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Aug 31, 2011 - Promoting Survival of Liver Cancer Stem Cells via an Epithelial–. Mesenchymal Transition-like Phenomenon. Ho Min Kim1, Naotsugu ...
Ann Surg Oncol (2012) 19:S539–S548 DOI 10.1245/s10434-011-2040-5

ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

Increased CD13 Expression Reduces Reactive Oxygen Species, Promoting Survival of Liver Cancer Stem Cells via an Epithelial– Mesenchymal Transition-like Phenomenon Ho Min Kim1, Naotsugu Haraguchi1, Hideshi Ishii1,2, Masahisa Ohkuma1,3, Miho Okano1, Koshi Mimori2, Hidetoshi Eguchi1, Hirofumi Yamamoto1, Hiroaki Nagano1, Mitsugu Sekimoto1, Yuichiro Doki1, and Masaki Mori1 Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Osaka, Japan; 2Department of Molecular and Cellular Biology, Division of Molecular and Surgical Oncology, Kyushu University, Medical Institute of Bioregulation, Beppu, Ohita, Japan; 3Department of Gastroenterological Surgery, Jikei Collage Graduate School of Medicine, Tokyo, Japan 1

ABSTRACT Background. Recently, it has been reported that a small population of cancer stem cells (CSCs) play a role in resistance to chemotherapy and radiation therapy. We reported that CD13? liver CSCs survive in hypoxic lesions after chemotherapy, presumably through increased expression of CD13/Aminopeptidase N, which is a scavenger enzyme in the reactive oxygen species (ROS) metabolic pathway. On the other hand, the concept of epithelial–mesenchymal transition (EMT) was indicated by a recent study showing an increased plasticity linked to the cellular ‘‘stemness’’ of CSCs. Methods. To study the relationship between CSCs and EMT, we examined biological characteristics of liver cancer cell lines with EMT by exposing transforming growth factor-b (TGF-b). Results. We showed that a TGF-b-induced EMT-like phenomenon is associated with increased CD13 expression in liver cancer cells. This phenomenon prevents further increases in the ROS level as well as the induction of apoptosis, promoting the survival of CD13? CSCs, Electronic supplementary material The online version of this article (doi:10.1245/s10434-011-2040-5) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2011 First Received: 14 March 2011; Published Online: 31 August 2011 H. Ishii e-mail: [email protected] M. Mori e-mail: [email protected]

whereas inhibition of CD13 stimulates apoptosis. Immunohistochemical analysis also indicated that after chemotherapy, CD13 was coexpressed with N-cadherin in surviving cancer cells within fibrous capsules. We have demonstrated that CD13 expression plays a role in supporting the survival of CSCs and that there is an EMTassociated reduction in ROS elevation. Conclusions. This novel and consistent linkage between functional CSC markers and the EMT phenomenon suggests a bona fide candidate for targeted therapy for EMTmediated invasion and metastasis of liver cancer.

Cancer stem cells (CSCs) are believed to share unique characters with normal stem cells, self-renewal ability, and produce differentiated cells.1,2 Additionally, CSCs play a key role in resistance to chemotherapy and radiotherapy. CSCs give rise to cancer recurrence even though most cancer cells have been disrupted after this therapy.3 Since the identification of CSCs in leukemia,4–6 CSCs have been reported in various solid tumors; brain, head and neck, breast, and colon.7–12 In studies of hepatocellular carcinoma, side population (SP) fraction, CD133, CD90, CD44, and epithelial cell adhesion molecules (EpCAMs) were reported as markers of CSCs or cancer-/tumor-initiating cells.13–20 We have reported CD13 as a novel liver CSCs marker and as candidate therapeutic target with some sets of functional analyses.21 We showed that CD13? cells mainly existed in SP fraction, contained potentially dye-long retaining cell, and had high tumorigenic activity. Additionally, CD13 ? CSCs showed strong chemoradiation resistance in vitro and in vivo, by protecting cells from DNA damage via regulation of the

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reactive oxygen species (ROS) level. Inhibition of CD13induced tumor cell apoptosis resulted in tumor disruption via blocking self-renewal activity. CD13 also has been associated with tumor invasion, regulation of angiogenesis, and resistance to apoptosis.22–24 Epithelial–mesenchymal transition (EMT) plays crucial roles in developmental processes, such as neural crest and mesoderm formation during embryogenesis, and has been focused as a key process in tumor invasion, metastasis, and tumorigenicity.25,26 EMT has been well characterized by numerous pathways of transcriptional factors, including Snail, Twist, and Zeb, which are generally promoted by transforming growth factor (TGF) b superfamily.25 In the study of mammary epithelial cells, it was reported that EMT generates normal mammary epithelial cells with properties of stem cells.27 In the study of breast cancer CSCs, it has reported that breast cancer cells increase malignant potential with increased CSCs phenotype by the treatment of EMT inducers.27 Furthermore, ROS have been proposed recently to be involved in tumor metastasis, which is a complicated process, including EMT of cancer cells and the microenvironment inside or around the tumor lesion.28,29 In the present study, we identify a TGF-b-induced EMTlike phenomenon is associated with increased CD13 expression in liver cancer cells and plays a role in the reduction of the intracellular ROS level, promoting the survival of liver CSCs. The present data indicate a novel and consistent linkage between CD13, a functional CSC marker, and the EMT phenomenon.

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Gene Expression Study Total RNA was prepared using TRIzol reagent (Invitrogen). Reverse transcription was performed with SuperScriptIII (Invitrogen). Quantitative real-time (qRT)-PCR was performed using a Light Cycler TaqMan Master kit (Roche Diagnostics). The expression of mRNA copies was normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. The PCR primers used for amplification were as follows: GAPDH, 50 -GTCAACGGATTTGGTC TGTATT-30 (forward) and 50 -AGTCTTCTGGGTGGCAGTGAT-30 (reverse); N-cadherin, 50 -ACAGTGGCCACCTA CAAAGG-30 (forward) and 50 -CCGAGATGGGGTTGA TAATG-30 (reverse); fibronectin, 50 -CAGTGGGAGACCTC GAGAAG-30 (forward) and 50 -TCCCTCGGAACATCA GAAAC-30 (reverse); vimentin, 50 -GAGTCCACTGAGTAC CGGAGAC-30 (forward) and 50 -TGTAGGTGGCAATCT CAATGTC-30 (reverse); E-cadherin, 50 -ACACCATCCTCAG CCAAGA-30 (forward) and 50 -CGTAGGGAAACTCTCT CGGT-30 (reverse); Bmi-1, 50 -TGTAAAACGTGTATTGTT CGTTAC-30 (forward) and 50 -CAATATCTTGGAGAGTT TTATCTGACC-30 (reverse); Notch-1, 50 -CGCACAAG GTGTCTTCCAG-30 (forward) and 50 -AGGATCAGTGG CGTCGTG-30 (reverse); Gclm, 50 -GAAGAAGATATTTT TCCTGTCATTGAT-30 (forward) and 50 -CCATTCATG TATTGAAGAGTGAATTT-30 (reverse); Gss, 50 -CCTGCTA GTGGATGCTGTCA-30 (forward) and 50 -TCATCCTGTTT GATGGTGCT-30 (reverse); c-Maf, 50 -AATACGAGAAGC TGGTGAGCAA-30 (forward) and 50 -CGGGAGAGGAAGG GTTGTC-30 (reverse).

MATERIALS AND METHODS Cell Culture

Flow Cytometric Analysis and Cell Sorting

Human hepatocellular carcinoma (HCC) cells, HuH7, and PLC/PRF/5, obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer (Tohoku University, Sendai, Japan) were cultured in an RPMI 1640 medium (Invitrogen) with 10% fetal bovine serum (FBS; GIBCO). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

The following antibodies were used to characterize cancer cells: allophycocyanin (APC)-conjugated antihuman CD133/1 (clone AC133, Miltenyi-Biotec), APCconjugated anti-human CD90 (clone 5E10, BioLegend), APC-conjugated anti-human CD13 (clone WM-15, eBioscience), APC-conjugated anti-human CD324 (E-cadherin, clone 67A4, Bio Legend), phycoerythrin (PE)-conjugated anti-human CD90 (BioLegend), PE-conjugated anti-human CD325 (N-cadherin, clone 8C11, eBioscience), and fluorescein isothiocyanate-conjugated anti-human CD13 (eBioscience). Dissociated cells were stained with anti-human CD13 with N-cadherin. The labeled cells were analyzed and sorted by the BD FACSAria II Cell Sorter System (Becton Dickinson), and the data were analyzed with Diva software (Becton Dickinson).

Induction and Reversal of EMT Cells were seeded and incubated in a standard medium for 48 h and then incubated in a serum-free medium supplemented with 5 lg/ml insulin and 10 ng/ml endothelial growth factor at 37°C in an atmosphere containing 5% CO2 for 48–72 hr with TGF-b1 (R&D) at concentrations of 0–5 ng/ml with daily replacement of the fresh culture medium.30

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Colony Formation Assay

RESULTS

Cells were plated in soft agar at a density of 1 9 103– 1 9 104 cells/3.5-cm dish. The base layer was 0.6% agar in the RPMI 1640 medium and the top was 0.2%. Cells were incubated for 30 days; colonies were stained with Diff-Quik kit (Sysmex).

TGF-b-Induced EMT Phenotypes in Liver Cancer Cells

Transplantation of Cancer Cells into Nonobese Diabetic/Severe Combined Immunodeficient (NOD/SCID) Mice Isolated cells were resuspended in 50 ll of PBS after sorting, and cell aliquots were diluted 1:1 with Growth Factor Reduced Matrigel Matrix (BD Biosciences) and injected subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice at densities of 5 9 102, 2.5 9 103, and 1 9 104 cells under anesthesia. After 15 weeks, all tumors were removed and analyzed. All of the animal studies were approved by ethics board of animal study of Osaka University. ROS Assay The ROS assay was performed as described previously.21 Cells were loaded with 10 lM of 20 ,70 -dichlorofluorescein diacetate (DCF-DA) at 37°C for 30 min, and cells were pretreated with 100–500 lg/ml of ubenimex (Nihon Kayaku) at 37°C for 24 h and stained with DCF-DA. To evaluate the effect of ROS to induce EMT, 0 to 10 lM of N-acetyl cysteine (NAC) (Wako Pure Chemical Industries), which has been known as a general antioxidant agent and ROS inhibitor, was added with or without TGF-b1. Immunohistochemistry Ten, fresh, HCC, surgical tissue specimens were obtained from Osaka University, Japan, with the patients’ informed consent and the approval of the Research Ethics Board of Osaka University. The VECTASTAIN Elite ABC Kit (Vector Laboratories) was used to detect signals from the antigen-antibody reactions. The primary anti-CD13 mouse polyclonal antibody (38C12; Neomarkers Laboratory Vision) and the primary anti-N-cadherin rabbit polyclonal antibody (YS; Immuno-Biological Laboratories) were used. Statistical Analysis Statistical analyses were performed using JMP 8.0.1 for Windows (SAS Institute). Possible differences between groups were analyzed using Student’s t test, v2 test, or Wilcoxon test. A probability level of 0.05 was chosen to indicate statistical significance.

To determine whether liver cancer cells (HuH7, PLC/ PRF/5) induced the EMT phenotype, we exposed liver cancer cells to TGF-b. Culturing the liver cancer cells with TGF-b changed their phenotype to spindle-shape morphology (Fig. 1a); this cell phenotype was detected at levels higher than 0.5 ng/ml for HuH7 and 1 ng/ml for PLC/PRF/5. An expression study using qRT-PCR indicated that the mesenchymal markers N-cadherin, fibronectin, and vimentin also were significantly elevated (Fig. 1b). FACS analysis showed that expression of the epithelial marker E-cadherin was decreased (Fig. 1c). Induced EMT Associated with Increased CD13 Expression in Liver Cancer Cells To assess whether TGF-b-induced EMT phenotype associate to liver cancer stem cell markers, HuH7 and PLC/ PRF/5 cells were incubated with or without TGF-b. By the treatment of TGF-b, expression of CD133 and CD90 was not changed in both HuH7 and PLC/PRF/5.15–19 In contrast, expression of CD13 was increased almost double in both HuH7 and PLC/PRF/5 (Fig. 2a).21 Multicolor analysis of N-cadherin and CD13 revealed that N-cadherin? cells were localized in CD13? cell fraction (Fig. 2b). Because the CD13 fraction was \3% in HuH7 and [30% in PLC/ PRF/5 cells under standard conditions, we used PLC cells in the subsequent studies. Tumorigenicity of TGF-b-Induced CD13? Cells To investigate colony formation ability, the soft agar assay was performed in CD13?/N-cadherin-, CD13?/ N-cadherin?, and CD13-/N-cadherin- cell fractions. The number of CD13-/N-cadherin? cells was not sufficient for analysis. The soft agar assay showed that after TGF-b stimulation, CD13?/N-cadherin-, and CD13?/N-cadherin? cells revealed significantly higher colony-forming ability compared with CD13-/N-cadherin- cells (P \ 0.001; Fig. 3a and b). There was no appreciable difference between N-cadherin? and N-cadherin- cells, which indicated that CD13? cells exhibited significantly higher colony-forming ability than CD13- cells. To investigate tumorigenic activity of CD13?/N-cadherin-, CD13?/ N-cadherin?, and CD13-/N-cadherin- cells, isolated 5 9 102, 2.5 9 103, and 1 9 104 cells of each fraction were inoculated into NOD-SCID mice. Serial transplantation experiment indicated that CD13?/N-cadherin- cells possess higher tumorigenic activity compared with CD13-/ N-cadherin- cells. CD13?/N-cadherin? cells formed tumor

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FIG. 1 TGF-b induces EMT in liver cancer cells. a Phase contrast microscopy of TGF-b treated (?) or untreated (-) cells of HuH7 and PLC/PRF/5 (PLC). The black arrow shows morphologically spindleshaped cells. b Expression analysis of N-cadherin (N-cad), vimentin (VIM), and fibronectin (FIB) by qRT-PCR. GAPDH was used as

internal control. c FACS analysis of E-cadherin with or without TGFb treatment. Control: cells were cultured in conditional medium containing with 10% FBS. TGF-b: cells were treated with (?) or without (-) TGF-b in serum-free medium. HuH7 cells: treated with 1 ng/ml of TGF-b. PLC cells: treated with 2 ng/ml of TGF-b

from 2.5 9 103 cells, which indicated that CD13?/ N-cadherin? cells possess higher tumorigenic activity than CD13-/N-cadherin- cells but lesser than that of CD13?/N-cadherin- (Fig. 3c and d). The data are compatible with the results of the in vitro soft agar assay (Fig. 3a and b).

TGF-b Increased the ROS Level in N-cadherin? Liver Cancer Cells It has been reported that the control of ROS is indispensable for hematopoietic stem cell maintenance.31,32 In cancer, low ROS levels and radiation-resistance in breast

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FIG. 2 TGF-b-induced EMT is associated with increased CD13 expression. a Expression of CD13 and CD133 in HuH7 cells (upper), and CD13 and CD90 in PLC cells (lower). Each number shows percentage of positive fraction of each marker. HuH7 cells: treated

with 1 ng/ml of TGF-b. PLC cells: treated with 2 ng/ml of TGF-b. Control: cells were cultured in conditional medium containing with 10% FBS. b Expression analysis of N-cadherin (N-cad) and CD13 with or without TGF-b treatment

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FIG. 3 CD13? liver cancer cells have higher tumorigenicity than CD13- cells. a Soft agar colony formation assay. After stimulation by TGF-b in culture, cells were isolated by FACS and indicated numbers of cells were subjected to assay. b Formed colonies (bar, 500 lm).

c Limiting dilution assay. The indicated numbers of CD13 ? PLC liver cancer cells were inoculated into NOD/SCID mice. d The resultant tumor volumes

CSCs and liver CSCs have been reported.21,33 To study the change of ROS status during EMT, intracellular ROS levels were measured by pro-oxidants using the 20 ,70 dichlorofluorescein diacetate (DCF-DA) stain. The data indicated that ROS level in CD13? cells was low compared with that in CD13- cells. ROS level of CD13?/N-cadherin? cells was higher than that of CD13?/N-cadherincells, suggesting that the TGF-b–induced EMT process is associated with an increase of ROS level (Fig. 4a). With regard to ROS scavengers in these cells, the qRT-PCR data indicated that the expression of GCLM and GSS was high in the CD13?/N-cadherin- cells compared with the CD13-/N-cadherin- cells (Fig. 4b). Interestingly, expression of GCLM and GSS was low in CD13?/N-cadherin? cells compared with CD13?/N-cadherin- cells (Fig. 4b), suggesting that TGF-b/EMT-induced ROS were metabolized to the appropriate level mainly via the CD13 function, rather than via the actions of the other scavengers, GCLM and GSS. We speculated that the survival of TGF-b–induced EMT cells might be dependent on CD13 function. To confirm the involvement of ROS in TGF-b-induced EMT cells, cells were treated with the antioxidant N-acetylcysteine (NAC) in culture and measured resulting ROS level. By TGF-b treatment, expression of the N-cadherin, fibronectin, and

vimentin was increased, but with NAC treatment, expression of N-cadherin and fibronectin was reduced (Fig. 4c). In contrast, expression of vimentin was increased by NAC with TGF-b treatment, suggesting that increased levels of ROS induce EMT, but should be controlled at suitable levels to sufficient induction of ROS. To assess the effect of CD13 inhibition on TGF-binduced EMT, cells were cultured in a medium containing ubenimex, which is an inhibitor of CD13. The data indicated that inhibition of CD13 by ubenimex elicited cell death and consequently suppressed N-cadherin in a dosedependent manner (Fig. 4d). Additionally, as CD13? cells were enriched by TGF-b treatment, they were reduced by TGF-b with NAC treatment (Fig. 4e). We concluded that CD13 plays a role in the TGF-b-induced EMT properties of CD13 ? liver cancer cells analyzed in the present study. TGF-b-Induced CD13? Cells with Stem Cell Properties We studied the expression of the stemness genes B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and translocation-associated notch protein TAN-1 (NOTCH1). The data indicated that BMI1 expression was significantly higher in CD13?/N-cadherin? cells than in

EMT in Hepatocellular Carcinoma FIG. 4 TGF-b increase intracellular ROS level and induce EMT. a ROS level was measured after TGF-b exposure in culture. ROS level was detected by DCF-DA in the CD13?, CD13-, CD13?N-cadherin-, and CD13?N-cadherin? fractions, as indicated. b qRT-PCR analysis of GSS and GCLM in CD13?N-cadherin-, CD13?N-cadherin-, and CD13?Ncadherin? fractions. c Expression analysis of N-cadherin (N-cad), fibronectin (FIB), and vimentin (VIM) by qRT-PCR. Cells were treated by NAC with or without TGF-b in culture as indicated concentration. d Cells were cultured in medium containing ubenimex, an inhibitor of CD13, as indicated concentration and expression of N-cadherin was assessed. Each number indicates the percentage of N-cadherin expressing cells in each condition. e Expression of CD13 after treatment of NAC with or without TGFb in culture as indicated concentration

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CD13-/N-cadherin- cells. Similarly, NOTCH1 expression was significantly higher in CD13?/N-cadherin? cells than in CD13?/N-cadherin- cells, although expression in CD13?/N-cadherin- cells was still significantly higher than that in CD13-/N-cadherin- cells (Fig. 5a). Taken

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together that CD13?/N-cadherin? cells have high tumorigenic ability next to CD13?/N-cadherin- cells but express highest BMI1 and NOTCH1 expression, it can be thought that CD13?/N-cadherin ? cells acquired stemness activity during EMT process induced by TGF-b.

S546 FIG. 5 CD13 and N-cadherin expression in human HCC sample. a Expression of BMI-1 and NOTCH1 in CD13?N-cadherin-, CD13?Ncadherin-, and CD13?N-cadherin? fractions. After stimulation by TGF-b in culture, cells were isolated by FACS, RNA was extracted and qRT-PCR was performed. b Immunohistochemical analysis of post-TACE HCC sample stained with anti-human CD13 (red) and DAPI (blue) for the nucleus (left) and N-cadherin (right). Lower panel shows a higher magnification (940) of the square in upper column (910)

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CD13?/N-cadherin? Cells are Expressed in TherapyResistant HCC Cells In the previous study, we have reported that CD13 ? cells form cellular cluster along to fibrous capsule, especially after post-transcatheter arterial lipiodol chemoembolization (TACE) therapy.21 To study N-cadherin expression in clinical hepatocellular carcinoma (HCC) sample, ten samples of HCC resected after TACE were used following study (Supplemental Table 1). Because CD13 was hard to detect in formalin-fixed sample, frozen sample was used to detect CD13 ? cells. Our data indicated that, in all of the post-TACE samples examined,

CD13 was found on the cell surface and N-cadherin expression was high (representative data shown in Fig. 5b). The surviving areas within the fibrous capsule of HCC tissue specimens taken after chemotherapy contained regions positive for both CD13 and N-cadherin; CD13 was expressed in adjacent regions of the HCC cells and formed bile canaliculi. Analysis of serial sections indicated that CD13 expression was combined with membrane expression of N-cadherin. The present data indicate that highly tumorigenic CD13?/N-cadherin? HCC cells survive within the fibrous capsule of HCC tissues, suggesting that they are involved in therapy resistance and the aggressive behaviors of tumor cells after TACE.

EMT in Hepatocellular Carcinoma

DISCUSSION Numerous reports have indicated that CSCs play a critical role in the generation of chemoradiation resistance.1,2 Hence the findings that the EMT process could induce stemness in CD24?/CD44 (low) CSC fractions in breast cancer, in CD24?/CD44?/CD133?/Aldehyde Dehydrogenase? CSCs in pancreatic adenocarcinomas, and in CD44?/CD24- CSCs in prostate cancer indicate a new epoch in cancer therapy.27,34,35 Although our understanding of the link between therapy resistance, EMT, and metastasis remains unclear, in the present study, we show for the first time the functional relevance of CSC markers in the EMT phenomenon; our data suggest that in liver cancer, the functional CSC marker CD13 plays a role in the reduction of intracellular ROS, which has been shown to contribute to drug resistance.21 Emerging evidence supports the notion that EMT is an important step in tumor invasion and metastasis and is intimately involved in de novo and acquired chemoradiation resistance.36 Consistent with this, the present data indicated that TGF-b exposure could induce the EMT phenotype but also elicit an increase in intracellular ROS and that this was reverted by the addition of a strong antioxidant (NAC) in culture. Recently, ROS have been found to be associated with tumor metastasis via tumor cell migration, invasion, and angiogenesis.36 Thus, our findings suggest that strong chemopreventive agents antagonizing ROS could be useful for targeted reversal of EMT, which could become a novel approach for the prevention of tumor progression and metastasis. However, this type of inhibition would not kill CSCs but just suppress the cells. We also noted that an acute blockade of CD13, a natural, mild scavenger of intracellular ROS, in CD13-dependent malignant CSCs resulted in the induction of apoptosis,21 suggesting the possible elimination of deleterious clones. The data suggest that a situation could arise wherein CSCs become accustomed to a relatively high level of intracellular ROS and are dependent on CD13 expression for the regulation of ROS levels; they survive by avoiding apoptosis. Thus, combining conventional chemotherapies, such as proliferation antagonizing 5-FU, with CSC-targeted reagents, such as ubenimex, may be beneficial for efficient tumor elimination. Although simultaneous administration of 5-FU and ubenimex has been reported to have a synergic effect on tumor growth, the biological function of CD13 in liver CSCs remains to be elucidated.21 To study the effect of CD13 inhibition on TGF-b-induced EMT, we cultured cells in a medium containing ubenimex, an inhibitor of CD13. Our data indicate that inhibition of CD13 by ubenimex elicited cell death and consequently resulted in dose-dependent suppression of N-cadherin, suggesting that

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CD13 plays a role in TGF-b-induced EMT properties in the CD13? liver cancer cells. As biosynthesis, our data indicate that TGF-b stimulation elicits N-cadherin expression (EMT) as well as CD13 expression, presumably via the involvement of glycolysis and oxidative stress-responsive transcription factor c-Maf upstream of the CD13 gene’s promoter (MAF-recognition element, MARE; Supplemental Fig. 1a and b), indicating that in the microenvironment of heterogeneous tumors, hypoxic conditions could increase the secretion of cytokines, such as IL6 and TGF-b from mesenchymal cells.37,38 In cancer cells, these stimulate the response of Stat3 and hypoxia-inducible factors (HIFs) downstream of IL6 and of Smad4 downstream of TGF-b.38 These in turn are involved in the transcriptional regulation of the proto-oncogene c-MAF, presumably via p300/CEBPbeta, AP1, and nuclear factor (NF) kappa B sites in its gene promoter.38–41 Consequently, the present study proposes a natural working hypothesis where the hypoxic and inflammatory conditions in the microenvironment elicit cellular responses to increased CD13 expression that are beneficial for the survival of EMTstimulated CSCs. Although the majority of the experiments in this study are based on cell lines, the expression and ROS analyses support the contention that PLC cells reflect clinical HCC and may hold promise for preclinical studies. This study also suggests that the future development of liver cancer therapy based on CSC and EMT concepts appears promising. We are undertaking further analyses of clinical HCC samples to provide the necessary confirmation of our contention using in vivo assays. ACKNOWLEDGMENT This study was supported in part by a grant from Core Research for Evolutional Science and Technology (CREST), a Grant-in-Aid for scientific research on Priority Areas (20012039), Grants-in-Aid for scientific research S (21229015) and C (20590313) from the Ministry of Education, Culture, Sports, Science, and Technology, and a grant from the Tokyo Biochemical Research Foundation, Japan.

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