Chemical approaches to targeting drug resistance in cancer stem cells.

Drug Discovery Today Volume 00, Number 00 May 2014

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Chemical approaches to targeting drug resistance in cancer stem cells Q1

Panagiota A. Sotiropoulou1,4, Michael S. Christodoulou2, Alessandra Silvani2, Christel Herold-Mende3,4 and Daniele Passarella2,4 1 Interdisciplinary Research Institute (IRIBHM), Universite´ Libre de Bruxelles (ULB) 808, route de Lennik, BatC, Bruxelles 1070, Belgium 2 Dipartimento di Chimica, Universita` degli Studi di Milano, Via Golgi 19, Milano 20133, Italy 3 Division of Experimental Neurosurgery, Department of Neurosurgery, Ruprecht-Karls-Universita¨t Heidelberg, Heidelberg, Germany

Cancer stem cells (CSCs) are a subpopulation of cancer cells with high clonogenic capacity and ability to reform parental tumors upon transplantation. Resistance to therapy has been shown for several types of CSC and, therefore, they have been proposed as the cause of tumor relapse. Consequently, much effort has been made to design molecules that can target CSCs specifically and sensitize them to therapy. In this review, we summarize the mechanisms underlying CSC resistance, the potential biological targets to overcome resistance and the chemical compounds showing activity against different types of CSC. The chemical compounds discussed here have been divided according to their origin: natural, natural-derived and synthetic compounds. Introduction The first link between cancer and stem cells was established during the 19th century based on histological similarities between tumors and embryonic tissues. The observation that teratocarcinomas comprised immature and more differentiated cells led to two key assumptions of the CSC hypothesis: (i) not all tumor cells are identical with regard to their phenotypic and biological behavior and (ii) tumors contain a subpopulation of tumor cells that are endowed with central properties of normal stem cells, such as self-renewal and the ability to produce more differentiated daughter cells. In addition, the CSC model suggests that tumors are organized similarly to normal tissues, with a cellular hierarchy in which CSCs drive tumor growth because they are endowed with increased therapeutic resistance [1,2].

CSC versus the stochastic tumor growth model The first convincing data demonstrating the existence of phenotypically distinct, hierarchically organized subpopulations of tumorigenic and nontumorigenic cells were obtained in acute Corresponding author: Passarella, D. ([email protected]) 4

These authors contributed equally to this article.

Peggy Sotiropoulou received her BSc in Biology from the University of Athens in 2000, followed by a PhD in cancer immunology from the Medical School of the University of Crete in 2005. Dr Sotiropoulou performed a postdoctoral fellowship at the Cancer Immunology and Immunotherapy Center of Athens, followed by a postdoctoral internship in the lab of Professor Blanpain in the Universite´ Libre de Bruxelles. Dr Sotiropoulou was appointed to a tenured position as chercheur qualifie´ of the FNRS in 2010. Her team is studying the mechanisms of genome maintenance in stem cells and cancer stem cells. Christel Herold-Mende graduated from the University of Heidelberg, Germany in biology and chemistry and did her PhD in the ENT Department of the University of Heidelberg in 1995. She has worked as a research fellow at the German Cancer Research Center, Department of Cytopathology and at the Department of Applied, Experimental and Interdisciplinary Oncology, Ruhr University, Bochum, Germany. In 1996, she started the Molecular Cell Biology Group in the ENT Department and the Molecular Laboratory at the Department of Neurosurgery of the University of Heidelberg, focusing on immunotherapies, identification of biomarkers and characterization of tumor stem cells in head and neck tumors and gliomas. In 2006, she became head of the Division of Neurosurgical Research and, in 2012, professor of Experimental Neurosurgery at the University of Heidelberg. Daniele Passarella has been an associate professor in the Department of Chemistry at the University of Milan since 2006. He obtained his PhD in Chemistry from the University of Milan in 1991. After a postdoctoral fellowship at the University of Barcelona, he obtained a permanent position as researcher in the University of Milan in 1993. His research focuses on organic and bioorganic chemistry with the specific aim of designing and preparing new anticancer compounds. From 2007 to 2011, he was the coordinator of European Cooperation in Science and Technology (COST) Action CM 0602 ‘Inhibitors of angiogenesis: design, synthesis and biological exploitation’ and is currently coordinating COST Action CM 1106 ‘Chemical approaches to targeting drug resistance in cancer stem cells’.

www.drugdiscoverytoday.com 1 Please cite this article in press as: P.A. Sotiropoulou, et al., Chemical approaches to targeting drug resistance in cancer stem cells, Drug Discov Today (2014), http://dx.doi.org/10.1016/ j.drudis.2014.05.002

1359-6446/06/$ - see front matter ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2014.05.002

Reviews KEYNOTE REVIEW

Teaser Reviewing the small molecules that showed efficacy against CSCs is of crucial importance to stimulate the design of new promising anticancer compounds.

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myeloid leukemia (AML). Extensive studies over decades and substantial technical progress led to the conclusion that leukemic stem cells from patients with AML not only are self-maintaining, but also reconstitute the full spectrum of phenotypes consistent with the CSC model (reviewed in [1]). Meanwhile, similar observations were reported for other tumor types, including chronic myeloid leukemia (CML), breast cancer and glioma (reviewed in [4]). Moreover, in a couple of recent elegant lineage-tracing experiments, the hierarchical organization was confirmed in epidermal and colon tumors by following the fate of individual tumor cells [2]. Moreover, Parada and coworkers demonstrated not only a similar hierarchical organization in a genetically engineered mouse glioma model, but also that the relative quiescent subset of tumor cells with stem-like properties gave rise to new tumor cells after treatment with the standard therapeutic drug temozolomide (TMZ); thus, this subset of cells is responsible for the wellknown drug resistance of gliomas [3]. Given that there is still controversy over whether the CSC hypothesis is able to explain fully tumor growth and heterogeneity in all tumor types, occasionally the more neutral terms ‘tumorinitiating’ (TIC) or ‘tumor-propagating’ cells are used only to refer to the increased tumorigenicity and the tumor-repopulating capacity observed after xenotransplantation of this immature tumor cell subpopulation. The so-called ‘stochastic model’ represents an alternative approach to explain tumor growth. In contrast to the CSC model, it claims that all tumor cells are equipotent and stochastically self-renew or differentiate, thus resulting in tumor heterogeneity. For example, in melanomas, the stochastic model rather than the CSC model might better explain the extraordinary high frequency of TICs [2,4]. The occurrence of clonal evolution, which is regarded as a central event in the development of tumor heterogeneity and is defined as the continuous accumulation of mutations responsible for survival advantages in individual tumor cells, is in agreement with both tumor growth models. As an inevitable consequence of clonal evolution, especially for latestage tumors, deep sequencing approaches revealed a high number of subclones that, in the case of CSC-driven tumors, indicates the generation of different types of CSC within the same tumor that are presumably endowed with different drug-resistance phenotypes. In addition, it strongly implicates the need to target all CSC subtypes simultaneously to achieve more effective tumor treatment [2,4].

Plasticity of CSCs The putative occurrence of several types of CSC in the same tumor complicates the situation in terms of promising treatment targets. Moreover, there is increasing evidence that the ability of CSCs to contribute to tumor growth is plastic. An intensively studied example of this plasticity is the reversible transition between epithelial and mesenchymal states in carcinomas (EMT), which can have a marked impact on their growth properties. The acquisition of mesenchymal properties is associated with the loss of adhesion molecules and increased migratory properties, and its occurrence in cancer cells results in more aggressive tumors. Although the functional role of EMT has not been fully elucidated, many EMT markers are expressed by cells with ex vivo stem cell properties. For instance, the EMT marker Twist-related protein 1 (TWIST1) increases mammosphere formation in vitro and growth 2


of secondary tumors after xenotransplantation in vivo. Accordingly, several studies on breast, pancreatic and colorectal cancers came to the conclusion that EMT generates cells with CSC characteristics and, thus, these acquire a multidrug resistance phenotype [2]. Importantly, the observation of plasticity in tumor cells does not contradict the CSC model because it can also be found in normal stem cells. For instance, in differentiated nontumor cells, expression of four transcription factors was sufficient to revert them fully to an immature embryonic stem cell-like state, which is in disagreement with an entirely irreversible, deterministic phenotype [1]. Additionally, it has been found that plucking-induced depletion of hair follicle stem cells in skin epidermis is followed by repopulation of more differentiated progenitor cells that reacquire stem cell properties [2]. Another indication that properties of CSCs are not necessarily irreversible comes from studies where CSCs were exposed to therapeutic drugs in vitro. The observed adaptive response from sensitive to resistant progenies during cultivation suggests that multidrug resistance also represents a plastic property. However, because most studies analyzing plasticity of CSCs were performed on cultivated cells, it is still not clear to what extent a transition from sensitive to resistant and nontumorigenic to a tumorigenic state occurs [4].

Influence of niches on stem cell properties Recently, the microenvironment of CSCs and normal stem cells has gained much attention because it is supposed to be an important regulator of stem cell properties and, therefore, an additional source of tissue heterogeneity. It has been shown that maintenance of an immature phenotype, the balance between selfrenewal and differentiation, as well as multidrug resistance is not only controlled by intrinsic signals, but can also be markedly influenced by direct interaction with neighboring cells or in an indirect manner by binding to proteins secreted from these cells. This specific microenvironment where CSCs and normal stem cells reside in is called the ‘niche’. Although the exact cellular and molecular composition of distinct CSC and normal stem cell niches is still under investigation, two types of niche, the perivascular and the hypoxic niches, seem to be of major importance for the maintenance of stem cell properties and, thus, the preservation of a multidrug resistance phenotype. In the perivascular niche, endothelial cells are one of the main cell types. For instance, in the brain, both normal neural stem cells and glioma CSCs are located close to blood vessels and their immature phenotype is dependent on this niche. In line with this observation, stem cell properties of glioblastoma CSCs can be markedly downregulated through an antiangiogenic treatment resulting in a disruption of the perivascular niche. Similar stemness-promoting effects of the perivascular niche were observed in epithelial tumors [2]. With regard to the hypoxic niche, a crucial step comprises the activation of soluble factors, such as hypoxia inducible factors (HIFs), through lowered oxygen concentration. HIFs were shown to be essential for long-term reconstitution abilities of hematopoietic stem cells, resistance to therapy and tumorigenicity of leukemic stem cells. In line with this, expansion of CSCs has been reported in response to HIFs in brain and pancreatic cancer [2].

www.drugdiscoverytoday.com Please cite this article in press as: P.A. Sotiropoulou, et al., Chemical approaches to targeting drug resistance in cancer stem cells, Drug Discov Today (2014), http://dx.doi.org/10.1016/ j.drudis.2014.05.002

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Besides typically representing a minority of total cancer cells, CSCs have been related to resistance to therapy, becoming a cornerstone of treatment failure and consequent relapse. This review summarizes the mechanisms used by CSCs to survive anticancer therapy, and the compounds that showed activity against CSCs, with the aim to offer suggestions for the design of new effective anticancer compounds.

Relevant biological targets for CSCs and mechanisms of resistance Here, we discuss the biological targets and the cellular mechanisms responsible for drug resistance in CSCs, as summarized in Table 1.

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Hoechst dye by members of the ABC family, was more tumorigenic in xenotransplantations and resistant to numerous chemotherapeutic drugs in vitro [7]. In addition, the side population from aerodigestive squamous cell carcinoma cell lines preferentially expressed ABCG2 and ABCC1 and was more clonogenic in vitro and tumorigenic in vivo. MDR1 inhibitors have already been in clinical trials, albeit with poor results [8,9]. This could be explained by the fact that cancer cells and CSCs express several members of the ABC family. Importantly, the development of new ABC inhibitors, potentially active against several ABC family members, could be effective in the clinical setting.

Antiapoptotic molecules Drug efflux

CHK1 and CHK2

DNA repair pathways

WEE1

DNA damage checkpoints

MGMT

Direct reversal DNA repair

DNA-PKcs

Nonhomologous end-joining

p21

p53 pathway, cell cycle checkpoint

Gamma-secretase, a-secretase, MAML1 and other Notch family members or ligands

Notch pathway

SMO, GLI1, GLI2, PTCH and other Hh family members

Hedgehog pathway

The apoptotic response to chemotherapy is regulated by the balance of pro-apoptotic (such as BAD, BAX and BAK) and anti- Q2 apoptotic (such as BCL2, MCL1 and BCL-XL) members of the BCL2 family of proteins, which regulate the permeabilization of the outer mitochondrial membrane. Overexpression of antiapoptotic molecules, conferring resistance to therapy, has been shown for several types of CSC. CD133+ glioblastoma CSCs from primary cultures express higher mRNA levels of the antiapoptotic genes BCL2, BCLXL, XIAP and FLIP compared with the CD133 cancer cells [10], and are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death via methylation of the caspase-8 promoter and subsequent suppression of caspase-8 expression [11]. Bcl-2-inhibition sensitized CSC-like cells from glioma cell lines to TRAIL treatment in vitro and in xenograft models [12]. Moreover, treatment of primary glioblastoma cell cultures with miR145 led to suppression of the antiapoptotic genes and sensitized them to temozolomide treatment [13]. Using siRNAs against c-FLIP in mammosphere assays, or cisplatin/TRAIL treatment in breast cancer cell line cultures targeted specifically CSC-like cells [14,15]. Although miRNAs and siRNAs cannot be envisaged in the clinical practice in the near future, these studies could be useful to predict novel druggable targets for the sensitization of the CSCs to therapy using small molecules. Prostate CSCs from primary cancer cell lines can be sensitized to apoptosis through pharmacological inhibition of antiapoptotic molecule expression, such as Bcl2, survivin and XIAP [16,17]. In clinical trials, monotherapy using death-receptor agonists showed limited efficacy, probably because cells expressing other antiapoptotic factors might drive tumor growth. However, combining these with chemotherapy and other targeted therapy, such as BCL2 antagonists and histone deacetylase inhibitors, holds promise for effectively sensitizing tumors to chemotherapy [18].

TCF4 and AKT

Wnt pathway

DNA-damage response and DNA repair

EGFR

EGF pathway

BMP and BMPR

BMP pathway

Radiotherapy and chemotherapeutic drugs induce genotoxic stress. Regardless of the type of DNA lesion, DNA damage is sensed by highly conserved signaling pathways that constitute the DNA damage response (DDR) pathway [19]. The maintenance of genomic integrity is indispensable for cell survival, yet normal tissues and tumors can differ in their DDR [20]. CSCs have been shown to have enhanced DDR activation as a mechanism to repair DNA damage effectively. One of the mechanisms that CSCs use is higher checkpoint activation and disruption of the ATM/Chk2/p53 pathway, both accelerate tumor growth and contribute to radiation resistance in glioma mouse models [21]. In a pioneer study, Bao

Chemotherapeutic hydrophobic compounds, such as antimetabolites, taxanes and topoisomerase inhibitors, are eliminated by the cell via the ATP-binding cassette (ABC) transporter family, comprising 49 transmembrane proteins, out of which three have been correlated with multidrug resistance, namely multidrug resistance protein 1 (MDR1; ABCB1), multidrug resistance-associated protein 1 (MRP1; ABCC1) and breast cancer resistance protein (BCRP; ABCG2) [5]. The selective expression of several members of the ABC transporter family has been reported in several types of CSC [6]. In lung cancer cell lines, the side population, that is, a selection of cells based on their ability to efflux the TABLE 1

Relevant biological targets responsible for resistance in CSCs Biological target

Pathway

ABCG2, ABCC1 and MDR1

Drug efflux

BCL2, MCL1, BCLXL, c-FLIP, XIAP and survivin

Apoptosis

BMI1

DNA damage response and DNA repair pathways

STAT3

JAK/STAT pathway

IGF1R

IGF pathway

NF-kB, IkBa, IKK and other NF-kB family members

NF-kB pathway

HIF1a and HIF2a

Hypoxia

ALDH1

Alcohol oxidation

Retinoic acid receptor

Retinoic acid pathway

a

Q6 For definitions, see main text.






and colleagues demonstrated that CD133+ CSCs from human gliomas preferentially activate ATM-dependent DNA damage checkpoint upon irradiation, conferring radioresistance to those cells. Importantly, this radioresistance could be reversed using Chk1/Chk2 inhibitors [22]. Indeed, Chk2-knockout glioma mouse models do not develop resistance to radiation therapy [21]. Moreover, CD24+/CD44+/ESA+ CSCs from pancreatic adenocarcinoma xenografts were sensitized to gemcitabine treatment when a Chk1 inhibitor was co-administered [23]. It would be interesting to determine whether WEE1 inhibitors, shown to induce mitotic catastrophe and subsequent cell death in glioblastoma cells exposed to temozolomide and to sensitize glioblastoma tumors to ionizing radiation in orthotopic and invasive glioblastoma mouse models [24], have a specific effect in CSCs. The polycomb group protein BMI1 is upregulated in glioblastoma and head and neck squamous cell carcinoma (HNSCC) CSCs, with a role in the recruitment of components of the DDR, such as ATM, 53BP1 and RNF8. BMI1 ablation sensitizes CSCs to irradiation with a concomitant higher number of DSBs [25,26]. Nonetheless, the outcome of checkpoint inhibition depends on p53 status. It was shown that suppression of ATM or Chk2 in tumor cells with functional p53 protects from genotoxic agent-induced apoptosis [27], whereas p53 status was proven to be a crucial regulator of radiation sensitivity upon BMI1 knockdown in a model of nasopharyngeal cancer [28]. The enhanced DDR and checkpoint activation offer a delay in the cell cycle, providing more time to repair the damaged DNA, as shown in glioblastoma CSC cultures, which presented more effective repair, albeit only because of enhanced checkpoint activation rather than to more effective DNA repair mechanisms per se [29]. However, certain types of CSC have been shown to also exhibit enhanced DNA repair mechanisms. Zhang and colleagues demonstrated that Lin-CD29HCD24H cells from a syngeneic p53 knockout mouse mammary gland tumor model represent CSCs and preferentially express genes involved in DNA repair, such as Brca1 and Xrcc5 [30]. Lin-CD29HCD24H cells exhibited accelerated DSB repair upon irradiation, and pharmacological inhibition of Akt sensitized CSCs to irradiation by inhibiting the Wnt pathway [31]. Higher expression of DNA repair-associated genes, sometimes accompanied by accelerated DNA repair, has also been shown in CSCs from glioblastoma and pancreatic cancer cell lines [10,32]. Stem like-gliomaspheres were resistant to irradiation with less-persistent DSBs, and this was reversed through inhibition of EGFR and Akt phosphorylation [33]. The combination of radiotherapy and the alkylation agent temozolomide is currently the first-line therapy against glioblastoma. Glioblastoma CSCs have been shown to exhibit higher levels of the DNA repair protein O6methylguanine-DNA methyltransferase (MGMT), and siRNA against MGMT sensitized malignant gliomas to temozolomide [34,35]. However, the MGMT-negative glioblastoma cell line KNS42 is resistant to temozolomide, demonstrating MGMT-independent mechanisms to temozolomide resistance [36]. Finally, short hairpin (sh)RNA-mediated inhibition of DNA-PKcs, which is a major component of the nonhomologous end-joining repair mechanism, enhanced radiation-induced autophagic cell death of glioblastoma CSCs from primary cultures [37]. These data demonstrate that compounds targeting DNA repair pathway components in CSCs could provide a means to overcome CSC resistance to therapy. 4


Quiescence Conventional chemo- and radiotherapy are more efficient against cells with high proliferative rates. However, in vitro and in vivo experimental evidence has shown that several types of tumor, including glioblastoma, melanoma, pancreatic, lung and breast cancer, contain slow cycling cells, which have often been correlated with drug resistance. CSCs from ovarian and breast tumors are relatively quiescent [38,39]. Activation of the cell cycle inhibitor p21 in slowly proliferating leukemia CSCs prevents DNA damage accumulation and subsequent exhaustion, and is indispensable for leukemogenesis [40]. In a seminal study, Chen and colleagues used a transgenic mouse model to label slow-cycling cells in gliomas, enabling their temporal-regulated ablation. They showed that slow-cycling cells survive classic temozolomide treatment and initiate relapse. More importantly, when those cells were eliminated, mouse survival was prolonged [3]. Label-retaining cells have also been identified in melanoma cell lines, where they are characterized by the expression of the H3K4 demethylase JARID1B. Knockdown of JARID1B in vitro and in vivo leads to culture exhaustion, reduced tumor growth and fewer pulmonary metastases [41]. Other studies have used dyes, such as DiI and CFSE, to label slow-cycling cells in cancer cell lines and showed that those cells were more resistant to common chemotherapeutic agents [42,43]. Given that quiescence emerges as an important factor of CSC resistance to therapy, novel regimens targeting slow-cycling cells or, conversely, factors inducing CSC proliferation could be proven efficient therapeutic regimens. Accordingly, leukemia CSCs in xenograft models have been sensitized to cell cycledependent chemotherapy with prior treatment with the mitogen GCSF [44] and histone deacetylase inhibitors [45], effectively inducing the CSCs to enter the cell cycle. Moreover, arsenic trioxide sensitized leukemia CSCs to cytarabine in vitro and in xenograft models, probably through induction of the exit from quiescence [46]. These studies indicate that a two-step regimen, including induction of dormant CSCs to enter the cell cycle, followed by chemotherapy, could overcome drug resistance.

Prosurvival signaling Similar to their normal counterparts, CSCs rely on specific signaling pathways, triggered by intrinsic and extrinsic stimuli, to regulate proliferation, survival and the balance between selfrenewal and differentiation. CSCs are capable of activating prosurvival signaling as an adaptive response to therapy. Several signaling pathways associated with cell survival, including the Notch, Hedgehog (Hh), Wnt, Janus kinase/signal transducers and activators of transcription (JAK/STAT), bone morphogenetic proteins (BMP), insulin growth factor (IGF) and nuclear factor (NF)-kB pathways, are preferentially activated in CSCs. Notch signaling regulates cell-to-cell interaction, cell proliferation, differentiation and apoptosis. Notch pathway inhibition has been accomplished by blocking its cleavage using g-secretase inhibitors, impeding ligand–receptor interaction or by interference with the Notch co-activator MAML1 [47]. When treated with g-secretase inhibitors, CSC-like cells from medulloblastoma cell lines underwent apoptosis [48]. Moreover, g-secretase inhibitor treatment enhanced apoptosis specifically in CSC-like cells in glioblastoma cell lines upon radiation, by reducing Akt activity and Mcl-1 expression [49]. These results show that brain tumor


DRUDIS 1405 1–16 Drug Discovery Today Volume 00, Number 00 May 2014

Hypoxic niches Hypoxia is a common characteristic of all solid tumors and is correlated with disease progression, poor prognosis and therapeutic resistance. Hypoxia-induced signaling regulates cell survival, proliferation and differentiation, angiogenesis, metabolism, invasion and metastasis, and is mediated mainly by HIF-1 and HIF-2a, regulators of O2 homeostasis[61]. Zhizhong and colleagues showed that knocking down HIF-1a and/or HIF-2a in primary human glioblastoma CSCs inhibited proliferation and induced apoptosis in vitro and suppressed tumor initiation in xenograft models in vivo [62]. In melanoma cell lines, hypoxia induced Octamer (Oct)-4 expression, which is correlated with decreased differentiation, spheroid formation and resistance to therapy [63], whereas in neuroblastoma, hypoxia stimulated DLK1 expression, which is important to maintain stemness and tumorigenicity [64]. Moreover, hypoxia led to increased expression of vascular endothelial growth factor (VEGF), interleukin (IL)-6 and CSC signature genes, and sphere formation in pancreatic and prostate cancer cell lines [65,66]. In addition, in HNSCC cell lines, hypoxia induced an increase in CD44high/ESAlow CSC-like cells, sphere formation and EMT profile[67]. An increasing number of HIF-1 inhibitors have been reported, several of which are currently been evaluated in clinical trials, whereas others are being tested in preclinical models [68].

Aldehyde dehydrogenase The aldehyde dehydrogenase (ALDH) superfamily comprises 19 enzymes, the ALDH1 family being the most important. ALDH enzymes catalyze aldehyde oxidization and ester hydrolysis, have nitrate reductase activity and are implicated in retinoid acid signaling. ALDH enzymatic activity safeguards the cell against cytotoxic drugs, and ALDH-mediated oxidation of aldophosphamide to carboxyphosphamide represents the main mechanism of cyclophosphamide detoxification [69]. ALDH has been indicated as a marker of CSCs in leukemia and several types of solid tumor, such as breast, prostate, head and neck, ovary, liver, pancreas, bladder and colon cancer [70]. Using tissue microarrays and immunostaining, it has been shown that ALDH expression in human breast and prostate tumors is correlated with poor prognosis [71–73]. More importantly, immunostaining on breast tumor tissues from patients before and after paclitaxel and/or epirubicin treatment showed that ALDH1 expression was associated with resistance to neoadjuvant chemotherapy [74]. Using xenograft models of colorectal tumors, it has been shown that ALDH1-expressing cells survive treatment with cyclophosphamide and Irinotecan, and knocking down ALDH1 with shRNA sensitizes cancer cells to cyclophosphamide [75]. Inhibition of ALDH restored the sensitivity of CSC-like cells from breast cancer cell lines to doxorubicin and/or paclitaxel [76], of glioblastoma cell lines to temozolomide [77] and sensitized a resistant AML cell line to 4-hydroperoxycyclophosphamide [78]. Given that ALDH1 is crucial for both CSC and normal stem cell function, uncovering the pathways linked to ALDH activity is crucial for the development of specific ALDH inhibitors that would mediate CSC sensitization to therapy.

Differentiation resistance Given that the ability to differentiate in various cell types is one of the key properties of normal and neoplastic stem cells, it has been



CSCs are selectively sensitive to Notch pathway modifications. Indeed, phase I and II clinical trials are currently evaluating Notch inhibitors in glioblastoma, leukemia and breast cancer [47]. Hh signaling is involved in stem cell maintenance and tissue polarity. Hyperactivation of the Hh pathway has been implicated in cancer initiation in several tissues, whereas it is specifically activated in CSCs in breast cancer and CML [47]. Blockade of the Hh pathway by cyclopamine reduces the sphere-forming ability and downregulates the stemness transcriptional signature in glioblastoma primary cultures and cell lines, whereas it inhibits tumor growth in xenograft models [50,51]. Numerous clinical trials using inhibitors of the Hh pathway, alone or in combination with classic chemotherapy, are ongoing in a variety of solid tumors, such as breast, prostate, small-cell lung cancer, medulloblastoma and glioblastoma [47]. Wnt signaling regulates cell fate determination during development and tissue selfrenewal during homeostasis, and has been associated with CSC activity [47]. Kendziorra and colleagues have shown that the Wnt transcription factor TCF4 is overexpressed in 5-fluoruracil-resistant colorectal cancer cell lines and siRNA-silencing of TCF4 sensitized them to radiation by compromising DSB repair and impairing the ability to arrest cell cycle [52]. Furthermore, Akt1 and Akt2 were increased upon irradiation specifically in CD44+/CD24 mammosphere-forming cells from the MCF-7 breast cancer cell line, and Akt-inhibitors sensitized exclusively MCF-7 mammosphere cells to radiation [53]. BMP, EGFR, JAK/ STAT, IGF and NF-kB signaling pathways have also been shown to be selectively activated in CSCs, and their inhibition led to abrogation of CSC tumorigenicity [47,54–60]. Specifically, blocking EGF signaling using tyrosine kinase inhibitors suppressed proliferation of CD133+ cells in glioblastoma cell lines [54], whereas forced expression of BMP receptor 1B induced astroglial differentiation and consequent reduction of the in vivo tumorigenicity of glioblastoma CSCs from human tumors [55]. Targeting the JAK/STAT pathway has been shown as a promising treatment in HNSCC and non-small cell lung cancer (NSCLC). Chen and colleagues showed that inhibition of STAT-3 impedes tumorigenicity, sphere formation and BCL-2 expression, and induces apoptosis in CD44+/ALDH1+ CSCs from HNSCC tumors [56]. The same results have been obtained using CD133+ cells from NSCLC specimens, which lost stemness and underwent apoptosis with concomitant decrease of BCL-2 and BCL-XL expression [57]. The IGF signaling pathway has also been implicated in NSCLC CSC resistance to therapy. Specifically, chemoradioresistant CD133+/ALDH1+ cells from NSCLC cell lines were sensitized to radiation upon treatment with IGF-1R siRNA [58]. Lastly, NF-kB inhibitors hindered the stemness properties of CSC-like cells from pancreatic cancer cell lines [59] and altered the stemness transcriptional profile of CSCs from glioblastoma cell lines [60]. Despite the promising results obtained with the use of the inhibitors mentioned above, these pathways rarely operate alone, at least during cancer development. Instead, signaling crosstalks construct complex networks. For example, interactions have been shown between Wnt and Hh, as well as between Notch and PI3K/ Akt and EGFR pathways [47]. These broader signaling networks might indicate the need for combination of inhibition regiments to target CSCs effectively.

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reasoned that eradication of CSCs through controlled, druginduced differentiation could either induce terminal differentiation or sensitize the cells toward standard therapies through a loss of their stem cell properties [6]. In particular, all-trans-retinoic acid (ATRA) has been studied intensively in this regard. However, so far it has only been successfully applied for the treatment of acute promyelocytic leukemia, whereas in other tumor types, tumorigenicity could not be completely abolished [6,79]. Possible reasons for the disturbed retinoic acid signaling, which show downregulation of retinoic acid receptors and upregulation of retinoic aciddegrading enzymes, have been identified in gliomas [80]. A similar impaired differentiation has been observed when applying BMPs because of epigenetic silencing of the respective receptor on CSCs [55]. Therefore, it is of major importance to investigate the molecular underpinnings keeping CSCs in an immature state and how this can be translated into therapeutic regimens.

Small molecules that target CSCs Around 70 compounds are reported to be able to interact with the biology of CSCs. In Table 2, these are listed and classified as natural (N), naturally derived (ND) or synthetic (S). For each compound, the biological target and type of CSCs treated are listed. The most cited biological targets belong to the Hh (9), Wnt (11) and Notch (11) pathways and to ABC transporters (7). Twenty different types Q3 of CSC are mentioned. We highlight in succession the compounds that attracted our attention and divide them into natural and synthetic compounds (Fig. 1).

Natural compounds and derivatives Thirty different natural products and semisynthetic products (Fig. 2) are reported in Table 2. They are congeners of different families of natural products and, thus, are worthy to be considered as lead compounds or as building blocks for the design and synthesis of new interesting molecules. Icaritin is a prenylflavonoid derivative from plants of the genus Epimedium that has long been used in Chinese traditional medicine. Icaritin has many pharmacological and biological activities, such as neuroprotective effects, stimulation of neuronal and cardiac differentiation, growth inhibition of human prostate carcinoma PC-3 cells and estrogen-like activities. Icaritin induced sustained activation of ERK signaling, cell cycle arrest and apoptosis. Unlike the wellknown antiestrogen tamoxifen, icaritin also inhibited the growth of breast cancer stem and/or progenitor-like cells [81]. Five different alkaloids have been reported to be active against five different CSC lines. Harmine has been reported to have the ability to inhibit selfrenewal and promote differentiation of glioblastoma stem-like cells (GSLCs). In particular, the authors reported that Har-hc inhibited neurosphere formation of human primary GSLCs. In vivo tests also confirmed that Har-hc decreased the tumorigenicity of GSLCs [82]. Glioblastoma CSCs are also the targets of the wellknown cyclopamine alkaloid that was recently reported to reduce significantly drug resistance of CD34+ leukemic cells to cytarabine [83]. HPMA copolymer–cyclopamine conjugate showed antiCSC efficacy on human prostate cancer epithelial cells (RC-92a/ hTERT). The copolymer showed selective inhibitory effect on prostate CSCs in an in vitro prostate cancer model [84]. Berberine, an alkaloid isolated from Coptis chinensis, Hydrastis canadensis and species of the family Berberidaceae, has positive antibacterial 6


activity and anti-inflammatory effects, and has been formulated into liposomes for targeted delivery to mitochondria of breast CSCs (BCSCs). The targeting berberine liposomes were shown to cross the CSC membrane, inhibit ABC transporters (ABCC1, ABCC2, ABCC3 and ABCG2) and selectively accumulate in the mitochondria. Furthermore, the proapoptotic protein BAX was activated, whereas the antiapoptotic protein BCL-2 was inhibited, resulting in apoptosis [85]. Seven different terpene or terpenoid compounds have been reported to block six different types of CSC. ATRA (a potent differentiating agent) has been reported to induce in vitro differentiation of stem-like glioma cells (SLGCs) and, consequently, to induce therapy-sensitizing effects, to impair the secretion of angiogenic cytokines and disrupt SLGCs motility. The use of ATRA suggested the potential of differentiation treatment to target the stem-like cell population in GSLCs [79]. More recently, ATRA has been reported to suppress the expression of the stem cell markers Oct4, Sox2, Nestin and CD44 in HNSC CSCs. ATRA resulted in the inhibition of the proliferation of HNSC CSCs in vitro and in vivo. The antitumour effects of ATRA could be associated with downregulation of Wnt/b-catenin signaling [86]. The macrocyclic tanespimycin (17-AAG) is a derivative of the antibiotic geldanamycin. It eliminates lymphoma CSCs in vitro and in vivo by disrupting the transcriptional function of HIF1a, a client protein of HSP90. 17-AAG preferentially induced apoptosis and eliminated the colony-formation capacity of mouse lymphoma CSCs and human AML CSCs. However, low concentrations of 17-AAG failed to eliminate highly proliferative lymphoma and AML cells (nonCSCs), in which the AKT-GSK3 signaling pathway is constitutively active. The heat shock transcription factor HSF1 is highly expressed in nonCSCs, but it is weakly expressed in lymphoma CSCs. However, siRNA-mediated attenuation of HSF1 abrogated the colony-formation ability of both lymphoma and AML CSCs [87]. Other macrocyclic compounds, for example telomestatin, were able to impair the maintenance of glioma stem cells (GSCs) by inducing apoptosis in vitro and in vivo. The migration potential of GSCs was also impaired by telomestatin treatment. By contrast, both normal neural precursors and nonGSCs were relatively resistant to telomestatin. Treatment of GSC-derived mouse intracranial tumors resulted in the reduction of tumor size in vivo without noticeable cell death in normal brains. iFISH revealed both telomeric and nontelomeric DNA damage caused by telomestatin in GSCs but not in nonGSCs [88]. Another potentially useful antibiotic is mithramycin. This is a polyauroleic acid isolated from Streptomyces that was initially evaluated as a chemotherapeutic agent in patients with cancer during the 1960s and 1970s, but was discontinued because of excessive systemic toxicities. More recently, renewed interest in clinical development resulted in remarkable findings. In particular, mithramycin showed efficacy in repressing ABCG2 and in inhibiting stem cell signaling in thoracic malignancies [89]. Three different simple compounds sharing a polyphenolic structure have been used to inhibit four different types of CSC (colon, pancreatic, breast and brain). Curcumin has been reported to be effective against colon (in combination with dasatinib [90]), breast [91] and brain (in combination with paclitaxel [92]) tumors. A difluorinated derivative was active against colon cancer stem-like



REVIEWS

TABLE 2

Compounda

Classb

Biological targets/ mechanism of action d

CSC typee

Clinical trial (phase, studies)f

Refs

17-AAG ABT-737

N

HIF1a

Lymphoma

Phase I, 1 study

[87]

S

Bcl-2

Glioma

No studies

[12]

Acetaminophen

S

Wnt/b-catenin

Breast

Phase II, 9 studies

[121]

Adamantyl retinoid-related (AHP3 and 3-Cl-AHPC)

S

IGF-1R and Wnt/ b-catenin

Pancreatic

No studies

[122]

PML

Leukemic

Phase I-IV, 7 studies

[46]

ROCK1 and FAK

Prostate

Phase II, 5 studies

[123]

Combined with (further indication)c

Arsenic trioxide Atorvastatin

S

AZD7762

S

Gemcitabine

Chk1

Pancreatic

No studies

[23]

Berberine

N

(liposome)

ABC transporters

Breast

No studies

[85]

Bortezomib

S

TRAIL

Apoptosis

Glioblastoma

Phase I-III, 122 studies

[107]

Cabozantinib

S

c-Met

Pancreatic

No studies

[124]

CCT129202

S

Aurora kinase and ABC transporters

CSC-likee

No studies

[125]

ATM/ATR and PKB/Akt

Lung

No studies

[126]

Doxorubicin, vincristine and paclitaxel

N Celecoxib

S

Wnt

Colon

Phase II–III, 5 studies

[127]

Cisplatin

S

TRAIL (lipoplatin)

Wnt/b-catenin Apoptosis

Breast Cervical

Phase I–IV, 106 studies No studies

[15] [128]

Cucurbitacin I

N

Radiation

Apoptosis

HNSC

No studies

[129]

Curcumin

N

Paclitaxel Piperine Dasatinib

Apoptosis ALDH and Wnt ALDH, CD133, CD44 and CD166

Brain Breast Colon

Phase II–III, 3 studies

[92] [91] [90]

Curcumin (difluorinated)

ND

ABCG2 transporter and NF-kB

Colon

No studies

[93]

Cyclopamine

N

Hedgehog Hedgehog Hedgehog

Glioblastoma CD34+ leukemic Prostate

No studies

[50] [83] [84]

Hedgehog and mTOR

Pancreatic

Cytarabine (HPMA copol. conjugate) Rapamycin

[130]

DAC (and AZA)

S

DNA methylation

Leukemic and epithelial

Phase I–IV, 21 studies (phase I–III, 31 studies)

[113]

Daidzein derivative

ND

Apoptosis

Ovarian

No studies

[131]

DAPT

S

g-Secretase and Notch g-Secretase and Notch Notch

Ovarian Glioma DCIS

No studies

[132] [49] [133]

EZH2

Hepatocellular

No studies

[118]

Radiation Lapatinib 3-Deazaneplanocin A

S

Debromohymenialdisine (DMH)

N

Radiation

Chk1

Glioblastoma

No studies

[22]

Disulfiram/copper

S

Paclitaxel Gemcitabine

ROS-MAPK and NF-kB ROS, ALDH and NF-kB

Breast Glioblastoma

Phase I, 1 study

[105] [106]

Dofequidar fumarate

S

Irinotecan

ABCG2 transporter

CSC-likee

No studies

[134]

Eriocalyxin B

N

NF-kB

Ovarian

No studies

[135]

Erismodegib (NVP-LDE-225)

S

Hedgehog

Prostate

Phase I–II, 2 studies

[17]

Galiellalactone

N

STAT3 and ALDH

Prostate

No studies

[97]

GANT-61

S

Gli transcription factor

Pancreatic

No studies

[136]

Guggulsterone

N

Ras/NF-kB and Hedgehog

Glioblastoma

No studies

[137]

Harmine hydrochloride

N

Akt phosphorylation

Glioblastoma

No studies

[82]

Honokiol

N

Notch

Colon

No studies

[95]

Icaritin

N

ERK and apoptosis

Breast

No studies

[81]

Imatinib

S

MAPK

Glioblastoma


[112]

SANT-1 Radiation



Small molecules that target CSCs



TABLE 2 (Continued )


Compounda

Classb

Imatinib (and nilotinib AMN107)

S

IMD-0354

S

L685,458

S

LY2109761

S

LLP-3

S

Metformin

S


CSC typee


Refs

ABCG2 transporter

Hematopoietic

Phase I-IV, 49 studies (phase I-IV, 10 studies)

[111]

Doxorubicin

NF-kB and apoptosis

Breast

No studies

[138]

Radiation

g-secretase and Notch

Glioma

No studies

[133]

Radiation

TGFbR-I kinase

Glioblastoma

No studies

[108]

Survivin–Ran protein complex

Glioma

No studies

[109]

ROS and AMPK/mTOR CD133+

Pancreatic Colorectal


[139] [140]

ABCG2 and multiple pathways

Lung and esophageal

Phase II, 1 study

[89]

g-secretase and Notch

Breast


[102]


5-FU Mithramycin

N

MK-0752

S

MK-5108

S

Aurora-A kinase and NF-kB

Ovarian

No studies

[115]

Morusin

N

NF-kB and apoptosis

Cervical

No studies

[141]

MRK-003

S

g-Secretase and Notch

Breast

No studies

[103]

MST312

S

Telomerase

Lung

No studies

[119]

Niclosamide

S

Apoptosis

Ovarian

No studies

[116]

NSC747854

S

PARP-1

Lung

No studies

[142]

Omacetaxine

ND

Mcl-1, apoptosis

CML

No studies

[143]

Oximatrine

N

Wnt/b-catenin

Breast

No studies

[144]

Parthenolide and andrographolide

N

NF-kB

MM

No studies

[145]

PD166285

S

Radiation

WEE1

Glioblastoma

No studies

[24]

PDTC

S

Paclitaxel

NF-kB

Breast

No studies

[146]

PF-03084014

S

Irinotecan

g-Secretase and Notch

Colorectal

No studies

[120]

Phosphosulindac (OXT-328)

S

Wnt/b-catenin

Breast

No studies

[104]

Quercetin

N

Apoptosis

Prostate

Phase I, 2 studies

[16]

Resveratrol

N

Pluripotency-maintaining factors and EMT

Pancreatic

No studies

[94]

Retinoic acid (all trans)

N

Glioma Glioblastoma HNSC

Phase I–IV, 23 studies

Cisplatin

Differentiation Notch Wnt/b-catenin

[79] [147] [86]

g-Secretase and Notch g-Secretase and Notch

Melanoma Breast


Radiation

[148] [149]

RO4929097

Docetaxel

EGCG

S

Rottlerin

S

PI3K/Akt/mTOR

Pancreatic

No studies

[150]

Salinomycin

N

Wnt and apoptosis Apoptosis

Endometrial HNSC

No studies

[151] [152]

Apoptosis Oxidative stress induction Differentiation

Leukemia Prostate Breast

Cisplatin and paclitaxel

[153] [154] [155]

SB-T-1214 (taxoid)

ND

Stem cell-related genes

Colon

No studies

[156]

Sorafenib

S

PI3K/Akt, MAPK and M Apoptosis and NF-kB

Glioblastoma Pancreatic

Phase I-II, 4 studies

[110] [117]

PI3K/Akt, JNK1/2 and MAPK

Breast

No studies

[157]

STAT3 and apoptosis

Glioblastoma

No studies

[158]

ALDH, Notch-1 and c-ReI

Phase not indicated, 1 study

[99]

Wnt/b-catenin Hedgehog (Gli) Hedgehog

Pancreas and prostate Breast Pancreatic Pancreatic

ABCG2 transporter

CSCs-likee

Phase I, 1 study

[159]

IL-6/STAT3/NF-kB

Breast

No studies

[160]

Sulforaphane Strigolactone analogues

ND

STX-0119

S

Sulforaphane

S

Tandutinib

S

Tanshinone IIA

N

8

Drugs

Mitoxantrone

[98] [100] [101]



REVIEWS

Classb


CSC typee


Refs

Telomestatin

N

Telomeric G-Quadruplex and c-Myb

Glioma

No studies

[88]

Thioridazine

S

Tranilast (ALDH)

S

Dopamine receptors

Breast

Phase 0, 1 study

[161]

Aryl hydrocarbon receptor

Breast

Phase 3, 1 study

[162]

Trifluoperazine

S

CD44, CD133, Wnt

Lung

No studies

[163]

Vismodegib (GDC-0449) WP1193

S

Hedgehog

Pancreatic


[164]

S

JAK2/STAT3 and apoptosis

Glioblastoma

No studies

[165]

Z-LLNle-CHO (GSI-1)

S

Notch

Ovarian

No studies

[114]


Doxorubicin Cisplatin and gefitinib

Cisplatin

a

The compounds are listed in alphabetical order. For definitions, see main text. b The compounds are characterized as natural (N), naturally derived (ND) or synthetic (S). c Combined treatment with any other general anticancer compound. d Biological target or the more general targeted mechanism. e Different types of treated CSCs. f From http://www.clinicaltrials.gov/.

cells [93]. Resveratrol has been reported to inhibit pancreatic CSC characteristics in humans and KrasG12D transgenic mice by inhibiting pluripotency-maintaining factors and epithelial–mesenchymal transition [94]. Honokiol, a biphenolic compound, has been

Antiapo ptot ic p ath wa Cell death ys

ABC

Hh

proliferation, survival...

M

G2

XIAP

BcIXL

Resistance

BcI2

Differentiation

McI1

Cancer stem cells: Mechanisms of resistance

nce sce Quie

Resistance

Cyclophosphamide

S

Carboxy phosphamide

Low O2 G1 ALDH G0

HIF1

HIF2a

Angiogenesis survival proliferation stemness...

Hy pox ic n iche s

G2

M

S

G1

Al de hy de deh ydro gena se

Notch

Fz R LP

Jag

Ptch1 SMO

Prosur viva l sig nal ing d

t Wn

Caspases

tion entia iffer dd rbe stu Di

ABC

x efflu ug r D

DII

used in traditional Chinese medicine for treating various ailments. It has been shown to be a potent inhibitor of colon cancer growth and able to target CSCs by inhibiting the g-secretase complex and the Notch signaling pathway [95].

DNA repair

se on p s e re mag a d A DN Drug Discovery Today

FIGURE 1

Molecular mechanisms of cancer stem cell resistance to therapy. www.drugdiscoverytoday.com 9 Please cite this article in press as: P.A. Sotiropoulou, et al., Chemical approaches to targeting drug resistance in cancer stem cells, Drug Discov Today (2014), http://dx.doi.org/10.1016/ j.drudis.2014.05.002


TABLE 2 (Continued ) Compounda



O O O O

O

S

O OH

N

N NH O

O O

O

OH O O

OH

O O

N

N

N

O O

OH HO

N H

OH

OH

O

NH2 O

Telomestatin 1

O

OH

H

OHH O H

O

O

O OH O

O

O

O

O

O

N

N

SB-T-1214 (taxoid)

OH O

O

N

O

O

H N

O

H

O

OH

OH

OH

O

HO

OH

17-AGG

EGCG

Salinomycin


OH O H

O H

HO

H

O

OCH3

OH O

HO

OH

O

O

O

HO

N H

H +

OH O

Cucurbitacin I

N

H3CO

OH O

N

OCH3

Icaritin

O

HO H

OH

H

O

O

H O OH OH O

O

OH O

Oximatrine

Berberine

OH

O O

OH

OH O

OH O

O

OH

Mitramycin

O O

HN H

H

O O H

O

HO

HO

Cyclopamine

O

H

O OH O

H

HO

O

O

H H

HO

N

O

Omacetaxine

H

H OH

O

Guggulsterone

OH

Curcumin

All-trans-retinoic acid

OH

O HO

HO O

OH

Quercetin

HO

OH

HO

OH O

Reservatrol

O

O

O

OH

Strigolactone

HO

O

O

O OH

O

OH O

OH

OH O

COOH OCH3

H3CO

O

Andrographolide

OH

HO

O H

Morusin

Tanshinone IIA

Honokiol H2N

O

H

H O H

O O OH

OH

Eriocalyxin B

N O

O

O

HO

N

N

O

Parthenolide

O

OH

N

Caffeine

O OH

Daidzein

O

H

O O

N

HN

H

Galiellalactone

H3CO

N H

Harmine

N

O S

HN NCS

Sulforaphane

NH O

DBH

Drug Discovery Today

FIGURE 2

Natural compounds with anticancer stem cell activity.

Salinomycin, a polyether ionophore antibiotic isolated from Streptomyces albus, has been shown to block CSCs in different types of human cancer, and is the subject of a recent review by Naujokat and Steinhart [96]. The activity of salinomycin is probably the result of interference with ABC drug transporters, the Wnt/bcatenin signaling pathway and other CSC pathways. Promising results from preclinical trials using human xenografts in mice and a few pilot clinical studies reveal that salinomycin is able to eliminate effectively CSCs and to induce partial clinical regression of heavily pretreated and therapy-resistant cancers. The ability of salinomycin to kill both CSCs and therapy-resistant cancer cells could define the compound as a novel and effective anticancer drug. Moving toward polyketides, galiellalactone has emerged as a promising compound for the development of prostate cancer drugs. Galiellalactone is a hexaketide metabolite produced by the fungus Galiella rufa. It is a highly potent and selective inhibitor of IL-6 signaling through STAT3, and is believed to 10

inhibit STAT3 signaling by blocking the binding of activated STAT3 to DNA. The effects of galiellalactone on ALDH-expressing prostate cancer cells have been studied to explore the expression of ALDH as a marker for cancer stem cell-like cells in human prostate cancer cell lines. Galiellalactone treatment decreased the proportion of ALDH+ prostate cancer cells and induced apoptosis of ALDH+ cells. The gene expression of ALDH1A1 was downregulated in vivo in galiellalactone-treated DU145 xenografts [97]. The plant compound isothiocynate sulforaphane is present in high concentration in broccoli and other cruciferous vegetables and, despite its structural simplicity, showed interesting activity against three different types of CSC. One of the first contributions in this field reported the inhibitory activity of sulforaphane against BCSCs and the downregulation of the Wnt/b-catenin self-renewal pathway [98]. The ability of sulforaphane to increase the effectiveness of various cytotoxic drugs without inducing additional toxicity in mice has been reported for pancreas and prostate CSCs



REVIEWS

O S

OH H N

Cl

pluripotency-maintaining factors (Nanog and Oct-4), and PDGFRa and Cyclin D1. Thus, sulforaphane has been highlighted as an inexpensive, safe and effective alternative for the management of pancreatic cancer [101].

O

N O

HN

N

N

S

N H

N

N

N

S

CN F

O

O P O

O

N

LLP-3 H N

O

N N H

N

N

OH

O

COOH

O S

N

O

Tandutinib

S

O

OH

MST312

OH

Rottlerin

F F

O

H N

O

OH

O

O

HN

S

O

N H

MK-5108

O OH

OH

HO

NH

HO

O S O NH

N H N

N

N

N

SANT-1

NSC747854

HO H N

O

N O

O

CCT129202

F Cl

N N

H N

N N

Imanatib

N

HN N

N N

N

O

Cl

O

O

Phospho-sulindac (P-S, OXT-328)

O

N

HN

O

O O


[99]. More recently, sulforaphane has been described as able to regulate selfrenewal of pancreatic CSCs through the modulation of the Sonic Hh–GLi pathway [100] and to inhibit downstream targets of Gli transcription by suppressing the expression of

HN

O

N

O

HN

N

N

N

HO

N

O

O

N N

HO

O

O

H OH

OH

N

RO4929097

MRK-003

OH

Azacytidine (AZA)

Decitabine (DAC)

N

O Cl

O

N

ABT-737

F

O

N H

N H

H N

N

H N

O

OH NH N

OH

OH

O

N

F

Celecoxib

Cl

F

N H

AHP3

N

Cl

NH O N

N

F

Nilotinib

N H

O

O

O HN

F

S

N

N H

NH

O

N

AZD7762

B

O

OH

O

N

STX-0019

Bortezomib

O

N

N O N

Trifluoperazine O

Cl

O

S O O

OH

NH

N

N

O

N

N

N

Cl

OH

N H OH

O

GANT-61

OH H N

Dofequidar

H N HO

O

Vismodegib

N

N

O

LY-2109761

N H

t-Bu

O N N

O

S

Ph O

H N

OH

H N

N

N N

N

N

O

Z-LLNle-CHO

O

F

H

N H

O

N

Cl

O

H N

Ph

NH

MK-0752

N

O

O

O S O

O

O

H N

DAPT

HO

HN N

IMD-0354

O O

HO

3-Cl-AHPC

Cl

F

N HO

N H

O

Erismodegib

COOH

N

N

O

Atorvastatin

COOH

O

N HN

S

N

PF-03084014

OH O

N

S O O

Thioridazine,THZ

NH

N N H

F

Sorafenib

N H N

Cl

N

O

H N

O

Cabozantinib

S

F

H N O

Tranilast

H N OH

N O

N

N

N

O

Cl

N

O

OH

N

Acetaminophen

Niclosamide

HO

N

Cl

Cl

3-Deazaneplanocin-A

PD166285

Ph H N O

O

O

i-Bu Ph N H

O

L-685, 458

N

N CN

S

S

O Br

N H

WP1193

S

S

N

SH N

S

Disulfiram

N

H N

PDTC

Cl Pt

NH

NH

Cl

Metformin

Cisplatin

Drug Discovery Today

FIGURE 3

Synthetic compounds with anticancer stem cell activity. www.drugdiscoverytoday.com 11 Please cite this article in press as: P.A. Sotiropoulou, et al., Chemical approaches to targeting drug resistance in cancer stem cells, Drug Discov Today (2014), http://dx.doi.org/10.1016/ j.drudis.2014.05.002


Synthetic compounds


In terms of synthetic small molecules (Fig. 3), a variety of chemical structures can be detected, mainly characterized by the presence of aromatic and heteroaromatic rings and the scarcity of stereogenic elements. The chiral 3-cyclohexylpropanoic acid derivative MK-0752 is known to be a moderately potent GSI. It was used in a study evaluating the impact of GSIs on the breast CSC (BCSC) population and the efficacy of combining GSI with docetaxel treatment. Treatment with GSI reduced the percentage of BCSCs by inhibition of the Notch pathway. The clinical trial showed the feasibility of combination GSI and chemotherapy and, together, these results encourage further study of Notch pathway inhibitors in combination with chemotherapy in breast cancer [102]. MRK-003, a potent and selective GSI bearing a chiral spiro[methanobenzo[8]annulene-thiadiazolidine] skeleton, acts as an antagonist of Notch signaling. Given that the Notch pathway is deregulated in human breast tumors, the supposition that it would be required for CSC activity has been investigated. It was found that MRK-003 administration to tumor-bearing mice eliminated tumor-initiating cells and resulted in rapid and durable tumor regression. MRK-003 also inhibited the proliferation of tumor cells and induced apoptosis and differentiation. These findings suggest that MRK-003 targets BCSCs and illustrate that eradicating these cells in breast tumors ensures long-term, recurrence-free survival [103]. Phosphosulindac, also known as OXT-328 or PS, is a phosphateester derivative of sulindac, a nonsteroidal anti-inflammatory drug of the arylalkanoic acid class. It selectively and effectively eliminates BCSCs both in vitro and in vivo. It has been found that long-term treatment of mixtures of cultured BCSCs and breast cancer cells with phosphosulindac preferentially eliminated the CSCs. This strong inhibitory effect against breast cancer seems to be result, at least in part, from suppression of the Wnt/b-catenin pathway [104]. Disulfiram is the common name of bis(diethylthiocarbamoyl) disulfide, a popular drug discovered during the 1920s and used to support the treatment of chronic alcoholism. Previous studies indicate that disulfiram is cytotoxic to cancer cell lines and reverses anticancer drug resistance, acting as an ALDH inhibitor. It has been found that, in copper-containing medium, disulfiram inhibits BCSCs and enhanced cytotoxicity of paclitaxel in breast cancer cell lines. This might be caused by simultaneous induction of reactive oxygen species (ROS) and inhibition of NF-kB [105]. Other studies highlight the cytotoxicity of disulfiram/Cu and the enhancing effect on the cytotoxicity of gemcitabine in glioblastoma multiforme (GBM) stem-like cells, which might be caused by induction of ROS and inhibition of both ALDH and the NF-kB pathway [106]. Bortezomib was the first therapeutic proteasome inhibitor to be tested in humans. In 2003, it was approved in the USA by the US Food and Drug Administration (FDA) for use in multiple myeloma. The drug is an N-protected dipeptide characterized by the substitution of a carboxylic acid with a boronic acid. From analysis of the effect of bortezomib on TRAIL-induced apoptosis signaling pathways, compelling evidence has been provided that the combination of bortezomib and TRAIL represents a promising novel strategy to trigger cell death in glioblastoma, including glioblastoma CSC [107]. 12


The quinoline derivative LY2109761, a transforming growth factor-b receptor (TGF-bR) I kinase inhibitor, has been found to be active against glioblastoma alone and to enhance the antitumor efficacy of radiation both in vitro and in vivo, in particular in GBM CSLCs. These findings offer a sound rationale for positioning TGFbR kinase inhibitors as radiosensitizers to improve the treatment of glioblastoma [108]. In a study aimed to provide evidence that both Survivin and Ran are expressed abundantly by GSCs in GBM, a novel small-molecule inhibitor, the dihydropyridinone compound LLP-3, was developed using a structure-based computational drug design. LLP-3 effectively inhibited the interaction of Survivin with the small GTPase Ran. Treatment of patient-derived GBM spheres with LLP3 selectively inhibited the growth of GSCs both in vitro and in vivo. These inhibitory effects of LLP-3 were dependent on the p53 status of the tumor cells. These data indicate that Survivin–Ran-directed therapeutics constitute a novel class of targeted agents for future GBM treatment strategies [109]. The 1-(4-hydroxyphenyl)urea derivative sorafenib is a small molecular inhibitor of several tyrosine protein kinases and Raf kinases. It is a drug approved for the treatment of advanced renal cell carcinoma, hepatocellular carcinoma, and radioactive iodineresistant advanced thyroid carcinoma. Evidence has been provided that sorafenib has a selective action on glioblastoma CSCs. It reduces proliferation of glioblastoma cultures, an effect that depends, at least in part, on the inhibition of PI3K/Akt and MAPK pathways, both involved in gliomagenesis. Given that current GBM therapy enriches the tumor CSC content, the evidence for a selective action of sorafenib on these cells is therapeutically relevant [110]. The 2-phenyl amino pyrimidine derivative imatinib is an attested drug used in the treatment of multiple cancers, most notably CML, owing to its specific inhibition of BCR-ABL kinase activity. Given that such kinase enzymes exist only in cancer cells and not in healthy cells, imatinib works as a form of targeted therapy, and represents one of the first cancer therapies to show the potential for such targeted action. Even if most patients with CML maintain durable responses to the drug, most of them will relapse after withdrawal of imatinib and patients with advancedstage CML often develop drug resistance. By means of photoaffinity-labeling experiments, it has been demonstrated that imatinib, together with the closely related and more potent nilotinib, interacts with the ATP binding-cassette transporter ABCG2, which is highly expressed on primitive hematopoietic stem cells, thus suggesting a role of ABC transporters in resistance to tyrosine kinase inhibitors in primitive hematopoietic stem cells and CML stem cells [111]. With regards to GBM, its highly complex and heterogeneous nature mostly accounts for the modest antitumor effect in patients treated by targeted agents including imatinib. However, it has been observed that long-term culture with imatinib mesylate against platelet-derived growth factor receptor (PDGFR) and c-Kit (stem cell factor receptor) resulted in reduced CSC ability in glioblastoma cells through cell differentiation. Such targeted inhibition seems to disturb glioma stem cell biology in subsets of GBM cells and, therefore, might have potential in clinical applications [112]. The cytidine analog azacytidine and its deoxy derivative decitabine are inhibitors of DNA methyltransferase, mainly used in the


DRUDIS 1405 1–16

treatment of myelodysplastic syndrome (MDS). It has been shown that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses of such demethylating agents produces an antitumor ‘memory’ response, including inhibition of subpopulations of cancer stem-like cells, without causing immediate cytotoxicity [113]. The Notch signaling pathway, and Notch3 in particular, are crucial for the regulation of CSCs and tumor resistance to platinum in ovarian cancer. It has been found that the norleucine derivative Z-LLNle-CHO (GSI-1), a g-secretase and Notch pathway inhibitor, depletes CSCs and increases tumor sensitivity to platinum. Most importantly, the cisplatin–GSI combination seems to be the only treatment that effectively eliminates both CSCs and the bulk of tumor cells, indicating that a dual combination therapy targeting both populations is needed for tumor eradication. Such combination therapy has a synergistic cytotoxic effect in Notchdependent tumor cells by enhancing the DNA damage response, G2/M cell cycle arrest and apoptosis [114]. MK-5108 is a chiral 4-hydroxycyclohexanecarboxylic acid derivative with potent inhibitory activity against Aurora-A kinase, which is one of the key regulators during mitosis progression and, therefore, a potential target for anticancer therapies. A study looking at the effect of Aurora-A inhibition in epithelial ovarian CSCs (EOCSCs) showed that MK-5108 decreased cell proliferation in EOCSCs by inducing cell cycle arrest and affecting the NF-kB pathway. Given that EOCSCs represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition effectively targets the CSC population in ovarian cancer [115]. The hydroxybenzamide derivative niclosamide belongs to the family of anthelmintics and is widely used in the treatment of worm infections. By subjecting stem-like ovarian tumor-initiating cells (OTIC) to high-throughput drug screening using more than 1200 clinically approved drugs, niclosamide was shown to target selectively OTICs in vitro and in vivo, disrupting multiple metabolic pathways. These studies support niclosamide as a promising therapy for ovarian cancer and warrant further preclinical and clinical evaluation of this safe drug for the management of ovarian cancer [116]. The 2-thiophenecarboxamide derivative AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials, has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents, supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings. It has been demonstrated that Chk1 inhibition in combination with gemcitabine reduces both the percentage and the tumor-initiating capacity of pancreatic CSCs. Furthermore, the finding that the Chk1-mediated DNA damage response was greater in CSCs than in nonCSCs suggests that Chk1 inhibition selectively sensitizes pancreatic CSCs to gemcitabine, thus making Chk1 a potential therapeutic target for improving pancreatic cancer therapy [23]. In a study aimed at targeting pancreatic CSCs with sorafenib, enhanced elimination of CSC characteristics was achieved by cotreatment with sulforaphane, the broccoli-derived isothiocyanate compound that was recently described to eliminate

REVIEWS

pancreatic CSCs by downregulation of NF-kB activity without inducing adverse effects. In vivo, combination therapy reduced tumor size in a synergistic manner as a result of the induction of apoptosis, inhibition of proliferation and angiogenesis, and downregulation of sorafenib-induced expression of proteins, thus suggesting that sulforaphane is suited to increase targeting of CSCs by sorafenib [117]. 3-Deazaneplanocin A is a cyclopentenyl analog of 3-deazaadenosine, originally synthesized as an inhibitor of S-adenosyl-Lhomocysteine hydrolase. Histone-lysine N-methyltransferase EZH2, acting mainly as a gene silencer, has been characterized as a general selfrenewal regulator in a range of normal stem cells. It has been demonstrated that pharmacological inhibition of EZH2, by means of 3-deazaneplanocin A, significantly reduced the number and tumorigenic potential of tumor-initiating hepatocellular carcinoma (HCC) cells. This effect might be attributed to the impaired selfrenewal capability of tumor-initiating HCC cells caused by interference with EZH2 [118]. The small molecule N,N0 -bis(2,3-dihydroxybenzoyl)-1,3-phenylenediamine is a telomerase inhibitor, known as MST 312. It was found that MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in all tumor cells. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (with short-term effects) and through decreasing telomere length (with long-term effects). These results suggest that antitelomeric therapy using MST312 mainly targets lung CSCs and represents a novel approach for effective treatment of lung cancer [119]. The N-(1H-imidazol-5-yl)pentanamide derivative PF-03084014 is a clinically relevant GSI. It has been tested in combination with irinotecan to identify the effects of treatment on tumor recurrence and the tumor-initiating population in a colorectal cancer (CRC) preclinical explant model. The results obtained indicated that the combination of PF-03084014 and irinotecan is effective in reducing tumor recurrence in patients with CRC whose tumors exhibit elevated levels of the Notch pathway [120].

Concluding remarks In this review, we have summarized current understanding of the biological targets that are at the base of CSC resistance and the chemical compounds showing potential activity against different types of CSC. The development of a ‘magic bullet’ that will eliminate CSCs and, therefore, prevent the development of drug resistance, metastasis and relapse sounds attractive. However, we are aware that the complexity of tumor organization and the plasticity of tumor cells and CSCs hinder progress in this direction. We hope that this review will stimulate new efforts toward new frontiers in the battle against cancer.

Acknowledgments This review has been developed under the umbrella of CM0602 COST Action ‘Chemical Approaches for Targeting Drug Resistance in Cancer Stem Cells’ (www.stemchem.org). The authors express their gratitude to all the members of the CM0602 COST Action for the interesting discussions during the drafting of this review. The authors would like to thank Andrea Karambelas for revision of the manuscript.






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Chemical Proteomic Approaches Targeting Cancer Stem Cells: A Review of Current Literature.

Getting to the heart of the matter in cancer: Novel approaches to targeting cancer stem cells.

Concise Review: Targeting Cancer Stem Cells Using Immunologic Approaches.

Therapeutic approaches to target cancer stem cells.

Colon cancer: cancer stem cells markers, drug resistance and treatment.

Targeting Lung Cancer Stem Cells with Antipsychological Drug Thioridazine.

Targeting the Achilles' heel of drug-resistant cancer stem cells.

Multiple drug resistance due to resistance to stem cells and stem cell treatment progress in cancer (Review).

Approaches of targeting Rho GTPases in cancer drug discovery.

Chemotherapy targeting cancer stem cells.

Mechanisms of cisplatin resistance and targeting of cancer stem cells: Adding glycosylation to the equation.

Clinical and laboratory approaches to drug resistance.

Anti-Cancer Phytometabolites Targeting Cancer Stem Cells.

Overcoming Multidrug Resistance in Cancer Stem Cells.

Targeting cancer stem cells with oncolytic virus.

Therapeutic strategies targeting cancer stem cells.

Targeting cancer stem cells with p53 modulators.

MicroRNAs targeting prostate cancer stem cells.

Oncolytic viral therapy: targeting cancer stem cells.

Systemic approaches identify a garlic-derived chemical, Z-ajoene, as a glioblastoma multiforme cancer stem cell-specific targeting agent.

Nanomaterials in Targeting Cancer Stem Cells for Cancer Therapy.

Targeting Cancer Stem Cells in Castration-Resistant Prostate Cancer.

Targeting Signaling Pathways in Cancer Stem Cells for Cancer Treatment.

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