Characteristics of liver cancer stem cells and clinical correlations

ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Mini-review

Characteristics of liver cancer stem cells and clinical correlations Zhuo Cheng a,b, Xiaofeng Li a,b, Jin Ding a,b,* a International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital/Institute, Second Military Medical University, Shanghai 200433, China b National Center of Liver Cancer, Shanghai 200433, China

A R T I C L E

I N F O

Article history: Received 22 June 2015 Received in revised form 17 July 2015 Accepted 18 July 2015 Keywords: Liver cancer Cancer stem cell Self-renewal Chemoresistance

A B S T R A C T

Liver cancer is an aggressive malignant disease with a poor prognosis. Patients with liver cancer are usually diagnosed at an advanced stage and thus miss the opportunity for surgical resection. Chemotherapy and radiofrequency ablation, which target tumor bulk, have exhibited limited therapeutic efficacy to date. Liver cancer stem cells (CSCs) are a small subset of undifferentiated cells existed in liver cancer, which are considered to be responsible for liver cancer initiation, metastasis, relapse and chemoresistance. Elucidating liver CSC characteristics and disclosing their regulatory mechanism might not only deepen our understanding of the pathogenesis of liver cancer but also facilitate the development of diagnostic, prognostic and therapeutic approaches to improve the clinical management of liver cancer. In this review, we will summarize the recent advances in liver CSC research in terms of the origin, identification, regulation and clinical correlation. © 2015 Elsevier Ireland Ltd. All rights reserved.

Introduction

Origin of liver cancer stem cells

Liver cancer is the sixth most common malignancy and is second only to lung cancer in mortality [1]. Hepatocellular carcinoma (HCC) is the major pathological type and accounts for approximately 80% of cases of liver cancer. Despite the enormous advances in the diagnosis and treatment of liver cancer during the past several decades, the therapeutic effect of this deadly disease remains disappointing. The first-line curative approaches for liver cancer mainly involve partial liver resection and liver transplantation. Unfortunately, most patients miss the opportunity for resection due to a late diagnosis [2–5]. Even after surgical resection, the long-term prognosis remains very poor due to frequent recurrence. Chemotherapy via either systemic treatment or transarterial chemoembolization is the secondline treatment, but the overall response rate is rather low due to the high chemoresistance of liver cancer [6]. Emerging evidence has shown that cancer stem cells (CSCs), also termed tumor-initiating cells (T-ICs), exist in solid tumors and are responsible for cancer relapse, metastasis and chemoresistance [7]. The existence of liver CSCs has been reported in numerous studies [8], and these cells are considered to account for the heterogeneous and hierarchical organizations of HCCs [9,10]. In this review, we present a brief and up-to date overview of the characteristics of liver CSCs and their clinical correlations.

Although the existence of liver CSCs has been widely accepted, the origin of these cells remains controversial. One possible origin of liver CSCs is the transformed liver stem/progenitor cells [11]. Hepatoblasts are the stem cells of the liver that retain the ability to self-renew and proliferate to provide liver progenitor cells (LPCs), which can differentiate into hepatocytes and cholangiocytes. Malignant transformation of hepatoblasts could be the most direct way to generate liver CSCs. LPCs, a subpopulation of small and ovalshaped cells, usually reside quiescently in the canals of Hering and bile canaliculi. It was reported that LPCs could be derived from hematopoietic stem cells or mesenchymal stem cells [12,13]. Most recently, Sahin et al. demonstrated that hepatocytes were the origin of LPCs [14]. LPCs are known to differentiate into hepatocytes or cholangiocytes and engage in liver regeneration when the replication of liver parenchymal cells is restricted [15]. Nevertheless, chronic liver disease alone with long-lasting inflammation and hepatocyte regeneration might facilitate the transformation of LPCs into liver CSCs [16,17]. Our previous study demonstrated that longterm TGF-β exposure drove the transformation of LPCs into liver CSCs, contributing to cirrhosis-elicited hepatocarcinogenesis [18]. Another possible origin of liver CSCs is transformed adult hepatocytes/ cholangiocytes through mutation and dedifferentiation. Distinct from other types of mature parenchymal cells, adult hepatocytes possess the ability to regenerate during liver damage and can differentiate into both hepatocyte and biliary lineages exhibiting stem cell properties [15]. Acquisition of stemness during or after the transformation of hepatocytes might give rise to liver CSC generation (Fig. 1). Recently, Thorgeirsson et al. stably transduced oncogenic H-Ras into

Zhuo Cheng and Xiaofeng Li contributed equally to this work. * Corresponding author. Tel.: +86 21 81875366; fax: +86 21 65566851. E-mail address: [email protected] (J. Ding). http://dx.doi.org/10.1016/j.canlet.2015.07.041 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.

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Fig. 1. Potential origin and generation of liver CSC.

murine hepatoblasts, LPCs and adult hepatocytes and found that all of these transformed cells acquired stem cell properties and converted into CSCs after the genetic/epigenetic alterations [19]. Additionally, phenotypic flexibility characterizes a subset of cancer cells possessing the capability of interconverting between differentiated and stem-like status via a continuum of cell fate specifications [20]. Recent studies have suggested that non-stem breast cancer cells dedifferentiated into breast CSCs following a typical EMT (epithelial–mesenchymal transition) process [21,22]. These findings implied that the plasticity of liver CSCs might be responsible for the heterogeneous organization of liver cancers and the distinct survival of patients. Identification of liver cancer stem cells In general, liver CSCs can be isolated and enriched based on their immunogenic or functional characteristics. The antigenic approach targets certain cell surface markers and has been utilized to achieve distinct liver CSC fractions from human HCCs. The functional isolation depends on the particular characteristics of liver CSCs, such as side population (SP), high aldehyde dehydrogenase (ALDH) activity and autofluorescence. Considering the plasticity and heterogeneity of the liver CSC origin, it is impossible to define liver CSCs by a single marker or one functional property alone. Therefore, a combination of antigenic and functional approaches could be more appropriate to identify liver CSCs.

to self-renew, differentiate and initiate tumors [26]. Yang et al. isolated CD90+ liver CSCs from MHCC97H and PLC/PRF/5 cell lines and unraveled their CSC properties, including self-renewal, tumor formation and metastatic capacity [27]. Unlike other known liver CSC markers, CD44 serves as a liver CSC marker only in combination with other CSC markers, such as CD133 [28] or CD90 [29]. In addition, a CD44 variant has been unveiled to regulate the redox status by stabilizing xCT and protecting CSCs from oxidative stress [30]. Our lab clarified that the CD133+ cell population was significantly enriched in OV6+ HCC cells and that OV6+ HCC cells possess greater tumorigenicity and chemoresistance compared with OV6− cells, indicating that OV6 could be another liver CSC marker [31]. Haraguchi et al. identified that CD13+ HCC cells were liver CSCs and were enriched in SP cells isolated from Huh-7, PLC/PRF/5 or Hep3B cells respectively. In further study, they showed that the CD13+ population existed predominantly in the G1/G0 phase and that CD13 expression reduced the cell damage triggered by genotoxic reagents, which was consistent with the quiescent characteristic of

Surface markers of liver CSCs Distinct cell surface proteins have been reported as liver CSC markers, including CD133, EpCAM, CD90, CD44, OV-6, CD13, CD24, DLK1, α2δ1, ICAM-1 and CD47 (Fig. 2). Moreover, some of these markers have been elucidated to possess a regulatory role in liver CSC, which include CD24, CD133, CD47, CD13, CD44 and ICAM-1 [23]. CD133, also known as prominin 1, is a transmembrane glycoprotein expressed in the adult stem cells. Ma et al. reported, for the first time, that CD133 was a liver CSC marker and was required for maintenance of liver CSCs through activation of neurotensin/IL-8/CXCL1 signaling [24,25]. Yamashita et al. proposed the utility of EpCAM for HCC classification and observed that EpCAM+ HCC cells possessed CSC phenotypes, including the capacity

Fig. 2. Liver CSC surface markers identified to date.

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CSCs [32]. CD24 and ICAM-1 were both verified to be functional liver CSC markers that drove HCC initiation and progression through the activation of stemness-associated factor Nanog [33,34]. Han et al. reported that DLK+ cells possessed robust chemoresistance and selfrenewal capacity in vitro and potent tumor initiating ability in vivo [35]. Recently, Zhao et al. sorted liver CSCs using a new antibody (1B50-1) recognizing isoform 5 of the cell surface calcium channel α2δ1 subunit. They also verified that α2δ1 potentiated liver CSCs by activating pro-survival pathways via a calcium-dependent mechanism [36]. Lee et al. demonstrated that CD47 was another liver CSC biomarker that was preferentially expressed in liver CSCs, contributing to tumor initiation and metastasis. They also found that CD47 expression significantly correlated with HCC recurrence and patient survival [37]. Functional isolation of liver CSCs Side population (SP) cells possess the ability to efflux Hoechst33342 through an ATP-binding cassette (ABC) membrane transporter. Chiba and colleagues demonstrated that SP cells sorted from HCC exhibited potent tumorigenicity in the primary and secondary transplanted tumors exerting their CSC properties [38]. They also clarified that Bmi-1 was a key regulator for the self-renewal and tumorigenicity of SP cells [39]. Unique from other approaches, isolation of liver CSCs by side population sorting reflects the active efflux of intracellular drugs, which is closely associated with the chemoresistant property of CSCs. Although SP cells did not provide a particular target for HCC therapy, overexpression of the ABC transporter implies the therapeutic potential of targeting the SP population [40]. ALDH belongs to a universally expressed enzyme family that is responsible for the oxidation of intracellular aldehydes [41]. Growing evidence has indicated that isolated cancer cells with high ALDH activity exhibit the CSC phenotype, and high ALDH activity confers chemoresistance to CSCs [41,42]. Ma and colleagues observed that CD133+ALDH+ cells are much more tumorigenic than their counterparts, and combination of CD133 and ALDH achieved a more ‘‘homogeneous” liver CSC fraction [42]. Similar to normal stem cells, CSCs maintain themselves through symmetric and asymmetric divisions. Identification of liver CSCs could be achieved by qualitatively and quantitatively determining the chromosomal co-segregation during the mitosis, resembling the process in various normal stem cells [43]. It has been observed that expression of the CSC marker CD133 co-segregated with the template DNA, whereas the differentiation markers presented in the opposing daughter cancer cells, suggesting that CSCs might be identified and isolated during the cell division process regarding their asymmetric division characteristics. Recently, a new functional approach was established to identify CSCs by Heeschen and colleagues. They identified an intrinsic autofluorescent characteristic of CSCs in diverse epithelial cancers including HCC and successfully isolated these cells based on this distinct feature. They also reported that autofluorescence was a result of the accumulation of fluorescent vitamin riboflavin in ABCG2coated vesicles exclusively located in the cytoplasm of CSCs. Moreover, these cells presented distinguished CSC features and phenotypes, such as conspicuous chemoresistance, long-term tumorigenicity and remarkable invasiveness [44]. Regulation of liver cancer stem cells Considerable advances in liver CSC research over the past decade have unraveled many factors that regulate liver CSCs, such as CSCassociated signaling pathways, stemness-related transcription factors, epigenetic regulators, non-coding RNAs and the liver CSC niche. Most of those regulators are also involved in the regulation of normal stem

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cells or CSCs in other types of cancer. Herein, we summarize the relatively specific mediators involved in the regulation of liver CSCs. Liver CSC-associated signaling pathway The Wnt/β-catenin pathway is important in development and differentiation, and disruption of Wnt/β-catenin cascade was detected in 20–40% of human HCCs [31]. Accumulating studies have indicated the hyperactivation of Wnt/β-catenin signaling in distinct liver CSC populations, including EpCAM+ [26], CD133+ [24] and OV6+ [31] liver CSCs. Among the currently identified liver CSC markers, EpCAM was ascertained to be a direct transcriptional target of β-catenin. Consistently, Wang et al. found that high tumorigenic and invasive EpCAM+AFP+ liver CSCs were regulated by Wnt/βcatenin signaling [26]. Moreover, our previous study also unraveled that Wnt/β-catenin signaling contributed to the activation of normal and transformed liver progenitor cells [31]. Increasing evidence has shown that Notch is essential for the maintenance and expansion of CSCs in many malignancies including HCC. It was reported that liver-specific Notch activation recapitulated the consecutive stages of human hepatocarcinogenesis and was associated with liver cancer metastasis [45–47]. Consistently, Notch and the Notch ligand Jagged were detected to be highly expressed in human HCC and in CD133+ liver CSCs [24,48]. Moreover, the tumor suppressor RUNX3 was reported to inhibit hepatocarcinogenesis by suppression of Jagged-mediated CSC expansion [49]. Activation of IL-6/STAT3 signaling is critical to the survival and self-renewal of stem cells [50]. Mishra et al. found that liver stem cells with hyperactivated STAT3 gave rise to liver cancer through IL-6-dependent signaling [17]. These data were further supported by our laboratory; we found that hepatitis B virus X protein (HBx)induced malignant transformation of PLCs facilitated hepatocarcinogenesis through activation of IL-6/STAT3 signaling in mice [51]. Additionally, a recent study demonstrated that tumorassociated macrophages (TAMs) produced IL-6, which promoted the expansion of CD44+ liver CSCs and facilitated tumorigenesis through STAT3 signaling pathway [52]. Ng and colleagues also demonstrated that STAT3 activation enhanced the self-renewal of CD24+ liver CSCs by upregulating Nanog expression [33]. Stemness-related transcription factors The maintenance and propagation of CSCs were reported to be regulated by distinct stemness-related transcription factors, including Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28, etc. These factors have been well-established as essential modulators in maintenance of stem cell pluripotency, and dysregulation of the factors promoted CSC propagation and tumorigenesis [53]. c-Myc is one of the most common oncogenes activated in human cancers, particularly HCC. Thorgeirsson et al. found that c-Myc activation was required for the oncogenic reprogramming of terminally differentiated hepatocytes into liver CSCs [19]. Recently, they established a direct connection between c-Myc activity and CSC properties, showing that c-Myc expression levels exhibited differential impact on liver CSC characteristics. At low levels, activation of c-Myc led to augmented proliferation and enhanced CSC properties. However, when surpassing a threshold level, c-Myc activation triggered a proapoptotic program and attenuated CSC properties [54]. Nanog has also been implicated in promoting the chemoresistance and invasion of liver cancer [55]. Shan and colleagues found that downregulation of Nanog in Nanog-positive liver CSCs decreased the expression of stemness genes and increased the mature hepatocyte-associated genes. Overexpression of Nanog in Nanognegative HCC cells restored the self-renewal capacity [56]. Moreover,

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stemness-related factors Oct4 [57], Sox2 [58] and Lin28 [16] were also reported to regulate liver CSCs. SALL4 is a C2H2 zinc-finger transcription factor that is encoded by the human homolog of the drosophila spalt homeotic gene [59]. SALL4 is known to regulate the stemness of embryonic and hematopoietic stem cells, and upregulated SALL4 expression was observed in those HCCs with stem cell features. A most recent study involving two independent large cohorts indicated that SALL4 could be a marker for the progenitor cell-derived HCC displaying an aggressive phenotype. Gene expression analysis unraveled the enrichment of a progenitor cell-associated expression profile in SALL4-positive HCCs [60]. Zeng and colleagues illustrated that SALL4 activation upregulated the expressions of CK19, EpCAM and CD44 and enhanced spheroid formation and invasion capacities in HCC cells. In addition, SALL4 expression status correlated with histone deacetylase activity, and the histone deacetylase inhibitor dramatically suppressed the proliferation of SALL4 positive HCC cells [61]. Most recently, Marquardt and colleagues showed that blockage of NFκB specifically suppressed CSC population in HCCs and proposed that combinational inhibition of NF-κB and HDAC signaling could be a promising strategy in HCC treatment [62]. Epigenetic regulation The importance of epigenetic regulation has been well illustrated by the fact that epigenome undergoes profound changes, whereas the genome remains unaltered from zygote to somatic tissues. Dysregulation of epigenetic machinery has been recognized as a fundamental mechanism involved in carcinogenesis and CSC regulation [63]. Marquardt and colleagues clarified the significance of epigenetic mechanisms in liver CSC using DNMT1 inhibitorted SP cells [64]. They found that inhibition of DNA methylation in HCC cells led to the increase of tumorigenic cells among the SP fraction. Bmi-1 belongs to the polycomb group gene (PcG) family, which is highly conserved throughout evolution. The PcG family acts as an epigenetic chromatin modifier in self-renewal process of embryonic and adult stem cells. Overexpression of Bmi-1 has been detected in sorted populations of CD133+ [24], EpCAM+ [26], CD24+ [33] and CD90+ [29] liver CSCs, suggesting the important role of Bmi-1 in liver CSCs. Moreover, Chiba and colleagues found that Bmi-1 was preferentially expressed in the SP subpopulation of Huh-7 and PLC/PRF/5 HCC cells in comparison with non-SP cells [39]. It was reported that forced Bmi-1 expression resulted in hepatic stem cell expansion and tumorigenesis in both Ink4a/Arf-dependent and Ink4a/Arf-independent manners [65]. Non-coding RNAs Noncoding RNAs (ncRNAs) are regulatory molecules extensively studied in the field of cancer research during the past decade. An increasing number of ncRNAs have shown abnormal expression patterns in various human cancers including HCC. Screening and identification of ncRNAs involved in liver CSC regulation should be important in the early diagnosis and treatment of HCC. MircoRNAs MircoRNAs (miRNAs) are small non-coding RNAs that have a posttranscriptional regulatory role in gene expression. Accumulating studies have established the important role of miRNAs in regulating various cellular processes including differentiation and stemness. Increasing evidence has suggested that miRNAs are involved in the regulation of CSCs including liver CSCs. Wang and colleagues found that miR-181 was highly expressed in the sorted EpCAM+ liver CSCs. MiR-181 promoted the self-renewal capacity of EpCAM+ liver CSCs through augmenting β-catenin activity via targeting NLK (a selective inhibitor Wnt/β-catenin signaling) and through repressing cell

differentiation via targeting CDX2 and GATA6 (two hepatocyte differentiation associated transcription factors) [66]. Through miRNA profiling analysis, Ma and colleagues found that miR-130b was preferentially expressed in CD133 + liver CSCs. Forced miR-130b expression increased the self-renewal ability of liver CSCs and facilitated liver cancer initiation and chemoresistance. Moreover, the tumor-suppressor gene TP53INP1 was identified as a direct target of miR-130b in mediating self-renewal and tumorigenicity of the CD133+ liver CSCs [67]. In addition, miR-150 levels were notably reduced in CD133+ liver CSCs compared to CD133− counterpart. Overexpression of miR-150 resulted in a significant decrease in the CD133+ cell population and the attenuated spheroid formation through inhibiting c-Myb expression [68]. Chai et al. reported that miR-142-3p directly targeted CD133 to regulate its ability to confer cancer stem cell-like features in HCC. Recently, our laboratory observed that miR-429 was upregulated in primary liver CSCs isolated from patient HCC tissues, and miR-429 enhanced liver CSC properties by targeting the Rb binding protein 4 (RBBP4)/E2F transcription factor 1 (E2F1) signaling [69]. LncRNAs In recent years, tremendous effort has been put into the functional study of long noncoding RNAs (lncRNAs) in human cancers, including HCC [70]. Aberrant expression of several lncRNAs was verified to be involved in the recurrence and metastasis of HCC [71–73]. The mechanism underlying the function of lncRNAs is the most difficult and least understood aspect of lncRNA research. Recently, Wang and colleagues conducted transcriptome microarray analysis and identified an lncRNA named lncTCF7 that is highly expressed in liver CSCs. Functional studies revealed that lncTCF7 was required for the self-renewal and propagation of liver CSCs. Mechanistically, lncTCF7 recruits the SWI/SNF complex to the TCF7 promoter and regulates the expression of TCF7 and the subsequent activation of Wnt signaling [74]. Recently, Sun et al. reported that expression of lncRNADANCR was enhanced in liver CSCs and correlated with patient prognosis. They also found that lncRNA-DANCR promoted the selfrenewal of liver CSCs by augmenting β-catenin activation through a microRNA-dependent mechanism [75]. Liver CSC niche The cancer cell niche has a complex architecture consisting of fibroblastic cells, immune cells, endothelial cells, extracellular matrix (ECM) components and numerous cytokines. It is hypothesized that CSCs reside in a particular niche, which not only maintains CSCs in an undifferentiated state but also reinforces their self-renewal abilities [76]. Accumulating studies have elucidated that CSCs release various factors into the CSC niche, which not only stimulate the selfrenewal of CSCs themselves but also induce angiogenesis and recruit immune and other stromal cells that secrete additional factors to facilitate tumor progression and chemoresistance. Mesenchymal stem cells (MSCs) were reported to secrete CXCL12, IL-6 and IL-8 to enhance the CSC stemness through NF-κB activation, and CSCs could secrete IL-6 to recruit more MSCs [77]. Tumor-associated macrophages (TAMs) were reported to produce TNF-α or TGF-β to enhance the CSC plasticity through induction of a NF-κB or TGFβ-dependent EMT process [78]. In terms of HCC, TAMs secreted TGF-β to augment the CSC-like properties in Hepa1-6 cells and increase their invasive capability [79]. Wan and colleagues found that TAMproduced IL6 promoted the expansion of CD44+ liver CSCs and facilitated tumorigenesis via a STAT3-dependent signaling pathway [52]. Furthermore, it has been reported that hypoxia promoted CSC survival upon chemotherapy and radiotherapy through ROSactivated stress response pathways and ROS-induced TGF-β and TNF-α signaling cascades [80]. Inhibition of HIF-1α by 3-(5′hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) suppressed the

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hypoxia-induced chemoresistance in liver CSCs through inhibition of the Akt/HIF-1α/PDGF autocrine loop [81]. Therefore, the CSC niche might be an important target for novel therapeutic strategies for liver cancer. Clinical correlations Conventional HCC therapies primarily target the rapidly growing differentiated HCC cells. However, emerging concept of CSC explain, at least partially, the disappointing outcomes of these therapies. Advances in liver CSC research have shed new light on the potential application of liver CSCs in the clinical management of liver cancer [82]. Detection of liver CSCs might contribute to the prediction of the postoperative survival of patients. The development of therapeutic strategies targeting liver CSCs might greatly improve HCC treatment. Diagnostic and prognostic potential Although the measurement of serum AFP is widely used in the early diagnosis of HCC, some liver CSC markers might be utilized in the diagnosis of HCC at a very early stage and in the prediction of patient survival. The FDA-approved CellSearch system, which was established to determine the EpCAM+ circulating tumor cells, has been successful in predicting patient prognosis [83]. Yang et al. identified CD45−CD90+ liver CSCs in blood samples from HCC patients, suggesting that examination of CD45−CD90+ cells in blood could be a novel diagnostic method for human HCC [29]. Liu and colleagues isolated ICAM-1+ hepatoma cells in the peripheral blood samples of HCC patients as circulating liver CSCs [34]. Therefore, detection of circulating hepatoma cells expressing liver CSC markers might be developed into novel diagnostic approaches for human HCC. According to the characteristics of liver CSCs, existence of these cells closely correlated with patient prognosis. For example, increased CD133 expression acted as an independent prognostic factor for HCC recurrence and poor patient survival [84]. Overexpression of CD90 in HCC was also associated with the poor prognosis of patients [85]. CD24 overexpression correlated well with aggressive HCC behavior and the poor clinical outcome of patients [33]. Increased numbers of ICAM-1+ liver CSCs in the serum of patients with HCC indicated a worse prognosis [34]. CD44 was reported to be correlated with enhanced Smad2 phosphorylation, EMT and poor prognosis of patients with HCC [86]. Gene expression profiles generally indicate the characteristics of the specific cell population. With the gene-profiling approach, CSCs are assumed to express stemnessassociated genes, such as Sall4, Sox2, Oct4, Nanog, etc. Consistently, growing studies have suggested that these genes highly expressed in HCC reflect the abundance of liver CSCs and usually indicate a poor prognosis [60,87,88]. Potential in clinical intervention Since the conventional cancer therapies fail to eliminate CSCs, which are the origin of cancer recurrence and chemoresistance resulting in the dismal outcome of current cancer therapy, exploration of novel therapeutic strategies targeting and eradicating CSCs would be greatly anticipated. Indeed, numerous studies have been conducted targeting the distinct characteristics of liver CSCs. Targeting surface markers of liver CSCs Despite the fact that liver CSCs account for a very small fraction in HCCs, these cells possess much greater tumorigenicity than the bulk of tumor cells. In immune deficient mice, 2 × 104 CD133+ liver CSCs isolated from patients with HCC were sufficient for tumor initiation [26,67]. One recent study revealed that as few as 4000 CD24+ liver CSCs triggered HCC formation [33]. An increasing number

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of studies suggested that directly targeting liver CSC surface markers, such as CD133, CD90, EpCAM, CD44, α2δ1 or CD47, might be an effective approach to specifically eradicate liver CSCs. Smith and colleagues targeted CD133+ liver CSCs by administrating a CD133specific antibody conjugated with a cytotoxic drug, monomethyl auristatin F. They found that this approach potently inhibited the propagation of hepatoma cells both in vitro and in vivo [89]. Yang et al. utilized a neutralizing antibody against CD44 to suppress CD90+ liver CSC-mediated tumor initiation and metastasis [27]. Zhao and colleagues found that treatment of HCC cells with 1B50-1 (an α2δ1 specific antibody) eliminated liver CSCs via the induction of cellular apoptosis. 1B50-1 not only depleted the α2δ1+ liver CSCs cells but also ablated the serial transplantation capacity of hepatoma cells in NOD/SCID mice [36]. Wang and colleagues demonstrated that RNAi-mediated EpCAM silencing dramatically inhibited the tumorigenicity and invasive capacity of liver CSCs [26]. Lee et al. found that blockade of CD47 using anti-CD47 antibody increased the sensitivity of HCC cells to sorafenib [90]. Mori et al. demonstrated that CD13 inhibition significantly impaired the self-renewal and tumor initiating capacity of liver CSCs, which suggests that CD13 could be a potential therapeutic target for human liver CSCs [32]. Anti-self-renewal of liver CSCs Self-renewal is one of the most distinct characteristics of liver CSCs and is closely associated with HCC maintenance and propagation. Therefore, blocking self-renewal of liver CSCs represents a rational therapeutic strategy for HCC treatment [91]. Wang and colleagues identified microRNA-181 as a direct downstream target of Wnt/β-catenin signaling in HCC [26,66]. Suppression of microRNA181 expression by delivering its specific inhibitor dramatically repressed the stemness gene expression and self-renewal of liver CSCs [66]. Moreover, knockdown of Bmi-1 expression remarkably decreased the SP proportion and abolished the tumor-initiating capacity of SP cells in NOD/SCID mice through impairing the selfrenewal of SP cells [39]. Ng et al. reported that Lup-20(29)-en-3βol (lupeol), a triterpene that exists in fruits and vegetables, interrupted the self-renewal capacity of liver CSCs in HCC cell lines and primary HCC cells. The same group also found that knockdown of CD24 could significantly reduce the self-renewal of liver CSCs both in vitro and in vivo [33]. All of these studies demonstrated that impairing the self-renewal of liver CSCs could be a promising approach in HCC targeted treatment. Eliminating chemoresistance of liver CSCs It is well accepted that CSCs are resistant to chemotherapeutic agents due to their characteristic of quiescence as well as their ability to transport drugs out of the CSCs [91]. Chemoresistance of liver CSCs also results in frequent postoperative recurrence and tumor metastasis. Ma and colleagues demonstrated that activation of Akt/ PKB signaling was preferentially required in the chemoresistance of CD133+ liver CSCs. The treatment of CD133+ liver CSCs with an Akt inhibitor abolished the chemoresistant nature of those liver CSCs and sensitized them to DOX and 5-FU administration [92]. Ng et al. reported that lupeol sensitized liver CSCs to chemotherapeutic agents through a PTEN/Akt/ABCG2-dependent manner [93]. They also found that inhibition of CD47 by morpholino suppressed HCC growth and exerted a chemosensitization effect via blockade of CTSS/PAR2 signaling [37]. CD13 attenuated ROS-triggered DNA damage after genotoxic chemotherapy and protected cancer cells from apoptosis. The combination of 5-FU and CD13 inhibitor exhibited a much stronger repressive effect on the xenografted HCC growth in comparison with 5-FU treatment alone [32]. Cheung and colleagues found that chemoresistant HCC cells expressing granulin–epithelin precursor (GEP) presented liver CSC markers, and GEP enhanced chemoresistance by upregulating the expression of ABCB5 drug transporter [94]. Moreover, GEP antibody sensitized the chemoresistant

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subpopulations to chemotherapy, and the combination of GEP antibody and chemotherapeutic agent could eradicate the intrahepatic xenografts [95]. Our study also revealed that β-catenin signaling was required for the protection of OV6+ liver CSCs from chemotherapeuticinduced cytotoxicity, and silencing β-catenin sensitized those liver CSCs to the chemotherapeutic agent [31]. Inducing the differentiation of liver CSCs Differentiation therapy for cancer has achieved certain therapeutic effects in quite a few cancer types, particularly in leukemia. Importantly, differentiation therapy not only induces the malignant cancer cell into a benign phenotype but also forces CSCs to differentiate and lose their self-renewal properties. Previous studies have revealed that all-trans retinoic acid (ATRA) treatment elicited the apoptosis of leukemic cells, presumably secondary to the differentiation process [96]. Zhang and colleagues found that ATRA could induce differentiation of EpCAM+ liver CSCs, as reflected by the notable reduction of CSC marker expression and the induction of liver-specific gene expression. The combinational treatment of ATRA and cisplatin achieved a more pronounced therapeutic effect than cisplatin treatment alone, indicating that the combination of differentiation therapy and conventional chemotherapy might be an effective strategy for HCC treatment [97]. Bone morphogenetic proteins (BMPs) are a subgroup of TGF-β super family members that play a critical role in embryonic liver development [98]. Li and colleagues showed that a high dose of exogenous BMP4 promoted the differentiation of CD133+ liver CSCs and inhibited the self-renewal, chemoresistance and tumorigenicity of these cells. Oncostatin M (OSM) is a cytokine known to induce the differentiation of hepatoblasts into hepatocytes by activating STAT3. Yamashita and colleagues indicated that OSM could induce the hepatocytic differentiation of EpCAM+ liver CSCs through OSMRdependent signaling. Moreover, a combination of OSM and the traditional chemotherapeutic 5-FU synergistically eliminated HCC by targeting both liver CSCs and non-CSC hepatoma cells [99]. Hepatocyte nuclear factor 4α (HNF-4α) is a liver-enriched transcription factor that plays a central role in regulating hepatocyte differentiation and maintaining hepatic function. Using an adenoviralbased approach, Xie et al. introduced HNF4α into both hepatoma cells and DEN-treated rat liver. Their data showed that HNF4α not only reduced the CD133 + /OV6 + liver CSC population and suppressed DEN-induced hepatocarcinogenesis but also induced the differentiation of both liver CSCs and non-CSC hepatoma cells [100,101]. This concept could be extended to the differentiation therapy for HCC using other hepatocyte-specific transcription factors. Targeting the liver CSC niche Since tumor microenvironment has the potential to reinforce stem cell-associated programs and support CSC plasticity, targeting CSC niche factors has been hypothesized to be a more powerful modality in cancer therapy than directly targeting the CSCs. Some attempts to target the CSC niche have already shed new light on certain malignancies. Fibronectin and hyaluronic acid facilitate the quiescent state of CSCs at the presence of chemotherapeutic agent. Indeed, antibodies specific to fibronectin receptor α4β1 integrin were shown to interrupt the interaction between the CSCs and their niche [102]. Targeting hypoxia is another option to interfere with the niche of quiescent and drug-resistant CSCs. HIF-1α and HIF-2α have been considered as promising targets for the treatment of glioma [103,104]. Moreover, blockage of Akt/HIF-1α/PDGF-BB autocrine signaling attenuated the chemoresistance of liver cancer cells as well as liver CSCs under hypoxic condition [81]. Anti-angiogenic therapy targeting VEGF was proved to suppress tumor vasculature formation and deplete self-renewing CSCs, which led to the tumor regression [105]. Collectively, these findings suggested that targeting

liver CSC niche is another optional therapeutic strategy for HCC treatment. Liver CSC-targeted immunotherapy In recent years, CSC-targeted immunotherapy has attracted increasing interest. Indeed, cytotoxic T lymphocyte (CTL)-based immunotherapy has emerged as a promising CSC-targeted therapeutic strategy [106,107]. Xu and colleagues found that glioblastoma multiforme (GBM) stem cells express high levels of tumor-associated antigens and major histocompatibility complex molecules. The vaccination with dendritic cells (DC) loaded with GBM stem cell antigen elicited the CTLs against GBM stem cells and significantly prolonged the survival of animals bearing GBM stem cell-derived tumors [108]. Weng and colleagues isolated ovarian cancer stem cells from patient and fused them with DCs. Their study further showed that DC-CSC fusion cells activated T cells to express IFN-γ with enhanced killing of ovarian cancer stem cells [109]. Morita and colleagues reported that DnaJ homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in colorectal cancer stem cells rather than in non-stem colorectal cancer cells. DNAJB8-derived antigenic peptide could induce a DNAJB8-specific CTL response, which led to a specific killing of colorectal cancer stem cells [110]. In terms of HCC, a most current study showed that Annexin A3 (ANXA3) was preferentially expressed in liver CSCs and played a pivotal role in promoting stem cell-like features through a dysregulated JNK pathway [111]. Moreover, ANXA3-transfected dendritic cells effectively activated T cells, resulting in a specific killing of liver CSCs, suggesting that ANXA3 might represent a potential target for CSCtargeted immunotherapy [112]. More recently, Xiao and colleagues have shown that CD47 blockade led to a significant increase in HCC cell phagocytosis by macrophages as well as increased migration of macrophages into the HCC mass. Furthermore, CD47 blockade suppressed tumor growth both in heterotopic and orthotopic xenograft models of HCC, suggesting that targeting CD47 by specific antibody possesses potential immunotherapeutic efficacy in patient HCC [113]. Perspectives and future challenges During the past several decades, identification and characterization of liver CSCs has laid the foundation for improving HCC diagnosis and prognosis prediction, assisting in patient stratification with the potential for personalized treatment. The heterogeneity of liver cancer could be due to the flexibility of liver CSCs expressing distinct cell surface markers. The heterogeneity of liver CSCs might be attributed to the distinct origin of liver CSCs. Therefore, one single marker might only represent a restricted subpopulation of liver CSCs. It would be more appropriate to identify liver CSCs by optimized combinations of the currently identified markers. Given that, whether all of the liver cancers originate from CSCs and whether CSCs exist in all liver cancers remains controversial. Some scientists still argue that liver CSCs could be just liver cancer cells with an extremely poor differentiation status. Actually, liver cancer development triggered by LPC or hepatoblast transformation fits the CSC concept well. As far as these liver CSCs are concerned, they are indeed the origin of liver cancer and exist persistently in tumor bulk. However, in those liver cancers which originate from hepatocyte or cholangiocyte transformation, whether the liver cancer cells could acquire stemness or not depends on the genetic and epigenetic alterations as well as the extracellular circumstances. If these cells really acquire stemness and convert into liver CSCs, they are more like a product of cancer evolution instead of an origin of cancer development. It has been gradually recognized that a comprehensive regulatory network is responsible for the maintenance and expansion of liver CSCs. HBV was reported to promote the “stemness” of liver CSCs

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by activating β-catenin and up-regulating miR-181, both of which target EpCAM [114]. Moreover, HBV-mediated DNA demethylation contributed to the up-regulated expression of EpCAM [115]. Several recent studies demonstrated that metabolism-dependent regulation of epigenetics plays an essential role in determining the fate of CSCs [116,117]. A current study showed that autophagy could regulate CSC maintenance by modulating IL-6 secretion, indicating that autophagy mediators might be optimized targets in CSC-targeted cancer therapy [118]. Most recently, an important milestone in the development of gene therapy has been reached with the approval of alipogene tiparvovec (Glybera®) for the treatment of familial lipoprotein lipase deficiency in Europe [119], which sheds new light on gene therapy for cancer. There has been increasing interest in developing gene therapy to interrupt CSC regulation and eliminate CSCs to cure cancer. Nevertheless, identifying a liver CSCspecific regulatory mechanism remains a great challenge. The current understanding of CSC regulatory mechanism focuses primarily on the stem cell features. The regulatory discrepancy between the normal stem cells and CSCs remains largely unknown. Disclosing the unique regulatory mechanism of CSCs will facilitate the development of optimal strategies in eradicating CSCs without sacrificing normal stem cells. Furthermore, as the CSC niche plays an essential role in CSC regulation, targeting specific liver CSC niche components is likely to be a promising therapeutic approach. As shown by PD-1 (programmed death 1) and CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) antibodies, immunotherapy has attracted tremendous interest in cancer therapy recently. However, it must be noted that these therapies might not effectively target CSCs because these cells could express distinct surface antigens. Genetically modifying T cells with chimeric antigen receptors (CARs) is the most promising strategy to achieve tumorspecific T cells [120]. Distinct from the normal T cells, CAR T cells could be genetically modified to recognize specific antigens on CSCs. Therefore, targeted immunotherapy would offer a new opportunity to eradicate CSCs. Nevertheless, the majority of current biomarkers and signaling pathways in liver CSCs exist in normal stem cells as well. Eliminating liver CSCs by targeting these markers or signaling molecules will inevitably affect the survival and function of normal stem cells. Therefore, identification of liver CSC-specific targets remains a great challenge in the future. Acknowledgements The authors apologize for not citing many other exciting papers in this review due to the space limitation. The research by the authors’ group was supported by grants from the National Natural Science Foundation of China (81222034 and 81372329) and Ministry of Science and Technology key program (2012ZX10002009 and 2013ZX10002010). Conflict of interest The authors declare that they have no conflict of interest. References [1] L. Laursen, A preventable cancer, Nature 516 (2014) S2–S3. [2] Z. Morise, N. Kawabe, H. Tomishige, H. Nagata, J. Kawase, S. Arakawa, et al., Recent advances in the surgical treatment of hepatocellular carcinoma, World J. Gastroenterol. 20 (2014) 14381–14392. [3] M. Maluccio, A. Covey, Recent progress in understanding, diagnosing, and treating hepatocellular carcinoma, CA Cancer J. Clin. 62 (2012) 394–399. [4] J. Bruix, M. Sherman, American Association for the Study of Liver Diseases, Management of hepatocellular carcinoma: an update, Hepatology 53 (2011) 1020–1022. [5] V. Mazzaferro, Y.S. Chun, R.T. Poon, M.E. Schwartz, F.Y. Yao, J.W. Marsh, et al., Liver transplantation for hepatocellular carcinoma, Ann. Surg. Oncol. 15 (2008) 1001–1007.

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