Arch. Pharm. Res. DOI 10.1007/s12272-014-0531-1

REVIEW

The molecular mechanisms underlying the therapeutic resistance of cancer stem cells Jun-Kyum Kim • Hee-Young Jeon Hyunggee Kim

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Received: 4 November 2014 / Accepted: 8 December 2014 Ó The Pharmaceutical Society of Korea 2014

Abstract Chemo-resistance and radio-resistance are a major cause of recurrence and progression of many cancers, regardless of improvements in therapies. Since cancer stem cells (CSCs) were identified as a rare population with the abilities of self-renewal; tumor initiation; aberrant differentiation, which contributes to tumor heterogeneity; and resistance to anticancer therapeutics, they have been considered a major cause of tumor recurrence post-therapy and a primary therapeutic target in relapse prevention. A number of studies have demonstrated the mechanisms underlying chemo-resistance and radioresistance of CSCs. In this review, we describe intrinsic and extrinsic factors underlying CSC chemo-resistance and radioresistance. The intrinsic factors regulate CSC signaling pathways involved in stem cell signaling, anti-apoptotic pathways, ABC transporter expression, and DNA damage repair systems. The extrinsic factors include the resistance mechanisms resulting from the interactions between CSCs and the microenvironment composed of vessels, fibroblasts, immune cells, extracellular matrix, and diverse soluble factors. Furthermore, we introduce diverse therapeutic agents used in experimental or clinical trials to target CSCs. Understanding how CSCs acquire resistance to anticancer therapeutics will give us opportunity to develop improved therapeutic approaches. Keywords Cancer stem cells
Chemo-resistance
Radioresistance
Tumor microenvironment
Tumor recurrence Jun-Kyum Kim and Hee-Young Jeon have contributed equally to this work. J.-K. Kim
H.-Y. Jeon
H. Kim (&) Department of Biotechnology, School of Life Sciences and Biotechnology, Korea University, 5-ga, Anam-dong, Seongbuk-gu, Life Science West Building 207, Seoul 136-713, Republic of Korea e-mail: [email protected]

Introduction Despite advances in basic and clinical cancer biology, cancer remains a major cause of death. Cancer consists of heterogeneous cells that contribute to progression and therapy failure. Upon a hierarchy model, intratumoral heterogeneity occurs due to small unique populations of tumor cells in the various human malignancies (Sun et al. 2011; Dick 2008). Recently, these populations have been called cancer stem cells (CSCs) and are considered a main cause of tumor recurrence after standard chemo- and radio-therapy. Since CSCs were first identified in human acute myeloid leukemia (AML) (Lapidot et al. 1994), several CSC populations were found in solid tumors, including brain, breast, prostate, and colon tumors (Ailles and Weissman 2007; Schatton et al. 2009). Although many studies have suggested that CSCs share molecular and cellular properties of normal stem cells, including self-renewal and differentiation potential (O’Brien et al. 2010), the therapy resistance mechanisms of CSCs are poorly understood. Therefore, many studies have focused on the molecular pathway associated with chemo-resistance and radio-resistance of CSCs that leads to tumor recurrence. In this review, we will discuss the mechanisms underlying therapeutic resistance of CSCs and introduce a number of novel therapeutic strategies targeting CSCs to prevent tumor relapse.

Chemo-resistance and radio-resistance of cancer stem cells Activation of stem cell signaling in cancer stem cells CSCs have the ability to self-renew and differentiate into multiple lineages such as normal stem cells (Reya et al.

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2001). Although the molecular mechanisms regulating stem cell signaling of CSCs are generally similar to normal stem cells, CSCs and normal stem cells exhibit different biological behaviors because stem cell signaling is aberrantly activated in CSCs (Verga Falzacappa et al. 2012). Self-renewal and differentiation of CSCs are mainly regulated by various developmental signaling pathways including Wnt, Notch, and Hedgehog signaling (Korkaya et al. 2009; Ward et al. 2009; Agarwal and Matsui 2010; Hoey et al. 2009; Harrison et al. 2010). Interestingly, these signaling pathways have been demonstrated to regulate not only self-renewal and differentiation, but also resistance to chemo-therapy in various CSC types (Fig. 1). Among them, aberrant Wnt/b-catenin signaling activation has been reported to mediate chemo-resistance in CSCs in ovarian cancer, hepatocellular carcinoma, leukemia, and neuroblastoma. In c-Kit? ovarian CSCs, resistance to cisplatin/paclitaxel is regulated by ABCG2 expression through Wnt/b-catenin activity. Further, ABCG2 was shown to be the transcriptional target of b-catenin by identifying LEF/ TCF binding sites within the ABCG2 gene promoter and verifying b-catenin-driven transcriptional regulation of

ABCG2 (Chau et al. 2013). Compared to OV6- cancer cell populations, less-differentiated stem/progenitor-like OV6? hepatocellular carcinoma cells showed markedly enhanced self-renewal and resistance to standard chemo-therapy, which can be attributed to active Wnt/b-catenin signaling. OV6? cell chemo-resistance was reversed by expression of miRNA targeting b-catenin (Yang et al. 2008). In MLL stem cells, the acquired chemo-resistance to GSK3 inhibitors was reversed by b-catenin inhibition (Yeung et al. 2010). In neuroblastoma, Wnt/b-catenin activity was implicated in chemo-resistance of the CD133? cancer cell population (Vangipuram et al. 2012). Notch signaling has been shown to regulate selfrenewal, angiogenesis, and epithelial-to-mesenchymal transition in various human malignancies (Ranganathan et al. 2011). Recently, several studies have suggested that Notch signaling contributes to chemo-resistance of CSCs, especially in glioma, prostate cancer, ovarian cancer, and pancreatic cancer. Using c-secretase inhibitors (GSI), the inhibition of Notch activity suppresses glioma tumorsphere formation in vitro as well as resistance and recurrence of temozolomide (TMZ)-treated gliomas in vitro and in vivo

Activated stemness signaling - Notch signaling - Hedgehog signaling - Wnt/ƅECVGPKPUKIPCNKPI

ABC transporters (Increased efflux)

Maintenance of low ROS levels Impaired apoptosis pathways (cFLIP, IAPs, Bcl-2 family)

DNA damage repair system (ATM, ATR, Chk1, MGMT, etc)

Cancer stem cell Fig. 1 The molecular mechanisms underlying chemo-resistance and radio-resistance of cancer stem cells. Developmental signaling pathways, such as Wnt, Notch, and Hedgehog signaling, can activate diverse target genes that mediate chemo-resistance and radio-resistance of CSCs. CSCs obtain chemo-resistance and radio-resistance capacity through overexpression of anti-apoptotic proteins including cFLIP, IAP family proteins, and Bcl-2 family. CSCs are capable of acquiring chemo-resistance by overexpressing ABC transporters

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which reduce intracellular drug levels through efflux of chemotherapeutic agents. CSCs possess relatively active DNA damage checkpoints. Thus, activation of DNA damage repair systems is another mechanism underlying chemo-resistance and radio-resistance of CSCs. CSCs exert chemo-resistance and radio-resistance by maintaining low intracellular ROS levels through activation of ROS-scavenging systems

Therapeutic resistance of cancer stem cells

(Gilbert et al. 2010). In hormone-refractory prostate cancer, a docetaxel-resistant subpopulation with tumor-initiating capacity showed increased Notch and Sonic Hedgehog (SHH) signaling activities. The inhibition of Notch and SHH signaling abrogated docetaxel resistance and resulted in depletion of resistant prostate cancer cells (DomingoDomenech et al. 2012). In ovarian cancer, ovarian CSCs, regulated by Notch signaling, are the main cause of resistance to platinum therapy. Targeting Notch signaling depleted ovarian CSCs and increased sensitivity to platinum (McAuliffe et al. 2012). SHH signaling plays a crucial role in maintenance and differentiation of various CSCs, including those from breast cancer, pancreatic cancer, colon cancer, glioblastoma, multiple myeloma, and chronic myeloid leukemia (CML) (Bar et al. 2007; Feldmann et al. 2008; Liu et al. 2006b; Jagani et al. 2010; Blotta et al. 2012; Varnat et al. 2009). Although the precise mechanisms have not been clearly demonstrated, SHH signaling has been suggested to confer chemo-resistance to CSCs. For instance, pharmacological inhibition of SHH signaling impaired growth of imatinib-resistant CML (Zhao et al. 2009). Thus, it is plausible that chemo-resistance of imatinib-treated CML might be abrogated by targeting essential stem cell maintenance pathways, such as SHH. The inhibition of the SHH signaling pathway in gastric CSCs reduced self-renewal and enhanced the efficacy of chemo-therapeutic drugs in vitro and in vivo (Song et al. 2011). Quiescence of cancer stem cells In general, ionizing radiation (IR) sensitivity is influenced by cell cycle stage. S-phase cells are the most sensitive to IR, while G1-phase cells are the most resistant to IR (Pawlik and Keyomarsi 2004). Because normal stem cells and CSCs grow slowly and are mostly quiescent (Moore and Lyle 2011; Masunaga et al. 1991), it is plausible that CSCs survive relatively well after IR treatment. After IR treatment, surviving CSCs reenter the cell cycle and accelerate tumor repopulation through activation of various developmental signaling pathways such as Wnt, Notch, and SHH (Withers et al. 1988; Boyer and Cheng 2008). Therefore, promoting CSC cell cycle progression and unregulated cell proliferation likely makes them more sensitive to IR treatment (Sun et al. 2011). Wnt/b-catenin signaling also maintains the CSC pool by controlling symmetric and asymmetric division (Piccin and Morshead 2011). Of interest, silencing of TCF4, a Wnt signaling-dependent transcription factor, improved sensitivity to IR by increasing the populations of cells in G2/M phase in rectal cancer, presumably independent of b-catenin activity (Kendziorra et al. 2011).

Impaired apoptosis pathways Because apoptotic cell death is a cell intrinsic barrier to oncogenic events, apoptotic signaling is dysregulated in many cancers (Lowe et al. 2004). Therefore, evasion of apoptosis is one of the hallmarks of cancer (Hanahan and Weinberg 2011). This feature is also associated with increased CSC survival in response to chemo-therapy (Fig. 1). There are two major routes to activate apoptotic pathways, the extrinsic and intrinsic pathways. The extrinsic pathway is activated by binding of death receptors such as the tumor necrosis factor (TNF) receptor superfamily, which includes FAS (also known CD95 and APO1) and TNF-related apoptosis inducing ligand (TRAIL) receptor, to their corresponding ligands. The intrinsic pathway is triggered by mitochondrial outer membrane permeabilization and subsequent release of cytochrome c into the cytoplasm under various stress conditions, such as anti-cancer drug treatment and cellular damage (Kruyt and Schuringa 2010; Fulda and Pervaiz 2010; Fulda 2013). The induction of apoptosis by the TNF superfamily is known to be mediated primarily through the activation of type I receptors (Rath and Aggarwal 1999). However, they can give rise to distinct phenotypes such as cell survival and cell death, according to cell context. Recent studies have reported that TNF superfamily members exhibit protumorigenic functions in various cancers and CSCs. For example, long-term treatment with TNFa conferred a CSC phenotype to oral squamous cell carcinoma cells, which included tumor sphere-forming ability, chemo-resistance, and tumorigenicity, through activation of the Notch1-Hes1 signaling pathway (Lee et al. 2012). In human breast cancer cells, TNFa treatment increased SLUG expression via NFjB/HIF signaling, leading to acquisition of CSC properties (Storci et al. 2010). In mouse models of ovarian cancer and liver cancer, CD95 signaling promoted tumor growth via the JNK and Jun signaling pathway (Chen et al. 2010), indicating that TNFa and FAS/CD95 signaling should be inhibited depending on the tumor type for therapy. Contrary to other TNF superfamily members, TRAIL can uniquely induce apoptosis in cancer cells in vitro and in vivo. However, since most primary cancer cells are resistant to TRAIL monotherapy, TRAIL has been used in combination therapy in clinical trials depending on tumor types (Lemke et al. 2014). Impaired apoptosis in cancer is mainly caused by aberrant expression of cellular FLICE-like inhibitory proteins (cFLIP, FLAME, or Usurpin), inhibitor of apoptosis (IAP) family proteins, Bcl-2 family members, and the NFjB signaling pathway (Fulda and Pervaiz 2010). cFLIP is a negative regulator of death-receptor-induced apoptosis and it is overexpressed in various cancers, including melanoma, colon cancer, lymphoma, and thyroid cancer

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(Dutton et al. 2006). In glioma, CD133? glioma stem cells are resistant to TMZ, carboplatin, paclitaxel, and etoposide, and they express anti-apoptotic genes, including cFLIP, more strongly than do the CD133- glioma cells (Liu et al. 2006a). The IAP protein family is comprised of eight members in humans: X chromosome-linked IAP (XIAP), cellular IAP1 (cIAP1), cellular IAP2 (cIAP2), survivin, livin/melanoma-IAP (ML-IAP), neuronal apoptosis inhibitory protein (NAIP), BIRC6 (apollon), and ILP-2 (Salvesen and Duckett 2002). IAP proteins block the intrinsic and extrinsic apoptotic pathways by binding to and inhibiting caspases. In particular, XIAP directly interacts with and inhibits caspases 3, 7, and 9 (Fulda and Pervaiz 2010). cIAP and ML-IAP induce XIAP-mediated caspase inhibition by binding to SMAC rather than inhibition of apoptosis by direct binding to caspases (Salvesen and Duckett 2002). In head and neck squamous cell carcinoma (HNSCC) stem cells expressing high CD44v3 and ALDH1, hyaluronan (HA) stimulated CD44v3 interaction with Oct4-Sox2-Nanog and increased expression of survival proteins such as cIAP1, cIAP2, and XIAP, leading to both self-renewal and cisplatin resistance (Bourguignon et al. 2012). In colon cancer, BIRC6 (apollon) was one of the proteins that was shown markedly higher expression in colon CSCs than in the non-stem-like colon cancer cells. BIRC6 knockdown led to sensitization of colon CSCs to cisplatin and oxaliplatin (Van Houdt et al. 2011). The Bcl-2 family proteins contain two distinct antiapoptotic (Bcl-2, Bcl-XL, Bcl-W, Bcl-A1A, and Mcl-1) and pro-apoptotic (BAX, BAK, and BH3 domain only molecules) subgroups. Therefore, the balance of anti- or pro-apoptotic proteins is an important determinant in regulating sensitivity to apoptosis (Fulda and Pervaiz 2010; Fulda 2013). In human cancers, anti-apoptotic Bcl-2 family members have been suggested to play a crucial role in tumorigenesis and cancer cell survival (Abdullah and Chow 2013). In breast cancer, Bcl-2 was highly expressed in CD44?/CD24-/low breast CSCs (Madjd et al. 2009). Altogether, these findings suggest that targeting antiapoptotic Bcl-2 family proteins may be a therapeutic strategy to ablate chemo-resistance of CSCs. For example, ABT-737, the Bcl-2 inhibitor, showed less efficient against glioma stem cells than in non-stem-like glioma cells. Since resistance was associated with high Mcl-1 expression, downregulation of Mcl-1 could partially reduce glioma stem cell resistance (Tagscherer et al. 2008). ATP-binding cassette transporters (ABC transporters) ABC transporters are transmembrane proteins that utilize adenosine triphosphate (ATP) to translocate various substrates including drugs, lipids, and metabolic products

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across the intracellular and extracellular membranes (Fletcher et al. 2010). ABC transporter expression is another resistance mechanism in human cancers because these proteins protect cancer cells through efflux of chemotherapeutic agents (Fig. 1), resulting in reduced intracellular drug levels (Fletcher et al. 2010). Among the 49 ABC transporters in humans, only three have been largely studied in multi-drug resistance of cancers (Fletcher et al. 2010; Holohan et al. 2013): ABCB1 (Multi-drug resistance protein 1; MDR1/P-glycoprotein), ABCC1 (MDR-associated protein1/MRP1), and ABCG2 (BCRP). ABCB1 is overexpressed and associated with chemo-resistance in many cancers including leukemia as well as liver, colon, ovarian, and breast cancers (Holohan et al. 2013; Triller et al. 2006; Calcagno et al. 2010). Lin-CD34?CD38CML stem cells had higher ABCB1 and ABCG2 expression than the more mature Lin-CD34?CD38? cells did, which is further evidence of imatinib mesylate resistance (Jiang et al. 2007). In ovarian cancer, the CSC population, represented by the drug-resistant side population, showed higher expression of ABCB1 and EZH2 than did the nonside population of cancer cells (Rizzo et al. 2011). ABCC1 overexpression is correlated with chemo-resistance in lung, prostate, and breast cancers (Holohan et al. 2013; Nooter et al. 1997). In prostate cancer, CD44?CD133? prostate CSCs were isolated using FACS and were more resistant to cisplatin than non-stem-like prostate cancer cells. This resistance was achieved through Notch1 direct binding to the ABCC1 promoter and increasing its expression (Liu et al. 2014). In breast cancer, the overexpression of Twist promoted generation of breast CSCs, identified by CD44?CD24-/low expression. These cells have elevated efflux rates of Hoechst 33342 and Rhodamine 123 due to increased ABCC1 expression (Vesuna et al. 2009). ABCG2 is also a potential marker that causes CSC chemo-resistance in ovarian cancer, breast cancer, hepatocellular carcinoma, and glioblastoma. In breast CSCs overexpressing caveolin-1, chemo-resistance is mediated by b-catenin/ ABCG2 signaling (Wang et al. 2014). ABCG2 overexpression enhances malignant behavior, including doxorubicin resistance of hepatocellular carcinoma (HCC). These results suggest that ABCG2 is a potential CSC marker for HCC (Zhang et al. 2013). In glioma stem cells, the PTEN/ PI3K/Akt pathway regulates ABCG2 activity, enhances the side population, and increases drug resistance to TMZ treatment (Bleau et al. 2009). Recently, ABCB5 was identified as a novel chemo-resistance mediator and molecular marker of CSCs in melanoma (Schatton et al. 2008). Moreover, ABCB5 can maintain melanoma-initiating cells through IL1b/IL8/CXCR1 cytokine signaling (Wilson et al. 2014). Because ABC transporters mediate the export and import of the drugs, lipids, and various metabolic products, they can influence CSC chemo-

Therapeutic resistance of cancer stem cells

resistance not only through direct drug efflux, but also through exporting cancer-promoting substrates in the tumor microenvironment. DNA damage repair systems As many chemo-therapeutic agents and IR induce DNA damage, the DNA repair pathways of cancer cells can influence the effectiveness of chemo- and radio-therapies. Various DNA repair pathways can be differentially activated based on the type of lesions. DNA mismatch repair (MMR) system repairs mismatched bases that occurred due to insertion, deletion, or misincorporation of bases during DNA replication and recombination. The nucleotide excision repair (NER) pathway removes bulky DNA adducts such as thymine dimers that were induced by ultraviolet radiation. The base excision repair (BER) removes damaged DNA bases using lesion-specific DNA glycosylases (Blanpain et al. 2011; Bouwman and Jonkers 2012). All of these systems are activated upon single-strand damage. For double-strand breaks (DSBs), there are three mechanisms of repair. The exchange between two similar or identical DNA strands is exerted via homologous recombination (HR). Non-homologous end-joining (NHEJ) does not require a homologous template and directly ligates broken ends. Microhomology-mediated end joining (MMEJ) repairs DNA breaks via base pairing between microhomologous sequences of about 5–25 nucleotides. However, MMEJ always results in deletions (Bouwman and Jonkers 2012; McVey and Lee 2008). Although these systems and DNA damage response genes play a role in tumor suppression, many studies have reported that CSCs utilize these DNA repair systems to acquire resistance to chemoand radio-therapy (Fig. 1). CD133? glioma stem cells activate DNA damage checkpoints after exposure to IR and repair damaged DNA (Bao et al. 2006; Rich 2007). Thus, CD133? glioma stem cells are more resistant to IR than CD133- glioma cells are. Notably, this IR resistance of CD133? glioma stem cells can be reversed by CHK1/2 inhibitors. Therefore, these results suggest that activation of damage checkpoints plays a crucial role in radio-resistance of CSCs. Glioma stem cells also regulate resistance to TMZ via O6-methylguanine DNA methyltransferase (MGMT). Since MGMT can reverse alkylation at the O6 position of guanine and attenuate the cytotoxic effects of TMZ, its expression increases resistance in glioma stem cells (Maugeri-Sacca et al. 2012; Qiu et al. 2014). CD44?/CD24- breast CSCs exhibited greater radio-resistance than the non-stem-like breast cancer cells through activation of ATM signaling, not by NHEJ mechanism (Yin and Glass 2011). Similarly, CD133? lung CSCs contributed to radio-resistance via alteration of DNA repair gene expression (Desai et al.

2014). In prostate cancer, repression of CHK1 abrogated G2/M arrest and increased apoptosis in prostate CSCs (Wang et al. 2012). In pancreatic cancer, CD24?CD44?ESA? pancreatic CSCs displayed greater sensitivity to CHK1 inhibition in combination with gemcitabine than did the non-stem-like pancreatic cancer cells (Venkatesha et al. 2012). Treatment of non-small-cell lung CSCs with chemotherapy combined with CHK1 inhibitors significantly reduced tumorigenicity in mouse xenografts (Bartucci et al. 2012). Altogether, these results indicate that CHK1 is a potential regulator of CSC chemo-resistance and radioresistance. Polycomb group (PcG) proteins are epigenetic modifiers involved in gene silencing and regulate DNA damage responses. In addition, PcG proteins are recruited and accumulate in DSBs, where they ubiquitinate histone H2A and other substrates (Vissers et al. 2012; Gieni et al. 2011; Ginjala et al. 2011; Hong et al. 2008; Rouleau et al. 2007). In particular, BMI1, a well-known PcG protein, accumulates at the chromatin of CD133? CSCs after IR treatment, and this is a critical event in activating DSB repair signaling. Consequently, repression of BMI1 in CSCs impairs the DSB response and increases radio-sensitivity (Vissers et al. 2012). Reactive oxygen species (ROS) levels ROS are a major cause of apoptosis induced by IR. Although ROS are important for cancer cell proliferation, metabolism, and tumorigenesis, increased ROS levels interrupt the redox balance and kill CSCs (Shi et al. 2012). Therefore, to balance ROS levels, cells use ROS-scavenging systems regulated by superoxide dismutases (SOD1, SOD2, and SOD3), peroxiredoxins, glutaredoxin, thioredoxin, catalase, and glutathione peroxidase (Trachootham et al. 2009). Interestingly, CSCs maintain lower intracellular ROS levels than non-CSCs, which contribute to radio-resistance (Fig. 1). CD44?/CD24- breast CSCs have significantly low levels of ROS through high activity of the ROS scavenger glutathione (GSH), which is a cellular reducing molecule and acts as an antioxidant (Phillips et al. 2006; Diehn et al. 2009). They have high levels of the GSH biosynthesis enzyme glutamate-cysteine ligase, which catalyzes the rate-limiting step of GSH synthesis (Diehn et al. 2009). Inhibition of GSH synthesis decreases CSC colony formation after IR and thus increased radio-sensitivity. Similarly, CD13 and ROS scavengers play a critical role in maintaining CSCs in liver cancer (Kim et al. 2012; Haraguchi et al. 2010). CD13 regulates both self-renewal and ROS level of CSCs through modulation of ROS scavenger synthesis (Haraguchi et al. 2010). CD44 and its variant CD44v enhance radio-resistance of CSCs by interacting

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with and stabilizing xCT, which is important for the cysteine uptake required for GSH synthesis (Ishimoto et al. 2011).

Chemo-resistance and radio-resistance and cancer stem cell microenvironment In addition to the intrinsic characteristics of cancer cells in acquiring chemo-resistance and radio-resistance, crosstalk between cancer cells and the microenvironment is important in tumor initiation, progression, and response to therapy (Hanahan and Weinberg 2011). The tumor microenvironment is composed of different cancer cells and various non-cancerous cells and cellular components such as vessels, fibroblasts, immune cells, extracellular matrix (ECM), and diverse soluble factors. Notably, the microenvironment also plays crucial roles in maintaining the self-renewal and chemo-resistance and radio-resistance of CSCs through formation of specific niches such as perivascular and hypoxic/necrotic niches (Cabarcas et al. 2011; Mannino and Chalmers 2011; Ghotra et al. 2009). CSCs are located near vessels to maintain their selfrenewal and proliferative capacities, and endothelial cells protect CSCs from chemo- and radio-therapy (Calabrese et al. 2007; Borovski et al. 2011; Garcia-Barros et al. 2003). Endothelial cells that are resistant to apoptosis maintain tumor growth after IR. Because ablation of blood vessels in tumors decreased the CSC population and tumor growth, disruption of CSC vascular niche environments might serve as a promising therapeutic modality (Fig. 2). In a murine Burkitt’s lymphoma model, IL-6 released from endothelial cells of thymus in response to a chemo-therapeutic agent created a ‘chemo-resistant niche’ resulting in the survival of minimal residual tumor cells and tumor relapse (Gilbert and Hemann 2010). In colorectal cancer, soluble Jagged-1 secreted from endothelial cells promoted the CSC phenotypes in colorectal cancer cells, including chemo-resistance (Lu et al. 2013). CXCR4 is a receptor for CXCL12 and is expressed on the surface of many different cell types, including hematopoietic stem cells, endothelial cells, and epithelial cells (Trautmann et al. 2014). CXCR4 level is increased in a variety of cancers and involved in cancer progression, metastasis, and stemness (Gelmini et al. 2008; Croker and Allan 2008; Orimo et al. 2005). In particular, interaction between CXCR4 and CXCL12 plays important roles in regulating the cancer microenvironment, angiogenesis, and the CSC niche (Trautmann et al. 2014). CXCL12 is mainly secreted from cancer-associated stromal cells and is generated by radiation-induced hypoxia (Kioi et al. 2010). Increased CXCL12 might promote stem cell signaling,

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monocyte recruitment, and changes in the tumor microenvironment, all of which lead to increased angiogenesis, resistance to IR, and tumor recurrence (Fig. 2). The CSC population might be enriched from the preexisting CSCs due to high activity of the DNA repair system, low levels of ROS, and increased cell survival signals as well as increased by generation of new CSC by ionizing radiation (Lagadec et al. 2012, 2013). Of particular interest, IR promotes reprogramming of non-stem-like cancer cells into CSCs, and this is dependent on Notch signaling in breast cancer. These induced CSCs express high levels of OCT4, SOX2, Nanog, and KLF4, and show increased stem cell properties such as mammosphere formation and tumorigenicity.

Targeting cancer stem cells and therapeutic approaches Since CSCs are more resistant to conventional anticancer therapies than non-stem-like cancer cells are, they are enriched after anticancer therapy, resulting in tumor recurrence and progression. Therefore, many studies have focused on targeting CSCs by understanding the various signaling pathways involved in their self-renewal ability and resistance to chemo- and radio-therapy. Multiple therapeutic target agents have been identified to inhibit self-renewal signaling (Wnt, Notch, and Hedgehog), ABC transporters, DNA repair enzymes, and the tumor microenvironment (Fig. 3). Targeting stem cell signaling A number of experimental agents have been assessed for their capacity to inhibit stem cell signaling pathways. ICG001, a preclinical agent targeting the Wnt signaling pathway, selectively inhibited binding of b-catenin to its transcriptional cofactor CBP, resulting in cancer cell-specific cell death (Emami et al. 2004). NSC668036 and FJ9 are also preclinically tested compounds that target the PDZ domain of dishevelled, a key protein linking the extracellular Wnt signal to cytoplasmic b-catenin activity (Shan et al. 2005; Fujii et al. 2007). Inhibition of Notch signaling has been performed by targeting c-secretase, which releases Notch intracellular domain (NICD) through proteolytic cleavage and activates Notch transcriptional function. RO4929097 and MK0752 are c-secretase inhibitors (GSI) in the phase I clinical trials (Deangelo et al. 2006). When MK0752 was initially tested in T cell acute lymphoblastic leukemia, it exhibited cytotoxic side effects. However, those toxicities were successfully reduced by coadministration of glucocorticoids and GSI in a mouse model of T-cell acute lymphoblastic leukemia (Real et al. 2009).

Therapeutic resistance of cancer stem cells Fig. 2 Acquisition of chemoresistance and radio-resistance of CSCs by tumor microenvironment. After chemo- and radio-therapy, the self-renewal and proliferative capacities of CSCs located near vessels are activated by Jagged1 and IL6. In addition, therapeutic resistance of CSCs is also regulated by CXCL12 that promotes stem cell signaling, monocyte recruitment, and change in the tumor microenvironment

CXCL12 CXCR4

Vasculogenesis stemness

Jagged

Cancer stem cell IL6

Cyclopamine, a Hedgehog pathway-specific inhibitor, is a steroidal alkaloid that binds to and inhibits SMO (Taipale et al. 2000). A number of preclinical studies showed that high-dose cyclopamine showed nonspecific effects. GDC0449, a small molecule SMO inhibitor, is in phase II clinical trials in patients with ovarian and colorectal cancer (LoRusso et al. 2008; Von Hoff et al. 2009; Takebe et al. 2011). Targeting ABC transporters ABCB1 (P-glycoprotein) inhibitors have been tested in phase I and phase II clinical trials. First-generation P-glycoprotein inhibitors, such as verapamil, quinine, and cyclosporine, were ineffective or toxic at the doses that inhibit P-glycoprotein function. Nevertheless, a randomized phase III clinical trial showed effectiveness in AML patients when a P-glycoprotein inhibitor were combined with anticancer drugs, such as cytarabine and daunorubicin (List et al. 2001). There are novel ABC transporter inhibitors such as MS-209, VX-710, and tariquidar that can suppress both P-glycoprotein and MRP1. Recent trials using MS-209 have shown promising outcomes in breast cancer with drug resistance. Combination treatment of tariquidar and docetaxel has been investigated for the treatment of ovarian, cervical, and lung cancers (Patil et al. 2009; Blanpain et al. 2011; Minderman et al. 2004).

CXCL12

IL6

Monocyte

Endothelial cell

CXCR4

Targeting the DNA damage checkpoint response Chemo- and radio-therapy generally activates the checkpoint transducers ATM and ATR, which phosphorylate the downstream effectors of the DNA damage repair response CHK2 and CHK1, respectively (Reinhardt and Yaffe 2009; Cimprich and Cortez 2008; Shiloh 2001). Therefore, specific inhibitors for these key factors have been developed to effectively eradicate CSCs. Debromohymenialdisine has been used to inhibit the checkpoint response in glioma stem cells by targeting CHK1/2 (Bao et al. 2006). AZD7762, an ATP-competitive inhibitor of the CHK family, enhances the anticancer therapeutic efficacy of DNA damaging agents (Zabludoff et al. 2008). A number of ATM, ATR, and CHK inhibitors are undergoing clinical development (Dai and Grant 2010). ROS generating mechanisms ROS play crucial roles in controlling resistance of cancer cells to chemo- and radio-therapy. Depletion of the antioxidant GSH and treatment with buthionine sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase, ablated the colony forming ability and radio-resistance of breast CSCs (Diehn et al. 2009). Combination therapy with CD13 inhibitor and an antibody disrupting the ROS pathway inhibited the self-renewal and cancer-initiating ability of CSCs (Haraguchi et al. 2010). In addition,

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Activated stemness signaling

Wnt/ƅECVGPKPKPJKDKVQTU - ICG-001, PNU-74654, NSC668036, FJ9

ABCB1 inhibitors - verapamil, quinene, cyclosporine, GF120918

- Wnt/ƅECVGPKPUKIPCNKPI - Notch signaling - Hedgehog UKIPCNKPI

Notch inhibitors - RO4929097, MK0752, MPC7869

ABC transporters (Increased efflux)

ABCB1 and ABCC1 inhibitors - MS-209, VX-710, tariquidar ABCG2 inhibitors - Ko143, GF120918,

Hedgehog inhibitors - Cyclopamine, GDC-0449, IPI926, BMS-833923 Maintenance of low ROS levels

Chk inhibitor - AZD7762

Impaired apoptosis pathways

GPx inhibitor - Arsenic trioxide(As2O3)

Smac mimetics

(cFLIP, IAPs, Bcl-2 family)

DNA damage repair system (ATM, ATR, Chk1, MGMT, etc)

Cancer stem cell Microenvironment CXCR4-CXCL12 signaling

CXCR4 antagonist AND3100, T14003 analogs

Fig. 3 Therapeutic approaches targeting CSCs. ICG-001 and PNU74654 prevent interaction of b-catenin with CBP, resulting in suppression of b-catenin-downstream gene expression. NSC668036 and FJ9 suppress Wnt/b-catenin signaling by inhibiting PDZ domain of deshevelled protein. RO4929097, MK0752, and MPC-7869 inhibit Notch signaling by suppressing c-secretase activity. Cyclopamine, GDC-0449, IPI-926, and BMS-833923 suppress Hedgehog signaling

by inhibiting SMO. Smac mimetics activate apoptosis of CSCs in combination with chemo-therapeutic agents. The cytotoxic effect of chemo-therapeutic agents on CSCs is activated by ABC transporter inhibitors. AZD7762 inhibit DNA damage checkpoints by targeting CHK1/2. Arsenic trioxide leads to increase of ROS by suppressing glutathione peroxidase (GPx)

silencing CD44 and its variant CD44v increased ROS levels in breast CSCs by suppressing levels of the antioxidant, and this effect was restored by administering the antioxidant N-acetyl L-cysteine (Ishimoto et al. 2011). Because chemo- and radio-therapy generates ROS directly or indirectly, combination therapy with ROSgenerating agents and conventional chemo- and radiotherapy can enhance therapeutic efficacy. Arsenic trioxide (As2O3) which is known to inhibit glutathione peroxidase (GPx), was approved by FDA for treatment of acute promyelocytic leukemia (Rao et al. 2013). Many ROSgenerating agents are currently in clinical trials (Trachootham et al. 2009).

tumor vessel normalization and can disrupt the CSC vascular niche (Burkhardt et al. 2012; Narita 2013). The CXCR4/CXCL12 pathway led to changes in the tumor microenvironment after chemo-and radio-therapy, which were crucial for tumor recurrence (Kioi et al. 2010). Plerixafor (AMD3100), a CXCR4 antagonist, blocks recruitment of monocytes, inhibits restoration of cancer vasculature, and prevents cancer recurrence. Several inhibitors of the CXCR4/CXCL12 pathway are currently in clinical trials (Burger and Peled 2009; Konopleva et al. 2009).

Conclusion Targeting microenvironment Because the vasculature plays crucial roles in tumor progression and in generating a vascular niche for CSCs, many studies have been conducted to develop therapeutic modalities targeting the tumor vasculature. A number of recent studies have demonstrated that Bevacizumab, a VEGF inhibitor, inhibits tumor progression by leading to

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Accumulating evidence indicates a direct role of CSCs in tumor relapse post-therapy. Based on the many studies introduced in this review, it is obvious that chemo-resistance and radio-resistance are regulated by not only CSC intrinsic mechanisms, but also by extrinsic factors activated through interactions between CSCs and the microenvironment. Despite numerous basic research studies and clinical

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trials, chemo- and radio-therapy provides limited effects on cancer patient survival, suggesting that new therapeutic modalities should be developed. Of interest, a number of recent studies have demonstrated that diverse intrinsic and extrinsic mechanisms controlling therapy-resistance might be activated in different CSC populations in the same tumor, leading to ‘heterogeneity of CSCs’ (Magee et al. 2012; Tang 2012). This may be a reason why cancers are resistant to various therapeutic modalities and are eventually recur. Recently, anticancer therapeutic treatments have moved toward molecular targeted therapies, which customize based on the diverse features of cancers observed across different patients and even within the same patient. Therefore, understanding the precise mechanisms underlying CSC resistance to therapeutics should help develop various novel therapeutic modalities for prevention of tumor relapse. Acknowledgments This work was supported by the National Nuclear Technology Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT and Future Planning (No. 2013M2A2A7042530 to H. Kim). H.-Y. Jeon was supported by the Kwanjeong Educational Foundation Domestic Scholarship.

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