J Hepatobiliary Pancreat Sci (2015) 22:531–537 DOI: 10.1002/jhbp.248
TOPIC
Cancer stem cells as therapeutic targets of hepato-biliary-pancreatic cancers Shinji Tanaka Published online: 14 April 2015 © 2015 Japanese Society of Hepato-Biliary-Pancreatic Surgery
Abstract Heterogeneity is one of the essential hallmarks of malignancies. Within bulk cancer cells, a striking variability differs in biological characteristics including the proliferation rate, cell–cell interaction, metastatic tendency and even sensitivity to anticancer therapies. Such diversity makes the investigation and treatment of the cancers complicated. Increasing evidence suggests this plasticity of cancers is a result of selfrenewing and differentiation of a small subpopulation of cancer cells with stem-like properties, called cancer stem cells (CSCs). More importantly, CSCs are believed to be responsible for the resistance to conventional therapies and metastatic abilities in clinical practice. This review summarizes the molecular pathogenesis of hepato-biliary-pancreatic CSCs on the basis of the recent studies, and promising strategy of novel therapy targeting the signal transduction pathways or potentially epigenetic addictions of CSCs. Keywords Asymmetric cell division · Epigenetic reprograming · Hedgehog · PI3K/Akt · Wnt Introduction Cancers are principally characterized by phenotypic and functional heterogeneity of the cell components cells with genetic and epigenetic alterations [1]. If every cell within a tumor has the same tumorigenic potential, stochastic accumulation of genetic and epigenetic factors determine their variable activities. Darwinian-like clonal evolution can lead to phenotypic and functional differences among the cancer cells within a single tumor (Fig. 1a). In contrast to the stochastic model of clonal evolution, if only rare initiating cells can recapitulate the tumor, these cells must comply with a stem-like hierarchical system, so-called cancer stem cell (CSC) system (Fig. 1b). Indeed, only approximately 0.01–1% of human cancer cells S. Tanaka (✉) Department of Molecular Oncology, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan e-mail: [email protected]
are able to form tumors in highly immunodeficient mice [2]. Tumorigenic CSCs lying at the apex of the hierarchy are intrinsically resistant to chemotherapeutic agents and irradiation, and function as a source to metastasizing and relapsing of cancers. CSCs have the capacity to both self-renew and to yield the various heterogeneous lineages that comprise the bulk tumor. As evidenced by substantial studies, however, the clonal evolution and CSC models are not mutually exclusive but seem to coexist in cancers that follow CSCs themselves would be expected to evolve clonally. The models of clonal evolution and CSCs constitute two major frameworks for interpreting the causes of phenotypic and functional heterogeneity in cancers. CSC markers in hepato-biliary-pancreatic cancers The concept of solid tumor CSCs was originally described in 1952 by Tomizo Yoshida, a Japanese pathologist known for discovering the chemical-induced hepatocarcinogenesis in rats [3]. Recently, a variety of CSC markers were identified by flow cytometry analysis scanning (FACS) for solid tumors including hepato-biliary-pancreatic cancers [4]. In hepatocellular carcinoma (HCC), the existence of CSCs was first reported as side population (SP), defined as cells that actively exclude dyes such as Hoechst 33342 [5]. Several cell surface molecules (EpCAM, CD133, CD24, CD90 and CD13) were also reported to be useful markers for isolation of HCC CSC subpopulations (Table 1) [6–13]. In the next sections, EpCAM and CD133 are described in detail. CD13 was identified by Mori and colleagues as a functional marker that can be used to isolate HCC CSCs resistant to anti-cancer treatment [10]. CD13+ HCC cells exhibited relatively slow proliferation in vitro, but higher tumorigenic potential in vivo. CD13 is also known as aminopeptidase N, a Zn2+ dependent membranebound ectopeptidase that degrades preferentially proteins and peptides with a N-terminal neutral amino acid. It is important that ubenimex (bestatin) specifically antagonizes the zincbinding site of the aminopeptidase N domain [10]. Through
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Fig. 1 Models constitute two major frameworks for interpreting the causes of phenotypic and functional heterogeneity in cancers. (a) Stochastic model of clonal evolution. (b) Hierarchical model of cancer stem cells
(a)
(b)
Table 1 Hepatocellular carcinoma (HCC) cancer stem cell (CSC) markers and therapeutic targets Subpopulation markers
Molecular backgrounds
Targeted agents
References
SP EpCAM+ CD133+ CD90+ CD13+ CD24+ Calcium channel α2δ+ Proteasomelow ROSlow
BMI1 Wnt, miRNA Notch, Hedgehog, Wnt, BMI1, Akt ROS inhibition STAT3/Nanog Calcium intake NF-κB, chemokines
ABC transporter inhibitor Wnt inhibitor Akt inhibitor Aminopeptidase N inhbitor STAT inhibitor Antibody 1B50-1 NF-κB inhibitor?
Hepatology, 2006 [5] Gastroenterology, 2009 [6] Gastroenterology, 2007 [7], Oncogene, 2008 [8] Cancer Cell, 2008 [9] J Clin Invest, 2010 [10] Cell Stem Cell, 2011 [11] Cancer Cell, 2013 [12] Hepatology, 2013 [13]
blocking CD13 function, ubenimex might be suitable for CSCs targeted therapy. Reportedly, several agents potentially targeting HCC CSCs are listed in Table 1. Similar approaches have been applied for isolation of CSC subpopulations of bile duct cancer (cholangiocarcinoma), but less druggable applications were reported (Table 2) [14–17]. CD44, an adhesion molecule that interacts with hyaluronic acid, is widely used as a CSC marker in various types of cancers [18]. CD44 gene transcription is stimulated by Wnt/beta-catenin signaling, and CD44 protein can activate Wnt/beta-catenin signal transduction by regulating localization of Wnt co-receptor LRP6. Interestingly, Saya and colleagues reported the variant isoforms CD44v stabilizes xCT, a subunit of the cystine-glutamate transporter, and contributes
to reactive oxygen species (ROS) defense in CSCs [19]. A specific xCT inhibitor sulfasalazine is one of the promising agents targeting CSCs. To isolate CSC subpopulations of pancreatic ductal adenocarcinoma, EpCAM, CD44, CD24, CD133, CXCR4, ALDH, c-MET and DCLK1 have been used for the markers (Table 3) [20–25]. Recently, DCLK1 was reported as the specific maker to identify functionally distinct subpopulations with CSC-like properties in pancreatic cancer cells [25] and intestinal tumor cells [26]. DCLK1 protein consists of N-terminal microtubule-associated domains required for neuronal migration to the cerebral cortex, and a C-terminal serine/threonine protein kinase domain homologous to Ca2 +/calmodulin-dependent protein kinase [27]. We found
Table 2 Biliary cancer stem cell (CSC) markers and therapeutic targets Subpopulation markers
Molecular backgrounds
Targeted agents
References
CD44+CD133+ CD44+CD24+EpCAM+ CD133+ CD274-
Hedgehog, ABCG2 Hegehog? Oct4, Nanog PD-L1 (immunosuppression)
Hedgehog or ABC transporter inhibitor? ALDH inhibitor?
Cancer Biol Ther, 2010 [14] Int J Cancer, 2011 [15] World J Gasroeterol, 2011 [16] Cancer Sci, 2014 [17]
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Table 3 Pancreatic cancer stem cell (CSC) markers and therapeutic targets Subpopulation markers
Molecular backgrounds
Targeted agents
References
CD44+CD24+EpCAM+ CD133+CXCR4+ ALDHhighCD44+CD24+ c-MethighCD44+ Proteasomelow DCLK1+
Hedgehog Chemokine receptor HGF receptor Microtubule polymerization
Hedgehog inhibitor? CXCR4 inhibitor ALDH inhibitor? c-Met inhibitor Quercetin (β-catenin inhibitor or epigenetic modulator?) Serine/threonine kinase inhibitor?
Cancer Res, 2007 [20] Cell Stem Cell, 2007 [21] J Natl Cancer Inst, 2010 [22] Gastroenterology, 2011 [23] Gastroenterology, 2012 [24] Gastroenterology, 2014 [25]
specific expression of DCLK1 related to invasive and metastatic potentials in pancreatic CSCs (unpubl. data). The cell motility might be regulated by the microtubule-polymerizing activity of DCLK1. As evidenced by the significance of axon guidance mutations in pancreatic carcinogenesis [28], DCLK1 is one of the attractive targets for treatment of pancreatic CSCs. Wnt signal transduction and targeted agents EpCAM is a type I transmembrane glycoprotein, a synonym for ESA and CD326 characterized as one of the specific markers of hepatic stem/progenitor cells [29]. We have previously reported that EpCAM might be a biomarker of HCC with poor prognosis [30] and an immunotoxin targeting EpCAM suppressed dramatically the CSC phenotypes as well as orthotopic HCC xenografts [31]. Yamashita et al. reported EpCAM as one of the direct downstream molecules of Wnt-activated beta-catenin signaling pathway in human HCC CSCs [6]. Wnt signals are known to transduce the patterning and growth during embryonic development, and also postembryonic regulation of stem cells in epithelia undergoing renewal [4]. The signaling is initiated by secreted Wnt proteins, which bind to a class of seven-transmembrane receptors Frizzled (Fig. 2). We isolated human Frizzled-7 as one of the therapeutic targets in various cancer cells [32]. In the absence of Wnt signals, beta-catenin phosphorylation by GSK3beta-APC-Axin complex allows recognition by beta-TRCP, an E3 ubiquitin ligase with subsequent degradation of beta-catenin by proteasome system. By contrast, Wnt-binding to the Frizzled receptor leads to dissociation of the GSK3beta complex, and results in the cytoplasmic accumulation of unphosphorylated beta-catenin, which translocates to the nucleus where it binds to CBP and engages the TCF family of transcription factors to activate genes like EpCAM and CD44. Several small molecule compounds (LGK974, PRI-724), recombinant proteins (OMP-54 F28) and antibodies (Vantictumab/OMP-18R5) are under intense study for the development of agents targeting Wnt/betacatenin pathway (Fig. 2). One important issue is the specificity of anti-cancer effects, since Wnt signals is indispensable for the maintenance of CSCs but also normal stem cells in
hepato-biliary-pancreatic tissues. Druggable targets should be identified for the CSC-specific therapy of hepato-biliarypancreatic cancers. Hedgehog signal transduction and targeted agents Not only Wnt, but also Hedgehog pathways play an essential role in regulating self-renewal potential of stem cells [33]. Overexpression of Sonic Hedgehog (Shh) in pancreas is sufficient to initiate precancerous lesions in the transgenic mice. Secreted Shh binds to the receptor Patched to activate Smoothened (SMO) seven-transmembrane protein, resulting in translocation of Kif7 and dissociation of SuFu-Gli complex (Fig. 2). Activated Gli1 translocates to the nucleus and activates transcription of target genes such as Gli1 and FoxM1 that induce cancer initiation through stem cell expansion. It should be noted that the crosstalk between Hedgehog and Wnt signal transductions is reported. Without hedgehog signaling, SuFu can bind to not only Gli but also beta-catenin [34], leading to blocking Wnt/β-catenin signal transduction. Gli inhibits Wnt signaling under overexpression of β-catenin, whereas β-catenin stimulates Hedgehog signaling under overexpression of Gli1. These crosstalk effects might be consistent with the previous report indicating the upregulation of Hedgehog pathway in CD44+CD24+EpCAM+ pancreatic CSCs [20]. As specific inhibitors of Hedgehog signaling, SMO antagonists such as vismodegib (GDC-0449), saridegib (IPI-926) and sonidegib (LDE225) have been examined in clinical trials hepato-biliary-pancreatic cancers. Since Shh inhibition induced mainly stromal depletion in several tumor models and clinical samples [33], a paracrine mechanism of Hedgehog-mediated carcinogenesis should clarify the microenvironmental role in generation of cancer stem niche. PI3K/Akt signal transduction and targeted agents CD133 is a five-transmembrane domain glycoprotein, a synonym for Prominin-1 initially identified in humans as a hematopoietic stem cell marker [35], is used as one of the common CSC markers in brain tumor and colon cancer [36, 37] as well as in hepato-biliary-pancreatic cancers (Tables 1–3). Ma et al. reported that CD133 is useful for isolation of CSC
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Fig. 2 Wnt and Hedgehog signaling pathways. Targeted agents for pancreatic cancer in italics
subpopulation in HCC cells [7], and the chemoresistance of CD133+ CSC through preferential activation of Akt signaling [8]. Indeed, the closed relationship between CD133 expression and PI3K/Akt activation has been recognized in CSCs derived from neuroblastoma and colon cancer [38, 39]. PI3K consists of p85 regulatory and p110 catalytic subunits (Fig. 3) [33]. Recently, the direct and functional interaction of CD133 cytoplasmic domain mediates with PI3K p85 regulatory subunit, resulting in preferential activation of PI3K/Akt pathway in CSCs [40]. PI3K/Akt pathway plays an essential role in self-renewal of normal stem cell fate [41]. PI3K phosphorylates PIP2 to generate PIP3 transducing Fig. 3 PI3K/Akt signaling pathways. Targeted agents for pancreatic cancer in italics
PDK, which in turn activates a serine/threonine kinase Akt. A tumor suppressor PTEN dephosphorylates PIP3 and reverses this pathway. Akt regulates multiple cellular target proteins including TSC complex and IKK. The TSC/Rheb pathway activates an evolutionarily-conserved mTOR serine/threonine kinase, and mTOR phosphorylates the ribosomal S6 protein, facilitating translation of a specific mRNA subset containing a 5’-polypyrimidine tract that encodes ribosomal proteins and elongation factors. In addition to S6K, mTOR exerts its effects by phosphorylating 4E-BP1. Phosphorylation of 4EBP1 leads to its release of eIF4E, allowing initiation of translation of 5’-cap-dependent mRNA. Genomic mutations in the
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PI3K/mTOR pathway were detected in 15% of sporadic pancreatic neuroendocrine tumors (PNETs); TSC2, PTEN, and PIK3CA encoding PI3K p110 catalytic subunit alpha. Actually, Everolimus (Afinitor, RAD001), an inhibitor of mTOR was received approval for use in patients with advanced PNETs (Fig. 3) [33]. IKK, another downstream protein of Akt, provokes the subsequent activation of NF-kappaB transcription factor, promoting cell survival by activating transcription of target genes such as cytokines and chemokines. Based on the essential role of chemokines in the microenvironmental interactions with CSCs of HCC [13] and pancreatic cancer [21], more intensive studies including other signaling networks should be carried out. Asymmetric cell divisions: dilemma and potential therapeutics targeting CSCs “Self-renewal” is theoretically based on asymmetric divisions of stem cells that give rise to one cell of the same potency, and another stimulated to further differentiate into non-stem cell types [42]. In general, the stemness is maintained by the quiescent dormancy that is critical for protecting the stem cell components from in vivo stress [43]. Such intrinsic fate of stemness should cause difficulty in the studies on CSCs. By utilizing multiple markers, even highly purified CSCs might divide in an asymmetric manner, and rapidly reconstruct mixed populations with their differentiated progeny in the
Fig. 4 Evidence of cancer stem cells (CSCs). (a) Visualization of asymmetric cell division of green fluorescent CSC observed by time-lapse microscopy [13, 24]. (b) Waddington’s epigenetic landscape [50]. The concept is proposed as a landscape of hills and valleys to represent the epigenetic process of cellular decision-making during development. The drawing program is originated from http://tex. stackexchange.com/questions/ 182638/how-to-get-a-waddingtonlandscape
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hierarchical fashion. The majority of bulk cancer cells become eventually occupied by non-CSCs dividing rapidly in a symmetric manner. Therefore, the validity of current studies remains unsatisfying in terms of evaluating therapeutic targets to pinpoint CSCs per se [44]. One of the sophisticated solutions to such a dilemma might be a monitoring system based on CSC-specific functions. In our recent studies, the proteasome-independency of dormant stem-cell fate was used for fluorescent visualization of CSC subpopulations in human HCC [13] and pancreatic cancer cells [24]. Using our system to distinguish CSCs from nonCSCs, the visualized CSCs demonstrated asymmetric divisions in a real-time approach, and remarkable tumorigenicity with heterogenic expansion in vivo (Fig. 4a). This real-time imaging system may allow better assessment of therapeutic approaches, because CSCs can be distinguished as visible and dynamic targets in and of themselves. Our system of synthetic lethal screening identified a polyphenol quercetin as one of the specific agents targeting human pancreatic CSCs [24]. Quercetin (3,30,40,5,7-pentahydroxyflavone) is classified into plant-based flavonoids enriched in various fruits and vegetables. Quercetin is known to function as an inhibitor of the nuclear translocation of beta-catenin, downstream of the Wnt signaling pathway [45]. Little effect of other Wnt inhibitors or beta-catenin siRNA, however, was detected in our assays. It implies other constitutive mechanisms for quercetin in the targeting of CSCs.
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Noteworthy, quercetin is characterized by an epigenetic drug promoting DNA demethylation [46] as well as histone H3 acetylation [47]. Epigenetic mechanisms are the key component of the dynamic programming that occurs along the process of differentiation from stem cells to non-stem cells [48]. Heterogeneous generation of cancers is accompanied with aberrant epigenetic changes to switch the CSC phenotype on and off [49]. Characterizing epigenetic landscape may thus help discriminate the CSC specific profiles (Fig. 4b) [50]. Such epigenetic characteristics including histone modification and chromatin remodeling are approved to directly connect to the essential mechanism to maintain the stemness in cancer cells [48, 49]. The Achilles heels of CSCs should be fatefully determined by the interaction with their microenvironments, which drive the epigenetic reprogramming of the cell nucleus. Further studies on their epigenetic and niche addictions must be actively engaged in developing a novel targeted therapy of CSCs. Acknowledgments I sincerely appreciate the opportunity to write this review article by recommendation of the Japanese Society of Hepato-Biliary-Pancreatic Surgery. I thank all of my colleagues and collaborators at Tokyo Medical and Dental University, Osaka University, Kyushu University, Tokushima University, and Brown Medical School. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Research (A), Project of Development of Innovative Research on Cancer Therapeutics from Ministry of Education, Culture, Sports, Science & Technology of Japan, and Health & Labour Sciences Research Grant from Ministry of Health Labour & Welfare of Japan.
Conflict of interest
None declared.
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TOPIC
Cancer stem cells as therapeutic targets of hepato-biliary-pancreatic cancers Shinji Tanaka Published online: 14 April 2015 © 2015 Japanese Society of Hepato-Biliary-Pancreatic Surgery
Abstract Heterogeneity is one of the essential hallmarks of malignancies. Within bulk cancer cells, a striking variability differs in biological characteristics including the proliferation rate, cell–cell interaction, metastatic tendency and even sensitivity to anticancer therapies. Such diversity makes the investigation and treatment of the cancers complicated. Increasing evidence suggests this plasticity of cancers is a result of selfrenewing and differentiation of a small subpopulation of cancer cells with stem-like properties, called cancer stem cells (CSCs). More importantly, CSCs are believed to be responsible for the resistance to conventional therapies and metastatic abilities in clinical practice. This review summarizes the molecular pathogenesis of hepato-biliary-pancreatic CSCs on the basis of the recent studies, and promising strategy of novel therapy targeting the signal transduction pathways or potentially epigenetic addictions of CSCs. Keywords Asymmetric cell division · Epigenetic reprograming · Hedgehog · PI3K/Akt · Wnt Introduction Cancers are principally characterized by phenotypic and functional heterogeneity of the cell components cells with genetic and epigenetic alterations [1]. If every cell within a tumor has the same tumorigenic potential, stochastic accumulation of genetic and epigenetic factors determine their variable activities. Darwinian-like clonal evolution can lead to phenotypic and functional differences among the cancer cells within a single tumor (Fig. 1a). In contrast to the stochastic model of clonal evolution, if only rare initiating cells can recapitulate the tumor, these cells must comply with a stem-like hierarchical system, so-called cancer stem cell (CSC) system (Fig. 1b). Indeed, only approximately 0.01–1% of human cancer cells S. Tanaka (✉) Department of Molecular Oncology, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan e-mail: [email protected]
are able to form tumors in highly immunodeficient mice [2]. Tumorigenic CSCs lying at the apex of the hierarchy are intrinsically resistant to chemotherapeutic agents and irradiation, and function as a source to metastasizing and relapsing of cancers. CSCs have the capacity to both self-renew and to yield the various heterogeneous lineages that comprise the bulk tumor. As evidenced by substantial studies, however, the clonal evolution and CSC models are not mutually exclusive but seem to coexist in cancers that follow CSCs themselves would be expected to evolve clonally. The models of clonal evolution and CSCs constitute two major frameworks for interpreting the causes of phenotypic and functional heterogeneity in cancers. CSC markers in hepato-biliary-pancreatic cancers The concept of solid tumor CSCs was originally described in 1952 by Tomizo Yoshida, a Japanese pathologist known for discovering the chemical-induced hepatocarcinogenesis in rats [3]. Recently, a variety of CSC markers were identified by flow cytometry analysis scanning (FACS) for solid tumors including hepato-biliary-pancreatic cancers [4]. In hepatocellular carcinoma (HCC), the existence of CSCs was first reported as side population (SP), defined as cells that actively exclude dyes such as Hoechst 33342 [5]. Several cell surface molecules (EpCAM, CD133, CD24, CD90 and CD13) were also reported to be useful markers for isolation of HCC CSC subpopulations (Table 1) [6–13]. In the next sections, EpCAM and CD133 are described in detail. CD13 was identified by Mori and colleagues as a functional marker that can be used to isolate HCC CSCs resistant to anti-cancer treatment [10]. CD13+ HCC cells exhibited relatively slow proliferation in vitro, but higher tumorigenic potential in vivo. CD13 is also known as aminopeptidase N, a Zn2+ dependent membranebound ectopeptidase that degrades preferentially proteins and peptides with a N-terminal neutral amino acid. It is important that ubenimex (bestatin) specifically antagonizes the zincbinding site of the aminopeptidase N domain [10]. Through
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Fig. 1 Models constitute two major frameworks for interpreting the causes of phenotypic and functional heterogeneity in cancers. (a) Stochastic model of clonal evolution. (b) Hierarchical model of cancer stem cells
(a)
(b)
Table 1 Hepatocellular carcinoma (HCC) cancer stem cell (CSC) markers and therapeutic targets Subpopulation markers
Molecular backgrounds
Targeted agents
References
SP EpCAM+ CD133+ CD90+ CD13+ CD24+ Calcium channel α2δ+ Proteasomelow ROSlow
BMI1 Wnt, miRNA Notch, Hedgehog, Wnt, BMI1, Akt ROS inhibition STAT3/Nanog Calcium intake NF-κB, chemokines
ABC transporter inhibitor Wnt inhibitor Akt inhibitor Aminopeptidase N inhbitor STAT inhibitor Antibody 1B50-1 NF-κB inhibitor?
Hepatology, 2006 [5] Gastroenterology, 2009 [6] Gastroenterology, 2007 [7], Oncogene, 2008 [8] Cancer Cell, 2008 [9] J Clin Invest, 2010 [10] Cell Stem Cell, 2011 [11] Cancer Cell, 2013 [12] Hepatology, 2013 [13]
blocking CD13 function, ubenimex might be suitable for CSCs targeted therapy. Reportedly, several agents potentially targeting HCC CSCs are listed in Table 1. Similar approaches have been applied for isolation of CSC subpopulations of bile duct cancer (cholangiocarcinoma), but less druggable applications were reported (Table 2) [14–17]. CD44, an adhesion molecule that interacts with hyaluronic acid, is widely used as a CSC marker in various types of cancers [18]. CD44 gene transcription is stimulated by Wnt/beta-catenin signaling, and CD44 protein can activate Wnt/beta-catenin signal transduction by regulating localization of Wnt co-receptor LRP6. Interestingly, Saya and colleagues reported the variant isoforms CD44v stabilizes xCT, a subunit of the cystine-glutamate transporter, and contributes
to reactive oxygen species (ROS) defense in CSCs [19]. A specific xCT inhibitor sulfasalazine is one of the promising agents targeting CSCs. To isolate CSC subpopulations of pancreatic ductal adenocarcinoma, EpCAM, CD44, CD24, CD133, CXCR4, ALDH, c-MET and DCLK1 have been used for the markers (Table 3) [20–25]. Recently, DCLK1 was reported as the specific maker to identify functionally distinct subpopulations with CSC-like properties in pancreatic cancer cells [25] and intestinal tumor cells [26]. DCLK1 protein consists of N-terminal microtubule-associated domains required for neuronal migration to the cerebral cortex, and a C-terminal serine/threonine protein kinase domain homologous to Ca2 +/calmodulin-dependent protein kinase [27]. We found
Table 2 Biliary cancer stem cell (CSC) markers and therapeutic targets Subpopulation markers
Molecular backgrounds
Targeted agents
References
CD44+CD133+ CD44+CD24+EpCAM+ CD133+ CD274-
Hedgehog, ABCG2 Hegehog? Oct4, Nanog PD-L1 (immunosuppression)
Hedgehog or ABC transporter inhibitor? ALDH inhibitor?
Cancer Biol Ther, 2010 [14] Int J Cancer, 2011 [15] World J Gasroeterol, 2011 [16] Cancer Sci, 2014 [17]
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Table 3 Pancreatic cancer stem cell (CSC) markers and therapeutic targets Subpopulation markers
Molecular backgrounds
Targeted agents
References
CD44+CD24+EpCAM+ CD133+CXCR4+ ALDHhighCD44+CD24+ c-MethighCD44+ Proteasomelow DCLK1+
Hedgehog Chemokine receptor HGF receptor Microtubule polymerization
Hedgehog inhibitor? CXCR4 inhibitor ALDH inhibitor? c-Met inhibitor Quercetin (β-catenin inhibitor or epigenetic modulator?) Serine/threonine kinase inhibitor?
Cancer Res, 2007 [20] Cell Stem Cell, 2007 [21] J Natl Cancer Inst, 2010 [22] Gastroenterology, 2011 [23] Gastroenterology, 2012 [24] Gastroenterology, 2014 [25]
specific expression of DCLK1 related to invasive and metastatic potentials in pancreatic CSCs (unpubl. data). The cell motility might be regulated by the microtubule-polymerizing activity of DCLK1. As evidenced by the significance of axon guidance mutations in pancreatic carcinogenesis [28], DCLK1 is one of the attractive targets for treatment of pancreatic CSCs. Wnt signal transduction and targeted agents EpCAM is a type I transmembrane glycoprotein, a synonym for ESA and CD326 characterized as one of the specific markers of hepatic stem/progenitor cells [29]. We have previously reported that EpCAM might be a biomarker of HCC with poor prognosis [30] and an immunotoxin targeting EpCAM suppressed dramatically the CSC phenotypes as well as orthotopic HCC xenografts [31]. Yamashita et al. reported EpCAM as one of the direct downstream molecules of Wnt-activated beta-catenin signaling pathway in human HCC CSCs [6]. Wnt signals are known to transduce the patterning and growth during embryonic development, and also postembryonic regulation of stem cells in epithelia undergoing renewal [4]. The signaling is initiated by secreted Wnt proteins, which bind to a class of seven-transmembrane receptors Frizzled (Fig. 2). We isolated human Frizzled-7 as one of the therapeutic targets in various cancer cells [32]. In the absence of Wnt signals, beta-catenin phosphorylation by GSK3beta-APC-Axin complex allows recognition by beta-TRCP, an E3 ubiquitin ligase with subsequent degradation of beta-catenin by proteasome system. By contrast, Wnt-binding to the Frizzled receptor leads to dissociation of the GSK3beta complex, and results in the cytoplasmic accumulation of unphosphorylated beta-catenin, which translocates to the nucleus where it binds to CBP and engages the TCF family of transcription factors to activate genes like EpCAM and CD44. Several small molecule compounds (LGK974, PRI-724), recombinant proteins (OMP-54 F28) and antibodies (Vantictumab/OMP-18R5) are under intense study for the development of agents targeting Wnt/betacatenin pathway (Fig. 2). One important issue is the specificity of anti-cancer effects, since Wnt signals is indispensable for the maintenance of CSCs but also normal stem cells in
hepato-biliary-pancreatic tissues. Druggable targets should be identified for the CSC-specific therapy of hepato-biliarypancreatic cancers. Hedgehog signal transduction and targeted agents Not only Wnt, but also Hedgehog pathways play an essential role in regulating self-renewal potential of stem cells [33]. Overexpression of Sonic Hedgehog (Shh) in pancreas is sufficient to initiate precancerous lesions in the transgenic mice. Secreted Shh binds to the receptor Patched to activate Smoothened (SMO) seven-transmembrane protein, resulting in translocation of Kif7 and dissociation of SuFu-Gli complex (Fig. 2). Activated Gli1 translocates to the nucleus and activates transcription of target genes such as Gli1 and FoxM1 that induce cancer initiation through stem cell expansion. It should be noted that the crosstalk between Hedgehog and Wnt signal transductions is reported. Without hedgehog signaling, SuFu can bind to not only Gli but also beta-catenin [34], leading to blocking Wnt/β-catenin signal transduction. Gli inhibits Wnt signaling under overexpression of β-catenin, whereas β-catenin stimulates Hedgehog signaling under overexpression of Gli1. These crosstalk effects might be consistent with the previous report indicating the upregulation of Hedgehog pathway in CD44+CD24+EpCAM+ pancreatic CSCs [20]. As specific inhibitors of Hedgehog signaling, SMO antagonists such as vismodegib (GDC-0449), saridegib (IPI-926) and sonidegib (LDE225) have been examined in clinical trials hepato-biliary-pancreatic cancers. Since Shh inhibition induced mainly stromal depletion in several tumor models and clinical samples [33], a paracrine mechanism of Hedgehog-mediated carcinogenesis should clarify the microenvironmental role in generation of cancer stem niche. PI3K/Akt signal transduction and targeted agents CD133 is a five-transmembrane domain glycoprotein, a synonym for Prominin-1 initially identified in humans as a hematopoietic stem cell marker [35], is used as one of the common CSC markers in brain tumor and colon cancer [36, 37] as well as in hepato-biliary-pancreatic cancers (Tables 1–3). Ma et al. reported that CD133 is useful for isolation of CSC
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Fig. 2 Wnt and Hedgehog signaling pathways. Targeted agents for pancreatic cancer in italics
subpopulation in HCC cells [7], and the chemoresistance of CD133+ CSC through preferential activation of Akt signaling [8]. Indeed, the closed relationship between CD133 expression and PI3K/Akt activation has been recognized in CSCs derived from neuroblastoma and colon cancer [38, 39]. PI3K consists of p85 regulatory and p110 catalytic subunits (Fig. 3) [33]. Recently, the direct and functional interaction of CD133 cytoplasmic domain mediates with PI3K p85 regulatory subunit, resulting in preferential activation of PI3K/Akt pathway in CSCs [40]. PI3K/Akt pathway plays an essential role in self-renewal of normal stem cell fate [41]. PI3K phosphorylates PIP2 to generate PIP3 transducing Fig. 3 PI3K/Akt signaling pathways. Targeted agents for pancreatic cancer in italics
PDK, which in turn activates a serine/threonine kinase Akt. A tumor suppressor PTEN dephosphorylates PIP3 and reverses this pathway. Akt regulates multiple cellular target proteins including TSC complex and IKK. The TSC/Rheb pathway activates an evolutionarily-conserved mTOR serine/threonine kinase, and mTOR phosphorylates the ribosomal S6 protein, facilitating translation of a specific mRNA subset containing a 5’-polypyrimidine tract that encodes ribosomal proteins and elongation factors. In addition to S6K, mTOR exerts its effects by phosphorylating 4E-BP1. Phosphorylation of 4EBP1 leads to its release of eIF4E, allowing initiation of translation of 5’-cap-dependent mRNA. Genomic mutations in the
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PI3K/mTOR pathway were detected in 15% of sporadic pancreatic neuroendocrine tumors (PNETs); TSC2, PTEN, and PIK3CA encoding PI3K p110 catalytic subunit alpha. Actually, Everolimus (Afinitor, RAD001), an inhibitor of mTOR was received approval for use in patients with advanced PNETs (Fig. 3) [33]. IKK, another downstream protein of Akt, provokes the subsequent activation of NF-kappaB transcription factor, promoting cell survival by activating transcription of target genes such as cytokines and chemokines. Based on the essential role of chemokines in the microenvironmental interactions with CSCs of HCC [13] and pancreatic cancer [21], more intensive studies including other signaling networks should be carried out. Asymmetric cell divisions: dilemma and potential therapeutics targeting CSCs “Self-renewal” is theoretically based on asymmetric divisions of stem cells that give rise to one cell of the same potency, and another stimulated to further differentiate into non-stem cell types [42]. In general, the stemness is maintained by the quiescent dormancy that is critical for protecting the stem cell components from in vivo stress [43]. Such intrinsic fate of stemness should cause difficulty in the studies on CSCs. By utilizing multiple markers, even highly purified CSCs might divide in an asymmetric manner, and rapidly reconstruct mixed populations with their differentiated progeny in the
Fig. 4 Evidence of cancer stem cells (CSCs). (a) Visualization of asymmetric cell division of green fluorescent CSC observed by time-lapse microscopy [13, 24]. (b) Waddington’s epigenetic landscape [50]. The concept is proposed as a landscape of hills and valleys to represent the epigenetic process of cellular decision-making during development. The drawing program is originated from http://tex. stackexchange.com/questions/ 182638/how-to-get-a-waddingtonlandscape
(a)
(b)
hierarchical fashion. The majority of bulk cancer cells become eventually occupied by non-CSCs dividing rapidly in a symmetric manner. Therefore, the validity of current studies remains unsatisfying in terms of evaluating therapeutic targets to pinpoint CSCs per se [44]. One of the sophisticated solutions to such a dilemma might be a monitoring system based on CSC-specific functions. In our recent studies, the proteasome-independency of dormant stem-cell fate was used for fluorescent visualization of CSC subpopulations in human HCC [13] and pancreatic cancer cells [24]. Using our system to distinguish CSCs from nonCSCs, the visualized CSCs demonstrated asymmetric divisions in a real-time approach, and remarkable tumorigenicity with heterogenic expansion in vivo (Fig. 4a). This real-time imaging system may allow better assessment of therapeutic approaches, because CSCs can be distinguished as visible and dynamic targets in and of themselves. Our system of synthetic lethal screening identified a polyphenol quercetin as one of the specific agents targeting human pancreatic CSCs [24]. Quercetin (3,30,40,5,7-pentahydroxyflavone) is classified into plant-based flavonoids enriched in various fruits and vegetables. Quercetin is known to function as an inhibitor of the nuclear translocation of beta-catenin, downstream of the Wnt signaling pathway [45]. Little effect of other Wnt inhibitors or beta-catenin siRNA, however, was detected in our assays. It implies other constitutive mechanisms for quercetin in the targeting of CSCs.
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Noteworthy, quercetin is characterized by an epigenetic drug promoting DNA demethylation [46] as well as histone H3 acetylation [47]. Epigenetic mechanisms are the key component of the dynamic programming that occurs along the process of differentiation from stem cells to non-stem cells [48]. Heterogeneous generation of cancers is accompanied with aberrant epigenetic changes to switch the CSC phenotype on and off [49]. Characterizing epigenetic landscape may thus help discriminate the CSC specific profiles (Fig. 4b) [50]. Such epigenetic characteristics including histone modification and chromatin remodeling are approved to directly connect to the essential mechanism to maintain the stemness in cancer cells [48, 49]. The Achilles heels of CSCs should be fatefully determined by the interaction with their microenvironments, which drive the epigenetic reprogramming of the cell nucleus. Further studies on their epigenetic and niche addictions must be actively engaged in developing a novel targeted therapy of CSCs. Acknowledgments I sincerely appreciate the opportunity to write this review article by recommendation of the Japanese Society of Hepato-Biliary-Pancreatic Surgery. I thank all of my colleagues and collaborators at Tokyo Medical and Dental University, Osaka University, Kyushu University, Tokushima University, and Brown Medical School. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Research (A), Project of Development of Innovative Research on Cancer Therapeutics from Ministry of Education, Culture, Sports, Science & Technology of Japan, and Health & Labour Sciences Research Grant from Ministry of Health Labour & Welfare of Japan.
Conflict of interest
None declared.
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