Arch. Pharm. Res. DOI 10.1007/s12272-015-0628-1

REVIEW

Existing drugs and their application in drug discovery targeting cancer stem cells Junfang Lv1 • Joong Sup Shim1

Received: 13 April 2015 / Accepted: 28 June 2015 Ó The Pharmaceutical Society of Korea 2015

Abstract Despite standard cancer therapies such as chemotherapy and targeted therapy have shown some efficacies, the cancer in many cases eventually relapses and metastasizes upon stopping the treatment. There is a small subpopulation of cancer cells within tumor, with specific characters similar to those found in stem cells. This group of cancer cells is known as tumor-initiating or cancer stem cells (CSCs), which have an ability to self-renew and give rise to cancer cell progeny. CSCs are related with drug resistance, metastasis and relapse of cancer, hence emerging as a crucial drug target for eliminating cancer. Rapid advancement of CSC biology has enabled researchers to isolate and culture CSCs in vitro, making the cells amenable to high-throughput drug screening. Recently, drug repositioning, which utilizes existing drugs to develop potential new indications, has been gaining popularity as an alternative approach for the drug discovery. As existing drugs have favorable bioavailability and safety profiles, drug repositioning is now actively exploited for prompt development of therapeutics for many serious diseases, such as cancer. In this review, we will introduce latest examples of attempted drug repositioning targeting CSCs and discuss potential use of the repositioned drugs for cancer therapy. Keywords Cancer stem cell  Drug repositioning  Niclosamide  Metformin  Choloroquine

& Joong Sup Shim [email protected] 1

Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, SAR 999078, China

Introduction Cancer is the commonest fatal disease, which is currently estimated to affect one in three people during their life. Although a significant progress has been made in developing cancer therapeutics during the last century, cancer remains one of the leading causes of death in the world (Siegel et al. 2014). Drug resistance is one of the commonest problems in cancer therapy, which impairs the effectiveness of anti-cancer drugs and exerts significant impact on survival rate of cancer patients. Various factors result in drug resistance, such as genetic mutation, metabolic changes and cancer stem cells (CSCs). CSCs are a small population of cancer cells within tumors, with an ability to self-renew and give rise to progeny that differentiate into diverse tumor cells to drive tumorigenesis (Rosen and Jordan 2009). Although the hypothesis of CSCs has been put forward from a long time ago, the actual existence of CSCs was experimentally proven in the early 1990s on the basis of advances in study of cell surface markers of stem cells (Spangrude et al. 1988). Lapidot et al. first isolated and identified the leukemia stem cells from human acute myeloid leukemia (AML) by using fluorescence-activated cell sorting (FACS) with the hematopoietic stem cell marker, CD34?/CD38- (Table 1). The enriched CD34?/CD38- fraction from human patients was able to produce large numbers of AML progenitor cells in severe-combined immunodeficient (SCID) mice (Lapidot et al. 1994). This study for the first time demonstrated that CSCs exist in cancer cell population and are capable of self-renewing and giving rise to differentiated cancer cells. By using similar techniques, CSCs in solid tumors have been identified from the early 2000s. In 2003, Al-Hajj et al. prospectively identified and isolated CSCs in human breast cancer cells. Since then CSCs have been

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J. Lv, J. S. Shim Table 1 Tumor types and cancer stem cell-related markers Tumor type

Cancer stem cell-related markers

References

Acute myeloid leukemia

CD34?CD38-

Lapidot et al. (1994)

Breast

CD44?CD24-/low Lineage-; CXCR4?; CD44?

Al-Hajj et al. (2003); DeCastro et al. (2015); Tseng et al. (2014)

Brain Skin

CD133? CD133?Nestin?

Singh et al. (2004) Sabet et al. (2014)

Head and neck

CD44?; ALDH1?

Prince et al. (2007)

Colon

CD133?; EpCAM(high)CD44?CD166?; ALDH1?

O’Brien et al. (2006); Ricci-Vitiani et al. (2006); Dalerba et al. (2007); Huang et al. (2009)

Pancreas

CD133? CXCR4?; CD44?CD24?ESA?

Hermann et al. (2007); Li et al. (2007)

Lung

CD133?; Sca-1?CD45-PECAM-

Eramo et al. (2008), Kim et al. (2005)

Bladder

MAGE-A3?

Yin et al. (2014)

Prostate

CD44?a2b1high; CD133?

Collins et al. (2005)

Liver

CD90?

Yang et al. (2008)

?

Ovarian

CD133

Melanoma

ABCB5?

Schatton et al. (2008)

Metastatic melanoma

CD20?

Fang et al. (2005)

Bone

Stro1?CD105?CD44?

Gibbs et al. (2005)

Nasopharynx

CD24?

Yang et al. (2014)

Curley et al. (2009)

found in many other solid tumors, such as prostate cancer, brain cancer, pancreatic cancer and colorectal cancer using the CSC-specific markers (Singh et al. 2004; Collins et al. 2005; O’Brien et al. 2006; Ricci-Vitiani et al. 2006; Li et al. 2007) (Table 1).

Drug resistance mechanisms in CSCs CSCs are resistant to the majority of anticancer chemotherapy and to many targeted therapy drugs, thus posing a significant clinical challenge. Using the leukemia targeted therapy drug, imatinib, as a model, Michor et al. (2005) elaborated how CSCs exert drug resistance and tumor relapse against the current therapies. Treatment of patients with imatinib with long-term follow-ups demonstrated that there was a biphasic decrease in the number of leukemia cells; a rapid decline in the number of differentiated leukemia cells and a slow rate of decline in the number of leukemia progenitor cells. However, leukemia stem cells have not been eradicated during the treatment course and caused rapid relapse of the leukemia by fully reconstituting heterogeneous cancer cell populations after the end of the treatment. The authors concluded that finding mechanisms for drug resistance in CSCs would be critical for the development of future drugs that are capable of completely eliminating cancer from patients.

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Extensive studies in CSC biology have revealed some critical mechanisms of drug resistance in CSCs (Visvader and Lindeman 2008; Abdullah and Chow 2013). Overexpression of ATP-binding cassette (ABC) transporters and aldehyde dehydrogenase (ALDH) was often observed in CSCs (Vinogradov and Wei 2012). ABC transporters are expressed mainly in the cell membrane and mediate CSC drug resistance by effluxing chemotherapy drugs from the cells (Dean 2009; Chen et al. 2013). ALDH was found to play an important role in self-protection, differentiation, and expansion of CSCs and confer resistance to specific chemotherapy drugs such as cyclophosphamide and gemcitabine (Ma and Allan 2011; Abdullah and Chow 2013). In addition to those proteins overexpressed, some key signaling pathways are critical for CSC survival and drug resistance, including JAK/STAT (Abubaker et al. 2014), Notch (Abel et al. 2014), Sonic Hedgehog (SHH) (Liu et al. 2006), WNT (Cai et al. 2013), MAPK/ERK (Ahn et al. 2013), NF-jB (Jiang et al. 2014) and TGF-b (Oktem et al. 2014) pathways (Table 2). Among them, Notch, WNT and SHH pathways are the most common in CSCs and are known to contribute drug resistance (Abdullah and Chow 2013). Hence, inhibition of these signaling pathways is important for eliminating CSCs and CSC-driven drug resistance. Several agents that specifically target key signaling pathways of CSC have shown promising anticancer activities by drug alone or combination with standard

Existing drugs and their application in drug discovery targeting cancer stem cells Table 2 Signaling pathways involved in self-renewal and drug resistance in cancer stem cells Pathways

Cancer types

Drug resistances

JAK/STAT

Ovarian cancer

Paclitaxel resistance

Notch

Lymphoblastic leukemia; breast cancer; colon cancer

Cisplatin, doxorubicin and paclitaxel resistance

SHH

Basal-cell carcinoma; glioma; pancreatic cancer; prostate cancer

Docetaxel, methotrexate and etoposide resistance

WNT

Lymphoblastic leukemia; colorectal cancer; liver cancer

Imatinib, cisplatin, doxorubicin and paclitaxel resistance

MAPK/ERK

Breast cancer, lung cancer; prostate cancer

Doxorubicin, paclitaxel and fluorouracil (5-FU) resistance

NF-jB

Breast cancer; ovarian cancer; epidermoid carcinoma

Paclitaxel and cisplatin resistance

TGF-b

Breast cancer; pancreatic cancer; chronic myelocytic leukemia

Paclitaxel, gemcitabine and imatinib resistance

chemotherapy in animal models and some of them are under clinical investigations (Chen et al. 2013). For example, vismodegib, a SHH inhibitor developed by Curis/ Genentech was recently approved by US Food and Drug Administration (FDA) for the treatment of basal-cell carcinoma (Axelson et al. 2013). Vismodegib is currently undergoing clinical trials for a variety of cancer types as a mono- or combination therapy, including medulloblastoma (ClinicalTrials.gov Identifier-CT Identifier: NCT01601184), metastatic castration-resistant prostate cancer (CT Identifier: NCT02115828), metastatic colorectal cancer (CT Identifier: NCT00636610), ovarian cancer (CT Identifier: NCT00739661), and lymphocytic leukemia (CT Identifier: NCT01944943).

Experimental models of CSCs and drug repositioning Extensive characterization of CSCs has enabled discovery and development of new drugs targeting CSCs. CSCs can be isolated and enriched from cancer cells or tissues by FACS using CSC-specific markers or non CSC-specific markers like Hoechst-exclusion based on overexpression of ABC transporter (Tirino et al. 2012). Although in vitro culture and maintaining CSCs are still challenging, several newly developed technologies have enabled the CSC culture, such as tumor spheroid culture in ultra-low adherent culture plates or 3D culture of CSCs in reconstituted basement membranes such as Matrigel (Kimlin et al. 2013; Weiswald et al. 2015). On the other hand, cells undergoing epithelial mesenchymal transition (EMT) have very similar characteristics to stem cells. These types of cells are resistant to the chemotherapy and have many CSC-like phenotypes such as high expression of stem cell markers and increased ability to form tumor spheres (Mani et al. 2008). Based on this idea, Gupta et al. generated EMT cells from HMLER breast cancer cells, and screened a chemical library containing 16,000 compounds to identify CSC inhibitors. They found potassium ionophores, including salinomycin and nigericin, as CSC-specific inhibitors

(Gupta et al. 2009). Although the finding of CSC-specific inhibitors is exciting, severe human poisoning have been reported with these ionophores, preventing them from further development as anticancer agents for human use (Story and Doube, 2004). Recently, drug repositioning has been gaining increasing attention from both pharmaceutical industry and scientists including cancer researchers. Drug repositioning utilizes existing drugs to identify new pharmacological activities and repurposes them into new disease indications (Ashburn and Thor 2004). As such, drug repositioning is also referred to drug repurposing, drug re-profiling, drug re-tasking or therapeutic switching (Mizushima 2011; Finsterer and Frank 2013; Strittmatter 2014). This approach significantly reduces time and costs associated with drug developmental process and increases success rate, as existing drugs have well-known pharmacokinetic properties and favorable human safety profiles. Particularly, drug repositioning has been increasingly applied for anticancer drug discovery where toxic side effects of conventional chemotherapy have been always an issue. Among such efforts, we have identified a number of candidate anticancer drugs from a library of existing drugs, the Johns Hopkins Drug Library (JHDL) (Shim et al. 2010; Xu et al. 2010; Shim et al. 2012). These candidate anticancer drugs are currently under clinical investigations for various types of cancer. Due to the growing demand for the existing drugs in anticancer drug discovery, a number of collections of existing drugs are now available commercially or noncommercially, which are amenable for high throughput screening (Chong et al. 2006; Diamandis et al. 2007; Doudican et al. 2008; Huang et al. 2011; Hothi et al. 2012; Robinson et al. 2013; Czyz et al. 2014; Ketley et al. 2014; Mei et al. 2014) (Table 3). To extend such drug discovery effort to CSCs, many cancer researchers are now looking into the existing drugs to target CSC signaling pathways to impede the cancer drug resistance and relapse. As this is a quickly growing area, a number of candidate drugs have been identified as CSC inhibitors from existing drugs. Among the candidates,

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J. Lv, J. S. Shim Table 3 Libraries of existing drugs and investigational therapeutics for drug repositioning Drug library

Institutions/companies

Contents

References

NCATS pharmaceutical collection (NPC)

NIH

More than 3000 approved drugs and investigational therapeutics

Huang et al. (2011); Ketley et al. (2014)

Johns Hopkins drug library (JHDL)

Johns Hopkins School of Medicine

More than 3000 approved drugs and investigational therapeutics

Chong et al. (2006); Shim et al. (2010); Shim et al. (2012)

Pharmakon-1600 (spectrum collection)

MicroSource

1600 drugs approved or in clinical studies

Doudican et al. (2008); Hothi et al. (2012)

Prestwick library

Prestwick

1280 approved drugs

Robinson et al. (2013)

LOPAC-1280 library

Sigma-Aldrich

1280 approved drugs and investigational therapeutics

Diamandis et al. (2007)

FDA-approved drug library

Selleckchem

1018 approved drugs

Mei et al. (2014)

SCREEN-WELLÒ FDA approved drug library V2

Enzo Life Sciences

786 approved drugs

Czyz et al. (2014)

we here introduce recent key examples of drug repositioning for anti-CSC drugs.

Niclosamide Niclosamide belongs to a teniacide family of anthelminthic drugs, which is specifically effective against the tapeworm infections. It was approved by US FDA for the treatment of worm infections and has been used in human for more than 50 years (Al-Hadiya 2004). The mechanism of anthelminthic activity of niclosamide has been well illustrated. Niclosamide is known to interfere with the production of adenosine triphosphate (ATP) by inhibiting mitochondria (Pearson and Hewlett 1985; Al-Hadiya, 2004). It uncouples oxidative phosphorylation in the parasite mitochondria, thus inhibiting ATP production without affecting the respiratory chain and ATP synthase (Frayha et al. 1997). In addition to its anthelminthic property, niclosamide has been recognized as an anticancer agent since it was reported to inhibit the growth of various types of cancer. In 2012, Yo and colleagues have isolated CSCs from drug-resistant ovarian cancer cells and grow them in spheroid cultures for drug screening. They screened 1200 existing drugs against the tumor spheroids and identified niclosamide as the most potent and selective inhibitor of ovarian CSCs (Yo et al. 2012). Niclosamide significantly inhibited the CSC growth in mice models and showed anticancer activity in differentiated tumor xenograft models. Similarly, Wang et al. screened the existing drug library against breast CSCs and identified niclosamide as a potential anti-CSC drug. Treatment of niclosamide significantly decreased the area of tumor spheroids and caused considerable increase in apoptosis in the breast CSCs (Wang et al. 2013). Furthermore, niclosamide showed promising anticancer activity in

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mice implanted with the breast CSCs without apparent systemic toxicity. Niclosamide is a multi-targeted drug, which inhibits several cellular signaling pathways important for CSC selfrenewal and expansion (Li et al. 2014). It is known to inhibit WNT signaling pathway by promoting the endocytosis of WNT receptor Frizzled (FZD), down-regulating Dishevelled (DVL) protein, and decreasing the stability of b-catenin and TCF/LEF-driven transcription in U2OS and colon cancer cells (Chen et al. 2009; Osada et al. 2011). In addition, niclosamide decreased the expression and phosphorylation of WNT co-receptor, LRP6 in prostate and breast cancer cells (Lu et al. 2011). On the other hand, niclosamide inhibited CBF-1-dependent transcription in Notch signaling and decreased the expression of target genes such as HES-1, cyclin D1, and c-Myc in leukemia and glioblastoma cells (Wang et al. 2009; Wieland et al. 2013). Niclosamide is also known to inhibit STAT3 signaling pathway by suppressing STAT3 phosphorylation and nuclear translocation in the prostate cancer cells (Ren et al. 2010). By inhibiting STAT3, niclosamide sensitizes antitumor effects of erlotinib and ionizing radiation in drug- and radiation-resistant cancer cells (Li et al. 2013; You et al. 2014). In addition to the inhibition of CSC signaling pathways, niclosamide was shown to inhibit mitochondrial functions. It induced the loss of mitochondrial membrane potential, uncoupled the oxidative phosphorylation and generated mitochondrial reactive oxygen species (ROS) in multiple myeloma (Khanim et al. 2011). A genome-wide gene expression analysis showed that niclosamide disrupted multiple metabolic pathways affecting biogenetics, biogenesis, and redox regulation, thereby inducing intrinsic mitochondrial apoptosis pathway and loss of tumor stemness in ovarian CSCs (Yo et al. 2012). Together, these results demonstrated that niclosamide is a potential anti-CSC drug that has inhibitory effects

Existing drugs and their application in drug discovery targeting cancer stem cells

on multiple CSC signaling pathways (Fig. 1). Based on its potential anticancer activities and favorable safety profile, prompt preclinical and clinical evaluations of niclosamide on cancer treatment are warranted. Metformin Metformin is a synthetic derivative of biguanide class of compounds that were isolated from a medicinal plant called French lilac (Galega officinalis). In the early 1950s, metformin was accidentally found to lower the blood glucose level and was named as ‘‘Glucophage’’ (Bailey and Day 2004). Since this finding, it was approved as an anti-diabetic drug in UK in 1958 and in Canada in 1972 (Lucis

1983). About 20 years later, metformin was approved by US FDA for the treatment of type 2 diabetes and marketed by Bristol-Myers Squibb (Bailey and Turner 1996). By virtue of its favorable safety profile and low cost, metformin has been widely considered as the first-line therapy for type 2 diabetes. However, the precise molecular mechanism to lower the blood glucose level has not been fully elucidated. Metformin was initially known to inhibit mitochondria respiratory complex I, thereby restraining hepatic gluconeogenesis while increasing glucose utilization in peripheral tissues (Owen et al. 2000). Later, it was reported to activate 50 -adenosine monophosphate (AMP)activated kinase (AMPK), an enzyme crucial for insulin signaling and the metabolism of glucose and lipids, and

Fig. 1 Signaling pathways in CSCs and inhibition by the existing drugs. WNT signaling: WNT binding to frizzled (FZD) and lipoprotein receptor-related protein (LRP) activates dishevelled (DVL), which in turn inhibits the b-catenin destruction complex containing adenomatous polyposis coli (APC), glycogen synthase kinase 3 and Axin. Inhibition of the destruction complex causes the accumulation of b-catenin that translocates into nucleus where it binds to the transcription factor T cell factor/lymphoid enhancing factor (TCF/LEF) and activates WNT target genes. Niclosamide is known to inhibit FZD, DVL and LRP and promote b-catenin degradation. Notch signaling: binding of delta-like (DLL) or jagged (JAG) to notch receptor activates Notch and promotes proteolytic cleavage of notch intracellular domain (NICD). NICD then translocates into nucleus, binds to the transcription factor CBF1–Su(H)–LAG1 (CSL) and activates Notch target genes. Metformin is known to downregulates Notch, whereas niclosamide was reported to inhibit CSL-dependent transcriptional activity. JAK/STAT signaling: cytokine binding to receptor induces receptor dimerization and JAK activation. Activated JAK induces STAT phosphorylation, dimerization and translocation into the nucleus to activate transcription. Chloroquine is known to inhibit the expression of JAK2, thereby suppressing JAK/STAT signaling. In addition, niclosamide was reported to inhibit STAT3 phosphorylation and nuclear translocation. SHH signaling: binding of SHH to the negative regulator patched (PTCH) induces smoothened (SMO) activation, which in turn activates Gli transcription factor. Chloroquine is known to inhibit SHH signaling by suppressing SMO production

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hence reduce the gluconeogenic gene transcription (Zhou et al. 2001; Shaw et al. 2005). More recently, Madiraju and colleagues reported that metformin non-competitively inhibited mitochondrial glycerophosphate dehydrogenase (mGPD) activity. The inhibition of mGPD caused an altered hepatocellular redox state and decreased the conversion of lactate and glycerol to glucose, hence inhibiting hepatic gluconeogenesis (Madiraju et al. 2014). Accumulating evidence suggested that metformin has an ability to inhibit CSC self-renewal and expansion. Bao et al. reported that metformin significantly inhibited clonogenicity and sphere-forming ability of drug-resistant pancreatic cancer cells. It also caused disintegration of pancreatospheres, decreased the expression of CSC markers such as epithelial cell adhesion molecule (EpCAM), EZH2, Notch-1, Nanog and Oct4, and reactivated the expression of miRNAs that are typically repressed in pancreatospheres (Bao et al. 2012). Lonardo and colleagues also reported that metformin decreased the expression of CSCs markers and pluripotency-associated proteins in pancreatic CSCs (Lonardo et al. 2013). An exciting observation was that metformin selectively diminished the expansion of pancreatic CSC clones by inducing apoptosis. In contrast, non-CSCs preferentially underwent cell cycle arrest and were not eliminated by the metformin treatment (Lonardo et al. 2013). A recent report showed that CSCs exhibited higher glycolytic activity compared to non-CSCs and high glucose in the microenvironment could increase the percentage of the CSC-like cells among the cancer cell population. Conversely, glucose starvation caused a rapid depletion of CSCs (Liu et al. 2014). These results strongly suggest that glucose homeostasis is critical for CSC survival and explain why CSCs show hypersensitivity to the metformin treatment. Based on strong anticancer and anti-CSC activities, metformin is actively investigated in more than hundreds of Phase I or II trials for the treatment of cancer (information available at https://clinicaltrials.gov/). Very recently, the City of Hope Medical Center in collaboration with the National Cancer Institute (NCI) started Phase I study of metformin combined with gemcitabine, paclitaxel and a standardized dietary supplement for the treatment of Stage IV Pancreatic Cancer (CT identifier: NCT02336087). The London Health Sciences Centre also initiated Phase II trials for muscle invasive bladder cancer with metformin in combination with simvastatin, a cholesterol lowering agent (CT identifier: NCT02360618). In addition, University of California, Davis in collaboration with the NCI initiated Phase I trial of enzalutamide and metformin combination in treating patients with castration-resistant prostate cancer (CT identifier: NCT02339168) in 2015.

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Chloroquine Chloroquine is a 4-aminoquinoline compound, which has been used to treat malaria. It was first developed by Hans Andersag, who worked in Bayer, in 1934 with strong antimalarial activity (Krafts et al. 2012). Later in 1947, it was introduced into clinical practice and has been widely used as a first-line therapy for the treatment and prevention of malaria. The mechanism of anti-malarial activity of chloroquine was well-illustrated. In red blood cells, the malarial parasites digest hemoglobin to obtain essential amino acids from the molecule. To avoid the metabolic toxicity, the parasite biocrystallizes the toxic byproduct heme to form nontoxic molecule hemozoin. This process occurs in an acidic organelle vacuole. Chloroquine, as a weak base, is accumulated in a vacuole and prevent biocrystallization of heme, thus causing heme toxicity to the parasite (Hempelmann 2007). In addition to antimalaria activity, chloroquine has the potential effects on other diseases, such as rheumatoid arthritis (Augustijns et al. 1992), lupus erythematosus (Meinao et al. 1996) and cancer (Solomon and Lee 2009). In mammalian cells, it is known to inhibit autophagy, a process of the breakdown of unnecessary cellular components, by blocking lysosomal degradation (Kimura et al. 2013). Recent evidences suggest that chloroquine has a strong anti-CSC activity. Choi et al. demonstrated that chloroquine has an ability to eliminate CSCs in triple negative breast cancer. This group analyzed gene expression signatures of the CD44?/CD24-/low cell populations and identified the candidate drugs targeting CSCs through in silico analysis. Chloroquine was amongst the candidates and chosen for further in vitro and in vivo validations due to its favorable safety profile. The drug sensitized paclitaxel treatment to the triple negative breast cancer and decreased CSC population in both preclinical and clinical settings (Choi et al. 2014). Inhibition of JAK/STAT3 signaling pathways was proposed as a potential mechanism of anti-CSC activity of chloroquine in triple negative breast cancer (Fig. 1). On the other hand, Balic and colleagues recently screened existing drugs to identify drugs that inhibit primary pancreatic CSCs. The screen identified chloroquine as a potential anti-CSC drug. Chloroquine significantly decreased the CSCs in vitro and diminished tumorigenicity and invasiveness of a large panel of pancreatic cancer in vivo (Balic et al. 2014). Moreover, combination of chloroquine with gemcitabine more effectively eradicated pancreatic cancer in vivo and improved overall survival of the animals. The drug was found to inhibit CXCL12/CXCR4 signaling, which led to the inhibition of STAT3. In addition, it strongly suppressed SHH signaling by decreasing the SMO production, which

Existing drugs and their application in drug discovery targeting cancer stem cells

resulted in the inhibition of EMT in the pancreatic CSCs (Fig. 1) (Balic et al. 2014). The promising anticancer activity of chloroquine and its inhibitory effects on CSC signaling pathways promptly brought the drug into multiple clinical investigations. Phase II clinical trial of chloroquine has started in 2015 for the treatment of invasive breast cancer by the Ottawa Hospital Research Institute (CT identifier: NCT02333890). Another Phase II trial of chloroquine in combination with taxane chemotherapy was recently initiated by the Methodist Hospital System for the treatment of advanced metastatic breast cancer patients who have failed anthracycline based therapy (CT identifier: NCT01446016). The University of Cincinnati also initiated Phase I trial of chloroquine in combination with standard chemotherapies, carboplatin and gemcitabine for the treatment of advanced solid tumors in 2014 (CT identifier: NCT02071537). In addition to these, more than 10 clinical studies are ongoing with the chloroquine as a mono- or combination therapy for the treatment of various types of human cancer.

Concluding remark CSCs are resistant to many of current standard chemo- and targeted therapies that target proliferating differentiated tumor cells. The survival of a small fraction of CSCs can eventually cause cancer relapses that are even more detrimental to the patients. In virtue of the explosive advances in stem cell biology during the past ten years, several key factors and signaling pathways critical for CSC self-renewal and drug resistance have been elucidated. Inhibition of such signaling pathways became a promising strategy not only to inhibit CSC survival, but also to completely eradicate tumor by combination with current standard therapies. Existing drugs that target such CSC signaling pathways would be the most suitable candidate for the combination partner of the current standard therapy to overcome drug resistance and eliminate cancer. However, there are many challenges for drug repositioning targeting CSCs. As the original development of existing drugs were not intended to target CSCs, dose limiting off-target effects would be an issue. In addition, since CSCs are buried in a large number of proliferating progenies and complex tumor microenvironment, efficient diffusion and penetration of the drugs to reach the CSCs are critical. Optimization of the repositioned drugs to improve the target selectivity and drug penetration without changing the bioavailability and safety profile would be necessary. Nevertheless, given that existing drugs are an increasing source for prompt drug discovery, which expand every year, drug repositioning will be continuously exploited for anticancer drug discovery targeting CSCs.

Acknowledgments This work was supported by the Science and Technology Development Fund (FDCT) of Macau SAR (FDCT/119/ 2013/A3), Matching Research Grant (MRG002/JSS/2015/FHS) and Multi-Year Research Grant (MYRG2015-00181-FHS) of the University of Macau. Compliance with Ethical Standards Conflict of interest The authors have declared that there are no conflict of interest.

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