosed with this disease at an early stage are prone to progress to a metastatic stage despite administered treatment such as chemotherapy and/or adjuvant therapies.² This suggests that despite much advancement in breast cancer treatment over the years, relapse of this disease with time (approximately 40% of all patients with breast cancer experience relapsed disease, with 60%-70% cases of relapse having metastasis) serves as a major roadblock to complete cure of this disease. The only established reason behind this is the underlying presence of a small population of stem-like cells called cancer stem cells (CSCs), ie, breast cancer stem cells (BCSCs). A recent hypothesis states that these CSCs originate from normal tissue stem cells; adult stem cells serve as ideal targets for malignant transformation because of their lengthy lifespan, and they are normally under tight control within a niche. Also these CSCs share certain properties with normal stem cells (noteworthy ones being the self-renewal capability), which leads to the generation of more CSCs and the ability to differentiate to form a variety of differentiated cells that are found in malignancy.³ Additionally, CSCs pose a threat in the form of invasion that is resistant to current chemotherapy/radiotherapy, as well as distant metastasis.

The concept of CSCs was first identified in hematologic malignancies and has been supported by abundant evidence over the years.4, 5 However, although it is an established concept that these tumors originate from cells having self-renewal capacity, an essential thrust for the future is understanding whether the CSCs exhibit this property on their own or acquire it during transitions, such as epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET). EMT is triggered through extracellular signaling of collagen or from growth factors such as fibroblast growth factors, epidermal growth factors, and platelet-derived growth factors (PDGFs) such as PDGF-A, PDGF-B, and PDGF-D. Through the process of EMT, molecular alterations in the form of loss of apical polarity, loss of epithelial cell-cell junctions, reorganization of actin cytoskeleton, acquisition of more spindle-shaped morphologic features, and upregulation and downregulation of mesenchymal markers (fibronectin, N-cadherin, vimentin) and epithelial markers (E-cadherin epithelial-specific antigen) are acquired by epithelial cells. All these alterations constitutively aid in tumor growth and metastasis by a gain in stem-like properties through CSC generation. Also, it has been reported that mesenchymal stem cells (MSCs) are responsible for the initiation of EMT and they promote the entry of breast cancer cells into the bone marrow, which are then recruited by the tumor microenvironment and thus might play a critical role in cancer progression. In addition, bone marrow–derived MSCs also promote tumor progression through cytokine and growth factor production.⁶ Further evidence of bone marrow–derived MSCs contributing to upregulation of EMT-specific markers in breast tumorigenesis has been reported in the recent past by Martin et al, elucidating the underlying role played by these MSCs in cancer progression.⁷ Conversely, MET induction by inhibition of tumor necrosis factor-β (TGF-β) and MEK-ERK pathways has resulted in improving the efficiency of induced pluripotent stem cell generation from fibroblasts.⁸ Moreover, in past years, 6 independent intrinsic molecular subtypes of breast tumor such as normal-like, human epidermal growth factor receptor 2 (HER2)-enriched, luminal A and B, basal A/basal-like, and basal B/claudin-low have been reported.9, 10, 11 Of these, basal B/claudin-low show increased expression of EMT gene signatures after cancer therapy,¹² which might provide resistance to present treatment-related therapeutic approaches. Another striking feature regarding the origin of BCSCs has been reported by Morel et al.¹³ Their experimental results indicate that generation of BCSCs can take place from nonmalignant primary human mammary epithelial cells through activation of the Ras/MAPK signaling pathway, which gets accelerated by EMT induction with a limited number of oncogenes and cancer-associated genes. Therefore therapeutic strategies targeting the aforesaid pathways without affecting normal tissue homeostasis can provide new dimensions to future treatment outcomes.

Another emerging theory that has come to notice over the past few years is that both chemotherapy and radiotherapy enrich BCSCs. In vitro studies conducted by Yu et al)¹⁴ pointed out a 14-fold increase in mammosphere formation from cells isolated from 5 first-degree patients with breast cancer who received neoadjuvant chemotherapy compared with 8 first-degree chemotherapy-naive patients after 15 days of culture. Surprisingly, 74% ± 7% of malignant cells from chemotherapy-treated patients represented BCSCs compared with just 9% ± 4% of cells from the chemotherapy-naive group, whose cells represented BCSCs. They further extended their research to in vivo studies by consecutive passaging of breast cancer cells (SKBR3 cell line) in chemotherapy-treated NOD/SCID mice to test the in vitro findings. The third time passaged cells (SK-3rd) underwent suspension culture for generation of mammospheres. The SK-3rd–generated tumors were further passaged into chemotherapy-treated (SK-4th[+]) and nontreated (SK-4th[–]) NOD/SKID mice. SK-4th[+] cells revealed a percentage of sphere-forming cells equal to the SK-3rd passage, compared with SK4th[–] cells, which revealed 8-fold less sphere production, highlighting the importance of chemotherapy in maintaining the volume of BCSCs in vivo. Further in vitro experiments by Fillmore and Kuperwasser¹⁵ on breast cancer cell lines (SUM-159, SUM-1315, and MDA-MB-231) also pointed out BCSC enrichment after chemotherapy by showing up to a 30-fold increase in the CSC population after 6 days of treatment with paclitaxel or 5-fluorouracil compared with control cultures. These results clearly elucidate that chemotherapeutic medications enhance BCSC survival in vivo, and researchers thus need to venture deep into further studies to bridge the gap of understanding of how and by what possible routes chemotherapy and BCSCs interplay, finding out prominent reasons for BCSC enrichment and maintenance by chemotherapy. However unlike chemotherapy, radiotherapy, which in general is considered to be ineffective against CSCs,16, 17, 18, 19, 20 was also believed to enrich the breast cancer–initiating cells²¹ until recently when this belief was partly opposed by Zielske et al.²² Their in vivo tests on patient-derived breast tumor (MC1 and UM2) xenografts in NOD/SCID mice showed an increase in the BCSC population in irradiated UM2 tumors compared with controls but surprisingly revealed a drop in BCSCs from 2.5% ± 0.7% in untreated controls to 0.31% ± 0.14% in irradiated MC1 tumors, giving rise to the concept of radiosensitive CSCs. This suggests that breast cancers may consist of radioresistant or radiosensitive CSCs. Thus understanding the exact properties that differentiate radiosensitive from radioresistant CSCs might help in developing future therapies aimed at altering the properties of resistance to radiotherapy to radiosensitivity of the CSCs for partial or complete knockout of the cancerous stem cells, looking toward better treatment outcomes.

A recent preclinical study by Conley et al²³ found that antiangiogenic agents (bevacizumab—a vascular endothelial growth factor [VEGF] neutralizing humanized antibody—and sorafenib/sunitinib—a tyrosine kinase inhibitor for the VEGF receptor), which were considered to be novel anti–breast cancer therapeutic agents, enhance tumor invasion and metastasis of breast cancer cells. Tests were conducted with sunitinib malate on human breast tumors (MDA-MB-231 and SUM-159); tumor growth was inhibited as usual but testing the cells from the same tumors using the ALDEFLUOR assay (STEMCELL Technologies, Vancouver, British Columbia, Canada) revealed a many-fold increase in positive (exhibiting stem cell properties) cell percentages. Similar results of increased positive cell percentages were reported in bevacizumab-treated tumors when compared with the control group. BCSC enrichment through postantiangiogenic treatment was further brought to notice by trypan blue exclusion, which pointed out no significant difference in cell viability between tumors from the control group and drug-treated tumors, implying an increase in absolute number and proportion of BCSCs. Moving further, their investigations revealed that these antiangiogenic agents enriched the BCSC populations through the generation of intratumoral hypoxia. Using Hypoxyprobe (Hypoxyprobe, Inc., Burlington, MA) staining, they found that tumors from the control group showed little or no hypoxia, whereas tumors from sunitinib-treated mice possessed multiple areas of intense hypoxia, leading to an 8-fold greater number of positive cells by ALDEFLUOR staining within the hypoxic regions, stating that this increase in cells with stem cell properties is mediated by hypoxia-inducible factor-1α and is also partly regulated by the activation of the Akt/β-catenin CSC signaling pathway. Thus antiangiogenic therapy that has been practiced for a long time in treating patients with breast cancer needs to be revised because of its limited effectiveness against the underlying hypoxic stress–mediated CSC stimulation.

Thus all these complex novel insights have opened up newer challenging avenues for stem cell–based breast cancer research to curb tumor progression from the roots within a shorter time span. Possibly the CSCs exhibit increased resistance to chemotherapy because of their resistance to apoptosis and their higher expression of adenosine triphosphate (ATP) binding cassette (ABC) transporters, resulting in efflux of drugs from cells and, more importantly, because chemotherapy mostly targets dividing cells; these CSCs easily escape because they remain mostly in the resting stage of the cell cycle.²⁴ Agents that have the potential to target the BCSCs must be developed for better treatment outcomes. The aforesaid novel findings over recent years suggest that these BCSCs can be targeted by inhibiting the self-renewal signaling pathways, targeting the CSC niche, and by unraveling and targeting the underlying mechanisms of BCSC enrichment by present chemotherapeutic medications. It is also hoped that the use of CSC targeting agents, if used in conjunction with chemotherapy and radiotherapy, might help in clearing these CSCs. In support for CSC targeting, many therapeutic antibodies such as MT110, a bispecific antibody construct (BiTE) that targets the stem cell surface antigen epithelial cell adhesion molecule (EpCAM), are currently making their mark in clinical trials for treatment of breast cancer.²⁵ With all these novel advancements, we can say that present and future breast cancer research demands a multitargeted approach to drive out the BCSCs from their roots, which will lead to long-term survival of the affected population in the future.

The surface marker proteins on membranes define cell types. Likewise, BCSCs are identified by the presence of cell surface marker protein CD44, with low levels of CD24,2, 26 Lin,²⁷ and B38.1.²⁸ CD24 along with CD44 are called adhesion molecules, whereas B38.1 is called a breast- or ovarian cancer–specific marker. Breast stem cells can be easily identified by their ability to grow in serum-free suspension cultures like the neural stem cell–forming aggregates called mammospheres.29, 30 Another approach is to identify cells retaining bromodeoxyuridine or H3-thymidine for a longer time than do cycling cells because they remain inactive in G0 phase.31, 32

As researchers were able to spot CSCs, the next objective was to identify the reason behind the development and identification of CSCs. Investigations suggest that breasts of young virgins at puberty are highly sensitive to carcinogenic exposure, which aids in a high susceptibility of breast stem cells to form tumors after many years.33, 34 According to Morrison et al,³⁵ epidemiology data on breast cancer suggests that on exposure to radiation, somatic breast stem cells undergo certain mutations that ultimately lead to some kind of transformation in cells that results in the formation of tumors. These CSCs maintain their self-renewal behavior based on Notch, Wnt/β-catenin, and hedgehog (Hh) pathways.36, 37, 38 In another approach, researchers used flow cytometry for isolation of cells that were positive or negative for each marker. On the basis of expression results of CD44⁺, CD24^−/low, and Lin⁻, which induced detectable tumor formation on injection into NOD/SCID mice, they could easily identify BCSCs.²⁶ A different study identified BCSCs by the expression of genotype CD44⁺/CD24⁻/ALDH1⁺ (aldehyde dehydrogenase).39, 40 Even though Al-Hajj et al pointed out that human BCSCs are enriched in CD44⁺/CD24^−/low,²⁸ some other studies revealed that among CD44⁺/CD24⁻ subpopulations, ALDH1⁺ cells (CD44⁺/CD24^−/low/ALDH1⁺) exhibit more enhanced tumor-initiating capacity. ALDH1 immunohistochemical analysis also revealed that the survival rate of patients expressing higher levels of ALDH1 was much lower than those who did not.41, 40 More recently, Zhu et al reported that BCSCs can also be identified by the EpCAM⁺/CD44⁺/CD24⁻ phenotype.⁴²

Essential stem cell signaling pathways involved in oncogenesis regulating CSC renewal and maintenance are the hedgehog (Hh), Notch, and Wnt pathways(Table 1). Abnormalities in these pathways in mammary glands play a vital role in tumorigenesis.43, 44, 45

The Hh signaling pathway is associated both with normal mammary gland development and development of tumorous outgrowth and progression of breast cancer. Vertebrates consist of 3 main Hh homologs: Indian hedgehog (Ihh), Desert hedgehog (Dhh), and Sonic hedgehog (Shh). These proteins, which undergo posttranslational modification before secretion,⁴⁶ belong to a family of major signaling molecules that play a vital role in stimulation of the target cells expressing Patched-1 (Ptc-1), an Hh receptor necessary for proliferation, differentiation, and cell fate.⁴⁷ Experimental findings state that self-renewal properties exhibited by mammary stem cells are regulated by a family of zinc-finger proteins containing Gli transcription factors also known as Gli proteins (Gli1, 2, and 3).36, 48 Based on the findings about the involvement of Bmi-1, a polycomb gene, in self-renewal features of neural and hematopoietic stem cells, Liu et al tested its role on self-renewal of the BCSCs by using small interfering RNA (siRNA) lentiviral vectors to cross-check the Bmi-1-mediated Hh effects, following which they confirmed Bmi-1 as a mediator for Hh effects on stem cell self-renewal. Further investigations using CD44⁺/CD24^−/low/Lin⁻ cells isolated from human metastatic breast cancer xenografts in NOD/SCID mice also revealed activation of the Hh pathway along with Bmi-1 in BCSCs.³⁶

Research over the years has also found the importance of the Wnt signaling pathway both in different stages of mammary gland development and in oncogenesis.49, 50, 51, 52 Its participation in deciding the fate of stem cells is also accountable for its role in the onset of cancer.⁵³ β-Catenin, an essential mediator of Wnt signaling, is involved in 2 distinct functions in the cell depending on its cellular localization. The membrane-localized β-catenin is sequestered by the epithelial cell-cell adhesion protein E-cadherin to maintain cell-cell adhesion, and the cytoplasmic accumulation of β-catenin and its subsequent nuclear translocation eventually leads to activation of Wnt target genes such as c-Jun, c-Myc, fibronectin, and cyclin D1. Activated Wnt/β-catenin signaling can be linked to “stemness.”⁵⁴ In the case of oncogenesis, many of the proteins involved in Wnt signaling are either oncogenes or tumor suppressors. Initially the discovery of WNT1 as an oncogene that has the ability to transform mice mammary cells unraveled the involvement of this signaling pathway in oncogenesis. After this discovery, it was found that adenomatous polyposis coli (APC), a tumor suppressor protein, on binding to β-catenin downregulates β-catenin.⁵⁵

In normal pathways, APC and Axin prevent β-catenin from reaching the nucleus, whereas during APC deficiency, mutated β-catenin and/or tumor suppressor gene Wnt5a can lead to formation of tumors by inhibiting the normal regulation of stem cell renewal and proliferation rates. However even though histologic evidence proves activation of β-catenin in breast cancer and an active Wnt pathway in human breast epithelia, mutations in APC, Axin, or for that matter β-catenin are not prevalent.⁵⁶ Hence recent focus is on epigenetics in the form of differential expression patterns of Wnt, soluble Wnt inhibitors, Wnt receptors, and transcription factors of the TCF/LEF family in breast cancers, which might contribute to malignancies in the mammary gland.

Again, Wnt signaling activities have been found in mammary stem cells situated in the basal layer of mammary ducts.⁵⁷ Usually, MMTV-wnt-1 mice serve as a model for unraveling the hidden mysteries of Wnt signaling in breast cancers and BCSCs. Shackleton et al revealed a 6.4-fold expansion of mammary stem cells in premalignant mammary tissue from MMTV-wnt-1 mice, inferring the involvement of Wnt1 in targeting mammary stem cells in tumorigenesis.⁵⁷ Recent research by Zeng and Nusse⁵⁸ showed a 7-fold increase in the number of secondary colonies in vitro on addition of Wnt3a protein in primary cultures of isolated mammary stem cells, and although there was a 50% reduction in secondary colony formation on incubation with Dkk1, cells cultured with Wnt3a showed continued expansion while going through multiple passages, producing increasing number of colonies. Moreover, to test the in vivo self-renewal and multipotency capabilities of the in vitro cultured cells, cultured colonies were transplanted in vivo, which revealed the ability to retain mammary gland reconstitution efficiency. Hence mammary stem cells can be directly targeted by Wnt proteins to promote self-renewal and expansion.

The Notch pathway is also associated with the regulation of cell fate at several distinct developmental stages of the mammary gland. Previous breast cancer studies have shown that disruption of this pathway is related to the development and maintenance of BCSCs and thus can drive tumorigenesis. Inhibition of this signaling pathway using γ-secretase to cleave Notch receptors results in promoting BCSC differentiation in progenitor cells, rendering them sensitive to chemotherapy.⁵⁹ A recent study used an antibody that binds specifically to a non–ligand-binding region of extracellular domain of a human Notch receptor, thereby inhibiting the growth of tumor cells.⁶⁰ Despite the advancements, there are concerns about the matter, since the Notch signaling pathway is a highly conserved cell signaling system and blocking it might have negative side effects.

Cell surface proteins such as CD44, CD24, CD47, CD133, and ALDH1 are the most prominent and frequently used markers of CSCs. For CSC targeted therapy, they first need to be isolated after identification for in vitro culturing and therapeutic testing. A range of experimental procedures that have been developed for their isolation and characterization includes the following: (1) a side population technique based on the overexpression of ATP binding molecules such as ATP-binding cassette half transporter (ABCG2)/breast cancer-resistant protein 1 (BCRP1)61, 62; (2) sphere-forming assays in suspension culture conditions in the presence of growth factors such as basic fibroblast growth factors or epidermal growth factors41, 63; (3) in vitro tumorosphere formation by breast cancer cells enriched with stem cells⁶⁴; (4) aldehyde dehydrogenase method based on the enzymatic activity of ALDH165, 66, 67—the use of the ALDEFLUOR assay by Ginestier et al to identify cells expressing ALDH1 from transplanted and passaged primary human breast tumor cells into NOD/SCID mice showed that tumors formed by ALDEFLUOR-positive cells have shown malignancy⁴⁰; and (5) clonogenic assays using semisolid media.⁴¹

Especially in the case of media supported with growth factors, research also found that if kept in this kind of medium, cells maintain their stem-like properties for longer periods. In many studies, CSCs have been isolated based on cell surface markers and enzymatic activity by flow cytometry. Thus isolating these cell lines is slowly revealing the inner concepts and complexities to overcome the barrier of self-renewal and treatment resistance properties.

Decades of research show that only a minority of cells possess the self-renewal capability that gives rise to differentiated cancer cells armed with rapid proliferation capacity. Recent studies have shown the presence of CSCs in human brain tumors⁶⁸ and breast tumors.³⁵ BCSCs in solid tumor were first reported in 2003 by scientists from the University of Michigan Comprehensive Cancer Center,⁶⁹ who concluded that only “a handful of CSCs” are required for the growth, maintenance, and invasiveness of breast cancer. These are responsible for metastases and resistance to general cancer treatment therapies. As we have reported here, conventional therapies used to kill cancer cells are effective only in killing cells that have limited proliferative capabilities but not CSCs; thus CSC-targeted therapeutic strategies need to be incorporated to restrict tumor maintenance and regrowth, which might lead to complete cure of this deadly disease.

Extensive research over the years has shown that these CSCs residing inside tumors drive both its growth and its recurrence28, 68, 69, 70, 71 and that they can also metastasize to other areas of the body. Multidrug-resistant properties shown by many cancer cell lines because of the ABC transporters encoded by multidrug-resistant (MDR) gene 1 (MDR1), MDR protein, and BCRP1 makes the treatment therapies much more complicated as they pump out the anticancer drugs out of cells.⁶¹ Killing the tumor alone does not wipe out this disease; if CSCs remain, new tumors form. Thus the primary objective of the next generation of treatment strategies toward breast and other types of cancers will be to switch off the CSCs permanently, which will totally eradicate the chance of relapse after successful treatment. However killing CSCs is not an easy task. In a recent study on human and mice BCSCs by Diehn et al, it was shown that these CSCs protect themselves by enhanced antioxidant protein expression that binds to and deactivates the reactive oxygen species; these CSCs not only have cellular DNA and protein damaging capabilities but also are important mediators of anticancer therapies such as chemotherapy and radiotherapy.⁷² Research further suggests that blocking glutathione (antioxidant) activity makes the CSCs more sensitive for killing by irradiation.

A different study by researchers from Massachusetts Institute of Technology and the Broad Institute in Cambridge, MA,² explored a systemic approach for the first time in screening agents for killing CSCs. They silenced a particular breast cancer gene and converted epithelial cells to a mesenchymal state by in vitro EMT induction, thereby increasing the CSC population in breast cancer cells. Among them, salinomycin, a polyether ionophore antibiotic from Streptomyces albus,73, 74 was reported to reduce BCSCs by a proportion greater than 100-fold compared with paclitaxel, a commonly used chemotherapeutic drug for breast cancer. Experiments conducted by these investigators showed a 20-fold decrease in the proportion of CD44⁺/CD24^−/low breast cancer cells after salinomycin treatment, whereas treatment with paclitaxel increased the proportion by 18-fold. Compared with paclitaxel, treatment with salinomycin revealed a 360-fold decrease of the CD44⁺/CD24^−/low proportion. Another experiment with breast cancer cells containing a higher percentage of CSCs revealed an approximately 75-fold reduction in the proportion of CSCs with treatment with salinomycin.² Fuchs et al⁷⁵ and Riccioni et al⁷⁶ reported that salinomycin induces apoptosis in resistant cells expressing higher levels of Bcl-2 and p-glycoprotein. Further research on salinomycin needs to be done, as Li et al reported that human exposure to salinomycin either by ingestion or inhalation can cause severe toxicity, thus bringing down the practical therapeutic use of this excellent compound.⁷⁴

Recently, another compound called parthenolide showed antiproliferative and apoptosis-inducing properties in leukemic stem cells,77, 78, 79 and Hassane et al⁸⁰ confirmed this to be effective in killing leukemic stem cells using throughput in silico screening techniques. In relation to this finding, BCSCs have also been reported as targets for parthenolide.81, 82

Marcato et al showed that oncolytic reovirus can infect, target, and kill BCSCs.⁸³ They also reported that human reovirus does not cause disease and targets and kills CSCs in breast cancer tissues. Treating tumorigenic cells with reovirus produced a decrease in the CSC population at a rate equivalent to the rate of decrease of non-CSCs within the tumor. Further in vivo experiments showed a proportionate reduction both in tumor mass and the CD44⁺/CD24^−/low population after treatment with reovirus and also confirmed that of a total 60% infected population, 85% of the CD44⁺/CD24^−/low population was affected by this virus. Absence of any enrichment for CD44⁺/CD24^−/low cells in the case of reovirus-treated tumors was also reported. In vitro experiments confirmed that ALDH⁺ BCSCs were infected by reovirus, which effectively triggered the apoptotic pathway, causing death.

Very recently, Lang et al used a specific gene therapy technique that induced apoptosis of BCSCs, thereby increasing the effects of some types of chemotherapy and also reducing the chances of relapse.⁸⁴ It was shown that BikDD, an active mutant form of the proapoptotic gene BIK, effectively reduces BCSC populations by blocking activity of Bcl-2, Bcl-x_L, and Mcl-1 of the Bcl-2 family of proteins essential for tumor growth and treatment resistance. This finding was coupled with an innovative versatile expression vector (VISA) delivery system for targeted gene therapy to BCSCs. This highly innovative delivery system consisted of a targeting agent called promoter, 2 components boosting BikDD gene expression in the target BCSCs, and a payload consisting of mutant gene BikDD for killing those cancer cells. Thus by using VISA-mediated BikDD delivery; they blocked the activity of the Bcl-2 family of proteins, killing and eliminating the CSCs.

Of late, broccoli, a vegetable rich in vitamin C and multiple nutrients with potential anticancer properties, has been reported to contain sulforaphane, a natural compound that downregulates the Wnt/β-catenin self-renewal pathway and thus has the ability to kill BCSCs.⁸⁵ In vitro experiments using the ALDEFLUOR assay and a mammosphere formation assay reported an inhibition of BCSCs, whereas in vivo experiments on NOD/SCID mice revealed elimination of BCSCs.

Another groundbreaking study by Ginestier et al contributed a novel identification of CXCR1, a receptor for interleukin-8 (IL-8) produced at the time of chronic inflammation and tissue injury that triggers growth of stem cells.⁸⁶ Tumors, on exposure to chemotherapy, produce IL-8, which stimulates replication of CSCs. In vitro experiments proved successful blockage of CXCR1 by CXCR1-specific blocking antibody or repertaxin (a drug originally developed for prevention of rejection of organ transplants), showing selective elimination of 2 human breast cancer cell lines. Further in vivo experiments in NOD/SCID mice revealed a 75% decrease in populations that stained positive with the ALDEFLUOR assay on treatment with repertaxin alone or with repertaxin in combination with docetaxel, preventing metastasis.

As discussed earlier in this article, the stem cell signaling pathways play important roles in tumorigenesis and CSC renewal and maintenance. Hence pathway inhibitors are constantly developed to check the progress of oncogenesis. Recently, Chia and Ma reported a phase I trial using a drug named GDC-0449 in combination with other molecules for inhibiting the Hh pathways for the treatment of breast cancer.⁸⁷ Also, Lu et al⁸⁸ reported that salinomycin inhibits the Wnt/β-catenin signaling pathway by blocking the phosphorylation of Wnt coreceptor LRP6 and inducing its degradation.

Although new chemically developed compounds are regularly making their way into CSC research, a group of researchers from Asia has reported some noteworthy results from medicinal herbs. A chemical component called ginsenoside F2 from a widely recognized medicinal herb called ginseng has been tested for its efficacy against BCSCs for the first time. Extensive research led to the findings that ginsenoside F2 suppressed BCSC proliferation through an intrinsic apoptotic pathway along with protective autophagy through phosphorylation of tumor suppressor p53. The cell death–inducing activity of F2 was compared with well-known phytochemicals such as quercetin, baicalein, tangeretin, and nobiletin in BCSCs and MCF-7 cell lines. Results showed that F2 kindled a dose-dependent reduction in BCSC viability at 24 hours, whereas the phytochemicals had a very weak suppressive effect on BCSC proliferation, thereby suggesting further large-scale investigations of F2-induced cell death activity in BCSC populations.⁸⁹

In recent years, pharmaceutical companies have also shown a great concern toward drug discovery targeting CSCs to eliminate them completely with time. In 2009, Merck & Co developed a new breast cancer experimental drug called MK-0752 that kills BCSCs that show resistance to radiation and chemotherapy.⁹⁰ Researchers also reported that a specific drug, MK-0752, a γ-secretase inhibitor, blocks the Notch pathway on which BCSCs are dependent for their survival. They injected mice with human breast cancer cells, and those mice grew tumors that were identical to the ones in women. Thereafter, with a combination of Merck's MK-0752 and chemotherapy, researchers were able to hit those CSCs. This led to a successful conclusion of the phase I clinical trial in which they administered this drug to 35 women with advanced breast cancer; breast biopsy samples obtained before and after MK-0752 treatment showed a significant reduction in the number of BCSCs. Researchers also mentioned that they need to test this drug in larger trials of women with advanced breast cancer to see the effectiveness of this drug in destroying the CSCs permanently.

Although a range of BCSC-targeted therapies are under way, with some currently being tested in large-scale clinical trials, targeting CSCs might be better achieved by nanobiotechnological approaches, because this mode of treatment provides a more targeted delivery of essential compounds to the underlying CSC populations without intoxicating the normal healthy cells, providing a faster mode of treatment. Recently, Zhang et al conducted tests in MCF-7 cancer stem cells, MCF-7 cancer stem cell mammospheres, and relapsed tumor by MCF-7 cancer stem cell xenografts into NOD/SCID mice by developing mitochondria-targeting daunorubicin plus quinacrine liposomes to target and prevent breast cancer relapse arising from MCF-7 cancer stem cells.⁹¹ Test results demonstrated important features such as high encapsulation efficiency, well-distributed particle size, delayed drug release preventing rapid drug leakage for timely release of drugs during blood/lymphatic circulation of liposomes. Also seen were increased mitochondrial drug uptake through selective accumulation into mitochondria, induced opening of mitochondrial permeability transition pores, decreased mitochondrial membrane potential, release of cytochrome C from mitochondria to cytosol followed by activation of caspase-9 and caspase-3; all of this occurred through the activation of proapoptotic Bax protein from the Bcl-2 family, leading to apoptosis of MCF-7 cancer stem cells. Comparison of the efficacy of mitochondrial targeting of daunorubicin plus quinacrine liposomes with other liposomes used in this study for relapsed tumor arising from MCF-7 cancer stem cells revealed tumor inhibitory ratios of 44.2% ± 6.0% (daunorubicin liposomes), 28.1% ± 7.7% (quinacrine liposomes), 44.9% ± 3.8% (antiresistant daunorubicin plus quinacrine liposomes), and 79.3% ± 1.7% (mitochondria-targeting daunorubicin plus quinacrine liposomes), thereby giving researchers a good amount of hope for clearing both cancer cells and CSCs and thereby preventing relapse from residual CSCs after therapy, motivating future large-scale investigations.

Similarly some researchers in a recent study reported developing octreotide (Oct)-modified paclitaxel (PTX)-loaded PEG-b-PCL polymeric micelles (Oct-M-PTX) and salinomycin (Sal)-loaded PEG-b-PCL polymeric micelles (M-SAL) for driving out both breast cancer cells and BCSCs. Various formulations of drug-loaded micelles (M-PTX, Oct-M-PTX, M-SAL) were used in this study for comparative analysis of somatostatin receptor (SSTR)-overexpressing MCF-7 breast cancer cells. Investigations showed a more effective BCSC suppression in vivo by M-SAL compared with free SAL. However the inhibitory effect of Oct-M-PTX was stronger compared with the M-PTX formulation, and this inhibition increased further with the use of combination therapy (Oct-M-PTX and M-SAL) against the MCF-7 cells. Furthermore, the efficacy of this combination therapy both in vitro and in vivo experiments revealed eradication of both nonstem cancer cells and CSCs, elucidating a possible use of this combination therapy for treating SSTR-overexpressing breast cancers.²⁴

Heat shock proteins (Hsps) such as Hsp90, which are well known as molecular chaperones, are often found to be overexpressed in a range of cancers, including MCF-7 breast cancers, as higher Hsp levels permit the cancerous cells to tolerate the lethal oncogenic mutations from within, thus making these Hsps an ideal target for anticancer therapies.⁹² Very recently, some researchers from Taiwan made an important discovery revealing the overexpression of Hsp90α (an isoform of Hsp90) in ALDH⁺ BCSCs; also suggesting a decrease in the ALDH⁺ population can be achieved by a siRNA gene silencing–mediated Hsp27 knockout in combination with geldanamycin (GA), a benzoquinoid ansamycin antibiotic. Previous investigators have reported the release of heat shock factor-1 (HSF-1) along with upregulation of some other Hsps such as Hsp27 and Hsp70 after Hsp90 inhibitory treatment, all of which are collectively thought to cause resistance to Hsp90 inhibitory treatment. Thus their siRNA approach of knocking down the Hsp27 expression demonstrated a 20.3% ALDH⁺ reduction that rose to 63.7% in combination with 40 nM GA, suggesting their possibilities in future clinical applications.⁹³

However despite the aforesaid innovative contributions toward eliminating BCSCs, there have been some very recent noteworthy explorations for carrying out BCSC targeting. Some researchers have reported the use of novel phosphosulindac (OXT-328; PS), a derivative of sulindac (a nonsteroidal antiinflammatory drug) that selectively kills BCSCs both in vitro and in vivo by targeting and inhibiting Wnt/β-catenin and EMT signaling pathways. Cell lines used in this study were HMLER/HMLER^TGFβ1, HMLER^shCTRL/HMLER^shECAD and MDA-MB-231; all of which were allowed to gain stem-like properties by different approaches, except the HMLER^shCTRL (HMLER cells transfected with control vector) and MDA-MB-231 (derived from triple-negative human breast cancer with an aggressive mesenchymal phenotype). In vitro results demonstrated impairment of mammosphere formation of BCSCs, leading to drastic reduction in the number of mammospheres formed. Further investigation revealed the downregulation of stem cell–related genes (Oct-4, Bmi-1, Sox-2, Nanog, nestin, Notch-1, ABCG2, and c-Myc) involved in maintaining the stem-like properties of CSCs by a greater percentage compared with salinomycin (used for targeting BCSCs). Reduction in β-catenin levels was seen after PS treatment. Expression of Snail (zinc finger transcription factor), which induces EMT, was also inhibited by PS through the enhancement of GSK-3β promoting ubiquitination and degradation of Snail. Further blocking of TGF-β1–mediated EMT induction by PS, inhibiting BCSC production, was also reported. The in vivo findings with regard to PS-mediated inhibition of BCSCs were somewhat similar to those of the in vitro results. Thus the ability of PS to inhibit BCSCs in a cytotoxicity-independent manner (an eye-catching feature) over a prolonged treatment period (12 days), leading to more selective and efficient killing, shows a bright future for upcoming large-scale trials as an anti–breast cancer therapy.⁹⁴

Very recently, some researchers found that basal/mesenchymal tumorospheres exhibit overexpressed mevalonate metabolic pathway enzymes (responsible for protein farnesylation, synthesis of cholesterol, protein geranylgeranylation [GG]). In vitro and in vivo findings suggested the involvement of protein GG in the maintenance of BCSCs. Evaluation of 2 important mevalonic acid metabolism-associated enzymes such as 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and farnesyl diphosphate farnesyltransferase 1 (FDFT1)) detected their presence in tumorosphere samples, suggesting a possibility of an important functioning basal/mesenchymal BCSC. After treatment with a small-molecule geranylgeranyl transferase (GGTI)-specific inhibitor GGTI-288 (GGTI), CSC assays found a reduction of BCSCs (< 0.3% staining positive by ALDEFLUOR assay in GGTI-treated cells) and also a reduction in the formation of primary and secondary tumorospheres. In vivo analysis of primary human breast cancers (T226, T214) in mice xenografts either with GGTI (100 mg/kg/d for 2 weeks) or docetaxel (10 mg/kg/wk for 2 weeks) or a combination of both revealed a decrease in cell populations staining positive by ALDEFLUOR assay by > 70% in GGTI-treated tumors compared with the docetaxel-treated xenografts in which an increase in positive cells by ALDEFLUOR assay was reported for T226 tumors. For the xenografts treated with combination therapy (GGTI + docetaxel), docetaxel-induced CSC expansion was inhibited by GGTI. Thus it was concluded that GGTI targeted the BCSCs specifically, and this was partly mediated by RHoA/P27^kip1, signaling thereby highlighting the identification and future evaluation of an important CSC pathway for therapeutic targeting.⁹⁵

Thus culmination of all these new discoveries and approaches is surely going to amplify the next generation of cancer research across several orders of magnitude, with the hope of a more targeted CSC therapeutic treatment for possible elimination of CSCs, making way to the complete cure of this disease. As said by Winquist et al,⁹⁶ we need to disengage the locomotive engine ie, the CSC population in this case, which would not bring down the apparent size of the freight train but would disable any further progress of the train ie, growth and metastasis.